U.S. patent application number 09/045020 was filed with the patent office on 2002-02-28 for novel tau/neurofilament protein kinases.
Invention is credited to INGRAM, VERNON M., RODER, HANNO M..
Application Number | 20020025942 09/045020 |
Document ID | / |
Family ID | 27114068 |
Filed Date | 2002-02-28 |
United States Patent
Application |
20020025942 |
Kind Code |
A1 |
INGRAM, VERNON M. ; et
al. |
February 28, 2002 |
NOVEL TAU/NEUROFILAMENT PROTEIN KINASES
Abstract
Novel TAU/neurofilament protein kinases, PK40 and PK36, are
essentially purified and characterized. Novel immunoassays relating
to the kinases and inhibitors for the kinases also are provided.
Finally, DNA sequences encoding the kinases and cell lines relating
to the kinases are provided.
Inventors: |
INGRAM, VERNON M.;
(CAMBRIDGE, MA) ; RODER, HANNO M.; (WUPPORTAL,
DE) |
Correspondence
Address: |
EDWARD R GATES
WOLF GREENFIELD AND SACKS
600 ATLANTIC AVENUE
BOSTON
MA
02210
|
Family ID: |
27114068 |
Appl. No.: |
09/045020 |
Filed: |
March 20, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09045020 |
Mar 20, 1998 |
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08480793 |
Jun 7, 1995 |
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5955444 |
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08480793 |
Jun 7, 1995 |
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07912293 |
Jul 10, 1992 |
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07912293 |
Jul 10, 1992 |
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07742880 |
Aug 9, 1991 |
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Current U.S.
Class: |
514/47 |
Current CPC
Class: |
C07K 16/40 20130101;
C12N 9/12 20130101 |
Class at
Publication: |
514/47 |
International
Class: |
A61K 031/70; A01N
043/04 |
Claims
1. A preparation containing an isolated, essentially pure,
nonskeletal-associated kinase, the kinase capable of
phosphorylating dephosphorylated NF-M to an extent sufficient to
cause a shift on SDS-PAGE of the apparent M.sub.r of
dephosphorylated NF-M toward that of native NF-M.
2. A preparation as claimed in claim 1 wherein the kinase is
capable of phosphorylating both KSP sites of TAU and is capable of
abolishing the TAU epitope on TAU.
3. A preparation as claimed in claim 1 wherein the kinase is
capable of phosphorylating and reconstituting phospho-epitopes on
completely dephosphorylated NF-triplet or purified dephosphorylated
NF-M.
4. A preparation as claimed in claim 1 wherein the kinase is
capable of being inhibited by excess ATP.
5. A preparation as claimed in claim 1 wherein the kinase has a
K.sub.m of about 93 for ATP.
6. A preparation as claimed in claim 1 wherein the kinase is
capable of phosphorylating completely dephosphorylated NF-M to an
extent sufficient to cause a complete shift and wherein the kinase
is capable of phosphorylating completely dephosphorylated NF-H to
an extent sufficient to cause a partial shift on SDS-PAGE of the
apparent M.sub.r of completely dephosphorylated NF-H toward that of
native NF-H.
7. A preparation as claimed in claim 1 wherein the kinase is
capable of phosphorylating completely dephosphorylated TAU to an
extent sufficient to cause a complete shift on SDS-PAGE of the
apparent M.sub.r of completely dephosphorylated TAU to that of
native TAU.
8. A prepartion as claimed in claim 1 wherein the kinase is capable
of phosphorylating TAU to an extent sufficient to result in an
SDS-PAGE pattern characteristic of human TAU proteins extracted
from PHF.
9. A preparation as claimed in claim 1 wherein the kinase has a
sequence characterized by sequence numbers 1 and 2.
10. A preparation as claimed in any one of claims 1-9 wherein the
kinase has an apparent molecular weight of 40 kD.
11. A preparation as claimed in claim 1 wherein the kinase is
capable of phosphorylating completely dephosphorylated NF-M to an
extent sufficient to cause at least a partial shift on SDS-PAGE of
the apparent M.sub.r of completely dephosphorylated NF-M toward
that of native NF-M.
12. A preparation as claimed in claim 11 wherein the kinase has a
K.sub.m of about 50 for ATP.
13. A preparation as claimed in claims 1 and 11-13 wherein the
kinase has an apparent M.sub.r of 36 kD.
14. A method for detecting a mammalian kinase comprising, preparing
a fraction of biological material derived from a mammal, it being
unknown whether the fraction contains the kinase, the fraction
being substantially free of epitopes that are characteristic of a
protein in a particular state of phosphorylation and that are
reactive with a test antibody, contacting the fraction with a
protein free of the epitope under conditions so as to permit the
phosphorylation of the neuroprotein if the mammalian kinase is
present, and testing for the presence of the epitope using the test
antibody.
15. A method for detecting a mammalian kinase comprising: preparing
a fraction of biological material derived from a mammal, it being
unknown whether the fraction contains the kinase, the fraction
being substantially free of epitopes that are characteristic of a
protein in a particular state of phosphorylation and that are
reactive with a test antibody, contacting the fraction with a
protein that includes the epitope under conditions so as to permit
the phosphorylation of the protein if the mammalian kinase is
present, and testing for the presence of the epitope using the test
antibody.
16. A method as claimed in claim 15 further characterized by
contacting the fraction with a neuroprotein.
17. A method as claimed in claims 14 or 15 further characterized by
contacting the fraction with a completely dephosphorylated
neuroprotein.
18. A method as claimed in claims 14 or 15 further characterized by
contacting the fraction with a completely dephosphorylated
NF-H.
19. A method as-claimed in claims 14 or 15 further characterized by
contacting the fraction with a completely dephosphorylated TAU.
20. A method as claimed in claims 14-15 wherein the test antibody
is a test antibody selected from the group consisting of SMI-31,
SMI 33 and SMI-34.
21. An immunoassay comprising: an antibody that is reactive with an
epitope characteristic of a particular state of phosphorylation of
a.-protein, and the protein in a state of phosphorylation that does
not bind to the antibody.
22. An immunoassay as claimed in claim 21 wherein the protein is a
neuroprotein.
23. An immunoassay as claimed in claim 21 wherein the protein is
dephosphorylated relative to the native protein.
24. An immunoassay as claimed in claim 21 wherein the protein is
TAU.
25. An immunoassay as claimed in claim 23 wherein the protein is
TAU.
26. An immunoassay employing dephosphorylated NF.
27. An immunoassay as claimed in claim 26 wherein the assay employs
completely dephosphorylated NF-triplet.
28. An immunoassay as claimed in claim 26 wherein the assay employs
completely dephosphorylated NF-M.
29. An immunoassay as claimed in claim 26 wherein the assay employs
completely dephosphorylated NF-H.
30. A monoclonal antibody selectively specific for PK40.
31. A monoclonal antibody capable of binding to and inhibiting the
kinase activity of PK40.
32. An immunoassay for detecting PK40 employing the monoclonal
antibody of claim 30.
33. A polyclonal antibody selectively specific for PK40.
34. A polyclonal antibody as claimed in claim 33 wherein the
antibody is capable of inhibiting the kinase activity of PK40.
35. An immunoassay for detecting PK40 employing the polyclonal
antibody of claim 33.
36. A monoclonal antibody selectively specific for PK36.
37. A monoclonal antibody capable of binding to and inhibiting the
kinase activity of PK36.
38. An immunoassay for detecting PK36 employing the monoclonal
antibody of claim 36.
39. A polyclonal antibody selectively specific for PK36.
40. A polyclonal antibody as claimed in claim 39 wherein the
antibody is capable of inhibiting the kinase activity of PK36.
41. An immunoassay for detecting PK36 employing the polyclonal
antibody of claim 39.
42. A method for inhibiting neuroprotein phosphorylation activity
in a cell comprising, introducing into a cell an inhibitor of PK40
or PK36 in an amount sufficient to inhibit phosphorylating activity
of the PK40 or PK36.
43. A method as claimed in claim 42 wherein a fragment of a
substrate of PK40 or PK36 is introduced into the cell.
44. A method as claimed in claim 42 wherein an antibody capable of
binding to PK40 is introduced into the cell.
45. A method as claimed in claim 42 wherein an antibody capable of
binding to PK36 is introduced into the cell.
46. A method as claimed in claim 42 wherein ATP or an analog
thereof is introduced into the cell.
47. A method as claimed in claim 42 wherein the inhibitor is
administered in a sufficient amount to prevent the formation of
neurofilament tangles.
48. A vector containing an oligonucleotide encoding PK40 or a
unique fragment thereof.
49. A vector as claimed in claim 48 wherein the oligonucleotide is
a CDNA.
50. A vector as claimed in claim 48 wherein the oligonucleotide
corresponds to human PK40-or a unique fragment thereof.
51. A vector containing an oligonucleotide encoding PK36 or a
unique fragment thereof.
52. A vector as claimed in claim 51 wherein the oligonucleotide is
cDNA.
53. A vector as claimed in claim 51 wherein the oligonucleotide
corresponds to human PK36 or a unique fragment thereof.
54. A cell line transformed or transfected with an oligonucleotide
encoding PK40 or a unique fragment thereof.
55. A cell line transformed or transfected with an oligonucleotide
encoding PK36 or a unique fragment thereof.
56. A kinase produced by the cell line of either of claims 54 or
55.
Description
[0001] The present application is a continuation-in-part of
co-pending application Ser. No. 07/742,880, filed Aug. 9, 1991, the
entire contents of which are incorporated herein by reference.
[0002] This invention relates to novel TAU/neurofilament protein
kinases, DNA sequences therefor and cell lines relating thereto, as
well as inhibitors of the kinases and immunoassays relating to the
kinases.
BACKGROUND OF THE INVENTION
[0003] Neurofilaments (NF), the intermediate filaments (IF)
specific for neurons, are an assembly of three subunits of apparent
Mr on SDS-PAGE of 68 kD, 160 kD and 200 kD, termed NF-L, NF-M and
NF-H, respectively. All three subunits contain a highly conserved
helical rod domain. The two heavier subunits also have extended
C-terminal tail domains which are heavily phosphorylated. The
cDNA-derived sequences of the two heavy NF-subunits have revealed
the presence of 5, 12 and 40 Lys-Ser-Pro (Val,Ala,X) repeats in the
C-terminal domains of rat NF-M, human NF-M and human NF-H,
respectively (Napolitano et al., 1987; Myers et al., 1987 and Lees
et al., 1988). These sequences form the epitopes of several
phosphoepitope-specific anti-NF-mAbs (Lee et al., 1988). The
physiological significance of NF and their phosphorylation is not
very well understood yet (reviewed by Matus, 1988); correlative
evidence suggests involvement in the regulation of axonal diameter
(Hoffman et al., 1987; Pleasure et al., 1989). Electron microscopic
studies in conjunction with antibody decoration (Hirokawa wt al.,
1984) and biochemical evidence (Minami et al., 1983) favor NF-H as
a component in interactions of the NF and microtubule networks. The
phosphorylation status of NF and their ability to promote tubulin
polymerization are correlated in vitro (Minami et al., 1985).
[0004] The existence of NF-kinase(s) not activated by common second
messengers and some of their expected properties were postulated
from in vivo phosphorylation studies on extruded axoplasm of the
giant axons of the squid (Pant et al., 1978, 1986) and of Myxicola
(Shecket et al., 1982). In vitro characterization of purified
NF-kinases has focused so far on activities that copurify with the
NF-cytoskeleton and can be dissociated under high salt conditions
(Runge et al., 1981; Toru-Delbauffe et al., 1983). There is
currently no evidence of second messenger dependence of any of
these activities. From a mixture of such kinases one 67 kD activity
has been purified to apparent homogeneity (Wible et al., 1989).
This kinase prefers NF-H as a substrate, but only if not completely
dephosphorylated. A cAMP-dependent kinase copurifying with
microtubules has been shown to phosphorylate preferentially NF-M in
NF-triplets (Leterrier et al., 1981). In no case are the
stoichiometry or the sites of phosphorylation known and no shift of
apparent Mr of NF-M and NF-H on SDS-PAGE has been demonstrated.
Such a shift is expected after incorporation of phosphate in high
stoichiometric ratios into the dephosphorylated subunits. A smaller
than expected gel shift associated with a heterogeneous state of
KSP-phosphorylation of NF-M is induced by uncharacterized kinases
in mouse L cells transfected with a human NF-M clone (Pleasure et
al., 1990).
[0005] A possible pathological role of aberrant NF-phosphorylation
was considered when the anti-rat-NF mAb 07-5 (commercially
available as SMI-34 from Sternberger-Meyer Immunochemicals of
Jarretsville, Md., U.S.A.) was found to stain neurofibrillary
tangles in brain tissue from Alzheimer's patients (Sternberger et
al., 1985), but did not stain normal human brain tissue, except for
cerebellar basket cell axons and certain motoneuron axons of
patients 60 years of age (Blanchard & Ingram, 1989). On the
other hand, there is a report that the localization of the SMI-34
epitope is exclusively perikaryonal, while most other mAbs reacting
with NF-phosphoepitopes stain axons preferentially (Sternberger et
al., 1983).
[0006] However, immunochemical evidence (Grundke-lqbal et al.,
1986; Kosik et al., 1986; Wood et al., 1986; Nukina et al., 1987)
concerning the crossreactivities of a series of mAbs with NFs,
microtubule associated protein TAU and the main component of
tangles and paired helical filaments (PHF) point to TAU as a major
constituent of PHFs. This deduction is reinforced by the isolation
from PHFs of TAU-derived peptides (Wischik et al, 1988), while no
NF-derived peptides (Kondo et al., 1988) were obtained. A number of
anti-NF mAbs crossreacting with TAU, among them SMI-31
(commercially available from Sternberger-Meyer Immunochemical) and
RT97, recognize the phosphorylated KSP-sequence repeat in NF
proteins (Lee et al., 1988). PHFs react strongly with RT97, but
only after prolonged treatment with SDS, suggesting the presence of
this phosphorylated epitope in PHF in a nonperipheral location
(Rasool et al., 1984). Several lines of evidence indicate an
abnormal level or an abnormal site of phosphorylation in the
C-terminal portion of the TAU molecule in Alzheimer's Disease (AD)
(Grundke-lqbal et al., 1986; Kondo et al., 1988; Iqbal et al.,
1989). If an abnormally phosphorylated form of TAU is responsible
for or involved in the development of neurological conditions
characterized by PHFs in neurofibrillary tangles, then it clearly
would be extremely important to identify the factor(s) which cause
that phosphorylation.
SUMMARY OF THE INVENTION
[0007] The invention provides preparations containing isolated,
essentially pure, nonskeletal-associated kinases, the kinases
capable of phosphorylating dephosphorylated NF-M to an extent
sufficient to cause a shift on SDS-PAGE of the apparent M.sub.r of
dephosphorylated NF-M toward that of native NF-M. The kinases
further are capable of phosphorylating both dephosphorylated and
native Tau and are capable of phosphorylating and reconstituting
phospho-epitopes on completely dephosphorylated NF-triplet or
purified dephosphorylated NF-M. The kinases also are inhibited by
excess ATP.
[0008] One kinase, PK40, has an apparent molecular weight of 40 kD
and is capable of phosphorylating completely dephosphorylated NF-M
to an extent sufficient to cause a complete shift of apparent
molecular weight from that of completely dephosphorylated NF-M to
that of native NF-M. This kinase also is capable of causing a
complete shift for TAU and a partial shift for NF-H. It in
particular can phosphorylate TAU to an extent that mimicks the
alterations characteristic of human TAU proteins extracted from
PHFs. It further can phosphorylate both KSP sites of TAU and can
abolish the TAU-1 epitope.
[0009] Another kinase, PK36 has an apparent molecular weight of 36
kD and is capable of phosphorylating completely dephosphorylated
NF-M to an extent sufficient to cause at least a partial shift of
apparent molecular weight from that of completely dephosphorylated
NF-M toward that of native NF-M.
[0010] According to another aspect of the invention, novel assays
are provided. One assay involves a method for detecting a mammalian
kinase. A fraction of biological material derived from a mammal is
prepared, it being unknown whether the fraction contains the kinase
of interest. The fraction is substantially free of epitdpes
characteristic of a phosphorylated protein and reactive with a test
antibody. The fraction is contacted with a dephosphorylated protein
free of the epitope under conditions so as to permit the
phosphorylation of the protein if the kinase is present. The
fraction then is tested for the presence of the epitope using the
test antibody. Preferably the fraction is contacted with a
completely dephosphorylated neuroprotein. The presence of the
epitope can be detected using an antibody reactive with an epitope
correlated with phosphorylated neuroproteins such as SMI-31
antibody or SMI-34 antibody, and reagents may be employed to
produce a color in the presence of an immunoprecipitate complex
between the antibodies and the epitope. The color produced then may
be measured as a quantitative measure of the presence of the
complex.
[0011] In a similar immunoassay, the fraction of biological
material derived from a mammal is prepared, being unknown whether
the fraction contains the kinase of interest. The fraction is
substantially free of epitopes that are characteristic of a protein
in a particular state of phosphoralation and that are reactive with
the test antibody. (Particular state of phosphoralation includes
completely dephosphoralated.) The fraction is contacted with a
protein that is characterized by the epitope under conditions so as
to permit the phosphoralation of the protein if the mammalian
kinase is present. The presence of the epitope that is tested for
using the antibody. If the epitope has dissappeared, then the
kinase is present. Examples of useful antibodies include SMI-33 and
TAU-l. Preferred substrates include neuroproteins that are reactive
with the foregoing antibodies, such as, for example, the
phosphoralated TAU.
[0012] Another novel immunoassay according to the invention
involves use of an antibody that is reactive with an epitope
characteristic of a particular state of phosphoralation of a
protein, and use of the protein in a state of phosphoralation that
does not bind to the antibody. Such immunoassays may be in the form
of a kit including a container with the first container containing
the antibody and the second container containing the protein.
Preferrably the protein is a neuroprotein and most preferrably the
protein is desphosphoralated TAU.
[0013] Yet another novel immunoassay according to the invention
employs dephosphorylated NF. Preferred embodiments involve assays
utilizing completely dephosphorylated NF-triplet, completely
dephosphorylated NF-M and completely dephosphorylated NF-H.
[0014] According to another aspect of the invention, antibodies to
the novel kinase of the invention are provided. Monoclonal and
polyclonal antibodies capable of binding to and, preferably
selectively specific for PK40 and PK36 are provided. Most
preferably the antibodies are capable of inhibiting the kinase
activity of either PK40 or PK36. The antibodies may be used among
other things for detecting the presence of PK40 or PK36.
[0015] The invention also provides methods for inhibiting
neuroprotein phosphorylation activity in a cell by introducing into
a cell an inhibitor of PK40 or PK36 in an amount sufficient to
inhibit the phosphorylation activity of the PK40 or PK36. Preferred
inhibitors include fragments of substrates of PK40 or PK36,
antibodies selectively specific for PK40 or PK36 and ATP or analogs
of ATP. Most preferably the inhibitor is administered in an amount
sufficient to prevent the formation of neurofilament tangles.
[0016] According to still another aspect of the invention, vectors
are provided containing oligbnucleotides encoding PK40 or unique
fragments thereof and PK36 or unique fragments thereof. Likewise,
cell lines are provided that are transformed or transfected with an
oligonucleotide encoding PK40 or a unique fragment thereof or PK 36
or a unique fragment thereof. Products of the cell line also are
provided.
[0017] These and other features of the invention are described in
greater detail below in connection with the detailed description of
the preferred embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a photograph of a stained gel (12% SDS-PAGE)
indicating-the presence of PK36 and PK40.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0019] The invention in one aspect involves the identification of
novel kinases, PK40 and PK36. PK40 and PK36 have been isolated from
bovine brain as described in Example 4 and are essentially pure. By
"essentially pure" it is meant that at least 40% of the material in
a preparation is the kinase of interest. Preferably the kinase
represents at least 80%, and most preferably the kinase represents
at least 90%, of the material in the preparation. In any event, the
preparations of the invention are sufficiently pure so as to permit
amino acid sequencing by conventional methods, and further, can be
made sufficiently pure to permit the generation and identification
of antibodies to the kinases of interest. PK40 and PK36 have
apparent molecular weights (M.sub.r) of 40 kD and 36 kD on
SDS-PAGE, respectively.
[0020] A unique characteristic of PK40 is its slight shift (C<1)
in apparant molecular weight during the purification procedures of
the invention. This is believed to be due to phosphorylation of
PK40 during purification. The kinases are
noncytoskeletal-associated. "By noncytoskeletal-associated it is
meant that the kinase does not co-purify with the NF-cytoskeleton
under high-salt extraction conditions." PK40, but not PK36,
normally is isolated in a tyrosine phosphorylated form.
[0021] The kinases are capable of phosphorylating a variety of
dephosphorylated native substrates. The native substrates have
characteristic mobilities on SDS-PAGE which change when the
substrates are dephosphorylated. Treatment of these
dephosphorylated substrates with the kinases of the invention under
conditions permitting phosphorylation of the substrates, may result
in a mobility shift on SDS-PAGE of the apparent M.sub.r of the
dephosphorylated substrate toward that of the native substrate,
depending upon the particular substrate and kinase selected, and
the conditions applied. A "shift" is any detectable change in
mobility. By "complete shift" it is meant that the mobility of the
previously dephosphorylated substrate, after treatment with the
kinase of the invention, is the same as that of the native
substrate. A "partial shift" means that the mobility has moved
between that of the dephosphorylated substrate and that of the
native substrate. "No shift" means no detectable change in mobility
after treatment of the dephosphorylated substrate with the kinase
of the invention.
[0022] PK40 is capable of phosphorylating
completely-dephosphorylated NF-M (cdNF-M) so as to cause a complete
shift on SDS-PAGE of the apparent Kr of the cdNF-M to that of
native NF-M. PK40 causes a partial shift of
completely-dephosphorylated NF-H (cdNF-H). PK40 further is capable
of causing a complete shift of completely-dephosphorylated native
bovine TAU or pure human TAU isoform expressed in E.coli from the
clone Htau 40 (Goedert et al. 1989). In this regard, under
saturation phosphorylation conditions, PK40 causes a change in the
isoform pattern that closely resembles the pattern of human TAU
proteins extracted from PHF. This and other pattern changes are
discussed in greater detail below. It also phosphorylates both KSP
sites of TAU and abolishes the TAU 1 epitope.
[0023] PK36 is capable of phosphorylating cdNF-M so as to cause at
least a partial shift on SDS-RAGE of the apparent M.sub.r of the
cdNF-M to that of native NF-M. It also can cause a partial shift
with respect to TAU.
[0024] Neither kinase is activated by the usual second messengers,
i.e., small molecules (such as cAMP, cGMP, Calcium, Ca.sup.+
Phosphatidyl Serine and Ca/CAM) that are produced inside the cell
when the outside of the cell membrane receives a signal or
stimulus, such as a peptide hormone%
[0025] The ATP dependence and inhibition of the activities of PK40
and PK36 were determined as described in Example 6. The apparent
K.sub.m value for ATP of PK40 is 93+12 pM and of PK36 is 50 pM.
These values reflect a requirement for relatively high ATP
concentrations. Both kinases, however, are strongly inhibited by an
excess of ATP, i.e., when ATP is in relatively small excess over
Mg.sup.2+. In addition, PK36, but not PK40, is inhibited by the
Walsh inhibitor.
[0026] Identification of these novel kinases was made possible by
the employment of a novel kinase immunoassay described herein. This
immunoassay required NF proteins devoid of immunoreactivity with
mAbs SMI-31 and SMI-34, in that the assay measures kinase activity
specific for epitopes recognized by these antibodies, i.e., the
repeated phosphorylated KSP sequences. In order to be devoid of
such immunoreactivity, the NF proteins were completely
dephosphorylated as described in Example 1. Thus, by "completely
dephosphorylated", it is meant nonreactive with SMI-31 and SMI-34
antibodies. With such dephosphorylated substrates, it is possible
to assay for kinase activity i.e., the rephosphorylation of the KSP
sequences, by measuring the reappearance of immunoreactivity with
SMI-31 and SMI-34. Thus, the completely-dephosphorylated NF
proteins were incubated with different ammonium sulfate fractions
from bovine brain supernatants, and reconstitution of the SMI-31
and SMI-34 epitopes was assayed in the different fractions, as
described in Example 2.
[0027] A colorimetric immunoassay also described in Example 2, can
be used to quantitatively measure levels of phosphorylating
activity. In such an assay, the presence of the epitopes
characteristic of phosphorylated NF proteins is tested using
reagents that produce a color in the presence of an
immunoprecipitate complex between antibodies such as SMI-31 or
SMI-34 and the phosphorylated NF proteins. The amount of color
produced is determined, thus providing a quantitative measurement
of the amount of complex formed. Such a measurement correlates with
the KSP-specific phosphorylating activity present in the sample
tested. Again, completely-dephosphorylated neuroprotein can be used
as a substrate, although there are instances that do not
necessarily require completely-dephosphorylated material as a
substrate.
[0028] The invention also pertains to the nucleic acids encoding
the human kinases corresponding to bovine PK40 and PK36, and to a
method for cloning DNA sequences encoding the human kinases. The
purified bovine kinases are sequenced as described in Example 11.
With this sequence information, oligonucleotide probes are
constructed and used to identify the gene encoding the human kinase
in a cDNA library. Due to degeneracy of the genetic code, most
amino acids are represented by more than one codon. Therefore, in
order to increase the proportion of codons on the probe that
actually Correspond to the codons in the genome, the amino acid
sequence chosen from the bovine kinase that is used to synthesize
the corresponding oligonucleotide probe will be from a region that
has a minimal amount of degeneracy. Specifically, a radiolabeled
synthetic oligonucleotide hybridization probe corresponding to the
least degenerate codon sequence of a peptide sequence for each of
kinase PK40 and PK36 is prepared and used to screen a cDNA library
from human cells as described in Example 12. For PK40 it is
preferred to use codon sequences-corresponding to the unigue
fragments: Sequence I.D. Nos. 1 and 2.
[0029] Clones are obtained whose codon order matches the amino acid
sequence of each of the kinases. From overlapping partial clones, a
full-length CDNA sequence for each of the human kinases, which
correspond to bovine PK40 and PK36, is thus identified, and
recombinant vector molecules containing the total cDNA sequences
are obtained. Such a cloning method can be utilized because each of
the corresponding human kinases is encoded by an oligonucleotide
with substantial homology to either bovine PK40 or PK36. Thus,
there is sufficient homology such that the human CDNA is capable of
being identified by the hybridization technology described
herein.
[0030] A vector containing an oligonucleotide means a vector
containing the CDNA sequence, but not necessarily expressing it.
For expression of the cDNA sequence, it must be operably linked to
a eukaryotic or prokaryotic expression control DNA sequence. Such
recombinant molecules are easily prepared and identified by one of
ordinary skill in the art using routine skill and without undue
experimentation. Cells transformed or transfected with these
recombinant vector molecules are capable of expressing the human
kinase, or fragments thereof. Alternatively, the human kinases can
be isolated according to the methods described in the Examples that
were used for isolating bovine PK40 and PK36.
[0031] The human PK40 and PK36 kinases are inhibited by excess ATP,
phosphorylate dephosphorylated neurofilament and TAU proteins, and
in particular, phosphorylate KSP sequences in these proteins. Human
kinases herein mean those nonskeletal-associated kinases identified
as described in this invention, including human PK40 and human
PK36. Except for the Examples, as used herein and in the claims,
PK40 and PK36 mean mammalian PK40 and PK36, naturally occurring and
cloned. In the Examples, unless specifically referred to otherwise,
PK40 and PK36 mean bovine PK40 and PK36. By human PK40 and PK36, is
meant the human kinases corresponding to bovine PK40 and PK36.
[0032] According to another aspect of the invention, antibodies,
both polyclonal and monoclonal, can be raised against the kinases
of the invention, and then, if desired, selected on the basis of
their ability to inhibit the phosphorylating activity of the
kinases. Monoclonal antibodies are obtained by the method described
by Milstein and Kohler. Such a procedure involves injecting an
animal with an immunogen, removing cells from the animal's spleen
and fusing them with myeloma cells to form a hybrid cell, called a
hybridoma, that reproduces in vitro. The population of hybridomas
is screened and individual clones are isolated, each of which
secretes a single antibody species to a specific antigenic site on
the immunogen. The monoclonal antibodies are useful for detecting
the presence or absence of the PK40 or PK36 kinases. In addition,
the monoclonal or polyclonal antibodies are useful as inhibitors of
the PK40 and PK36 kinases.
[0033] Because PK40 has sequence homology with ERK kinases, it will
be understood that certain preferred antibodies will be selected so
as not to react with such ERK kinases. Preparing such antibodies is
well within the level of skill in the art.
[0034] The invention also involves the identification of inhibitors
of PK40 and/or PK36. An inhibitor of PK40 or PK36 is a molecule
that is capable of binding to PK40 or PK36 in a manner so as to
inhibit the phosphorylating activity of PK40 or PK36. This
invention discloses that PK40 and PK36 are strongly inhibited by an
excess of ATP. Other inhibitors may be identified by those of
ordinary skill in the art using the assays as described herein,
e.g., adding the putative inhibitor to the kinase and subjecting
the mixture to the quantitative calorimetric immunoassay described
in Example 2. Thus, various analogs and conjugates of ATP may be
screened for their ability to inhibit the phosphorylating activity
of PK40 or PK36. Examples of analogs are readily available in the
literature and can be accessed using various data-bases, including
full-text patent data-bases. The inhibitors thus can resemble the
molecular structure of ATP, especially in the distribution of
charged groups. The inhibitors can be modified to enable them to
enter neurons in a variety of ways. For example, the charged groups
of ATP analogs can be modified by esterification by analogy with
dibutyryl-cyclic-AMP.
[0035] Tens of thousands of putative inhibitors may be screened,
first in mixtures containing, for example, 1000 candidates, and
then, after inhibition by a mixture is established, in submixtures
containing, for example, 100, then 10, and then one inhibitor.
Other inhibitors may include, but are not limited to, KSP binding
site proteins, or proteins which bind to one of the kinases of this
invention, e.g., substrates, fragments of substrate, antibodies,
fragments of antibodies, and peptides such as single chain antibody
constructs or structural analogs of any of these. A substrate of
PK40 or PK36 is a protein that is acted upon by PK40 and/or PK36 in
vivo. An inhibiting fragment of a substrate of PK40 and/or PK36 as
used herein is a peptide that is a structural analog of at least a
portion of the substrate and that is capable of binding to PK40
and/or PK36, so as to titrate out the phosphorylating activity of
PK40 and/or PK36 for the native substrate. Such fragments may be
identified and prepared by cleaving substrates of PK40 and/or PK36,
e.g., neurofilament or TAU protein, and testing the ability of the
fragments produced thereby to interfere with the phosphorylating
activity of the kinase for native substrate. Alternatively,
structural analogs of the PK40 or PK36 substrates may be prepared
which contain at least one KSP site and are resistant to
degradation by cytoplasmic, proteolytic enzymes. Such fragments are
easily prepared and identified by one of ordinary skill in the art
using routine skill and without undue experimentation. For example,
they can be prepared from known sequence information of substrates
of the kinases of the invention.
[0036] A use of this invention is to administer to a cell an
inhibitor of one of the kinases of the invention. This can act to
reduce the phosphorylation activity in the cell and also to reduce
or prevent the formation of paired helical filaments or tangles.
This permits the analysis, for example, of the contribution of such
phosphorylation activity to cell maintenance as well as to
neurocellular states characteristic of neurodegenerative disease
and aging.
[0037] A therapeutic use of this invention is to administer to a
subject in need of such treatment an inhibitor of one of the
kinases of this invention in order to treat neurodegenerative
conditions characteristic of Alzheimer's disease and normal aging.
Such an inhibitor can reduce the formation of paired helical
filaments.
[0038] The inhibitor is administered to a subject in a
therapeutically acceptable amount. The term "subject" is intended
to include mammals. The term "therapeutically acceptable amount" is
that amount which is capable of ameliorating or delaying
progression of the diseased or degenerative condition in the
subject. A therapeutically acceptable amount can be determined on
an individual basis and will be based, at least in part, on
consideration of the subject's size, severity of symptoms to be
treated, results sought, and the specific inhibitor used. A
therapeutically acceptable amount can be determined by one of
ordinary skill in the art employing such factors and using no more
than routine experimentation.
[0039] As discussed herein, inhibitors include, but are not limited
to, ATP, analogs of ATP, KSP binding site proteins, or proteins
which bind to one of the kinases of this invention, e.g.,
substrates, fragments of substrate, antibodies, fragments of
antibodies, and peptides such as single chain antibody
constructs.
[0040] Administration of the inhibitor of this invention may be
made by any method which allows the inhibitor to reach the target
cells. Typical methods include oral, rectal; peritoneal,
subcutaneous, intravenous and topical administration of the
inhibitor. Other delivery systems can include sustained release
delivery systems. Preferred sustained release delivery systems are
those which can provide for release of the inhibitor of the
invention in sustained release pellets or capsules. Many types of
sustained release delivery systems are available. These include,
but are not limited to: (a) erosional systems in which the
inhibitor is contained in a form within a matrix, found in U.S.
Pat. No. 4,452,775 (Kent) and U.S. Pat. No. 4,667,014 (Nestor et
al.); and (b) diffusional systems in which an active component
permeates at a controlled rate through a polymer, found in U.S.
Pat. No. 3,832,252 (Higuchi et al.) and U.S. Pat. No. 3,854,480
(Zaffaroni). In addition, a pump-based hardware delivery system can
be used, some of which are adapted for implantation directly into
the brain.
[0041] A particular problem which must be overcome for those
systems which deliver inhibitor via the bloodstream is to cross the
blood-brain barrier, which controls the exchange of materials
between the plasma and the central nervous system. Many substances
are unable to pass through this barrier. One way to accomplish
transport of the inhibitor across the blood-brain barrier is to
couple the inhibitor to a secondary molecule, a carrier, which is
either a peptide or a non-proteinaceous moiety. The carrier is
selected such that it is able to penetrate the blood-brain barrier.
Examples of carriers are fatty acids, inositol, cholesterol, and
glucose derivatives. Alternatively, the carrier can be a compound
which enters the brain through a specific transport system in brain
endothelial cells, such as transport systems for transferring
insulin, or insulin-like growth factors I and II. This combination
of inhibitor and carrier is called a prodrug. Upon entering the
central nervous system, the prodrug may remain intact or the
chemical linkage between the carrier and inhibitor may be
hydrolyzed, thereby separating the carrier from the inhibitor.
[0042] An alternative method for transporting the inhibitor across
the blood-brain barrier is to use liposomes. Liposomes are single
or multi-compartmented bodies obtained when lipids are dispersed in
aqueous suspension. The walls or membranes are composed of a
continuous lipid bilayer which enclose an inner aqueous space. Such
vesicles can be used to encapsulate and deliver therapeutic agents.
International Patent No. WO 91/04014 (Collins et al.) describes a
liposome delivery system in which the therapeutic agent is
encapsulated within the liposome, and the outside layer of the
liposome has added to it molecules that normally are transported
across the blood-brain barrier. Such liposomes can target
endogenous brain transport systems that transport specific ligands
across the blood-brain barrier, including but not limited to,
transferring insulin, and insulin-like growth factors I and II.
Alternatively, antibodies to brain endothelial cell receptors for
such ligands can be added to the outer liposome layer. U.S. Pat.
No. 4,704,355 (Bernstein) also describes methods for coupling
antibodies to liposomes. In addition, Pat. No. 4,704,355 describes
preparing liposomes which encapsulate ATP
[0043] The invention also describes a novel assay that can be used
as a diagnostic test for early Alzheimer's disease. The assay
measures the level of neuroprotein phosphorylation activity in a
human cell by human kinases corresponding to PK40 and PK36. Skin
fibroblasts are grown in vitro from a normal and from a test
subject. Varying concentrations of an uncoupler of oxidative
phosphorylation from ATP production are added to the skin
fibroblasts and the presence of immunological epitopes that are
correlated with phosphorylated neuroproteins are determined.
Fibroblasts from Alzheimer's patients show this effect at lower
concentrations of uncoupling agent than fibroblasts from normal
subjects. The appearance of such epitopes will indicate the release
from inhibition of kinases PK40 and PK36.
[0044] Antibodies selectively specific for PK40 or PK36 also can be
used to evaluate the level of neuroprotein phosphorylation
activity, by quantifying the amount of PK40 or PK36 present in a
tissue sample. It is believed that diseased states will be
characterized by a higher level of kinase. present.
EXAMPLE 1
[0045] The novel kinase immunoassays required NF proteins devoid of
immunoreactivity with mabs SMI-31 and SMI-34. These immunoassays
measure kinase activity specific for these epitopes, i.e., the
repeated KSP sequences. Such specificity was required because crude
brain extracts contain a very large number of protein kinases. In
order to be devoid of such immunoreactivity, the NF proteins must
be completely dephosphorylated. NF-triplet protein and individual
NF-subunits were prepared, and subsequently dephosphorylated, as
follows.
[0046] NF-triplet protein was prepared by one of two methods:
"native" or "reconstituted." The preparation of "native" NF-triplet
was a modification of previously described procedures (Tokutake et
al., 1983; Lee et al., 1987). A freshly obtained bovine spinal cord
(100-150 g, Arena & Sons, Hopkinton, MA) was desheathed, minced
with a razor blade and left for 2 hours in 3 l of 10 EM Tris, pH
7.0, 50 MM NaCl, 2 mM EGTA, 1 mM DTT, 0.1 mM PMSF at 4.degree. C.
far swelling. The supernatant was decanted anh the swollen tissue
was homogenized for 1 minute in 200 ml of a similar buffer
containing 150 mM NaCl (isotonic buffer) with an Ultra-Turax at 2/3
speed. After 15 minutes centrifugation at 12,000.times.g the
precipitate was twice rehomogenized in 200 ml isotonic buffer for 1
minute at full speed. Supernatants of the centrifugations
were-combined and made 0.85 M in sucrose by adding solid sucrose (1
mole/l). Centrifugation at 100,000.times.g for 4 hours yielded
about 200 mg of gelatinous precipitate which was dissolved (aided
by slow Ultra Turax homogenization) in 100 ml adsorption buffer: 10
mM potassium phosphate, pH 7.4, 8 M urea (deionized for 1-2 hours
over mixed bed ion exchanger AG 501-X8 (D), Bio-Rad), 0.5%
.beta.-mercapto-ethanol (B-ME). NFs were absorbed by shaking this
solution for 10 minutes at 4.degree. C. with hydroxyapatite (HTP,
40 g, dry weight, Bio-Rad), preequilibrated in adsorption buffer.
The adsorbent was sedimented for 10 minutes at 15,000.times.g and
washed (10 minutes each) subsequently with 100 ml adsorption
buffer, 3.times.85 ml 130 mM KPO.sub.4, pH 7.0, 8 M urea, 0.5% B-ME
and once each with 50 ml 300 mM and 250 MM KPO.sub.4, pH 7.0, 8 M
Urea, 0.5% B-ME. The supernatants of the latter two washes
contained the bulk amount of NF-L, NF-M and NF-H and were combined
for reconstitution of the NF-triplet by dialysis into 3 changes of
1 liter of 10 mM MES, pH 6.8, 100 mM NaCl, 1 mM MgCl.sub.2 and 1 mM
EGTA. After 30 minutes of incubation at 37.degree. C. and
centrifugation for 6 hours at 120,000.times.g, 40-60 mg of
NF-triplet proteins were obtained. The gelatinous precipitate was
rehomogenized in 40% glycerol with a glass-teflon homogenizer to
form suspensions of 2.5-3 mg/ml and stored at -20.degree. C.
[0047] For separation of the individual NF-subunits a previously
described procedure (Tokutake, 1984) was modified. The native
NF-triplet precipitate was taken up (0.5-1 ml/mg NF protein) in 10
mM sodium phosphate, pH 6.8, 6 M urea, 0.5% B-ME (starting buffer),
centrifuged at 100,000.times.g for 1 hour and loaded onto a
40.times.1.5 cm DEAE-Sephacel column (Pharmacia). NF subunits were
eluted at room temperature with 600 ml of a linear gradient formed
by starting buffer and 400 mM sodium phosphate, pH 6.8, 6 M urea,
0.5% B-ME at 10-15 ml/hour. Fractions were collected (120
fractions, 5 ml each) and fractions 41-48, 71-80 and 85-94 were
pooled; these contained pure NF-H, SNF-M, and NF-L, respectively,
according-to analysis by SDS-PAGE. The three fractions were
concentrated to 2-3 ml by vacuum dialysis and dialysed into water.
NF-L was obtained as a clear gelatinous precipitate after
centrifugation for 1 hour at 100,000.times.g; NF-M and NF-H were
precipitated by ammonium sulfate. For storage at -20.degree. C. the
pure subunits were homogenized (NF-L) or dissolved (NF-M, NF-H) in
40% glycerol to form stock concentrations of about 1 mg/ml of
protein. Alternatively, NF-subunits were separated by FPLC on a
Mono Q 5/5 column (Karlsson et al., 1987).
[0048] The "reconstituted" NF-triplet was reconstituted from the
three purified subunits after recombination of the appropriate
column fractions, in a manner similar to that described above for
reconstitution of "native" NF-triplet protein.
[0049] Dephosphorylation of NF-triplet was accomplished with E.
coli alkaline phosphatase. One ml (2.5-3 mg) of NF-triplet stock
solution was incubated for 5 days at 37.degree. C. with 10 units
(about 400 .mu.g) E. coli alkaline phosphatase (type III-N, Sigma
Chemicals) in a total volume of 2 ml, containing 50 mM Tris pH 8.5,
100 mM NaCl, 0.5 mM MgSO.sub.4, 0.5 mM ZnSO.sub.4, 1 mM PMSF and 5
.mu.g leupeptin. The NF triplet protein was separated from the
phosphatase by centrifugation for 1 hour at 100,000.times.g,
4.degree. C. The pellet was washed twice by rehomogenization in 2
ml water. The final pellet (yield 40-50%) was resuspended by a
glass-teflon homogenizer in 40% glycerol to form a stock solution
of about 0.5 mg/ml, stored at -20.degree. C. Dephosphorylated
NF-triplet tended to aggregate over several weeks of storage. After
analytical SDS-PAGE of dephosphorylation reactions, phosphatase and
accompanying impurities were removed by subjecting the gel for 6
hours to a "Western-blot electrophoresis" in an SDS-free buffer
prior to staining.
[0050] Dephosphorylation of subunit NF-M was accomplished by
incubating NF-M (0.5 g) with 2 units (80 .mu.g) E. coli alkaline
phosphatase for 5 days in a total volume of 1 ml under the same
buffer conditions as used for the NF-triplet. The phosphatase was
removed by gel filtration of the mixture on a 50.times.1N5 cm
Sephadex G200 column (50-120 .mu.m, 10 ml/hr flow rate),
equilibrated with 10 mM BisTris, pH 7.0, 100 mM NaCl. Fractions
were analyzed by SDS-PAGE. NF-M containing fractions around the
exclusion volume were pooled (4 ml), dialyzed into water,
concentrated in a SpeedVac and stored at -20.degree. C. as a 0.3
mg/ml stock solution containing 40% glycerol. The yield was 270
.mu.g (54%).
[0051] Dephosphorylation of subunit NF-H was accomplished by
incubating NF-H (1.05 mg) with 120 .mu.g calf intestinal alkaline
phosphatase for 6 days at 37.degree. C. in a total volume of 1.5 ml
containing 50 mM Tris, pH 8.5, 1 mM MgSO.sub.4, 1 mM PMSF and 15
.mu.g leupeptin. Separation from the phosphatase, concentration and
storage were as described for NF-M. The yield was 700 .mu.g
(67%).
[0052] The dephosphorylation reactions for both NF-subunits were
monitored by spotting 1-1.5 .mu.g of NF-protein on nitrocellulose.
Blocking, staining with SMI-31 and SMI-34 and development of the
blots were performed as described for Immuno-dotblot assays.
[0053] The shift of apparent M.sub.r on SDS-PAGE (1.5 mm gels
(Laemmli, 1970), 7.5% acrylamide, accompanying dephosphorylation of
NF-M and NF-H in the "native" triplet was virtually completed
within minutes. Five days of incubation, however, was necessary to
completely abolish the SMI-31 and SMI-34 immunoreactivity and
create substrates suitable for the immunoassays of the
invention.
[0054] The phosphatase was removed quantitatively by repeated
sedimentation of the dephosphorylated triplet. Dephosphorylation of
the NF-M and EF-H subunits in the NF-triplet which had been
"reconstituted" from FPLC-purified subunits, occurred much more
slowly as monitored by gel shift, removal of SMI-31 reactivity and
generation of the SMI-33 epitope. mAb SMI-33 (Sternberger--Meyer
Immunochemicals) is specific for the non-phosphorylated KSP
sequence, Lee et al., 1988. Loss of SMI-31 reactivity was not
complete even after five days of incubation.
[0055] FPLC-purified NF-M, but not FPLC-purified NF-H, was
dephosphorylated with E. coli alkaline phosphatase so as to be
unreactive to SMI-31 and SMI-34 under conditions similar to those
used for the NF-triplet. For the FPLC-purified NF-H, the shift of
apparent M.sub.r on SDS-PAGE and the removal of SMI-31 and SMI-34
immunoreactivity remained incomplete even after five days of
incubation with high concentrations of E. coli alkaline
phosphatase. The immunoreactivity of FPLC-purified NF-H with SMI-31
and SMI-34, however, was removed with calf intestinal alkaline
phosphatase (special molecular biology grade, Boehringer Mannheim
Biochemicals) after five days of incubation. NF-M was completely
dephosphorylated with either phosphatase. The phosphatases were
removed by gel filtration. Heat treatment and freezing of the NF
were avoided because the proteins tended to aggregate.
EXAMPLE 2
[0056] The preferred method for the immunoassay for detecting
KSP-phosphorylating kinases is as follows. Dephosphorylated
NF-triplet protein was incubated with the 35-45% ammonium sulfate
fraction of the brain supernatant (see section B.). Immuno-dotblot
assays were performed in 50 mM HEPES, pH 7.0, 2 mM MgCl.sub.2, 1 mM
ATP, 2 mM DTT in a total volume of 30 .mu.l with 5 .mu.g of
dephosphorylated native NF-triplet or 1.2 .mu.g of dephosphorylated
pure subunits NF-M or NF-H as substrates together with a control
assay lacking NFs. After incubation at 37.degree. C. for 18 hours,
assays were diluted to 100 .mu.l with 10 mM PBS, pH 7.2, and
aliquots of 50 .mu.l were spotted on nitrocellulose (0.22 .mu.m,
Schleicher and Schull). Blots were blocked by 1 hour incubation
with 3% BSA in 10 mM PBS, pH 7.2, and washed once in 0.5%
Triton-X100/10 mM PBS. Antibodies were diluted in sterile 10 mM
PBS, pH 7.2, 0.5% Triton-X100, 10% fetal calf serum. Blots were
incubated with SMI mAbs for at least 2 hours. The blots were then
washed five times. Mouse mAbs were detected by reaction with
horseradish-peroxidase-linked goat-anti-mouse antibody (Cappel Co.)
in 1:200 dilution and by staining with 0.05% 4-chloro-1-naphthol
(Sigma) and 0.05% H.sub.2O.sub.2 in 50 mM TBS, pH 7.5, 33% ethanol
for 5-20 minutes. All incubations and washes were at room
temperature. Incubations were sealed in plastic bags with 50 .mu.l
of solution/cm membrane.
[0057] The SMI-31 and SMI-34 epitopes were reconstituted. The
activity was NF-specific, since control immunoassays lacking
dephosphorylated NF-triplet were negative. These site specific
kinase immunoassays, while only semiquantitative, nevertheless
allowed for the estimation of some of the properties of the kinases
while still in crude form.
[0058] The foregoing procedure involved parameters that were
optimized as follows. Immuno-dotblot-assays (0.5 mM Mg.sup.2+, 0.5
mM ATP) were performed using various ammonium sulfate fractions of
whole brain supernatant to determine the fraction containing the
desired activity. The control assays did not contain NF proteins. A
35-45% fraction was found to contain the strongest activity for
reconstituting SMI-31 and SMI-34 epitopes. This activity was
NF-specific, since the corresponding control immunoassay which
lacked dephosphorylated native NF-triplet was almost negative for
this fraction. An additional less permanent NF-specific activity
was detected in the 40-55% and 55-70% AS-fractions of cytoskeletal
extract with 0.8M KCl where the main NF-kinase activity had been
expected.
[0059] The soluble nature of the kinases in the 35-45% fraction was
confirmed when the activity did not cosediment under low salt
conditions (10 mM HEPES Buffer pH7) after 15 minutes of incubation
with the phosphorylated native NF-triplet at 37.degree. C.
orassembled cold solubilized microtubules according to the method
of Shelanski et al (1973) [4M glycerol, 1 mM GTP, 37.degree. C., 30
minutes], in the absence or presence of 5 m Mg/ATP.
[0060] The optimal incubation time, pH and NaCl concentration for
conducting the assay was determined using the 35-45% fraction. The
assays were performed with 0.5 mM Mg.sup.2+ (unless indicated
otherwise). At an ATP concentration of 1 mM, an 18 hour incubation
time was optimal; the assays for pH and NaCl were performed at the
18 hour incubation time. The assay responses were optimal at pH
7.0, low salt conditions (50 mM or less;, 10, 20, 50, 75 and 100 mM
tested) and 1 mM ATP.
[0061] Immuno-dotblot-assays were conducted to determine the
optimal Mg.sup.2+ and ATP concentrations using the SMI-31 and
SMI-34 antibodies. The amount of enzyme-was varied as follows:
0.04, 0.09, 0.13, 0.18, 0.22, 0.33 and 0.44 micrograms of crude
enzyme. Control assays were without NFs using 0.4 micrograms crude
enzyme. Mg.sup.2+ and ATP concentrations were at 1.0, 2.0, and 4.0
mM.
[0062] The optimal Mg.sup.2+ and ATP concentrations were found to
be 2 mM and 1 mM, respectively. ATP [5 mM] inhibited the kinase
activity. Mn.sup.2+ was about twice as effective as Mg.sup.2+. GTP
could not substitute for ATP. Concentrations of NaCl greater than
20 mM diminished the assay response. This effect was attributable
to ionic strength rather than specifically to sodium or chloride
ions, since the same decline was seen with (NH.sub.4).sub.2SO.sub.4
at comparable ionic strength.
[0063] Alternatively, a quantitative calorimetric immunoassay of
PK40 and PK36 can be used. Such an assay measures binding of mAbs
to dephosphorylated human or other specie neurofilaments, TAU
protein, or recombinant TAU protein by kinases PK40 and PK36. The
kinases PK40 and PK36, alone or together, were assayed by a
quantitative ELISA-type assay based on the mAbs SMI-31, SMI-34
(Sternberger-Meyer Immunochemicals, Jarretsville, Md.) as primary
antibody. The secondary antibody was horseradish-peroxidase-linked
goat anti-mouse antibody (Cappel Co.), followed by color
development with H.sub.2O.sub.2 and "ABTS"
(2,2'-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)
.2NH4.sup.+), and color extraction and measurement. Different
amounts of PK40 and/or PK36 were incubated with 6 .mu.l 250 mM
HEPES buffer, pH 7.0/10 MM MgSO4, 1.2 .mu.l 25 mM ATP and 40-160 ug
of dephosphorylated bovine neurofilament triplet protein in a total
volume of 30 .mu.l. Incubation was for 18 hours at 37.degree. C.
and was followed by dilution to 150 .mu.l with 10 mM PBS. Each
assay (20 .mu.l) was applied to a nitrocellulose membrane as a dot,
the membrane was blocked with bovine serum albumin and individual
dots were punched out. They were next incubated with SMI-31 mAb
(1:500, 100 .mu.l) for 6 hours at 25.degree. C. Each dot was washed
5.times. with 1 ml of PBS containing Triton X-100. The second
incubation was with the goat anti-mouse Ab, 1:200 for 6 hours at
25.degree. C., followed by washing 5.times. with 1 ml PBS/Triton
X-100. Color development was carried out by shaking each dot
individually with a 0.1% ABTS in citrate pH 4.0/0.03%
H.sub.2O.sub.2 at room temperature for 30 minutes. The reaction
produced a soluble color in the supernatant which was. measured at
415 nm.
EXAMPLE 3
[0064] An alternative method for assaying the phosphorylating
activity of the kinases was by .sup.32P assays. Radioactive assays
in the same buffer system as for immunoassays contained 5 .mu.g of
HTP-purified native NF-triplet as substrate (3.mu.g of substrate
proteins other than NFs) and 150-250 cpm/pmole gamma-.sup.32P-ATP.
Incubation'times were 15 minutes at 37.degree. C. for activities up
to about 1 pmole/min/assay, since the assay responses were linear
within these time intervals. Assays were stopped by cooling on ice,
addition of 20 .mu.l 25 mM EDTA and immediate transfer of the
mixture onto glass filters (Whatman GF/A) wetted with 10% TCA/2%
sodium pyrophosphate (PPA). The glass filters were washed twice for
1 hour and once for at least 3 hours in 10% TCA/2% PPA and finally
in ethanol and were air-dried. Radioactivity was assessed by
scintillation counting (Beckman LS 230) with 5 ml "Liquiscint"
(National Diagnostics) for 20 minutes. Assays were routinely
carried out in triplicate except for some duplicate assays in a few
explicitly mentioned cases; a control assay lacking NFs was
subtracted from the mean value.
[0065] A preferred substrate for this assay is TAU, prepared as
described in the procedure of Example 9, below.
[0066] Assays to be analyzed on SDS-PAGE were stopped with an
equivalent amount of sample buffer, boiled for 3 minutes and run on
7.5% gels. After staining with Coomassie Blue, destaining and
drying on Whatman 3MM paper, autoradiography was performed with a
DuPont Cronex screen intensifier at -70.degree. C. For quantitative
measurements, radioactive bands of individual NF-subunits were cut
out, placed in an Eppendorf vial immersed in 20 ml water and the
Cerenkov radiation of the sample was counted. Counting efficiency
was about 30%.
EXAMPLE 4
[0067] The method for purifying the kinases was optimized by
exposing the 35-45% AS-fraction to a variety of chromatography
media at 4.degree. C. The activity as assayed using the SMI
antibodies was lost in almost every case. These losses occurred
even in the presence of 4M NaCl, which by itself did not affect
enzyme survival in controlled experiments. (NaCl was added to
prevent binding of the kinase or possible essential subunits to the
chromatography media). It was discovered, however, that inclusion
of Mg-ATP in the solution stabilized the activity on some media. In
order of decreasing survival of activity, Sephadex Agarose,
CM-Sepharose and quarternary ammonium ion exchangers were found to
be useful chromatography media in the presence of Mg-ATP.
[0068] The preferred method for purifying the KSP-phosphorylating
kinases is as follows. Step I: A fresh bovine brain (350-450 g wet
weight) was cleared from meninges and blood vessels and homogenized
at 4.degree. C. in 350 ml homogenization buffer (10 mM Bis Tris, pH
7.0, 150 mM NaCl, 2 mM EGTA, 1 mM DTT, 0.1 MM PMSF, 5 .mu.g/ml
leupeptin) with an UltraTurax or a Sorvall Omni-Mixer for 3
minutes. The pellet after centrifugation at 20,000.times.g for 20
minutes was extracted twice with 300 ml homogenization buffer. The
turbid supernatants were clarified by centrifugation at
100,000.times.g for 1 hour. Solid ammonium sulfate was added'slowly
over about 1 hour while keeping the pH at 8.0-8.5 with ammonia. The
precipitate obtained between 35% and 45% saturation was collected
by centrifugation at 20,000.times.g for 20 minutes, redissolved in
20 ml 10 mM HEPES, pH 7.0, 1 MM MgCl.sub.2, 1 mM EGTA and 1 mM DTT,
and dialyzed extensively against this buffer to form a "crude
enzyme" stock solution of about 20 mg/ml protein, which could be
stored for several weeks at 4.degree. C. with little loss of
activity.
[0069] Step II: 20 ml of crude enzyme was dialyzed into
CM-Sepharose starting buffer (5 mM magnesium acetate, 5 mM ATP, 1
mM DTT, 10% glycerol, 0.02% sodium azide, adjusted to pH 6.0 with
BisTris) and loaded onto a 3.times.2.5 cm CM-Sepharose column
equilibrated with starting buffer. The column was washed with 60 ml
starting buffer at about 50 ml/hr, then the kinases were eluted in
one step with 85 mM magnesium acetate, 5 nm ATP, 1 mM DTT, 10%
glycerol, 0.02% sodium azide, pH 6.0 as a fraction of 15 ml
volume.
[0070] Step III: The combined fractions of the CM-Sepharose
chromatography containing the bulk of the activity were dialyzed
into 10 mM HEPES, pH 7, 1 mM EDTA and 1 mM DTT. The protein then
was concentrated to about 3 ml in a SpeedVac and loaded onto a
95.times.2.5 cm column of Sephadex G200 Superfine (Pharmacia) and
were eluted with a filtration buffer (48 m? BisTris, pH 7.0, 5 mM
MgCl.sub.2, 5 mM ATP, 1 mM DTT, 0.02% sodium azide). After elution
of 155 ml at a flow rate of 1.5-2 ml/hr, fractions of 5 ml were
collected.
[0071] The collected 5 ml fractions (10-28) were tested in
immuno-dotblot-assays using SMI-31 and SMI-34 antibodies after 18
hours of incubation with NF-triplet. The fractions also were
subjected to .sup.32P-assays (30 minutes of incubation with
NF-triplet) to test for the presence of kinase activity. The kinase
activity eluted as a very broad peak (Fractions 14-23). No
significant NF-specific phosphatase activity could be detected in
the relevant kinase fractions by monitoring the liberation of
phosphate under assay conditions from .sup.32P-labeled
dephosphorylated native NF-triplet, prepared by phosphorylation
with partially purified NF-kinase. The fractions further were
subjected to 12% SDS-PAGE gel electrophoresis. The PK40 and PK36
bands are identifiable (arrows, FIG. 1), with the PK40 being most
prominent in fraction 17-19 and PK36 being most prominent-in
fractions 21 and 22. These fractions then were used in the assay
method.
[0072] Step IV: Gel filtration fractions containing do significant
amounts of PK40 (17-19) and PK36 (21-22), according to SDS-PAGE
analysis, were pooled, dialyzed into Mono Q starting buffer (20 mM
Tris, pH 8.0, 20 mM MgCl.sub.2, 5 mM ATP, 1 mM DTT, 0.02% sodium
azide) and loaded on an HR 5/5 Mono Q FPLC-column (Pharmacia)
equilibrated with starting buffer. Elution of PK40 at a flow rate
of 1 ml/min started with 5 ml starting buffer followed by a linear
gradient of 7 ml up to 60 mM MgCl.sub.2, 7 ml isocratic elution at
60 mM MgCl.sub.2 and finally a linear gradient up to 110 mM
MgCl.sub.2 formed with elution buffer (20 mM Tris, pH 8.0, 110 mM
MgCl.sub.2, 5 mM ATP, 1 mM DTT, 0.02% sodium azide). The gradient
profile for PK36 was identical.
[0073] The various fractions were subjected to a variety of assays,
including: a dotblot assay; a .sup.32P-assay; and 12% SDS-PAGE gel
electrophoresis. The activity within the fractions was demonstrated
by the immuno-dotblot-assay and .sup.32P-assay.
[0074] The activity of the PK40 fractions in the dotblot assay and
.sup.32P-assay were correlated with the particular fraction run on
the SDS-PAGE gel and, importantly with the particular band revealed
by the SDS-PAGE. The activity of the fractions correlated with
presence of the gel electrophoresis 40 kD band. The assays revealed
relatively prominent phosphorylation of NF-M and NF-H by PK40.
[0075] The activity of-the PK36 fractions in the dotblot assay and
.sup.32P-assay correlated with the presence of the gel
electrophoresis 36 kD band. The assays revealed relatively
prominent phosphorylation of NF-M and NF-L by PK36.
[0076] Peak fractions of the NF-kinases (PK40: 11-12; PK36: 12-13)
were pooled, dialyzed into storage buffer (20 mM BisTris, pH 7.0, 2
mM MgCl.sub.2, 2 mM ATP, 1 mM DTT, 0.02% sodium azide) and
concentrated about 10-fold in microconcentrators (Amicon 10) for
storage purposes. The enzyme is stable when frozen. Activity was
retained for several days at 4.degree. C. and after 5 cycles of
freeze-thawing with little loss. These pooled fractions were used
in the preparations described below.
[0077] A relatively pure mix of PK36 and PK40 as used for the
identification of 36 kD and 40 kD proteins as kinases was obtained
after pooling of gel filtration fractions 16-23 and elution from
Mono Q with an uninterrupted linear gradient of 17 ml from 20 mM to
110 mm MgC.sub.2.
[0078] To further confirm the identity of PK40 and PK36 as protein
kinases, a highly enriched mixture of both kinases obtained as
described (refer to above procedure for elution from Mono Q) was
separated on polyacrylamide gels (PAGE) and the migration of both
proteins was correlated with NF-kinase activity eluted from gel
slices as follows:
[0079] The PK40/36 mixture was electrophoresed on a 10% PAGE
containing SDS, 1 mM DTT and 5 mM MgATP. Gel slices of about 2 mm
size were eluted overnight into small amounts of water. Aliquots of
the supernatants were assessed for NF-kinase activity and activity
by the .sup.32P-assay followed by SDS-PAGE of NF and
autoradiography. A comparatively weak NF-kinase activity correlated
precisely with the presence of a 36 kD protein in the respective
gel slices (as analyzed on 12% SDS-PAGE) while the 40 kD kinase
apparently could not be renatured successfully from SDS.
[0080] To also prove the identity of the 40 kD protein as a kinase
a similar experiment was performed whereby the PK40/36 mixture was
separated on a non-denaturing 7.5% PAGE. In this case the 40 kD
protein appeared as the dominating kinase very well correlated with
activity.
[0081] Step V: The best preparations of PK40 were obtained after
PK40 was run preparatively on nondenaturing 7.5% PAGE and was
eluted from gel slices in an electro-eluter (model UEA,
International Biotechnologies, New Haven, Conn.) in two consecutive
30 minute runs at 120 V and 4.degree. C. into a trapping buffer
consisting of 7.5 M ammonium acetate, 10 mM Mg-ATP, 2 mM DTT and a
trace of bromophenol blue. The elution buffer contained 25 mM Tris
pH 8.3, 192 mM glycine, 2 mm Mg-ATP and 1 mM DTT. The kinase was
dialyzed into a storage buffer of 20 mM BisTris pH 7.0, 2 mM
Mg-ATP, 1 mM DTT, and concentrated about 10-fold in a
microconcentrator (Amicon 10).
[0082] Table 1, below, details the enrichment of PK40 and PK36.
1TABLE 1 Enrichment of PK40 and PK36 through various
chromatographic steps spec. total activity.sup.c activity.sup.d
[mg] Step PK40 PK36 PK40 PK36 PK40 PK36 I) AS-Fractionation
--.sup.a -- -- -- 290 II) CM-Sepharose 0.55 23.6 43 III) Sephadex
G200 1.45 1.15 3.80 2.27 2.6 2.0 IV) Mono Q FPLC 3.8 2.8 0.73 0.39
0.19 0.14 V) Prep. Gel- 5.2 --.sup.b 0.67 -- 0.13 --
electrophoresis .sup.anot determined. NF-specific activity too low
against background. .sup.bElectroelution of PK36 from a preparative
SDS-gel was unsuccessful. .sup.cnmoles 32P-P04 transferred/min/mg
protein. .sup.dnmoles 32P-P04 transferred/min.
[0083] The Mono Q fractions containing PK40 was further subjected
to a two-dimensional SDS-PAGE gel separation procedure (O'Farrell
procedure). A Western Blot assay was performed using a polyclonal
anti-ERK antibody, Anti-rat MAP Kinase R2 (Upstate Biotechnology,
Inc., N.Y. Cat #406-182). Western analysis revealed the presence of
only one ERK protein, although three isoforms could be
distinguished.
EXAMPLE 5
[0084] The-substrate specificity of PK40 and PK36 was determined as
follows. Among neuronal proteins tested, the specificity of PK40
for dephosphorylated NF-M was most striking. Other substrates were
less efficient. The order of specificity was: dephosphorylated
NF-M>>TAU>NF-M=NF-L>dephosphorylated NF-H>NF-H. PK36
had a lower specific activity than PK40, the substrate specificity
being: NF-L=TAU=dephosphorylated
NF-M>NF-M>>NF-H=dephosphorylated NF-H. Some
microtubule-associated proteins were also good substrates for both
kinases.
[0085] MAP2 in a crude microtubule-preparation was a substrate for
PK40 and PK36 comparable to, or better than, TAU proteins,
especially for PK40. MAP#2 is phosphorylated by both kinases above
background level (PK40:2.5x; PK36:1.5x; determined by
CERENKOV-counting). Background labeling of MAP2 was due to a second
messenger independent activity intrinsic to cycled microtubules.
Lysine-rich histone type III (calf thymus, Sigma Chemicals) was the
most preferred substrate for both PK36 and PK40. This is a feature
in stark contrast to all other known ERK kinases, which do not
phosphorylate lysine-rich histone type III very well, and which
phosphorylate microtubule associated proteins including TAU and
MAP2 to a substantially greater extent than lysine-rich histone
type III. The acidic protein phosvitin (Sigma Chemicals) and
tubulin (from calf brain, gift of Dr. F. Solomon, Dept. of Biology,
MIT) were very poor substrates for either PR36 or PK40.
EXAMPLE 6
[0086] The ATP dependence and inhibition of the activities of PK40
and PK36 were determined at 2 mM Mg.sup.2+ with soluble
dephosphorylated NF-M as second substrate to avoid uncertainties
arising from the aggregation state of NF-triplet in suspension. The
optima were at 0.5-1 mM ATP for both kinases. Apparent K.sub.m
values for ATP of both kinases were estimated from Woolf-Hanes
plots (Dixon and Webb, 1979) for a range of ATP concentrations
sufficiently below the onset of inhibition. Three determinations of
the K.sub.m of PK40 at three different concentrations of the second
substrate, NF-M, gave similar values (mean .+-.S.D.: 93.+-.12
.mu.M), indicating little influence of the concentration of NF-M on
ATP-affinity of PK40. The apparent K.sub.m of PK36 was
approximately 50 .mu.M.
[0087] PK36 and particularly PK40 were strongly inhibited to 14%
and 7%, respectively, of the control level in the presence of 5 mm
ATP, amounting to 3 mM excess of free (uncomplexed) ATP over
Mg.sup.2+. In contrast, with excess Mg.sup.2+ (5 mM over 1 mM ATP
or 5 mM over 5 mM ATP), little or no inhibition was observed for
PK40, while the activity of PK36 was significantly reduced.
[0088] The activity of the kinases was also reduced to 27% (PK40)
and 40% (PK36) in the presence of 150 mM NaCl. Inhibition by the
Walsh inhibitor was seen only for PK36, with an estimated IC.sub.50
of 50 micromolar.
[0089] The foregoing is summarized in TABLE 2, below. Table 2.
Effect of sodium chloride, excess magnesium and ATP and of the
Walsh inhibitor on the relative activity [%] of PK40 and PK36
2TABLE 2 Effect of sodium chloride, excess magnesium and ATP and of
the Walsh inhibitor on the relative activity [%] of PK40 and PK36
PK40.sup.a PK36.sup.a Control 2 mM Mg.sup.2 100.sup.b .+-. 6.9.sup.
100 .+-. 1.12 mM 1 mM ATP 2 mM Mg.sup.2 7.2 .+-. 6.9 14 .+-. 0.5 5
mM ATP 5 mM Mg.sup.2 107 .+-. 3.5 40 .+-. 0.2 1 mM ATP 5 mM
Mg.sup.2 78 .+-. 7.1 38 .+-. 2.2 5 mM ATP NaCl 27 .+-. 1.8 40 .+-.
0.8 150 mM Walsh Inhibitor [M] 4.5 102 .+-. 5.3 103 .+-. 0.5 1.5
107 .+-. 3.7 87 .+-. 0.6 45 101 .+-. 1.1 58 .+-. 3.3 The values
represent the mean of 3 assays (.+-.S.D.); except for the Walsh
inhibitor assays, which were carried out in duplicate.
.sup.apreparations of PK40: see Table 1, step V; preparations of
PK36: see Table 1, step IV. .sup.bAll values represent relative
activities in % of the control.
EXAMPLE 7
[0090] A comparison of the phosphorylating activity of PK40 and
PK36 with other kinases was performed. Phosphorylation of the KSP
sequence in dephosphorylated NF-triplet and dephosphorylated NF-M,
using the SMI-31 immunoassay, i.e., measuring reconstitution of the
SMI-epitopes, was achieved with a mixture.of PK40 and PK36; but not
with PKC, calcium/calmodulin dependent kinase II, cAMP-dependent
kinase or second messenger-independent microtubule-associated
kinase.
[0091] Phosphorylations with Ca.sup.2+/calmodulin-dependent kinase
II and protein zza kinase C were performed at 37.degree. C. in 30
microliters of 50 mM HEPES, pH 7.5, 10 mM Mg.sup.2+, 5 m Ca.sup.2+,
1 mm EGTA, 2 mM DTT, 1 mM ATP and 50 micrograms/milliliter
calmodulin and phosphatidylserine, respectively, and 5 micrograms
NF-triplet protein.
EXAMPLE 8
[0092] PK40 and PK36 induced mobility shifts of the heavy
NF-subunits on SDS-PAGE and incorporated phosphate in high molar
ratios. To determine the maximum number of phosphates incorporated
into the heavy NF-subunits by PK40 and PK36 the purified activities
of step V and step IV above were incubated in increasing
concentrations with dephosphorylated NF-M and dephosphorylated
NF-H. The stoichiometry of phosphorylation was determined by
assuming that the correct molecular masses of NF-M and NF-H are 110
kD and 140 kD, respectively, as determined by Kaufmann et al.
(1984), since SDS-PAGE considerably overestimates their M.sub.r.
The saturation phosphorylation of completely dephosphorylated NF-M
and dephosphorylated NF-H by PK40 and PK36 was measured by
assaying: (i) increasing amounts of enzyme activity measured
against extent of phosphorylation in 18 hour assays as monitored by
.sup.32P-incorporation (mole PO.sub.4 per mole NF-M); (ii) gel
mobility shift on 7.5% SDS-PAGE; and (iii) SMI-31 and SMI-34
immunoassays. A mixture of PK40 and 36 also was tested.
[0093] PK40 incorporated up to 15 phosphate groups into NF-M which
corresponds well to the number of phosphates found in isolated
bovine NF-M (Wong et al., 1984) and induced a complete shift of the
NF-M band on SDS-PAGE to the higher apparent M.sub.r of native
NF-M. In contrast, only a partial shift of NF-H was achieved with a
maximum of 7 phosphates introduced into a molecule with presumably
about 40 KSP-sites. The phosphorylation of NF-M with PK36 appeared
to be saturated at 10 moles phosphate/mole NF-M with a substantial
gel mobility shift; however, the NF-M band remained diffuse,
possibly due to a heterogeneous phosphorylation state. NF-H was not
phosphorylated very well by PK36 and showed virtually no gel shift,
in correlation with its poor substrate properties for PK36. Both
kinases reconstituted the SMI-epitopes, but only weakly in the case
of NF-H and PK36. The maximal phosphorylation of NF-M was not
significantly higher with a mixture of the two kinases, indicating,
that PK40 and PK36 might have a largely overlapping
site-specificity on NF-M. After incorporation of 7-13 phosphates,
NF-M had a gel mobility comparable to native NF-M. The
SMI-immunoassay responses were correlated with the gel mobility
shift, but did not respond at lower levels of phosphorylation,
i.e., <5 moles PO.sub.4/mole NF-M. The SMI-34 immuno-assay
required a higher level of phosphorylation than the SMI-31
assay.
EXAMPLE 9
[0094] PK40 can induce a variety of changes in properties on both
dephosphorylated and native TAU, which changes are of pathological
interest.
[0095] To further confirm the effect of PK40 on TAU, bovine TAU was
prepared. The procedure used is preferred as it can be carried out
at 4.degree. C. and results in a product that is free of
phosphorylation that ordinarily would occur at higher
temperatures.
[0096] TAU was prepared from fresh bovine brain according to
Baudier et al. (1987). Briefly, 1 kg of brain tissue was
homogenized in 1 liter of 100 mM potassium phosphate (KPO.sub.4)
buffer, pH 6.5, containing 2 mM each of EDTA and EGTA, 1 mM DTT,
0.1 mM PMSF and 5 mg/l each of aprotinin, leupeptin, antipain,
antichymotrypsin and pepstatin A. Tissue debris was removed by
centrifugation at 15,000.times.g and reextracted with 1 liter of
KPO4 buffer. Supernatants were made 45% in ammonium sulfate, the
precipitate was collected after centrifugation at 20,000.times.g,
rehomogenized and dialyzed extensively into KPO.sub.4 buffer. The
dialysate was adsorbed onto 8 g of preswollen CM-Sephadex, unbound
material was removed by washing with KPO.sub.4 buffer, and the TAU
containing fraction was eluted with KPO.sub.4/0.5 M NaCl, pH 6.5.
HClO.sub.4 was added to 3%, precipitated material was removed by
centrifugation and crude TAU in the Tris-neutralized supernatant
was precipitated by 45% ammonium sulfate. The pellet was taken up
in 5 ml water, dialyzed into 50 mM HEPES, 1 mM EDTA, 1 mM DTT, pH
6.9 (Mono S starting buffer). FPLC was performed on a 5/5 Mono S
column (Pharmacia) with a linear gradient of 30 ml from 0 to 500 mM
NaCl, whereby TAU eluted as a broad peak around 250 mM NaCl. TAU
containing fractions were dialyzed into water and minor
contaminating protease activities were destroyed by boiling for 5
minutes.
[0097] Dephosphorylated TAU protein was prepared by incubation of
300 .mu.g native bovine TAU with 60 .mu.g calf intestinal alkaline
phosphatase (Boehringer Mannheim) and 20 .mu.g E. coli alk.
phoshpatase (Sigma type III-N) in 350 .mu.l of 50 mM Tris, each 0.5
mM MgSO.sub.4 and ZnSO.sub.4, 0.1M PMSF, 10 .mu.g/ml each of
aprotinin, leupeptin, antipain, pepstatin and antichymotrypsin (all
Sigma), pH 8.0, for 3 days at 37.degree. C. Phosphates were removed
by precipitation with 10 .mu.l HClO.sub.4 at room temperature. The
supernatant was neutralized with Tris and dialyzed into water.
[0098] Gel mobility shifts of bovine and human TAU proteins induced
by PK40 and PK36 phosphorylation was measured by incubating 10
.mu.g of native or dephosphorylated bovine TAU (prepared according
to the above alternative procedure) and 3 .mu.g of bacterially
expressed human TAU isoform hTau40 for 18 hr at 37.degree. C. with
about 25 pmole/min of PK40 or PK36. Analysis was performed by 10%
SDS-PAGE and autoradiography.
[0099] Similar results were obtained with a pure 42 KD human TAU
isoform expressed in E. coli from the clone Htau 40 (Goedert et
al., 1989), kindly supplied as protein by Dr. E. M. Mandelkow.
Under saturating conditions as described above, PK40 incorporated
up to 14 phosphates into the 42 kD TAU isoform. PK36 induced a
partial mobility shift in TAU protein, as in the case of NF-M.
[0100] Saturating phosphorylation by PK40 of dephosphorylated
bovine TAU and of TAU native bovine TAU resulted in significantly
reduced mobility on 10% SDS-PAGE as well as a characteristic change
of the isoform patterns. The alterations produced in vitro result
in a pattern that very closely resembles the pattern of human TAU
proteins extracted from PHF, whether native or dephosphorylated TAU
was used. The shift in the pattern of human TAU proteins extracted
from PHF relative to TAU from normal human brains is depicted
clearly in (Goedert et al., 1992), which shift was shown to be
entirely due to hyperphosphorylation.
[0101] On 10% SDS PAGE, multiple isoforms of native bovine TAU are
converted to a 3 isoform pattern. This is the same 3 isoform
apparent pattern produced when the six isoforms of native human TAU
are phosphorylated. In some preparations of bovine TAU, the isoform
with the least mobility was less prominent after phosphorylation,
essentially creating a pattern of two isoforms which has been
observed in some PHF-extracts. The level of phosphorylation and the
concomitant conformational changes are approximately equal with
partially phosphorylated native TAU and dephosphorylated TAU as
substrates.
[0102] The bacterially expressed 42 kD human TAU isoform from the
clone hTau40 behaves in a way similar to bovine TAU upon PK40
phosphorylation. The gel mobility shift corresponds to about 15 kD
of increased apparent size. PK36 was not able to phosphorylate
hTau40 to the same extent and consequently induced only a partial
mobility shift; The heterogeneous appearance of HTau40 probably
reflects the end state of PK36 phosphorylation since the pattern
was not altered at threefold higher concentrations of the kinase.
Pretreatment with PK40 prior to PK36 phospherylation produced the
same completely shifted band as PK40 alone (not shown).
[0103] Treatment of bovine TAU with PK40, but not PK36, also
results in phosphorylation of TAU to an extent that yields a
phosphorylated-TAU protein that is immunochemically similar to the
hyperphosphorylated TAU of PHF-TAU. Dephosphorylated bovine TAU,
and native bovine TAU were incubated with 25 pmole/min PK40 or 15
pmole/min PK36 for 18 hours and probed with monoclonal antibodies
TAU-1 (SIGMA) (0.15 .mu.g TAU as antigen); SMI33 (0.5 .mu.g TAU as
antigen); SMI31 (1 .mu.g-TAU as antigen); and SMI34 (2 .mu.g TAU as
antigen). TAU-1 does not directly recognize a phosphorylated
epitope but PHF-TAU protein treated with phosphates, will bind to
TAU-1 (Grundke-Iqbal et al. 1986). SMI-33 also does not directly
recognize phosphorylated epitopes but does recognize an epitope on
dephosphorylated TAU and on native TAU. This epitope is believed to
be the unphosphorylated KSP sequence (Lee et al., 1988), which
occurs twice in the sequence of all bovine TAU isoforms (Himmler et
al, 1989). Coversely, the SMI-31 epitope is not present in native
or dephosphorylated TAU, in agreement with the KSP sites not
normally being phosphorylated.
[0104] As determined by Western blotting, treatment of native
bovine TAU with PK40 completely abolished imnunoreactivity towards
TAU-1 and SMI-33, but resulted in very strong antibody binding
reactions with SMI-31 and SMI-34, both of which detect
phosphorylated epitopes. This indicates phosphorylation of the KSP
sites of TAU as it occurs in PHF. (The reduction in electrophoetic
mobility was also detected on these Western blots. The effect of
PK40 treatment on TAU-1 binding is of particular interest since the
TAU-1 epitope is masked by phosphorylation in tangles in situ as
well as in PHP-TAU on Western blots (Grundke-Iqbal et al.
1986).
[0105] In contrast, the only significant immunochemical alteration
induced by PK36 treatment of TAU was a strongly reduced SMI 33
activity, the TAU-1 activity being substantially unchanged from
diphosphorylated bouine TAU. PK36 is a less efficient
phosphorylating kinase of TAU than PK40.
[0106] Several kinases have previously been suggested to play a
role in the conversion of TAU proteins into PHF-TAU. However,
unlike PK40, none confer all the known pathological changes on TAU.
Thus, by the criteria of conformational and immunochemical
alterations as well as stoichiometry of phosphorylation, PK40, a
new member of the ERK-family, can perform abnormal
TAU-hyperphosphorylations characteristic of Alzheimer's Disease
(AD), Down's Syndrome (DS) and normal aging. PK36 with a partly
overlapping site-specificity could not induce TAU-changes to a
similar extent.
[0107] A variety of immunohistochemical and biochemical studies
point to TAU-hyperphosphorylation as an event that precedes tangle
formation. PK40 plays a key role in the hyperphosphorylation of
TAU. Chronic upregulation of PK40 might cause neurite degeneration
and interference with the functional integrity of neurons.
EXAMPLE 10
[0108] Uncoupling of oxidative phosphorylation from ATP production
by chemical means causes the appearance of immunological epitopes
in fibroblast cells from healthy patients, cultured under special
conditions. (Blass et al., 1990). This observation is used for the
diagnosis of early Alzheimer's disease by linking the appearance of
these epitopes to the activity of kinases PK40 and PK36, which are
released from inhibition when ATP levels fall, as is the case when
oxidative phosphorylation is uncoupled from ATP production.
Uncoupling is achieved by the use of an uncoupling reagent, e.g.,
CCCP (carbonyl cyanide m-chlorophenylhydrazone), or the deprivation
of oxygen. The diagnostic test for early neuronal degeneration is
applicable for various conditions where neurons degenerate, e.g.,
Alzheimer's disease, Parkinson's disease, Huntington's chorea,
normal aging, and brain infarcts.
[0109] A diagnostic test for early Alzheimer's disease is described
using kinases PK40 and PK36. Primary cultures of skin fibroblasts
are obtained from the patient to be tested. These are grown in
Dulbecco's modified Eagle's medium containing 0.1 mM dibutyryl
cyclic-AMP, 0.1 ug/ml 7S nerve growth factor, 10 ug/ml mixed bovine
gangliosides and 5% chick embryo extract. In the presence of an
uncoupler of oxidative phosphorylation from ATP production, e.g.,
CCCP (carbonyl cyanide m-chlorophenyl hydrazone), or with the
deprivation of oxygen, the cells show immunological epitopes
(Alz-50), PHF-epitopes ,SMI-31/SMI-34-positive TAU/neurofilament
epitopes), indicating the release from inhibition of kinases PK40
and PK36. Cells from Alzheimer patients show this effect at lower
concentrations of uncoupler, compared to normal cells. Thus, cells
from patients to be tested are "titrated" with increasing
concentrations of uncoupling agent or with decreasing oxygen
concentrations. They are distinguished from cells from normal
individuals by their lower resistance to the effects of decreasing
the ATP concentration.
EXAMPLE 11
[0110] Protein Sequencing of PK40
[0111] To obtain pure PK40 approximately 150 .mu.g of total protein
from the Mono Q FPLC fraction containing enriched kinase was loaded
into a 50 mm wide slot and was separated on a 1.5 mm thick 12%
SDS-PAGE. The major proteins were made visible by brief immersion
into 1M KCl at 4.degree. C. The band corresponding to PK40 was cut
out, mninced and homogenized in 3 ml of water containing 1 mM DTT
with a Mini-Turax. The suspension was sealed into a dialysis bag
and shaken with water, 1 mM DTT overnight. The gel fragments were
removed by centrifugation and the supernatant was lyophilized.
[0112] Peptide Mapping of Kinase
[0113] The lyophilisate, containing 20 .mu.g of an apparently
homogeneous 40 kD protein on SDS-PAGE. 20 .mu.g kinase was
dissolved in 500 .mu.L reduction buffer (6M guangidine
hydrochloride/0.5M tris-(hydroxymethyl)-aminomethane pH 8.6). The
pH was adjusted to 8.6 with ammonia. 15 .mu.L 1M dithiothetreitol
was added; The reduction was performed for 2 h at 52.degree. C.
under nitrogen. Then 30 .mu.L .mu.L 1M sodium iodoacetate solution
was added and the sample was incubated for 30 min. at room
temperature in the dark. Excess reagents were removed by dialysis
(cut off 1 kD) against 500 mL 0.5M urea/0.1M NH.sub.4HCO.sub.3 pH
8.6 overnight (complete buffer change after 4 h). After dialysis,
sequence grade trypsin (Boehringer Mannheim) was added in the ratio
1:20, The protein was cleaved for 18 h at 37.degree. C. The
solution was then concentrated to about 250 .mu.L. The reaction was
stopped by cooling to 4.degree. C. After concentration the peptides
were separated HPLC.
[0114] Separation of Tryptic Peptides by HPLC
[0115] A sample was injected onto an HP 1090 HPLC-system equipped
with an Nucleosil RP-18 HPLC-column (CS-Chromatographie Service)
250 mm.times.4.6 mm; 5.mu. material; 300 Angstrom), and samples
were collected at 0.7 ml/min., 40 C with 0.1% TFA and a gradient
from 0 to 70% acetonitrile. The single peptides were collected as
0.5 min. fractions using a fraction collector from LKB/Pharmacia
Superrac model 2211. Elution of peptides was monitored at 210, 280
and 295 nm and 0.35 ml fractions were collected. The single
fractions were lyophilized after separation.
[0116] Chromatographic conditions: flow 0.7 mL/min, column
temperature 40.degree. C., detection 210 nm, 280 nm and 295 nm,
solvent A 0.1% TFA, solvent B 60% acetonitril/0.1% TFA, gradient
Smin O%B, 120 min 70%B, 125 min 100%B, 130 min 0%B, 140 min
O%B.
[0117] All chemicals used were of biochemical or analytical quality
and were purchased from Bio-Rad (D-8000 Munich), Pharacia/LKB
(D-7800 Freiburg), Merck (D-6100 Darmstadt), Serva (D-6900
Heidelberg), Applied Biosystems (sequencer chemicals; D-6108)
Weiterstadt or Pierce (PTH-amino acid standard; Rockford, Ill.
61105 U.S.A.), Boehringer (D-6800 Mannheim) for sequence grade
trypsin.
[0118] The dialysis tube was from Reichelt Chemietechnik (D-6900
Heidelberg).
[0119] The gas phase protein sequencer model 470A was purchased
from Applied Biosystems. A slightly modified standard sequencer
program was used. The sequencer is described in detail in the
respective instrument manual.
[0120] For detection of phenylthiohydantion amino acids (PTH-aa) a
HPLC system from Hewlett Packard PH 1082 and an autosampler from
Waters (D-5090 Leverkusen) WISP 7208 equipped with a Kontron
(D-4000 Dusseldorf) datasystem MT 450 were used. The HPLC-columns
used for PTH-detection (250 mm.times.4.6 mm) filled with
Superspher.TM. material from Merck were purchased from
CS-Chromatographie Service (D-5163 Langerwehe).
[0121] Peptide maps were run on an HPLC-system from Hewlett Packard
HP 1090 equipped with a diode array detector HP1040A and.a
chemstation with the integration software. The-HPLC-column for
peptide mapping was purchased from CS-Chromatrographie Service
(Nucleiosil RP-19; 250 mm.times.4.6 mm; 5.mu. material; 300
Angstrom).
[0122] N-Terminal sequence analysis
[0123] N-terminal sequence analysis was performed using the gas
phase protein sequencer 470A from Applied Biosystems. The standard
sequencer program was used with a slight modification instead of
20% trifluoro acetic acid (TFA) 1M methanolic HCl was taken for
conversion of PTH-amino acids. 50-100 pmol of kinase peptides were
used for sequencing.
[0124] For the identification of PTH-amino acids a HPLC-system
based on a Merck Superspher.TM. column was used (Lottspeich F.
1985. "Microscale isocratic separation of phenylthiohydantoin amino
acid derivatives", J. Chromatography 326: 321-327). The system was
run isocratically.
[0125] Conditions: flow 1.5 mL/min, detection 269 nm, oven
temperature 61.degree. C., mobile phase 68.5% 10 mm sodium acetate,
pH 4.9/31.5% acetonitril supplemented with 5 mL dichloromethane per
L.
[0126] The calibration was performed with a 25 pmol standard of all
PTH-amino acids before each sequencer run.
[0127] The purified tryptic digests are given as Sequence I.D. Nos.
1-15.
[0128] The sequences of these tryptic digests (Seq. I.D. Nos. 1-15)
were compared to the known amino acid sequences of the ERK1 and
ERK2 proteins derived from rat brain cDNA clones (Boulton et al.,
1991). Sequence I.D. Nos. 3-15 matched very closely the ERK-kinase
family proteins. ERK's have the highest homology (about 40%) to
cell cycle-associated cdc2 kinase and thus PK40 can be considered
to be a member of the cell cycle-associated ERK kinase family.
Sequence I.D. Number 6 contains the consenses site of
serine/threonine kinases for nucleotide bi'ding, Gly Glu Gly Ala
Tyr Gly (Hanks et al., 1988).
[0129] Sequence I.D. Numbers 1 and 2 were not homologous to any of
the ERK proteins.
EXAMPLE 12
[0130] A cloning procedure for cDNAs encoding kinases PK40 and PK36
is described. A radiolabeled synthetic oligonucleotide
hybridization probe corresponding to the most unique codons of the
peptide sequence for each of the PK40 and PK36 kinases is
prepared.
[0131] Specifically, probes corresponding to Sequence I.D. Numbers
1 and 2 are most suitable for the cloning procedure for CDNA
encoding PK40.
[0132] The oligonucleotide probes (e.g. Sequence I.D. Nos. 1 and 2)
for PK40 and oligonucleotide probes for PK36 are used to screen
lambda gt11 cDNA libraries prepared from poly(A).sup.+ RNA from
human fetal brain cells, commercially available from a variety of
sources. Hybridization conditions are as described by Cate et al.
(1986), except that the final wash in tetramethyl ammonium chloride
is omitted. DNA inserts from positive plaques are subcloned
directly into the plasmid vector pBlue-script SKM13+ (Stratagene,
Inc. San Diego, Calif.). Positive plasmid subdlones are identified
by colony hybridization, with the use of the same oligonucleotide
hybridization probe. Minipreparations of plasmid DNA are prepared
from positive colonies.
[0133] The nucleotide sequence immediately upstream from the
oligonucleotide binding site is determined by double strand
sequencing (Chen and Seeburg, 1985), using .sup.32P end-labeled
oligonucleotide as sequencing primer and non-radioactive
nucleotides in the extension reactions. Subclones whose codon order
upstream from the priming site match the known amino acid sequence
are sequenced in their entirety by the dideoxy chain termination
method, with either the Klenow fragment of Escherichia coli DNA
polymerase I or modified bacteriophage T7 DNA polymerase
(Sequenase; United States Biochemicals) in the extension reactions.
Subclones are sequenced from their termini, from both directions
from a set of restriction sites. Clones are obtained whose codon
order matches the amino acid sequence of each of the kinases. A
full-length cDNA sequence is assembled from the overlapping partial
clones for each of the kinases.
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Sequence CWU 1
1
* * * * *