Inhibitory Properties of the Regulatory Domains of Human Protein Kinase Calpha and Mouse Protein Kinase Cepsilon *

Amadeo M. ParissentiDagger §, Angie F. KirwanDagger , Sandra A. Kim, Concettina M. Colantonio, and Bernard P. Schimmerpar

From the Dagger  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

    ABSTRACT
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Abstract
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Materials & Methods
Results
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References

Two fusion proteins in which the regulatory domains of human protein kinase Calpha (Ralpha ; amino acids 1-270) or mouse protein kinase Cepsilon (Repsilon ; 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 Calpha (PKCalpha ) and other protein kinases in vitro. Both GST-Ralpha and GST-Repsilon but not GST itself potently inhibited the activities of lipid-activated rat brain PKCalpha . 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-Ralpha and GST-Repsilon , on a molar basis, were 100-200-fold more potent inhibitors of PKCalpha activity than was the pseudosubstrate peptide PKC19-36. In addition, a GST-Ralpha fusion protein in which the first 32 amino acids of Ralpha were deleted (including the pseudosubstrate sequence from amino acids 19-31) was an effective competitive inhibitor of PKCalpha activity. The three GST-R fusion proteins also inhibited protamine-activated PKCalpha and proteolytically activated PKCalpha (PKM), two lipid-independent forms of PKCalpha ; 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 PKCalpha 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 PKCalpha catalytic activity through direct interactions. These data indicate that the R domains of PKCalpha and PKCepsilon are specific inhibitors of protein kinase Calpha activity and suggest that regions of the R domain outside the pseudosubstrate sequence contribute to autoinhibition of the enzyme.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
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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 PKCalpha 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 PKCalpha (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 PKCalpha R domain inhibited PKC-mediated phenotypes in intact yeast cells and suggested that PKCalpha 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 PKCalpha and mouse PKCepsilon inhibit PKC activity. We demonstrate that the R domains of PKCalpha and PKCepsilon potently inhibit PKCalpha 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 Ralpha does not markedly diminish its ability to inhibit PKC holoenzyme or proteolytically activated PKCalpha (PKM) activity. On this basis, we suggest that R domain sequences outside the pseudosubstrate region of PKC may contribute significantly to enzyme autoinhibition.

    MATERIALS AND METHODS
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Introduction
Materials & Methods
Results
Discussion
References

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 PKCalpha substrate [Ser-25]PKC19-31 (RFARKGSLRQKNV) and the PKCalpha pseudosubstrate peptide PKC19-36 (RFARKGALRQKNVHEVKN) were obtained from Canadian Life Technologies Inc. (Burlington, Ontario, Canada). LRRASLG (Kemptide), PMA, purified rat brain PKCalpha , and isopropylthio-beta -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 [gamma -32P]ATP (111 TBq/mmol) was from Mandel Scientific (Guelph, Ontario, Canada).

Preparation of GST-Ralpha , GST-Ralpha Delta 1-32, and GST-Repsilon -- A fusion protein in which amino acids 1-270 of human PKCalpha was fused in frame to GST (GST-Ralpha ) 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 Ralpha were deleted (GST-Ralpha Delta 1-32) was prepared by cloning a DraIII-EcoRI fragment of the human PKCalpha 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 PKCepsilon (amino acids 1-385) was prepared by cloning an NcoI-PvuII fragment from mouse PKCepsilon cDNA in pMT2epsilon (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 DH5alpha 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-Ralpha , GST-Ralpha Delta 1-32, and GST-Repsilon 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 PKCepsilon (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-- PKCalpha catalytic activity was measured by monitoring the transfer of 32P from [gamma -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 [gamma -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 PKCalpha . 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.

Other Ser/Thr kinase activities and src Tyr kinase activity were assayed by measuring the transfer of 32P from [gamma -32P]ATP to acceptor peptide substrates under standard conditions for each enzyme. In each case, peptide substrate concentrations were equal to experimentally determined Km values. cAMP-dependent protein kinase activity was measured by incubating the catalytic subunit of the enzyme (25 ng) with 20 µM Kemptide (15) for 5 min at 30 °C in a reaction (50 µl) containing 5 mM MgCl2, 1 mM EGTA, 100 µM [gamma -32P]ATP (1 µCi), and 20 mM Tris-HCl, pH 7.5. cGMP-dependent protein kinase assays were conducted under identical conditions to that described for cAMP-dependent protein kinase except that 4 µM Kemptide and 2 µM cGMP were present in the reaction. Mitogen-activated protein kinase activity was assayed by incubating the enzyme (13.5 ng) with 1.5 mM mitogen-activated protein kinase substrate peptide for 5 min at room temperature in a reaction (25 µl) containing 12.5 µM beta -glycerolphosphate, 7.5 mM MgCl2, 0.5 mM EGTA, 2 mM dithiothreitol, 50 µM NaF, 0.5 mM sodium orthovanadate, 50 µM [gamma -32P]ATP (1 µCi), and 12.5 mM 3-(N-morpholino)propanesulfonic acid, pH 7.2 (16). Myosin light chain kinase activity was measured by incubating the enzyme (25 ng) with 20 µM K-MLC11-23 substrate peptide (17) for 4 min at 30 °C in a reaction (100 µl) containing 4 mM MgCl2, 60 mM KCl, 150 µM CaCl2, 1 mg of calmodulin, 200 µM [gamma -32P]ATP (0.8 µCi), and 25 mM Tris-HCl, pH 7.5. Casein kinase II activity was measured by incubating the enzyme (0.5 milliunit) with 500 µM substrate peptide (18) for 5 min at 37 °C in a reaction (50 µl) containing 130 mM KCl, 10 mM MgCl2, 4.8 mM dithiothreitol, 50 µM [gamma -32P]ATP (1.2 µCi), and 20 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.9. src kinase assays were conducted by incubating 6 units of purified enzyme with 100 µM substrate peptide (cdc26-20) for 10 min at room temperature in a reaction (50 µl) containing 2.5 mM MnCl2, 0.5 mM EGTA, 0.625 mM sodium orthovanadate, 31 mM sodium acetate, 31 mM MgCl2, 112 µM [gamma -32P]ATP (1 µCi), and 25 mM Tris-HCl, pH 7.2. Reactions were terminated and processed as described above for PKC.

    RESULTS
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Abstract
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Materials & Methods
Results
Discussion
References

Isolation and Characterization of GST-Ralpha , GSTRalpha Delta 1-32, and GST-Repsilon -- Purified preparations of GST-Ralpha , GST-Ralpha Delta 1-32, and GST-Repsilon 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-Ralpha , 52 kDa for GST-Ralpha Delta 1-32, and 68 kDa for GST-Repsilon ) including 26 kDa for the GST component (Fig. 1). GST-Ralpha and GST-Ralpha Delta 1-32 also reacted with the antibody that recognizes the alpha , beta , and gamma  isoforms of rat PKC (14), whereas GST-Repsilon reacted with the PKCepsilon antibody (Fig. 1), thus confirming the identity of the fusion proteins. The GST-Repsilon 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-Repsilon , as it is strongly immunoreactive with the monoclonal PKCepsilon antibody and has a molecular weight equal to the R domain of PKCepsilon without GST attached.


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Fig. 1.   Electrophoretic and immunoblot analysis of GST-R fusion proteins. In A, preparations of GST-Ralpha , GST-Ralpha Delta 1-32, and GST-Repsilon were electrophoresed on SDS-polyacrylamide gels and stained with Coomassie Brilliant Blue. Marker proteins (M) of known molecular mass were included as size standards. In B, samples electrophoresed on SDS-polyacrylamide gels were transferred to nitrocellulose and immunoblotted with either a rat brain PKC polyclonal antibody (Anti-PKC alpha ,beta ,gamma serum) or a monoclonal PKCepsilon antiserum (Anti-PKC epsilon  serum).

Effects of GST-Ralpha and GST-Repsilon on PKCalpha Activity and on the Activities of Various Other Ser/Thr and Tyr Kinases-- Previously we demonstrated that GST-Ralpha is a potent competitive inhibitor of yeast-expressed bovine PKCalpha and yeast-expressed rat PKCbeta -I (12). To assess whether the inhibition of protein kinase activity by GST-Ralpha was PKC-specific, we examined the ability of GST-Ralpha to inhibit the activities of several Ser/Thr and Tyr kinases. As shown in Fig. 2A, GST-Ralpha potently inhibited the activity of purified rat brain PKCalpha (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-Ralpha stimulated the activities of cGMP-dependent protein kinase and myosin light chain kinase, although the mechanism of this activation by GST-Ralpha is unknown. To determine whether the effects of GST-Ralpha were isozyme-specific, we examined the effects of GST-Repsilon on PKCalpha activity. GST-Repsilon (Fig. 2B) also inhibited PKCalpha 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-Ralpha and GST-Repsilon each competitively inhibited PKCalpha 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 PKCalpha 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 PKCalpha and PKCepsilon 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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Fig. 2.   Effects of GST-Ralpha and GST-Repsilon on the catalytic activities of various protein kinases. Increasing concentrations of purified GST-Ralpha (A) and GST-Repsilon (B) were examined for their abilities to inhibit the catalytic activities of purified rat brain PKCalpha (bullet ), cAMP-dependent protein kinase (black-square), casein kinase II (black-triangle), cGMP-dependent protein kinase (square ), myosin light chain kinase (black-diamond ), mitogen-activated protein kinase (diamond ), and src kinase (triangle ) at substrate concentrations equal to the experimentally determined Km for each enzyme. Activities are presented as percentages of the total activity present in the absence of fusion protein and are representative of three independent experiments.


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Fig. 3.   Competitive inhibition of PKCalpha by GST-R fusion proteins and the pseudosubstrate peptide PKC19-36. GST-Ralpha (A), GST-Repsilon (B), and GST-Ralpha Delta 1-32 (C) were evaluated for their abilities to inhibit PKCalpha as a function of substrate ([Ser-25]PKC19-31) concentration. Purified preparations of each fusion protein (bullet ) or GST alone (open circle ) were added to enzymatic assays for PKCalpha activity at final concentrations of 1.0 µM. Results are expressed as double-reciprocal plots of substrate concentration (µM) versus enzymatic activity expressed as picomoles of 32P transferred from [gamma -32P]ATP to [Ser-25]PKC19-31/min/µg of protein.


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Fig. 4.   Inhibitory effects of GST-R fusion proteins and the pseudosubstrate peptide PKC19-36 on PKCalpha activity. The catalytic activity of purified rat brain PKCalpha was measured in the presence of increasing concentrations of GST-Ralpha (black-triangle), GST-Repsilon (square ), GST-Ralpha Delta 1-32 (black-square), PKC19-36 (bullet ), or GST alone (open circle ). PKC activity is expressed as a percentage of the maximal activity (340 ± 35 nmol of 32P transferred from [gamma -32P]ATP to [Ser-25]PKC19-31/min/mg of protein, n = 4) obtained in the absence of inhibitor.

Role of the Pseudosubstrate Sequence in the Inhibition of PKCalpha by GST-Ralpha -- Although the R domains of PKCalpha and PKCepsilon have different pseudosubstrate sequences, their lack of selectivity for PKC isozymes (Fig. 2) could be reconciled, as four amino acid residues within the PKCalpha and PKCepsilon pseudosubstrate sequences are conserved (19), including Arg-22, which is essential for the inhibitory activity of the PKCalpha pseudosubstrate peptide (20). On a molar basis, however, the GST-Ralpha and GST-Repsilon 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 PKCalpha 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 PKCalpha R domain were deleted and the remaining sequence (amino acids 33-270) was linked in frame with GST. This protein, GST-Ralpha Delta 1-32, which lacks the pseudosubstrate sequence, retained the ability to inhibit the activity of purified rat brain PKCalpha over a concentration range similar to that seen for GST-Ralpha and GST-Repsilon (Fig. 4); inhibition of PKCalpha activity by GST-Ralpha Delta 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 PKCalpha Activities-- We next examined the effects of GST-Ralpha , GST-Ralpha Delta 1-32, and GST-Repsilon on protamine-activated PKCalpha and on PKM to distinguish direct effects of the fusion proteins on PKCalpha 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 PKCalpha 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-Ralpha inhibited protamine-activated PKCalpha in a concentration-dependent manner with an IC50 value of 650 ± 80 nM (n = 3). GST-Ralpha Delta 1-32 and GST-Repsilon 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 PKCalpha . 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 PKCalpha 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 PKCalpha than was the pseudosubstrate peptide PKC19-36, as the pseudosubstrate peptide did not inhibit protamine-activated PKCalpha (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 PKCalpha activity with potencies that are 100-200-fold greater than PKC19-36. GST-Ralpha Delta 1-32 and GST-Repsilon 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 PKCalpha 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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Fig. 5.   Inhibitory effects of GST-R fusion proteins on protamine-activated PKCalpha and PKM activities. Increasing concentrations of GST-Ralpha (black-triangle), GST-Repsilon (square ), GST-Ralpha Delta 1-32 (black-square), or PKC19-36 (bullet ) were evaluated for their abilities to inhibit protamine-activated PKCalpha from rat brain (A). Reactions (100 µl) contained 100 µM [gamma -32P]ATP (0.5 µCi), 10 mM MgCl2, 250 µM EGTA, 30 mM NaCl, 4 µM GST, 2.1 µM protamine, and 5 milliunits of purified PKCalpha . Samples were assayed for PKC activity as described under "Materials and Methods." Results are expressed as a percentage of the maximal activity obtained in the absence of inhibitor ± SE (n = 3). Increasing concentrations of GST-Ralpha Delta 1-32 (black-square) and GST-Repsilon (square ) also were tested for their effects on the activity of the proteolytically activated form of PKCalpha , PKM (B). PKM assays were conducted as described under "Materials and Methods." Results are expressed as nanomoles of 32P transferred from [gamma -32P]ATP to [Ser-25]PKC19-31/min/mg of protein.

    DISCUSSION
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Materials & Methods
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As shown in this in vitro study, GST-Ralpha and GST-Repsilon 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 PKCalpha 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 PKCalpha 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 PKCalpha (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 PKCalpha (Fig. 5), indicating that the GST fusion proteins can directly inhibit the catalytic domain of the protein. Furthermore, in the absence of lipid, GST-Ralpha inhibited PKCalpha activity with a 2-fold greater potency than GST-Repsilon or GST-Ralpha Delta 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 PKCalpha 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 PKCalpha 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 PKCalpha , 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 Ralpha , Rbeta 1, and Rdelta were shown to affect the growth of rat embryo fibroblasts (31, 32), whereas an expression vector encoding Repsilon 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 Ralpha and Repsilon exhibited only modest PKC isoform selectivity in vitro (Fig. 5), it remains possible that Ralpha and Repsilon 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, Ralpha and Repsilon 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).

    ACKNOWLEDGEMENTS

We thank Dr. John Knopf (Genetics Institute, Cambridge, MA) for the cDNAs for human PKCalpha and mouse PKCepsilon and Dr. Michael Walsh (University of Calgary, Alberta) for purified preparations of myosin light chain kinase and calmodulin.

    FOOTNOTES

* 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.

par 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 PKCalpha ; GST, glutathione S-transferase; PMA, phorbol 12-myristate 13-acetate.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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