U.S. patent application number 11/011557 was filed with the patent office on 2005-08-25 for isozyme-specific antagonists of protein kinase c.
Invention is credited to Mochly-Rosen, Daria.
Application Number | 20050187156 11/011557 |
Document ID | / |
Family ID | 34699955 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050187156 |
Kind Code |
A1 |
Mochly-Rosen, Daria |
August 25, 2005 |
Isozyme-specific antagonists of protein kinase C
Abstract
A method of changing or otherwise converting the biological
activity of a PKC peptide agonist to a peptide antagonist is
described. The method involves substituting one or more amino acid
residues so as to effect a change in charge in the peptide and/or
to otherwise make the sequence similar to a sequence derived from
the PKC binding site on the RACK protein for the respective PKC
enzyme. Methods of inhibiting the activity of a PKC enzyme, and
various peptide antagonists of .epsilon.PKC are also disclosed.
Inventors: |
Mochly-Rosen, Daria; (Menlo
Park, CA) |
Correspondence
Address: |
PERKINS COIE LLP
P.O. BOX 2168
MENLO PARK
CA
94026
US
|
Family ID: |
34699955 |
Appl. No.: |
11/011557 |
Filed: |
December 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60529223 |
Dec 11, 2003 |
|
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Current U.S.
Class: |
514/1.7 ;
514/16.6; 514/17.9; 514/7.3; 530/324 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 7/02 20180101; A61P 11/06 20180101; A61P 43/00 20180101; A61P
17/06 20180101; A61P 31/04 20180101; A61P 37/02 20180101; A61P
37/00 20180101; A61P 1/16 20180101; C12N 9/1205 20130101; A61P 1/04
20180101; A61P 11/00 20180101; A61P 5/16 20180101; A61P 19/02
20180101; A61P 3/10 20180101; A61P 17/00 20180101; A61P 1/00
20180101; A61P 29/00 20180101 |
Class at
Publication: |
514/012 ;
530/324 |
International
Class: |
A61K 038/54; C07K
014/47 |
Goverment Interests
[0002] This work was supported in part by The National Institutes
of Health Grant HL52141. Accordingly the United States government
may have certain rights in this invention.
Claims
What is claimed is:
1. A method of converting a protein kinase C (PKC) agonist peptide
or peptidomimetic to a PKC antagonist peptide or peptidomimetic,
comprising substituting at least one amino acid in said agonist
peptide or peptidomimetic with an amino acid that converts the PKC
agonist peptide or peptidomimetic into a PKC antagonist peptide or
peptidomimetic.
2. The method of claim 1, wherein said at least one amino acid is a
charged amino acid which is substituted with an uncharged amino
acid.
3. The method of claim 2, wherein said charged amino acid is
aspartic acid and said uncharged amino acid is asparagine.
4. The method of claim 1, wherein said PKC agonist peptide is a
classical PKC agonist, a novel PKC agonist or an atypical PKC
agonist.
5. The method of claim 1, wherein said PKC agonist peptide is an
.epsilon.PKC agonist peptide.
6. The method of claim 5, wherein said .epsilon.PKC agonist peptide
is a .psi..epsilon.RACK peptide having an amino acid sequence set
forth in SEQ ID NO:3.
7. The method of claim 5, wherein said .epsilon.PKC agonist peptide
has an amino acid sequence set forth in SEQ ID NO:12; SEQ ID NO:20;
SEQ ID NO:16; or SEQ ID NO:21.
8. The method of claim 1, wherein said PKC antagonist peptide is an
.epsilon.PKC antagonist peptide having an amino acid sequence set
forth in SEQ ID NO:50; SEQ ID NO:51; SEQ ID NO:52; SEQ ID NO:53;
SEQ ID NO:54; SEQ ID NO:55; SEQ ID NO:56; or SEQ ID NO:57.
9. The method of claim 1, wherein said PKC agonist peptide is an
.epsilon.PKC agonist peptide and wherein said at least one amino
acid is a charged amino acid which is substituted with an uncharged
amino acid.
10. A method of inhibiting the activity of a protein kinase C (PKC)
enzyme, comprising contacting the enzyme with a PKC inhibitor
peptide or peptidomimetic, said peptide derived from a PKC agonist
peptide or peptidomimetic wherein at least one amino acid in the
agonist peptide or peptidomimetic is substituted with another amino
acid sufficient to convert the agonist peptide or peptidomimetic
into an antagonist peptide or peptidomimetic.
11. The method of claim 10, wherein said PKC agonist peptide is
derived from the .psi.-RACK sequence of said PKC enzyme.
12. The method of claim 10, wherein said .psi.-RACK sequence is a
.psi..epsilon.RACK sequence.
13. The method of claim 10, wherein said protein kinase C is
.epsilon.PKC and said PKC agonist peptide is a derivative of a
.psi.RACK sequence of said PKC enzyme.
14. The method of claim 13, wherein said PKC agonist peptide is a
.psi..epsilon.RACK agonist peptide having an amino acid sequence
set forth in SEQ ID NO:3.
15. The method of claim 13, wherein said PKC agonist peptide has an
amino acid sequence set forth in SEQ ID NO:12; SEQ ID NO:16; SEQ ID
NO:20 or SEQ ID NO:21.
16. The method of claim 13, wherein said PKC agonist peptide is a
.psi..epsilon.RACK peptide having an amino acid sequence set forth
in SEQ ID NO:3 and said PKC inhibitor peptide has an amino acid
sequence that differs from SEQ ID NO:3 in that said at least one
amino acid residue is substituted with another amino acid that has
a decreased electrical charge compared to said at least one amino
acid.
17. The method of claim 16, wherein said at least one amino acid is
negatively charged and said another amino acid is uncharged.
18. The method of claim 17, wherein said at least one amino acid is
aspartic acid and said another amino acid is asparagine.
19. The method of claim 16, wherein said PKC inhibitor peptide has
the amino acid sequence set forth in SEQ ID NO:55.
20. The method of claim 10, wherein said PKC inhibitor peptide has
an amino acid sequence set forth in SEQ ID NO:50; SEQ ID NO:51; SEQ
ID NO:52; SEQ ID NO:53; SEQ ID NO:54; SEQ ID NO:55; SEQ ID NO:56;
or SEQ ID NO:57.
21. A peptide, comprising a peptide having PKC antagonistic
activity, said peptide derived from a PKC agonist peptide wherein
at least one amino acid in the agonist peptide is substituted with
another amino acid sufficient to convert the agonist peptide into
an antagonist peptide.
22. The peptide of claim 21, wherein said PKC is .epsilon.PKC and
wherein said PKC agonist peptide is an .epsilon.PKC agonist
peptide.
23. The peptide of claim 22, wherein said peptide has an amino acid
sequence set forth in SEQ ID NO:55.
24. The peptide of claim 21, wherein said antagonist peptide has an
amino acid sequence set forth in SEQ ID NO:50; SEQ ID NO:51; SEQ ID
NO:52; SEQ ID NO:53; SEQ ID NO:54; SEQ ID NO:55; SEQ ID NO:56; or
SEQ ID NO:57.
25. The peptide of claim 21, wherein said at least one amino acid
is substituted with an amino acid that provides a change of
electrical charge at the substituted position.
26. A peptide having PKC antagonistic activity, comprising a
peptide having an amino acid sequence set forth in SEQ ID NO:50;
SEQ ID NO:51; SEQ ID NO:52; SEQ ID NO:53; SEQ ID NO:54; SEQ ID
NO:55; SEQ ID NO:56; or SEQ ID NO:57.
27. A treatment method, comprising administering to a patient in
need thereof a therapeutically effective amount of an .epsilon.PKC
antagonist peptide having the amino acid sequence set forth in SEQ
ID NO:50; SEQ ID NO:51; SEQ ID NO:52; SEQ ID NO:53; SEQ ID NO:54;
SEQ ID NO:55; SEQ ID NO:56; or SEQ ID NO:57, or a combination
thereof.
28. The method of claim 27, wherein said treatment is for a disease
or condition modulated by .epsilon.PKC.
29. The method of claim 28, wherein said disease or condition is a
fibrotic disease or an inflammatory disease.
30. The method of claim 29, wherein said fibrotic disease is
scleroderma, liver fibrosis or lung fibrosis.
31. The method of claim 29, wherein said inflammatory disease is an
autoimmune disease or a lung disease.
32. The method of claim 31, wherein said autoimmune disease is
multiple sclerosis, Guillain-Barre syndrome, psoriasis, Grave's
disease, rheumatoid arthritis and Type 1 diabetes mellitus.
33. The method of claim 31, wherein said lung disease is chronic
obstructive pulmonary disease or asthma.
34. The method of claim 29, wherein said inflammatory disease is
septic shock or inflammatory bowel disease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/529,223, filed Dec. 11, 2003, which
is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to a method of converting the
biological action of an agonist to an antagonist, and in particular
a method of converting an agonist of protein kinase C to an
antagonist of protein kinase C. The present invention also relates
to a peptide composition for isozyme-specific modulation of protein
kinase C and to a method of antagonizing the action of specific
isozymes of protein kinase C.
BACKGROUND OF THE INVENTION
[0004] Protein kinase C ("PKC") is a key enzyme in signal
transduction involved in a variety of cellular functions, including
cell growth, regulation of gene expression, and ion channel
activity. The PKC family of isozymes includes at least 11 different
protein kinases that can be divided into at least three subfamilies
based on their homology and sensitivity to activators. Each isozyme
includes a number of homologous ("conserved" or "C") domains
interspersed with isozyme-unique ("variable" or "V") domains.
Members of the "classical" or "cPKC" subfamily, .alpha.,
.beta..sub.I, .beta..sub.II, and .gamma.PKC, contain four
homologous domains (C1, C2, C3 and C4) and require calcium,
phosphatidylserine, and diacylglycerol or phorbol esters for
activation. In members of the "novel" or "nPKC" subfamily, .delta.,
.epsilon., .eta. and .theta.PKC, a C2-like domain preceeds the C1
domain. However, that C2 domain does not bind calcium and therefore
the nPKC subfamily does not require calcium for activation.
Finally, members of the "atypical" or ".alpha.PKC" subfamily,
.zeta. and /.sub.IPKC, lack both the C2 and one-half of the C1
homologous domains and are insensitive to diacylglycerol, phorbol
esters and calcium.
[0005] Studies on the subcellular distribution of PKC isozymes
demonstrate that activation of PKC results in its redistribution in
the cells (also termed translocation), such that activated PKC
isozymes associate with the plasma membrane, cytoskeletal elements,
nuclei, and other subcellular compartments (Saito, N. et al., Proc.
Natl. Acad. Sci. USA 86:3409-3413 (1989); Papadopoulos, V. and
Hall, P. F. J. Cell Biol. 108:553-567 (1989); Mochly-Rosen, D., et
al., Molec. Biol. Cell (formerly Cell Reg.) 1:693-706, (1990)). The
unique cellular functions of different PKC isozymes are determined
by their subcellular location. For example, activated
.beta..sub.IPKC is found inside the nucleus, whereas activated
.beta..sub.IIPKC is found at the perinucleus and cell periphery of
cardiac myocytes (Disatnik, M. H., et al., Exp. Cell Res.
210:287-297 (1994)). .epsilon.PKC, a member of the novel PKC family
independent from calcium but requiring phospholipids for
activation, is found in primary afferent neurons both in the dorsal
root ganglia as well as in the superficial layers of the dorsal
spinal cord.
[0006] The localization of different PKC isozymes to different
areas of the cell in turn appears due to binding of the activated
isozymes to specific anchoring molecules termed Receptors for
Activated C-Kinase ("RACKs"). RACKs are thought to function by
selectively anchoring activated PKC isozymes to their respective
subcellular sites. RACKs bind only fully activated PKC and are not
necessarily substrates of the enzyme. Nor is the binding to RACKs
mediated via the catalytic domain of the kinase (Mochly-Rosen, D.,
et al., Proc. Natl. Acad. Sci. USA 88:3997-4000 (1991)).
Translocation of PKC reflects binding of the activated enzyme to
RACKs and the binding to RACKs is required for PKC to produce its
cellular responses (Mochly-Rosen, D., et al., Science 268:247-251
(1995)). Inhibition of PKC binding to RACKs in vivo inhibits PKC
translocation and PKC-mediated function (Johnson, J. A., et al., J.
Biol. Chem. 271:24962-24966 (1996); Ron, D., et al, Proc. Natl.
Acad. Sci. USA 92:492-496 (1995); Smith, B. L. and Mochly-Rosen,
D., Biochem. Biophys. Res. Commun. 188:1235-1240 (1992)).
[0007] In general, translocation of PKC is required for proper
function of PKC isozymes. Peptides that mimic the RACK-binding site
on PKC [Ron, D., et a., Proc. Natl. Acad. Sci. USA 92:492-496
(1995); Johnson, J. A., et al., J. Biol. Chem. 271:24962-24966
(1996)] are isozyme-specific translocation inhibitors of PKC that
selectively inhibit the function of the enzyme in vivo. Such
isozyme-selective inhibitors of PKC have been identified based on
their ability to selectively inhibit the interaction of the
activated isozymes with their respective anchoring proteins (RACKs)
(Souroujon, M. and Mochly-Rosen, D., Nature Biotechnol. 16:919-924,
(1998)). These short peptide inhibitors (7-12 amino acids long)
have been shown to selectively interfere with the functions of
individual isozymes (Mochly-Rosen, D., et al., Proc. Natl. Acad.
Sci. USA 88:3997-4000, (1991); Ron, D., et al., J. Biol. Chem.
270:24180-24187, (1995); Johnson, J. A., et al., J. Biol. Chem.
271:24962-24966, (1996); Zhang, Z., et al., Biophys. J. 70(2, part
2):A391, (1996); Gray, M. O., et al., J. Biol. Chem.
272:30945-30951, (1997)).
[0008] Translocation agonist peptides of .beta. and .epsilon.PKC,
as well as other PKC isozymes, have also been identified [Ron, D.
and Mochly-Rosen, D., Proc. Natl. Acad. Sci. USA 92:492-496,
(1995); Dorn, G. W., et al., Proc. Natl. Acad. Sci. USA
96:12798-12803, (1999)]. Peptides that mimic the PKC-binding site
on RACKs [Mochly-Rosen, D., et al., J. Biol. Chem. 226:1466-1468
(1991); Mochly-Rosen, D., et al., Science 268:247-251 (1995)] are
isozyme-specific translocation activators of PKC that selectively
inhibit the function of the enzyme in vivo (Mochly-Rosen, D., Proc.
Natl. Acad. Sci. USA 92:492-496, (1995); Dorn, G. W., et al., Proc.
Natl. Acad. Sci. USA 96:12798-12803, (1999)). These 6-8 amino acid
peptides derived from PKC are homologous to a sequence within their
corresponding RACK and hence they were termed pseudo-.beta.RACK
(.psi..beta.RACK) and pseudo-.epsilon.RACK (.psi..epsilon.RACK),
respectively. Introduction of .psi..beta.RACK or .psi..epsilon.RACK
into cells causes a selective translocation of the corresponding
isozymes and increases their catalytic activity as measured by
substrate phosphorylation in vitro and in vivo (Ron, D. and
Mochly-Rosen, D., Proc. Natl. Acad. Sci. USA 92:492-496, (1995);
Dorn, G. W., et al., Proc. Natl. Acad. Sci. USA 96:12798-12803,
(1999)). The position in the C2 or C2-like domain of the .PSI.TRACK
sequence in isozymes whose RACK has not been identified yet (e.g.,
.delta. and .theta.PKC) was also found to correspond to
translocation agonist peptides [Chen et al., Proc. Natl. Acad. Sci.
USA 98:1114-1119, (2001)]. For example, introduction of
.PSI..delta.RACK into cells causes a selective translocation of the
.delta.PKC and increases its catalytic activity [Chen et al., Proc.
Natl. Acad. Sci. USA 98:1114-1119, (2001)]. These peptides have
also been used to identify the role of .beta.PKC, .delta.PKC and
.epsilon.PKC in cells and in vivo [Ron, D. and Mochly-Rosen, D.,
Proc. Natl. Acad. Sci. USA 92:492-496, (1995); Chen et al., Proc.
Natl. Acad. Sci. USA 98:1114-1119, (2001); Dorn, G. W., et al.,
Proc. Natl. Acad. Sci. USA 96:12798-12803, (1999)).
[0009] From a therapeutic perspective, individual isozymes of PKC
have been implicated in the mechanisms of various disease states,
including the following: cancer (alpha and delta PKC); cardiac
hypertrophy and heart failure (beta I and beta II PKC) nociception
(gamma and epsilon PKC); ischemia including myocardial infarction
(epsilon and delta PKC); immune response, particularly T-cell
mediated (theta PKC); and fibroblast growth and memory (delta and
zeta PKC). Various PKC isozyme- and variable region-specific
peptides have been previously described (see, for example, U.S.
Pat. No. 5,783,405). The role of .epsilon.PKC in pain perception
has recently been reported (WO 00/01415; U.S. Pat. No. 6,376,467)
including therapeutic use of the .epsilon.V1-2 peptide, a selective
inhibitor of .epsilon.PKC first described in the U.S. Pat. No.
5,783,405.
[0010] It is clear that PKC isozymes are involved in a variety of
disease states, and there continues to be a need for methods of
modulating the action of specific PKC isozymes to develop
therapeutic agents to treat human disease.
SUMMARY OF THE INVENTION
[0011] It has been discovered that substituting at least one amino
acid in a protein kinase C (PKC) agonist peptide with another amino
acid to alter the charge distribution in the peptide, such as by
changing the electrical charge at the substituted position, and/or
such that the peptide more closely resembles a sequence within the
protein kinase C binding site on the respective receptor for
activated C kinase (RACK) protein will produce a PKC antagonist
that may be used to inhibit the activity of the PKC enzyme.
Accordingly, methods of converting a protein kinase C agonist
peptide or peptidomimetic into a protein kinase C antagonist
peptide or peptidomimetic are provided. In one form, a method
includes substituting at least one amino acid in the agonist
peptide or peptidomimetic with an amino acid that converts the PKC
agonist peptide or peptidomimetic into a PKC antagonist peptide or
peptidomimetic. In certain forms of the invention, the peptide
antagonist is a selective inhibitor of the isozyme from which it
was derived.
[0012] In yet another aspect of the invention, methods of
inhibiting the activity of a PKC enzyme are also provided. In one
form, a method includes contacting the enzyme with a PKC inhibitor
peptide or peptidomimetic, wherein the peptide is derived from a
PKC agonist peptide or peptidomimetic and wherein at least one
amino acid in the agonist peptide or peptidomimetic is substituted
with another amino acid sufficient to convert the agonist peptide
or peptidomimetic into an antagonist peptide or peptidomimetic. In
certain forms of the invention, the substitution results in a
change of charge at the residue position substituted.
[0013] Treatment methods are also provided. In one form, a method
includes administering a therapeutically effective amount of an
.epsilon.PKC antagonist peptide or peptidomimeitc to a patient in
need thereof. The methods may be used to treat a wide variety of
diseases or conditions by modulating the activity of an
.epsilon.PKC enzyme.
[0014] Peptides or peptidomimetics having PKC antagonistic activity
are also provided. In one form of the invention, the peptide or
peptidomimetic is derived from a PKC agonist peptide or
peptidomimetic wherein at least one amino acid in the agonist
peptide or peptidomimetic is substituted with another amino acid
sufficient to convert the agonist peptide or peptidomimetic into an
antagonist peptide or peptidomimetic. In yet other forms of the
invention, the peptide has the sequence as indicated herein.
[0015] It is an object of the invention to provide methods of
converting a PKC agonist peptide or peptidomimetic into a protein
kinase C antagonist peptide or peptidomimetic.
[0016] It is yet another object of the invention to provide methods
of inhibiting the activity of a PKC enzyme.
[0017] It is yet another object of the invention to provide
treatment methods using the PKC antagonists described herein.
[0018] It is a further object of the invention to provide peptides
or peptidomimetics having .epsilon.PKC antagonistic activity.
[0019] These and other objects and features of the invention will
be more fully appreciated when the following description of the
invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1A is a graph showing the number of contractions
measured in a 15 second interval in neonatal rat cardiac myocytes
as a function of time in cells treated with a control, carrier
peptide (squares) and in cells treated with .psi..epsilon.RACK
peptide (triangles), where both cell populations (control and
.psi..epsilon.RACK peptide treated) were treated with phorbol
12-myristate 13-acetate (PMA) at 30 minutes as more fully described
in Example 1.
[0021] FIG. 1B is a bar graph showing the contraction rate of
neonatal rat cardiac myocytes, expressed as the percent decrease in
contraction rate relative to the basal contraction rate, after
treatment with (i) the .epsilon.PKC agonist peptide
(.psi..epsilon.RACK peptide), (ii) phorbol 12-myristate 13-acetate
(PMA), (iii) PMA and .psi..epsilon.RACK peptide, (iv) PMA,
.psi..epsilon.RACK peptide, and FV1-2 peptide, or (v) PMA,
.psi..epsilon.RACK peptide, and chelerythrine (Che) as more fully
described in Example 1.
[0022] FIGS. 2A-2C are graphs of the contraction rate of neonatal
rat cardiac myocytes, expressed as the percent decrease in
contraction rate relative to the basal contraction rate, as a
function of time as more fully described in Example 1; where FIGS.
2A-2B show the data for cells not treated with the ePKC antagonist
peptide derived from the .psi..epsilon.RACK peptide
(N-.psi..epsilon.RACK peptide) (squares) and for cells treated with
N-.psi..epsilon.RACK (SEQ ID NO:2, diamonds), where both cell
populations were treated with 1 nM (FIG. 2A) or 5 nM (FIG. 2B) PMA
15-20 minutes after administration of the N.psi..epsilon.RACK
peptide; and FIG. 2C shows the data for cells treated with
N-.psi..epsilon.RACK peptide (diamonds) and then with
.psi..epsilon.RACK (SEQ ID NO:1 ) and with 1 nm PMA, and for cells
treated with .psi..epsilon.RACK (triangles) and then with 1 nM
PMA.
[0023] FIGS. 3A-3B shows Western blot analysis of PMA-induced
translocation of .epsilon.PKC (FIG. 3A) and .alpha.PKC (FIG. 3B)
from the cell soluble (S) to cell particulate (P) fractions of
cardiac myocytes treated with a control, carrier peptide (Lane nos.
1), treated with 1 nM PMA (Lane nos. 2), treated with PMA and
.psi..epsilon.RACK (SEQ ID NO:1, Lane nos. 3) or treated with
N-.psi..epsilon.RACK, .psi..epsilon.RACK, and PMA (Lane nos. 4) as
more fully described in Example 2.
[0024] FIG. 4 is a bar graph showing the percentage of cell damage
to rat cardiac myocytes resulting from an ischemic episode, where
myocytes not subjected to ischemia are represented by the clear
area in each bar, and the percent of ischemic damage represented by
the filled area; cells were pretreated prior to the ischemic
episode with .psi..epsilon.RACK, N-.psi..epsilon.RACK,
A-.psi..epsilon.RACK, .epsilon.V1-2, and various combinations of
these peptides as indicated as more fully described in Example
3.
[0025] FIG. 5A depicts a Western blot analysis showing the rate of
degradation of .PSI..epsilon.RACK mutants by the protease, Arg C,
using anti-.epsilon.PKCV5 antibodies as more fully described in
Example 4. Wt, wild type; D(86)A, 6PKC mutant wherein D at position
86 of .psi..epsilon.RACK on .epsilon.pKC is substituted with an A;
D(86)N, .epsilon.pKC mutant wherein D at position 86 of
.psi..epsilon.RACK in .epsilon.pKC is substituted with an N.
[0026] FIG. 5B depicts a graph showing the rate of degradation of
the .PSI..epsilon.RACK .PSI..epsilon.PKC mutants by Arg C as a
function of time, wherein the data represents the average of five
independent experiments as more fully described in Example 4. Data
were normalized to the initial amount of enzyme, and are expressed
as percent of full length .epsilon.PKC. Wt (closed squares); D(86)A
(open triangles.), D(86)N (open circles); *p<0.05 using T
test.
[0027] FIG. 5C depicts a Western blot analysis showing binding of
.OMEGA..epsilon.RACK .epsilon.PKC mutants to GST-.epsilon.RACK in
the presence and absence of PL as determined using
anti-.epsilon.PKC V5 antibodies as more fully described in Example
5. PL, phospholipid activators (phosphatidylserine and Sn-1,2
dioleoylglycerol.
[0028] FIG. 5D depicts a graph showing normalized binding in PKC
wild type and mutants (average of four independent experiments) for
binding of .PSI..epsilon.RACK .epsilon.PKC mutants to
GST-.epsilon.RACK, in the absence (plain bars) or presence of PL
(filled bars) (*p<0.05 using T test) as more fully described in
Example 5. PL, phospholipid activators (phosphatidylserine and
Sn-1,2 dioleoylglycerol.
[0029] FIG. 6A shows a Western blot analysis of Immunoprecipitated
GFP-.epsilon.PKC mutants detected with anti-.epsilon.PKC V5
antibodies (upper panel) and their catalytic activity measured by
autoradiography of [.gamma..sup.32P] labeled myelin basic protein
(lower panel, a representative experiment) as more fully described
in Example 6.
[0030] FIG. 6B shows a graph of myelin basic protein (MBP)
phosphorylation in .epsilon.PKC wild type or mutants as more fully
described in example 6 (average of three independent kinase
reactions showing equal activity of the .epsilon.PKC mutants upon
activation as seen by myelin basic protein phosphorylation in the
presence of PL (reactions were carried out for 3 and 15
minutes).
[0031] FIG. 6C depicts a Western blot analysis showing
translocation of CHO cells transfected with GFP-.epsilon.PKC
.PSI..epsilon.RACK mutants and treated with 100 nM PMA for 10
minutes as more fully described in Example 6. GFP-.epsilon.PKC was
detected with anti-.epsilon.PKC V5 antibodies.
[0032] FIG. 6D shows a Western blot analysis depicting
translocation of GFP-.epsilon.PKC mutants in MCF-7 cells upon
stimulation with 10 nM PMA for 10 minutes as more fully described
in Example 6.
[0033] FIG. 6E shows a graph of GFP-.epsilon.PKC translocation in
Wt, or D(86)N mutants or D(86)A mutants after PMA stimulation as
more fully described in Example 6. An average of 4 independent
experiments of translocation of GFP-.epsilon.PKC .PSI..epsilon.RACK
in MCF-7 control (plain bars) and cells stimulated with 10 nM PMA
for 10 minutes (filled bars) (*p<0.05 using T test) is shown in
the figure.
[0034] FIG. 7A depicts confocal images of .PSI..epsilon.RACK
.epsilon.PKC mutants at different time points upon stimulation with
100 nM PMA as more fully described in Example 6. An arrow within a
panel indicates the time at which translocation to the cell
periphery began to be apparent for each .epsilon.PKC mutant.
[0035] FIG. 7B depicts a typical line-intensity profile showing the
distribution of PKC between the cell periphery and cytosol for
representative transfected cells at different time points
(indicated by a line in A, left panels) as more fully described in
Example 7.
[0036] FIG. 7C depicts a graph of percent fluorescence in the
cytosol of Wt cells or D(86)A or D(86)N .psi..epsilon.RACK
.epsilon.PKC mutants as a function of time after stimulation with
100 nM PMA as more fully described in Example 6: Wt (closed
squares), D(86)A (open triangle); D(86)N(open circle). Average
translocation rates were expressed by normalizing the initial
fluorescence intensity to 100% (average of at least three
independent experiments, with at least three cells analyzed for
each experiment). The time courses for the mutants and Wt enzymes
were statistically different from each other by 2 way ANOVA with
p<0.001.
[0037] FIGS. 8A-8D depicts mathematical modeling analysis of D(86)A
and analyzed as a graphical representation of percent fluorescence
as a function of time after PMA stimulation as more fully described
in Example 7. Similar results were obtained with D(86)N and Wt
.epsilon.PKC. FIG. 8A depicts non-linear regression analysis using
the single exponential equation; FIG. 8B depicts non-linear
regression analysis using the bi-exponential equation; FIG. 8C is a
graphical representation of fit between curves of the raw data for
D(86)A. The curves were obtained by nonlinear regression with a
bi-exponential equation; FIG. 8D is a graphical representation of
the fit between curves of the raw data for D(86)A. The curves were
obtained by a differential equation using the values for k1, k-1,
k2 and k-2 provided in Table 1. The residual error for all curves
fitted data was similar to the one obtained with a non-linear
regression using a bi-exponential equation.
[0038] FIG. 9A depicts confocal images of CHO cells, transfected
with both YFP-.epsilon.PKC D(86)A and CFP-.epsilon.PKC Wt, at
different times after stimulation with100 nM PMA as more fully
described in Example 8.
[0039] FIG. 9B depicts confocal images of CHO cells, transfected
with D(86)A and D(86(N) mutants, as a function of time after being
stimulated with 1 mM ATP as more fully described in Example 9. In
comparison to wild type, the D(86)A mutant had a similar
translocation rate and the RACK D(86)N mutant had a slower
translocation rate upon stimulation with 1 mM ATP. The arrows
indicate the time at which translocation to the cell periphery
began to be apparent for each .epsilon.PKC enzyme.
[0040] FIG. 9C depicts fluorescence intensity as a function of time
after PMA stimulation in cells transfected with D(86)A and D(86(N)
as more fully described in Example 9. Translocation rates were
analyzed by measuring the loss of fluorescence in the cytoplasm
relative to time after addition of 1 mM ATP: Wt (closed square),
D(86)A (open triangle), D(86)N (open circle). Data are averages of
at least three independent experiments with at least three cells in
each experiment. The time course for the D(86)N mutant was
statistically different from either D(86)A or Wt .epsilon.PKCs
using a 2 way ANOVA test with p<0.001.
[0041] FIG. 9D depicts fluorescence intensity as a function of time
after ATP stimulation of CHO cells transfected CFP-.epsilon.PKC
Wt.(closed square) and YFP-.epsilon.PKC D(86)A (open triangle) as
more fully described in Example 8. Levels of the YFP-.epsilon.PKC
D(86)A mutant in the cytoplasm decrease faster than levels of
CFP-.epsilon.PKC Wt.
[0042] FIG. 10 depicts a pictorial showing the first steps in
.epsilon.PKC translocation to the membrane upon activation; a
two-step process, as more fully described in the Discussion section
of the Examples. An additional step in the process of translocation
includes binding to the RACK (not shown in the scheme). For
simplicity, only the intramolecular interaction between the
.PSI..epsilon.RACK and the .epsilon.RACK-binding site are
shown.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0043] For the purposes of promoting an understanding of the
principles of the invention, reference will now be made to
preferred embodiments and specific language will be used to
describe the same. It will nevertheless be understood that no
limitation of the scope of the invention is thereby intended, such
alterations and further modifications of the invention, and such
further applications of the principles of the invention as
illustrated herein, being contemplated as would normally occur to
one skilled in the art to which the invention relates.
[0044] The present invention provides methods of converting a PKC
agonist peptide or peptidomimetic into a PKC antagonist peptide or
peptidomimetic. It has been discovered that substituting at least
one amino acid in a PKC agonist peptide or peptidomimetic with
another amino acid such that the charge distribution of the peptide
or peptidomimetic is changed and/or such that the peptide or
peptidomimetic more closely resembles a sequence within the PKC
binding site on the respective RACK protein will produce an
antagonist that may be used to inhibit the activity of the PKC
enzyme. In one form, a method includes substituting at least one
amino acid in the agonist peptide or peptidomimetic with an amino
acid that converts the PKC agonist peptide or peptidomimetic into a
PKC antagonist peptide or peptidomimetic. In yet other forms of the
invention, the substitution may be made by synthesizing a peptide
or peptidomimetic having the different amino acid or by modifying
an amino acid in a pre-existing peptide or peptidomimetic such that
the agonist peptide or peptidomimetic is converted into an
antagonist peptide or peptidomimetic. The peptidomimetic herein
includes a small molecule that selectively binds to the
intermolecular binding site on the respective PKC enzyme.
[0045] Methods of inhibiting the activity of a protein kinase C
(PKC) enzyme are also provided. In one form, a method includes
contacting the PKC enzyme with a PKC inhibitor peptide or
peptidomimetic derived from a PKC agonist peptide or peptidomimetic
wherein at least one amino acid in the agonist peptide or
peptidomimetic is substituted with another amino acid sufficient to
convert the agonist peptide or peptidomimetic into an antagonist
peptide or peptidomimetic.
[0046] Treatment methods are also provided. In one form, a method
includes administering a therapeutically effective amount of an
.epsilon.PKC antagonist peptide or peptidomimetic to a patient in
need thereof. The methods may be used to treat a wide variety of
diseases or conditions by modulating the activity of an
.epsilon.PKC enzyme.
[0047] Peptides or peptidomimetics having PKC antagonistic activity
are also provided. In certain forms of the invention the peptide or
peptidomimetic is derived from a PKC agonist peptide or
peptidomimetic wherein at least one amino acid in the agonist
peptide or peptidomimetic is substituted with another amino acid
sufficient to convert the agonist peptide or peptidomimetic into an
antagonist peptide or peptidomimetic.
[0048] In one aspect of the invention, methods of converting a PKC
enzyme agonist peptide or peptidomimetic into a PKC enzyme
antagonist peptide or peptidomimetic are provided. In one form, a
method includes substituting at least one amino acid in a PKC
agonist peptide or peptidomimetic with another amino acid
sufficient to convert the PKC agonist into a PKC antagonist.
"Peptide" and "polypeptide" as used herein refer to a compound made
up of a chain of amino acid residues linked by peptide bonds.
Unless otherwise indicated, the sequence for peptides is given in
the order from the amino terminus to the carboxyl terminus.
[0049] The method is applicable to a wide variety of PKC enzymes.
As described above, PKC enzymes are classified into three families
based on the homology of their regulatory domains: conventional PKC
(cPKC; including .alpha., .beta..sub.I, .beta..sub.II, and
.gamma.), novel PKC (nPKC; including .epsilon., .delta., .eta., and
.theta.) and atypical PKC (.alpha.PKC; including .zeta., i, and
.lambda.). As previously mentioned herein, in order for the PKC
enzymes to exert their biological effects, they must be activated
and bind to their respective RACK proteins. As an example, an
.epsilon.PKC binding site exists on .epsilon.RACK (the amino acid
sequence of which is set forth in SEQ ID NO:1 as NNVALGYD). PKC
binding sites also exist on other RACKS for binding interactions
with their respective PKC enzymes. For example, the .beta.PKC
binding site on RACK1 is set forth in SEQ ID NO:2 as SIKIWD. This
sequence is identical between .beta..sub.I and .beta..sub.II since
they differ only in the last 50 amino acids and is identical in
.alpha. and .gamma.PKC. In one form of the invention when designing
a particular antagonist from a specified agonist, these binding
site sequences can be referred to in order to ensure the at least
one amino acid is substituted with an amino acid that makes the
peptide or peptidomimetic more closely resemble the PKC binding
site on the respective PKC enzymes or to otherwise increase the
binding affinity of the peptide or peptidomimetic to the binding
site. In yet other forms of the invention, substituting at least
one charged amino acid in the peptide agonists or peptidomimetics
described herein with an uncharged amino acid is expected to
convert the PKC agonist into a PKC antagonist.
[0050] A wide variety of PKC peptide or peptidomimetic agonists may
be modified or otherwise converted into a PKC antagonist peptide or
peptidomimetic and consequently find use in the invention. The PKC
agonists may include a sequence of at least about 4 to about 30
amino acids or at least about 5 to about 15 amino acids. It is
realized that the PKC agonists may be composed of sequences longer
than about 30 amino acids. By "PKC agonist", it is meant herein a
compound that activates a PKC to form an activated PKC, facilitates
or allows PKC to perform its biological functions, or mimics the
activity of a PKC to allow the mimic to carry out one or more of
the biological functions of PKC. The agonists may, for example,
allow activated PKC to be translocated to specific areas of the
cell so that it may exert its biological effect. As known in the
art, the PKC family of enzymes are serine/threonine kinases and are
involved in a myriad of cellular process, including cell growth,
regulation of gene expression, and ion channel activity. By "PKC
antagonist" or "PKC inhibitor", it is meant herein a compound that
inhibits a PKC enzyme to form a deactivated PKC enzyme, prevents or
facilitates prevention of PKC from performing its biological
functions, or mimics the activity of a PKC antagonist to allow the
mimic to inhibit the biological functions of PKC. The antagonists
may, for example, prevent activated PKC from being translocated to
specific areas of the cell so that the PKC may be prevented from
exerting its biological effect. It is noted that the terms
"antagonist" and "inhibitor" are used interchangeably herein.
[0051] Such agonists include cPKC agonists, such as .alpha.PKC
agonists, .beta..sub.IPKC agonists, .beta..sub.IIPKC agonists, and
.gamma.PKC agonists; nPKC agonists, including .epsilon.PKC
agonists, .delta.PKC agonists, .eta.PKC agonists, and .theta.PKC
agonists; and .alpha.PKC agonists, including .zeta.PKC agonists,
.tau.PKC agonists and .lambda.PKC agonists. The respective PKC
agonists may be derived from the .psi.RACK sequences present in the
respective PKC enzymes (e.g., .psi..alpha.RACK,
.psi..beta..sub.IRACK, .psi..epsilon.RACK, .psi..lambda.RACK,
etc.). A peptide or peptide fragment is "derived from" a parent
peptide or polypeptide if it has an amino acid sequence that is
identical or otherwise has a specified percent identity to the
amino acid sequence of the parent peptide or polypeptide, including
at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90% and at least about 95% identity. Such a definition
includes peptides or peptidomimetics that have at least one amino
acid substitution therein when compared to the parent peptide,
polypeptide or peptidomimetic. Although not being limited by
theory, these .psi.RACK sequences have been found herein to be
involved in at least inhibitory intramolecular interactions with
the respective RACK binding site in a PKC enzyme as more fully
discussed in the Examples herein.
[0052] Percent identity may be determined, for example, by
comparing sequence information using the advanced BLAST computer
program, including version 2.2.9, available from the National
Institutes of Health. The BLAST program is based on the alignment
method of Karlin and Altschul. Proc. Natl. Acad. Sci. USA
87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol.
Biol. 215:403-410 (1990); Karlin And Altschul, Proc. Natl. Acad.
Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids
Res. 25:3389-3402 (1997). Briefly, the BLAST program defines
identity as the number of identical aligned symbols (i.e.,
nucleotides or amino acids), divided by the total number of symbols
in the shorter of the two sequences. The program may be used to
determine percent identity over the entire length of the proteins
being compared. Default parameters are provided to optimize
searches with short query sequences in, for example, blastp with
the program. The program also allows use of an SEG filter to
mask-off segments of the query sequences as determined by the SEG
program of Wootton and Federhen, Computers and Chemistry 17:149-163
(1993).
[0053] A wide variety of such .psi.RACK sequences are known in the
art in a variety of species including the following human
sequences, such as .psi..epsilon.RACK (HDAPIGYD, SEQ ID NO:3);
.psi..delta.RACK (MRAAEEPM, SEQ ID NO:4); .psi..theta.RACK
(KGKNVDLI, SEQ ID NO:5); .psi..eta.RACK (HETPLGYD, SEQ ID NO:6);
and .psi..beta.RACK (SVEIWD, SEQ ID NO:7). RACK sequences were
identified by searching for regions of homology between each PKC
isozyme and its RACK. In .epsilon.PKC, for example, the
.psi..epsilon.RACK sequence HDAPIGYD (SEQ ID NO:3; .epsilon.PKC
85-92) has approximately 75% homology with a sequence in
.epsilon.RACK consisting of amino acids NNVALGYD (SEQ ID NO:1;
.epsilon.RACK285-292). Peptides corresponding to the .PSI.RACK
sequences, including .epsilon.PKC, function as an
.epsilon.PKC-selective agonist.
[0054] The agonist may be a derivative of the sequences described
above, including fragments or modifications thereof. Modifications
include conservative amino acid substitutions in the amino acid
sequences to obtain derivatives of the peptides that may
advantageously be utilized as agonists in the present
invention.
[0055] Conservative amino acid substitutions are substitutions
which do not result in a significant change in the activity (e.g.,
.epsilon.PKC-agonist activity or .psi..epsilon.RACK-agonist
activity) or tertiary structure of a selected peptide or
polypeptide. Such substitutions typically involve replacing a
selected amino acid residue with a different residue having similar
structure, size or other physico-chemical properties. For example,
substitution of Glu for Asp is considered a conservative
substitution since both are similarly-sized negatively-charged
amino acids. Groupings of amino acids by physico-chemical
properties are known to those of skill in the art. For example, the
amino acids within each of the following groups may be interchanged
with other amino acids in the same group: amino acids having
aliphatic side chains, including glycine, alanine, valine, leucine
and isoleucine; amino acids having non-aromatic,
hydroxyl-containing side chains, such as serine and threonine;
amino acids having acidic side chains, such as aspartic acid and
glutamic acid; amino acids having amide side chains, including
glutamine and asparagine; basic amino acids, including lysine,
arginine and histidine; amino acids having aromatic ring side
chains, including phenyialanine, tyrosine and tryptophan; and amino
acids having sulfur-containing side chains, including cysteine and
methionine.
[0056] Accordingly, suitable .epsilon.PKC agonists derived from the
.PSI..epsilon.RACK sequence of .epsilon.PKC, include, for example,
HDAPIGYD (SEQ ID NO:3) and modifications and/or fragments thereof,
including the following sequences: SEQ ID NO:8 (HEADIGYD); SEQ ID
NO:9 (HDAPIGYE); SEQ ID NO:10 (HDAPVGYE); SEQ ID NO:11 (HDAPLGYE);
SEQ ID NO:12 (HDAPIGDY); SEQ ID NO:13 (HDAPIGEY); SEQ ID NO:14
(ADAPIGYD); SEQ ID NO:15 (HDGPIGYD); SEQ ID NO:16 (HDMIGYD); SEQ ID
NO:17 (AEAPVGEY); SEQ ID NO:18 (HEAPIGDN); SEQ ID NO:19 (HDGDIGYD);
SEQ ID NO:20 (HDAPIG) and SEQ ID NO:21 (HDAPIPYD).
[0057] Suitable .delta.PKC agonists derived from the
.PSI..delta.RACK sequence of .delta.PKC include, for example, SEQ
ID NO:4 (MRAAEEPV) and modifications and/or fragments thereof,
including the following sequences: SEQ ID NO:22 (MRVAEEPV); SEQ ID
NO:23 (MRWEEPV); SEQ ID NO:24 (MRAADEPV); SEQ ID NO:25 (MRAAEEP);
SEQ ID NO:26 (MRLLEEPV); SEQ ID NO:27 (MRLAEEPV); and SEQ ID NO:28
(MRAAEE).
[0058] Exemplary IPKC agonists derived from the .PSI..eta.RACK
sequence on .eta.PKC include, for example, HETPLGYD (SEQ ID NO:6)
and modifications and/or fragments thereof, including the following
sequences: SEQ ID NO:29 (HDTPLGYD); SEQ ID NO:30 (HDTPLG); SEQ ID
NO:31 (HDTPIGYD); SEQ ID NO:32 (HETPAGYD); SEQ ID NO:33 (HETPAGYE);
SEQ ID NO:34 (KETPAGYD); SEQ ID NO:35 (KETPVGYD) and SEQ ID NO:36
(KETPVG).
[0059] Exemplary .theta.PKC agonists derived from the
.PSI..theta.RACK sequence on .theta.PKC include, for example,
KGKNVDLI (SEQ ID NO:5) and modifications and/or fragments thereof,
including the following sequences: SEQ ID NO:37 (RGKNVELA); SEQ ID
NO:38 (KNVDLI); SEQ ID NO:39 (RGRNVDLI; SEQ ID NO:40 (KGRNADLI; SEQ
ID NO:41 (KGKNVELI); SEQ ID NO:42 (KGKNVELA); SEQ ID NO:43
(KGKQVDLI) and SEQ ID NO:44 (RGKNLDLI).
[0060] Exemplary .beta.PKC agonists derived from the
.psi..beta.RACK sequence on .beta.PKC include, for example, SVEIWD
(SEQ ID NO:7) and modifications and/or fragments thereof, including
the following sequences: SEQ ID NO:45 (SAEIWD); SEQ ID NO:46
(SVELWD); SEQ ID NO:47 (TVEIWD); SEQ ID NO:48 (SVEIWE) and SEQ ID
NO:49 (SVEIW).
[0061] It is appreciated that other suitable agonists include those
have at least about 50% identity, further at least about 60%
identity, at least about 70% identity, further at least about 80%
identity and further at least about 90% identity to the amino acid
sequences of the agonists described herein, including SEQ ID
NOs:3-49 and that function as agonists for the respective PKC
enzymes.
[0062] It will be also appreciated that the .psi.RACK sequences
described herein may also include one or more amino acid residues
beyond the residues shown herein. For example, with respect to the
.psi..epsilon.RACK sequence at positions 85-92 (corresponding to
the sequence HDAPIGYD; SEQ ID NO:3), SEQ ID NO:93
(TDVCNGRKIELAVFHDAPIGYDDFVA- NCTI) shows the sequence of residues
from amino acids 71-100 in .epsilon.PKC. Regarding the
.psi..epsilon.RACK sequence at positions 74-81 (corresponding to
the sequence MRAAEEPV; SEQ ID NO:4), SEQ ID NO:94
(IQIVLMRAAEEPVSEVTV) shows the sequence of residues from amino
acids 69-86 in 5PKC. Regarding the .psi..theta.RACK sequence at
positions 75-82 (corresponding to the sequence KGKNVDLI; SEQ ID
NO:5), SEQ ID NO:95 (MQIIVKGKNVDLISETTV) shows the sequence of
residues from amino acids 70-87 in .theta.PKC. Regarding the
.psi..eta.RACK sequence at positions 88-95 (corresponding to the
sequence HETPLGYD; SEQ ID NO:6), SEQ ID NO:96 (ELAVFHETPLGYDHFVAN)
shows the sequence of residues from amino acids 83-100 in .eta.PKC.
Regarding the .psi..beta.RACK sequence at positions 241-246
(corresponding to the sequence SVEIWD; SEQ ID NO:7), SEQ ID NO:97
(KDRRLSVEIWDWDWDL) shows the sequence of residues from amino acids
236-251 in PPKC. Sequences derived from SEQ ID NOs:93-96 and having
activity as an .epsilon.PKC, .delta.PKC, .theta.PKC and .eta.PKC
agonist, for example, can be modified according to the teachings
herein to convert the biological activity to that of an antagonist
by selecting replacement amino acids that effect a change of charge
in the peptide at the location of the substitution, such as by
decreasing or increasing the electrical charge at the location of
substitution. For example, a negatively or positively-charged amino
acid may be substituted for an uncharged amino acid. In certain
forms of the invention, the modified sequence more closely
resembles the charge distribution of the native RACK for the
respective enzyme.
[0063] The amino acid present in the PKC agonist peptide that is
sufficient to convert the PKC agonist into a PKC antagonist is, in
one form of the invention, an amino acid that makes the agonist
peptide more closely resemble a sequence within the PKC binding
site on the respective RACK protein. Accordingly, the substitution
will be at a particular location on the agonist peptide and with an
amino acid that increases the percent identity between the PKC
agonist and the reference sequence within the PKC binding site on
the respective RACK protein when compared to the percent identity
between the two sequences prior to the substitution. Such an amino
acid that replaces the substituted amino acid may include one that
would provide a non-conservative amino acid substitution in the
peptide.
[0064] The reference sequence on the RACK protein that the peptide
sequence is compared to is one that is derived from optimally
aligning the two sequences being compared using the BLAST program
previously described herein or other similar programs known in the
art, and may represent, for example, a sequence of about 4 to about
30 amino acids or about 5 to about 15 amino acids. It is realized
that reference sequences longer than 30 amino acids in the PKC
binding site of the respective RACKs may also be utilized when
comparing the peptide antagonist being constructed to the reference
sequence, including in situations wherein the length of the peptide
antagonist is longer than about 30 amino acids. Examples of such
sequences wherein reference sequences may be selected from for
various isotypes of PKC discussed herein has been previously
enumerated herein. The amino acid that is substituted can be any
amino acid in the peptide that, when substituted with another
appropriate amino acid as described herein, will result in a
peptide sequence having increased percent identity to a sequence
within the PKC binding site on the RACK protein of the PKC enzyme
and, consequently, will result in conversion of the PKC agonist
peptide into a PKC antagonist peptide. This appropriate amino acid
includes an amino acid that increases the binding affinity between
the peptide or peptidomimetic and the PKC binding site on the RACK
protein of the PKC enzyme.
[0065] In other forms of the invention, the amino acid that is
sufficient to convert the PKC agonist into a PKC antagonist is an
amino acid that allows the peptide or peptidomimetic to more
closely approximate the charge distribution in a reference peptide
chosen from the PKC binding site on its respective RACK protein or
where one substitutes a charged amino acid with an uncharged amino
acid. Such an amino acid that replaces the substituted amino acid
may be one that would provide a non-conservative amino acid
substitution in the peptide. As an example, if a reference peptide
of 8 amino acids has an uncharged amino acid at position 2, and the
agonist has a charged amino acid in the same position when
optimally aligned using the BLAST program, the amino acid in the
agonist may be substituted with an uncharged amino acid in order to
allow the agonist peptide to more closely approximate the charge at
a particular location in the reference peptide and convert the PKC
agonist into an antagonist. Therefore, one may substitute a
negatively charged (e.g., aspartic acid or glutamic acid) amino
acid in the PKC agonist with a polar, uncharged amino acid (e.g.,
asparagine or glutamine), thereby decreasing the electrical charge
at the location of substitution in the peptide. It will be
appreciated that in some cases, a polar, uncharged residue in a
given sequence may be replaced with a charged residue, such as a
positively or negatively-charged amino acid residue, thereby
increasing the electrical charge at the location of substitution in
the peptide to effect a change in peptide function.
[0066] At least one amino acid in the agonists is substituted. In
certain forms of the invention, 2, 3, or 4 amino acids or more may
be substituted. The number, or percentage, of amino acids that may
be substituted in the agonists may also depend on the number of
amino acids in the peptide or peptidomimetic. The longer the length
of the agonist, and/or the greater the difference in percent
identity or charge distribution between the agonist peptide and a
sequence within the PKC binding site in the respective RACK, would
allow for a greater number of substitutions. A suitable number of
substitutions in an agonist of about 8 amino acids includes no more
than about 4. As an example, at least about 20%, at least about
30%, further at least about 40%, further at least about 50%, and at
least about 60% of the amino acids in the agonist peptide or
peptidomimetic may be substituted.
[0067] Exemplary peptides that may have EPKC antagonistic activity
that are derived from .epsilon.PKC agonist peptides according to
the methods of the present invention include those set forth in SEQ
ID Nos: 50-57. These antagonists are based on alternative
.psi..epsilon.RACK sequences identified as SEQ ID NOs: 12
(HDAPIGDY), 20 (HDAPIG), 16 (HDMIGYD), and 21 (HDAPIPYD), which are
disclosed in U.S. Pat. No. 6,165,977 and may be modified to, for
example, look more similar to a sequence within the .epsilon.PKC
binding site on .epsilon.RACK, such as, for example, NNVALGYD (SEQ
ID NO:1) and/or to include a change of charge at selected amino
acid residues. These alternative .psi..epsilon.RACK sequences may
be modified according to the method disclosed herein, wherein one
or more amino acid residues can be replaced with an amino acid
residue to effect a change of charge in the peptide. For example,
SEQ ID NO:12 (HDAPIGDY) may be modified by substitution of N for D
to arrive at SEQ ID NO:50 (HNAPIGDY) and SEQ ID NO:51 (HDAPIGNY).
SEQ ID NO:20 (HDAPIG) may be modified by substitution of N for D to
arrive at SEQ ID NO:52 (HNAPIG). SEQ ID NO:21 (HDAPIPYD) may be
modified by substitution of N for D to arrive at SEQ ID NO:53
(HNAPIPYD) and SEQ ID NO:54 (HDAPIPYN). Additionally, SEQ ID NO:3
(HDAPIGYD) may be modified by substituting the first D for N to
arrive at SEQ ID NO:55 (HNAPIGYD). SEQ ID NO:16 (HDAAIGYD) may be
modified by substituting either the first or second D with an N, to
arrive at SEQ ID NO:56 (HNAAIGYD) and SEQ ID NO:57 (HDAAIGYN),
respectively.
[0068] Exemplary peptides that may have 8PKC antagonistic activity
that are derived from .delta.PKC agonist peptides according to the
methods of the present invention include those set forth in SEQ ID
Nos: 58-65. These antagonists may be based on alternative
.psi..delta.RACK sequences described above and may be modified to
form antagonists according to the present invention. For example,
SEQ ID NO:4 (MRVAEEPV) may be modified by changing R to D or E to
arrive at SEQ ID NO:58 (MDVAEEPV) and SEQ ID NO:59 (MEVAEEPV),
respectively. Alternatively, the second E in SEQ ID NO:4 (MRVAEEPV)
may be substituted with N or Q to arrive at SEQ ID NO:60 (MRVAENPV)
and SEQ ID NO:61 (MRVAEQPV). Similar modifications can be made, for
example, to SEQ ID NO:27 (MRLAEEPV) to arrive at SEQ ID NO:62
(MDLAEEPV) (changing R to D); SEQ ID NO:63 (MELAEEPV) (changing R
to E); SEQ ID NO:64 (MRLAENPV) (changing the second E with N); and
SEQ ID NO:65 (MRLAEQPV) (changing the second E to Q).
[0069] Exemplary peptides that may have .eta.PKC antagonistic
activity that are derived from .eta.PKC agonist peptides according
to the methods of the present invention include those set forth in
SEQ ID Nos:66-69. These antagonists may be based on the
.psi..eta.RACK sequence described above as well as the alternative
.psi..eta.RACK sequences and may be modified to form antagonists
according to the present invention. For example, SEQ ID NO:6
(HETPLGYD) and SEQ ID NO:34 (KETPAGYD) may be modified by
substituting E for Q or N to arrive at SEQ ID NO:66 (HQTPLGYD); SEQ
ID NO:67 (KNTPAGYD); SEQ ID NO:68 (KQTPAGYD); and SEQ ID NO:69
(KNTPAGYD).
[0070] Exemplary peptides that may have .theta.PKC antagonistic
activity that are derived from .theta.PKC agonist peptides
according to the methods of the present invention include those set
forth in SEQ ID Nos:70-78. These antagonists may be based on the
.psi..theta.RACK sequence described above as well as the
alternative .psi..theta.RACK sequences and may be modified to form
antagonists according to the present invention. For example, SEQ ID
NO:5 (KGKNVDLI) may be modified by substituting the second K with
either D or E to arrive at SEQ ID NO:70 (KGDNVDLI) and SEQ ID NO:71
(KGENVDLI); substituting N for E to arrive at SEQ ID NO:72
(KGKEVDLI); or substituting D for N to arrive at SEQ ID NO:73
(KGKNVNLI); Similarly, SEQ ID NO:37 (RGKNVELA) may be modified by
substituting K with either D or E to arrive at SEQ ID NO:74
(RGDNVELA) and SEQ ID NO:75 (RGENVELA), respectively. Additionally,
SEQ ID NO:43 (KGKQVDLI) may be modified by substituting D for N to
arrive at SEQ ID NO:76 (KGKQVNLI) or by substituting the second K
with D or E to arrive at SEQ ID NO:77 (KGDQVNLI) and SEQ ID NO:78
(KGEQVNLI), respectively.
[0071] Exemplary peptides that may have .beta.PKC antagonistic
activity that are derived from .beta.PKC agonist peptides according
to the methods of the present invention include those set forth in
SEQ ID Nos:79-85. These antagonists may be based on the
.psi..beta.RACK sequence described above as well as the alternative
.psi..beta.RACK sequences and may be modified to form antagonists
according to the present invention. For example, SEQ ID NO:7
(SVEIWD), SEQ ID NO:45 (SAEIWD); SEQ ID NO:46 (SVELWD); SEQ ID
NO:47 (TVEIWE); SEQ ID NO:48 (SVEIWE) and SEQ ID NO:49 (SVEIW) may
all be modified by substituting E with K, to arrive at SEQ ID NO:80
(SVKIWD); SEQ ID NO:81 (SAKIWD); SEQ ID NO:82 (SVKLWD); SEQ ID
NO:83 (TVKIWE); SEQ ID NO:84 (SVKIWE) and SEQ ID NO:85 (SVKIW).
[0072] It is understood that this is not an exhaustive list of the
antagonists that may be produced by the various agonists described
herein. Other similar PKC agonist sequences, or alternative
.psi.RACK sequences for the respective PKC isozymes described
herein and/or otherwise known to the art, may be modified using the
methods described herein to produce a PKC antagonist. It will also
be appreciated from the description herein that the methodology
extends beyond the specific substitutions described herein.
Substitution of negatively charged or positively charged amino acid
residues in a peptide with polar, uncharged amino acid residues is
contemplated to effect a change in the activity of the peptide or
peptidomimetic, as are substitutions of polar, uncharged amino
acids with positively or negatively charged amino acids.
Substitution of positively charged amino acids with negatively
charged amino acids, and substitution of negatively charged amino
acids with positively charged amino acids is also envisioned.
Positively charged amino acids include lysine, arginine and
histidine. Negatively-charged amino acids include aspartic acid and
glutamic acid. Polar, uncharged amino acids that can substitute for
selected amino acids include serine, threonine, cysteine, tyrosine,
asparagine, and glutamine. The amino acids are expected to have the
aforementioned charges at least at physiological pH (e.g., about pH
7). Accordingly, for example, lysine, arginine and histidine can be
substituted with aspartic acid or glutamic acid. Aspartic acid and
glutamic acid can be substituted with, for example, asparagine,
glutamine, lysine or arginine. Serine and threonine can be
substituted with, for example, glutamic acid, aspartic acid,
glutamine or asparagine.
[0073] The PKC peptide agonists or antagonists described herein may
be obtained by methods known to the skilled artisan. For example,
the protein agonist and/or antagonist may be chemically synthesized
using various solid phase synthetic technologies known to the art
and as described in, for example, Williams, Paul Lloyd, et al.
Chemical Approaches to the Synthesis of Peptides and Proteins, CRC
Press, Boca Raton, Fla., (1997). Additionally, methods of
converting a peptide into a peptidomimetic are also well known to
the skilled artisan.
[0074] Alternatively, the protein agonist or antagonist may be
produced by recombinant technology methods as known in the art and
as described, for example, in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Springs Harbor laboratory, 2.sup.nd ed.,
Cold Springs Harbor, N.Y. (1989); Martin, Robin, Protein Synthesis:
Methods and Protocols, Humana Press, Totowa, N.J. (1998); and
Current Protocols in Molecular Biology (Ausubel et al., eds.), John
Wiley & Sons, which is regularly and periodically updated. For
example, an expression vector may be used to produce the desired
peptide agonist or antagonist in an appropriate host cell and the
product may then be isolated by known methods. The expression
vector may include, for example, the nucleotide sequence encoding
the desired peptide wherein the nucleotide sequence is operably
linked to a promoter sequence.
[0075] As defined herein, a nucleotide sequence is "operably
linked" to another nucleotide sequence when it is placed in a
functional relationship with another nucleotide sequence. For
example, if a coding sequence is operably linked to a promoter
sequence, this generally means that the promoter may promote
transcription of the coding sequence. Operably linked means that
the DNA sequences being linked are typically contiguous and, where
necessary to join two protein coding regions, contiguous and in
reading frame. However, since enhancers may function when separated
from the promoter by several kilobases and intronic sequences may
be of variable length, some nucleotide sequences may be operably
linked but not contiguous. Additionally, as defined herein, a
nucleotide sequence is intended to refer to a natural or synthetic
linear and sequential array of nucleotides and/or nucleosides, and
derivatives thereof. The terms "encoding" and "coding" refer to the
process by which a nucleotide sequence, through the mechanisms of
transcription and translation, provides the information to a cell
from which a series of amino acids can be assembled into a specific
amino acid sequence to produce a polypeptide.
[0076] The agonists or antagonists may include natural amino acids,
such as the L-amino acids or non-natural amino acids, such as
D-amino acids. The amino acids in the peptide may be linked by
peptide bonds or, in modified peptides, including peptidomimetics
described herein, by non-peptide bonds.
[0077] A wide variety of modifications to the amide bonds which
link amino acids may be made and are known in the art. Such
modifications are discussed in general reviews, including in
Freidinger, R. M. "Design and Synthesis of Novel Bioactive Peptides
and Peptidomimetics" J. Med. Chem. 46:5553 (2003), and Ripka, A.
S., Rich, D. H. "Peptidomimetic Design" Curr. Opin. Chem. Biol.
2:441 (1998). These modifications are designed to improve the
properties of the peptide in one of two ways: (a) increase the
potency of the peptide by restricting conformational flexibility;
(b) increase the half-life of the peptide by introducing
non-degradable moieties to the peptide chain.
[0078] Examples of strategy (a) include the placement of additional
alkyl groups on the nitrogen or alpha-carbon of the amide bond,
such as the peptoid strategy of Zuckerman et al, and the alpha
modifications of, for example Goodman, M. et. al. [Pure Appl. Chem.
68:1303 (1996)]. The amide nitrogen and alpha carbon may be linked
together to provide additional constraint [Scott et al, Org. Letts.
6:1629-1632 (2004)].
[0079] Examples of strategy (b) include replacement of the amide
bond by, for instance, a urea residue [Patil et al, J. Org. Chem.
68:7274-7280 (2003)] or an aza-peptide link [Zega and Urleb, Acta
Chim. Slov. 49:649-662 (2002)]. Other examples such as introducing
an additional carbon ["beta peptides", Gellman, S. H. Acc. Chem.
Res. 31:173 (1998)] or ethene unit [Hagihara et al, J. Am. Chem.
Soc. 114:6568 (1992)] to the chain, or the use of hydroxyethylene
moieties [Patani, G. A., Lavoie, E. J. Chem. Rev. 96:3147-3176
(1996)] are also well known. One or more amino acids may be
replaced by an isosteric moiety such as, for example, the
pyrrolinones of Hirschmann et al [J. Am. Chem. Soc. 122:11037
(2000)], or tetrahydropyrans [Kulesza, A. et al., Org. Letts.
5:1163 (2003)].
[0080] In yet another aspect of the invention, methods of
modulating the activity of a PKC enzyme, such as .epsilon.PKC, by
administering in vitro or in vivo a PKC antagonist are provided.
The PKC antagonist can be a peptide, i.e., an amino acid sequence,
or a peptidomimetic organic molecule selected to simulate the
action of the peptide. As an example, methods of inhibiting the
activity of a protein kinase C (PKC) enzyme are provided. In one
form, a method includes contacting a PKC enzyme with a PKC
inhibitor peptide or peptidomimetic, wherein the peptide or
peptidomimetic is derived from a PKC agonist peptide or
peptidomimetic and wherein at least one amino acid in the agonist
peptide or peptidomimetic is substituted with another amino acid
sufficient to convert the agonist peptide or peptidomimetic into an
antagonist peptide or peptidomimetic. The agonist peptides suitable
for use are as previously described herein. The amino acid
substitutions amenable to convert the agonist peptide or
peptidomimetic into an antagonist peptide or peptidomimetic are
also previously described herein.
[0081] In yet another aspect of the invention, methods of treating
conditions modulated by a PKC enzyme are provided. In one form, a
method includes administering to a patient in need thereof a
therapeutically effective amount of a peptide, or peptidomimetic,
that acts as an antagonistic modulator of a corresponding PKC
isozyme. One PKC antagonist, or a combination of PKC antagonists,
may be administered. Such antagonists are those previously
described herein.
[0082] A wide variety of diseases or conditions may be treated that
are modulated by PKC. The diseases or conditions that are treated
herein are typically those which are associated with increased
activity of the respective PKC isozyme and that would benefit from
administration of a PKC antagonist. As one example, various
inflammatory diseases and fibrotic diseases have been associated
with an increased activity of .epsilon.PKC [Aksoy, E., et al., Int.
J. Biochem. Cell. Biol. 36:183-188 (2004); Fang, Q. et al., Eur
Respir. J. 24:918-924 (2004)].
[0083] Accordingly, conditions amenable for treatment with an
.epsilon.PKC antagonist include, for example, fibrotic diseases,
including pulmonary fibrosis, and scleroderma; and inflammatory
diseases, including, for example, inflammatory bowel disease,
septic shock, allergic rhinitis; lung diseases, including chronic
obstructive pulmonary disease, and asthma; autoimmune diseases,
including multiple sclerosis, Guillain-Barre syndrome, psoriasis,
Grave's disease, rheumatoid arthritis and immune-mediated diabetes,
including Type 1 diabetes mellitus.
[0084] The inhibitors described herein may be modified by being
part of a fusion protein. The fusion protein may include a protein
or peptide that functions to increase the cellular uptake of the
peptide inhibitors, has another desired biological effect, such as
a therapeutic effect, or may have both of these functions. For
example, it may be desirable to conjugate, or otherwise attach, an
.epsilon.PKC or other peptide or peptidomimetic antagonist to a
cytokine or other protein that elicits a desired biological
response. The fusion protein may be produced by methods known to
the skilled artisan. The inhibitor peptide may be bound, or
otherwise conjugated, to another peptide in a variety of ways known
to the art. For example, the inhibitor peptide or peptidomimetic
may be bound to a carrier peptide or other peptide described herein
via cross-linking wherein both peptides of the fusion protein
retain their activity. As a further example, the peptides may be
linked or otherwise conjugated to each other by an amide bond from
the C-terminal of one peptide to the N-terminal of the other
peptide. The linkage between the inhibitor peptide and the other
member of the fusion protein may be non-cleavable, with a peptide
bond, or cleavable with, for example, an ester or other cleavable
bond known to the art.
[0085] Furthermore, in other forms of the invention, the carrier
protein or peptide that may increase cellular uptake of the peptide
antagonist may be, for example, a Drosophila Antennapedia
homeodomain-derived sequence which is set forth in SEQ ID NO:91
(CRQIKIWFQNRRMKWKK), and may be attached to the inhibitor by
cross-linking via an N-terminal Cys-Cys bond as discussed in
Theodore, L., et al. J. Neurosci. 15:7158-7167 (1995); Johnson, J.
A., et al. Circ. Res 79:1086 (1996). Alternatively, the inhibitor
may be modified by a Transactivating Regulatory Protein
(Tat)-derived transport polypeptide (such as from amino acids 47-57
of Tat shown in SEQ ID NO:92; YGRKKRRQRRR) from the Human
Immunodeficiency Virus, Type 1, as described in Vives, et al., J.
Biol. Chem, 272:16010-16017 (1997), U.S. Pat. No. 5,804,604 and
Genbank Accession No. AAT48070; or with polyarginine as described
in Mitchell, et al. J. Peptide Res. 56:318-325 (2000) and Rolhbard,
et al., Nature Med. 6:1253-1257 (2000). The inhibitors may be
modified by other methods known to the skilled artisan in order to
increase the cellular uptake of the inhibitors.
[0086] The inhibitors may be advantageously administered in various
forms. For example, the inhibitors may be administered in tablet
form for sublingual administration, in a solution or emulsion. The
inhibitors may also be mixed with a pharmaceutically-acceptable
carrier or vehicle. The carrier may be a liquid, suitable, for
example, for parenteral administration, including water, saline or
other aqueous solution, or may be an oil. The carrier may be
selected for intravenous or intraarterial administration, and may
include a sterile aqueous or non-aqueous solution that may include
preservatives, bacteriostats, buffers and antioxidants known to the
art. In tablet form, a solid carrier may include, for example,
lactose, starch, carboxymethyl cellulose, dextrin, calcium
phosphate, calcium carbonate, synthetic or natural calcium
allocate, magnesium oxide, dry aluminum hydroxide, magnesium
stearate, sodium bicarbonate, dry yeast or a combination thereof.
The tablet preferably includes one or more agents which aid in oral
dissolution. The inhibitors may also be administered in forms in
which other similar drugs known in the art are administered.
[0087] The inhibitors may be administered to a patient by a variety
of routes. For example, the inhibitors may be administered
parenterally, including intraperitoneally, intravenously,
intraarterially, subcutaneously, or intramuscularly. The inhibitors
may also be administered via a mucosal surface, including rectally,
and intravaginally; intranasally, including by inhalation;
sublingually; intraocularly and transdermally. Combinations of
these routes of administration are also envisioned.
[0088] A therapeutically effective amount of the .epsilon.PKC or
other inhibitor peptide is provided. As used herein, a
therapeutically effective amount of the inhibitor is the quantity
of the inhibitor required to decrease or eliminate the symptoms
associated with a particular disease or condition. Such clinical
endpoints are well known in the art for a wide variety of diseases
or conditions. This amount includes the amount which is effective
in modulating, such as decreasing, the activity of the respective
PKC enzyme. For example, the therapeutically effective amount
includes an amount effective in affecting the intracellular
activity of the PKC enzyme, including kinase activity, PKC enzyme
translocation activity and/or otherwise preventing PKC from
performing its biological function. This amount will vary depending
on the route of administration, the duration of treatment, the
specific inhibitor used, the particular disease or condition and
the health of the patient as known in the art. The skilled artisan
will be able to determine the optimum dosage. Generally, the amount
of inhibitor typically utilized may be, for example, about 0.0005
mg/kg body weight to about 20 mg/kg body weight, but is preferably
about 0.001 mg/kg to about 5 mg/kg.
[0089] The patient to be treated is typically one in need of such
treatment, including one that is affected by a particular disease
or condition amenable to treatment with a PKC antagonist. The
patient is furthermore typically a vertebrate, preferably a mammal,
and including a human. Other animals which may be treated include
farm animals, such as horse, sheep, cattle, and pigs. Other
exemplary animals that may be treated include cats, dogs; rodents,
including those from the order Rodentia, such as mice, rats,
gerbils, hamsters, and guinea pigs; members of the order
Lagomorpha, including rabbits and hares, and any other mammal that
may benefit from such treatment. The patient is preferably treated
in vivo.
[0090] Thus, the PKC peptide antagonists, including the
.epsilon.PKC antagonists, described herein find use as therapeutic
agents for treatment of conditions involving translocation or other
activity of .epsilon.PKC. Administration of the PCK antagonist
will, for example, inhibit translocation and the subsequent cascade
of signaling events. The antagonist also finds use in studies on
the effect of PKC isozymes in ischemia and other conditions, and
can also be used as a screening aid in designing therapeutic agents
with antagonistic activity or that act as mimics of the
peptide.
[0091] Reference will now be made to specific examples illustrating
the invention described above. It is to be understood that the
examples are provided to illustrate preferred embodiments and that
no limitation to the scope of the invention is intended thereby.
Additionally, all documents cited herein are indicative of the
level of skill in the art and are hereby incorporated by reference
in their entirety.
EXAMPLE 1
Effect of N-.psi..epsilon.RACK on Cardiac Myocytes
[0092] A. Peptide Preparation and Delivery to Cells
[0093] The following peptides were synthesized and purified
(>95%) by conventional techniques:
1 .psi..epsilon.RACK (SEQ ID NO:3, HDAPIGYD, .epsilon.PKC amino
acid residues 85-92); N-.psi..epsilon.RACK (SEQ ID NO:55,
HNAPIGYD); A-.psi..epsilon.RACK (SEQ ID NO:79, HAAPIGYD);
.epsilon.V1-2 (SEQ ID NO:86, EAVSLKPT; .epsilon.PKC amino acid
residues 14-21 and described in U.S. Pat. No. 6,165,977);
.psi..beta.RACK (SEQ ID NO:7, SVEIWD, .beta.PKC amino acids
241-246).
[0094] The peptides were either unmodified or were cross-linked via
an N-terminal Cys-Cys bond to the Drosophila Antennapedia
homeodomain-derived carrier peptide (SEQ ID NO:6,
C-RQIKIWFQNRRMKWKK) (Derossi, D., et al., J. Biol. Chem.
269:10444-10450, (1994); Derossi, D., et al., J. Biol. Chem.
271:18188-18193, (1996); Theodore, L., et al, J. Neurosci.
15:7158-7167, (1995)). This carrier peptide (SEQ ID NO:6) was used
as a control peptide.
[0095] Primary cardiac myocyte cell cultures (90-95% pure) were
prepared from newborn rats as previously described (Gray, M. O., et
al., J. Biol. Chem. 272:30945-30951, (1997); Disatnik, M.-H., et
al., Exp. Cell Res. 210:287-297, (1994)). Peptides (100 nM-1 .mu.M
applied concentration) were introduced into cells by transient
permeabilization (Johnson, J. A., et al., Circ. Res. 79:1086-1099,
(1996)) with sham permeabilization as control, or as
carrier-peptide conjugates (30 nM-1 .mu.M) (Derossi, D. et al.,
surpa (1994); Derossi, D. et al., surpa (1996); Theodore, L., et
al., surpa (1995)) with a carrier-carrier dimer as a control.
[0096] Cells were treated for 10-20 minutes in the absence or
presence of peptide followed by an additional incubation with or
without 1 nM phorbol 12-myristate 13-acetate (PMA) for 10 or 20
minutes. Alternatively, cells were incubated for 10 minutes with
100 nM PMA (positive control) or in the absence of PMA.
[0097] B. Cardiac Myocyte Contraction Rate Measurement
[0098] Measurements of cardiac myocyte contraction rate were
carried out essentially as previously described (Johnson, J. A., et
al., J. Biol. Chem. 271:24962-24966, (1996). In brief,
cardiomyocytes cultured on 35 mm plates were placed in a
temperature-regulation apparatus at 37.degree. C. (Medical Systems
Corp.) and positioned on the stage of an inverted microscope (Carl
Zeiss Inc.). The contraction rates of four cells in one microscopic
field were determined every 2 minutes for 15 seconds each. The
basal contraction rate (.about.300 beats/minute) was stable over
many hours.
[0099] Results
[0100] As described above, the peptides represented by SEQ ID
NOs:1-5 were synthesized and purified. Specifically,
.psi..epsilon.RACK (SEQ ID NO:3, HDAPIGYD), N-.psi..epsilon.RACK
(SEQ ID NO:55, HNAPIGYD), A-.psi..epsilon.RACK (SEQ ID NO:79,
HAAPIGYD); .epsilon.V1-2 (SEQ ID NO:86, EAVSLKPT); and
.psi..epsilon.RACK (SEQ ID NO:7, SVEIWD) were prepared. The
peptides were either unmodified or were cross-linked via an
N-terminal Cys-Cys bond to the Drosophila Antennapedia
homeodomain-derived carrier peptide (SEQ ID NO:81,
C-RQIKIWFQNRRMKWKK).
[0101] Peptide SEQ ID Nos:3, 55 and 79 were introduced into cardiac
myocyte cell cultures prepared from newborn rats by transient
permeabilization, with sham permeabilization as control, or as
carrier-peptide conjugates with a carrier-carrier dimer as control.
The cells were treated for 10-20 minutes in the absence or presence
of peptide followed by an additional incubation with or without
phorbol 12-myristate 13-acetate (PMA) for 10 or 20 minutes.
Alternatively, cells were incubated for 10 minutes with 100 nM PMA
(positive control) or in the absence of PMA. The effect of the
peptide and/or PMA was determined by measuring the contraction rate
of the myocytes, as described in Example 1B.
[0102] FIG. 1A shows the results for cells treated with
.psi..epsilon.RACK (triangles) and for cells treated with a control
peptide (squares), followed by incubation with PMA which was added
to the culture about 30 minutes after initiation of the experiment
(addition of PMA is indicated by the arrows on graph in FIG. 1A).
About 10 minutes after .psi..epsilon.RACK peptide application to
the cells, a reduction in the contraction rate of cardiomyocytes
was induced, as compared to the control cells. The reduction
observed in the presence of .psi..epsilon.RACK was greatly enhanced
by the addition of PMA, which is known to induce reduction in the
contraction rate of cardiac myocytes (Johnson, J. A. et al., J.
Biol. Chem., 271:24962 (1996)).
[0103] FIG. 1B shows the percent decrease in contraction rate of
neonatal rat cardiac myocytes, where the percent decrease is
relative to the basal contraction rate of the cells which
spontaneously and stably beat at about 300 beats per minute. Cells
treated with .psi..epsilon.RACK peptide had a 9.+-.2% decrease in
contraction rate from the basal rate for .psi..epsilon.RACK, 20
minutes after peptide addition, as compared to 2.+-.1% for cells
treated with a control peptide. Cells treated with
.psi..epsilon.RACK together with PMA experienced a decrease of
44.+-.6% from the basal contraction rate, whereas treatment with
PMA alone caused a decrease of only 11.+-.4%.
[0104] With continuing reference to FIG. 1B, cells were also
treated with an .epsilon.PKC-specific inhibitor peptide,
.epsilon.V1-2, to determine whether the .psi..epsilon.RACK effect
on contraction rate was due to its ability to induce .epsilon.PKC
translocation, since, if this is the case, then an
.epsilon.PKC-selective translocation inhibitor should inhibit
translocation. Indeed, the .psi..epsilon.RACK effect on contraction
of cardiac myocytes was abolished by prior application of the
.epsilon.PKC-specific inhibitor peptide .epsilon.V1-2, as seen in
FIG. 1B. In addition, if the .psi..epsilon.RACK effect is due to an
increase in the catalytic activity of .epsilon.PKC, this effect
should be abolished by an inhibitor of the catalytic activity. As
seen, the non-selective PKC inhibitor chelerythrine (Che) inhibited
.psi..epsilon.RACK-induced negative chronotropy. These data
demonstrate that .epsilon.PKC activation is required and sufficient
to induce negative chronotropy in neonatal cardiac myocytes.
[0105] The results in FIG. 1A-1B establish that .psi..epsilon.RACK
stimulates suppression of the contraction rate in cardiac myocytes.
The methodology provides a means to determine the effect of other
peptides and peptidomimetics on translocation of .epsilon.PKC.
Thus, a peptide having the sequence HNAPIGYD (SEQ ID NO:55) was
designed, which is referred to herein as N-.psi..epsilon.RACK as
the sequence is identical to .psi..epsilon.RACK (HDAPIGYD, SEQ ID
NO:3) except for substitution of aspartate (D), a negatively
charged amino acid, to asparagine (N), a polar, uncharged amino
acid. The effect of N-.psi..epsilon.RACK on the contraction rate of
cardiac myocytes was studied and the results are shown in FIGS.
2A-2C.
[0106] FIG. 2A shows the contraction rate of neonatal rat cardiac
myocytes as a function of time after treatment with a control
peptide (squares) or with N-.psi..epsilon.RACK (diamonds). The
contraction rate is expressed as the percent decrease in
contraction rate relative to the basal contraction rate, discussed
above and in Example 1. In contrast to .psi..epsilon.RACK,
N-.psi..epsilon.RACK had no agonistic effect on contraction rate of
cardiac myocytes as seen by the fact that the contraction rate of
the cells treated with N-.psi..epsilon.RACK was approximately the
same as the cells treated with a control peptide, during the first
20 minutes of the data presented in FIGS. 2A-2B, prior to PMA
treatment. The cells were treated with PMA about 20 minutes after
peptide treatment, and as seen in FIGS. 2A-2B at times greater than
about 20 minutes, N-.psi..epsilon.RACK acted as an antagonist of
.epsilon.PKC. That is, N-.psi..epsilon.RACK was able to reduce the
decrease in contraction rate induced by PMA. As seen in FIG. 2B,
.psi..epsilon.RACK reduced the decrease in contraction rate induced
by 5 nM PMA. In a study not shown here, N-.psi..epsilon.RACK
reduced the decrease in contraction rate induced by 10 nM PMA from
47.+-.3% to 24.+-.5% 40 minutes after PMA addition (n=4;
p<0.05).
[0107] FIG. 2C shows the percent decrease in contraction rate for
cardiac myocytes treated with N-.psi..epsilon.RACK (diamonds).
About 15 minutes after treatment with N-.psi..epsilon.RACK, the
cells were additionally treated with .psi..epsilon.RACK, as
indicated by the arrows at the 15 minute time point. Control cells,
treated with .psi..epsilon.RACK at the 15 minutes time point
(triangles), showed an immediate decrease in contraction rate;
however the cells treated with both N-.psi..epsilon.RACK and
.psi..epsilon.RACK had little change in contraction rate. Treatment
of the cells with PMA at the 30 minute time point led to a further
decrease in contraction rate in the control cells not treated with
N-.psi..epsilon.RACK. However, cells treated with
N-.psi..epsilon.RACK had less than a 10% decrease in contraction
rate, indicating the N-.psi..epsilon.RACK was able to almost
completely abolish the combined effect of .psi..epsilon.RACK and
PMA in decreasing the contraction rate. Thus, N-.psi..epsilon.RACK
acts as an antagonist of .epsilon.PKC function in cardiac
myocytes.
[0108] Although not being limited by theory, it is contemplated
that both .psi..epsilon.RACK and N-.psi..epsilon.RACK bind to the
RACK-binding site in .epsilon.PKC. The .psi..epsilon.RACK acts by
reversibly interfering with the intramolecular interaction between
the RACK binding site and the .psi..epsilon.RACK sequence, whereas
N-.psi..epsilon.RACK acts by interfering with the intermolecular
interaction between the RACK binding site and .epsilon.RACK. It is
also possible that the peptides affect access of the enzyme to its
substrate or post-translational modifications of .epsilon.PKC such
as phosphorylation; phosphorylation modulates activation and
translocation of the conventional PKC isozymes. In the examples
described herein, a charged amino acid was substituted with an
uncharged amino acid to arrive at a peptide that more closely
resembled an endogenous anchoring protein. The sequence of a
cognate receptor for any sequence provides guidance in ascertaining
the substitution.
EXAMPLE 2
Effect of N-.psi..epsilon.RACK on Translocation of of
.epsilon.PKC
[0109] Translocation of specific PKC isozymes in rat neonatal and
adult cardiac myocytes was assessed after peptide treatment by
using PKC isozyme-specific antibodies in western blot analysis
(Santa Cruz Biotechnology, Inc.) of cytosolic and particulate
fractions of treated cells, as previously described (Johnson, J. A.
and Mochly-Rosen, D., Circ. Res. 76:654-663, (1995)). In brief,
after peptide treatment the cells were homogenized, fractionated,
and analyzed by Western blot. Blots were first probed with
anti-.epsilon.PKC (FIG. 3A), followed by stripping and re-probing
for anti-.alpha.PKC (FIG. 3B). Percent translocation (amount of PKC
in the particulate fraction over total amount of PKC in the cell)
is shown in the histograms of FIGS. 3A-3B.
[0110] Results
[0111] Western blot analysis was used to evaluate the translocation
of .epsilon.PKC from cytostolic to particulate subcellular
fractions, a hallmark of PKC activation. N-.psi..epsilon.RACK or
.psi..epsilon.RACK peptides were introduced into the cells by
transient permeabilization in the presence of a dose of PMA (1 nM).
As described in Example 2, the cells were then homogenized,
fractionated, and analyzed by Western blot. Blots were first probed
with anti-.epsilon.PKC, and is shown in FIG. 3A. The blots were
then stripped and reprobed with anti-.epsilon.PKC, as shown in FIG.
3B. The percent translocation is shown in the histogram in FIGS.
3A-3B, taken as amount of PKC in the particulate fraction over
total amount of PKC in the cell. The Western blot shows that
N-.psi..epsilon.RACK inhibited PMA-induced translocation and is,
therefore, an antagonist of PMA and .psi..epsilon.RACK-induced
.epsilon.PKC translocation, and of .epsilon.PKC function (FIG.
2).
EXAMPLE 3
Effect of N-.psi..epsilon.RACK on .psi..delta.RACK-Inducted
Protection from Ischemia
[0112] Cardiac myocytes were isolated from adult male rats as
previously described (Chen et al., Proc. Natl. Acad. Sci.,
96(22):12784 (1999)). Myocytes in incubation buffer were treated
with a peptide or combination of peptides, prepared as described in
Example 1, for 15 minutes. Myocytes were then pelleted at 1000 rpm
for 1 minute in a degassed incubation buffer saturated with
nitrogen and incubated at 37.degree. C. for 3 hours to simulate
ischemia. Cell damage was assessed after the 3 hours by trypan blue
exclusion and percent cell with damage was determined as previously
described (Chen et al., Proc. Natl. Acad. Sci., 96(22):12784
(1999)).
[0113] Results
[0114] .psi..epsilon.RACK (SEQ ID NO:1) when administered to cells
confers protection against ischemia (Dorn et al., Proc. Natl. Acad.
Sci., 96(22):12798 (1999)). In this example, the effect of
N-.psi..epsilon.RACK on cells exposed to an ischemic episode was
evaluated. Additionally, a peptide having the sequence HAAPIGYD
(SEQ ID NO:3), referred to as A-.psi..epsilon.RACK, was prepared to
determine the effect of changing the aspartate (D) residue in
.psi..epsilon.RACK (HDAPIGYD; SEQ ID NO:3), since, if this residue
is important for the action of the peptide as an agonist, its
substitution to alanine (A), an uncharged, nonpolar amino acid,
should render the peptide inactive. The effect of this peptide on
cardiac protection was also evaluated. As described in this
example, cardiac myocytes were treated with .psi..epsilon.RACK,
N-.psi..epsilon.RACK, A-.psi..epsilon.RACK, .epsilon.V1-2 peptides
and various combinations of these peptides before simulated
ischemia. After a simulated ischemic episode, the cell damage was
evaluated by trypan blue dye exclusion. The results are shown in
FIG. 4.
[0115] FIG. 4 is a bar graph showing the percentage of cell damage
to rat cardiac myocytes resulting from an ischemic episode, where
myocytes not subjected to ischemia are represented by the clear
area in each bar, and the percent of ischemic damage represented by
the filled area. As a control for PKC activity, myocytes were
treated with a combination of chelerythrine (Che) and
.psi..epsilon.RACK peptide. Treatment with .psi.RACK peptide
reduced ischemia-induced cell damage by 60%. However,
N-.psi..epsilon.RACK and A-.psi..epsilon.RACK had no effect on
response to ischemia, indicating that a change of aspartate to
asparagine or alanine results in loss of agonist activity of
.psi..epsilon.RACK. Moreover, a combination of N-.psi..epsilon.RACK
and .psi..epsilon.RACK abolished protection from ischemic damage.
Inhibition of .epsilon.PKC with its selective inhibitor
.epsilon.V1-2 or with an inhibitor of PKC catalytic activity, Che,
reversed .psi..epsilon.RACK-induced protective effect.
N-.psi..epsilon.RACK abolished .psi..epsilon.RACK-induced
protection, whereas the inactive peptide, A-.psi..epsilon.RACK did
not, indicating that N-.psi..epsilon.RACK is an .epsilon.PKC
inhibitor.
[0116] These findings show that a single amino acid substitution,
increasing the resemblance of .psi..epsilon.RACK to the
.epsilon.RACK sequence resulted in a loss of the agonist activity
and a gain of antagonist activity. The .psi..epsilon.RACK peptide
agonist interferes with the intramolecular interaction in PKC,
without altering the intermolecular interaction of PKC with its
RACK or with lipids. In addition, this peptide exposes at least
part of the RACK-binding site on .epsilon.PKC, and is then
displaced by .epsilon.RACK to obtain full activation. In contrast,
the N-.psi..epsilon.RACK peptide interferes not only with the
intramolecular interaction between the RACK-binding site and
.psi..epsilon.RACK, but also with the intermolecular interaction
between .epsilon.PKC and .epsilon.RACK. Since the interaction
between .epsilon.PKC and .epsilon.RACK is inhibited, translocation
and .epsilon.PKC function is consequently also inhibited.
EXAMPLE 4
Sensitivity to Protease Degradation of the Different
.PSI..epsilon.RACK .epsilon.PKC Mutants
[0117] If the D86 in the .PSI..epsilon.RACK site is engaged in an
intramolecular interaction with the RACK-binding site, the D(86)N
mutant may reside more in the closed state, and therefore be more
resistant to proteolysis. In contrast, the D(86)A mutant should
favor an open conformation and therefore be more susceptible to
proteolysis. To test this hypothesis, .epsilon.PKC Wt and mutants
expressed in insect cells were subjected to proteolysis by the
endopeptidase, Arg C, as previously shown for .epsilon.PKC [Orr, J.
W. et al., J. Biol. Chem. 267:15263-15266 (1992)]. Degradation by
Arg C was monitored by the decrease of full-length
.epsilon.PKC.
[0118] Materials
[0119] Restriction enzymes were from New England Biolabs.
Anti-.epsilon.PKC V5 antibodies were from Santa Cruz
Biotechnology.
[0120] Cell Cultures
[0121] CHO-Hir cells (kindly provided by BioImage A/S, Soeborg,
Denmark) were kept in culture in F-12(HAM) nutrient mixture
supplemented with glutamax (Gibco-Brl), 10% fetal bovine serum (US
qualified, Gibco-Brl) and antibiotics (100 units/ml penicillin
and100 .mu.g/ml Streptomycin sulfate, Gibco-Brl). MCF7 cells (a
gift of James Ford, Stanford University) were kept in RPMI1640
media (Gibco-Brl) supplemented with 5% FBS and antibiotics. Cells
were cultured at 37.degree. C. 5% CO2. Insect cells were grown at
26.degree. C., Sf9 cells were cultured in SF900II SFM media
(Gibco-Brl) supplemented with 10% fetal bovine serum (Gibco-Brl),
and antibiotics. Hi5 insect cells were cultured in Insect Xpress
(Bio Whittaker) containing antibiotics.
[0122] Constructs
[0123] The .epsilon.GFP-.epsilon.PKC construct used in these
studies was kindly provided by Biolmage A/S, Soeborg, Denmark. This
construct contained two amino acid substitutions [Q(34)A and
S(336)G], when compared to the human .epsilon.PKC sequence
deposited in the gene bank, accession number X65293. Site-directed
mutagenesis of this clone was performed using the Quick Change site
directed mutagenesis kit (Stratagene) according to the manufactures
instructions. Primers used for the site directed mutagenesis were:
for D(86)N forward primer (5'-GTAGCCTATGGGGGCATTGTGAAAGACAGCCAG-3')
(SEQ ID NO:87) and reverse primer (5'-CTGGCTGTCTTTCACMTGCCCCCATAGGC
TAC-3') (SEQ ID NO:88). For D(86)A forward primer
(5'-CTGGCTGTCTTTCACGCTGCCCCCATAGGCTAC) (SEQ ID NO:89) and reverse
primer. (5'-GTAGCCTATGGGGGCAGCGTGAAA GACAGCCAG-3') (SEQ ID NO:90).
The .epsilon.PKC mutants were fully sequenced and subcloned by
cutting and re-ligating into the XhoI and BamHI (New England
Biolabs) sites into pEYFPC1 and pECFP C1 (Clontech). For expression
in the baculovirus expression system GFP-.epsilon.PKC was cut with
XhoI and BamHI to release the insert, filled in with Klenow (New
England Biolabs) and cloned into pvL1392 (BD Biosciences) digested
with SmaI (New England Biolabs). Human .epsilon.RACK construct
cloned into PZeoSV used in these studies was kindly provided by
Biolmage A/S, Soeborg, Denmark. The .epsilon.RACK insert was
released by cutting with ScaI (New England Biolabs) and XhoI, blunt
ended with Klenow, and religated into the SmaI site of PacG3.times.
(BD Biosciences). .epsilon.RACK was expressed as a GST fusion
protein.
[0124] Transient Transfection:
[0125] Transfections of CHO-Hir, MCF-7 and Hi5 cells was carried
out using FUGENE 6 (Roche) according to the manufacturers
instruction.
[0126] Insect Cell Protein Expression
[0127] Baculovirus was produced in Sf9 cells and all proteins were
expressed in Hi5 cells. Expression of all constructs was optimal at
72 h post-transfection. Infected Hi5 cells were lysed in
homogenization buffer [20 mM Tris-HCl pH 7.5, 2 mM EDTA, 10 mM
EGTA, 0.25M sucrose, 12 mM PME, and protease inhibitors: leupeptin
(25 .mu.g/ml), aprotinin (25 .mu.g/ml), PMSF (17 .mu.g/ml), SBTI
(20 .mu.g/ml), E64 (25 .mu.g/ml) (Sigma)]. The soluble fraction was
isolated after a 30-minute spin at 49000 g and the supernatant was
stored in 50% glycerol at -20.degree. C. until further use.
[0128] Arg C Proteolysis Assay
[0129] ArgC digests were performed as described [Orr, J. W. et al.,
J. Biol. Chem. 267:15263-15266 (1992)]. Crude insect cell lysate
containing approximately 70 ng of PKC [determined by Western Blot
comparison with standardized (PKC (PanVera)] was digested in 520
.mu.l of 20 mM Tris pH 7.4 with 20 .mu.l of endoproteinase ArgC
(Boehringer Mannheim 25 units/ml) at room temperature. Aliquots
were removed at the indicated time points run on 8% SDS-PAGE gels,
followed by Western blot using anti-.epsilon.PKC antibodies.
[0130] Statistical Analysis
[0131] For quantitative analysis autoradiographs were scanned and
quantified using NIH image software. Analysis of fluorescence data
was performed using MetaMorph.RTM. (Universal Imaging Corporation).
To determine statistical significance al tail type 2 T-test
(Microsoft excel) was used. Significance of time course curves was
determined using two way ANOVA test (Stat View.RTM.).
[0132] Results
[0133] Although under these experimental conditions D(86)A mutation
did not alter susceptibility of the enzyme to degradation by Arg C
when compared to the wild type (Wt) enzyme, the D(86)N mutant was
significantly more resistant to proteolysis than either D(86)A or
Wt (FIGS. 5A, B).
EXAMPLE 5
Binding of the .epsilon.PKC Mutants to .epsilon.RACK
[0134] RACKs bind active PKC [Mochly-Rosen, D. & Gordon, A. S.,
FASEB J. 12:35-42 (1998); Mochly-Rosen, D. et al., Molec. Biol.
Cell. (formerly Cell Regulation) 1:693-706 (1990). If the
intramolecular interaction between .psi..epsilon.RACK and the
RACK-binding site stabilizes the inactive closed form, increasing
or decreasing the affinity of this intramolecular interaction
should cause a corresponding decrease or increase in the ability of
the enzyme to bind to its RACK. To test whether increasing or
decreasing the affinity of the intramolecular interaction between
.psi..epsilon.RACK and the RACK-binding site proposed herein to
stabilize the inactive closed form will cause a decrease or
increase in the ability of the enzyme to bind its RACK, the binding
of insect cell-expressed .epsilon.PKC (Wt and .psi..epsilon.RACK
mutants) to immobilized GST-.epsilon.RACK in the presence and
absence of phospholipid (PL) activators was determined.
[0135] .epsilon.RACK Binding Assay
[0136] Insect cells expressing either .epsilon.PKC or
GST-.epsilon.RACK were lysed as described in Example 4.
GST-.epsilon.RACK, 10 ng was immobilized on Glutathion-Sepharose 4B
beads (Amersham-Pharmacia), and washed thoroughly with wash buffer
(20 mM Tris-HCl pH 7.5, 2 mM EDTA, 100 mM NaCl, 12 mM pME, and 0.1%
TritonX-100). Immobilized GST-.epsilon.RACK was then incubated with
the soluble fraction of insect cell lysates containing 100 ng Wt,
D(86)A, or D(86)N .epsilon.PKC protein, in the presence or absence
of phospholipid activators (PL) (phosphatidylserine 12
.mu.g/reaction and, Sn-1,2 dioleoylglycerol 400 ng/reaction) for 1
hour at 4.degree. C. Upon thorough washing with wash buffer, bound
proteins were eluted in SDS-PAGE sample buffer and proteins
separated on 8% SDS-PAGE gels. The amount of .epsilon.PKC
interacting with .epsilon.RACK was determined by Western blot
probing for .epsilon.PKC with anti .epsilon.PKC-V5 antibodies.
Blots were then re-probed with ant-GST (Santa Cruz) to verify that
the same amount of GST-.epsilon.RACK was present in each binding
assay. All other methods for this Example were performed as
described, for example, in Example 4.
[0137] Results
[0138] Binding of D(86)A .epsilon.PKC mutant to .epsilon.RACK in
the absence of PL activators was at least two fold greater than
binding of either D(86)N or Wt enzymes (FIGS. 5C, D). Binding of
D(86)N and of Wt enzyme to .epsilon.RACK was significantly
increased in the presence of PL, whereas binding of the D(86)A
mutant to .epsilon.RACK was not increased (FIGS. 5C, D). In the
presence of an equal concentration of PL, there was less
.epsilon.RACK binding of D(86)N .epsilon.PKC than either D(86)A or
Wt .epsilon.PKCs. These results are consistent with the prediction
that the .epsilon.RACK-binding site in the D(86)A mutant is already
available for binding to .epsilon.RACK, whereas this site is masked
in both Wt or D(86)N .epsilon.PKCs and becomes accessible for
binding only upon activation.
EXAMPLE 6
Rate of Translocation of .epsilon.PKC and .epsilon.PKC Mutants in
Cells
[0139] The in vitro studies of .PSI..epsilon.RACK mutants and Wt
.epsilon.PKC described herein support the hypothesis proposed
herein that .PSI..epsilon.RACK is involved in an intramolecular
interaction that stabilizes the inactive closed state. To further
test this hypothesis, the rate of translocation of
.PSI..epsilon.RACK mutants and Wt .epsilon.PKC in cells in response
to stimulation was examined. It was hypothesized herein that part
of the process of translocation requires disruption of the
intramolecular interaction between the .PSI..epsilon.RACK site and
the .epsilon.RACK binding site. To investigate whether modulation
of this intramolecular interaction could affect translocation rates
of .epsilon.PKC, .epsilon.PKC .PSI..epsilon.RACK mutants were fused
at their amino-termini to GFP, CFP or YFP proteins and
translocation was analyzed both by cell fractionation and by
real-time imaging.
[0140] Kinase Assay
[0141] The ability of the different .epsilon.PKC mutants to
phosphorylate substrates was assayed by following the incorporation
of [.gamma..sup.32P]ATP into myelin basic protein according to a
method modified from Kikkawa, U. et al., Biochem. Biophys. Res.
Commun. 135:636-643 (1986). Myelin basic protein phosphorylation
was measured either by liquid scintillation or for
immunoprecipitation experiments, by autoradiography. For kinase
reaction of immunoprecipitated enzyme, beads were resuspended in 20
.mu.l of a kinase reaction buffer composed of 20 mM Tris pH 7.5,
[[.gamma..sup.32P]ATP (Amersham) 0.3 .mu.Ci/reaction, ATP (Sigma) 9
.mu.M/reaction, myelin basic protein (Sigma) 12 .mu.g/reaction and
MgCl2 50 mM/reaction. 4 .mu.l phospholipids were added per reaction
when needed. Phopsholipids were prepared as described above. Kinase
reactions were stopped with SDS sample buffer and boiling. Samples
were then run on a 12% SDS PAGE and transferred to nitrocellulose
exposed for autoradiography. The same nitrocellulose was then
developed with anti .epsilon.PKC (V5) antibodies (Santa Cruz
Biotechnology) to verify the amount of fusion protein
immunoprecipitated.
[0142] Analysis of PKC Translocation by Western Blot
[0143] After 24 hours of transfection cells were serum starved for
an additional 24 hours and incubated with phorbol 12-myristate
13-acetate (PMA) (LC Laboratories) for the indicated times and
concentrations at room temperature and subsequently fractionated as
previously described [Schechtman, D & Mochly-Rosen, D., Methods
Enzymol. 345:470-489 (2002)]. To assess PKC distribution the
different cell fractions were run on SDS PAGE, transferred for
Western blot analysis and probed with anti-.epsilon.PKC V5
antibodies. Lysates of overexpressed GFP-.epsilon.PKC were diluted
to approximately 0.2 .mu.g/well. At this concentration endogenous
.epsilon.PKC was not detected. All other methods for this Example
were performed as described, for example, in Example 4.
[0144] Microscopy and Analysis
[0145] CHO cells were grown on glass coverslips, and serum-starved
as described in Example 4. For each experiment, cells were
transferred to a commercially available metal coverslip holder
(Molecular Probes) in which the coverslip formed the bottom of a 1
ml bath. The media was then replaced with extracellular buffer (120
mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1.5 mM MgCl2, 20 mM HEPES and 30
mM glucose). Cells were stimulated by either PMA (100 nM), or with
ATP (1 mM) in extracellular buffer. Fluorescence images of
GFP-tagged constructs were obtained using the 488 nm excitation
line of a laser scanning confocal microscope (Pascal, Zeiss) and
emission was collected through a 505-550 nm band pass filter. Cells
were imaged on the stage of an inverted microscope (Axiovert 100M)
using a 40.times. Zeiss Plan-apo oil immersion objective with 1.2
numerical aperture (NA). For dual color imaging of CFP and YFP
fusion proteins, a spinning disk Nipkow confocal microscope was
used. Cells were viewed using an inverted Olympus IX70 microscope
with a 40.times. oil immersion Olympus objective (1.35 NA) and
images were acquired with a CCD camera (Hamamatsu) and 2.times.2
pixel binning. CFP was excited with the 442 nm laser line of a
helium-cadmium laser (Kimmon) whereas YFP was imaged with the 514
nm line of an argon ion laser (Melles-Griot). To selectively
photobleach YFP labeled proteins in local regions of the cytoplasm
for FRAP experiments, the 514 nm line of an Enterprise laser
(Coherent) at maximal power (400 mW) was used. For these
experiments, a 60.times. oil immersion objective was used (1.4 NA).
Under these conditions, and by placing an iris in the beam path, it
was possible to bleach 80% of the YFP fluorescence in 500 ms in an
area of the cytosol measuring approximately 35 um2. Real time
confocal images were acquired every 10-15 seconds for 20 minutes
for experiments utilizing PMA, and every 5 seconds for a total
duration of 3 minutes in the case of cells stimulated with ATP.
Reagents were added to the cell chamber after the fifth image in
each time series. Control time lapses were acquired using the same
imaging conditions used in the experiments to check that the level
of general dye photobleaching did not exceed 10-30%. All images
were acquired at room temperature. Images were exported as 12 or 16
bit files and changes in fluorescence intensity were measured using
MetaMorph.RTM. data analysis software (Universal Imaging Corp.). To
monitor the translocation of PKC, a small region of interest was
selected in the cytosol of each cell and fluorescence intensity
values graphed against time after subtraction of background values
and normalized so that the initial fluorescence was 100%. Averages
of 10-15 cells from three independent experiments were used. To
determine statistical significance, a 1 tail type 2 T-test
(Microsoft excel) was used.
[0146] Immunoprecipitation
[0147] CHO-Hir cells were washed two times in serum free media and
serum starved 12-24 hrs after transfection. Cells were then kept in
serum free media for approximately 12 hours before the experiment.
Cells were lysed in homogenization buffer [20 mM Tris-HCl, pH 7.5,
2 mM EDTA, 10 mM EGTA, 0.25M sucrose, 12 mM p-mercapthoethanol,
PMSF (17 .mu.g/ml), soybean trypsin inhibitor (SBTI), (20
.mu.g/ml), leupeptin (25 .mu.g/ml), aprotinin (25 .mu.g/ml) and
0,1% Triton X 100 (Sigma)] and centrifuged at 1000.times.g for 30
minutes. The supernatant was then used for immunoprecipitation
experiments. Immunoprecipitation was performed with anti-GFP
monoclonal antibodies 3E6 (Molecular Probes) according to the
manufacturers instruction. Briefly, lysates (approximately 2 mg/ml)
were precleared in protein G agarose beads (25 .mu.l packed volume
Invitrogen) for at least 30 minutes and spun at 1000.times.g. The
supernatant was then incubated with 1 .mu.g anti-GFP monoclonal
antibodies for at least two hours. Protein G agarose beads (25
.mu.l packed volume) was then added to the lysate-antibodies
complex and incubated for at least two hours (all incubations were
done at 4.degree. C.). Beads were subsequently washed three times
with Buffer 1 (50 mM Tris, 0.15 M NaCl, 1 mM EDTA, 0.1% Triton
X100, pH 7.5), once with Buffer2 (50 mM Tris, 0.15 M NaCl, 0.1%
Triton X100, pH 7.5) and once with Buffer 3 (50 mM Tris, 0.1%
Triton X100, pH 7.5). For kinase assays, beads were washed an
additional time with 20 mM Tris-HCl, pH 7.5.
[0148] Results
[0149] In an in vitro kinase assay, it was first confirmed that the
GFP-fusion proteins had similar catalytic activity. As seen in
FIGS. 6A and B, immunoprecipitated GFP-.epsilon.PKC mutants
phosphorylated myelin basic protein to a similar extent upon
activation.
[0150] It was also confirmed that the Wt and .PSI..epsilon.RACK
.epsilon.PKC mutants were sensitive to activation by phorbol ester
and that they translocated from the soluble to the particulate
fraction of the cell upon activation. Translocation of the
.epsilon.PKC mutants was determined using two different cell lines,
MCF-7 and CHO. [CHO cells were stimulated for 10 minutes with 100
nM PMA (FIG. 6C). MCF-7 cells were stimulated with 10 nM PMA for 10
minutes (FIGS. 6D, 6E), since stimulation of MCF-7 cells with
higher doses of PMA caused a detachment of the cells]. After
treatment with PMA, cells were fractionated into soluble and
particulate fractions, and GFP-.epsilon.PKC was detected and
quantified by Western blot analysis using anti-.epsilon.PKC. Only
GFP-.epsilon.PKC fusion proteins ( about 110 Kda) and not the
endogenous .epsilon.PKC (about 80 Kda) were detected when 20 ng of
protein was loaded per lane and all the mutants had a similar level
of expression (FIGS. 6C-6E). On PMA treatment, there was a greater
increase in D(86)A .epsilon.PKC in the particulate fraction than
either D(86)N or Wt .epsilon.PKCs (FIGS. 6C, D). This is not due to
PKC degradation; the total amount of .epsilon.PKC was not changed
upon activation. Therefore, GFP-.epsilon.PKC .PSI..epsilon.RACK
mutants were catalytically active and responded to activation by
PMA by translocating from the soluble to the particulate fraction
of the cell. Using two different cell lines, it was found that
after 10 minutes of treatment with PMA, more D(86)A .epsilon.PKC
compared to Wt or D(86)N .epsilon.PKCs translocated to the
particulate fraction.
[0151] Since cell fractionation experiments are not suitable for
dynamic studies, the GFP tag was used to follow the rate of
translocation of the different .epsilon.PKCs by real-time
microscopy in CHO cells (FIGS. 7A-7C). Translocation of
.epsilon.PKC was seen as a decrease of fluorescence intensity in
the cytoplasm concomitant with an increase in fluorescence
intensity at the cell periphery. All of the .epsilon.PKC mutants
translocated to the same place (cell periphery) when stimulated by
PMA (FIG. 7A). Translocation of the D(86)A mutant to the cell
periphery began within one minute of PMA stimulation, whereas
translocation of the Wt enzyme became apparent only after 2-3
minutes of PMA stimulation (FIG. 7A, arrows). In contrast,
translocation of the D(86)N mutant became apparent only after 5
minutes of stimulation. A typical line intensity profile showing
the distribution of each .epsilon.PKC between the cell periphery
and cytosol is shown for a representative cell at different time
points (FIG. 7B).
[0152] The amount of fluorescence decrease in the cytoplasm was
proportional to the amount of fluorescence increase at the cell
periphery [Raghunath, A. et al. Biochem. J. 370:901-912 (2003)],
since the total fluorescence did not change. To quantitatively
monitor translocation of the different .epsilon.PKC enzymes, the
decrease of fluorescence intensity was measured in a region of the
cytoplasm upon PMA stimulation using MetaMorph.RTM. software
(Universal Imaging Corp.). A graphical comparison of .epsilon.PKC
translocation obtained by the decrease of fluorescence in the
cytoplasm indicated that the rates of translocation were
significantly different between the two mutants and Wt .epsilon.PKC
(p<0.0001; FIG. 7C). The D(86)A mutant translocated at a faster
rate than either the Wt or D(86)N .epsilon.PKC mutant. The Wt
enzyme reaches a similar steady state level (the level at which
there was no further accumulation of .epsilon.PKC at the cell
periphery) as the D(86)A mutant, but did so at a slower rate. In
contrast, the D(86)N mutant achieved a steady state at a higher
level than either the Wt or D(86)A mutant. Therefore, the D(86)A
mutant translocated at a faster rate than either the D(86)N or Wt
.epsilon.PKCS, and the amount of D(86)N .epsilon.PKC mutant that
reached the cell periphery was lower than the amounts of either
D(86)A or Wt .epsilon.PKCs.
EXAMPLE 7
Mathematical Modeling of .epsilon.PKC Translocation Suggests that
.epsilon.PKC Translocation is at Least a Two Step Process
[0153] To further characterize the initial process of translocation
of the different .epsilon.PKC .PSI..epsilon.RACK mutants, the
decrease of fluorescence in the cytoplasm was fitted to a
mathematical model.
[0154] Mathematic Modeling
[0155] Mathematical modeling was obtained from data of cells
expressing similar levels of the different .epsilon.PKC
.PSI..epsilon.RACK mutants. Non-linear least squares curve fitting
analysis was performed using the EViews software (QMS) and
differential equation analysis was performed using Berkeley
Madonna.TM.. All other methods for this Example were performed as
described, for example, in Examples 4 and 6.
[0156] Fluorescence time courses were analyzed by a non-linear
regression analysis with single and biexponential equations (1 and
2) as previously done by Nalefski and Newton Nalefski, E. A. &
Newton, A. C., Biochemistry 40:13216-13229 (2001)], where I(t) is
the concentration of observed molecules in the cytosol at time (t),
and C.sub.1-5 are constants.
I(t)=C.sub.1+C.sub.2e.sup.(-C3(t)) 1.
I(t)=C.sub.1+C.sub.2e.sup.(C3(t))+C.sub.4e.sup.(-C5(t)) 2.
[0157] Residual error graphs obtained using single exponential
equations (FIG. 8A) and using biexponential equations (FIG. 8B) for
the D(86)A .epsilon.PKC mutant translocation process is shown in
FIGS. 8A-8D. Residual error analysis for D(86)N and Wt
.epsilon.PKCs showed similar results (data not shown). Since a
superior fit, with a smaller and more equally distributed error was
obtained with a bi-exponential equation, a two-step model was
adopted to illustrate the initial process of .epsilon.PKC
translocation where the first step is the opening of .epsilon.PKC
and disruption of the intramolecular interaction between the
.PSI..epsilon.RACK and the .epsilon.RACK binding-site, and the
second step is .epsilon.PKC binding to the membrane (binding to the
membrane in this case may be binding to either lipids or proteins).
This process can be described as follows: 1
[0158] Where k1 is the rate of .epsilon.PKC opening in the cytosol,
k-1 is the rate of closing in the cytosol, k2 is the rate of
.epsilon.PKC binding to the membrane, and k-2 is the rate of
detachment from the membrane. Using the bi-exponential equation,
values for the constants C1, C3 and C5 (Table 1) were
estimated.
2TABLE I shows estimated constants using a bi-exponential equation
and calculated values for k1, k - 1, k2 and k - 2 .epsilon.PKC
D(86)A Wt D(86)N C.sub.1 30.59 .+-. 0.286 30.46 .+-. 0.991 38.38
.+-. 0.254 C.sub.3 0.016 .+-. 0.001 0.016 .+-. 0.004 0.010 .+-.
0.0003 C.sub.5 0.040 .+-. 0.007 0.009 .+-. 0.001 0.034 .+-. 0.004 k
- 2/(k2 + k - 0.306 .+-. 0.002 0.305 .+-. 0.010 2) = C.sub.1/100
(k2 + k - 2) = C.sub.3 0.016 .+-. 0.001 0.016 .+-. 0.004 k - 2
0.005 .+-. 0.0004 0.005 .+-. 0.0011 0.0052 k2 0.011 .+-. 0.003
0.011 .+-. 0.002 0.01138 *k1 = C.sub.5 0.040 .+-. 0.007 0.009 .+-.
0.001 0.0135 k - 1 .about.0 .about.0 0.005 *For D(86)N the
relations k1 = c5 is not valid since the corresponding values for
k1, k - 1, k2 and k - 2 were obtained by simulations using
differential equations.
[0159] The value of C1 corresponds to the level of .epsilon.PKC in
the cytosol at steady state. It was next determined whether the
hypothesis that the values obtained for C1 and C3 of D(86)A and Wt
.epsilon.PKCs are statistically equivalent. Using WALD's parameter
test, and using F-test and Xi-Squared Test, p values of 0.9 and
0.7, respectively, were obtained. It was therefore assumed that the
steady state level for A and D, C1, is the same. It was also
assumed that the second step (binding to the membrane) should not
differ between the .epsilon.RACK .epsilon.PKC mutants,since C3 was
equal for both Wt and D(86)A .epsilon.PKC. Therefore, if the steady
state level (C1) was the same for D(86)A and Wt .epsilon.PKCs and
the second step (binding to the membrane) was also the same, it is
plausible that k-1 (rate of closing of .epsilon.PKC) for both
D(86)A and D(86)N .epsilon.PKCs would be negligible. When k-1 is
negligible, the following equations (3-5) hold and can be used to
calculate k-2 (equation 6) and K2 (Table 1).
(C1/100)=k-2/(k2/+k-2) 3.
C3=(k2+k-2) 4.
C5=k1 5.
k-2=[(C1/100)(C3] 6.
[0160] If I.sub.o(t) equals the concentration of closed
.epsilon.PKC in the cytosol at time (t), I.sub.1(t) equals the
concentration of open .epsilon.PKC in the cytosol at time (t),
I.sub.2(t) equals the concentration of .epsilon.PKC at the membrane
at time (t) and I(t) equals the concentration of open and closed
.epsilon.PKC in the cytosol at time (t) (I.sub.o(t)+I.sub.1(t)),
the following differential equations (7-9) can be used to describe
this model:
dI.sub.o(t)/dt=-(K1)(Io(t))+(K-1)(I1(t)) 7.
dI.sub.1(t)/dt=-(K-1+K2)I.sub.1(t)+(K1)(I.sub.o(t))+(K-2)(I.sub.2(t))
8.
dI.sub.2(t)=-(K-2)(I.sub.2(t))+(K2)(I.sub.1(t)) 9.
[0161] The differential equations were next solved for I(t) using
the Runge-Kulta algorithm (Berkeley-Madonna) and the solutions were
compared to the experimental data (loss of fluorescence in the
cytoplasm). I(t) was solved for and the calculated parameters were
used for k1, k-1, k2 and k-2 (Table 1) assuming that the initial
amount of closed .epsilon.PKC in the cytoplasm is equal to 100%
[I.sub.o(0)=100]. FIG. 8C shows the fit between curves of the raw
data for D(86)A .epsilon.PKC, and the curve obtained by nonlinear
regression with a bi-exponential equation. FIG. 8D shows the fit
between curves of the raw data for D(86)A and the curve obtained by
the differential equations solving for I(t). Similar fitting
results were obtained for D(86)N and Wt .epsilon.PKCs (data not
shown). The residual error for all curve fitting data was similar
to the one obtained with a non-linear regression, using a
bi-exponential equation (FIG. 4B; data not shown).
[0162] The steady state level for D(86)N was higher than for either
D(86)A or Wt (Table 1), and therefore it is not possible to assume
that k-1 for this mutant was negligible. In this case, the
Runge-Kutta algorithm was also used to solve differential
equations. Because the rate of the second step, binding to the
membrane, should not be altered for either of the .epsilon.PKC
mutants, it was assumed that the values of k2 and k-2 should be in
the range of the ones obtained for D(86)A and Wt. Together, the
experimental data herein and mathematical modeling suggest a
two-step translocation process of .epsilon.PKC to the cell membrane
upon activation. Whereas the second step was independent of
intramolecular interactions, the first step, which was predicted
herein to involve opening of the enzyme, was greatly dependent on
these intramolecular interactions.
EXAMPLE 8
Co-Transfection of D(86)A T .epsilon.RACK Mutant and Wt
.epsilon.PKC in the Same Cell
[0163] To further investigate the behavior of the .epsilon.PKC
mutants within a single cell, two different .epsilon.PKC constructs
were cotransfected into CHO cells, fused to either YFP or CFP. All
methods for this Example were performed as described, for example,
in Examples, 4, 6 and 7.
[0164] Results
[0165] A representative cell that co-expresses a YFP-A and a CFP-D
.epsilon.PKC at similar levels is shown in FIG. 9A. Upon
stimulation with PMA, the D(86)A mutant translocated faster than
the Wt enzyme (FIG. 9A). Quantitative analysis, expressing the
decrease of fluorescence intensity in the cytoplasm is shown in
FIG. 9C. The D(86)A mutant was found at the cell periphery at 1.5
minutes after PMA stimulation whereas translocation of Wt
.epsilon.PKC was still minimal even after 10 minutes (FIG. 9A).
Therefore, even when the D(86)A .PSI..epsilon.RACK mutant and Wt
.epsilon.PKC were in the same cell, D(86)A translocated faster than
Wt .epsilon.PKC.
EXAMPLE 9
Translocation Rates of the .PSI..epsilon.RACK .epsilon.PKC Mutants
Upon Cell Stimulation by a G Protein Coupled Receptor
[0166] It was next determined whether differences in translocation
rates of the different .epsilon.PKCs were also observed when
translocation was stimulated via receptor signaling rather than by
PMA. ATP has been previously used to activate PKC in CHO cells by
stimulating purinergic G protein-coupled receptors [Maasch, C. et
al., FASEB J. 14:1653-1663 (2000)]. All methods for this Example
were performed as described, for example, in Examples 4, 6 and
7.
[0167] Results
[0168] It was found herein that translocation of GFP-.epsilon.PKC
upon stimulation with ATP to the cell periphery was faster than
PMA-induced translocation (FIG. 9B vs. FIGS. 7A-7C). Translocation
of the D(86)A and Wt .epsilon.PKCs was already apparent 10 seconds
after stimulation, whereas translocation of the D(86)N .epsilon.PKC
enzyme occurred after 40 seconds of stimulation reaching a steady
state at significantly higher levels than either D(86)A or Wt
.epsilon.PKCs (FIG. 9B). Quantitative analysis, expressing the
decrease of fluorescence intensity in the cytoplasm is in FIG.
9D.
EXAMPLE 10
Diffusion Rates of the .epsilon.PKC .PSI..epsilon.RACK Mutants
[0169] Different translocation rates may reflect differences in
overall mobility (diffusion) of the .epsilon.PKCs in cells.
Therefore, fluorescence recovery after photobleaching (FRAP) of the
cytoplasmic enzyme was measured; mobility of .epsilon.PKCs was
measured by monitoring the time required for the fluorescence to
recover in a bleached region. All methods for this Example were
performed as described, for example, in Examples 4, 6 and 7.
[0170] Fifty percent FRAP was reached at similar times for all
.epsilon.PKCs. (The average of at least 10 cells/each .epsilon.PKC
was: Wt=9.0.+-.1.2, D(86)A=9.2.+-.1.1 and D(86)N=9.1.+-.1.6
seconds.) By comparing the fluorescence in the bleached region
after full recovery (F) with that observed before bleaching (Fi)
and just after bleaching (FO), the mobile
fraction=(F.infin.-F0)/(Fi-F0) was determined. This was important
to determine, since the mobile fraction may be affected by
differences in interactions of the Wt and the mutant .epsilon.PKCs
with other proteins and membranes. It was found herein that the
mobile fraction was the same for all .epsilon.PKCs (63.+-.2% for
Wt, 63.+-.4 for D(86)A and 64.+-.2.5% for D(86)N, averages of at
least 10 cells/each). Therefore, differences in translocation rates
were not due to differences in mobility of the inactive enzyme, but
rather, to modulation of the intramolecular interaction between the
.PSI..epsilon.RACK and RACK-binding site.
[0171] Discussion
[0172] A key component in signal transduction is the inherent
mechanism by which the enzymes remain inactive in the absence of
extracellular stimuli. This mechanism involves the use of
intramolecular interactions that stabilize a closed conformation
with unexposed active site. Upon stimulation, the enzyme adopts an
open conformation whereby intramolecular interactions are
interrupted and binding sites for intermolecular interactions that
stabilize the open form are exposed, resulting in a catalytically
active enzyme. In the case of PKC, the intermolecular interaction
sites are the binding sites for phospholipids and anchoring
proteins [Mochly-Rosen, D & Gordon, A. S. FASEB J. 12:35-42
(1998); Oancea, E. et al., J. Cell. Biol. 140:485-498 (1998)].
Here, it is demonstrated that alterations in the intramolecular
interaction between the .epsilon.RACK binding site and the
.PSI..epsilon.RACK site affected the translocation rate of the
enzyme, further supporting a role of this intramolecular
interaction in .epsilon.PKC translocation and signaling.
[0173] As noted earlier, the .PSI..epsilon.RACK sequence in
.epsilon.PKC is about 25% different from the sequence in
.epsilon.RACK. It was suggested herein that the charge change (N-D)
contributed to the difference in strength of the intramolecular
interaction within .epsilon.PKC as compared to the intermolecular
interaction between .epsilon.PKC and its RACK, .epsilon.RACK
[Souroujon, M. C. & Mochly-Rosen, D. Nat. Biotechnol.
16:919-924 (1998); Dorn, G. W. 2.sup.nd, et al. Proc. Natl. Acad.
Sci. U.S.A. 96:12798-12803 (1999)]. By mutating the D(86) in the
.PSI..epsilon.RACK sequence of .epsilon.PKC to an N, an enzyme that
translocates slower than the wild type enzyme has been created,
presumably because of the increase in the intramolecular
interaction between the .PSI..epsilon.RACK and the RACK-binding
site in .epsilon.PKC. Mutating D(86) to an A in .epsilon.PKC
abolished the intramolecular interaction between the
.PSI..epsilon.RACK and the RACK-binding site, and resulted in an
enzyme that translocated at a faster rate than the wild type
enzyme.
Mutations in the .PSI..epsilon.RACK site in .epsilon.PKC affect
intrinsic properties of .epsilon.PKC
[0174] Three criteria were used to demonstrate the role of the
.PSI..epsilon.RACK site in the intramolecular interaction within
.epsilon.PKC and the role of D86 in this interaction. The first
criterion was the sensitivity of the enzyme to proteases; an open
enzyme should be more susceptible to protease degradation after
activation (Orr, J. W. et al., J. Biol. Chem. 267:15263-15266
(1992)]. It was shown herein that the D(86)N mutant was more
resistant to proteolysis; D(86)N mutant required twice the time for
the same extent of degradation of either the D(86)A or Wt enzymes
(FIG. 5). Therefore, D(86)N mutant is a more closed or inactive
enzyme. Because sensitivity to proteolysis of the A mutant was the
same as wild type D(86) .epsilon.PKC, it could not be determined
whether it was conformationally different from the wild type enzyme
using this method.
[0175] The second criterion examined PL-dependent binding of wild
type and mutant enzymes to .epsilon.RACK. If D(86) is critical for
an intramolecular interaction, D(86)A should be less dependent on
lipid activation for .epsilon.RACK-binding. Indeed, the single
amino-acid substitution modulated the intramolecular interaction
between the .PSI..epsilon.RACK and the .epsilon.RACK binding site;
the D(86)N mutant had a greater similarity to the .epsilon.RACK
sequence, and a reduced ability to bind to its .epsilon.RACK,
indicating that the D(86)N mutant is in a more closed conformation.
In contrast, the D(86)A .epsilon.PKC mutant was less dependent on
activators for RACK binding, indicating that it is in a more open
conformation.
Mutations in the .PSI..epsilon.RACK Site and Translocation Rates of
.epsilon.PKC in Cells
[0176] A third criterion demonstrating a critical role of the
.PSI..epsilon.RACK site in intramolecular interactions examined the
rate of translocation of the enzyme upon activation in cells. The
D(86)A .epsilon.PKC mutant translocated significantly faster than
either D(86)N or Wt .epsilon.PKCs, as measured by cell
fractionation studies. Using real-time confocal microscopy it was
demonstrated herein that the D(86)A mutant translocated at a faster
rate than wild type .epsilon.PKC, which in turn translocated faster
than the D(86)N mutant. Together, it appears likely that the
.PSI..epsilon.RACK site mediates a critical intramolecular
interaction that stabilizes the closed conformation in .epsilon.PKC
in the absence of stimulation.
[0177] A scheme for the mechanism of translocation of the different
.epsilon.PKC mutants is shown in FIG. 10. Disruption of an
intramolecular interaction between the .PSI..epsilon.RACK site and
the .epsilon.RACK-binding site must precede binding to the membrane
and is a rate-limiting step in the process. Modulating this
intramolecular interaction by mutating D(86) altered the
translocation rates of .epsilon.PKC, by affecting the first step of
the translocation process. Mathematical modeling of the data herein
further elucidated the molecular events leading to translocation
(see FIG. 8). Using non-linear regression analysis, an equation
with two exponents gave a better fit than a single exponential
equation, indicating that PMA-induced translocation involves at
least two steps (FIG. 7). It was proposed herein that the first
step represents the opening and closing processes of the enzyme and
the second step represents binding of the open enzyme to the cell
membrane. Importantly, the steady state level of the D(86)N mutant
was higher than that of either D(86)A or of Wt PKC, indicating that
the amount of D(86)N that reached the cell periphery was lower than
the amount of either Wt or D(86)A .epsilon.PKCS. The D(86)A mutant
translocated significantly faster than either the D(86)N or Wt
.epsilon.PKCs. Since the steady state level of D(86)A in the
particulate fraction was similar to the steady state level of the
Wt enzyme, and the second step of translocation (binding to the
membrane) was the same for all mutants, the rate of closing of an
enzyme, once it was open, could be considered negligible. Ochoa et
al [J. Mol. Bol. 311:837-849 (2001)] demonstrated that the binding
of the V1 domain to PL is not altered by D(86)A mutation,
supporting the hypothesis proposed herein that the second step of
translocation (binding to the membrane) is not altered [Ochoa et al
[J. Mol. Bol. 311:837-849 (2001)]. However, for the N mutant, the
k1 (rate of PKC opening) was slower than that of D(86)A and similar
to Wt .epsilon.PKC, and the k-1 (rate of 8PKC closing) was no PKC
longer negligible.
[0178] Similar to Shirai et al. [J. Cell. Biol. 143:511-521
(1998)], it was found herein that ATP-induced translocation of
.epsilon.PKC was much faster than PMA-induced translocation (FIGS.
9B vs. 7). Since ATP-mediated translocation was a fast process,
differences between the Wt and D(86)A mutants were not observed at
the time intervals analyzed. However, the translocation of the
D(86)N mutant was significantly slower than either the wild type or
A mutant, further supporting the importance of the .epsilon.RACK
site and the disruption of intramolecular interaction for PKC
translocation.
[0179] Schaefer et al., suggested that differences in translocation
between classical and novel PKCs are due to differences in
diffusion rates, and collision efficiencies with the membrane
Schaefer, M. et al., FASEB J. 15:1634-1636 (2001; Lenz, J. C. et
al., J. Cell. Biol. 159:291-302 (2002)]. Although diffusion and
collision with the membrane are likely factors in the translocation
rate, the data herein demonstrate that conformational changes in
the enzyme also occur, leading to at least a two-step process.
[0180] In the case of the novel PKC (calcium-independent) isozymes,
it is still not clear what triggers the opening of the enzyme.
Here, it has been shown that disrupting the intramolecular
interaction between the .epsilon.RACK and RACK-binding site is a
critical step in activation that precedes translocation and
anchoring to the cell periphery. Whether this anchoring is mainly
mediated by binding to membranes, whether it involves anchoring
proteins, and whether binding to lipids precedes binding to
proteins could not be determined in cells overexpressing
.epsilon.PKC. However, it has previously been demonstrated that
translocation of endogenous .epsilon.PKC results in its
co-localization with its .epsilon.RACK [Csukai, M. et al., J. Biol.
Chem. 272:29200-29206 (1997)] and that disruption of binding to
.epsilon.RACK in cells with a peptide corresponding to one of the
RACK-binding sites in .epsilon.PKC (.epsilon.V1-1 peptide) inhibits
.epsilon.PKC translocation and colocalization with .epsilon.RACK
[Johnson, J. A., et al., J. Biol. Chem. 271:24962-24966 (1996);
Mochly-Rosen, D. et al., Circ. Res. 86:1173-1179 (2000)].
Importantly, it has been previously shown that a peptide
corresponding to the .PSI..epsilon.RACK sequence induces
.epsilon.PKC translocation and co-localization with .epsilon.RACK
and triggers .epsilon.PKC function [Dorn, G. W., 2.sup.nd, et al.,
Proc. Natl. Acad. Sci. U.S.A. 96:12798-12803 (1999)].
[0181] When the enzyme is over-expressed, as it is in this study,
it is likely that the binding proteins including RACKs are no
longer present in stochiometric amounts to the enzyme. Indeed, the
over-expressed wild type .epsilon.PKC and the endogenous
.epsilon.RACK did not co-localize in the cells, whereas the
endogenous .epsilon.PKC co-localized with the .epsilon.RACK
following activation in non-transfected cells (not shown). Because
many attempts to co-express .epsilon.RACK with .epsilon.PKC have
failed, the translocation experiments that performed in the present
examples reflect mainly the interaction of the GFP-enzyme with
lipids in the cell membrane.
[0182] Where in .epsilon.PKC is this RACK-binding site? The V1
domain of .epsilon.PKC is homologous to the C2 domain of the
.beta.PKC [Dekker, L. V., et al. Curr. Opin. Struct. Biol.
5:396-402 (1995)]. However, there is an additional RACK-binding
site in the V5 region of .beta.PKC (30) and molecular dynamics
studies with the C2 region of .beta.PKC showed that an
intramolecular interaction between the .PSI.RACK and the
RACK-binding site in the C2 region is not possible [Banci, L. et
al., J. Biol. Chem. 277:12988-12997 (2002)]. Instead it is
suggested herein that the intramolecular interaction between the
.epsilon.RACK and the RACK-binding site in .beta.PKC is likely to
occur between the C2 and V5 regions in .beta.PKC. This may also be
the case for .epsilon.PKC. Recently Stubbs and collaborators have
demonstrated that in .alpha.PKC there is also an additional
intramolecular interaction between the C1 and C2 domains that
maintains the enzyme in its inactive state [Slater, S. J., et al.,
J. Biol. Chem. 277:15277-15285 (2002)]. In addition, they have
suggested that .alpha.PKC forms dimers through an intermolecular
interaction between the C1 and C2 domains. The hypothesis that this
is also the case for the .epsilon.RACK binding site and the
.PSI..epsilon.RACK can not be rejected [Slater, S. J., et al., J.
Biol. Chem. 277:15277-15285 (2002)].
[0183] By mutating the .PSI..epsilon.RACK site in the intact
.epsilon.PKC, it has been demonstrated herein the importance of
interrupting the intramolecular interaction between the
.PSI..epsilon.RACK and the .quadrature.RACK-binding site in the
process of .epsilon.PKC translocation. To the inventor's knowledge,
this is the first time that a single charge change made outside of
the (.PSI.-substrate) site or the catalytic site, or of calcium
binding sites evoked a change in translocation rate. Together, it
is concluded herein that disruption of the interaction between the
.PSI..epsilon.RACK and the RACK-binding site is a critical and rate
limiting step in PKC translocation.
[0184] From the foregoing, it can be seen how various objects and
features of the invention are met. For example, a method for
converting a PKC peptide agonist to a PKC peptide antagonist by
changing an amino acid residue to effect a charge change in the
agonist, such that, in one form of the invention, the modified
agonist peptide more closely resembles the RACK, is described. The
approach currently used to identify PKC isozyme-selective
antagonists and agonists capitalizes on the availability of large
databases containing the primary sequences of many proteins. Here,
the approach was used to convert an isozyme specific translocation
agonist to an antagonist by taking a peptide agonist, including
.psi..epsilon.RACK, and changing one amino acid residue (D to N).
This change increased the peptide's resemblance to the RACK-derived
sequence and also effected a charge change in the peptide. The
change in activity of the modified peptide is illustrated in the
Examples described herein by showing that regulation of
cardiomyocyte contraction rate, an .epsilon.PKC-mediated function
that can be induced by .psi..epsilon.RACK, was not induced by the
peptide with an asparagine (N-.psi..epsilon.RACK) or alanine
(A-.psi..epsilon.RACK) in the residue position of aspartate (D) in
the .PSI..epsilon.RACK. Moreover, N-.psi..epsilon.RACK inhibited
PMA or .psi..epsilon.RACK regulation of contraction as well as PMA-
or .psi..epsilon.RACK-induced .epsilon.PKC translocation.
Therefore, a single amino acid substitution, causing a change of
charge, increased the resemblance of the peptide to the RACK
sequence and resulted in loss of agonist activity and gain of
antagonist activity.
[0185] Although the invention has been described with respect to
particular embodiments, it will be apparent to those skilled in the
art that various changes and modifications can be made without
departing from the invention. Unless otherwise indicated, all terms
herein have the same meaning as they would to one skilled in the
art of the present invention. Practitioners are particularly
directed to Current Protocols in Molecular Biology (Ausubel, F. M.
et al., John Wiley and Sons, Inc., Media Pa.), which is regularly
and periodically updated, for definitions and terms of the art.
Sequence CWU 0
0
* * * * *