From the Department of Research, Northeastern Ontario
Regional Cancer Center, Sudbury, Ontario P3E 5J1 and Department of
Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario P3E
2C6, Canada, and the ¶ Banting and Best Department of Medical
Research and Department of Pharmacology, University of Toronto,
Toronto, Ontario M5G 1L6, Canada
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ABSTRACT |
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Two fusion proteins in which the
regulatory domains of human protein kinase C (R
; amino acids
1-270) or mouse protein kinase C
(R
; amino acids 1-385) were
linked in frame with glutathione S-transferase (GST) were
examined for their abilities to inhibit the catalytic activities of
protein kinase C
(PKC
) and other protein kinases in
vitro. Both GST-R
and GST-R
but not GST itself potently
inhibited the activities of lipid-activated rat brain PKC
. In
contrast, the fusion proteins had little or no inhibitory effect on the
activities of the Ser/Thr protein kinases cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein
kinase II, myosin light chain kinase, and mitogen activated protein
kinase or on the src Tyr kinase. GST-R
and GST-R
, on
a molar basis, were 100-200-fold more potent inhibitors of PKC
activity than was the pseudosubstrate peptide PKC19-36. In
addition, a GST-R
fusion protein in which the first 32 amino acids
of R
were deleted (including the pseudosubstrate sequence from amino acids 19-31) was an effective competitive inhibitor of PKC
activity. The three GST-R fusion proteins also inhibited
protamine-activated PKC
and proteolytically activated PKC
(PKM),
two lipid-independent forms of PKC
; however, the IC50
values for inhibition were 1 order of magnitude greater than the
IC50 values obtained in the presence of lipid. These
results suggest that part of the inhibitory effect of the GST-R fusion
proteins on lipid-activated PKC
may have resulted from sequestration
of lipid activators. Nonetheless, as evidenced by their abilities to
inhibit the lipid-independent forms of the enzyme, the GST-R fusion
proteins also inhibited PKC
catalytic activity through direct
interactions. These data indicate that the R domains of PKC
and
PKC
are specific inhibitors of protein kinase C
activity and
suggest that regions of the R domain outside the pseudosubstrate
sequence contribute to autoinhibition of the enzyme.
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INTRODUCTION |
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The protein kinase C (PKC)1 family is
composed of Ca2+- and phospholipid-dependent
isozymes that play important roles in signal transduction in both lower
and higher eukaryotic cells. In mammalian cells the PKC family has been
implicated in the regulation of a host of cellular processes including
growth, secretion, ion channel conductance, gene expression, and
receptor regulation (1-3). Each PKC isozyme contains a catalytic (C)
domain that catalyzes the phosphorylation of specific Ser and Thr
residues and an regulatory (R) domain that inhibits the activity of the C domain via intramolecular interactions (for review, see
Ref. 1). Some forms of PKC can be activated by receptor-mediated production of diacylglycerol, which binds to cysteine-rich sites within
the R domain (4-6), and by Ca2+, which acts through
high-affinity Ca2+-binding sites in the R domain (7). The
binding of diacylglycerol, phosphatidylserine, and Ca2+ to
the R domain induces a conformational change that relieves the
inhibitory effect of a pseudosubstrate-like sequence on catalytic activity. Evidence supporting this hypothesis comes from experiments in
which a small peptide corresponding to the pseudosubstrate-like sequence within the R domain of PKC significantly inhibited PKC catalytic activity (8), antibodies raised against this peptide constitutively activated the enzyme (9), and mutagenesis of sequences
within the pseudosubstrate site of the PKC R domain resulted in partial
activation of the enzyme (10). Furthermore, allosteric activation of
the enzyme has been shown to expose the pseudosubstrate region of PKC
to proteolytic attack consistent with its removal from the active site
of the enzyme (11).
Recently we examined the effects of the entire R domain of PKC on PKC
activity in vitro and demonstrated that the R domain of
human PKC (amino acids 1-270), when expressed as a fusion protein
with GST, behaved as a potent competitive inhibitor of PKC catalytic
activity (12). We also showed that the PKC
R domain inhibited
PKC-mediated phenotypes in intact yeast cells and suggested that PKC
R might provide a useful reagent to achieve specific, dominant
inhibition of PKC (12). In this study, we have examined the specificity
with which the R domains from human PKC
and mouse PKC
inhibit PKC
activity. We demonstrate that the R domains of PKC
and PKC
potently inhibit PKC
activity but do not appreciably inhibit the
activities of other Ser/Thr or Tyr kinases tested. We also find that
the deletion of the pseudosubstrate sequence from PKC R
does not
markedly diminish its ability to inhibit PKC holoenzyme or
proteolytically activated PKC
(PKM) activity. On this basis, we
suggest that R domain sequences outside the pseudosubstrate region of
PKC may contribute significantly to enzyme autoinhibition.
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MATERIALS AND METHODS |
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Reagents--
Mitogen-activated protein kinase (from
Pisaster ochraceus), human recombinant src
kinase, and their peptide substrates (APRTPGGRR and KVEKIGEGTYGVVYK,
respectively) were purchased from Upstate Biotechnology Inc. (Lake
Placid, NY). The PKC substrate [Ser-25]PKC19-31 (RFARKGSLRQKNV) and the PKC
pseudosubstrate peptide
PKC19-36 (RFARKGALRQKNVHEVKN) were obtained from Canadian
Life Technologies Inc. (Burlington, Ontario, Canada). LRRASLG
(Kemptide), PMA, purified rat brain PKC
, and
isopropylthio-
-D-galactoside were obtained from Sigma,
and PKM was obtained from Calbiochem-Novabiochem (San Diego, CA).
Recombinant human casein kinase II and casein kinase II substrate
peptide (RRREEETEEE) were from New England Biolabs (Missisauga,
Ontario, Canada). The catalytic subunit of cAMP-dependent protein kinase purified from bovine heart and
cGMP-dependent protein kinase from bovine aorta were
purchased from Promega Corp. (Madison, WI). The myosin light chain
kinase-specific substrate K-MLC11-23 (KKRPQRATSNVFS) was
from Peninsula Laboratories (Belmont, CA). Chicken gizzard myosin light
chain kinase and bovine brain calmodulin were generous gifts from
Michael P. Walsh (University of Calgary, Alberta, Canada). Radiolabeled
[
-32P]ATP (111 TBq/mmol) was from Mandel Scientific
(Guelph, Ontario, Canada).
Preparation of GST-R, GST-R
1-32, and
GST-R
--
A fusion protein in which amino acids 1-270 of human
PKC
was fused in frame to GST (GST-R
) was prepared as described
by Parissenti et al. (12) except that one tablet containing
a wide spectrum of protease inhibitors (CompleteTM,
Boehringer Mannheim) was added per 50 ml of bacterial cell lysate and
elution off glutathione-Sepharose columns was by several overnight incubations with 50 mM glutathione at 4 °C. A GST fusion
protein in which the first 32 amino acids of PKC R
were deleted
(GST-R
1-32) was prepared by cloning a
DraIII-EcoRI fragment of the human PKC
R
domain in pBluescript SK+ (12) into the GST fusion protein
expression vector pGEX-3X (Amersham Pharmacia Biotech) using standard
cloning techniques. A GST fusion protein containing the R domain of
PKC
(amino acids 1-385) was prepared by cloning an
NcoI-PvuII fragment from mouse PKC
cDNA in
pMT2
(a gift from Dr. John Knopf, Genetics Institute, Cambridge, MA)
into the SmaI and EcoRI sites of the GST fusion vector pGEX-2T (Amersham Pharmacia Biotech) using standard cloning techniques. Construction of each of the expression vectors was confirmed by restriction endonuclease digestion and DNA sequencing. These fusion proteins also were purified from Escherichia
coli DH5
as described above. The GST-R fusion proteins were
dialyzed in 1 mM EDTA, 1 mM dithiothreitol, and
10 mM Hepes, pH 7.5, and stored frozen at
70 °C.
Immunoblot Analysis--
Samples of GST-R,
GST-R
1-32, and GST-R
in SDS sample buffer were
electrophoresed on 10% polyacrylamide gels in the presence of SDS as
described by Laemmli (13), and electrophoretically blotted onto
nitrocellulose membranes. The membranes were then immunoblotted using a
goat anti-rat brain PKC antibody (14) or a mouse monoclonal antibody
specific for PKC
(Transduction Laboratories, Lexington, KY);
antigen-antibody reactivity was detected using horseradish
peroxidase-labeled secondary antibodies using the ECL detection system
(Amersham Pharmacia Biotech).
Measurement of PKC, PKM, and Other Protein Kinase
Activities--
PKC catalytic activity was measured by monitoring
the transfer of 32P from [
-32P]ATP to the
peptide substrate [Ser-25]PKC19-31 (8) in 30-min
reactions at 30 °C. Unless otherwise indicated, reactions (100 µl)
contained 2 µM [Ser-25]PKC19-31, 164 µM [
-32P]ATP (0.5 µCi), 18 mM MgCl2, 2 mM CaCl2,
46.4 µg/ml phosphatidylserine, 2.5 µM PMA, 20 mM Tris-HCl, pH 7.5, and 5 milliunits of purified rat brain
PKC
. The phosphatidylserine and PMA were added together as a
sonicated emulsion to form small unilamellar vesicles. Assays of PKM
activity were conducted under similar conditions except that the
concentration of [Ser-25]PKC19-31 was 1 µM, and 1 mg/ml bovine serum albumin was added to
stabilize the enzyme, and reactions were for 12 min. Reactions were
terminated by spotting 90 µl of sample onto P81 phosphocellulose
paper filters. Filters were washed with 1% phosphoric acid and counted
by liquid scintillation spectrometry.
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RESULTS |
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Isolation and Characterization of GST-R,
GSTR
1-32, and GST-R
--
Purified preparations
of GST-R
, GST-R
1-32, and GST-R
yielded major
protein bands on SDS-polyacrylamide gels that migrated with apparent
molecular weights in reasonable agreement with their predicted masses
(56 kDa for GST-R
, 52 kDa for GST-R
1-32, and 68 kDa for GST-R
) including 26 kDa for the GST component (Fig.
1). GST-R
and
GST-R
1-32 also reacted with the antibody that
recognizes the
,
, and
isoforms of rat PKC (14), whereas
GST-R
reacted with the PKC
antibody (Fig. 1), thus confirming the
identity of the fusion proteins. The GST-R
preparation contained a
small amount of contaminating low molecular weight material that was
visible on stained gels; presumably, this material represents a
proteolytic product of GST-R
, as it is strongly immunoreactive with
the monoclonal PKC
antibody and has a molecular weight equal to the
R domain of PKC
without GST attached.
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Effects of GST-R and GST-R
on PKC
Activity and on the
Activities of Various Other Ser/Thr and Tyr Kinases--
Previously we
demonstrated that GST-R
is a potent competitive inhibitor of
yeast-expressed bovine PKC
and yeast-expressed rat PKC
-I (12). To
assess whether the inhibition of protein kinase activity by GST-R
was PKC-specific, we examined the ability of GST-R
to inhibit the
activities of several Ser/Thr and Tyr kinases. As shown in Fig.
2A, GST-R
potently
inhibited the activity of purified rat brain PKC
(IC50 = 40 nM) but did not inhibit the activities of
cAMP-dependent protein kinase, cGMP-dependent protein kinase, or myosin light chain kinase and only marginally inhibited the activities of casein kinase II, mitogen activated protein
kinase, and src kinase. In fact, GST-R
stimulated the activities of cGMP-dependent protein kinase and myosin
light chain kinase, although the mechanism of this activation by
GST-R
is unknown. To determine whether the effects of GST-R
were
isozyme-specific, we examined the effects of GST-R
on PKC
activity. GST-R
(Fig. 2B) also inhibited PKC
activity
(IC50 = 60 nM) while having little or no effect
on the other Ser/Thr or Tyr kinases described above. As determined from
double-reciprocal plots of enzyme versus substrate concentration, GST-R
and GST-R
each competitively inhibited PKC
activity with Ki values of 0.5 ± 0.03 µM (Fig. 3A) and
0.8 ± 0.4 µM (Fig. 3B), respectively.
GST alone at concentrations up to 10 µM did not inhibit
PKC
activity (Fig. 4), nor did it inhibit the activities of any of the other protein kinases tested above
(data not shown). These data indicate that the R domains of PKC
and
PKC
are selective, competitive inhibitors of PKC activity but do not
exhibit PKC isoform selectivity when assayed in vitro in the
presence of activating lipids.
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Role of the Pseudosubstrate Sequence in the Inhibition of PKC by
GST-R
--
Although the R domains of PKC
and PKC
have
different pseudosubstrate sequences, their lack of selectivity for PKC
isozymes (Fig. 2) could be reconciled, as four amino acid residues
within the PKC
and PKC
pseudosubstrate sequences are conserved
(19), including Arg-22, which is essential for the inhibitory activity of the PKC
pseudosubstrate peptide (20). On a molar basis, however,
the GST-R
and GST-R
fusion proteins inhibited PKC activity 100-200 times more potently than did the PKC pseudosubstrate peptide PKC19-36 (Fig. 4), which has a Ki for
PKC
of 26 µM (data not shown). These observations
raised the possibility that regions within the PKC R domain but outside
the pseudosubstrate site play important roles in the inhibition of PKC
activity. To test this hypothesis further, a fusion protein was
prepared in which amino acids 1-32 of the PKC
R domain were deleted
and the remaining sequence (amino acids 33-270) was linked in frame
with GST. This protein, GST-R
1-32, which lacks the
pseudosubstrate sequence, retained the ability to inhibit the activity
of purified rat brain PKC
over a concentration range similar to that
seen for GST-R
and GST-R
(Fig. 4); inhibition of PKC
activity
by GST-R
1-32 was competitive (Fig. 3C;
Ki = 0.25 ± 0.12 µM). These observations strongly suggest that regions within the PKC R domain but
outside the pseudosubstrate sequence play a significant role in the
inhibition of PKC catalytic activity.
Effects of GST-R Fusion Proteins on PKM and Protamine-activated
PKC Activities--
We next examined the effects of GST-R
,
GST-R
1-32, and GST-R
on protamine-activated
PKC
and on PKM to distinguish direct effects of the fusion proteins
on PKC
from indirect effects due to sequestration of lipids.
Protamine serves both as a substrate for PKC and as an allosteric
activator of the enzyme, thus obviating the need for
phosphatidylserine, PMA, and Ca2+ in the assay (21). PKM is
a purified proteolytic product of PKC
that contains only the
catalytic half of the protein and neither requires nor is responsive to
phosphatidylserine, phorbol ester, or Ca2+ (22). As shown
in Fig. 5A, GST-R
inhibited
protamine-activated PKC
in a concentration-dependent
manner with an IC50 value of 650 ± 80 nM
(n = 3). GST-R
1-32 and GST-R
also
inhibited protamine-activated PKC but with somewhat higher
IC50 values of 1050 ± 60 nM
(n = 3) and 1300 ± 100 nM
(n = 3), respectively. GST alone did not inhibit
protamine-activated PKC
. The inhibitory effects of the GST-R fusion
proteins could be overcome with higher concentrations of protamine
(data not shown), suggesting that the GST-R fusion proteins were
competitive with substrate; however, the double-reciprocal plots of
velocity versus protamine concentration were parabolic in
the absence or presence of PKC
R, consistent with positive
cooperative effects of protamine on PKC activity observed previously
(12, 21). The GST-R fusion proteins were considerably more potent
inhibitors of protamine-activated PKC
than was the pseudosubstrate
peptide PKC19-36, as the pseudosubstrate peptide did not
inhibit protamine-activated PKC
(Fig. 5A) even at
concentrations as high as 50 µM. This finding is
consistent with the data presented in Fig. 4 which shows that the GST-R
fusion proteins inhibit PKC
activity with potencies that are
100-200-fold greater than PKC19-36. GST-R
1-32 and GST-R
also inhibited PKM activity
with IC50 values of approximately 500 nM (Fig.
5B). As expected, phosphatidylserine, PMA, and
Ca2+ had little effect on PKM activity (data not shown).
These observations thus indicate that the inhibition of PKC
activity
by GST-R fusion proteins may result at least in part from direct
effects of the fusion proteins on the catalytic domain of the
enzyme.
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DISCUSSION |
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As shown in this in vitro study, GST-R and
GST-R
inhibited lipid-activated PKC activity effectively and with
equal potency but did not inhibit the activities of several other
Ser/Thr protein kinases or the src Tyr kinase (Figs. 2 and
4). Thus the GST-R fusion proteins displayed a high degree of
specificity for PKC but lacked appreciable PKC isozyme selectivity
under these in vitro conditions. Although a number of
observations indicate that the R domains of PKC contribute to the
selective actions of different PKC isozymes, isozyme selectivity seems
to depend upon the responsiveness of the PKC isoforms to different
activating ligands and the targeting of the PKCs to different
subcellular compartments (23-25), factors that were not assessed in
the in vitro assays of PKC activity described here.
The inhibition of lipid-activated PKC by the GST-R fusion proteins
may have reflected competition between GST-R and substrate for the
active site of PKC (8) or may have been secondary to the sequestration
of lipid cofactors. The sequestration of lipids might impact on PKC
activity by reducing the effective concentrations required for
activation of PKC, by reducing the electrostatic potential of the
vesicles thereby preventing PKC binding to lipid surfaces (1), or by
inhibiting delivery of the PKC substrate peptide to the enzyme (26).
Indeed, lipids appeared to contribute to the inhibitory potency of the
GST-R fusion proteins, because the IC50 values of the GST-R
fusion proteins were 1 order of magnitude lower in
lipid-dependent assays of PKC
activity compared with the
lipid-independent assays of PKC activity (Fig. 4 versus Fig. 5). Although the concentrations of phosphatidylserine (approximately 50 µM) in the PKC assays were approximately 1000-fold higher
than the concentrations of fusion proteins required for 50% inhibition of lipid-ativated PKC
(approximately 50 nM), the
regulatory domain constructs may have sequestered up to 12 times the
amount of phosphatidylserine as estimated from direct PKC-lipid binding
assays (27) and may have sterically masked access to 100 times the
amount of lipid as estimated from light scattering and fluorescent
energy transfer measurements (28). Reductions in surface electrostatic
surface potential may have had additional effects on the interaction of PKC with lipid surfaces. Therefore, it is possible that the increased inhibitory potency of the GST-R fusion proteins on lipid-activated PKC
activity resulted from a nonselective sequestration of lipids required
for enzyme activation. Nonetheless, the GST fusion proteins inhibited
lipid-independent activated forms of PKC
(Fig. 5), indicating that
the GST fusion proteins can directly inhibit the catalytic domain of
the protein. Furthermore, in the absence of lipid, GST-R
inhibited
PKC
activity with a 2-fold greater potency than GST-R
or
GST-R
1-32 (Fig. 5), suggesting a modest degree of
isotype selectivity and a modest role for the pseudosubstrate region in
the inhibition of PKC activity by GST-R under these in vitro
conditions.
Although there is considerable evidence that the pseudosubstrate region
of PKC plays a role in the autoinhibition of the enzyme, our
observations raise the possibility that other regions in the PKC R
domain have significant PKC inhibitory activity. We find that the R
domain of PKC is a substantially more potent PKC inhibitor than is
the pseudosubstrate peptide PKC19-36 either in the presence of absence of lipid activators and that the R domain of PKC
retains significant PKC inhibitory activity after removal of the
pseudosubstrate site (Figs. 4 and 5). Additional support for this
hypothesis comes from observations of Riedel et al. (29). This group found that deletion of the N-terminal 153 amino acids of
bovine PKC
, including the pseudosubstrate and phorbol ester-binding domains, led to increased PKC constitutive activity and a loss of
phorbol ester-activated enzyme activity, consistent with an autoinhibitory function for the pseudosubstrate site. They also observed, however, that the truncated enzyme could be further activated
2.5-3-fold by the addition of Ca2+. Although they did not
comment on the significance of this finding, their results are
consistent with our suggestion that domains outside the pseudosubstrate
site contribute to the negative regulation of PKC by its R domain. One
candidate region that might function to inhibit PKC activity is the
pseudoRACK site, which is found in the R domains of different PKC
isoforms (30). The pseudoRACK site has sequence similarity to receptors
for activated PKC (RACKS) that participate in the targeting of
different PKC isoforms to selective substrates and is conserved in
different PKC isoforms. These pseudoRACK sites are proposed to interact
with RACK binding sites which overlap parts of the catalytic domain of
PKC and may contribute to enzyme autoinhibition.
Recently expression vectors encoding the regulatory domains of
different PKC isoforms have been used in transfection studies to assess
the roles of PKC in signal transduction. Expression vectors encoding
R, R
1, and R
were shown to affect the growth of rat embryo
fibroblasts (31, 32), whereas an expression vector encoding R
inhibited Golgi functions in mouse fibroblasts (33). In these studies,
the mechanisms by which the R domain proteins exerted their effects
were not explored, although they were presumed to act as dominant
inhibitors of PKC by interfering with PKC substrate utilization or with
interactions of PKC with its binding proteins. As demonstrated here,
PKC R domain proteins inhibit substrate phosphorylation with marked
specificity through direct effects on the enzyme and possibly through
effects associated with sequestration of lipid activators. Although
R
and R
exhibited only modest PKC isoform selectivity in
vitro (Fig. 5), it remains possible that R
and R
might
behave as isoform-specific inhibitors of PKC in vivo, for
example by preventing the translocation of the corresponding PKC
isozyme to specific subcellular compartments. Interestingly, R
and
R
each contain regions corresponding to RACK binding sites that have
been shown previously to interfere with the subcellular localization of
specific PKC isoforms (23, 34).
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ACKNOWLEDGEMENTS |
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We thank Dr. John Knopf (Genetics Institute,
Cambridge, MA) for the cDNAs for human PKC and mouse PKC
and
Dr. Michael Walsh (University of Calgary, Alberta) for purified
preparations of myosin light chain kinase and calmodulin.
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FOOTNOTES |
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* This work was supported by grants from the National Cancer Institute of Canada (with funds from the Canadian Cancer Society) and the Canadian Cystic Fibrosis Foundation (to B. P. S.), by a grant from the Northern Cancer Research Foundation (to A. M. P.), and by funds from the Ontario Cancer Treatment and Research Foundation (to A. M. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a postdoctoral fellowship award from the Canadian Cystic Fibrosis Foundation.
To whom correspondence should be addressed: Banting and Best
Dept. of Medical Research, University of Toronto, 112 College St.,
Toronto, Ontario, Canada M5G 1L6. Tel.: 416-978-6088; Fax: 416-978-8528.
1
The abbreviations used are: PKC, protein kinase
C; R domain, regulatory domain; C domain, catalytic domain; PKM,
proteolytically activated PKC; GST, glutathione
S-transferase; PMA, phorbol 12-myristate 13-acetate.
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REFERENCES |
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