U.S. patent application number 10/823433 was filed with the patent office on 2005-03-10 for rac-pk as a therapeutic agent or in diagnostics, screening method for agents and process for activating rac-pk.
Invention is credited to Alessi, Dario, Andjelkovic, Mirjana, Cohen, Philip, Cron, Peter David, Cross, Darren, Hemmings, Brian A..
Application Number | 20050053594 10/823433 |
Document ID | / |
Family ID | 46301967 |
Filed Date | 2005-03-10 |
United States Patent
Application |
20050053594 |
Kind Code |
A1 |
Alessi, Dario ; et
al. |
March 10, 2005 |
RAC-PK as a therapeutic agent or in diagnostics, screening method
for agents and process for activating RAC-PK
Abstract
The invention concerns RAC-PK and fragments thereof, as well as
activators and inhibitors of RAC-PK for use as medicaments,
particularly in the treatment of diseases concerned with
abnormalities in processes modulated by insulin, such as cellular
proliferation, insulin deficiency and/or excess blood sugar levels.
Moreover, the invention provides RAC-PK for use in screening
potential mimics or modulators thereof. A method for screening for
agents capable of affecting the activity of GSK3 is also disclosed.
The invention further provides a screening kit comprising the
RAC-PK as an active principle, and a method for screening compounds
which are candidate mimics or modulators of RAC-PK activity
comprising detecting specific interactions between the candidate
compounds and RAC-PK. There is also provided a process for
activating RAC-PK comprising treatment thereof with a phosphatase
inhibitor.
Inventors: |
Alessi, Dario; (Dundee,
GB) ; Andjelkovic, Mirjana; (Basel, CH) ;
Cohen, Philip; (Invergowrie, GB) ; Cron, Peter
David; (Basel, CH) ; Cross, Darren;
(Macclesfield, GB) ; Hemmings, Brian A.;
(Bettingen, CH) |
Correspondence
Address: |
NOVARTIS
CORPORATE INTELLECTUAL PROPERTY
ONE HEALTH PLAZA 104/3
EAST HANOVER
NJ
07936-1080
US
|
Family ID: |
46301967 |
Appl. No.: |
10/823433 |
Filed: |
April 12, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10823433 |
Apr 12, 2004 |
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10673091 |
Sep 26, 2003 |
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10673091 |
Sep 26, 2003 |
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09845667 |
Apr 30, 2001 |
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09845667 |
Apr 30, 2001 |
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09091763 |
Jun 19, 1998 |
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09091763 |
Jun 19, 1998 |
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PCT/GB96/03186 |
Dec 20, 1996 |
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10823433 |
Apr 12, 2004 |
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10147123 |
May 16, 2002 |
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10147123 |
May 16, 2002 |
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09542646 |
Apr 3, 2000 |
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09542646 |
Apr 3, 2000 |
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09091109 |
Jun 11, 1998 |
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09091109 |
Jun 11, 1998 |
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PCT/EP96/04811 |
Nov 5, 1996 |
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10823433 |
Apr 12, 2004 |
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09970000 |
Oct 3, 2001 |
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09970000 |
Oct 3, 2001 |
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09068702 |
May 13, 1998 |
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09068702 |
May 13, 1998 |
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PCT/EP96/04810 |
Nov 5, 1996 |
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Current U.S.
Class: |
424/94.5 ;
435/194 |
Current CPC
Class: |
C12N 9/12 20130101; C12Q
1/48 20130101; C12Q 1/42 20130101; A61K 38/00 20130101; A61P 35/00
20180101; C07K 14/47 20130101; C12N 9/1205 20130101; A61P 3/00
20180101 |
Class at
Publication: |
424/094.5 ;
435/194 |
International
Class: |
A61K 038/48; C12N
009/12 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 16, 1995 |
GB |
9523379.7 |
Dec 15, 1995 |
GB |
9525704.4 |
Dec 15, 1995 |
GB |
9525702.8 |
Dec 20, 1995 |
GB |
9526083.2 |
May 16, 1996 |
GB |
9610272.8 |
Jul 18, 1996 |
GB |
9615066.9 |
Claims
We claim:
1. A method of treating a disease related to glycogen metabolism
and/or protein synthesis comprising administering a composition of
RAC-PK, its analogues, isoforms, inhibitors, activators and/or the
functional equivalents thereof to a mammal in need thereof.
2. The method of claim 1 for the treatment of disease states where
glycogen metabolism and/or protein synthesis exhibits
abnormality.
3. The method of claim 1, wherein the disease related to glycogen
metabolism is diabetes.
4. The method of claim 1, wherein the disease related to protein
synthesis is cancer.
5. The method of claim 4, wherein the cancer is breast, pancreatic
or ovarian cancer.
6. The method of claim 1, wherein RAC-PK is RAC-PK.alpha., .beta.
or .gamma., an analogue, isoform, inhibitor, activator or a
functional equivalent thereof.
7. The method of claim 6, wherein RAC-PK, its analogue, isoform or
functional equivalent is modified at one or both of amino acids 308
and 473 by phosphorylation and/or mutation.
8. A pharmaceutical composition comprising RAC-PK, its analogues,
isoforms, inhibitors, activators and/or the functional equivalents
thereof.
9. A peptide comprising an amino acid sequence
Arg-Xaa-Arg-Yaa-Zaa-Ser/Thr- -Hyd, where Xaa is any amino acid, Yaa
and Zaa are any amino acid, and Hyd is a large hydrophobic residue,
or a functional equivalent of such a peptide.
10. The peptide of claim 9, wherein Hyd is Phe or Leu, or a
functional equivalent thereof.
11. A peptide as claimed in claim 9, wherein Yaa or Zaa or both are
an amino acid other than glycine.
12. A peptide as claimed in claim 9, having the amino acid sequence
as set forth in SEQ ID NO: 5, or a functional equivalent
thereof.
13. A method of identifying agents able to influence the activity
of GSK3, said method comprising: (a) exposing a test substance to a
substrate of GSK3; and (b) detecting whether said substrate has
been phosphorylated.
14. A method of identifying agents which influence the activity of
RAC-PK, comprising: (a) exposing a test substance to a sample
containing RAC-PK, to form a mixture; and (b) exposing said mixture
to the peptide of claim 9.
15. The method of claim 14, comprising the additional step of
detecting whether said peptide has been phosphorylated.
16. The method of claim 15, wherein the phosphorylation state(s) of
one or both of amino acids 308 and 473 on RAC-PK is determined.
17. The method of claim 13, wherein the test substance is an
analogue, isoform, inhibitor or activator of RAC-PK.
18. The method of claim 13, wherein steps (a) or (b), or both, are
carried out in the presence of divalent cations and ATP.
19. An agent capable of influencing the activity of RAC-PK, its
isoforms, analogues and/or functional equivalents, by modifying
amino acids 308 and/or 473 by phosphorylation or mutation.
20. A method of determining the ability of a substance to affect
the activity or activation of RAC-PK, the method comprising
exposing the substance to RAC-PK and phosphatidyl inositol
polyphosphate and determining the interaction between RAC-PK and
the phosphatidyl inositol polyphosphate.
21. A method of determining the ability of a substance to combat
diabetes, cancer or any disorder which involves irregularity of
protein synthesis or glycogen metabolism, the method comprising
exposing the substance to RAC-PK and phosphatidyl inositol
polyphosphate and determining the interaction between RAC-PK and
the phosphatidyl inositol polyphosphate.
22. The method of claim 20, wherein the interaction between RAC-PK
and the phosphatidyl inositol polyphosphate is measured by
assessing the phosphorylation state of RAC-PK.
23. The method of claim 22, wherein the phosphorylation state of
RAC-PK at Thr308 and/or Ser473 is assessed.
24. A method of identifying activators or inhibitors of GSK3
comprising exposing the substance to be tested to GSK3 and
determining the state of activation of GSK3.
25. A method as claimed in claim 24, wherein the state of
activation of GSK3 is determined by assessing its
phosphorylation.
26. A method of determining the suitability of a test substance for
use in combatting diabetes, cancer or any disorder which involves
irregularity of protein synthesis or glycogen metabolism, the
method comprising exposing the substance to be tested to GSK3 and
determining the state of activation of GSK3.
27. A method for screening for inhibitors or activators of enzymes
that catalyze the phosphorylation of RAC-PK, the method comprising
exposing the substance to be tested to one or more enzymes upstream
of RAC-PK and nucleoside triphosphate and determining whether (and
optionally to what extent) the RAC-PK has been phosphorylated on
Thr308 and/or Ser473.
28. A method for screening potential modulators of insulin-mediated
intracellular signalling comprising the steps of: (a) incubating
RAC-PK or a fragment thereof with the compound to be screened; and
(b) detecting interaction between the compound and RAC-PK or its
fragment.
29. The method according to claim 28, wherein RAC-PK is
activated.
30. Method according to claim 28, wherein the RAC-PK fragment is
selected from the PH domain, the catalytic domain and the
C-terminal domain.
31. A modulator of insulin-mediated intracellular signalling when
identified by a method according to claim 28.
32. A modulator according to claim 31, which is selected from the
group consisting of IMPDH, GSK-3 and a polypeptide comprising SEQ
ID NO: 1.
33. A kit comprising: (a) RAC, or a fragment thereof; (b) means for
incubating RAC-PK or its fragment with a compound to be screened;
and (c) means for detecting an interaction between RAC-PK or its
fragment and the compound.
34. A RAC-PK polypeptide which is activated by effecting one or
both of the mutations Thr308.fwdarw.D and Ser473.fwdarw.D.
35. A process for producing an active kinase of a signalling
pathway comprising treatment thereof with a phosphatase
inhibitor.
36. A process according to claim 35, which is carried out in vitro
and comprises the steps of: (a) incubating together a kinase of a
signalling pathway (b) an agent capable of phosphorylating the
kinase in order to activate it and a phosphatase inhibitor; and (c)
purifying the kinase from the incubation mixture.
37. A process according to claim 36, wherein the phosphorylating
agent is a kinase of the signalling pathway which is capable of
phosphorylating the kinase of interest, thereby activating it.
38. A process according to claim 35, which is performed in cells
which contain kinases of the signalling pathway.
39. A process for screening candidate modulators of a signalling
pathway comprising: (a) incubating together a kinase of a
signalling pathway and a phos phatase inhibitor; (b) adding the
candidate signalling pathway modulator; and (c) determining the
activity of the kinase.
40. A process according to claim 39, wherein steps (a) and (b) are
performed contemporaneously.
41. A process according to claim 39, wherein the phosphatase
inhibitor is okadaic acid.
42. The process of claim 39, wherein the phosphatase inhibitor is
vanadate.
43. The process of claim 39, wherein the kinase of the signalling
pathway is RAC-PK.
44. The process of claim 39, wherein the signalling pathway is an
insulin-dependent signalling pathway.
Description
[0001] This application is a Continuation In Part of application
(I) Ser. No.10/673,091, Filing Date Sep. 26, 2003, pending, which
is a continuation of Ser. No. 09/845,667, filed Apr. 30, 2001, now
abandoned, which is a continuation of Ser. No. 09/091,763, filed
Jun. 19, 1998, now abandoned, which is a National Stage of
PCT/GB96/03186, filed Dec. 20, 1996; (II) of application Ser.
No.10/147,123, filed May 16, 2002, which is a continuation of Ser.
No. 09/542,646, filed Apr. 3, 2000, now abandoned, which is a
continuation of Ser. No. 09/091,109, filed Jun. 11, 1998, now
abandoned, which is a National Stage of PCT/EP96/0481 1, filed
November 5, 1996; and (III) of application Ser. No. 09/970,000,
pending, which is a Continuation of Ser. No. 09/068,702, filed May
13, 1998, now abandoned, which is a National Stage of
PCT/EP96/04810, filed Nov. 5, 1996.
[0002] The present invention relates to the control of glycogen
metabolism and protein synthesis, in particular through the use of
insulin. Particularly, the present invention is related to the use
of RAC protein kinase (RAC-PK) as a therapeutic agent, as a ligand
for screening molecules for a possible interaction with RAC-PK and
to a method for identifying molecules involved in signal
transduction. Further, the present invention relates to a method
for producing an active form of a kinase involved in an
insulin-dependent signalling pathway.
BACKGROUND OF THE INVENTION
[0003] Many people with diabetes have normal levels of insulin in
their blood, but the insulin fails to stimulate muscle cells and
fat cells in the normal way (type II diabetes). Currently it is
believed that there is a breakdown in the mechanism through which
insulin signals to the muscle and fat cells.
[0004] Protein phosphorylation and dephosphorylation are
fundamental processes for the regulation of cellular functions.
Protein phosphorylation is prominently involved in signal
transduction, where extracellular signals are propagated and
amplified by a cascade of protein phosphorylation and
dephosphorylation. Two of the best characterized signal
transduction, where extracellular signals are propagated and
amplified by a cascade of protein phosphorylation and
dephosphorylation. Two of the best characterized signal
transduction pathways involve the c-AMP-dependant protein kinase
(PKA) and protein kinase C (PKC). Each pathway uses a different
second messenger molecule to activate the protein kinase, which, in
turn, phosphorylates specific target molecules.
[0005] A novel subfamily of serine (Ser)/threonine (Thr) kinases
has been recently identified and cloned, termed herein the RAC-PK
[see Jones et al., Proc Natl Acad Sci USA, Vol. 88, No. 10, pp.
4171-4175 (1991); and Jones, Jakubowicz and Hemmings, Cell Regul,
Vol. 2, No. 12, pp. 1001 -1009 (1991)], but also known as RAC-PK or
Akt. RAC kinases have been identified in two closely-related
isoforms, RAC.alpha. and RAC.beta., which share 90% homology at the
gene sequence. Mouse RAC.alpha. (c-akt) is the cellular homologue
of the viral oncogene v-akt, generated by fusion of the Gag protein
from the AKT8 retrovirus to the N-terminus of murine c-akt. Human
RAC.beta. is found to be involved in approximately 10% of ovarian
carcinomas, suggesting an involvement of RAC kinases in cell growth
regulation.
[0006] Another kinase implicated in cell growth control is S6
kinase, known as p70S6K. S6 kinase phosphorylates the 40S ribosomal
protein S6, an event which up-regulates protein synthesis and is
believed to be required in order for progression through the G1
phase of the cell cycle. The activity of p70S6K is regulated by
Ser/Thr phosphorylation thereof, and it is itself a Ser/Thr kinase.
The p70S6K signaling pathway is believed to consist of a series of
Ser/Thr kinases, activating each other in turn and leading to a
variety of effects associated with cell proliferation and growth.
RAC-PK is believed to lie on the same signaling pathway as p70S6K,
but upstream thereof.
[0007] RAC kinases contain an amino-terminal pleckstrin homology
(PH) domain. See Haslam, Koide and Hemmings, Nature, Vol. 363, No.
6427, pp. 309-310 (1993). The PH domain was originally identified
as an internal repeat, present at the amino and carboxy-termini of
pleckstrin, a 47 kDa protein which is the major PKC substrate in
activated platelets. See Tyers et al., Nature, Vol. 333, No. 6172,
pp. 470-473 (1988). The superfamily of PH domain containing
molecules consists of over 90 members including Ser/Thr kinases,
e.g., RAC, Nrk, .beta.-adrenergic receptor kinase (.beta.ARK) and
PKC.mu.; tyrosine kinases, e.g., Bruton's tyrosine kinase (Btk),
Tec and Itk; GTPase regulators, e.g., ras-GAP, ras-GRF, Vav, SOS
and BCR; all known mammalian phospholipase Cs; cytoskeletal
proteins, e.g., .beta.-spectrin, AFAP-110 and syntrophin; "adapter"
proteins, e.g., GRB-7 and 3BP2; and kinase substrates, e.g.,
pleckstrin and IRS-1.
[0008] While the PH domain structure has been solved for
.beta.-spectrin, dynamin and pleckstrin's amino-terminal domain,
its precise function remains unclear. The presence of PH domains in
many signaling and cytoskeletal proteins implicates it in mediating
protein-protein and membrane interactions. Indeed, the PH domain of
the .beta.ARK appears partly responsible for its binding to the
.beta..gamma.-subunits of the heterotrimeric G-proteins associated
with the .beta.-adrenergic receptor, while the PH domain of the Btk
appears to mediate an interaction with PKC. Several PH domains have
been shown to be able to bind
phosphatidyl-inositol-4-5-bisphosphate in vitro, although
weakly.
[0009] IMPDH is a highly-conserved enzyme (41% amino acid identity
between bacterial and mammalian sequences) involved in the
rate-limiting step of guanine biosynthesis. In mammals there are
two isoforms, 84% identical, called type I and type II which are
differentially-expressed. See Natsumeda et al., J Biol Chem, Vol.
265, No. 9, pp. 5292-5295 (1990). Type I is
constitutively-expressed at low levels while the type II mRNA and
protein levels increase during cellular proliferation. IMPDH
activity levels are also elevated during rapid proliferation in
many cells. See Collart and Huberman, J Biol Chem, Vol. 263, No.
30, pp. 15769-15772 (1988).
[0010] By measuring the metabolic fluxes, the proliferative index
of intact cancer cells has been shown to be linked with the
preferential channelling of IMP into guanylate biosynthesis.
Inhibition of cellular IMPDH activity results in an abrupt
cessation of DNA synthesis and a cell-cycle block at the G.sub.1-S
interface. The specific inhibition of IMPDH by tiazofurin and the
subsequent decline in the GTP pool, results in the down regulation
of the G-protein ras, which is involved in many signal transduction
pathways leading to cellular proliferation. For review see Avruch,
Zhang and Kyriakis, Trends Biochem Sci, Vol. 19, No. 7, pp. 279-283
(1994).
[0011] Interestingly, p53 has been implicated in regulating IMPDH
activity levels. See Sherley, J Biol Chem, Vol. 266, No. 36, pp.
24815-24828 (1991). Here a moderate over-expression of p53 (3- to
6-fold) induces a profound growth arrest which is rescued by purine
nucleotide precursors. Indeed, the p53 over-expression induces a
specific block in IMP to XMP conversion, and a diminished activity
level of IMPDH. The p53 block does not affect the rate of RNA
synthesis, nor is the phenotype rescued by deoxynucleotides
indicating that a lack of precursors for DNA synthesis is also not
the cause of the block. It would seem most likely that this effect
is mediated through a down-regulation of the GTP pool required by
G-proteins, such as ras.
[0012] The above observations suggest that IMPDH type II is
primarily involved in producing XMP which is channelled into the
GTP pool which is crucial for the regulation of G-proteins involved
in signal transduction, such as ras. It may be that the type I
enzyme provides a basal level of XMP that is channelled into the
GTP/dGTP pools required for RNA and DNA synthesis. Changes in IMPDH
type II activity would alter the GTP/GDP ratio by specifically
altering the GTP component which could greatly affect ras
signalling pathways as ras is sensitive to small changes in the
GTP/GDP ratio.
[0013] Glycogen synthase kinase-3 (GSK3) is implicated in the
control of several processes important for mammalian cell
physiology, including glycogen metabolism and the control of
protein synthesis by insulin, as well as the modulation of activity
of several transcription factors, such as AP-1 and CREB. GSK3 is
inhibited in vitro by serine phosphorylation caused by MAP kinase
and p70.sup.S6K, kinases which lie on distinct insulin-stimulated
signalling pathways.
[0014] GSK3 is responsible for serine phosphorylation in glycogen
synthase, whose dephosphorylation underlies the stimulation of
glycogen synthesis by muscle. Thus, GSK3 inactivates glycogen
synthase, resulting in an increase in blood sugar levels. Insulin
inhibits the action of GSK3, which, in combination with the
concomitant activation of phosphatases which dephosphory late
glycogen synthase, leads to the activation of glycogen synthase and
the lowering of blood sugar levels.
[0015] GSK3 is inhibited in response to insulin with a half-time of
2 minutes, slightly slower than the half-time for activation of
RAC-PK.alpha. (1 minute). Inhibition of GSK3 by insulin results in
its phosphorylation at the same serine residue (serine 21) which is
targeted by RAC-PK.alpha. in vitro. Like the activation of
RAC-PK.alpha., the inhibition of GSK3 by insulin is prevented by
phosphatidyl inositol (PI-3) kinase inhibitors wortmannin and LY
294002. The inhibition of GSK3 is likely to contribute to the
increase in the rate of glycogen synthesis [see Cross et al.,
Biochem J, Vol. 303, Pt. 1, pp. 21-26 (1994)] and translation of
certain mRNAs by insulin. See Welsh et al., Biochem J, Vol. 303,
Pt. 1, pp. 15-20 (1994).
[0016] We have used the yeast two-hybrid system [see Fields and
Song, Nature, Vol. 340, No. 6230, pp. 245-246 (1989); and Chien,
Bartel, Sternglanz and Fields, Proc Natl Acad Sci USA, Vol. 88, No.
21, pp. 9578-9582 (1991)] to determine if RAC-PK could function by
forming specific interactions with other proteins. We have
identified RAC-PK as interacting with human inosine-5'
monophosphate dehydrogenase (IMPDH) type II, and with a novel
protein termed RAC-PK Carboxy-Terminal Binding Protein (CTBP).
RAC-PK stimulates IMPDH type II activity. In conjunction with the
known role of IMPDH in GTP biosynthesis, our findings suggest a
role for RAC-PK in the regulation of cell proliferation.
[0017] Moreover, using a peptide derived from GSK3 and GSK3 itself,
we have been able to show that RAC-PK interacts with,
phosphorylates and inactivates GSK3. This implicates RAC-PK in the
regulation of insulin-dependent signalling pathways, which control
cellular proliferation. Taken together, these results suggest a
major involvement for RAC-PK in the control of insulin action.
[0018] Many growth factors trigger the activation of
phosphatidylinositol (PI) 3-kinase, the enzyme which converts PI
4,5 bisphosphate (PIP2) to the putative second messenger PI 3,4,5
trisphosphate (PIP3) and RAC-PK lies downstream of PI 3-kinase. See
Franke et al., Cell, Vol. 81, No. 5, pp. 727-736 (1995).
RAC-PK.alpha. is converted from an inactive to an active form with
a half-time of about 1 minute when cells are stimulated with PDGF
[see Franke et al. (1995), supra], EGF or basic FGF [see Burgering
and Coffer, Nature, Vol. 376, No. 6541, pp. 599-602 (1995)] or
insulin [see Cross et al. (1995), supra; and Kohn, Kovacina and
Roth, EMBO J, Vol.14, No. 17, pp. 4288-4295 (1995)] or
perpervanadate. See Andjelkovic et al., Proc Natl Acad Sci USA,
Vol. 93, No. 12, pp. 5699-5704 (1996). Activation of RAC-PK by
insulin or growth factors is prevented if the cells are
pre-incubated with inhibitors of PI 3-kinase (wortmannin or LY
294002) or by over-expression of a dominant negative mutant of PI
3-kinase. See Burgering and Coffer (1995), supra. Mutation of the
tyrosine residues in the PDGF receptor that when phosphorylated
bind to PI 3-kinase also prevent the activation of RAC-PK.alpha..
See Burgering and Coffer (1995), supra; and Franke et al. (1995),
supra.
[0019] When isolated from natural sources, especially convenient
sources, such as tissue culture cells, RAC-PK and other signaling
kinases are normally in the inactive state. In order to isolate
active PKs, it is necessary to stimulate cells in order to switch
on the signaling pathway to yield active kinase. Moreover, when
cells expressing kinase enzymes are used in kinase activity assays,
it is necessary to employ activating agents prior to conducting the
assay. Thus, cells are normally stimulated with mitogens and/or
activating agents, such as IL-2, platelet-derived growth factor
(PDGF), insulin, epidermal growth factor (EGF) and basic fibroblast
growth factor (bFGF). Such agents are expensive and, when it is
desired to produce active kinases or to activate cells in large
amounts, the use of such agents is disadvantageous.
[0020] Screening of candidate compounds for activity as inhibitors
of RAC-PK, or other signaling kinases in order to identify
candidate immunosuppressive or anti-proliferative agents requires a
plentiful supply of PK. Using modern day technology, it is possible
to produce large quantities of virtually any desired protein in
recombinant DNA expression systems. In the case of kinases, such as
those with which we are presently concerned, however, such systems
are unsatisfactory because the proteins produced would be
unphosphorylated and therefore inactive. There is therefore a
requirement to identify a cost-effective way to produce
phosphorylated PKs which can be employed in screening
procedures.
[0021] It is known [see Jano et al., Biochemistry, Vol. 85, pp.
406-410 (1988)] that vanadate can activate p70S6K itself. The
mechanism of this activation, however, is not known. We have now
found that vanadate acts generally on signaling kinases, activating
them and preventing deactivation by phosphatases. Moreover, we have
found that okadaic acid, a different class of compound from
vanadate which interacts with different proteins, may be used to
similar effect.
SUMMARY OF THE INVENTION
[0022] According to the invention, there is provided RAC-PK and
fragments, analogues, isoforms and functional equivalents thereof,
as well as activators and inhibitors of RAC-PK for use in the
treatment of diseases concerned with abnormalities in processes
modulated by insulin, such as cellular proliferation, insulin
deficiency and/or excess blood sugar levels, e.g., in the treatment
of type II diabetes and cancer, such as ovarian, breast and
pancreatic cancer. Moreover, the invention provides RAC-PK for use
in screening potential mimics or modulators thereof. The invention
further provides a screening kit comprising the RAC-PK as an active
principle, and a method for screening compounds which are candidate
mimics or modulators of RAC-PK activity comprising detecting
specific interactions between the candidate compounds and
RAC-PK.
[0023] The present invention also provides a novel peptide
comprising the amino acid sequence Arg-xaa-Arg-Yaa-zaa-Ser/Thr-Hyd,
where Xaa is any amino acid, Yaa and Zaa are any amino acid
[preferably not glycine (Gly)] and Hyd is a large hydrophobic
residue, such as Phe or Leu, or a functional equivalent thereof.
The invention also provides a method for screening for substances
which inhibit the activation of RAC-PK in vivo by preventing its
interaction with PIP3 or P13,4-bisP. Thus the invention also
provides a method of determining the ability of a substance to
affect the activity or activation of RAC-PK. The method of the
invention can also be used for identifying activators or inhibitors
of GSK3. The invention also provides a method for screening for
inhibitors or activators of enzymes that catalyse the
phosphorylation of RAC-PK.
[0024] There is also provided a process for producing an active
form of a kinase involved in an insulin depedent signalling
pathway.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0025] In a first aspect, the invention provides RAC-PK or a
fragment thereof, or a modulator thereof except vanadate and
wortmannin, for use as a medicament.
[0026] Vanadate, which term as used herein includes various forms
thereof, such as ortho- and metavanadate, pervanadate and other
related vanadium ions, is known as a therapeutic agent in the
treatment of diabetes. See, e.g., U.S. Pat. No. 5,421,125, European
Patent Application Nos. 0521787, 0264278 and 0245979. In UK Patent
Application No. 9525702.8 (Ciba-Geigy AG), filed Dec. 15, 1995, it
is disclosed that vanadate is a potent activator of kinases of the
insulin-stimulated signalling pathways and of RAC-PK in particular.
Accordingly vanadate exerts its therapeutic effect on diabetes by
stimulating RAC, which phosphorylates GSK3 and deactivates it,
leading to a lowering in blood sugar levels.
[0027] RAC-PK and activators thereof are therefore useful in the
treatment of diabetes and other diseases where blood sugar levels
are excessive. Conversely, inhibitors of RAC-PK, such as wortmannin
and okadaic acid are useful in the treatment of diseases involving
insufficiency in blood sugar levels.
[0028] In the present invention, RAC-PK may be any isoform of
RAC-PK as described in the literature, from any species. Human
RAC-PK is preferred. Human RAC-PK.alpha. is represented in SEQ ID
NO. 3. Domains of RAC-PK are the individual functional portions
thereof, such as the PH domain, the catalytic domain and the
C-terminal domain. Fragments of RAC, which include domains of RAC,
are functionally-active portions of the RAC-PK which may be used in
the present invention in place of RAC. Fragments of RAC-PK are
preferably the domains thereof, advantageously the PH domain, the
catalytic domain and the C-terminal domain. In each case, the
terminology used embraces mutants and derivatives of RAC-PK and its
fragments which can be created or derived from the
naturally-occurring protein according to available technology. For
instance, nucleic acids encoding RAC-PK may be mutated without
affecting the nature of the peptide encoded thereby, according to
the degeneracy of the amino acid code. Moreover, conservative amino
acid substitutions may be made in RAC-PK or a fragment thereof,
substantially without altering its function. Further additions,
deletions and/or substitutions which improve or otherwise alter the
function of RAC-PK or a fragment thereof are envisaged and included
within the scope of the invention.
[0029] The invention also provides the use of RAC-PK or a fragment
thereof, or a modulator of RAC-PK activity, except vanadate, for
the preparation of a medicament for use in the treatment of
diseases involving an anomaly in blood sugar levels, such as
diabetes.
[0030] Moreover, the invention provides the use of RAC-PK or a
fragment thereof, or a modulator of RAC-PK activity, except
wortmannin, for the preparation of a medicament for use in the
treatment of abnormalities in cellular proliferation.
[0031] The antibiotic wortmannin, which is known to inhibit
phosphatidylinositol 3-OH kinase (PI-3K) activation, targets signal
transduction and indirectly inactivates inter alia RAC, possibly
via PI-3K. Wortmannin has been indicated in the treatment of
neoplastic conditions. However, the broad involvement of the
various isoforms of RAC-PK in mitogenic signal transduction, as
well as insulin-dependent signalling has hitherto not been known.
We have now shown that RAC-PK is involved in the regulation of both
GSK3 and IMPDH, a factor involved in growth control. It can be
concluded, therefore, that RAC-PK plays a central role in growth
control.
[0032] Therapeutic agents according to the invention may be
formulated conventionally, according to the type of agent. Where
the agent is a salt, such as vanadate, it is conveniently
formulated in aqueous solution at neutral pH and administered
orally at room temperature. In the case of a peptide medicament,
such as RAC-PK itself, more elaborate delivery techniques, such as
liposomal delivery, may be required in order to introduce the
peptide into target cells. Delivery systems for peptide
therapeutics are documented in the art.
[0033] The identification of RAC-PK as a major mediator in growth
control permits the design of screening systems to identify
putative therapeutic agents for use in treating anomalies of growth
control. Thus, in a second aspect of the invention there is
provided a method for screening potential modulators of
intracellular signalling comprising the steps of:
[0034] (a) incubating RAC-PK or a fragment thereof with the
compound to be screened; and
[0035] (b) detecting interaction between the compound and RAC.
[0036] The screening may be carried out using complete RAC-PK or a
fragment thereof. In particular, it has been shown that the PH
domain of RAC-PK is important in mediating many of its effects, as
set out, e.g., in UK patent application No. 9525703.6 (Ciba-Geigy
AG), filed Dec. 15, 1995. Moreover, as disclosed hereinbelow,
RAC-PK interacts with IMPDH via the PH domain. The interaction is
not observable in the yeast two-hybrid system if complete RAC-PK is
used, although in vitro binding of RAC-PK to IMPDH occurs.
[0037] Interactions also occur between other fragments of RAC-PK
and its physiological targets and regulators. For example, GSK3
binds to RAC-PK via the catalytic domain, as evidenced by the
phosphorylation of GSK3 by RAC. CTBP, on the other hand, does not
bind the catalytic or PH domains but binds specifically to the
carboxy terminal domain of RAC.
[0038] Preferably, therefore, the invention includes incubating the
compound to be screened with a fragment of RAC, which is
advantageously the PH domain, the catalytic domain or the carboxy
terminal domain.
[0039] RAC-PK fragments for use in the method of the present
invention may be in the form of isolated fragments, or in the form
of the fragment complexed with further polypeptides. For example,
in the case of the two-hybrid system, the fragment is complexed to
a DNA binding or transcriptional activation domain derived from
another protein, such as the yeast activator GAL4.
[0040] Moreover, the RAC-PK used in the method of the invention may
be in the form of a mutant thereof, e.g., a constitutively
activated kinase. An important activating residue is T308, present
in the so-called T-loop between subdomains 7 and 8 of the kinase. A
general guide to kinase structure is given in Woodgeft, Protein
Kinases, IRL Press, UK (1994). Substitution of T308 with aspartic
acid results in a clear increase in basal activity of the kinase,
which however retains a potential for further activation. The
invention therefore provides a RAC-PK in which Thr308 has been
mutated to Asp.
[0041] Preferably, Ser473 is additionally mutated to Asp.
Phosphorylation of this residue is required for full activation of
RAC-PK in vivo, and the T308/S473 double mutant (both residues
converted to Asp) shows a constitutive activity 18-fold higher than
native RAC-PK. The double mutant is not susceptible to further
activation.
[0042] The mutations may be carried out by means of any suitable
technique. Preferred, however, is in vitro site-directed
mutagenesis of a nucleotide sequence encoding RAC and subsequent
expression of RAC in a recombinant DNA expression system. This
method is an in vitro mutagenesis procedure by which a defined site
within a region of cloned DNA can be altered. See Zoller and Smith,
Methods Enzymol, Vol. 100, pp. 468-500 (1983); and Botstein and
Shortle, Science, Vol. 229, No. 4719, pp. 1193-1201 (1985). Methods
for site-directed mutagenesis are well-known to those of skill in
the art, as exemplified by Sambrook et al., Molecular Cloning--A
Laboratory Manual, Cold Spring Harbor, N.Y., USA (1989), and the
number of commercially-available in vitro mutagenesis kits.
[0043] The compound to be screened may be present in essentially
pure, uncomplexed form, or may be complexed with chemical groups or
further polypeptides. In the case of the two hybrid system, it is
complexed to a DNA binding or transcriptional activation domain, in
order to complement the PH domain.
[0044] Isolated PH domain for use in the present invention may be
prepared as set forth in UK patent application No. 9525705.1
(Ciba-Geigy AG), filed Dec. 15, 1995. Where a small quantity of PH
domain suffices, however, PH domain may be obtained by expressing a
nucleic acid sequence encoding it in bacterial cell culture in the
form of a fusion protein which is subsequently cleaved according to
techniques known in the art. For example, amino acids 1-131 of RAC,
which encode the PH domain, may be expressed as a fusion protein,
advantageously with glutathione-S-transferase (GST), subsequently
cleaving the fusion protein with thrombin and isolating the domain
by protein purification techniques, such as FPLC. This method gives
a relatively small yield of pure soluble PH domain.
[0045] Carboxy and kinase domains are likewise advantageously
synthesised as fusion proteins, for instance as GST fusions.
[0046] RAC-PK or a fragment thereof for use in the present
invention may be prepared as set forth in UK patent application No.
9525702.8. Alternatively, RAC-PK may be expressed in recombinant
cell culture. Baculovirus vectors, specifically intended for insect
cell culture, are especially preferred and are widely obtainable
commercially, e.g., from Invitrogen and Clontech. Other virus
vectors capable of infecting insect cells are known, such as
Sindbis virus. See Hahn, Hahn, Braciale and Rice, Proc Natl Acad
Sci USA, Vol. 89, No. 7, pp. 2679-2683 (1992). The baculovirus
vector of choice [reviewed by Miller, Ann Rev Microbiol, Vol. 42,
pp. 177-199 (1988)] is Autographa californica multiple nuclear
polyhedrosis virus (AcMNPV).
[0047] Typically, the heterologous gene replaces at least in part
the polyhedrin gene of AcMNPV, since polyhedrin is not required for
virus production. In order to insert the heterologous gene, a
transfer vector is advantageously used. Transfer vectors are
prepared in E. coli hosts and the DNA insert is then transferred to
AcMNPV by a process of homologous recombination. Baculovirus
techniques useful in the present invention are standard and
well-known in the art. See O'Reilly et al., Baculovirus expression
vectors; A laboratory manual, Oxford University Press Inc., New
York (1994), as well as in literature published by suppliers of
commercial baculovirus kits, e.g., Pharmingen.
[0048] Incubation conditions will vary according to the precise
method used to detect the interaction between the PH domain and the
screened compound. In the case of transcription activation
detection systems, such as the yeast two-hybrid system, incubation
conditions are suitable for gene transcription, such as those
prevailing inside a living cell. Other detection systems, however,
will require different incubation conditions. For example, if the
detection of interaction is based on relative affinity in a
chromatographic assay, e.g., as is known in affinity
chromatography, conditions will be adjusted to promote binding and
then gradually altered, such that the point at which the screened
compound no longer binds to the RAC-PK PH domain may be
determined.
[0049] The detection method may employ the natural fluorescence of
tryptophan at position 22 (Trp.sup.22) in the RAC-PK PH domain,
which is inhibited by certain interactions with the domain, as set
forth in UK patent application No. 9525703.6. Briefly, fluorescence
of the amino-terminal Trp residue in the PH domains of certain PH
domain containing proteins may be detected by exciting the molecule
to fluoresce at the appropriate frequency and monitoring the
emission. The N-terminal Trp.sup.22 of RAC, e.g., fluoresces at 345
nm when excited at 290 nm. Techniques for monitoring protein
fluorescence are widely-known in the art. We have shown that the PH
domain of RAC-PK binds phospholipid with high affinity, which
suggests that RAC-PK may be membrane-bound in vivo via the PH
domain. Binding of phospholipid to the RAC-PK PH domain quenches
the natural fluorescence of the N-terminal Trp.sup.22. Interaction
of the PH domain with the cell membrane is believed to be important
for the stable interaction of RAC-PK with membrane bound partners
in signalling pathways, such that disruption of this interaction
will lead to modulation of the signalling effect through the
dissociation of the signalling molecule from the cell membrane. The
modulation could be either down-regulating, e.g., if the otherwise
stable interaction of the molecule with membrane-bound partners is
a stimulatory interaction, or up-regulating, in the event that the
interaction is an inhibitory interaction. Accordingly, a compound
which is a candidate modulator of signal response may be screened
for by means of a method comprising the steps of:
[0050] (a) incubating the compound with the PH domain of a
signalling molecule which is capable of fluorescing; and
[0051] (b) determining the phospholipid-induced modulation in the
fluorescence of the PH domain, an alteration of the fluorescence in
the presence of the compound being indicative of a functional
interaction between the compound and the PH domain.
[0052] In this case, the incubation conditions will be adjusted to
facilitate the detection of fluorescence at 345 nm when the PH
domain is excited at a frequency of 290 nm.
[0053] Incubation according to the invention may be achieved by a
number of means, but the basic requirement is for RAC-PK or a
fragment thereof and the screened compound to be able to come into
contact with each other. This may be achieved by admixing RAC-PK or
a fragment thereof and the compound, or by producing them in situ,
such as by expression of nucleic acids encoding them. Where the
RAC-PK or RAC-PK fragment and/or the compound are in the form of
fusions with other polypeptides, they may be expressed as such in
situ.
[0054] Preferably, the method of the invention is based on a
two-hybrid system. Such systems detect specific protein:protein
interactions by exploiting transcriptional activators having
separable DNA-binding and transcription activating domains, such as
the yeast GAL4 activator. A reporter gene is operatively linked to
an element responsive to the transcriptional activator being used,
and exposed to RAC-PK or a fragment thereof and the compound to be
screened, one of which is complexed to the transcription activating
domain of the transcriptional activator and the other of which is
joined to the DNA binding domain thereof. If there is a specific
interaction between RAC-PK or a fragment thereof and the compound,
the DNA binding and transcription activating domains of the
transcriptional activator will be brought into juxtaposition and
transcription from the reporter gene will be activated.
[0055] Alternatively, the detection may be based on observed
binding between RAC-PK or a fragment thereof, such as its PH domain
or its catalytic domain, and the screened compound, or a fragment
thereof. For example, the interaction between RAC-PK and the
insulin mediator GSK3 is detected hereinbelow by monitoring the
interaction of a peptide surrounding the major phosphorylation site
of GSK3 known to be responsible for its inactivation with RAC-PK.
In a similar manner, the involvement of RAC-PK on the activation or
inactivation of a particular compound may be screened for by
monitoring the interaction of a portion thereof known to be
involved in modulation events with RAC.
[0056] RAC-PK or a fragment thereof may be used to screen for
compounds which bind thereto by incubating it with the compound to
be screened and subsequently "pulling down" RAC-PK complexes with a
RAC-specific antibody. Antibodies suitable for immunoprecipitation
or immuno-affinity chromatography may be prepared according to
conventional techniques, known to those of ordinary skill in the
art, and may be monoclonal or polyclonal in nature. For example,
see Lane et al., EMBO J, Vol. 11, No. 5, pp. 1743-1749 (1992).
After the RAC-compound complex has been isolated by affinity, the
compound may be dissociated from the RAC-PK antibody and
characterised by conventional techniques.
[0057] The interaction of RAC-PK or a fragment thereof with the
screened compound may also be observed indirectly. For example, an
inhibitor or activator of RAC-PK function may be detected by
observing the effects of RAC-PK on a substrate in the presence or
absence of the compound.
[0058] The activity of RAC-PK or the catalytic domain thereof may
be assessed by means of a kinase activity assay, employing a
substrate for the kinase. For example, myelin basic protein (MBP)
may be used, in accordance with established assay procedures.
Physiological substrates, such as GSK3, may also be used.
Alternatively, RAC-PK activity may be assessed by determining the
degree of activating phosphorylation of RAC-PK itself.
Advantageously, phosphorylation on residues normally implicated in
kinase activation is assessed. RAC-PK, as disclosed in UK patent
application No. 9525702.8, is preferentially activated by
phosphorylation at Ser and Thr residues.
[0059] The assay of the invention may be used to measure the direct
effect of the candidate compound on RAC, or it may be used to
determine the effect of the compound on a kinase acting upstream
thereof in a signalling pathway. In the latter situation, RAC-PK
acts as a substrate for the upstream kinase and the activity of the
upstream kinase is assessed by determining the phosphporylation
state or the activity of RAC-PK.
[0060] In order to obtain a meaningful result, the activity of
RAC-PK exposed to the candidate immunosuppressive or
antiproliferative agent should be compared to the activity of
RAC-PK not exposed to the agent, a modulation of RAC-PK activity
being indicative of potential as a modulator of cell proliferation
and/or insulin signal transduction.
[0061] Promising compounds may then be further assessed by
determining the properties thereof directly, for instance, by means
of a cell proliferation assay. Such an assay preferably involves
physical determination of proliferation in cells which have been
subjected to kinase activation by a phosphatase inhibitor, exposed
to the candidate RAC-PK modulator and optionally subsequently
stimulated with a mitogen, such as a growth factor, IL-2 or PMA.
More simply, the assay may involve exposure of unstimulated cells
to the candidate modulator, followed by stimulation with a
phosphatase inhibitor.
[0062] The invention further comprises the use of RAC-PK or a
fragment thereof in a screening system. The screening system is
preferably used to screen for compounds which are modulators of
insulin activity, particularly where that activity is related to
glycogen metabolism or cell proliferation.
[0063] Kits useful for screening such compounds may be prepared,
and will comprise essentially RAC-PK or a fragment thereof together
with means for detecting an interaction between RAC-PK and the
screened compound. Preferably, therefore, the screening kit
comprises one of the detection systems set forth hereinbefore.
[0064] RAC-PK for use in kits according to the invention may be
provided in the form of a protein, e.g., in solution, suspension or
lyophilised, or in the form of a nucleic acid sequence permitting
the production of RAC-PK or a fragment thereof in an expression
system, optionally in situ. Preferably, the nucleic acid encoding
RAC-PK or a fragment thereof encodes it in the form of a fusion
protein, e.g., a GST fusion.
[0065] In a still further embodiment, the invention provides a
compound which interacts directly or indirectly with RAC-PK or a
fragment thereof. In the case of indirectly acting compounds,
agents, such as insulin and wortmannin, are excluded. Such a
compound may be inorganic or organic, e.g., an antibiotic, and is
preferably a proteinaceous compound involved in intracellular
signalling. For example, the compound may be CTBP (SEQ ID NOs: 1
and 2).
[0066] Compounds according to the invention may be identified by
screening using the techniques described hereinbefore, and prepared
by extraction from natural sources according to established
procedures, or by synthesis, especially in the case of low
molecular weight chemical compounds. Proteinaceous compounds may be
prepared by expression in recombinant expression systems, e.g., a
baculovirus system as described hereinbefore or in a bacterial
system, e.g., as described in UK patent application No. 9525705.1.
Proteinaceous compounds are mainly useful for research into the
function of signalling pathways, although they may have a
therapeutic application.
[0067] Low molecular weight compounds, on the other hand, are
preferably produced by chemical synthesis according to established
procedures. They are primarily indicated as therapeutic agents. Low
molecular weight compounds and organic compounds in general may be
useful as insulin mimics or anti-proliferative agents.
[0068] The present invention further provides the use of RAC-PK,
its analogues, isoforms, inhibitors, activators and/or the
functional equivalents thereof to regulate glycogen metabolism
and/or protein synthesis, in particular, in disease states where
glycogen metabolism and/or protein synthesis exhibits abnormality,
e.g., in the treatment of type II diabetes; also in the treatment
of cancer, such as ovarian, breast and pancreatic cancer. A
composition comprising such agents is also covered by the present
invention, and the use of such a composition for treatment of
disease states where glycogen metabolism and/or protein synthesis
exhibit abnormality.
[0069] The present invention also provides a novel peptide
comprising the amino acid sequence Arg-xaa-Arg-Yaa-zaa-Ser/Thr-Hyd,
where Xaa is any amino acid, Yaa and Zaa are any amino acid
(preferably not Gly), and Hyd is a large hydrophobic residue, such
as Phe or Leu, or a functional equivalent thereof. Represented in
single letter code, a suitable peptide would be RXRX'X'S/TF/L,
where X' can be any amino acid, but is preferably not Gly; Gly can
in fact be used, but other amino acids are preferred. Typical
peptides include GRPRTSSFAEG (SEQ ID NO: 5), RPRAATC (SEQ ID NO: 6)
or functional equivalents thereof. The peptide is a substrate for
measuring RAC-PK activity.
[0070] The invention also provides a method for screening for
substances which inhibit the activation of RAC-PK in vivo by
preventing its interaction with PIP3 or PI3,4-bisP.
[0071] Thus the invention also provides a method of determining the
ability of a substance to affect the activity or activation of
RAC-PK, the method comprising exposing the substance to RAC-PK and
phosphatidyl inositol polyphosphate, i.e., PIP3 or PI3,4-bisP, etc)
and determining the interaction between RAC-PK and the phosphatidyl
inositol polyphosphate. The interaction between RAC-PK and the
phosphatidyl inositol polyphosphate can conveniently be measured by
assessing the phosphorylation state of RAC-PK, preferably at T308
and/or S473, e.g., by measuring transfer of radiolabelled .sup.32P
from the PIP3, e.g., to the RAC-PK and/or by SDS-PAGE.
[0072] The method of the invention can also be used for identifying
activators or inhibitors of GSK3, such a method can comprise
exposing the substance to be tested to GSK3, and optionally, a
source of phosphorylation, and determining the state of activation
of GSK3, optionally by determining the state of its
phosphorylation. This aspect of the invention can be useful for
determining the suitability of a test substance for use in
combatting diabetes, cancer, or any disorder which involves
irregularity of protein synthesis or glycogen metabolism.
[0073] The invention also provides a method for screening for
inhibitors or activators of enzymes that catalyse the
phosphorylation of RAC-PK, the method comprising exposing the
substance to be tested to:
[0074] (a) one or more enzymes upstream of RAC-PK;
[0075] (b) RAC-PK; and optionally
[0076] (c) nucleoside triphosphate and determining whether, and
optionally to what extent the RAC-PK has been phosphorylated on
T308 and/or S473.
[0077] Also provided is a method of identifying agents able to
influence the activity of GSK3, said method comprising:
[0078] (a) exposing a test substance to a substrate of GSK3;
and
[0079] (b) detecting whether, and optionally, to what extent said
peptide has been phosphorylated.
[0080] The test substance may be an analogue, isoform, inhibitor or
activator of RAC-PK, and the above method may be modified to
identify those agents which stimulate or inhibit RAC-PK itself.
Thus such a method may comprise the following steps:
[0081] (a) exposing the test substance to a sample containing
RAC-PK, to form a mixture;
[0082] (b) exposing said mixture to a peptide comprising the amino
acid sequence defined above or a functional equivalent thereof
(usually in the presence of Mg.sub.2+ and ATP); and
[0083] (c) detecting whether, and optionally, to what extent said
peptide has been phosphorylated.
[0084] In this aspect, the method of the invention can be used to
determine whether the substance being tested acts on RAC-PK or
directly on GSK3. This can be done by comparing the phosphorylation
states of the peptide and RAC-PK; if the phosphorylation state of
GSK3 is changed but that of RAC-PK is not then the substance being
tested acts directly on GSK3 without acting on RAC-PK. In a further
aspect, the present invention provides a method of treatment of the
human or non-human, preferably mammalian, animal body, said method
comprising administering RAC-PK, its analogues, inhibitors,
stimulators or functional equivalents thereof to said body. Said
method affects the regulation of glycogen metabolism in the treated
body.
[0085] The method of treatment of the present invention may be of
particular use in the treatment of type II diabetes, where
desirably an activator of RAC-PK is used, so that the
down-regulation of GSK3 activity due to the action of RAC-PK is
enhanced.
[0086] The method of treatment of the present invention may
alternatively be of particular use in the treatment of cancer, such
as ovarian cancer, where desirably an inhibitor of RAC-PK is used,
so that the down-regulation of GSK3 activity due to the action of
RAC-PK is depressed. Other cancers associated with irregularities
in the activity of RAC-PK and/or GSK3 may also be treated by the
method, such as pancreatic cancer and breast cancer.
[0087] Stimulation of RAC-PK with insulin increases activity
12-fold within 5 minutes and induces its phosphorylation at Thr308
and Ser473. RAC-PK transiently-transfected into cells can be
activated 20-fold in response to insulin and 46-fold in response to
IGF-1 and also became phosphorylated at Thr308 and Ser473. The
activation of RAC-PK and its phosphorylation at both Thr308 and
Ser473 can be prevented by the phosphatidylinositol (P1) 3-kinase
inhibitor wortmannin. The phosphorylation of Thr308 and Ser473 act
synergistically to activate RAC-PK.
[0088] MAPKAP kinase-2-phosphorylated RAC-PK at Ser473 in vitro
increases activity 7-fold, an effect that can be mimicked (5-fold
activation) by mutating Ser473 to Asp. Mutation of Thr308 to Asp
also increases RAC-PK activity 5-fold and subsequent
phosphorylation of Ser473 by MAPKAP kinase-2 stimulates activity a
further 5-fold, an effect mimicked (18-fold activation) by mutating
both Thr308 and Ser473 to Asp. The activity of the Asp308/Asp473
double-mutant was similar to that of the fully phosphorylated
enzyme and could not be activated further by insulin. Mutation of
Thr308 to alanine (Ala) did not prevent the phosphorylation of
transfected RAC-PK at Ser473 after stimulation of 293 cells with
insulin or IGF-1, but abolished the activation of RAC-PK.
Similarly, mutation of Ser473 to Ala did not prevent the
phosphorylation of transfected RAC-PK at Thr308 but greatly reduced
the activation of transfected RAC-PK. This demonstrates that the
activation of RAC-PK by insulin or IGF-1 results from the
phosphorylation of Thr308 and Ser473 and that phosphorylation of
both residues is preferred to generate a high level of RAC-PK
activity in vitro or in vivo. Also, phosphorylation of Thr308 in
vivo is not dependent on the phosphorylation of Ser473 or vice
versa, that the phosphorylation of Thr308 and Ser473 are both
dependent on PI 3-kinase activity and suggest that neither Thr308
nor Ser473 phosphorylation is catalyzed by RAC-PK itself.
[0089] Thus, it is preferred that the present invention
incorporates the use of any agent which affects phosphorylation of
RAC-PK at amino acids 308 and/or 473, e.g. insulin, inhibitors of
PI 3-kinase, such as wortmannin or the like. The use of RAC-PK,
itself altered at amino acids 308 and/or 473, e.g., by
phosphorylation and/or mutation, is also suitable.
[0090] In a variation of the method of the present invention,
stimulation or inhibition of RAC-PK may be assessed by monitoring
the phosphorylation states of amino acids 308 and/or 473 on RAC-PK
itself.
[0091] Different isoforms of RAC-PK may be used or targeted in the
present invention, e.g., RAC-PK.alpha., .beta. or .gamma..
[0092] We have observed that modulation of RAC-PK activity appears
to be effected by reversible phosphorylation, in which the
equilibrium of the phosphorylation/dephosphorylation reaction is
shifted in order to change the levels of active RAC-PK with respect
to its inactive form. Build-up of the active form may therefore be
promoted by inhibition of the dephosphorylation reaction, achieved
by treatment with a phosphatase inhibitor.
[0093] A surprising aspect of the present invention is that
tyrosine phosphatase inhibitors, such as vanadate, are able to
activate RAC-PK notwithstanding the fact that, as is disclosed
herein, this kinase is activated by phosphorylation at Ser and Thr
residues.
[0094] It is known that vanadate activates p70S6K. The invention
accordingly does not extend to the use of vanadate to activate
p70S6K. However, the use of phosphatase inhibitors, such as okadaic
acid, which acts through a quite different mechanism, is part of
the present invention.
[0095] As referred to herein, the signalling pathways are the
activation cascades which ultimately regulate signal transduction
and kinases of these pathways are kinases whose in vivo targets
include at least one entity which contributes to such signal
transduction. Preferably, the signalling pathways of the invention
are insulin-dependent signalling pathways, which are responsible
for transduction of signals from insulin and other growth factors.
Without in any way wishing to place any limitation on the present
invention, one such a pathway is believed to be triggered in vivo
by binding of growth factors, such as insulin and the like to their
receptors, which stimulates inter alia PI-3K. PI-3K in turn
directly or indirectly phosphorylates RAC-PK, which indirectly
leads to the eventual phosphorylation of p70S6K.
[0096] Treatment of kinases according to the invention in order to
activate them requires the exposure of the kinase to a
phosphorylating agent, such as another kinase of the signaling
pathway and the phosphatase inhibitor. This may be accomplished,
e.g., in vitro by:
[0097] (a) incubating together a kinase of a signalling pathway, an
agent capable of phosphorylating the kinase in order to activate
it, and a phosphatase inhibitor; and
[0098] (b) purifying the kinase from the incubation mixture.
[0099] The phosphorylating agent should be effective to
phosphorylate the kinase on residues which lead to activation
thereof. In the case of RAC-PK, the phosphorylating agent
advantageously targets Ser and Thr residues.
[0100] Preferably, the phosphorylating agent is one or more kinases
of the signalling pathway which act, in the presence of suitable
activating factors, to phosphorylate and thereby activate the
kinase of interest. Preferably, this is accomplished by recovering
active kinase enzyme form phosphatase-inhibitor treated cells,
which contain the required signaling pathway kinases.
[0101] In the context of the present invention, in vitro signifies
that the experiment is conducted outside a living organism or cell.
In vivo includes cell culture. Treatment of cells in vivo with
phosphatase inhibitors is especially effective for the preparation
of active RAC-PK. However, since RAC-PK and other signaling
kinases, e.g., p70S6K, are on the same pathway, activation of
RAC-PK results in the activation of other kinases on the same
signalling pathway, e.g., p70S6K itself. The invention therefore
includes a method for activating kinases on signalling pathways in
general, except for p70S6K, especially where such kinases are
downstream of RAC-PK in the pathway.
[0102] Cells which produce kinases which may be used in the present
invention generally include any cell line of mammalian origin,
especially fibroblast cell lines, such as RAT-1, COS or NIH 3T3.
Swiss 3T3 cells are particularly preferred. Where human cell lines
are used, human embryonic kidney 293 cells are preferred.
[0103] Phosphatase inhibitors are agents which inhibit protein
dephosphorylation by inhibiting the activity of phosphatase
enzymes. A phosphatase has essentially the inverse activity of a
kinase, and removes phosphate groups.
[0104] Examples of phosphatase inhibitors are vanadate and okadaic
acid, with vanadate being the more effective agent in the case of
RAC-PK. However, the action of vanadate is believed to be indirect,
since it is a specific tyrosine phosphatase inhibitor and RAC-PK
does not appear to be stimulated by tyrosine phosphorylation.
Okadaic acid, on the other hand, which is known to act directly on
phosphatase PP2A, appears to directly inhibit dephosphorylation of
RAC-PK.
[0105] The use of other phosphatase inhibitors is envisaged and
limited only by the suitability of such inhibitors for
administration to the particular cell line being used. Vanadate and
okadaic acid are believed to be generally applicable, but those of
skill in the art will recognize that other phosphatase inhibitors
are available and that their activity and suitability may easily be
determined by routine empirical testing. For example, phosphatase
inhibitors which may be suitable in the present invention include
calyculin A, cantharidic acid, cantharidin, DTX-1, microcystin,
nodularin and tautomycin. These and other phosphatase inhibitors
are available commercially, e.g., from Calbiochem.
[0106] The phosphatase inhibitor is administered to cells in their
normal growth medium, which may be serum free. Serum is itself
observed to stimulate kinase activity, but is expensive and its
function may be substituted by a phosphatase inhibitor according to
the present invention. Suitable concentrations of phosphatase
inhibitors include levels from 0.01-10 mM, preferably 0.1-1 mM. The
most preferred concentration for vanadate is 0.1 mM.
[0107] The method of the invention may comprise additional steps
intended to isolate the desired active kinase from the cells in
which it is produced. Such steps are conventional procedures
familiar to those skilled in the art and may be substituted for
equivalent processes within the scope of the invention. The
preferred process, however, comprises the steps of homogenizing the
cells, removing cell debris, e.g., by centrifugation, and
separating the desired kinase by affinity purification.
[0108] Homogenization may be carried out in a standard isotonic
lysis buffer, advantageously containing a proteinase inhibitor,
such as phenylmethyl sulphonyl fluoride (PMSF) and a phosphatase
inhibitor in order to inhibit deactivation of the kinase during the
purification procedure. The cells are disrupted, thereby releasing
the cytoplasmic and nuclear contents thereof into the lysis
buffer.
[0109] Cell debris is then advantageously removed from the lysed
cellular preparation, preferably by centrifuging the mixture in
order to pellet all particulate matter. Only the soluble fraction
remains in the supernatant.
[0110] The supernatant can then be subjected to standard protein
purification techniques in order to isolate the kinase of interest
if desired. Preferred methods, especially for relatively low volume
preparations, involve affinity chromatography. Such techniques may
employ an anti-kinase antibody or antiserum immobilised to a
suitable matrix. Other immobilized binding agents, such as
substrate analogues, may be employed.
[0111] Antibodies useful for immunoseparation of activated kinases
according to the invention may be prepared according to techniques
known in the art. In order to prepare a polyclonal serum, e.g., an
antigenic portion of the desired kinase, consisting of a peptide
derived therefrom, such as a C-terminal peptide, or even the whole
kinase, optionally in the presence of an adjuvant or conjugated to
an immunostimulatory agent, such as keyhole limpet haemocyanin, is
injected into a mammal, such as a mouse or a rabbit, and antibodies
are recovered therefrom by affinity purification using a
solid-phase bound kinase or antigenic portion thereof. Monoclonal
antibodies may be prepared according to established procedures.
[0112] Alternatively, and especially for larger scale preparations,
separation procedures not involving affinity chromatography may be
used.
[0113] For example, numerous methods are available in the art for
separating polypeptides on the basis of size, such as
chromatography and gel electrophoresis. Preferred are methods which
perform a purification function, as well as a size separating
function, while not introducing unacceptable contaminants. Thus,
methods, such as step or continuous gradient centrifugation,
particularly using sucrose gradients, dialysis techniques using
controlled-pore membranes and membrane (Amicon) centrifugation, are
preferred. Especially preferred, however, is size exclusion
chromatography, typically performed using porous beads as the
chromatographic support. Size exclusion chromatography is, e.g.,
described by Stellwagen in Deutscher, Ed., Guide to Protein
Purification, Academic Press, Inc., San Diego, Calif., pp. 317-328
(1990).
[0114] Alternative purification methods, described in general in
Deutscher (1990), supra, include chromatography based on separation
by charge difference, such as ion exchange chromatography using an
exchange group, such as DEAE or CM bound to a solid phase packing
material, such as cellulose, dextran, agarose or polystyrene. Other
methods include hydroxyapatite column chromatography [see, e.g.,
Gorbunoff, Methods Enzymol, Vol. 117, pp. 370-380 (1985)], and
general affinity chromatography using glass beads or reactive dyes
as affinity agents.
[0115] Advantageously, cation exchange chromatography may be
employed, such that protein elution can be tailored to take into
account the known or estimated PI of the kinase in question. The PI
for any kinase may be determined experimentally, by isoelectric
focusing. In this manner, it is possible selectively to elute from
the cation exchange resin those proteins having a PI at or around
that of the kinase, which results in a high degree of
purification.
[0116] The invention further provides the use of an active kinase
prepared according to the invention in a method for screening
potential modulators of signalling pathways. Thus, the claimed
method may comprise the additional step of exposing the kinase to a
potential inhibitor and subsequently assessing the activity of the
kinase in order to determine the effectiveness of the
modulator.
[0117] The invention accordingly provides a method for screening
candidate modulators of signalling pathways comprising:
[0118] (a) incubating together a kinase of a signalling pathway and
a phosphatase inhibitor;
[0119] (b) adding a candidate modulator of the signalling pathway;
and
[0120] (c) determining the activity of the kinase.
[0121] The exposure to the modulator may be performed on the
activated or inactivated kinase either in a cell-free environment,
optionally after purification of the kinase from the crude cellular
preparation, or in situ in the cells which produce the kinase,
after phosphatase inhibitor activation. Steps (a) and (b) may
therefore be reversed, or conducted contemporaneously.
[0122] In step (a), especially if the assay is to be performed in
vitro, an agent capable of phosphorylating the kinase may be added
to the incubation mixture. Phosphatase inhibitors activate kinases
by preventing dephosphorylation, so a phosphorylating agent will be
required. Advantageously, the phosphorylating agent is a kinase of
an insulin-dependent signalling pathway or an analogue thereof.
Moreover, factors may be required to initiate or assist signal
transduction in the signalling pathway. For example, it the
compound being tested is a rapamycin analogue which binds FKBP,
FKBP will be required in the incubation mixture.
[0123] Preferably, however, the procedure is carried out in vivo in
cells containing kinases of the signalling pathway. In such an
assay, the phosphatase inhibitor replaces serum or other agents
previously employed as external stimulating agents to activate
kinases of the signalling pathway.
[0124] The activity of the kinase may be assessed by means of a
kinase activity assay, employing a substrate for the kinase. For
example, MBP may be used, in accordance with established assay
procedures. Physiological substrates, such as the 40S ribosomal
subunit, or S6, may also be used. Alternatively, kinase activity
may be assessed by determining the degree of phosphorylation of the
kinase. Advantageously, phosphorylation on residues normally
implicated in kinase activation is assessed. The identification of
such residues, which is part of the present invention, is set forth
below.
[0125] The assay of the invention may be used to measure the direct
effect of the candidate compound on the assayed kinase, or it may
be used to determine the effect of the compound on a kinase acting
upstream thereof in the signalling pathway. In the latter
situation, the assayed kinase acts as a substrate for the upstream
kinase and the activity of the upstream kinase is assessed by
determining the phosphporylation state or the activity of the
assayed kinase.
[0126] In order to obtain a meaningful result, the activity of the
assayed kinase exposed to the candidate modulator of the signalling
pathway should be compared to the activity of the kinase not
exposed to the agent, an inhibition of kinase activity being
indicative of potential as an immunosuppressive or
anti-proliferative.
[0127] Compounds which demonstrate elevated levels of kinase
inhibition may then be further assessed by determining the
immunosuppressive or anti-proliferative properties thereof
directly, for instance, by means of a cell proliferation inhibition
assay. Such an assay preferably involves physical determination of
T-cell proliferation in cells which have been subjected to kinase
activation by a phosphatase inhibitor, exposed to the candidate
kinase inhibitor and optionally subsequently stimulated with a
mitogen, such as a growth factor, IL-2 or PMA. More simply, the
assay may involve exposure of unstimulated cells to the candidate
inhibitor, followed by stimulation with a phosphatase
inhibitor.
[0128] According to a further aspect of the invention, we have been
able to determine which sites are important for the phosphorylation
of kinases, particularly those of the p70.sup.S6K/RAC-PK family.
Surprisingly, the majority of activating phosphorylation appears to
take place on Ser and Thr residues. It is known that
phosphorylation may in certain cases be mimicked by replacement of
the phosphorylated amino with an acidic amino acid, such as
aspartic acid or glutamic acid.
[0129] The invention accordingly provides a recombinant RAC-PK
protein wherein at least one threonine residue involved in
activation of the kinase through phosphorylation in vivo is
replaced with an acidic amino acid residue. Moreover, the invention
provides a method for screening compounds which inhibit signalling
by RAC-PK comprising exposing cells treated with the constitutively
active recombinant RAC-PK to the compounds.
[0130] For example, an important activating residue is Thr308,
present in the so-called T-loop between subdomains 7 and 8 of the
kinase. A general guide to kinase structure is given in Woodgett
(1994), supra. Substitution of Thr308 with aspartic acid results in
a clear increase in basal activity of the kinase, which however
retains a potential for further activation. The invention therefore
provides a RAC-PK in which Thr308 has been mutated to Asp.
[0131] Preferably, Ser473 is additionally mutated to Asp.
Phosphorylation of this residue is required for full-activation of
RAC-PK in vivo, and the Thr308/Ser473 double-mutant (both residues
converted to Asp) shows a constitutive activity 18-fold higher than
native RAC-PK. The double-mutant does not retain the capability for
further activation.
[0132] Constitutively active kinases according to the invention may
be employed in place of the phosphatase inhibitor activated kinase
in screening techniques as described herein. Advantageously, such
constitutively activated kinases require no external stimulating
agents.
[0133] The present invention will now be described in more detail
in the accompanying examples which are provided by way of
non-limiting illustration, and with reference to the accompanying
drawings.
EXAMPLE 1
[0134] Specific Interaction of RAC-PK with IMPDH
[0135] a. Bacterial and Yeast Strains
[0136] All yeast strains and plasmids for two-hybrid experiments
are obtained from Clontech (Palo Alto, Calif.) as components of the
MATCHMAKER Two Hybrid System or from Dr. Nathans, Howard Hughes
Medical Institute, Baltimore, Md. Yeast strains SFY526, e.g., MATa,
Ura3-52, His3-200, Ade2-101, Lys2-801, Trp1-901, Leu2-3, 112,
can.sup.r, Gal4-542, Gal80-538 and Ura3::GAL1-lacZ; HF7c, e.g.,
MATa, Ura3-52, His3-200, Lys2-801, Ade2-101, Trp1-901, Leu2-3, 112,
Gal4-542, Gal80-538, Lys2::Gal1-His3 and Ura3::(Gal4
17-mer).sub.3-CYC1 -LacZ; and PCY2 [see Chevray and Nathans, Proc
Natl Acad Sci USA, Vol. 89, No. 13, pp. 5789-5793 (1992)], e.g.,
MAT.alpha., His3-200, Ade2-101, Lys2-801, Trp1-63, Leu2-3,
Gal4-542, Gal80-538 and Ura3::Gal1-LacZ, are used to assay for
protein-protein interactions. Yeast strain HF7c is used for library
screening. SFY526 and PCY2 have the upstream activating sequence
and TATA sequence of the GAL1 promoter fused to the LacZ gene. In
HF7c, His3 is fused to a Gal1 promoter sequence and LacZ is fused
to three copies of a 17-mer Gal4 consensus sequence plus the TATA
sequence of the CYC1 promoter. Both His3 and LacZ are responsive to
Gal4 activation. Yeast techniques including transformation are
performed according to the instructions in the MATCHMAKER Two
Hybrid System and as described Ausubel et al., Current Protocols in
Molecular Biology, John Wiley and Sons, New York, N.Y. (1994). The
bacterial strains XL1 -blue (Statagene) and JM109 are employed in
the cloning of plasmids and the production of GST fusion proteins.
The bacterial strains JM109(DE3), BL21(DE3)pLysS and BL21(DE3)pLysE
(Invitrogen) are used for the production of (His).sub.6-tagged
proteins. General molecular biological techniques are performed as
previously described in Sambrook et al. (1989), supra; and Davis et
al., Basic Methods in Molecular Biology, Elsevier Science
Publishing Co., New York, N.Y. (1986).
[0137] b. Plasmid Construction
[0138] Yeast vector plasmids containing the Gal4 DNA binding domain
(amino acids 1-147, pGBT9) and the Gal4 activation domain (amino
acids 768-881, pGAD424), as well as the control plasmids pCL1
(full-length Gal4 gene), pVA3 (p53 gene), pTD1 (SV40 large T
antigen), and pLAM5' (human lamin C gene) are from Clontech. The
yeast vector pPC62, containing the Gal4 DNA binding domain, is from
Dr. Nathans. The GST fusion vector pGEX-2T is from Pharmacia. The
baculovirus transfer vector (pVL1392) and the (His).sub.6-tag
vector (pRSET-A) are from Invitrogen. pGBT-PH127, pGBT-PH150,
pGBT-PHI-III and pGBT-PHIII-VI contain in-frame fusions of amino
acids 1-127, 1-150, 1-47 and 47-127 of the human RAC.alpha. PH
domain, respectively, with the Gal4 DNA binding domain. They are
constructed by subcloning PCR fragments generated with specific
oligonucleotides into the EcoRI-BamHI sites of pGBT9. pGEX-PH131,
pGEX-PH-KIN, pGEX-PH-KIN-CT, pGEX-KIN-CT, pGEX-KIN and pGEX-CT
contained in-frame fusions of amino acids 1-131, 1-411, 1-480,
147-480, 147-411 and 411-480 of human RAC.alpha., respectively,
with GST. They are constructed by subcloning PCR fragments
generated with specific oligonucleotides into the BamHI-EcoRI sites
of pGEX-2T, pGBT-PH-KIN, pGBT-PH-KIN-CT, pGBT-KIN-CT, pGBT-KIN and
pGBT-CT contain in-frame fusions of amino acids 1-411, 1-480,
147-480, 147-411 and 411-480 of human RAC.alpha., respectively,
with the Gal4 DNA binding domain. They are constructed by
subcloning the appropriate BamHI-EcoRI fragments from the
corresponding pGEX constructs into the PstI-XbaI sites of pPC62
using PstI-BamHI and EcoRI-XbaI adapters. The XhoI-XbaI fragments
from the resultant pPC62 plasmids are then isolated and subcloned
into the XhoI-EcoRI sites of pGBT9 using a XbaI-EcoRI adapter.
pGAD-IMPDH1-481, pGAD-IMPDH1-427, pGAD-IMPDH1-325,
pGAD-IMPDH28-514, pGAD-IMPDH70-514, pGAD-IMPDH140 and
pGAD-IMPDH428-514 contain in-frame fusions of amino acids 1-481,
1-427, 1-325, 28-514, 70-514, 1-40 and 428-514 of human IMPDH type
II, respectively, with the Gal4 activation domain. They are
constructed by subcloning PCR fragments generated with specific
oligonucleotides into the BamHI-SalI sites of pGAD424, pGEX-IMPDH
contains an in-frame fusion of the complete human IMPDH type II
with GST. It is constructed by subcloning the SmaI-XhoI IMPDH
fragment from pGADGH-IMPDH into the SmaI site of pGEX-2T using a
XhoI-SmaI adapter. pVL1392-hRAC.alpha. contained the EcoRI fragment
from WI38xRAC71 [see Jones et al. (1991), supra] encompassing the
full-coding region of human RAC.alpha.. pRSET-PHQKKK contains an
in-frame fusion of amino acids 1-116 of human RAC.alpha. with an
amino-terminal (His).sub.6-tag and the addition of 3 lysines at the
carboxy terminus. It is constructed by subcloning an NdeI-PflMI
fragment from pRK-RAC [see Jones et al. (1991), supra] into the
BamHI-EcoRI sites of pRSET-A using BamHI-NdeI and PflMI-EcoRI
adapters. All plasmid constructions are confirmed by restriction
fragment analysis and sequencing.
[0139] c. Library Screening
[0140] To determine if RAC's PH domain could interact with other
proteins we fuse it to the Gal4 DNA binding domain and screen a
HeLa complementary DNA (cDNA) library fused to the Gal4
transcriptional activation domain in the yeast reporter strain
HF7c. The human HeLa S3 MATCHMAKER cDNA library is purchased from
Clontech. pGBT-PH127 is transformed into HF7c with and without the
control plasmids (pGAD424, pCL1 and pTD1). Colonies from this
transformation are tested for His3 and LacZ expression to confirm
that the PH domain alone does not activate transcription. The HF7c
transformant containing just pGBT-PH127 is then transformed with
enough of the HeLa S3 cDNA library inserted into the 2-hybrid
activation vector pGADGH to produce 1.0.times.10.sup.6 yeast
Leu.sup.+/Trp.sup.+ transformants. Doubly-transformed cells are
plated onto Leu.sup.-, Trp.sup.- and His.sup.- plates and incubated
at 30.degree. C. for 3-8 days. Positive colonies are picked,
re-streaked onto triple minus plates and assayed for LacZ activity
by the filter assay. Library clones that are His.sup.+ and
LacZ.sup.+ are then cured of the pGBT-PH127 plasmid and tested
again for His auxotrophy and LacZ activity. Cured clones that are
negative in both assays are then mated to transformants of PCY2
containing either pGBT9, pGBT-PH127, pLAM5' or pTD1. The activation
clones corresponding to the diploids which become positive for both
His auxotrophy and LacZ activity only in the presence of pGBT-PH127
are chosen for sequencing.
[0141] In our screen of 1.0.times.10.sup.6 primary transformants we
identify 37 clones which show specific interaction with RAC's PH
domain, by activation of the reporters for His auxotrophy and LacZ
activity. These clones could be subdivided into 6 different cDNA
classes, based on the size of the cDNA insert. Upon sequencing all
clones were found to encode human IMPDH type II inclusive of the
initiator methionine through to the termination codon of the
previously cloned cDNA. See Collart and Huberman (1988), supra.
[0142] The interaction requires a complete PH domain as constructs
containing either subdomains I-III (amino acids 1-47) or subdomains
IV-VI (amino acids 47-127) alone do not show any interaction with
IMPDH. The lack of interaction with subdomains IV-VI is significant
as this region has previously been shown to interact weakly with
the .beta..gamma.-subunits of heterotrimeric G-proteins. This
interaction of IMPDH and RAC's PH domain is however inhibited in
the 2-hybrid system with constructs containing RAC's kinase domain
juxtaposed to RAC's PH domain as occurs in its natural context.
This could be due to an intramolecular interaction of RAC's PH
domain with itself or another region of RAC. However, inclusion of
amino acids between the PH and kinase domains (including the first
four amino acids of the kinase domain) didn't inhibit the
interaction. We also fuse RAC's PH domain to the Gal4 activation
domain to test if it could interact with any of the human
RAC.alpha. Gal4 DNA binding constructs. We can detect no such
interaction, indicating that the PH domain does not self associate
or form a complex with other regions of the RAC-PK molecule in this
system. The inhibition of interaction observed above would thus
appear to be due to steric hindrance.
[0143] We construct nested amino and carboxyl-terminal deletions of
IMPDH to determine the region of the molecule responsible for
interaction with RAC's PH domain. This indicates that an almost
intact IMPDH molecule is required for the interaction. The
amino-terminal boundary of the PH interaction domain is found to
lie between amino acids 28 and 70, while the carboxyl-terminal
boundary lies between amino acids 427 and 481.
[0144] d. In Vitro Binding Studies
[0145] To test if IMPDH could interact directly with RAC's PH
domain we employ an in vitro binding assay system using GST
fusions. GST fusions produced from the plasmids pGEX-2T, pGEX-PH131
and pGEX-IMPDH are expressed in E. coli XL-1 blue cells by
induction with 0.1 mM IPTG for 2 hours at 24.degree. C. The fusion
proteins are purified as described [see Smith and Johnson, Gene,
Vol. 67, No. 1, pp. 31-40 (1988)] except that the cells are lysed
in a French Press. The human RAC.alpha. PH domain (His).sub.6
tagged fusion produced by B121(DE3)pLysS cells transformed with
pRSET-PHQKKK is expressed and purified as described in UK patent
application No. 9525705.1. Briefly, cells are induced with 0.2 mM
IPTG for 2 hours at 24.degree. C. before harvesting. Cell pellets
are lysed in a French Press and the soluble PH domain is purified
sequentially on Ni(II) affinity, cation exchange and gel filtration
columns. Binding studies are performed using GST fusions (2.5
.mu.g) coupled to glutathione-agarose beads in binding buffer (20
mM phosphate buffer, pH 7.2, 150 mM NaCl, 1% Triton X-100, 5 mM
DTT) containing 2.5 .mu.g of (His).sub.6-tagged PH domain or
baculovirus produced human RAC.alpha. in a total volume of 100
.mu.L. The samples are incubated at 4.degree. C. for 1-2 hours with
agitation every 5 minutes. The beads are then washed 3.times. with
buffer (20 mM phosphate buffer, pH 7.2, 150 mM NaCl) before being
analyzed by SDS-PAGE and stained with coomassie blue R-250 [for
(His).sub.6-tagged PH domain binding] or SDS-PAGE followed by
Western blot analysis using a human RAC.alpha. specific antiserum
(for human RAC.alpha. binding). See Jones et al., Jakubowicz and
Hemmings (1991), supra. The secondary antibody is a horseradish
peroxidase coupled anti-rabbit antibody (Amersham) which is
detected using the ECL method (Amersham) by autoradiography. In
this assay we see that the (His).sub.6-tagged PH domain can bind to
the GST-IMPDH fusion but not to GST alone.
[0146] We also employ this assay system to test if full-length
baculovirus purified human RAC.alpha. could directly interact with
IMPDH. Full-length human RAC.alpha. is expressed and purified from
the baculovirus system as described in UK patent application No.
9525702.8. Briefly, a baculovirus is constructed by co-transfection
of Sf9 cells with pVL1 392-hRAC.alpha. and wild-type (WT)
baculovirus AcMNPV DNA and purified by limiting dilution and
detected by dot-blot hybridization. The purified virus is used to
produced human RAC.alpha. in Sf9 cells. The human RAC.alpha. is
purified by sequential anion exchange, phospho-cellulose and gel
filtration chromatography. Here we see specific interaction of the
full-length RAC-PK molecule with GST-IMPDH and not GST alone or the
GST-PH fusion.
[0147] e. In Vivo Pull-Down Assay
[0148] Using GST-IMPDH in a pull-down assay with MCF-7 cell
extracts we see a specific association of human RAC.alpha. with the
GST-IMPDH and not with GST. MCF-7 cells are lysed in buffer (50 mM
Tris-HCl, pH 8.0,120 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM EGTA, 30 mM
pNPP, 25 mM .beta.-glycerol phosphate, 15 mM PPi, 25 mM NaF, 1 mM
vanadate, 20 .mu.M PAO, 1 mM benzamidine, 0.1 mM PMSF) using 12
strokes of a dounce homogenizer. Soluble protein from the
supernatant of lysates centrifuged at 14,000.times.g for 15 minutes
at 4.degree. C. are added to GST, GST-PH or GST-IMPDH protein (5
.mu.g) attached to glutathione beads and incubated at 4.degree. C.
for 2 hours with continuous agitation. The beads are then washed
4.times. with lysis buffer before being analyzed by Western
blotting as described above with the human RAC.alpha.-specific
antiserum. See Jones, Jakubowicz and Hemmings (1991), supra] or an
IMPDH-specific antiserum. See Collart and Huberman (1988), supra.
We could also perform the converse experiment, pulling down IMPDH
from cell lysates using the GST-PH domain fusion protein. Thus we
have shown the existence of an association between human RAC.alpha.
and human IMPDH type II in 3 heterologous systems.
[0149] f. Enzyme Assays
[0150] We then assay the effect of this interaction on the activity
of IMPDH. The addition of soluble PH domain as a GST fusion
produced an activation of IMPDH compared to the addition of GST
alone. Assays for IMPDH activity are performed essentially [see
Antonio and Wu, Biochem, Vol. 33, pp. 1753-1759 (1994)], monitoring
the production of XMP by absorbance at 286 nm. The IMPDH is
produced as a GST fusion purified on glutathione beads and then
eluted as soluble protein with reduced glutathione. IMPDH activity
is tested in the presence of either soluble GST (produced from
pGEX-2T) or PH domain (produced from pGEX-PH131) at a molar ratio
of IMPDH to GST/PH domain of 1:5. RAC kinase assays with the
baculovirus produced human RAC.alpha. using various substrates,
e.g., myelin basic protein, GST or GST-IMPDH, are performed
essentially as described. See Jones, Jakubowicz and Hemmings
(1991), supra. When IMPDH is tested in RAC-PK assays using the
baculovirus produced human RAC.alpha. to see if it is a substrate
we could detect no significant phosphorylation of the IMPDH.
EXAMPLE 2
[0151] Inhibition of GSK3
[0152] Two major kinases known to be involved in regulating the
insulin-dependent signalling pathways are MAPKAP kinase-1 and
p70.sup.S6K. These kinases are respectively inhibited by PD98059
and rapamycin. Both of these agents fail to inhibit phosphorylation
of GSK3, suggesting that the kinase responsible for GSK3
inactivation is not MAPKAP kinase-1 or p70.sup.S6K.
[0153] L6 myotubes are incubated with both compounds and the
stimulated with insulin, as follows. Monolayers of L6 cells are
grown in 6 cm petri dishes to the stage of myotubes, deprived of
serum overnight and then incubated for 1 hour in 20 mM Hepes/NaOH,
pH 7.4, 0.14 M NaCl, 5 mM KCl, 2.5 mM MgSO.sub.4, 1 mM CaCl.sub.2,
25 mM glucose (HBS buffer), in the presence or absence of 50 .mu.M
PD98059 or 100 .mu.M LY294002. Two (2) mM 8-Br-cAMP or 0.1 .mu.M
rapamycin, when added, are included for the last 15 minutes. The
cells are stimulated for 5 minutes with 0.1 .mu.M insulin, or for
time courses of up to 10 minutes. The medium is removed by
aspiration, the cells placed on ice and lysed in 0.2 mL of ice-cold
buffer A, 50 mM Tris-HCl, pH 7.5, 20.degree. C., 1 mM EDTA, 1 mM
EGTA, 1% (.sup.w/.sub.v) Triton X-100, 1 mM sodium orthovanadate,
10 mM sodium glycerophosphate, 50 mM sodium fluoride, 5 mM sodium
pyrophosphate, 2 .mu.M microcystin, 0.1% (.sup.v/.sub.v)
2-mercaptoethanol, leupeptin 4 .mu.g/mL, 1 mM benzamidine, 1 mM
phenylmethane sulphonyl fluoride, 30 .mu.g/mL aprotinin, 30
.mu.g/mL antipain, 10 .mu.g/mL pepstatin.
[0154] Precipitation of p42.sup.MAPK, MAPKAP kinase-1 or GSK3 from
the cell lysates by immunoprecipitation and analysis of their
activity with specific protein or peptide substrates [see Cross et
al. (1994), supra] shows that insulin inactivation of GSK3 is not
affected by agents (2 mM 8-Br-cAMP or 0.1 .mu.M rapamycin) which
inhibit classical MAP kinase or p70.sup.S6K signalling
pathways.
[0155] In order to identify the kinase which inhibits GSK3 in the
presence of rapamycin and PD98059, cells are lysed as above and the
cell lysates (0.3 mg) chromatographed on a 5.times.0.16 cm column
of Mono Q [see Sutherland and Cohen, FEBS Lett, Vol. 338, No.1, pp.
37-42 (1994)] except that the buffer additionally contains 1 mM
EGTA, 0.1 mM sodium orthovanadate and 0.5% (.sup.w/.sub.v) Triton
X-100.
[0156] The fractions (0.05 mL) assayed with the synthetic peptide
GRPRTSSFAEG SEQ ID NO: 5, which corresponds to the sequence of GSK3
surrounding the serine (bold type) whose phosphorylation triggers
the inactivation of GSK 3.alpha. (Ser21) and GSK3.beta. (Ser9).
Three peaks of activity which result in phosphorylation of this
peptide are eluted. These peaks are absent if insulin stimulation
is not given, or if cells are incubated with 0.1 .mu.M wortmannin
prior to insulin stimulation. The inactivating effect of insulin on
GSK3 is known to be prevented by administering this concentration
of wortmannin, or 100 .mu.M LY294002, a structurally-unrelated
inhibitor of PI-3K.
[0157] All 3 peaks of phosphorylating activity can be
immunoprecipitated with an anti-RAC antibody using a polyclonal
rabbit antiserum directed against the peptide FPQFSYSASSTA SEQ ID
NO: 7 raised by injecting rabbits subcutaneously with the peptide
and purified by precipitation using 50% (NH.sub.4).sub.2SO.sub.4
followed by affinity chromatography on RAC-peptide coupled
Affigel.RTM. 10 column (Bio-Rad).
[0158] In contrast, immunoprecipitating with an anti-MAPKAP
kinase-1 antibody fails to deplete any peptide phosphorylating
activity from the Mono Q column.
[0159] In order to determine that complete GSK3 can be inactivated
by RAC, GSK3.alpha. and GSK3.beta. are partially purified from
rabbit skeletal muscle [see Sutherland and Cohen (1994), supra] and
assayed with a specific peptide substrate. See Cross et al. (1994),
supra. Each GSK3 isoform is diluted to 15 U/mL and GSK3 activity
measured after incubation for 20 minutes with MgATP in the presence
or absence of RAC. The incubation is the made 20 mM in EDTA to stop
the kinase reaction, incubated for 20 minutes with 5 mU/mL PP2A, to
reactivate GSK3, made 2 .mu.M in okadaic acid to inactivate PP2A,
and then assayed for GSK3 activity.
[0160] In the absence of RAC, GSK3 is stably active throughout the
experiment. Otherwise, however, RAC-PK successfully inhibited GSK3
activity and,this inactivation was sensitive to PP2A.sub.1, which
restored GSK3 activity. The absence of insulin stimulation, the
presence of wortmannin or the disruption of RAC-PK
immunoprecipitation by incubation of the anti-RAC antibody with the
peptide immunogen all result in a lack of GSK3 inactivation in this
experiment.
EXAMPLE 3
[0161] To determine if RAC-PK's domain with its carboxyl-terminal
extension could interact with other proteins we fused it to the
Gal4 DNA binding domain and screened a HeLa cDNA library fused to
the Gal4 transcriptional activation domain in the yeast reporter
strain HF7c following the procedure of Example 1. Briefly, amino
acids 147-480 of RAC.alpha. are fused in frame to GST in the
expression vector pGEX-2T (see Example 1). Appropriate BamHI-EcoRI
fragment is then subcloned into the PstI-XbaI sites of yeast vector
pPC62 (Dr. Nathans) using PstI-BamHI and EcoRI-XbaI linkers in
order to create a Gal4 DNA binding domain-RAC-PK and C-terminal
domain fusion. XhoI-XbaI fragments are then subcloned into pGBT9
(Clontech). The HeLa S3 MATCHMAKER cDNA library is used as
before.
[0162] In our screen of 1.5.times.10.sup.6 primary transformants,
we identify 7 clones which show specific interaction with RAC-PK's
domain plus the carboxyl-terminal extension, by activation of the
reporters for His auxotrophy and LacZ activity. A detailed analysis
of the specific region of RAC-PK that the clone interacts with
shows that the carboxyl-terminal 69 amino acids are all that is
required to confer the interaction. We thus denote this molecule
carboxyl-terminal binding protein (CTBP). None of the clones shows
an interaction with the kinase domain alone. This interaction is
seen in all constructs containing the carboxyl-terminal extension
including a full-length RAC-PK construct, indicating that the
interaction is not inhibited by another region of the RAC-PK
molecule. Interestingly, the C-terminal domain of RAC-PK is
phosphorylated in response to insulin activation, suggesting a role
for CTBP as a modulator of insulin action.
[0163] All 7 specific interacting clones contain identical cDNA
inserts of 1.3 kb in length with an ALU repeat of .about.300 nt at
the 3' end (SEQ ID NO: 1). Searches of the Gene-EMBL nucleotide
database using the cDNA sequence without the ALU repeat with the
FASTA programme (GCG Package) identify no significant homologies.
The deduced amino acid sequence of the CTBP cDNA produces a short
47-residue polypeptide rich in alanines (21%) and arginines (21%).
Searches of the PIR, Swiss-Prot and GP protein databases using
FASTA (GCG Package) and the Gene-EMBL nucleotide database using
TFASTA (GCG Package) reveal no significant homologies to the CTBP
protein sequence.
[0164] The sequence of CTBP identified is believed to represent the
3' end of the complete CTBP molecule.
[0165] To test if the novel protein CTBP could interact directly
with RAC-PK we employ an in vitro binding assay system using GST
fusions, as described in Example 1. In this assay we see specific
interaction of the full-length RAC, produced in the baculovirus
system with GST-CTBP and not GST alone.
[0166] To test if this interaction occurs in vivo we employ a
pull-down assay using the GST-CTBP fusion protein and MCF-7 cell
extracts, as described in Example 1. Here we see the specific
association of the MCF-7 RAC.alpha. with the GST-CTBP but not GST
alone.
EXAMPLE 4
[0167] RAC-PK Influences GSK3 Activity
[0168] FIG. 1 (a)--L6 myotubes were incubated for 15 minutes with 2
mm 8-bromocyclic-AMP (8Br-cAMP) and then with 0.1 .mu.M insulin (5
minutes). Both GSK3 isoforms were co-immunoprecipitated from the
lysates and assayed before (black bars) and after (white bars)
reactivation with PP2A. See Cross et al. (1994), supra. The results
are presented relative to the activity in unstimulated cells, which
was 0.08.+-.0.006 U mg.sup.-1 (n=10).
[0169] (b and c)--The inhibition of GSK3 by insulin (0.1 .mu.M) is
unaffected by rapamycin (0.1 .mu.M) and PD 98059 (50 .mu.M), but
prevented by LY 294002 (100 .mu.M).
[0170] (b)--L6 myotubes were stimulated with insulin for the times
indicated with (filled triangle) or without (filled circles) a 15
minutes pre-incubation with LY 294002, and GSK3 measured as in (a).
The open circles show experiments from insulin-stimulated cells
where GSK3 was assayed after reactivation with PP2A. See Cross et
al. (1994), supra.
[0171] (c)--Cells were incubated with rapamycin (triangles) or
rapamycin plus PD 98059 (circles) before stimulation with insulin,
and GSK3 activity measured as in (a), before (filled symbols) and
after (open symbols) pretreatment with PP2A.
[0172] (d and e)--L6 myotubes were incubated with 8Br-cAMP (15
minutes), PD 98059 (60 minutes) or LY 294002 (15 minutes) and then
with insulin (5 minutes) as in (a-c). Each enzyme was assayed after
immunoprecipitation from lysates, and the results are presented
relative to the activities obtained. In the presence of insulin and
absence of 8Br-cAMP, which were 0.04.+-.0.005 U mg.sup.-1 (p42 MAP
kinase, n=6) and 0.071.+-.0.004 U mg.sup.-1 (MAPKAP Kinase.sup.-1,
n=6).
[0173] All the results (.+-.s.e.m.) are for at least 3
experiments.
[0174] Monolayers of L6 cells were cultured, stimulated and lysed
as described previously. See Cross et al. (1994), supra. p42 MAP
kinase, MAPKAP kinase 1 or GSK3-.alpha. plus GSK3-.beta. were then
immunoprecipitated from the lysates and assayed with specific
protein or peptide substrates as described previously. See Cross et
al. (1994), supra. One unit of PK activity was that amount which
catalyzed the phosphorylation of 1 nmol of substrate in 1 minute.
Where indicated, GSK3 in immunoprecipitates was reactivated with
PP2A. See Cross et al. (1994), supra.
[0175] FIG. 2--Identification of RAC-PK as the insulin-stimulated,
wortmannin-sensitive and PD 98059/rapamycin-insensitive Crosstide
kinase in L6 myotubes.
[0176] (a)--Cells were incubated with 50 .mu.M PD 98059 (for 1
hour) and 0.1 .mu.M rapamycin (10 minutes), then stimulated with
0.1 .mu.M insulin (5 minutes) and lysed. See Cross et al. (1994),
supra. The lysates (0.3 mg protein) were chromatographed on Mono Q
(5.times.0.16 cm) and fractions (0.05 mL) were assayed for
Crosstide kinase (filled circles). In separate experiments insulin
was omitted (open circles) or wortmannin (0.1 .mu.M) added 10
minutes before the insulin (filled triangles). The broken line
shows the NaCl gradient.
[0177] Similar results were obtained in 6 experiments.
[0178] (b)--Pooled fractions (10 .mu.L), 31-34 (lane 1), 35-38
(lane 2), 39-42 (lane 3), 43-45 (lane 4), 46-49 (lane 5) and 50-53
(lane 6), from a were electrophoresed on a 10% SDS/polyacrylamide
gel and immunoblotted with the C-terminal anti-RAC-PK.alpha.
antibody. Marker proteins are indicated. No immunoreactive species
were present in fractions 1-30 or 54-80.
[0179] (c)--L6 myotubes were stimulated with 0.1 .mu.M insulin and
RAC-PK immunoprecipitated from the lysates (50 .mu.g protein)
essentially as described previously [see Lazar et al., J Biol Chem,
Vol. 270, No. 35, pp. 20801-20807 (1995)], using the anti-PH domain
antibody and assayed for Crosstide kinase (open circles). In
control experiments, myotubes were incubated with 0.1 .mu.M
rapamycin plus 50 .mu.M PD 98059 (open triangles) or 2 mM 8Br-cAMP
(open squares), or 0.1 .mu.M wortmannin (filled circles) or 100
.mu.M LY 294002 (filled triangles) before stimulation with
insulin.
[0180] (d)--As (c) except that MAPKAP kinase-1 was
immunoprecipitated from the lysates and assayed with S6 peptide
(filled circles). In control experiments, cells were incubated with
0.1 .mu.M rapamycin plus 50 .mu.M PD 98059 (filled triangles) or
with 2 .mu.M 8BR-cAMP (open circles) before stimulation with
insulin. In (c) and (d), the error bars denote triplicate
determinations, and similar results were obtained in 3 separate
experiments.
[0181] Mono Q chromatography was performed as described [see
Burgering and Coffer (1995), supra], except that the buffer also
contained 1 mM EGTA, 0.1 mM sodium orthovanadate and 0.5%
(.sup.w/.sub.v) Triton X-100. Two RAC-PK.alpha. antibodies were
raised in rabbits against the C-terminal peptide FPQFSYSASSTA (SEQ
ID NO: 7) and bacterially-expressed PH domain of RAC-PK.alpha.. The
C-terminal antibody was affinity purified. See Jones et al. (1991),
supra. The activity of RAC-PK towards Crosstide is threefold higher
than its activity towards histone H2B and 11-fold higher than its
activity towards myelin basic protein, the substrates used
previously to assay RAC-PK. Other experimental details and units of
protein kinase activity are given in FIG. 1.
[0182] FIG. 3--GSK3 is inactivated by RAC-PK from
insulin-stimulated L6 myotubes.
[0183] (a)--Cells were stimulated for 5 minutes with 0.1 .mu.M
insulin, and RAC-PK immunoprecipitated from 100 .mu.g of cell
lysate and used to inactivate GSK3 isoforms essentially as
described previously. See Sutherland, Leighton and Cohen, Biochem
J, Vol. 296, Pt. 1, pp. 15-19 (1993); and Sutherland and Cohen
(1994), supra. The black bars show GSK3 activity measured after
incubation with MgATP and RAC-PK as a percentage of the activity
obtained in control incubations where RAC-PK was omitted. In the
absence of RAC-PK, GSK3 activity was stable throughout the
experiment. The white bars show the activity obtained after
reactivation of GSK3 with PP2A. See Embi, Rylatt and Cohen, Eur J
Biochem, Vol. 107, No. 2, pp. 519-527 (1980). No inactivation of
GSK3 occurred if insulin was omitted, or if wortmannin (0.1 .mu.M )
was added 10 minutes before the insulin, or if the anti-RAC-PK
antibody was incubated with peptide immunogen (0.5 mM) before
immunoprecipitation. The results (.+-.s.e.m.) are for 3 experiments
(each carried out in triplicate).
[0184] (b)--Inactivation of GSK3-.beta. by HA-RAC-PK.alpha.. cDNA
encoding HA-RAC-PK.alpha. was transfected into COS-1 cells, and
after stimulation for 15 minutes with 0.1 mM sodium pervanadate the
tagged protein kinase was immunoprecipitated from 0.3 mg of lysate
and incubated for 20 minutes with GSK3-.beta. and MgATP. In control
experiments, pervanadate was omitted, or WT RAC-PK.alpha. replaced
by vector (mock translation) or by a kinase-inactive mutant of
RAC-PK.alpha. in which Lys179 was mutated to Ala (K179A). Similar
results were obtained in 3 separate experiments. The levels of WT
and K179A-RAC-PK.alpha. in each immunoprecipitate were similar in
each transfection.
[0185] In a GSK3-.alpha. and GSK3-.beta. were partially-purified,
assayed, inactivated by RAC-PK and reactivated by PP2A from rabbit
skeletal muscle as described previously. See Sutherland, Leighton
and Cohen, Biochem J (1993), supra; and Sutherland and Cohen
(1994), supra. There was no reactivation in control experiments in
which okadaic acid (2 .mu.M) was added before PP2A.
[0186] FIG. 4--Identification of the residues in GSK3
phosphorylated by RAC-PK in vitro and in response to insulin in L6
myotubes.
[0187] (a)--GSK3-.beta. was maximally inactivated by incubation
with RAC-PK and Mg-[.gamma.-.sup.32P]ATP and after SDS-PAGE, the
.sup.32P-labelled GSK3-.beta. (M.sub.r 47K) was digested with
trypsin.sup.11 and chromatographed on a C18-column. See Sutherland,
Leighton and Cohen, Biochem J (1993), supra. Fractions (0.8 mL)
were analyzed for .sup.32P-radioactivity (open circles), and the
diagonal line shows the acetonitrile gradient.
[0188] (b)--The major phosphopeptide from a (400 c.p.m.) was
subjected to solid-phase sequencing [see Sutherland, Leighton and
Cohen, Biochem J (1993), supra], and .sup.32P-radioactivity
released after each cycle of Edman degradation is shown.
[0189] (c)--GSK3-.alpha. and GSK3-.beta. were co-immunoprecipitated
from the lysates of .sup.32P-labelled cells, denatured in SDS,
subjected to SDS-PAGE, transferred to nitrocellulose and
autoradiographed. See Saito, Vandenheede and Cohen, Biochem J
(1994). Lanes 1-3, GSK3 isoforms immunoprecipitated from
unstimulated cells; lanes 4-6, GSK3 isoforms immunoprecipitated
from insulin-stimulated cells.
[0190] (d)--GSK3 isoforms from (c) were digested with trypsin, and
the resulting phosphopeptides separated by isoelectric focusing
[see Saito, Vandenheede and Cohen (1994), supra] and identified by
auto-radiography. Lanes 1 and 4 show the major phosphopeptide
resulting from in vitro phosphorylation of GSK3-.beta. by RAC-PK
and MAPKAP kinase-1, respectively; lanes 2 and 5, the
phosphopeptides obtained from GSK3-.beta. and GSK3-.alpha.,
immunoprecipitated from unstimulated cells; lanes 3 and 6, the
phosphopeptides obtained from GSK3-.beta. and GSK3-.alpha.
immunoprecipitated from cells stimulated for 5 minutes with 0.1
.mu.M insulin; the arrow denotes the peptides whose phosphorylation
is increased by insulin. The PI values of two markers, Patent Blue
(2.4) and azurin (5.7) are indicated.
[0191] In (a), RAC-PK.alpha. was immunoprecipitated with the
C-terminal antibody from the lysates (0.5 mg protein) of
insulin-stimulated L6 myotubes and used to phosphorylate
GSK-.beta.. In (c), three 10 cm diameter dishes of L6 myotubes were
incubated for 4 hours in HEPES-buffered saline [see Cross et al.
(1994), supra] containing 50 .mu.M PD 98059, 100 nM rapamycin and
1.5 mCl ml-1 .sup.32P-orthophosphate- , stimulated for 5 minutes
with insulin (0.1 .mu.M) or buffer and GSK3 isoforms
co-immunoprecipitated from the lysates as in FIG. 1.
[0192] Discussion
[0193] Inhibition of GSK3 induced by insulin in L6 myotubes in FIG.
1 (a-c) was unaffected by agents which prevented the activation of
MAPKAP kinase-1 (8-bromo-cyclic AMP, or PD 98059) [see Alessi et
al., J Biol Chem, Vol. 270, No. 46, pp. 27489-27494 (1995)], FIG. 1
(d and e) and/or p70.sup.S6k rapamycin [see Kuo et al., Nature,
Vol. 358, No. 6381, pp. 70-73 (1992); and Cross et al. (1994),
supra], suggesting that neither MAPKAP kinase-1 nor p70.sup.S6k are
essential for this process. However, the phosphorylation and
inhibition of GSK3-.beta. after phorbol ester treatment [see
Stambolic and Woodget, Biochem J, Vol. 303, Pt. 3, pp. 701 -704
(1994)] is enhanced by co-expression with MAPKAP kinase-1 in HeLa
S3 cells, whereas in NIH 3T3 cells the EGF-induced inhibition of
GSK3-.alpha. and GSK3-.beta. [see Saito, Vandenheede and Cohen
(1994), supra] is largely suppressed by expression of a
dominant-negative mutant of MAP kineas kinase-1. See
Eldar-Finkelman, Seger, Vandenheede and Krebs, J Biol Chem, Vol.
270, No. 3, pp. 987-990 (1995). MAPKAP kinase-1 may therefore
mediate the inhibition of GSK3 by agonists which are much more
potent activators of the classical MAP kinase pathway than is
insulin.
[0194] To identify the insulin-stimulated protein kinase (ISPK)
that inhibits GSK3 in the presence of rapamycin and PD 98059, L6
myotubes were incubated with both compounds and stimulated with
insulin. The lysates were then chromatographed on Mono Q and the
fractions assayed with "Crosstide" GRPRTSSFAEG (SEQ ID NO:5), a
peptide corresponding to the sequence in GSK3 surrounding the Ser
(underlined) phosphorylated by MAPKAP kinase-1 and p70.sup.S6k
(Ser21 in GSK3-.alpha.) [see Sutherland and Cohen (1994), supra]
and Ser9 in GSK3-.beta.. See Sutherland, Leighton and Cohen,
Biochem J, Vol. 296, Pt. 1, pp. 15-19 (1993). Three peaks of
Crosstide kinase activity were detected, which were absent if
insulin stimulation was omitted or if the cells were first
preincubated with the PI 3-kinase inhibitor wortmannin. See FIG. 2
(a). Wortmannin [see Cross et al. (1994),supra; and Welsh et al.
(1994), supra], and the structurally-unrelated PI 3-kinase
inhibitor LY 294002; FIG. 1 (b), both prevent the inhibition of
GSK3 by insulin.
[0195] The PKs RAC-PK-.alpha., RAC-PK-.beta. and RAC-PK.gamma. are
Ser-/Thr-specific and cellular homologues of the viral oncogene
v-akt. See Coffer and Woodgett, Eur J Biochem, Vol. 201, No. 2, pp.
475-481 (1991); Jones et al. (1991), supra, Ahmed et al., Mol Cell
Biol, Vol. 15, pp. 2304-2310 (1995); and Cheng et al., Proc Natl
Acad Sci USA, Vol. 89, No. 19, pp. 9267-9271 (1992). These enzymes
have recently been shown to be activated in NIH 3T3, Rat-1 or Swiss
3T3 cells in response to growth factors or insulin, activation
being suppressed by blocking the activation of PI 3-kinase in
different ways. See Franke et al., (1995), supra; and Burgering and
Coffer (1995), supra. All three peaks of Crosstide kinase [see FIG.
2 (a)], but no other fraction from Mono Q, showed the
characteristic multiple bands of RAC-PK (relative molecular mass,
Mr 58K, 59K or 60K) that have been observed in other cells, when
immunoblotting was performed with an antibody raised against the
carboxyl-terminal peptide of RAC-PK-.alpha.. See FIG. 2 (b). The
more slowly migrating forms represent more highly-phosphorylated
protein, and are converted to the fastest migrating species by
treatment with phosphatases. Phosphatase treatment also results in
the inactivation of RAC-PK [see Burgering and Coffer (1995), supra]
and the complete loss of Crosstide kinase activity (data not
shown). Of the Crosstide kinase activity in peaks 2 and 3 from Mono
Q, 70-80% was immunoprecipitated by a separate antibody raised
against the amino-terminal PH domain of RAC-PK-.alpha.. The
C-terminal antibody also immunoprecipitated RAC-PK activity
specifically from peaks 2 and 3, but was less effective than the
anti-PH-domain antibody. Peak-1 was hardly immunoprecipitated by
either antibody and may represent RAC-PK.beta.. An
immunoprecipitating anti-MAPKAP kinase-1 antibody [see Cross et al.
(1994), supra] failed to deplete any of the Crosstide kinase
activity associated with peaks 1, 2 or 3.
[0196] Insulin stimulation of L6 myotubes increased RAC-PK activity
by more than 10-fold [see FIG. 2 (c)], and activation was blocked
by wortmannin or LY 294002, but was essentially unaffected by
8-bromo-cyclic AMP or rapamycin plus PD 98059. See FIG. 2 (c). The
half-time (t0.5) or activation of RAC-PK (1 minute) was slightly
faster than that for inhibition of GSK3 (2 minutes). See Cross et
al. (1994), supra. In contrast, the activation of MAPKAP kinase-1
[see FIG. 2 (d)] and p70.sup.s6k (not shown) was slower (t0.5>5
minutes). Activation of MAPKAP kinase-1 was prevented by
8-bromo-cyclic AMP or PD 98059 [see FIG. 2 (d)], and activation of
p70.sup.S6k by rapamycin. See Cross et al. (1994), supra. Akt/RAC
phosphorylated the Ser in the Crosstide equivalent to Ser21 in
GSK3-.alpha. and Ser9 in GSK3-.beta. (data not shown).
[0197] RAC-PK from insulin-stimulated L6 myotubes (but not from
unstimulated or wortmannin-treated cells) inactivated GSK3-.alpha.
and GSK3-.beta. in vitro, and inhibition was reversed by the
Ser-Thr-specific protein phosphatase PP2A. See Embi, Rylatt and
Cohen (1980) and FIG. 3 (a). To further establish that inactivation
was catalyzed by RAC-PK, and not by a co-immunoprecipitating PK,
hemagglutonin-tagged RAC-PK-.alpha. (HA-RAC-PK) was transfected
into COS-1 cells and activated by stimulation with pervanadate,
which is the strongest inducer of RAC-PK activation in this system.
The HA-RAC-PK inactivated GSK3-.beta., but not if treatment with
pervanadate was omitted or if WT HA-RAC-PK was replaced with a
"kinase inactive" mutant. See FIG. 3 (b).
[0198] The inactivation of GSK3-.beta. by RAC-PK in vitro was
accompanied by the phosphorylation of one major tryptic peptide
[see FIG. 4 (a)] which co-eluted during C18-chromatography [See
Sutherland, Leighton and Cohen, Biochem J, (1993), supra] and
isoelectric focusing with that obtained after phosphorylation by
MAPKAP kinase-1. See FIG. 4 (d). Stimulation of L6 myotubes with
insulin (in the presence of rapamycin and PD 98059) increased the
.sup.32P-labelling of GSK3-.alpha. and GSK3-.beta. by 60-100% [see
FIG. 4 (c)] and increased the .sup.32P-labelling of the same
tryptic peptides labelled in vitro. See FIG. 4 (d). Sequence
analyses established that the third residue of these, corresponding
to Ser9 (GSK3-.beta.) or Ser21 (GSK3-.alpha.), was the site of
phosphorylation in each phosphopeptide, both in vitro [see FIG. 4
(b)] and in vivo (not shown). The .sup.32P-labelling of other (more
acidic) tryptic phosphopeptides was not increased by insulin. See
FIG. 4 (d). These peptides have been noted previously in GSK3 from
A431 cells and shown to contain phosphoserine and phosphotyrosine.
See Saito, Vandenheede and Cohen, Vol. 303 (1994), supra.
[0199] PKC-delta (.delta.), epsilon (.epsilon.) and zeta (.zeta.)
are reported to be activated by mitogens, and PKC-.zeta. activity
is stimulated in vitro by several inositol phospholipids, including
PI(3, 4, 5)P3 the product of the PI 3-kinase reaction. See
Andjelkovic et al., Proc Natl Acad Sci USA, Vol. 93, No. 12, pp.
5699-5704 (1995). However, purified PKC-.epsilon. [see Palmer et
al., J Biol Chem, Vol. 270, No. 38, pp. 22412-22416 (1995)],
PKC-.epsilon. and PKC-.zeta. (data not shown) all failed to inhibit
GSK3-.alpha. or GSK3-.beta. in vitro. Moreover, although
PKC-.alpha., .beta.1 and .gamma. inhibit GSK3-.beta. in vitro [see
Palmer et al. (1995), supra], GSK3-.alpha. is unaffected, while
their downregulation in L6 myotubes by prolonged incubation with
phorbol esters abolishes the activation of MAPKAP kinase-1 in
response to subsequent challenge with phorbol esters, but has no
effect on the inhibition of GSK3 by insulin (not shown).
[0200] Taken together, our results identify GSK3 as a substrate for
RAC-PK. The stimulation of glycogen synthesis by insulin in
skeletal muscle involves the dephosphorylation of Ser residues in
glycogen synthase that are phosphorylated by GSK3 in vitro. See
Parker, Candwell and Cohen, Eur J Biochem, Vol. 130, No. 1, pp.
227-234 (1983). Hence the 40-50% inhibition of GSK3 by insulin,
coupled with a similar activation of the relevant glycogen synthase
phosphatase [see Goode, Hughes, Woodgett and Parker, J Biol Chem,
Vol. 267, No. 24, pp. 16878-16882 (1992)], can account for the
stimulation of glycogen synthase by insulin in skeletal muscle [see
Parker, Candwell and Cohen (1983), supra] or L6 myotubes. See
Goode, Hughes, Woodgett and Parker (1992), supra. The activation of
glycogen synthase and the resulting stimulation of glycogen
synthesis by insulin in L6 myotubes is blocked by wortmannin, but
not by PD 98059 [see Dent et al., Nature, Vol. 348, pp. 302-308
(1990)], just like the activation of Akt/RAC and inhibition of
GSK3. However, GSK3 is unlikely to be the only substrate of RAC-PK
in vivo, and identifying other physiologically relevant substrates
will be important because RAC-PK.beta. is amplified and
over-expressed in many ovarian neoplasms. See Cheng et al. (1992),
supra.
EXAMPLE 5
[0201] Activation of RAC-PK by Insulin in L6 Myotubes is
Accompanied by Phosphorylation of Residues Thr308 and Ser473
[0202] Insulin induces the activation and phosphorylation of
RAC-PK.alpha. in L6 myotubes. Three 10 cm dishes of L6 myotubes
were .sup.32P-labelled and treated for 10 minutes with or without
100 nM wortmannin and then for 5 minutes with or without 100 nM
insulin. RAC-PK.alpha. was immunoprecipitated from the lysates and
an aliquot (15%) assayed for RAC-PK.alpha. activity. See FIG. 5
(a). The activities are plotted .+-.SEM for 3 experiments relative
to RAC-PK.alpha. derived from unstimulated cells which was 10
mU/mg. The remaining 85% of the immunoprecipitated RAC-PK.alpha.
was alkylated with 4-vinylpyridine, electrophoresed on a 10%
polyacrylamide gel (prepared without SDS to enhance the
phosphorylation-induced decrease in mobility) and autoradiographed.
The positions of the molecular mass markers glycogen phosphorylase
(97 kDa), bovine serum albumin (66 kDa) and ovalbumin (43 kDa) are
marked.
[0203] Under these conditions, insulin stimulation resulted in a
12-fold activation of RAC-PK.alpha. [see FIG. 5 (a)] and was
accompanied by a 1.9.+-.0.3-fold increase in .sup.32P-labelling (4
experiments) and retardation of its mobility on SDS-polyacrylamide
gels. See FIG. 5 (b). The activation of RAC-PK.alpha., the increase
in its .sup.32P-labelling and reduction in electrophoretic
migration were all abolished by prior incubation of the cells with
100 nM wortmannin. Phosphoamino acid analysis of the whole protein
revealed that .sup.32P-labelled RAC-PK.alpha. was phosphorylated at
both serine and threonine residues and that stimulation with
insulin increased both the .sup.32P-labelling of both phosphoamino
acids (data not shown).
[0204] FIG. 6--Insulin stimulation of L6 myotubes induces the
phosphorylation of two peptides in RAC-PK.alpha.. Bands
corresponding to .sup.32P-labelled RAC-PK.alpha., from FIG. 5 (b),
were excised from the gel, treated with 4-vinylpyridine to alkylate
cysteine (Cys) residues, digested with trypsin and chromatographed
on a Vydac 218TP54 C18-column (Separations Group, Hesperia, Calif.)
equilibrated with 0.1% (by vol) trifluoroacetic acid (TFA) and the
columns developed with a linear acetonitrile gradient (diagonal
line). The flow rate was 0.8 mL/min. and fractions of 0.4 mL were
collected.
[0205] (a)--Tryptic peptide map of .sup.32P-labelled RAC-PK.alpha.
from unstimulated L6 myotubes.
[0206] (b)--Tryptic peptide map of .sup.32P-labelled RAC-PK.alpha.
from insulin-stimulated L6 myotubes.
[0207] (c)--Tryptic peptide map of .sup.32P-labelled RAC-PK.alpha.
from L6 myotubes treated with wortmannin prior to insulin. The two
major .sup.32P-labelled peptides eluting at 23.7% and 28%
acetonitrile are named Peptide A and Peptide B, respectively.
Similar results were obtained in 4 (a and b) and 2 (c)
experiments.
[0208] No major .sup.32P-labelled peptides were recovered from
.sup.32P-labelled RAC-PK.alpha. derived from unstimulated L6
myotubes [see FIG. 6 (a)] indicating that, in the absence of
insulin, there was a low level phosphorylation at a number of
sites. However, following stimulation with insulin, two major
.sup.32P-labelled peptides were observed, termed A and B [see FIG.
6 (b)], whose .sup.32P-labelling was prevented if the myotubes were
first preincubated with wortmannin. See FIG. 6 (c).
[0209] FIG. 7--Identification of the phosphorylation sites in
Peptides A and B.
[0210] (a)--Peptides A and B from FIG. 5 (b) (1000 cpm) were
incubated for 90 minutes at 110.degree. C. in 6 M HCl,
electrophoresed on thin layer cellulose at pH 3.5 to resolve
orthophosphate (Pi), phosphoserine (pS), phosphthreonine (pT) and
phosphotyrosine (pY) and autoradiographed.
[0211] (b)--Peptide A [see FIG. 5 (b3)] obtained from 50 10 cm
dishes of .sup.32P-labelled L6 myotubes was further purified by
chromatography on a microbore C18-column equilibrated in 10 mM
ammonium acetate pH 6.5 instead of 0.1% TFA. A single peak of
.sup.32P-radioactivity was observed at 21% acetonitrile which
coincided with a peak of 214 nm absorbance. Eighty percent (80%) of
the sample (1 pmol) was analysed on an Applied Biosystems 476A
sequencer to determine the amino acid sequence, and the
phenylthiohydantoin (Pth) amino acids identified after each cycle
of Edman degradation are shown using the single letter code for
amino acids. The residues in parentheses were not present in
sufficient amounts to be identified unambiguously. To identify the
site(s) of phosphorylation, the remaining 20% of the sample (600
cpm) was then coupled covalently to a Sequelon arylamine membrane
and analysed on an Applied Biosystems 470A sequencer using the
modified program described by Stokoe et al., EMBO J, Vol. 11,
No.11, pp. 3985-3994 (1992). .sup.32P-radioactivity was measured
after each cycle of Edman degradation.
[0212] (c)--Peptide B from FIG. 2 (b) (800 cpm) was subjected to
solid phase sequencing as in (b).
[0213] Peptide A was phosphorylated predominantly on Ser while
Peptide B was labelled on Thr. See FIG. 7(a). Amino acid sequencing
established that Peptide A commenced at residue 465. Only a single
burst of .sup.32P-radioactivity was observed after the eighth cycle
of Edman degradation [see FIG. 7 (b)], demonstrating that insulin
stimulation of L6 myotubes had triggered the phosphorylation of
RAC-PK.alpha. at Ser473, which is located 9 residues from the
C-terminus of the protein. Phosphopeptide B was only recovered in
significant amounts if .sup.32P-labelled RAC-PK.alpha. was treated
with 4-vinylpyridine prior to digestion with trypsin, indicating
that this peptide contained a Cys residue(s), and a single burst of
.sup.32p-radioactivity was observed after the first cycle of Edman
degradation. See FIG. 7 (c). This suggested that the site of
phosphorylation was residue 308, since it is the only Thr in
RAC-PK.alpha. that follows a Lysine (Lys) or Arginine (Arg) residue
and which is located in a tryptic peptide containing a Cys residue
(at position 310). The acetonitrile concentration at which
phosphopeptide B is eluted from the C18-column (28%) and its
isoelectric point (4.0) are also consistent with its assignment as
the peptide comprising residues 308-325 of RAC-PK.alpha.. The poor
recoveries of Peptide B during further purification at pH 6.5
prevented the determination of its amino acid sequence, but further
experiments described below using transiently transfected 293 cells
established that this peptide does correspond to residues 308-325
of RAC-PK.alpha..
[0214] FIG. 8--Mapping the phosphorylation sites of RAC-PK.alpha.
in transiently transfected 293 cells. Two hundred ninety-three
(293) cells were transiently transfected with DNA constructs
expressing WT RAC-PK.alpha., or a hemagglutonin (HA) epitope-tagged
RAC-PK.alpha. encoding the human protein, such as HA-KD
RAC-PK.alpha., HA-473A RAC-PK.alpha. or HA-308A RAC-PK.alpha..
After treatment for 10 minutes with or without 100 nM wortmannin,
the cells were stimulated for 10 minutes with or without 100 nM
insulin or 50 ng/mL IGF-1 in the continued presence of wortmannin.
RAC-PK.alpha. was immunoprecipitated from the lysates and assayed,
and activities corrected for the relative levels of expression of
each HA-RAC-PK.alpha.. The results are expressed relative to the
specific activity of WT HA-RAC-PK.alpha. from unstimulated 293
cells (2.5.+-.0.5 U/mg).
[0215] (b)--Twenty (20) .mu.g of protein from each lysate was
electrophoresed on a 10% SDS/polyacrylamide gel and immunoblotted
using monoclonal HA-antibody. The molecular markers are those used
in FIG. 5 (b).
[0216] FIG. 9--IGF-1 stimulation of 293 cells induces the
phosphorylation of two peptides in transfected HA-RAC-PK.alpha..
Two hundred ninety-three (293) cells transiently transfected with
WT HARAC-PK.alpha. DNA constructs were .sup.32P-labelled, treated
for 10 minutes without (a and b) or with (c) 100 nM wortmannin and
then for 10 minutes without (a) or with (b and c) 50 ng/mL IGF-1.
The .sup.32p-labelled HA-RAC-PK.alpha. was immunoprecipitated from
the lysates, treated with 4-vinylpyridine, electrophoresed on a 10%
polyacrylamide gel, excised from the gel and digested with trypsin.
Subsequent chromatography on a C18-column resolved 4 major
phosphopeptides termed C, D, E and F. Similar results were obtained
in 6 separate experiments for (a) and (b), and in 2 experiments for
(c).
[0217] Stimulation with insulin and IGF-1 resulted in 20-fold and
46-fold activation of transfected RAC-PK.alpha., respectively [see
FIG. 8 (a)], the half time for activation being 1 minute, as found
with other cells. Activation of RAC-PK.alpha. by insulin or IGF-1
was prevented by prior incubation with wortmannin [see FIG. 8 (a)]
and no activation occurred if 293 cells were transfected with
vector alone and then stimulated with insulin or 1 GF-1 (data not
shown).
[0218] Two prominent .sup.32P-labelled peptides were present in
unstimulated 293 cells. See FIG. 9 (a). One, termed Peptide C,
usually eluted as a doublet at 20-21% acetonitrile while the other,
termed Peptide F, eluted at 29.7% acetonitrile. Stimulation with
insulin or IGF-1 did not affect the .sup.32P-labelling of Peptides
C and F [see FIG. 9 (a and b)], but induced the .sup.32P-labelling
of 2 new peptides, termed D (23.4% acetonitrile) and E (28%
acetonitrile), which eluted at the same acetonitrile concentrations
as Peptides A and B from L6 myotubes [see FIG. 6 (b)] and had the
same isoelectric points (7.2 and 4.0, respectively). Treatment of
293 cells expressing HA-RAC-PK.alpha. with 100 nM wortmannin, prior
to stimulation with IGF-1, prevented the phosphorylation of
Peptides D and E, but had no effect on the .sup.32P-labelling of
Peptides C and F. See FIG. (c).
[0219] Peptides C, D, E and F were further purified by
re-chromatography on the C18-column at pH 6.5 and sequenced.
Peptides C gave rise to three separate (but closely eluting)
.sup.32P-labelled peptides (data not shown). Amino acid sequencing
revealed that all 3 commenced at residue 122 of RAC-PK.alpha. and
that Ser124 was the site of phosphorylation. See FIG. 10 (a).
Peptide D only contained phosphoserine and, as expected,
corresponded to the RAC-PK.alpha. tryptic peptide commencing at
residue 465 that was phosphorylated at Ser473. See FIG. 10 (b).
Peptide E, only contained phosphothreonine and amino acid
sequencing demonstrated that it corresponded to residues 308-325,
the phosphorylation site being Thr308. See FIG. 10 (c). Peptide F
only contained phosphothreonine and corresponded to the peptide
commencing at residue 437 of RAC-PK.alpha. phosphorylated at
Thr450. See FIG. 10 (d).
[0220] In the presence of phosphatidylserine, RAC-PK.alpha. binds
to PIP3 with submicromolar affinity. See James et al., Biochem J,
Vol. 315, Pt. 3, pp. 709-713 (1996); and Frech, Andjelkovic, Falck
and Hemmings, in preparation (1996). Phosphatidyl 4,5-bisphosphate
and phosphatidyl 3,4 bisphosphate bind to RAC-PK.alpha. with lower
affinities and PI 3,5 bisphosphate and PI 3 phosphate did not bind
at all under these conditions. See James et al. (1996), supra. The
region of RAC-PK.alpha. that interacts with PIP3 is almost
certainly the PH domain, because the isolated PH domain binds PIP3
with similar affinity to RAC-PK.alpha. itself [see Frech,
Andjelkovic, Falck and Hemmings (1996), supra] and because the PH
domain of several other proteins, such as the PH-domains of,
.beta.-spectrin and phospholipase Cl, are known to interact
specifically with other phosphoinositides. See Hyvonen et al., EMBO
J, Vol. 14, No. 19, pp. 4676-4685 (1995); and Lemmon et al., Proc
Natl Acad Sci USA, Vol. 92, No. 23, pp. 10472-10476 (1995).
[0221] The experiments described above were repeated using insulin
instead of IGF-1. The results were identical, except that the
.sup.32P-labelling of Peptides D and E was about 50% of the levels
observed with IGF-1 (data not shown). This is consistent with the
two-fold lower level of activation of RAC-PK.alpha. by insulin
compared with IGF-1 (FIG. 7A).
EXAMPLE 6
[0222] MAPKAP Kinase-2 Phosphorylates Ser473 of RAC-PK.alpha.
Causing Partial Activation
[0223] Ser473 of RAC-PK.alpha. lies in a consensus sequence
Phe-x-x-Phe/Tyr-SerfThr-Phe/Tyr found to be conserved in a number
of PKs that participate in signal transduction pathways. See
Pearson et al., EMBO J, Vol. 14, No. 21, pp. 5279-5287 (1995). In
order to Identify the Ser473 kinase(s) we therefore chromatographed
rabbit skeletal muscle extracts on CM-Sephadex, and assayed the
fractions for protein kinases capable of phosphorylating a
synthetic peptide corresponding to residues 465-478 of
RAC-PK.alpha.. These studies identified an enzyme eluting at 0.3 M
NaCl which phosphorylated the peptides 465-478 at the residue
equivalent to Ser473 of RAC-PK.alpha.. The Ser473 kinase co-eluted
from CM-Sephadex with MAPKAP kinase-2 [see Stokoe et al. (1992),
supra], which is a component of a stress and cytokine-activated MAP
kinase cascade. See Rouse et al., Cell, Vol. 78, No. 6, pp.
1027-1037 (1994); and Cuenda et al., FEBS Lett, Vol. 364, No. 2,
pp. 229-233 (1995). The Ser473 kinase continued to cofractionate
with MAPKAP kinase-2 through phenyl-Sepharose, heparin-Sepharose,
Mono S and Mono Q and was immunoprecipitated quantitatively by an
anti-MAPKAP kinase-2 antibody [see Gould, Cuenda, Thomson and
Cohen, Biochem J, Vol. 311, pp. 735-738 (1995)] demonstrating that
MAPKAP kinase-2 was indeed the Ser473 kinase we had purified.
[0224] FIG. 11--HA-RAC-PK.alpha. was immunoprecipitated from the
lysates of unstimulated COS-1 cells expressing these
constructs.
[0225] (a)--0.5 .mu.g of immunoprecipitated HA-RAC-PK.alpha. was
incubated with MAPKAP kinase-2 (50 U/mL), 10 mM magnesium acetate
and 100 mM [.gamma..sup.32P]ATP in a total of 40 .mu.L of Buffer B.
At various times, aliquots were removed and either assayed for
RAC-PK.alpha. activity (open circles) or for incorporation of
phosphate into RAC-PK.alpha. (closed circles). Before measuring
RAC-PKa activity, EDTA was added to a final concentration of 20 mM
to stop the reaction, and the immunoprecipitates washed twice with
1.0 mL of buffer B containing 0.5 M NaCl, then twice with 1.0 mL of
buffer B to remove MAPKAP kinase-2. The results are presented as
.+-.SEM for 6 determinations (2 separate experiments) and
RAC-PK.alpha. activities are presented relative to control
experiments in which HA-RAC-PK.alpha. was incubated with MgATP in
the absence of MAPKAP kinase-2 (which caused no activation).
Phosphorylation was assessed by counting the .sup.32P-radioactivity
associated with the band of RAC-PK.alpha. after SDS/polyacrylamide
gel electrophoresis. The open triangles show the activity of
immunoprecipitated HA-KD RAC-PK.alpha. phosphorylated by MAPKAP
kinase-2.
[0226] (b)--HA-RAC-PK.alpha. phosphorylated for 1 hour with MAPKAP
kinase-2 and 32P-.gamma.-ATP as in (a) was digested with trypsin
and chromatographed on a C18-column as described in the legend for
FIG. 2 (c). The major .sup.32P-labelled peptide from (b) was
analyzed on the 470A sequencer as in FIG. 3 to identify the site of
phosphorylation.
[0227] Bacterially-expressed MAPKAP kinase-2 phosphorylated WT
HA-RAC-PK.alpha. or the catalytically-inactive mutant
HA-RAC-PK.alpha. in which Lys179 had been mutated to Ala (data not
shown) to a level approaching 1 mol per mole protein. See FIG. 11
(a). Phosphorylation of WT RAC-PK.alpha. was paralleled by a 7-fold
increase in activity, whereas phosphorylation of the
catalytically-inactive mutant did not cause any activation. See
FIG. 11 (a). No phosphorylation or activation of WT
HA-RAC-PK.alpha. occurred if MAPKAP kinase-2 or MgATP was omitted
from the reaction (data not shown). WT HA-RAC-PK.alpha. that had
been maximally-activated with MAPKAP kinase-2, was completely
dephosphorylated and inactivated by treatment with protein
phosphatase 2A (data not shown).
[0228] HA-RAC-PK.alpha. that had been maximally-phosphorylated with
MAPKAP kinase-2 was digested with trypsin and C18-chromatography
revealed a single major .sup.32P-labelled phosphoserine-containing
peptide. See FIG. 11 (b). This peptide eluted at the same
acetonitrile concentration [see FIG. 11 (b)] and had the same
isoelectric point of 7.2 (data not shown) as the .sup.32P-labelled
tryptic peptide containing Ser473 [compare FIG. 11 (b) and FIG. 6
(b)]. Solid phase sequencing gave a burst of .sup.32P-radioactivity
after the eighth cycle of Edman degradation [see FIG. 11 (c)],
establishing that Ser473 was the site of phosphorylation. The same
.sup.32P-peptide was obtained following tryptic digestion of
catalytically inactive HA-KD RAC-PK.alpha. that had been
phosphorylated with MAPKAP kinase-2 (data not shown).
EXAMPLE 7
[0229] Phosphorylation of Thr308 and Ser473 Causes Synergistic
Activation of RAC-PK.alpha.
[0230] The experiments described above demonstrated that
phosphorylation of Ser-473 activates RAC-PK.alpha. in vitro but did
not address the role of phosphorylation of Thr-308, or how
phosphorylation of Thr-308 might influence the effect of Ser-473
phosphorylation on activity, or vice versa. We therefore prepared
HA-tagged RAC-PK.alpha. DNA constructs in which either Ser473 or
Thr308 would be changed either to Ala (to block the effect of
phosphorylation) or to Asp (to try and mimic the effect of
phosphorylation).
[0231] FIG. 12--Activation of HA-RAC-PK.alpha. mutants in vitro by
MAPKAP kinase-2.
[0232] (a)--WT and mutant HA-RAC-PK.alpha. proteins were
immunoprecipitated from the lysates of unstimulated COS-1 cells
expressing these constructs and incubated for 60 minutes with MgATP
in the absence (-, filled bars) or presence (+, hatched bars) of
MAPKAP kinase-2 and MgATP (50 U/mL). The RAC-PK.alpha. protein was
expressed as similar levels in each construct and specific
activities are presented relative to WT HA-RAC-PK.alpha. incubated
in the absence of MAPKAP kinase-2 (0.03 U/mg). The results are
shown as the average .+-.SEM for 3 experiments.
[0233] (b)--Twenty (20) .mu.g of protein from each lysate was
electrophoresed on a 10% SDS/polyacrylamide gel and immunoblotted
using monoclonal HA-antibody.
[0234] All the mutants were expressed at a similar level in
serum-starved COS-1 cells (data not shown) and the effects of
maximally phosphorylating each of them at Ser473 is shown in FIG.
12 (a). Before phosphorylation with MAPKAP kinase-2 the activity of
HA-473A RAC-PK.alpha. was similar to that of unstimulated WT
HA-RAC-PK.alpha. and, as expected, incubation with MAPKAP kinase-2
and MgATP did not result in any further activation of HA-473A
RAC-PK.alpha.. In contrast, the activity of HA-473D RAC-PK.alpha.
was 5- to 6-fold higher than that of unstimulated WT
HARAC-PK.alpha. protein, and similar to that of WT HA-RAC-PK.alpha.
phosphorylated at Ser473. As expected, HA-473D RAC-PK.alpha. was
also not activated further by incubation with MAPKAP kinase-2 and
MgATP. The activity of HA-308A RAC-PK.alpha. was about 40% that of
the unstimulated WT enzyme, and after phosphorylation with MAPKAP
kinase-2 is activity increased to a level similar to that of WT
HA-RAC-PK.alpha. phosphorylated at Ser473. Interestingly, HA-308D
RAC-PK.alpha. which (like HA473D PK) was 5-fold more active than
dephosphorylated WT HA-RAC-PK.alpha., was activated dramatically by
phosphorylation of Ser473. After incubation with MAPKAP kinase-2
and MgATP, the activity of HA-308D RAC-PK.alpha. was nearly 5-fold
higher than that of WT HA-RAC-PK.alpha. phosphorylated at Ser473.
See FIG. 12 (b). These results suggested that the phosphorylation
of either Thr308 or Ser473 leads to partial activation of
RAC-PK.alpha. in vitro, and that phosphorylation of both residues
results in a synergistic activation of the enzyme. This idea was
supported by further experiments in which both Thr308 and Ser473
were changed to Asp. When this double-mutant was expressed in COS-1
cells it was found to possess an 18-fold higher specific activity
than the dephosphorylated WT protein. As expected, the activity of
this mutant was not increased further by incubation with MAPKAP
kinase-2 and MgATP. See FIG. 12 (b).
EXAMPLE 8
[0235] Phosphorylation of Both Thr308 and Ser473 is Required for a
High Level of Activation of RAC-PK.alpha. In Vivo
[0236] FIG. 9--Effect of mutation of RAC-PK.alpha. on its
activation by insulin in 293 cells.
[0237] (a)--Two hundred ninety-three (293) cells were transiently
transfected with DNA constructs expressing WT RAC-PK.alpha.,
HA-D473-RAC-PK.alpha. and HA-308D/473D-RAC-PK.alpha.. After
treatment for 10 minutes with or without 100 nM wortmannin, cells
were stimulated for 10 minutes with or without 100 nM insulin in
the continued presence of wortmannin. RAC-PK.alpha. was
immunoprecipitated from the lysates and assayed, and activities
corrected for the relative levels of HA-RAC-PK.alpha. expression as
described in the methods. The results are expressed relative to the
specific activity of WT HA-RAC-PK.alpha. obtained from unstimulated
293 cells.
[0238] (b)--Twenty (20) .mu.g of protein from each lysate was
electrophoresed on a 10% SDS/polyacrylamide gel and immunoblotted
using monoclonal HA-antibody.
[0239] The basal level of activity of HA-473A RAC-PK.alpha. derived
from unstimulated cells was similar to that of WT RAC-PK.alpha..
See FIG. 8 (a). Stimulation of 293 cells expressing HA-473A
RAC-PK.alpha. with insulin or IGF-1 increased the activity of this
mutant 3- and 5-fold, respectively; i.e., to 15% of the activity of
WT HA-RAC-PK.alpha. which had been transiently-expressed and
stimulated under identical conditions. The basal activity of
HA-308A RAC-PK.alpha. in unstimulated cells was also similar to
that of WT HA-RAC-PK.alpha. derived from unstimulated cells, but
virtually no activation of this mutant occurred following
stimulation of the cells with insulin or IGF-1. These data are
consistent with in vitro experiments and indicate that maximal
activation of RAC-PK.alpha. requires phosphorylation of both Ser473
and Thr308 and that phosphorylation of both residues results in a
synergistic activation of the enzyme. Consistent with these
results, HA-473D RAC-PK.alpha. displayed 5-fold higher activity and
the HA-308D/HA473D double-mutant 40-fold higher activity than WT
HA-RAC-PK.alpha. when expressed in unstimulated cells. Following
stimulation with insulin, HA-473D RAC-PKA was activated to a level
similar to that observed with the WT enzyme, while the
HA-308D/HA-473D double-mutant could not be activated further. See
FIG. 13. As expected, activation of HA-473D RAC-PK.alpha. by
insulin was prevented by wortmannin, and the activity of the
HA-308D/ HA-473D double-mutant was resistant to wortmannin. See
FIG. 13.
EXAMPLE 9
[0240] Phosphorylation of Thr308 is Not Dependent on
Phosphorylation of Ser473 or Vice Versa (in 293 Cells)
[0241] FIG. 10--A 10 cm dish of 293 cells were transfected with
either HA-308A RAC-PK.alpha. or HA-473A RAC-PK.alpha.,
.sup.32P-labelled, then stimulated for 10 minutes with either IGF-1
(50 ng/mL) or buffer. The .sup.32P-labelled RAC-PK.alpha. mutants
were immunoprecipitated from the lysates, treated with
4-vinylpyridine, electrophoresed on a 10% polyacrylamide gel,
excised from the gel and digested with trypsin, then
chromatographed on a C18-column. The tryptic peptides containing
the phosphorylated residues Ser124, Thr308, Thr450, Ser473 are
marked and their assignments were confirmed by phosphoamino acid
analysis and sequencing to identify the sites of phosphorylation
(data not shown). The phosphopeptides containing Thr308 and Ser473
were absent if stimulation with IGF-1 was omitted, while the
phosphopeptides containing Ser124 and Thr450 were present at
similar levels as observed with WT RAC-PK.alpha.. See FIG. 9 (a).
Similar results were obtained in 3 separate experiments.
[0242] These experiments demonstrated that IGF-1 stimulation
induced the phosphorylation of HA-473A RAC-PK.alpha. at Thr308, and
the phosphorylation of HA-308A RAC-PK.alpha. at Ser473. Similar
results were obtained after stimulation with insulin rather than
IGF-I.
EXAMPLE 10
[0243] IGF-1 or Insulin Induces Phosphorylation of Thr308 and
Ser473 in a Catalytically Inactive Mutant of RAC-PK.alpha.
[0244] FIG. 15--The catalytically-inactive RAC-PK.alpha. mutant
(HA-KD-RAC-PK.alpha.) expressed in 293 cells is phosphorylated at
Thr308 and S er473 after stimulation with IGF-1. Each 10 cm dish of
293 cells transiently-transfected with HA-KD-RAC-PK.alpha. DNA
constructs was .sup.32P-labelled and incubated for 10 minutes with
buffer (a), 50 ng/mL IGF-1 (b) or 100 nM insulin (c). The
.sup.32P-labelled HA-KD-RAC-PK.alpha. was immunoprecipitated from
the lysates, treated with 4 vinylpyridine, electrophoresed on a 10%
polyacrylamide gel, excised from the gel and digested with trypsin,
then chromatographed on a C18-column. The tryptic peptides
containing the phosphorylated residues Ser124, Thr308, Thr450 and
Ser473 are marked. Similar results were obtained in 3 separate
experiments for (b) and (b), and in 2 experiments for (c).
[0245] This "kinase dead" mutant of RAC-PK.alpha., termed
HA-KD-RAC-PK.alpha., in which Lys179 was changed to Ala (see above)
was transiently expressed in 293 cells and its level of expression
found to be several-fold lower than that of WT HA-RAC-PK.alpha.
expressed under identical conditions. See FIG. 8 (b). As expected,
no RAC-PK.alpha. activity was detected when 293 cells expressing
HA-KD-RAC-PK.alpha., were stimulated with insulin or IGF-1. See
FIG. 7 (a).
[0246] Two hundred ninety-three (293) cells that had been
transiently transfected with HA-KD-RAC-PK.alpha. were
.sup.32P-labelled, then stimulated with buffer, insulin or IGF-1
and sites on RAC-PK.alpha. phosphorylated under these conditions
were mapped. In contrast to WT HA-RAC-PK.alpha. from unstimulated
293 cells (see FIG. 9), HA-KD RAC-PK.alpha. was phosphorylated to a
much lower level at Ser124, but phosphorylated similarly at Thr450.
See FIG. 15 (a). Following stimulation with IGF-1 [see FIG. 15 (b)]
or insulin [see FIG. 14 (c)], HA-KD-RAC-PK.alpha. became
phosphorylated at the peptides containing Thr308 and Ser473, the
extent of phosphorylation of these sites being at least as high as
WT RAC-PK.alpha.. Amino acid sequencing of these peptides
established that they were phosphorylated at Thr308 and Ser473,
respectively.
[0247] The above examples establish that RAC-PK influences GSK3
activity; that Thr308 and Ser473 are the major residues in
RAC-PK.alpha. that become phosphorylated in response to insulin or
IGF-1 (see FIGS. 2 and 5) and that phosphorylation of both residues
is required to generate a high level of RAC-PK.alpha. activity.
Thus, mutation of either Thr308 or Ser473 to Ala greatly decreased
the activation of transfected RAC-PK.alpha. by insulin or IGF-1 in
293 cells. See FIG. 8. Moreover, RAC-PK.alpha. became partially
active in vitro when either Thr308 or Ser473 were changed to Asp or
when Ser473 was phosphorylated by MAPKAP kinase-2 in vitro, and far
more active when the D308 mutant of RAC-PK.alpha. was
phosphorylated by MAPKAP kinase-2 or when Thr308 and Ser473 were
both mutated to Asp. See FIG. 12. Moreover, the D308/D473
double-mutant could not be activated further by stimulating cells
with insulin. See FIG. 13. These observations demonstrate that the
phosphorylation of Thr308 and Ser473 act synergistically to
generate a high level of RAC-PK.alpha. activity.
[0248] Thr308, and the amino acid sequence surrounding it, is
conserved in rat RAC-PK.beta. and RAC-PK.gamma. but, interestingly,
Ser473 (and the sequence su rrounding it) is only conserved in
RAC-PK.beta.. In rat RAC-PK.gamma., Ser473 is missing because the
C-terminal 23 residues are deleted. This suggests that the
regulation of RAC-PK.gamma. may differ significantly from that of
RAC-PK.alpha. and RAC-PK.beta. in the rat.
[0249] Thr308 is located in subdomain VIII of the kinase catalytic
domain, 9 residues upstream of the conserved Ala-Pro-Glu motif, the
same position as activating phosphorylation sites found in many
other PKs. However, Ser473 is located C-terminal to the catalytic
domain in the consensus sequence
Phe-Xaa-Xaa-Phe/Tyr-Ser/Thr-Phe/Tyr which is present in several
protein kinases that participate in growth factor-stimulated kinase
cascades, such as p70 S6 kinase, PKC and p9orsk. See Pearson et al.
(1995), supra. However, it is unlikely that a common PK
phosphorylates this motif in every enzyme for the following
reasons. Firstly, phosphorylation of the equivalent site in p70 S6
kinase is prevented by the immunosuppressant drug rapamycin [see
Pearson et al. (1995), supra] which does not prevent the activation
of RAC-PK.alpha. by insulin [see Cross et al., Nature, Vol. 378,
No. 6559, pp. 785-789 (1995)] or is phosphorylation at Ser473. See
D. Alessi, unpublished work. Secondly, the equivalent residue in PK
cascade is phosphorylated constitutively and not triggered by
stimulation with growth factors. See Tsutakawa et al., J Biol Chem,
Vol. 270, No. 45, pp. 26807-26812 (1995).
[0250] MAPKAP kinase-2 is a component of a PK cascade which becomes
activated when cells are stimulated with interleukin-1 or tumour
necrosis factor or exposed cellular stresses. See Rouse et al.
(1994), supra; and Cuenda et al. (1995), supra. MAPKAP kinase-2
phosphorylates RAC-PK.alpha. stoichiometrically at Ser473 (see FIG.
11) and this finding was useful in establishing the role of Ser473
phosphorylation in regulating RAC-PK.alpha. activity. However,
although MAPKAP kinase-2 activity is stimulated to a small extent
by insulin in L6 cells, no activation could be detected in 293
cells in response to insulin or IGF-1. Moreover, exposure of L6
cells or 293 cells to a chemical stress (0.5 mM sodium arsenite)
strongly activated MAPKAP kinase-2 (see D. Alessi, unpublished
work) as found in other cells [see Rouse et al. (1994), supra; and
Cuenda et al. (1995), supra], but did not activate RAC-PK.alpha. at
all. Furthermore, the drug SB 203580 which is a specific inhibitor
of the PK positioned immediately upstream of MAPKAP kinase-2 [see
Cuenda et al. (1995), supra], prevented the activation of MAPKAP
kinase-2 by arsenite but had no effect on the activation of
RAC-PK.alpha. by insulin or IGF-1. Finally, the activation of
RAC-PK.alpha. was prevented by wortmannin (see FIGS. 6 and 9), but
wortmannin had no effect on the activation of MAPKAP kinase-2 in L6
or 293 cells. It should also be noted that the sequence surrounding
Ser473 of RAC-PK.alpha. (HFPQFSY) does not conform to the optimal
consensus for phosphorylation by MAPKAP kinase-2 which requires Arg
at position n-3 and a bulky hydrophobic residue at position n-5,
where n is the position of the phosphorylated residue. The Km for
phosphorylation of the synthetic peptide comprising residues
465-478 is nearly 100-fold higher than the Km for the standard
MAPKAP kinase-2 substrate peptide (data not shown). It is therefore
unlikely that MAPKAP kinase-2 mediates the phosphorylation of
Ser473 in vivo.
[0251] The enzyme(s) which phosphorylates Thr308 and Ser473 in vivo
does not appear to be RAC-PK.alpha. itself. Thus incubation of the
partially active AsP-308 mutant with MgATP did not result in the
phosphorylation of Ser473, phosphorylation of the latter residue
only occurring when MAPKAP kinase-2 was added. See FIG. 11 (a) and
FIG. 12. Similarly, Thr308 did not become phosphorylated when
either the partially-active D473 mutant or the partially-active
Ser473 phosphorylated form of RAC-PK.alpha. were incubated with M
gATP. RAC-PK.alpha. when bound to lipid vesicles containing
phosphatidylserine and PIP3 also fails to activate upon incubation
with MgATP [see Alessi et al. (1996), supra] and after transfection
into 293 cells, a "kinase dead" mutant of RAC-PK.alpha. became
phosphorylated on Thr308 and Ser473 in response to insulin or
IGF-1. See FIG. 14. Furthermore, HA-RAC-PK.alpha. from either
unstimulated or insulin-stimulated 293 cells failed to
phosphorylate the synthetic C-terminal peptide comprising amino
acids 467-480.
[0252] In unstimulated L6 myotubes, the endogenous RAC-PK.alpha.
was phosphorylated at a low level at a number of sites [see FIG. 6
(a)], but in unstimulated 293 cells the transfected enzyme was
heavily phosphorylated at Ser124 and Thr450. See FIG. 10. Ser124
and Thr450 are both followed by praline (Pro) residues suggesting
the involvement of "Pro-directed" PKs. Although, the
phosphorylation of Ser124 was greatly decreased when "kinase dead"
RAC-PK.alpha. was transfected into 293 cells (see FIG. 14), it
would be surprising if Ser124 is phosphorylated by RAC-PK.alpha.
itself because the presence of a C-terminal Pro abolishes the
phosphorylation of synthetic peptides by RAC-PK.alpha. (see D.
Alessi, unpublished work). Since transfected RAC-PK.alpha. is
inactive in unstimulated 293 cells (see FIG. 12), phosphorylation
of Ser124 and Thr450 clearly does not activate RAC-PK.alpha.
directly. Ser 24 is located in the linker region between the PH
domain and the catalytic domain of the mammalian RAC-PK.alpha.
isoforms but, unlike Thr450, is not conserved in the Drosophila
homologue. See Andjelkovic et al., Proc Nat Acad Sci USA (1995),
supra.
[0253] While we do not wish to be bound by hypotheses, the results
described suggest that agonists which activate PI 3-kinase are
likely to stimulate RAC-PK.alpha. activity via one of the following
mechanisms. Firstly, PIP3 or P13,4-bisP may activate one or more
protein kinases which then phosphorylate RAC-PK.alpha. at Thr308
and Ser473. Secondly, the formation of PIP3 may lead to the
recruitment of RAC-PK.alpha. to the plasma membrane where it is
activated by a membrane-associated PK(s). The membrane associated
Thr308 and Ser473 kinases might themselves be activated by PIP3 and
the possibility that Thr308 and/or Ser473 are phosphorylated
directly by PI 3-kinase has also not been excluded, because this
enzyme is known to phosphorylate itself [see Dhand et al., EMBO J,
Vol.13, No. 3, pp. 522-533 (1994)] and other proteins [see Lam et
al., J Biol Chem, Vol. 269, No. , pp. 20648-20652 (1994)] on serine
residues.
EXAMPLE 11
[0254] Molecular Basis for Substrate Specificity of RAC-PK
[0255] RAC-PK.alpha. has been shown to influence GSK3 activity.
GSK3.alpha. and GSK3.beta. are phosphorylated at Ser21 and Ser9,
respectively, by 2 other insulin-stimulated PKs, namely p70 S6
kinase and MAP kinase-activated PK-1 (MAPKAP-K1, also known as p90
S6 kinase). However, these enzymes are not rate-limiting for the
inhibition of GSK3 by insulin in L6 myotubes because specific
inhibitors of their activation (rapamycin-p70 S6 kinase; PD
98059-MAPKAP kinase-1) have no effect. See Cross et al. (1995),
supra.
[0256] The activation of PI 3-kinase is essential for many of the
effects of insulin and growth factors, including the stimulation of
glucose transport, fatty acid synthesis and DNA synthesis,
protection of cells against apoptosis and actin cytoskeletal
rearrangements. Reviewed in Carpenter and Cantley, Curr Opinion
Cell Biol, Vol. 8, No. 2, pp. 153-158 (1996). These observations
raise the question of whether RAC-PK.alpha. mediates any of these
events by phosphorylating other proteins. To address this issue we
characterized the substrate specificity requirements of
RAC-PK.alpha.. We find that the optimal consensus sequence for
phosphorylation by RAC-PK.alpha. is the motif
Arg-Xaa-Arg-Yaa-Zaa-Ser/Thr-Hy, where Yaa and Zaa are small amino
acids (other than Gly) and Hyd is a large hydrophobic residue, such
as Phe or Leu. We also demonstrate that RAC-PK.alpha.
phosphorylates histone H2B (a substrate frequently used to assay
RAC-PK.alpha. in vitro) at Ser36 which lies in an
Arg-Xaa-Arg-Xaa-Xaa-Ser-Hyd motif. These studies identified a
further RAC-PK.alpha. substrate (Arg-Pro-Arg-Ala-Ala-Thr-Phe) that,
unlike other peptides, is not phosphorylated to a significant
extent by either p70 S6 kinase or MAPKAP-K1.
[0257] Results
[0258] Preparation of PK-B.alpha.
[0259] In order to examine the substrate specificity of
RAC-PK.alpha., it was first necessary to obtain a kinase
preparation that was not contaminated with any other PK activities.
Two hundred ninety-three (293) cells were therefore
transiently-transfected with a DNA construct expressing HA-tagged
RAC-PK.alpha., stimulated with IGF-1 and the HA-RAC-PK.alpha.
immunoprecipitated from the lysates. IGF-1 stimulation resulted in
a 38-fold activation of RAC-PK.alpha. (see FIG. 16) and analysis of
the immunoprecipitates by SDS-polyacrylamide gel electrophoresis
revealed that the 60 kDa RAC-PK.alpha. was the major protein
staining with coomassie Blue apart from the heavy- and light-chains
of the HA monoclonal antibody. See FIG. 16, Lanes 2 and 3. The
minor contaminants were present in control immunoprecipitates
derived from 293 cells transfected with an empty pCMV5 vector but
lacked HA-RAC-PK activity. See FIG. 16, Lane 4. Furthermore, a
catalytically inactive mutant HA-RAC-PK.alpha. immunoprecipitated
from the lysates of IGF-1 stimulated 293 cells had no Crosstide
kinase activity. See Alessi et al. (1996), supra. Thus, all the
Crosstide activity in HA-RAC-PK immunoprecipitates is catalyzed by
RAC-PK.alpha..
[0260] Identification of the residues in histone H2B phosphorylated
by RAC-PK.alpha.. Currently, 3 substrates are used to assay
RAC-PK.alpha. activity in different laboratories, histone H2B, MBP
and Crosstide. RAC-PK.alpha. phosphorylated Crosstide with a Km of
4 .mu.M and a Vmax of 260 U/mg (see Table 7.1 A, peptide 1),
histone H2B with a Km of 5 .mu.M and a Vmax of 68 U/mg and MBP with
a Km of 5 .mu.M and a Vmax of 25 U/mg. Thus the Vmax of histone H2B
and MBP are 4- and 10-fold lower than for Crosstide. In order to
identify the residue(s) in histone H2B phosphorylated by
RAC-PK.alpha., .sup.32P-labelled histone H2B was digested with
trypsin (see Methods) and the resulting peptides chromatographed on
a C18-column at pH 1.9. Only one major .sup.32P-labelled peptide
(termed T1) eluting at 19.5% acetonitrile was observed. See FIG. 17
(a). The peptide contained phosphoserine (data not shown), its
sequence commenced at residue 34 of histone H2B and a single burst
of radioactivity occurred after the third cycle of Edman
degradation [see FIG. 17 (b)], demonstrating that RAC-PK.alpha.
phosphorylates histone H2B at Ser36 within the sequence
Arg-Ser-Arg-Lys-Glu-Ser-Tyr. Thus, like the serine phosphorylated
in Crosstide, Ser36 of histone H2B lies in an
Arg-Xaa-Arg-Xaa-Xaa-Ser-Hyd motif (where Hyd is a bulky hydrophobic
residue-Phe in Crosstide, Tyr in H2B).
[0261] Molecular Basis for the Substrate Specificity of
RAC-PK.alpha.
[0262] To further characterize the substrate specificity
requirements for RAC-PK.alpha., we first determined the minimum
sequence phosphorylated efficiently by RAC-PK.alpha. by removing
residues sequently from the C-terminal and N-terminal end of
Crosstide. Removal of the N-terminal Gly and up to 3 residues from
the C-terminus had little effect on the kinetics of phosphorylation
by RAC-PK.alpha.. See Table 7.1A, comparing peptides 1 and 5.
However any further truncation of either the N- or C-terminus
virtually abolished phosphorylation. See Table 7.1A, peptides 8 and
9. The minimum peptide phosphorylated efficiently by RAC-PK.alpha.
(Arg-Pro-Arg-Thr-Ser-Ser-Phe) was found to be phosphorylated
exclusively at the second Ser residue as expected. Consistent with
this finding, a peptide in which this Ser was changed to Ala was
not phosphorylated by RAC-PK.alpha.. See Table 7.1A, peptide 7. All
further studies were therefore carried out using variants of
peptide 5 in Table 7.1A (see below).
[0263] A peptide in which the second Ser of peptide 5 (see Table
7.1 A) was replaced by Thr was phosphorylated with a Km of 30 .mu.M
and an unchanged Vmax. See Table 7.1, peptide 6. All the
.sup.32P-radioactivity incorporated was present as phosphothreonine
and solid phase sequencing revealed that the peptide was only
phosphorylated at the second Thr residue, as expected (data not
shown). Thus RAC-PK.alpha. is capable of phosphorylating Thr, as
well as Ser residues, but has a preference for Ser.
[0264] We next changed either of the two Arg residues in peptide 5
to Lys. These substitutions drastically decreased the rate of
phosphorylation by RAC-PK.alpha. (see Table 7.1A, peptides 10 and
11), demonstrating a requirement for Arg (and not simply any basic
residue) at both positions.
[0265] We then examined the effect of substituting the residues
situated immediately C-terminal to the phosphorylated Ser in
peptide 5, Table 7.1 B. The data clearly demonstrate that the
presence of a large hydrophobic residue at this position is
critical for efficient phosphorylation, with the Km increasing
progressively with decreasing hydrophobicity of the residue at this
position. See Table 7.1 B, peptides 14. Replacement of the
C-terminal residue with Lys increased the Km 18-fold and a
substitution at this position with either Glu or praline (Pro)
almost abolished phosphorylation. See Table 7.1B, peptides 5-7.
[0266] Replacement of the Thr situated 2 residues N-terminal to the
phosphorylated Ser increased the Km with any amino acid tested. See
Table 7.1C. Substitution with Ala only increased Km by 2- to
3-fold, but substitution with other residues was more deleterious
and with Asn (a residue of similar size and hydrophilicity to Thr)
phosphorylation was almost abolished. See Table 7.1C. Replacement
of the Ser situated 1 residue N-terminal to the phosphorylated Ser
also increased the Km with any amino acid tested, but the effects
were less severe than at position n-2. See Table 7.1C. When
residues n-2 and n-1 were both changed to Ala, the resulting
peptide RPRAASF (SEQ ID NO: 8) was phosphorylated by RAC-PK.alpha.
with a Km only 5-fold higher than RPRTSSF (SEQ ID NO: 9). In
contrast the peptides RPRGGSF (SEQ ID NO: 10), RPRAGSF (SEQ ID NO:
11) and RPRGASF (SEQ ID NO: 12) were phosphorylated less
efficiently. See Table 7.1C.
[0267] Comparison of the substrate specificity of RAC-PK.alpha.
with MAPKAP kinase-1, and p70 S6 kinase. Since MAPKAP-K1 and p70 S6
kinase phosphorylate the same residue in GSK3 phosphorylated by
RAC-PK.alpha., and studies with synthetic peptides have established
that MAPKAP-K1 and p70 S6 kinase also preferentially phosphorylate
peptides in which basic residues are present at positions n-3 and
n-5 [see Leighton et al., FEBS Lett, Vol. 375, No. 3, pp. 289-293
(1995)], we compared the specificities of MAPKAP-K1, p70 S6 kinase
and RAC-PK.alpha. in greater detail.
[0268] MAPKAP kinase-1 and p70 S6 kinase phosphorylate the peptides
KKKNRTLSVA (SEQ ID NO: 13) and KKRNRTLSVA (SEQ ID NO: 14) with
extremely low Km values of 0.2-3.3 .mu.M, respectively. See Table
7.2. However, these peptides were phosphorylated by RAC-PK.alpha.
with 50- to 900-fold higher Km values. See Table 7.2A, peptides 1
and 2. The peptide KKRNRTLTV (SEQ ID NO: 15), which is a relatively
specific substrate for p70 S6 kinase [see Leighton et al. (1995),
supra] was also phosphorylated very poorly by RAC-PK.alpha.. See
Table 7.2A, peptide 4.
[0269] Crosstide is phosphorylated by p70 S6 kinase and MAPKAP
kinase-1 with similar efficiency to RAC-PK.alpha.. See Leighton et
al. (1995), supra; Table 7.2B, peptide 1; and FIG. 18. However,
truncation of Crosstide to generate the peptide RPRTSSF (SEQ ID NO:
9) was deleterious for phosphorylation by MAPKAP-K1 and even worse
for p70 S6 kinase. See Table 7.2B, peptides 1 and 2; and FIG. 18.
Moreover, changing the phosphorylated Ser in RPRTSSF (SEQ ID NO: 9)
to Thr increased the Km for phosphorylation by p70 S6 kinase much
more than for RAC-PK.alpha. and almost abolished phosphorylation by
MAPKAP-K1. See Table 7.2B, peptide 3; and FIG. 18. The peptide
RPRAASF (SEQ ID NO: 8), was phosphorylated by MAPKAP-K1 with
essentially identical kinetics to that of RAC-PK.alpha.; however
phosphorylation by p70 S6 kinase was virtually abolished. See Table
7.2B, peptide 4; and FIG. 18. Based on these observations we
synthesized the peptide RPRAATF (SEQ ID NO: 16). This peptide was
phosphorylated by RAC-PK.alpha. with a Km of 25 .mu.M and similar
Vmax to RPRTSSF (SEQ ID NO: 9), but was not phosphorylated to a
significant extent by either MAPKAP-K1 or p70 S6 kinase. See Table
7.2B, peptide 5; and FIG. 18. In FIG. 18, the PK concentration in
the assays towards Crosstide were 0.2 U/mL, and each peptide
substrate was assayed at a concentration of 30 .mu.M. Filled bars
denote RAC-PK.alpha. activity, hatched bars MAPKAP kinase-1
activity, and grey bars p70 S6 kinase activity. The activities of
each PK are given relative to their activity towards Crosstide
(100). The results are shown .+-.SEM for 2 experiments each carried
out in triplicate.
[0270] Discussion
[0271] We have established that the minimum consensus sequence for
efficient phosphorylation by RAC-PK.alpha. is
Arg-Xaa-Arg-Yaa-Zaa-Ser-Hy, where Xaa is any amino acid, Yaa and
Zaa are small amino acid other than Gly (Ser, Thr and Ala) and Hyd
is a bulky hydrophobic residue (Phe and Leu). See Table 7.1. The
heptapeptide with the lowest Km value was RPRTSSF (SEQ ID NO: 9),
its Km of 5 .mu.M being comparable to many of the best peptide
substrates identified for other PKs. The Vmax for this peptide (250
nmoles min-1 mg-1) may be an underestimate because the
RAC-PK.alpha. was obtained by immunoprecipitation from extracts of
IGF-1 stimulated 293 cells over-expressing this PK, and a
significant proportion of the RAC-PK.alpha. may not have been
activated by IGF-1 treatment.
[0272] The requirement for Arg residues at positions n-3 and n-5
(where n is the site of phosphorylation) seems important, because
substituting either residue with Lys decreases phosphorylation
drastically. Ser and Thr residues were preferred at positions n-1
and n-2, although the Km value was only increased about 5-fold if
both of these residues were changed to Ala. Ser was preferred at
position n, since changing it to Thr caused a 6-fold increase in
the Km. The peptide RPRAATF (SEQ ID NO: 16), which was
phosphorylated with a Km of 25 .mu.M and similar Vmax to RPRTSSF
(SEQ ID NO: 9), may therefore be a better substrate for assaying
RAC-PK.alpha. in partially-purified preparations, because unlike
Crosstide, it contains only one phosphorylatable residue and is not
phosphorylated significantly by MAPKAP-K1 or p70 S6 kinase. See
Table 7.2; FIG. 18; and see below.
[0273] The Pro at position n-4 was not altered in this study
because it was already clear that this residue was not critical for
the specificity of RAC-PK.alpha.. Residue n-4 is Pro in GSK3.beta.
but Ala in GSK3.alpha.. Both GSK3 isoforms are equally good
substrates for RAC-PK.alpha. in vitro [see Cross et al. (1995),
supra], and the peptide GRARTSSFA (SEQ ID NO: 17), corresponding to
the sequence in GSK3a, is phosphorylated by RAC-PK.alpha. with a Km
of 10 .mu.M and Vmax of 230 U/mg. See Table 7.1A, peptide 2.
Moreover, in histone H2B, the residue located 4 amino acids
N-terminal to the RAC-PK.alpha. phosphorylation site is Serine. See
FIG. 17. The presence of Glu and Lys at positions n-1 and n-2 may
explain why histone H2B is phosphorylated by RAC-PK.alpha. with a
4-fold lower Vmax than the peptide RPRTSSF (SEQ ID NO: 9).
[0274] Two other PKs which are activated by insulin and other
growth factors, p70 S6 kinase and MAPKAP-K1, require basic residues
at positions n-3 and n-5 [see Leighton et al. (1995), supra],
explaining why they also phosphorylate and inactivate GSK3 in
vitro. See Sutherland, Leighton and Cohen, Biochem J (1993), supra.
Indeed, there is evidence that MAPKAP-K1 plays a role in the
inhibition of GSK3 by EGF because, unlike inhibition by insulin
which is prevented by inhibitors of PI 3-kinase, the inhibition of
GSK3 by EGF is only suppressed partially by inhibitors of PI
3-kinase. Moreover, in NIH 3T3 cells, the inhibition of GSK3.alpha.
and GSK3.beta. by EGF is largely prevented by expression of a
dominant negative mutant of MAPKAP kinase-1. See Eldar-Finkelman,
Seger, Vandenheede and Krebs (1995), supra. In contrast, p70S6
kinase is not rate limiting for the inhibition of GSK3 in the cells
that have been examined so far because rapamycin, which prevents
the activation of p70 S6 kinase by EGF or insulin, has no effect on
the inhibition of GSK3 by these agonists. See Cross et al. (1995),
supra; and Saito, Vandenheede and Cohen, Biochem J (1994),
supra.
[0275] Additional similarities between p70 S6 kinase, MAPKAP-K1 and
RAC-PK.alpha. include the failure to phosphorylate peptides
containing Pro at position n+1 and dislike of a Lys at the same
position. This suggests that, in vivo, these kinases are unlikely
to phosphorylate the same residues as MAP kinases (which
phosphorylates Ser/Thr-Pro motifs) or PK C, which prefers basic
residues C-terminal to the site of phosphorylation. However, the
present work has also revealed significant differences in the
specificities of these enzymes. In particular, MAPKAP-K1 and (to a
lesser extent) p70 S6 kinase can tolerate substitution of the Arg
at position n-5 by Lys, whereas RAC-PK.alpha. cannot. See Tables
7.1A and 7.2A; and Leighton et al. (1995), supra. MAPKAP-K1 and p70
S6 kinase can also tolerate, to some extent, substitution of Arg at
position n-3 by Lys. For example, the peptide KKRNKTLSVA is
phosphorylated by MAPKAP-K1 and p70 S6 kinase with Km values of 17
.mu.M and 34 .mu.M, respectively, as compared to Km values of 0.7
.mu.M and 1.5 .mu.M for the peptide KKRNRTLSVA (SEQ ID NO: 14). See
Table 7.2A. In contrast, RAC-PK.alpha. does not phosphorylate the
peptide KKRNKTLSVA (see Table 7.2A) or any other peptide that lacks
Arg at position n-3. RAC-PK.alpha. and p70 S6 kinase, but not
MAPKAP-K1, phosphorylate Thr, as well as Ser (see Table 7.1 A) and
can phosphorylate peptides lacking any residue at position n+2 [see
Leighton et al. (1995), supra; and Table 7.2A], while RAC-PK.alpha.
and NAPKAP-K1, but not p70 S6 kinase, can tolerate substitution of
both the n-1 and n-2 positions of the peptide RPRTSSF (SEQ ID NO:
9) with Ala. See Table 7.2B. These differences explain why the
peptide RPRAATF (SEQ ID NO: 16) is a relatively specific substrate
for RAC-PK.alpha..
[0276] One of the best peptide substrates for MAPKAP-K1 and p70 S6
kinase KKRNRTLSVA (SEQ ID NO: 14) was a poor substrate for
RAC-PK.alpha. (see Table 7.2, peptide 2), despite the presence of
Arg at positions n-3 and n-5. The presence of Leu at position n-1
and Val at position n+1 are likely to explain the high Km for
phosphorylation, because RAC-PK.alpha. prefers a small hydrophilic
residue at the former position and a larger hydrophobic residue at
the latter position. See Tables 7.1 and 7.2.
EXAMPLE 12
[0277] This example demonstrates that coexpression of GSK3 in 293
cells with either the WT or a constitutively-activated RAC-PK
results in GSK3 becoming phosphorylated and inactivated. However
coexpression of a mutant of GSK3 in which Ser9 is mutated to an Ala
residue is not inactivated under these conditions. These
experiments provide further evidence that RAC-PK.alpha. activation
can mediate the phosphorylation and inactivation of GSK3 in a
cellular environment, and could be used as an assay system to
search for specific RAC-PK inhibitors.
[0278] Monoclonal antibodies recognising the sequence EFMPME (EE)
(SEQ ID NO: 18) antibodies and the EQKLISEEDL (SEQ ID NO: 19) c-Myc
purchased from Boehringer, Lewis, UK.
[0279] Construction of expression vectors and transfections into
293 cells. HA-RAC-PK.alpha., HA-KD-RAC-PK and 308D/473D
HA-RAC-PK.alpha. as was described previously. See Alessi et al.
(1996), supra.
[0280] A DNA construct expressing human GSK3B with the EFMPME (EE)
(SEQ ID NO: 18) epitope tag at the N-terminus was prepared as
follows. A standard PCR reaction was carried out using as a
template the human GSK3.beta. cDNA clone in the pBluescript
SK+vector and the oligonucleotides
GCGGAGATCTGCCACCATGGAGTTCATGCCCATGGAGTCAGG GCGGCCCAGAACC (SEQ ID
NO: 20) and GCGGTCCGGMCATAGTCCAGCACCAG (SEQ ID NO: 21) that
incorporate a bgl II site (underlined) and a Bspe I site (double
underlined). A 3-way ligation was then set up in which the
resulting PCR product was subcloned as a Bgl II-Bspe I fragment
together with the C-terminal Bspe I-Cla I fragment of GSK3.beta.
into the Bgl II-Cla I sites of the pCMV5 vector. See Andersson et
al., J Biol Chem, Vol. 264, No. 14, pp. 8222-8229 (1989). The
construct was verified by DNA sequencing and pu rified using the
Quiagen plasmid Mega kit according to the manufacturers protocol.
The c-Myc GSK3, BA9 construct encodes GSK3.beta. in which Ser9 is
mutated to Ala and possesses a c-myc epitope tag at the C-terminus
and was prepared as described in Sperber, Leight, Goedert and Lee,
Neurosci Lett, Vol. 197, No. 2, pp. 149-153 (1995). The c-Myc
GSK3.beta. A9 gene was then subcloned into xba I/ECOR I sites of
the pCMV5 eukaryotic expression vector.
[0281] Cotransfection of GSK3.beta. with RAC-PK.alpha. and its
assay. 293 cells growing on 10 cm diameter dishes were transfected
with 10 .mu.g of DNA constructs expressing EE-GSK3, Myc-GSK3A9 in
the presence or absence of HA-RAC-PK, HA-KD-RAC-PK or
HA-308D/473D-RAC-PK exactly as described in Alessi et al. (1996),
supra. The cells were grown in the absence of serum for 16 hours
prior to lysis, and then lysed in 1.0 mL of ice-cold buffer A (50
mM Tris/HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% (by vol) Triton X100,
1 mM sodium orthopervanadate, 10 mM sodium glycerophosphate, 50 mM
NaF, 5 mM sodium pyrophosphate, 1 uM Microcystin-LR, 0.27 M
sucrose, 1 mM benzamidine, 0.2 mM phenylmethylsulphonyl fluoride,
10 .mu.g/mL leupeptin and 0.1% (by vol) 2-mercaptoethanol). The
lysate was centrifuged at 4.degree. C. for 10 minutes at
13,000.mu.g and an aliquot of the supernatant (100 .mu.g protein)
was incubated for 30 minutes on a shaking platform with 5 .mu.L of
Protein G-Sepharose coupled to lug of EE monoclonal antibody. The
suspension was centrifuged for 1 minute at 13,000.times.g, the
Protein G-Sepharose-antibody-EE-GSK3.beta. complex washed twice
with 1.0 mL of buffer A containing 0.5 M NaCl, and three times with
Buffer B (50 mM Tris, pH 7.5, 0.1 mM EGTA, 0.01% (by vol) Brij-35
and 0.1% (by vol) 2-mercaptoethanol), and the immunoprecipitate
assayed for GSK3 activity after incubation with either PP2A or
microcystin inactivated PP2A as described previously. See Cross et
al. (1994), supra.
[0282] Results
[0283] Cotransfection of GSK3.beta. with RAC-PK.alpha. in 293 cells
results in GSK3 phosphorylation and inactivation human embryonic
kidney 293 cells were transfected with a DNA construct expressing
EE-epitope tagged GSK3.beta. either in the presence or absence of
DNA constructs expressing WT-RAC-PK.alpha., a catalytically
inactive RAC-PK.alpha. or a constitutively active
HA-(308D/473D)-RAC-PK.alpha.. Cells were serum starved for 16
hours. Thirty-six (36) hours post-transfection, the cells were
lysed, and the GSK3.beta. immunoprecipitated from the lysates using
monoclonal EE antibodies and the GSK3.beta. activity measured
before and after treatment with PP2A. When EE-GSK3.beta. was
expressed alone or in the presence of a catalytically inactive
RAC-PK.alpha., treatment of the EE-GSK3.beta. with PP2A only
resulted in about a 12% increase in activity. See FIG. 19 (a).
However when EE-GSK3.beta. was coexpressed with either the WT
RAC-PK.alpha. or the constitutively activated
308D/473D-HA-RAC-PK.alpha., treatment of the EE-GSK3 from these
cell lysates with PP2A resulted in a 68% and 85% increase in the
GSK3 activity, respectively. Coexpression of Myc-GSK3.beta. A9 with
HA-RAC-PK or the constitutively active 308D/473D-HA-RAC-PK.alpha.
did not result in any significant inactivation of this mutant of
GSK3 as judged by its ability to be reactivated by PP2A. See FIG.
19 (b). These data demonstrate that even in a cellular environment,
RAC-PK.alpha. is capable of phosphorylating GSK3.beta. at Ser9 and
inactivation of the enzyme. To estimate the relative levels of
EE-GSK3.beta. and RAC-PK.alpha., EE-GSK3 and HA-RAC-PK.alpha. were
immunoprecipitated from equal volumes of cell lysate, and the
immunoprecipitates run on an SDS-polyacrylamide gel, and the gel
stained with Coomassie Blue. These experiments revealed that both
the WT HA-RAC-PK.alpha. and the 308D/473D-RAC-PK.alpha. were
expressed at a 20- to 30-fold higher level than GSK3a, whereas
KD-RAC-PK.alpha. is expressed at a level that is about 5-fold lower
than that of the WT RAC-PK.alpha.. Under the conditions used for
the immunoprecipitations, no RAC-PK.alpha. was
co-immnuoprecipitated with GSK3.beta., or no GSK3.beta. was
co-immunoprecipitated with the RAC-PK.alpha. (data not shown).
Coexpression of EE-GSK3.beta. with all forms of RAC-PK.alpha.
resulted in about a 2- to 3-fold decrease in the level of
expression on EE-GSK3.beta. compared to when it is expressed alone
in cells.
EXAMPLE 13
[0284] Basic Assay for Identifying Agents which Affect the Activity
of RAC-PK
[0285] A 40 .mu.L assay mix was prepared containing PK (0.2 U/mL)
in 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 0.1% (by vol)
2-mercaptoethanol, 2.5 .mu.M PKI, PK substrate (30 .mu.M), and the
indicated concentration of Ro-318220 or GC 109203X (test
inhibitors). After incubation on ice for 10 minutes, the reaction
was started by the addition of 10 .mu.L of 50 mM magnesium acetate
and 0.5 mM [.gamma..sup.32P]ATP (100-200 cpm/pmol). For the assay
of mixed isoforms of PKC 20 .mu.M diacylglycerol, 0.5 mM CaCl.sub.2
and 100 .mu.M phosphatidylsene were also present in the
incubations. The assays were carried out for 15 minutes at
30.degree. C., then terminated and analyzed as described. See
Alessi et al., Methods Enzymol, Vol. 255, pp. 279-290 (1995). One
unit of activity was that amount of enzyme that catalyzed the
phosphorylation of 1 nmol os substrate in 1 minute. The final
concentration of DMSO in each assay was 1% (by vol). This
concentration of DMSO does not inhibit any of these enzymes. Mixed
isoforms of PKC were assayed using histone H1 as substrate, while
MAPKAP-K1.beta. and p70 S6 kinase were assayed using the peptide
KKRNRTLSVA (SEQ ID NO: 14). See Leighton et al. (1995), supra. PK B
was assayed with the peptide GRPRTSSFAEG [9] (SEQ ID NO: 5) and
MAPKAP-K2 was assayed with the peptide KKLNRTLSVA (SEQ ID NO: 27).
See Stokoe, Caudwell, Cohen and Cohen, Biochem J, Vol. 296, Pt. 3,
pp. 843-849 (1993). p42 MAP kinase was assayed using MBP, and
MAPKK-1 and c-Rafl were assayed as described in Alessi et al.,
Methods Enzymol (1995), supra.
[0286] Results
[0287] Effect of Ro 318220 and GF 109203X on PKs activated by
growth factors, cytokines and cellular stresses. The mixed isoforms
of PKC were potently inhibited by Ro 318220, with an IC.sub.50 of 5
nM in our assay. See FIG. 20 (a). In contrast, a number of PKs
activated by growth factors (c-Rafl, MAPKK-1 and p42 MAP kinase)
and 1 PK that is activated by cellular stresses and proinflammatory
cytokines (MAPKAP-K2) were not inhibited significantly by Ro 318022
in vitro. See FIG. 20 (a). PK B, an enzyme that is activated in
response to insulin and growth factors was inhibited by Ro 318220
(IC.sub.50 of 1 .mu.M) (see FIG. 20 (b) similar to the IC.sub.50
for PK.alpha.. However, to our surprise, MAPKAP-K1.beta. an enzyme
which lies immediately downstream of p42 and p44 MAP kinases and
which is activated in response to every agonist that stimulates
this pathway, was inhibited by Ro 318220 even more potently than
the mixed PKC isoforms (IC.sub.50=3 nm). See FIG. 20 (b). The p70
S6 kinase, which lies on a distinct growth factor-stimulated
signalling pathway from MAPKAP-K1, was also potently inhibited by
Ro 318220 (IC.sub.50=15 nM). See FIG. 20 (b).
[0288] Similar results were obtained using GF 109203X instead of Ro
3318220. As reported previously [see Toullec et al., J Biol Chem,
Vol. 266, No. 24, pp.15771-15781 (1991)], GC 109203X inhibited the
mixed isoforms of PKC (IC.sub.50=30 nM) without inhibiting PK B
(see FIG. 21) or c-Raf, MAPKK-1 and p42 MAP kinase (data not
shown). However MAPKAP-K1 B and p70 S6 kinase were potently
inhibited by this compound with IC.sub.50 values of 50 nM and 100
nM, respectively. See FIG. 21.
[0289] General Materials and Methods
[0290] Tissue culture reagents, MBP, microcystin-LR, and IGF-1 were
obtained from Life Technologies Inc. (Paisley, UK), insulin from
Novo-Nordisk (Bagsvaerd, Denmark), phosphate free Dulbecco's
minimal essential medium (DMEM) from (ICN, Oxon, UK), Protein
G-Sepharose and CH-Sepharose from Pharmacia (Milton Keynes, UK),
alkylated trypsin from Promega (Southampton, UK), 4-vinylpyridine,
wortmannin and fluroisothiocyanante-labelled antimouse IgG from
goat from Sigma-Aldrich (Poole, Dorset, UK). Polyclonal antibodies
were raised in sheep against the peptides RPHFPQFSYSASGTA (SEQ ID
NO: 22), corresponding to the last 15 residues of rodent
RAC-PK.alpha., and MTSALATMRVDYEQIK (SEQ ID NO: 23), corresponding
to residues 352-367 of human MAPKAPkinase-2, and affinity purified
on peptide-CH-Sepharose. Monoclonal HA antibodies were purified
from the tissue culture medium of 12CA5 hybridoma and purified by
chromatography on Protein G-Sepharose. The peptide RPRHFPQFSYSAS
(SEQ ID NO: 24), corresponding to residues 465478 of RAC-PK.alpha.,
was synthesized on an Applied Biosystems 430A peptide synthesizer.
cDNA encoding residues 46-400 of human MAPKAP kinase-2 was
expressed in E. coli as a GST fusion protein and activated with
p38/RK MAP kinase by Mr A. Clifton (University of Dundee) as
described previously. See Ben-Levy et al., EMBO J, Vol. 14, No. 23,
pp. 5920-5930 (1995).
[0291] Monoclonal antibodies recognizing the HA epitope sequence
YPYDVPDYA (SEQ ID NO: 25), Protein G-Sepharose and histone H2B were
obtained from Boehringer (Lewes, UK). MAPKAP kinase-1 [see
Sutherland, Leighton and Cohen, Biochem J (1993), supra] and p70 S6
kinases [see Leighton et al. (1995), supra] were purified from
rabbit skeletal muscle and rat liver, respectively.
[0292] Construction of Expression Vectors
[0293] The pECE constructs encoding the human HARAC-PK.alpha. and
kinase-dead (K179A) HA-KD-RAC-PK.alpha. have already been
described. See Andjelkovic et al. (1996), supra. The mutants at
Ser473 (HA-473A RAC-PK.alpha. and HA-473D RAC-PK.alpha. were
created by PCR using a 5' oligonucleotide encoding amino acids
406-414 and mutating 3' oligonucleotide encoding amino acids
468-480, and the resulting PCR products subcloned as Celil-EcoRI
fragment into pECE.HA-RAC-PK.alpha.. The Thr308 mutants (HA-308A
RAC-PK.alpha. and HA308D RAC-PK.alpha.) were created by the 2-stage
PCR technique [see No et al., Gene, Vol. 77, pp. 51 -59 (1989)] and
subcloned as Notl-EcoRI fragments into pECE HA-RAC-PK. The
double-mutant HA-308D/473D RAC-PK was made by subcloning the
CelII-EcoRI fragment encoding 473D into pECE HA-308D RAC-PK.alpha..
For construction of cytomegalovinus-driven expression constructs,
BglII-XbaI fragments from the appropriate pECE constructs were
subcloned into the same restriction sites of the pCMVS vector. See
Andersson et al. (1989), supra.
[0294] All constructs were confirmed by restriction analysis and
sequencing and purified using Quiagen Plasmid Maxi Kit according to
the manufacturer's protocol. All oligonucleotide sequences are
available upon request.
[0295] .sup.32P-labelling of L6 myotubes and immunoprecipitation of
PKR.alpha.. L6 cells were differentiated into myotubes on 10 cm
diameter dishes. See Hundal et al., Endocrinology, Vol. 131, pp.
1165-1171 (1992). The myotubes were deprived of serum overnight in
DMEM, washed three times in phosphate free DMEM and incubated for a
further 1 hour with 5 mL of this medium. The myotubes were then
washed twice with phosphate free DMEM and incubated for 4 hours
with carrier-free [.sup.32P]orthophosphate (1 mCi/mL). Following
incubation in the presence or absence of 100 nM wortmannin for 10
minutes, the myotubes were stimulated for 5 minutes at 37.degree.
C. in the presence or absence of 100 nM insulin and placed on ice.
The medium was aspirated, the myotubes washed twice with ice-cold
DMEM buffer and then lysed with 1.0 mL of ice-cold buffer A (50 mM
Tris/HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% (by vol) Triton X100, 1
mM sodium orthopervanadate, 10 mM sodium glycerophosphate, 50 mM
NaF, 5 mM sodium pyrophosphate, 1 .mu.M Microcystin-LR, 0.27 M
sucrose, 1 mM benzamidine, 0.2 mM phenylmethylsulphonyl fluoride,
10 pg/mL leupeptin, and 0.1% (by vol) 2-mercaptoethanol). The
lysates were centrifuged at 4.degree. C. for 10 minutes at
13,000.times.g and the supernatants incubated for 30 minutes on a
shaking platform with 50 .mu.L of Protein G-Sepharose coupled to 50
.mu.g of preimmune sheep IgG. The suspensions were centrifuged for
2 minutes at 13,000.times.g and the supernatants incubated for 60
mintues with 30 .mu.L of Protein G-Sepharose covalently coupled to
60 .mu.g of RAC-PK.alpha. antibody. See Harlow and Lane,
Antibodies--A Laboratory Manual, Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y. (1988). The Protein
G-Sepharose-antibody-RAC-PK.alpha- . complex was washed eight times
with 1.0 mL of buffer A containing 0.5 M NaCl, and twice with 50 mM
Tris/HCl, pH 7.5, 0.1 mM EGTA and 0.1% (by vol) 2-mercaptoethanol
(buffer B).
[0296] Assay of immunoprecipitated RAC-PK.alpha. and protein
determinations. Three aliquots of each immunoprecipitate (each
comprising only 5% of the total immunoprecipitated RAC-PK.alpha.)
were assayed for RAC-PK.alpha. activity towards the peptide GRPRTSS
FAEG (SEQ ID NO: 5) as described previously. See Cross et al.
(1995), supra. One unit of activity was that amount which catalyzed
the phosphorylation of 1 nmol of substrate in 1 minute. Protein
concentrations were determined by the method of Bradford, Anal
Biochem, Vol. 72, pp. 248-254 (1976).
[0297] Tryptic digestion of in vivo phosphorylated RAC-PK.alpha..
The immunoprecipitated RAC-PK.alpha. was added to an equal volume
of 2% (by mass) SDS and 2% (by vol) 2-mercaptoethanol, and
incubated for 5 minutes at 100.degree. C. Aftercooling to room
temperature, 4-vinylpyridine was added to a final concentration of
2% (by vol) and the mixture was incubated for 1 hour at 30.degree.
C. on a shaking platform, followed by electrophoresis on a 10%
polyacrylamide gel. After autoradiography, the 60 kDa band
corresponding to rat RAC-PK.alpha. was excised and the gel piece
homogenized in 5 vols of 25 mM N-ethylmorpholine HCl, pH 7.7,
containing 0.1% (by mass) SDS and 5% (by vol) 2-mercaptoethanol.
The suspension was incubated for 1 hour at 37.degree. C. on a
shaking platform, then centrifuged for 1 minute at 13,000.times.g
and the supernatant collected. The pellet was incubated for a
further 1 hour with 5 vols of the same buffer and centrifuged for 1
minute at 13,000.times.g. The 2 supernatants, containing 80-90% of
the .sup.32P-radioactivity, were combined, 0.2 vols of 100% (by
mass) trichloroacetic acid added, and the sample incubated for 1
hour on ice. The suspension was centrifuged for 10 minutes at
13,000.times.g, the supernatant discarded and the pellet washed 5
times with 0.2 mL of water. The pellet was then incubated at
30.degree. C. with 0.3 mL of 50 mM Tris/HCl, pH 8.0, 0.1% (by vol)
Triton X100 containing 1 .mu.g of alkylated trypsin. After 3 hours,
another 1 .mu.g of trypsin was added and the suspension left for a
further 12 hours. Guanidinium hydrochloride (8 M) was added to
bring the final concentration to 1.0 M in order to precipitate any
residual SDS and, after standing on ice for 10 minutes, the
suspension was centrifuged for 5 minutes at 13,000.times.g. The
supernatant containing 90% of the .sup.32P-radioactivity was
chromatographed on a Vydac C18-column as described in the legend to
FIG. 2.
[0298] Transfection of 293 cells and immunoprecipitation of
HA-tagged RAC-PK.alpha.. Human embryonic kidney 293 cells were
cultured at 37.degree. C. in an atmosphere of 5% CO.sub.2, on 10 cm
diameter dishes in DMEM containing 10% fetal calf serum (FCS).
Cells were split to a density of 2.times.10.sup.6 per 10 cm dish,
and after 24 hours at 37.degree. C., the medium was aspirated and
10 mL of freshly-prepared DM EM containing 10% FCS added. Cells
were transfected by a modified calcium phosphate method [see Chen
and Okayama, Biotechniques, Vol. 6, No. 7, pp. 632-638 (1988)] with
1 .mu.g/mL DNA per plate. Ten (10) .mu.g of plasmid DNA in 0.45 mL
of sterile water was added to 50 .mu.L of sterile 2.5 M CaCl.sub.2,
and then 0.5 mL of a sterile buffer composed of 50 mM
N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid/HCl, pH 6.96,
0.28 M NaCl and 1.5 mM Na.sub.2HPO.sub.4 was added. The resulting
mixture was vortexed for 1 minute, allowed to stand at room
temperature for 20 minutes, and then added dropwise to a 10 cm dish
of 293 cells. The cells were placed in an atmosphere of 3%
CO.sub.2, for 16 hours at 37.degree. C., then the medium was
aspirated and replaced with fresh DMEM containing 10% FCS. The
cells were incubated for 12 hornus at 37.degree. C. in an
atmosphere of 5% CO.sub.2, and then for 12 hours in DMEM in the
absence of serum. Cells were preincubated for 10 minutes in the
presence of 0.1% DMSO or 100 nM wortmannin in 0.1% DMSO and then
stimulated for 10 minutes with either 100 nM insulin or 50 ng/mL
IGF-1 in the continued presence of wortmannin. After washing twice
with ice-cold DMEM the cells were lysed in 1.0 mL of ice-cold
buffer A, the lysate was centrifuged at 4.degree. C. for 10 minutes
at 13,000.times.g and an aliquot of the supernatant (10 .mu.g
protein) was incubated for 60 minutes on a shaking platform with 5
.mu.L of Protein G-Sepharose coupled to 2 .mu.g of HA monoclonal
antibody. The suspension was centrifuged for 1 minute at
13,000.times.g, the Protein G-Sepharose-antibody-HA-RAC-PK.alpha.
complex washed twice with 1.0 mL of buffer A containing 0.5 M NaCl,
and twice with buffer B, and the immunoprecipitate assayed for
RAC-PK.alpha. activity as described above.
[0299] .sup.32P-Labelling of 293 Cells Transfected with
HA-RAC-PK.alpha.
[0300] Two hundred ninety-three (293) cells transfected with
HA-RAC-PK.alpha. DNA constructs. were washed with phosphate-free
DMEM, incubated with [.sup.32p] orthophosphate (1 mCi/mL) as
described for L6 myotubes, then stimulated with insulin or IGF1 and
lysed, and RAC-PK.alpha. immunoprecipitated as described above. The
NP-labelled HA-RAC-PK.alpha. immunoprecipitates were washed,
alkylated with 4-vinylpyridine, electrophoresed and digested with
trypsin as described above for the endogenous RAC-PK.alpha. present
in rat L6 myotubes.
[0301] Transfection of COS-1 Cells and Immunoprecipitation of
HA-RAC-PK.alpha.
[0302] COS-1 cells were maintained in DMEM supplemented with 10%
FCS at 37.degree. C. in an atmosphere of 5% CO.sub.2. Cells at
70-80% confluency were transfected by a DEAE-dextran method [see
Seed and Aruffo, Proc Natl Acad Sci USA, Vol. 84, pp. 3365-3369
(1987)], and 48 hours later serum-starved for 24 hours. Cells were
lysed in a buffer containing 50 mM Tris-HCl, pH 7.5,120 mM NaCl, 1%
Nonidet P-40, 25 mM NaF, 40 mM sodium-, .beta.-glycerophosphate,
0.1 mM sodium orthopervanadate, 1 mM EDTA, 1 mM benzamidine, 1 mM
phenylmethylsulphonyl fluoride and lysates centrifuged for 15
minutes at 13,000.times.g at 4.degree. C. Supernatants were
pre-cleared once for 30 minutes at 4.degree. C. with 0.1 vols of
50% Sepharose 4B/25% Pansorbin (Pharmacia and Calbiochem,
respectively) and HA-RAC-PK.alpha. immunoprecipitated from 1 mg of
extract using the 12CA5 antibody coupled to Protein A Sepharose
beads. Immunoprecipitates were washed twice with lysis buffer
containing 0.5 M NaCl and once with lysis buffer.
[0303] Immunoblotting and Quantification of Levels of PK.alpha.
Expression.
[0304] Cell extracts were resolved by 7.5% SDS-PAGE and transferred
to Immobilon membranes (Millipore). Filters were blocked for 30
minutes in a blocking buffer containing 5% skimmed milk in
1.times.TBS, 1% Triton X-100 and 0.5% Tween 20, followed by a 2
hours incubation with the 12CA5 supernatant 1000-fold diluted in
the same buffer. The secondary antibody was alkaline (Alk)
conjugated anti-mouse Ig from goat (Southern Biotechnology
Associates, Inc), 1000-fold diluted in the blocking buffer.
Detection was performed using AP color development reagents from
Bio-Rad according to the manufacturer's instructions.
Quantification of levels of RAC-PK.alpha. expression was achieved
by chemiluminescence, using fluroisothiocyanante-labelled antimouse
IgG from goat as the secondary antibody and the Storm 840/860 and
ImageQuant software from Molecular Dynamics.
[0305] All peptides used to assay RAC-PK.alpha., and
TTYADFIASGRTGRRNAIHD (SEQ ID NO: 26), the specific peptide
inhibitor of cyclic AMP dependent PK--PKI, were synthesized on an
Applied Biosystems 431A peptide synthesizer. Their purity (>95%)
was established by HPLC and electrospray mass spectrometry, and
their concentrations were determined by guantitative amino acid
analysis.
[0306] Preparation and Assay of RAC-PK.alpha.
[0307] The construction of cytomegalovirus vectors (pCMV5) of the
human HA epitope-tagged WT (HA-RAC-PK.alpha.) was described
previously. See Alessi et al. (1996), supra. Two hundred
ninety-three (293) cells grown on 10 cm dishes were transfected
with a DNA construct expressing HA-RAC-PK.alpha. using a modified
calcium phosphate procedure. See Alessi et al. (1996), supra. The
cells were deprived of serum for 16 hours prior to lysis and, where
indicated, were stimulated for 10 minutes in the presence of 50
ng/mL IGF-1 to activate RAC-PK.alpha.. The cells were lysed in 1.0
mL ice-cold buffer A (50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA,
1% (by vol) Triton X-100, 1 mM sodium orthovanadate, 10 mM sodium
.beta.-glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 1
.mu.M Microcystin-LR, 0.27 M sucrose, 1 mM benzamidine, 0.2 mM
phenylmethylsulphonyl fluoride, 10 .mu.g/mL leupeptin and 0.1% (by
vol) 2-mercaptoethanol) the lysate centrifuged at 4.degree. C. for
10 minutes at 13,000.times.g and the supernatant obtained from one
10 cm dish of cells (2-3 mg protein) was incubated for 60 minutes
on a shaking platform with 20 p1 of Protein G-Sepharose coupled to
10 .mu.g of HA monoclonal antibody. The suspension was centrifuged
for 1 minute at 13,000.times.g, the Protein
G-Sepharose-antibody-HA-RAC-PK.alpha. complex washed twice with 1.0
mL of buffer A containing 0.5 M NaCl, and twice with buffer B (50
mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 0.01% (by vol) Brij-35 and 0.1%
(by vol) 2-mercaptoethanol). The RAC-PK.alpha. immunoprecipitates
were diluted in buffer B to an activity of 2.0 U/mL towards the
Crosstide peptide GRPRTSSFAEG (SEQ ID NO: 5) and 0.1 mL aliquots
snap frozen in liquid nitrogen and stored at -80.degree. C. No
significant loss of RAC-PK.alpha. activity occurred upon thawing
the RAC-PK.alpha. immunoprecipitates or during storage at
-80.degree. C. for up to 3 months. The standard RAC-PK.alpha._assay
(50 .mu.L) contained: 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 0.1% (by
vol) 2-mercaptoethanol, 2.5 .mu.M PKI, 0.2 U/ml RAC-PK.alpha.,
Crosstide (30 .mu.M), 10 mM magnesium acetate and 0.1 mM
[Y.sup.32P]ATP (100-200 cpm/pmol). The assays were carried out for
15 minutes at 30.degree. C., the assay tubes being agitated
continuously to keep the immunoprecipitate in suspension, then
terminated and analyzed as described. See Alessi et al. (1995),
supra. One unit of activity was that amount of enzyme which
catalyzed the phosphorylation of 1 nmol of Crosstide in 1 minute.
The phosphorylation of other peptides, histone H2B and MBP were
carried out in an identical manner. All the Crosstide activity in
HA-RAC-PK.alpha. immunoprecipitates is catalysed by RAC-PK.alpha.
(see Results) and the RAC-PK.alpha. concentration in the
immunoprecipitates was estimated by densitometric scanning of
Coomassie blue-stained polyacrylamide gels, using bovine serum
albumin as a standard. Protein concentrations were determined by
the method of Bradford using bovine serum albumin as standard. See
Bradford (1976), supra. Michaelis constants (Km) and Vmax values
were determined from double reciprocal plots of 1/V against 1/S,
where V is the initial rate of phosphorylation, and S is the
substrate concentration. The standard errors for all reported
kinetic constants were within <.+-.20%, and the data is reported
as mean values for 3 independent determinations. FIG. 16 shows the
results relative to those obtained for unstimulated
RAC-PK.alpha..
[0308] Tryptic Digestion of Histone 2B Phosphorylated by
RAC-PK.alpha.
[0309] Histone H2B (30 .mu.M) was phosphorylated with 0.2 U/mL
HA-RAC-PK.alpha.. After 60 minutes, 0.2 vol of 100% (by mass)
trichloroacetic acid was added, and the sample incubated for 1 hour
on ice. The suspension was centrifuged for 10 minutes at
13,000.times.g, the supernatant discarded and the pellet washed 5
times with 0.2 mL of ice-cold acetone. The pellet was re-suspended
in 0.3 mL of 50 mM Tris/HCl, pH 8.0, 0.1% (by vol) reduced
Triton-X100 containing 2 pg of alkylated trypsin and, after
incubation for 16 hours at 30.degree. C., the digest was
centrifuged for 5 minutes at 13,000.times.g. The supernatant,
containing 95% of the .sup.32P-radioactivity, was chromatographed
on a Vydac C18-column equilibrated with 0.1% (by vol) TFA in water.
With reference to the results shown in FIG. 17, the columns were
developed with a linear acetonitrile gradient (diagonal line) at a
flow rate of 0.8 mL/min. and fractions of 0.4 mL were
collected.
[0310] (a) Tryptic peptide map of .sup.32P-labelled histone H2B,
70% of the radioactivity applied to the column was recovered from
the major .sup.32P-peptide eluting at 19.5% acetonitrile.
[0311] (b) A portion of the major .sup.32P-peptide (50 pmol) was
analyzed on an Applied Biosystems 476A sequencer, and the Pth amino
acids identified after each cycle of Edman degradation are shown
using the single-letter code for amino acids. A portion of the
major .sup.32P-peptide (1000 cpm) was then coupled covalently to a
Sequelon arylamine membrane and analyzed on an Applied Biosystems
470A sequencer using the modified program. See Stokoe et al.
(1992), supra. .sup.32P-radioactivity was measured after each cycle
of Edman degradation.
1TABLE 7.1 Molecular basis for the substrate specificity of
RAC-PK.alpha. Peptides Km (.mu.M) Vmax (U/mg) V (0.1 mM) A 1.
GRPRTSSFAEG 4 250 100 2. RPRTSSFA 8 305 109 3. GRPRTSSF 8 385 129
4. RPRTSSF 5 260 105 5. RPRTSTF 30 243 78 6. RPRTSAF -- 0 7. PRTSSF
-- 0 8. RPRTSS >500 ND 2 9. KPRTSSF >500 ND 4 10. RPKTSSF
>500 ND 2 B 1. RPRTSSF 5 260 105 2. RPRTSSL 8 278 104 3. RPRTSSV
21 300 102 4. RPRTSSA 250 265 30 5. RPRTSSK 80 308 67 6. RPRTSSE
>500 ND 9 7. RPRTSSPA* -- 0 C 1. RPRTSSF 5 260 105 2. RPRASSF 12
230 89 3. RPRVSSF 25 273 77 4. RPRGSSF 60 163 37 5. RPRNSSF >500
ND 21 6. RPRTASF 20 213 83 7. RPRTGSF 25 233 77 8. RPRTVSF 30 365
89 9. RPRTNSF 30 300 81 10. RPRAASF 25 215 77 11. RFRGGSF 105 345
55 12. RPRGASF 105 160 37 13. RPRAGSF 49 114 70 The phosphorylated
residue is shown in boldface type The altered residue is
underlined. V(100 .mu.M) is the relative rate of phosphorylation at
0.1 mM peptide relative to peptide 1. ND = not determined. *An
alanine residue was added to the C-terminal of the peptide RPRTSSP,
since we have experienced difficulty in synthesizing peptides
terminating in Pro.
[0312]
2TABLE 7.2. Comparison of the Substrate Specificities of
RAC-PK.alpha., MAPKAP Kinase-1 and p70S6 Kinase Protein MAPKAP
kinase B.alpha. kinase-1 p70 S6 kinase K.sub.m V.sub.max K.sub.m
V.sub.max Km V.sub.max Peptide (mM) (U/mg) (mM) (U/mg) (mM) (U/mg)
A 1. KKKNRTLSVA 185 270 0.2* 1550* 33* 890* 2. KKRNRTLSVA 80 300
0.7* 1800* 1.5* 1520* 3. KKRNKTLSVA >500 ND 17* 840* 34* 760* 4.
KKRNRTLTV 388 330 40* 270* 4.8* 1470* B 1. GRPRTSSFAEG 4 250 2 790
3 1270 2. RPRTSSF 5 260 12 840 125 705 3. RPRTSTF 30 240 >500 ND
211 590 4. RPRAASF 25 215 20 1020 >500 ND 5. RPRAATF 25 230
>500 ND >500 ND Peptides 1 and 2 are very good substrates for
MAPKAP kinase-1 and p70 S6 kinase, and Peptide 3 is a relatively
specific substrate for p70 S6 kinase. *Data reported previously. ND
= not determined.
EXAMPLE 14
[0313] Mitogenic Stimulation and Phosphorylation of RAC-PK
[0314] The Swiss 3T3 cell line [see {haeck over (S)}u{haeck over
(s)}a and Thomas, Proc Natl Acad Sci USA, Vol. 87, pp. 7040-7044
(1990)] is utilized to investigate the possible involvement of
RAC-PK in growth factor signalling. Quiescent Swiss 3T3 cells are
serum-starved for 24 hours, followed by stimulation with 10%
FCS.
[0315] (a) Kinase activity is assessed by immunoprecipitating
RAC-PK and assaying the kinase using MBP as a substrate. Briefly,
cell free extracts are prepared by scraping preconfluent cells into
ice-cold TBS, lysing the cells in a buffer containing 50 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 1.0% Triton X-100, 2 mM EGTA, 1 mM
PMSF, 20 .mu.M leupeptin, 20 .mu.M aprotenin and 10 .mu.M
molybdate. Lysates are centrifuged for 15 minutes at 12,000.times.g
at 4.degree. C. RAC-PK.alpha. is immunoprecipitated from
pasorbin-cleared extracts using a rabbit polyclonal antibody
specific for the conserved C-terminus (anti-RAC.sup.469-480) [see
Jones et al. (1991), supra] raised by injecting rabbits
subcutaneously with the peptide FPQFSYSASSTA (SEQ ID NO: 7) coupled
to keyhole limpet haemocyanin and purified by precipitation using
50% (NH.sub.4).sub.2SO.sub.4 followed by affinity chromatography on
RAC-PK coupled Affigel.RTM. 10 column (Bio-Rad). These antisera
also recognize the b/AKT2 isoform, because its C-terminus differs
from that of RAC-PK.alpha. in the last 3 amino acids. RAC-PK
activity is assayed as described previously using MBP as substrate.
See Jones et al. (1991), supra. The extracts are incubated for 2
hours at 4.degree. C. with the antiserum (2 .mu.g/100 .mu.L
extract) the immunoprecipitates collected using Protein A sepharose
and washed with lysis buffer. The protein sepharose beads are
resuspended in 100 .mu.L of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 10 pm
molybdate and 35 .mu.L used for the kinase assay, as follows.
[0316] Reaction mixtures in a final volume of 50 .mu.L contain 50
mM Tris-HCl, pH 7.5, 10 mM MgCl.sub.2, 1 mM DTT, 1 mM PK inhibitor,
PKI peptide, 25 pg of MBP (Sigma), 50 .mu.M (.gamma.-32P) ATP (3500
cpm/pmol) and 35 .mu.L of immunoprecipitate from cell free extracts
or purified fractions of RAC-PK. After incubation at 30.degree. C.
for 10, 30 or 60 minutes samples are analyzed by 12% SDS/PAGE
followed by autoradiography and quantified by scintillation
counting of the phosphorylated MBP bands.
[0317] Immunoprecipitated RAC-PK activity is found to be 2- to
4-fold higher in serum-stimulated cells versus quiescent cells.
Activation occurs within 5 minutes and kinase activity remains
elevated for at least 120 minutes.
[0318] (b) Activation coincides with decreased mobility of RAC-PK
on SDS-PAGE. In order to determine which forms are present on
SDS-PAGE gels, immunoblotting is performed using anti-RAC-PK
antisera prepared as above. Cell extracts and immunoprecipitates
are resolved by 7.5% SDS-PAGE, transferred to Immobilon-P membranes
(Millipore) and incubated with the anti-RAC.sup.469-480 antibody.
Detection is performed using Alk phosphatase-conjugated anti-rabbit
antibody.
[0319] At least 3 different forms can be detected by immunoblot
analysis, termed a, b and c. The kinase from quiescent cells
migrates as a doublet of the a and b forms and during stimulation a
slower migrating form c appears, followed by disappearance of form
a. These results suggest that RAC-PK activity is modulated by
reversible phosphorylation.
[0320] (c) To test this possibility the in vivo effects of
phosphatase inhibitors okadaic acid and vanadate on RAC-PK from
Swiss 3T3 cells are examined. Cells are serum-starved for 24 hours,
followed by stimulation with 1 .mu.M okadaic acid, or 0.1 mM
vanadate prepared with 0.1 mM H.sub.2O.sub.2 [see Posner et al., J
Biol Chem, Vol. 269, pp. 4596-4604 (1994)], optionally in
conjunction with 10% FCS. Treatment of cells with okadaic acid, a
specific inhibitor of PP2A and PP1, induces a 3-fold increase in
RAC-PK activity and decreases electrophoretic mobility.
Simultaneous treatment with 1 .mu.M okadaic acid and 10% serum
causes a 5-fold activation and a larger alteration of the
electrophoretic mobility. An 11-fold activation is observed
following treatment with 0.1 mM vanadate, which converts the major
part of the protein into the slowest-migrating form c.
[0321] In order to confirm that multiple electrophoretic mobility
forms reflect different phosphorylation states of the kinase,
RAC-PK is immunoprecipitated from .sup.32P-labelled quiescent and
vanadate-stimulated Swiss 3T3 cells. Swiss 3T3 cells are arrested
in phosphate-free DMEM/FCS as described [see {haeck over
(S)}u{haeck over (s)}a and Thomas (1990), supra] and serum-starved
for 16 hours prior to labelling with [.sup.32P]orthophosphate for
6-10 hours (2 mCi per 15 cm dish). Stimulation is performed 0.1 mM
vanadate. Quantification of phosphorylation is performed using the
ImageQuant software. Vanadate treatment leads to a 3- to 4-fold
increase in phosphorylation, demonstrating that the mobility forms
b and c represent phosphorylated RAC-PK.
[0322] (d) In order to determine which residues are phosphorylated
in activated RAC-PK, phosphoamino acid analysis is carried out on
cells labelled as above according to Boyle et al., Methods Enzymol,
Vol. 201, pp. 110-149 (1991). The kinase from arrested cells
appears phosphorylated mainly on Ser residues, and at low levels on
Thr, with a ratio of 12:1. Vanadate stimulation leads to an
increase in phosphoserine and, in particular, in phosphothreonine
content, reducing the ratio to 4:1. Phosphotyrosine is not detected
after vanadate stimulation, either by phosphoamino acid analysis,
or by immunoblot analysis using an anti-phosphotyrosine antibody.
These results show that RAC-PK is activated by a phosphorylation
mechanism. Furthermore, we conclude that RAC-PK activation mediated
by vanadate is probably indirect, since vanadate is known to be an
inhibitor of tyrosine phosphatases.
EXAMPLE 15
[0323] Inactivation of RAC-PK by Protein Phosphatase 2A In
Vitro
[0324] To confirm that RAC-PK is regulated by phosphorylation the
effects of PP2A treatment on the kinase immunoprecipitated from
quiescent and vanadate-stimulated Swiss 3T3 cells are investigated.
As treatment of cells with 1 mM okadaic acid for 2 hours
preferentially inactivates PP2A rather than PP1, RAC-PK is
incubated either with the purified PP2A catalytic subunit (PP2Ac),
or PP2A dimer consisting of the catalytic and regulatory PR65
subunit (PP2A.sub.2).
[0325] Immunoprecipitated RAC-PK is incubated with 0.3 U/mL of
porcine muscle PP2Ac or 1.7 U/mL of rabbit muscle PP2A.sub.2 in 30
mL buffer containing 50 mM Tris-HCl, pH 7.5, 1% b-mercaptoethanol,
1 mM MnCl.sub.2, 1 mM benzamidine and 0.5 mM phenylmethylsulfonyl
fluoride at 30.degree. C. for 60 minutes (1 U is defined as 1 nmol
of Pi released from phosphorylase a per min.). The reactions are
stopped by addition of 50 nM calyculin A. The immune complexes
formed are washed with 50 mM Tris-HCl, pH 7.5, 1 mM benzamid,ine,
0.5 mM phenylmethylsulfonyl fluoride and 50 nM calyculin A and
RAC-PK is assayed as described above.
[0326] Dephosphorylation of the activated RAC-PK in vitro by PP2Ac
results in an 84% reduction of kinase activity and concomitant
change in electrophoretic mobility, converting it from form c to b.
PP2A.sub.2 treatment leads to a 92% reduction of activity and
restores the protein mobility on SDS-PAGE to the a/b doublet. These
results confirm that the activity changes observed are achieved by
a reversible phosphorylation mechanism. Moreover, PP2A is indicated
as a potential regulator of RAC-PK activity in vivo.
EXAMPLE 16
[0327] RAC-PK.alpha. Stimulates p70.sup.s6k Activity
[0328] In Swiss 3T3 cells RAC-PK is activated by insulin
(4.5-fold), comparable to levels detected for p70.sup.s6k. In
contrast, insulin has little or no effect on p42.sup.mapk and
p44.sup.mapk in these cells, suggesting that RAC-PK and p70.sup.s6k
may reside on the same signalling pathway, which is a different
pathway to the MAPK pathway.
[0329] In order to investigate this possibility the effects of
wortmannin and rapamycin on serum induced activation of the two
kinases is examined. Wortmannin, an inhibitor of PI 3-kinase, and
immunosuppressant rapamycin block the activation of p70.sup.s6k by
affecting the same set of phosphorylation sites.
[0330] Stimulation of quiescent Swiss 3T3 fibroblasts leads to a
.about.4-fold induction of RAC-PK activity, whereas wortmannin
treatment preceding serum stimulation almost completely blocks the
activation. On the other hand, rapamycin pretreatment does not
exert any significant effect on RAC-PK activation. Wortmannin also
blocks the appearance of slowest RAC-PK mobility form that is
observed following serum treatment, while rapamycin does not affect
RAC-PK mobility. In the same experiment wortmannin and rapamycin
pretreatment abolish p70s6k activation.
[0331] These results suggest that RAC-PK may lie upstream of
p70.sup.s6k on the p70.sup.s6k signalling pathway, which is
inhibited upstream of RAC-PK by wortmannin and downstream thereof
by rapamycin. To examine this possibility, the regulation of
p70.sup.s6k is investigated in a transient cotransfection assay
using human 293 cells. RAC-PK.alpha. constructs are prepared by
ligating the RAC-PK.alpha. cDNA [see Jones et al. (1991), supra]
in-frame to the initiator methionine, in the mammalian expression
vector pECE. The construct is also subcloned into a CMV
promoter-driven expression vector. The construct is confirmed by
restriction analysis and sequencing. Constructs expressing
Myc-tagged p70.sup.s6k are obtained from Dr. G. Thomas, Friederich
Miescher Institut, Basel, Switzerland. Constructs are transfected
into COS cells using standard procedures. Coexpression of
RAC-PK.alpha. with p70.sup.s6k-MyC results in a 3.5- and 3-fold
increase of basal and insulin-stimulated p70.sup.s6k-Myc activity,
respectively.
Sequence CWU 1
1
27 1 1302 DNA Artificial Sequence CDS (2)...(145) Molecule Type
cDNA to mRNA 1 g aat tcg gca cga gct aga gca agc gcg gcc ccg cgg
ccc gga gcc atg 49 Asn Ser Ala Arg Ala Arg Ala Ser Ala Ala Pro Arg
Pro Gly Ala Met 1 5 10 15 ctg agg agc tgc gcc gcg cgc ctc cgc acg
ctg ggg gct ctg tgc cgg 97 Leu Arg Ser Cys Ala Ala Arg Leu Arg Thr
Leu Gly Ala Leu Cys Arg 20 25 30 ccg cca gta ggc cgg cgc ctg ccg
gaa gcg acc cgc gac ccg agc tga 145 Pro Pro Val Gly Arg Arg Leu Pro
Glu Ala Thr Arg Asp Pro Ser * 35 40 45 ggtcattttc ttctgaggaa
gtcattctta aggactgttc tgtccccaac cccagctgga 205 acaaggacct
aagactgctc tttgaccagt ttatgaagaa atgtgaagat ggctcctgga 265
aacgtttgcc ttcatataaa cgtacaccta ctgaatggat tcaagacttc aaaacccatt
325 ttcttgaccc aaagcttatg aaagaagaac aaatgtcaca ggcccagctc
ttcaccagaa 385 gctttgatga tggcctgggc tttgaatacg tgatgttcta
caatgacatt gagaaaagga 445 tggtttgctt atttcaagga ggcccttacc
tggaagacca cctggattca ttcatggagg 505 tgccattgca accatgattg
atgctactgt tggtatgtgt gcaatgatgg ctgggggaat 565 cgtcatgact
gccaatctca acatcaatta tccccgacct atccctcttt gttctgttgt 625
tatgataaat agccaacttg ataaagttga aggaaggaaa ttttttgttt cctgtaatgt
685 tcagagtgtt gatgagaaga ccctatactc agaggcgaca agcttattat
aaagtcgaat 745 cctgctaaaa gtcttgatcg ataaagagtc gtcggtgaac
tccatctcat tctcgcccct 805 ccagaagaag gcagttgtcc cccaaatact
ctgctccctc actgctgaat cctgtaggga 865 gaagcctgcc aacagtgacc
ttccgaaaca gccttctgaa tacaaagagg attcagtttc 925 catcttctca
actttttaac acagaaacac ttcctgcgag actatcgaca actctcgggc 985
caggcgctgt ggctcacacc tgtaatccca gcactttagg aggccgaggc aggcggattg
1045 cctgagctca ggagttgaag atcagtctgg gcaacacgat gaaactccgt
ctctactaaa 1105 atacaaaaaa ttatccaggc atggtggcgt acgcctgtag
tcccagctac tcaggaggct 1165 gaggcaggag aatcgcttga acccaggagg
aagaggttgc agtgagccaa gatcatgcca 1225 catcactcca acctgggcaa
cagaacaaga acccatctca aacaaacaac aaacaaaaaa 1285 aaaaaaaaaa actcgag
1302 2 47 PRT Artificial Sequence Carboxy-Terminal-Binding Protein
of RAC-PK 2 Asn Ser Ala Arg Ala Arg Ala Ser Ala Ala Pro Arg Pro Gly
Ala Met 1 5 10 15 Leu Arg Ser Cys Ala Ala Arg Leu Arg Thr Leu Gly
Ala Leu Cys Arg 20 25 30 Pro Pro Val Gly Arg Arg Leu Pro Glu Ala
Thr Arg Asp Pro Ser 35 40 45 3 2610 DNA Homo Sapiens source
(0)...(0) Clone Human RAC-PK alpha 3 atcctgggac agggcacagg
gccatctgtc accaggggct tagggaaggc cgagccagcc 60 tgggtcaaag
aagtcaaagg ggctgcctgg aggaggcagc ctgtcagctg gtgcatcaga 120
ggctgtggcc aggccagctg ggctcgggga gcgccagcct gagaggagcg cgtgagcgtc
180 gcgggagcct cgggcacc atg agc gac gtg gct att gtg aag gag ggt tgg
231 Met Ser Asp Val Ala Ile Val Lys Glu Gly Trp 1 5 10 ctg cac aaa
cga ggg gag tac atc aag acc tgg cgg cca cgc tac ttc 279 Leu His Lys
Arg Gly Glu Tyr Ile Lys Thr Trp Arg Pro Arg Tyr Phe 15 20 25 ctc
ctc aag aat gat ggc acc ttc att ggc tac aag gag cgg ccg cag 327 Leu
Leu Lys Asn Asp Gly Thr Phe Ile Gly Tyr Lys Glu Arg Pro Gln 30 35
40 gat gtg gac caa cgt gag gct ccc ctc aac aac ttc tct gtg gcg cag
375 Asp Val Asp Gln Arg Glu Ala Pro Leu Asn Asn Phe Ser Val Ala Gln
45 50 55 tgc cag ctg atg aag acg gag cgg ccc cgg ccc aac acc ttc
atc atc 423 Cys Gln Leu Met Lys Thr Glu Arg Pro Arg Pro Asn Thr Phe
Ile Ile 60 65 70 75 cgc tgc ctg cag tgg acc act gtc atc gaa cgc acc
ttc cat gtg gag 471 Arg Cys Leu Gln Trp Thr Thr Val Ile Glu Arg Thr
Phe His Val Glu 80 85 90 act cct gag gag cgg gag gag tgg aca acc
gcc atc cag act gtg gct 519 Thr Pro Glu Glu Arg Glu Glu Trp Thr Thr
Ala Ile Gln Thr Val Ala 95 100 105 gac ggc ctc aag aag cag gag gag
gag gag atg gac ttc cgg tcg ggc 567 Asp Gly Leu Lys Lys Gln Glu Glu
Glu Glu Met Asp Phe Arg Ser Gly 110 115 120 tca ccc agt gac aac tca
ggg gct gaa gag atg gag gtg tcc ctg gcc 615 Ser Pro Ser Asp Asn Ser
Gly Ala Glu Glu Met Glu Val Ser Leu Ala 125 130 135 aag ccc aag cac
cgc gtg acc atg aac gag ttt gag tac ctg aag ctg 663 Lys Pro Lys His
Arg Val Thr Met Asn Glu Phe Glu Tyr Leu Lys Leu 140 145 150 155 ctg
ggc aag ggc act ttc ggc aag gtg atc ctg gtg aag gag aag gcc 711 Leu
Gly Lys Gly Thr Phe Gly Lys Val Ile Leu Val Lys Glu Lys Ala 160 165
170 aca ggc cgc tac tac gcc atg aag atc ctc aag aag gaa gtc atc gtg
759 Thr Gly Arg Tyr Tyr Ala Met Lys Ile Leu Lys Lys Glu Val Ile Val
175 180 185 gcc aag gac gag gtg gcc cac aca ctc acc gag aac cgc gtc
ctg cag 807 Ala Lys Asp Glu Val Ala His Thr Leu Thr Glu Asn Arg Val
Leu Gln 190 195 200 aac tcc agg cac ccc ttc ctc aca gcc ctg aag tac
tct ttc cag acc 855 Asn Ser Arg His Pro Phe Leu Thr Ala Leu Lys Tyr
Ser Phe Gln Thr 205 210 215 cac gac cgc ctc tgc ttt gtc atg gag tac
gcc aac ggg ggc gag ctg 903 His Asp Arg Leu Cys Phe Val Met Glu Tyr
Ala Asn Gly Gly Glu Leu 220 225 230 235 ttc ttc cac ctg tcc cgg gaa
cgt gtg ttc tcc gag gac cgg gcc cgc 951 Phe Phe His Leu Ser Arg Glu
Arg Val Phe Ser Glu Asp Arg Ala Arg 240 245 250 ttc tat ggc gct gag
att gtg tca gcc ctg gac tac ctg cac tcg gag 999 Phe Tyr Gly Ala Glu
Ile Val Ser Ala Leu Asp Tyr Leu His Ser Glu 255 260 265 aag aac gtg
gtg tac cgg gac ctc aag ctg gag aac ctc atg ctg gac 1047 Lys Asn
Val Val Tyr Arg Asp Leu Lys Leu Glu Asn Leu Met Leu Asp 270 275 280
aag gac ggg cac att aag atc aca gac ttc ggg ctg tgc aag gag ggg
1095 Lys Asp Gly His Ile Lys Ile Thr Asp Phe Gly Leu Cys Lys Glu
Gly 285 290 295 atc aag gac ggt gcc acc atg aag acc ttt tgc ggc aca
cct gag tac 1143 Ile Lys Asp Gly Ala Thr Met Lys Thr Phe Cys Gly
Thr Pro Glu Tyr 300 305 310 315 ctg gcc ccc gag gtg ctg gag gac aat
gac tac ggc cgt gca gtg gac 1191 Leu Ala Pro Glu Val Leu Glu Asp
Asn Asp Tyr Gly Arg Ala Val Asp 320 325 330 tgg tgg ggg ctg ggc gtg
gtc atg tac gag atg atg tgc ggt cgc ctg 1239 Trp Trp Gly Leu Gly
Val Val Met Tyr Glu Met Met Cys Gly Arg Leu 335 340 345 ccc ttc tac
aac cag gac cat gag aag ctt ttt gag ctc atc ctc atg 1287 Pro Phe
Tyr Asn Gln Asp His Glu Lys Leu Phe Glu Leu Ile Leu Met 350 355 360
gag gag atc cgc ttc ccg cgc acg ctt ggt ccc gag gcc aag tcc ttg
1335 Glu Glu Ile Arg Phe Pro Arg Thr Leu Gly Pro Glu Ala Lys Ser
Leu 365 370 375 ctt tca ggg ctg ctc aag aag gac ccc aag cag agg ctt
ggc ggg ggc 1383 Leu Ser Gly Leu Leu Lys Lys Asp Pro Lys Gln Arg
Leu Gly Gly Gly 380 385 390 395 tcc gag gac gcc aag gag atc atg cag
cat cgc ttc ttt gcc ggt atc 1431 Ser Glu Asp Ala Lys Glu Ile Met
Gln His Arg Phe Phe Ala Gly Ile 400 405 410 gtg tgg cag cac gtg tac
gag aag aag ctc agc cca ccc ttc aag ccc 1479 Val Trp Gln His Val
Tyr Glu Lys Lys Leu Ser Pro Pro Phe Lys Pro 415 420 425 cag gtc acg
tcg gag act gac acc agg tat ttt gat gag gag ttc acg 1527 Gln Val
Thr Ser Glu Thr Asp Thr Arg Tyr Phe Asp Glu Glu Phe Thr 430 435 440
gcc cag atg atc acc atc aca cca cct gac caa gat gac agc atg gag
1575 Ala Gln Met Ile Thr Ile Thr Pro Pro Asp Gln Asp Asp Ser Met
Glu 445 450 455 tgt gtg gac agc gag cgc agg ccc cac ttc ccc cag ttc
tcc tac tcg 1623 Cys Val Asp Ser Glu Arg Arg Pro His Phe Pro Gln
Phe Ser Tyr Ser 460 465 470 475 gcc agc agc acg gcc tga ggcggcggtg
gactgcgctg gacgatagct 1671 Ala Ser Ser Thr Ala * 480 tggagggatg
gagaggcggc ctcgtgccat gatctgtatt taatggtttt tatttctcgg 1731
gtgcatttga gagaagccac gctgtcctct cgagcccaga tggaaagacg tttttgtgct
1791 gtgggcagca ccctcccccg cagcggggta gggaagaaaa ctatcctgcg
ggttttaatt 1851 tatttcatcc agtttgttct ccgggtgtgg cctcagccct
cagaacaatc cgattcacgt 1911 agggaaatgt taaggacttc tacagctatg
cgcaatgtgg cattgggggg ccgggcaggt 1971 cctgcccatg tgtcccctca
ctctgtcagc cagccgccct gggctgtctg tcaccagcta 2031 tctgtcatct
ctctggggcc ctgggcctca gttcaacctg gtggcaccag atgcaacctc 2091
actatggtat gctggccagc accctctcct gggggtggca ggcacacagc agccccccag
2151 cactaaggcc gtgtctctga ggacgtcatc ggaggctggg cccctgggat
gggaccaggg 2211 atgggggatg ggccagggtt tacccagtgg gacagaggag
caaggtttaa atttgttatt 2271 gtgtattatg ttgttcaaat gcattttggg
ggtttttaat ctttgtgaca ggaaagccct 2331 cccccttccc cttctgtgtc
acagttcttg gtgactgtcc caccggagcc tccccctcag 2391 atgatctctc
cacggtagca cttgaccttt tcgacgctta acctttccgc tgtcgcccca 2451
ggccctccct gactccctgt gggggtggcc atccctgggc ccctccacgc ctcctggcca
2511 gacgctgccg ctgccgctgc accacggcgt ttttttacaa cattcaactt
tagtattttt 2571 actattataa tataatatgg aaccttccct ccaaattct 2610 4
480 PRT Homo Sapiens 4 Met Ser Asp Val Ala Ile Val Lys Glu Gly Trp
Leu His Lys Arg Gly 1 5 10 15 Glu Tyr Ile Lys Thr Trp Arg Pro Arg
Tyr Phe Leu Leu Lys Asn Asp 20 25 30 Gly Thr Phe Ile Gly Tyr Lys
Glu Arg Pro Gln Asp Val Asp Gln Arg 35 40 45 Glu Ala Pro Leu Asn
Asn Phe Ser Val Ala Gln Cys Gln Leu Met Lys 50 55 60 Thr Glu Arg
Pro Arg Pro Asn Thr Phe Ile Ile Arg Cys Leu Gln Trp 65 70 75 80 Thr
Thr Val Ile Glu Arg Thr Phe His Val Glu Thr Pro Glu Glu Arg 85 90
95 Glu Glu Trp Thr Thr Ala Ile Gln Thr Val Ala Asp Gly Leu Lys Lys
100 105 110 Gln Glu Glu Glu Glu Met Asp Phe Arg Ser Gly Ser Pro Ser
Asp Asn 115 120 125 Ser Gly Ala Glu Glu Met Glu Val Ser Leu Ala Lys
Pro Lys His Arg 130 135 140 Val Thr Met Asn Glu Phe Glu Tyr Leu Lys
Leu Leu Gly Lys Gly Thr 145 150 155 160 Phe Gly Lys Val Ile Leu Val
Lys Glu Lys Ala Thr Gly Arg Tyr Tyr 165 170 175 Ala Met Lys Ile Leu
Lys Lys Glu Val Ile Val Ala Lys Asp Glu Val 180 185 190 Ala His Thr
Leu Thr Glu Asn Arg Val Leu Gln Asn Ser Arg His Pro 195 200 205 Phe
Leu Thr Ala Leu Lys Tyr Ser Phe Gln Thr His Asp Arg Leu Cys 210 215
220 Phe Val Met Glu Tyr Ala Asn Gly Gly Glu Leu Phe Phe His Leu Ser
225 230 235 240 Arg Glu Arg Val Phe Ser Glu Asp Arg Ala Arg Phe Tyr
Gly Ala Glu 245 250 255 Ile Val Ser Ala Leu Asp Tyr Leu His Ser Glu
Lys Asn Val Val Tyr 260 265 270 Arg Asp Leu Lys Leu Glu Asn Leu Met
Leu Asp Lys Asp Gly His Ile 275 280 285 Lys Ile Thr Asp Phe Gly Leu
Cys Lys Glu Gly Ile Lys Asp Gly Ala 290 295 300 Thr Met Lys Thr Phe
Cys Gly Thr Pro Glu Tyr Leu Ala Pro Glu Val 305 310 315 320 Leu Glu
Asp Asn Asp Tyr Gly Arg Ala Val Asp Trp Trp Gly Leu Gly 325 330 335
Val Val Met Tyr Glu Met Met Cys Gly Arg Leu Pro Phe Tyr Asn Gln 340
345 350 Asp His Glu Lys Leu Phe Glu Leu Ile Leu Met Glu Glu Ile Arg
Phe 355 360 365 Pro Arg Thr Leu Gly Pro Glu Ala Lys Ser Leu Leu Ser
Gly Leu Leu 370 375 380 Lys Lys Asp Pro Lys Gln Arg Leu Gly Gly Gly
Ser Glu Asp Ala Lys 385 390 395 400 Glu Ile Met Gln His Arg Phe Phe
Ala Gly Ile Val Trp Gln His Val 405 410 415 Tyr Glu Lys Lys Leu Ser
Pro Pro Phe Lys Pro Gln Val Thr Ser Glu 420 425 430 Thr Asp Thr Arg
Tyr Phe Asp Glu Glu Phe Thr Ala Gln Met Ile Thr 435 440 445 Ile Thr
Pro Pro Asp Gln Asp Asp Ser Met Glu Cys Val Asp Ser Glu 450 455 460
Arg Arg Pro His Phe Pro Gln Phe Ser Tyr Ser Ala Ser Ser Thr Ala 465
470 475 480 5 11 PRT Artificial Sequence Synthetic Peptide 5 Gly
Arg Pro Arg Thr Ser Ser Phe Ala Glu Gly 1 5 10 6 7 PRT Artificial
Sequence Synthetic peptide 6 Arg Pro Arg Ala Ala Thr Cys 1 5 7 12
PRT Homo Sapiens PEPTIDE (469)...(480) C-Terminal peptide of Human
RAC-PK 7 Phe Pro Gln Phe Ser Tyr Ser Ala Ser Ser Thr Ala 1 5 10 8 7
PRT Artificial Sequence Synthetic peptide 8 Arg Pro Arg Ala Ala Ser
Phe 1 5 9 7 PRT Artificial Sequence Synthetic peptide 9 Arg Pro Arg
Thr Ser Ser Phe 1 5 10 7 PRT Artificial Sequence Synthetic peptide
10 Arg Pro Arg Gly Gly Ser Phe 1 5 11 7 PRT Artificial Sequence
Synthetic peptide 11 Arg Pro Arg Ala Gly Ser Phe 1 5 12 7 PRT
Artificial Sequence Synthetic peptide 12 Arg Pro Arg Gly Ala Ser
Phe 1 5 13 10 PRT Artificial Sequence Synthetic peptide 13 Lys Lys
Lys Asn Arg Thr Leu Ser Val Ala 1 5 10 14 10 PRT Artificial
Sequence Synthetic peptide 14 Lys Lys Arg Asn Arg Thr Leu Ser Val
Ala 1 5 10 15 9 PRT Artificial Sequence Synthetic peptide 15 Lys
Lys Arg Asn Arg Thr Leu Thr Val 1 5 16 7 PRT Artificial Sequence
Synthetic peptide 16 Arg Pro Arg Ala Ala Thr Phe 1 5 17 9 PRT
Artificial Sequence Synthetic peptide 17 Gly Arg Ala Arg Thr Ser
Ser Phe Ala 1 5 18 6 PRT Artificial Sequence Synthetic peptide 18
Glu Phe Met Pro Met Glu 1 5 19 10 PRT Artificial Sequence Synthetic
peptide 19 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 20 55 DNA
Artificial Sequence Oligonucleotides Primers 20 gcggagatct
gccaccatgg agttcatgcc catggagtca gggcggccca gaacc 55 21 27 DNA
Artificial Sequence Oligonucleotides Primers 21 gcggtccgga
acatagtcca gcaccag 27 22 15 PRT Artificial Sequence Sythetic
Peptide 22 Arg Pro His Phe Pro Gln Phe Ser Tyr Ser Ala Ser Gly Thr
Ala 1 5 10 15 23 16 PRT Artificial Sequence Sythetic Peptide 23 Met
Thr Ser Ala Leu Ala Thr Met Arg Val Asp Tyr Glu Gln Ile Lys 1 5 10
15 24 13 PRT Artificial Sequence Sythetic Peptide 24 Arg Pro Arg
His Phe Pro Gln Phe Ser Tyr Ser Ala Ser 1 5 10 25 9 PRT Artificial
Sequence Haemagglutonin (HA) epitope 25 Tyr Pro Tyr Asp Val Pro Asp
Tyr Ala 1 5 26 20 PRT Artificial Sequence Synthetic Peptide 26 Thr
Thr Tyr Ala Asp Phe Ile Ala Ser Gly Arg Thr Gly Arg Arg Asn 1 5 10
15 Ala Ile His Asp 20 27 10 PRT Artificial Sequence Synthetic
Peptide 27 Lys Lys Leu Asn Arg Thr Leu Ser Val Ala 1 5 10
* * * * *