Regulation of protein kinase C by the cytoskeletal protein calponin.

Previous studies from this laboratory have shown that, upon agonist activation, calponin co-immunoprecipitates and co-localizes with protein kinase Cepsilon (PKCepsilon) in vascular smooth muscle cells. In the present study we demonstrate that calponin binds directly to the regulatory domain of PKC both in overlay assays and, under native conditions, by sedimentation with lipid vesicles. Calponin was found to bind to the C2 region of both PKCepsilon and PKCalpha with possible involvement of C1B. The C2 region of PKCepsilon binds to the calponin repeats with a requirement for the region between amino acids 160 and 182. We have also found that calponin can directly activate PKC autophosphorylation. By using anti-phosphoantibodies to residue Ser-660 of PKCbetaII, we found that calponin, in a lipid-independent manner, increased auto-phosphorylation of PKCalpha, -epsilon, and -betaII severalfold compared with control conditions. Similarly, calponin was found to increase the amount of (32)P-labeled phosphate incorporated into PKC from [gamma-(32)P]ATP. We also observed that calponin addition strongly increased the incorporation of radiolabeled phosphate into an exogenous PKC peptide substrate, suggesting an activation of enzyme activity. Thus, these results raise the possibility that calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PKC.

noprecipitates with ERK1 and with PKC⑀ in ferret aorta homogenates, co-localizes in cells with ERK1 and PKC⑀, and binds activated PKC⑀ in a gel overlay assay (5). Calponin was speculated to function as an adaptor protein connecting the PKC cascade to the ERK cascade.
Three isoforms of calponin have been described in mammalian tissues. The isoform found in smooth muscle, h1 or basic calponin, is unusual in that it has an isoelectric point of 9.6. All isoforms of calponin contain an N-terminal conserved region called the calponin homology domain (CH domain). This region has been implicated in actin binding of many cytoskeletal proteins, but in the case of calponin, it has only been shown to bind acidic phospholipid (6,7) and ERK (8). The central region of calponin, which has homology to troponin I, is the primary actin binding region of basic calponin. The C-terminal half of calponin contains three repeats that have been shown to be composed of a second actin-binding site (9, 10) but otherwise have no known function.
Protein kinase C is a family of kinases regulated by calcium (conventional isoforms ␣, ␤, and ␥), by diacylglycerol (conventional isoforms and novel isoforms ␦, ⑀, , and ), and by less characterized means (atypical isoforms (i/ and ) and PKD/) (11). Other than atypical isoforms, the predominant isoform in ferret portal vein smooth muscle cells is PKC␣, whereas the dominate isoform in ferret aorta smooth muscle cells is PKC⑀ (12). Interestingly, ferret aorta, but not portal vein, can undergo contraction in response to PKC agonists in the absence of calcium. This contraction does not result from an increase in myosin light chain phosphorylation (5). While the exact targets of PKC that allow contraction in absence of increased intracellular calcium and myosin light chain phosphorylation are not known, the mitogen-activated protein kinase signaling pathways are one possibility (13).
All PKC isoforms contain catalytic and regulatory domains, as reviewed in Newton (11). The regulatory domain contains a pseudosubstrate region that binds the catalytic cleft inhibiting phosphotransferase activity of PKC. The regulatory domain also contains a C2 region that binds acidic phospholipid as well as calcium in the case of the conventional isoforms. The C1 region binds diacylglycerol in conventional and novel isoforms. It is binding of lipid to the C1 and C2 domains that releases the pseudosubstrate region from the catalytic cleft. Arginine-rich basic proteins, however, have been shown to activate PKC in the absence of lipid agonists (14). In the presence of lipid, basic peptides such as polylysine and polyarginine can inhibit PKC phosphorylation of substrate, the greatest inhibition being toward basic substrates with neutral substrates less affected (15).
The purpose of the present study was to identify the domains * This work was supported by United States Public Health Service Grants HL10026 (to B. L.), HL42293, and HL31704 (to K. G. M.) and Medical Research Council of Canada Grant MT-15037 (to A. M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ ‡ To whom correspondence and reprints should be addressed: Boston Biomedical Research Institute, 64 Grove St., Watertown, MA 02472. Tel.: 617-658-7761; Fax: 617-972-1759; E-mail: morgan@bbri.org. 1 The abbreviations used are: PKC, protein kinase C; GSTregPKC⑀-(V1/C2), expressed 45-kDa fragment of GSTregPKC⑀; PS, phosphatidylserine; CH domain, calponin homology domain; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; HPLC, high pressure liquid chromatography; BSA, bovine serum albumin; DPBA, 12-deoxyphorbol 13-isobutyrate; RACK, receptor for activated protein kinase C. of PKC and calponin that interact and, by doing so, clarify whether it is a kinase-substrate interaction or whether calponin might regulate the activation/targeting of PKC. It is shown here that calponin can interact with the regulatory domains of PKC and directly activate kinase activity in a lipid-independent manner, as assayed by 32 P incorporation into peptide substrates and by autophosphorylation experiments using a phospho-specific PKC antibody. These results, in combination with past studies, suggest that calponin has the potential to be an in vivo protein modulator of PKC activity.

MATERIALS AND METHODS
Reagents-Human recombinant PKC␣ and PKC⑀, produced from baculovirus in insect cells, were purchased from Panvera (Madison, WI). Rat brain PKC␣ was obtained from Sigma; Ser-PKC19-31 was obtained from Life Technologies Inc., and histone F2b was from U. S. Biochemical Corp. The phospho-PKCII␤ (Ser-660) antibody was purchased from New England Biolabs (Beverly, MA). Antibodies against the regulatory domains of PKC␣ and PKC⑀ were purchased from Life Technologies, Inc., and Transduction Laboratories (Lexington, KY), respectively. A monoclonal antibody raised against gizzard calponin (catalog number 6047) was obtained from Sigma and used in all cases except where calponin fragments were studied. In the latter case, a polyclonal antibody raised to gizzard calponin was provided by Dr. Katsuhide Mabuchi. This antibody recognized all calponin fragments. Expression vectors encoding the regulatory domain of human PKC␣ (16) and murine PKC⑀ (17) fused to glutathione S-transferase (GST) were supplied by Drs. Bernard P. Schimmer and Amadeo Parissenti. Phosphatidylserine was purchased from Avanti Polar Lipids (Birmingham, AL).
Calponin Proteolysis-Calponin was digested with ␣-chymotrypsin (Sigma) (19) with the Staphylococcus aureus V8 protease (Roche Molecular Biochemicals). V8 fragments were purified by reverse phase HPLC using a Vydac C18 column developed with a 0 -100% water acetonitrile gradient containing 0.1% trifluoroacetic acid. In some cases, HPLC-purified peptides were identified by N-terminal sequence analysis using an Applied Biosystems model 477A Pulsed Liquid Protein Sequencer and/or matrix-assisted laser desorption ionization-mass spectrometry on a Perspective Biosystems Voyager Elite Biospectrometry Workstation.
PKC Autophosphorylation and Kinase Assay-PKC autophosphorylation was carried out in the following solution A: 150 mM NaCl, 10 mM imidazole, 2 mM MgCl 2 , 0.5 mM CaCl 2 , 0.01% NaN 3 , 1 mM ATP, and 15 mM ␤-mercaptoethanol. PKC␤, PKC␣, or PKC⑀ (at a 20 nM concentration) was incubated in solution A with either 1 mg/ml PS and 10 Ϫ5 M DPBA, 25 M BSA, 25 M protamine, or 25 M calponin for 15 s at 37°C. The reaction was stopped by the addition of sample buffer followed by boiling. Samples were resolved by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride, and processed with antibody raised against phosphoserine 660. After visualization using enhanced chemiluminescence, blots were stripped and processed with a non-phosphospecific antibody. These values were used to correct for minor variations in loading. The effect of calponin and other agents on PKC autophosphorylation and the phosphorylation of exogenous PKC substrates were also monitored by analysis of kinase reaction products by gel electrophoresis and autoradiography using [␥-32 P]ATP as the phosphate donor. Assays were conducted in the absence or presence of 0.5 mM CaCl 2 and varying concentrations of calponin, histone, BSA, and the PKC substrate peptide Ser-PKC19-31. Each assay contained 50 mM Tris-HCl, pH 7.5, as buffer, 10 mM MgCl 2 , 100 M ATP, and 0.5 Ci of [␥-32 P]ATP. Reactions were stopped by addition of boiling 6ϫ gel loading buffer (300 mM Tris-HCl, pH 6.8, 0.6 M dithiothreitol, 12% SDS, 0.6% bromphenol blue, 60% glycerol) and loaded onto a large 12% acrylamide gel for resolution by SDS-polyacrylamide gel electrophoresis. Gels were fixed in ethanol/acetic acid/water (5:1:5) for 3 h with rocking and washed 6 times for 3 h with rocking in 10% acetic acid to remove unincorporated [␥-32 P]ATP. The gel was then placed in a heatsealable bag, sealed, and exposed to Kodak X-Omat film for varying times at Ϫ80°C with an intensifying screen.

RESULTS
Calponin Binds to the Regulatory Domain of PKC⑀ and PKC␣-As discussed "Materials and Methods," expression of the 68-kDa GSTregPKC⑀ construct also yielded an approximately 45-kDa by-product due to partial proteolysis. In overlay assays calponin bound to the 68-kDa full-length GSTregPKC⑀ (Fig. 1A, lane 3) and to an approximately 45-kDa fragment (Fig. 1A, lane 2). The 45-kDa fragment was determined by N-terminal sequencing (MSPIL) combined with mass spectroscopy (44.1 kDa) to contain full-length GST plus amino acids 1 to 154 -157 of PKC⑀. This corresponds to the V1 and C2 regions of the molecule but does not include the pseudosubstrate site or the C1 region. We have termed the 45-kDa fragment GSTreg-PKC⑀(V1/C2). Binding was minimal to GST alone (Fig. 1A, lane 1). Full-length (56 kDa) GSTregPKC␣ was also expressed. GSTregPKC␣ is shown in the left panel of Fig. 1B combined with a negative control, GST. The right panel suggests that calponin interacts far less with the 56-kDa GSTregPKC␣ band compared with that for GSTregPKC⑀(V1/C2).
Calponin Binds regPKC⑀ under Native Conditions-Co-sedimentation of GSTregPKC⑀(V1/C2) and calponin with lipid vesicles was used to test whether an association between the two proteins could be seen under native conditions. GSTregPKC⑀-(V1/C2) had a moderate affinity for phosphatidylserine (PS) vesicles and hence came down in the pellet (  The PKC Binding to Calponin Requires Residues 160 -182 of Calponin-In order to define the domains of calponin important for PKC binding, chymotryptic and V8 digests of calponin were performed. Whole calponin, limited (1:1000, 5 or 15 min) and extended (1:100, 1 h) chymotrypsin digest of calponin, and V8 (1:50, 4 h) digests were used in overlay assays to detect GSTregPKC⑀(V1/C2) binding. The identity of all fragments used was confirmed by N-terminal sequencing and mass spectrometry (Table I and Fig. 3B). Fig. 3A shows the proteins used in the overlay phase as follows: lane 1, whole calponin; lane 2, a limited chymotryptic digest calponin-(7-152) that migrates at 20 kDa and calponin-(183-261)/calponin-(183-290) that migrates between 13 kDa and 14 kDa; lane 3, an exhaustive chymotryptic digest containing mostly calponin-(7-91) that migrates at 13 kDa; and lane 4, HPLC purified calponin-(160 -291) from a V8 digest that migrates at about 14 kDa. The concentration of whole calponin or calponin fragments used in the overlay solutions ( Fig. 3C) was approximately 1.5 M. Fig.  3C shows overlay assays using the 45-kDa GSTregPKC⑀(V1/ C2) fragment on the membrane. Panel 1 demonstrates binding of whole calponin to GSTregPKC⑀(V1/C2). In contrast, although the limited chymotrypsin digest contained all fragments of the parent molecule and was added at the same concentration as whole calponin, no binding to GSTregPKC⑀(V1/C2) could be detected at these film exposures (Fig. 3C, panel 2). Similarly, essentially no binding to calponin-(7-91) containing most of the CH domain could be detected (Fig. 3C, panel 3). Binding was also not detected between GSTregPKC⑀(V1/C2) and the V8 fragment calponin-(1-154) ( Table I). On the other hand, when 1.5 M calponin-(160 -291) from the V8 digest was used in the overlay assay (Fig. 3C, panel 4), binding to GSTregPKC⑀(V1/ C2) was almost as great as that observed for whole calponin (Fig. 3C, panel 1). Since the calponin-(183-290) chymotryptic fragment did not bind GSTregPKC⑀(V1/C2), these results indicate that the region from 160 to 182 is required for binding the regulatory domain of PKC⑀. It is also interesting to note that no binding was observed to the CH domain, a region that has previously been shown to bind ERK (Fig. 3C, panel 3).
When the assay was reversed (Fig. 3D), i.e. the calponin fragments were on the membrane and GSTregPKC⑀(V1/C2) was in the overlay buffer and an anti-PKC⑀ regulatory domain antibody was used for detection, a similar localization of the PKC-binding domain could be made. GSTregPKC⑀(V1/C2) was found to consistently bind to the V8 fragment of calponin-(160 -291) (Fig. 3D, lane 1) and to a 31-kDa fragment of calponin (amino acids 1-272) only obtained upon very limited digestion of the molecule (Fig. 3D, lane 2). This 31-kDa fragment was N-terminally blocked, and the assumption was made that it started with the first residue of calponin. By using this as-sumption, it was calculated that the fragment extended to residue 272 at most. Removal of 20 amino acids would result in an approximately 2-kDa decrease in molecular mass; thus we referred to it here as calponin-(1-272) because this is the maximum possible length for an N-terminally blocked chymotryptic fragment of calponin that migrates with an apparent molecular mass of 31 kDa. Lane 2 of Fig. 3D also shows an approximately 20-kDa band that shows some binding to GSTregPKC⑀(V1/C2) in the corresponding overlay. The digest used for Fig. 3D, however, differed from that used in Fig. 3A. When the 20-kDa band from lane 2 of Fig. 3D was excised and sequenced, the results gave two amino acid residues in approximately equal amounts at each Edman cycle, indicating that the band was heterogeneous. Comparison of the amino acids released to the calponin sequence and the molecular weight by mass spectrometry (Table I) indicated that there were two major peptides in the band, calponin-(37-221) and calponin-(82-266).
To confirm that calponin-(160 -182) binds regPKC⑀, a peptide of this region was synthesized and added in increasing amounts to whole calponin in the overlay buffer. The overlay buffer was applied to a membrane containing GSTregPKC⑀(V1/ C2). As is seen in Fig. 3E, full-length calponin binding to GSTregPKC⑀(V1/C2) was inhibited at calponin:peptide ratios between 1:3 and 1:30. In contrast, a scrambled version of calponin-(160 -183) showed no competition with respect to the binding of calponin to GSTregPKC⑀(V1/C2).
Calponin Binds to the C2 Region of PKC␣ and PKC⑀-As shown in Fig. 1, calponin binds well to GSTPKC⑀(V1/C2), whereas binding to the whole GSTregPKC␣ construct is comparatively poor. The C2 domain of PKC⑀ is unusual in that is does not bind calcium and shows little homology to the C2 domains of conventional isoforms. Nevertheless, our results suggest that the V1 and/or C2 domain of PKC⑀ (but not PKC␣) may be involved in the binding of calponin. Alternatively, the possibility also exists that a strong C1-C2 interaction in PKC␣ prevents interaction with calponin. This hypothetical interaction may or may not exist in PKC⑀ because the C2 domain is N-terminal of the C1 domain in novel isoforms of PKC. In order to pursue further the issue of the calponin-binding domain within regPKC, truncated versions of the regulatory domain of PKC␣ were expressed and compared with GSTregPKC⑀(V1/C2) in an overlay assay. Since we had identified calponin-(160 -291) from the V8 digest to contain the domain binding reg-PKC⑀, membranes containing these constructs were incubated with an overlay solution containing 1.5 M calponin-(160 -291) (Fig. 4A). In other experiments whole calponin was used in the overlay buffer. Densitometry was performed on the bands in the overlays, normalizing for the intensity of the corresponding bands in the Coomassie-stained gels. The overlay experiment was performed twice with calponin-(160 -291) in the overlay solution and three times with whole calponin. With the calponin fragment, the staining with GSTregPKC⑀ averaged 2.9 times that for GSTregPKC␣ (Fig. 4A, 2nd versus 3rd lanes). With whole calponin in the overlay solution, the staining with GSTregPKC⑀ averaged 5.2 times that for GSTregPKC␣. Thus, the binding of both the peptide and calponin was consistently stronger for GSTregPKC⑀ than for GSTregPKC␣.
The binding of calponin-(160 -291) to the C1A, C1B/V2, C2, and C1/V2 fragments of GSTregPKC␣ averaged 0.8, 1.4, 1.5, FIG. 2. GSTregPKC⑀(C2) and calponin interact under native conditions. GSTregPKC⑀(V1/C2) binds to phosphatidylserine vesicles as seen by approximately equal amounts in the supernatant (s) and pellet (p) (1st set of lanes). The binding of calponin to phosphatidylserine vesicles is less pronounced (2nd set of lanes). However increased binding to phosphatidylserine vesicles is seen when both GSTregPKC⑀(V1/C2) and calponin are added to vesicles (3rd set of lanes). Results are typical of those obtained in five separate experiments. and 1.7 times that for GSTregPKC␣. The binding of whole calponin to the C1A, C1B/V2, C2, and C1/V2 fragments averaged 1.9, 3.8, 5.6, and 5.1 times that for GSTregPKC␣. Thus, consistently there was a pattern of lesser binding to the C1A fragment but greater binding to the C1B/V2 and C2 fragments.
The above results rule out the possibility that regPKC␣ lacks the calponin-binding site found in regPKC⑀; both C1-and C2based PKC␣ fragments clearly bound calponin. Given that the full PKC␣ regulatory domain shows less calponin binding in both full-length calponin and V8 fragment overlays, C1/C2 interactions may block the calponin-binding sites. This interpretation is consistent with the fact that the GSTregPKC⑀(V1/ C2) fragment binds well because it is a fragment of the full PKC⑀ regulatory domain where the C2 (calponin-binding) region is exposed. These data suggest that the calponin repeats may bind to the V1 or C2 regions of PKC⑀ and the C1B and C2 regions of PKC␣.
In order to determine the relative involvement of the V1 variable region of GSTregPKC⑀(V1/C2) fragment, a 100-fold excess of a peptide derived from the V-1 region of PKC⑀ (EAVS-LKPT; PKC⑀-(14 -21)) was used in an attempt to compete away calponin (Fig. 4C). This peptide has previously been shown to inhibit translocation of novel PKCs (20) and to inhibit contraction of ferret aorta cells (12). Neither the V1 peptide nor a scrambled V1 (LSETK PAV) peptide at a 100-fold excess were successful in reducing the amount of calponin bound to GSTregPKC⑀(V1/C2).
Calponin Increases PKC Autophosphorylation-Some basic proteins are able to increase PKC phosphotransferase activity in the absence of lipid activators (14). Since calponin binds to  the regulatory domain of PKC, the question arises as to whether calponin might activate PKC activity. Autophosphorylation has been described as a step required for maximal activation of PKC (19,20). Autophosphorylation was used as one assay for PKC activation. An antibody raised against a Ser-660 phosphorylation site of PKC␤II (phospho-pan-PKC antibody, New England Biolabs) was used to detect autophosphorylation. PKC was incubated with ATP for 15 s after which time the reaction was stopped by addition of sample buffer and boiling. Calponin, in the absence of lipids, induced a dramatic increase in autophosphorylation in PKC␤II during the first 15 s (Fig. 5A). Similar results were seen with protamine. Average densitometry results from three to four experiments are shown in Fig. 5C. This antibody is reported to cross-react with the homologous site on PKC␣ (New England Biolabs product literature). We also observed that it cross-reacted with PKC⑀ (Fig.  5B), although with somewhat lower affinity. To confirm specificity, we stained blots containing recombinant PKC␣, PKC␤II, and PKC⑀ with the phospho-pan-PKC antibody both in the absence (Fig. 5B, lanes 1-3) and in the presence of the peptide to which the antibody was raised (Fig. 5B, lanes 4 -6). The peptide was effective in competing away the signal in the presence of all three isoforms of PKC, consistent with the antibody recognizing the sites homologous to Ser-660 in all three cases. Similar results to those seen upon addition of calponin to PKC␤II were seen with PKC⑀ (Fig. 5C) and PKC␣ (Fig. 5D). Note that in all cases calponin increased PKC autophosphorylation as much as or more than DPBA plus PS.
Thus, it appears that calponin, in the absence of lipid cofactors, promotes the autophosphorylation of PKC at a key regulatory site homologous to Ser-660 in PKC␤II. To confirm that calponin promotes autophosphorylation of the kinase as well as to address the question of whether this leads to enhanced kinase activity with respect to exogenous substrates, we monitored the incorporation of 32 P into these proteins in vitro as shown in Fig. 6A. In order to minimize phosphorylation of calponin, these experiments employed the use of a mutant S175A calponin (a kind gift of Dr. Michael Walsh, University of Calgary). This mutant lacks the physiologically relevant phosphorylation site; however, it is reported that there are up to five PKC phosphorylation sites in calponin (21) explaining the phosphorylation observed in these experiments. Bands were obtained at the expected molecular weight for PKC, calponin, and the peptide. Note that the recombinant PKC␣ does possess a low level of basal activity consistent with the low level of basal autophosphorylation described in Fig. 5. However, the addition of calponin results in a clear increase in 32 P incorporation into not only PKC and calponin but also the peptide substrate PKC19 -31 (compare lanes 3 and 4 with lanes 5 and 6 in Fig. 6A). The phosphorylation of calponin to some degree also indicates that indeed an exogenous substrate can be phosphorylated, but also raises the question of whether the phenomenon might simply be a substrate effect.
Since calponin is a very basic protein and highly basic PKC substrate peptides are known to activate PKC kinase activity, the experiment shown in Fig. 6B was performed. In this experiment, a constant amount of Ser-PKC19-31 (2 M) was present in all lanes. As a reference, lanes 11-14 show the addition of PKC, calcium, and BSA at increasing concentrations. Lane 1 shows the addition of only PKC and lane 2 only PKC and calcium. Lanes 3-6 depict the effect of adding increasing amounts of mutant calponin to the reactions. Mutant calponin increased the phosphorylation of Ser-PKC19-31 as well as the phosphorylation of calponin itself and PKC (apparent in longer exposures, Fig. 6C). In comparison, when equivalent additional amounts of Ser-PKC19-31 were added in lanes 7-10, there was also increased phosphorylation of substrate peptide but no incorporation of 32 P into PKC itself (even at long exposures, Fig. 6C). Ser-PKC19-31 is a highly, positively charged peptide substrate that like protamine has been described previously to be capable of activating PKC in the absence of lipid cofactors (14,22). The experiment shown in Fig. 6D was performed using purified rat brain PKC␣. Interestingly, by using this source of PKC␣, the addition of mutant calponin (lanes 3 and 4) caused phosphorylation of the substrate peptide well above that caused by equimolar BSA (lanes 5 and 6), and in contrast, there was little incorporation of phosphate into mutant calponin. Lanes 7 and 8 show that histone, another very basic PKC substrate, has similar effects to that of calponin in equimolar amounts with respect to inducing phosphorylation of peptide substrate (but not with respect to inducing autophosphorylation of PKC-data not shown).

Calponin and GSTregPKC⑀ Do Not Enhance or Interfere with
Binding of Actin to Each Other-Imaging studies from our laboratory suggest that calponin translocates to cortical actin upon activation (3,4). PKC⑀ has also been reported to bind actin via a region in C1 (23,24). The non-actin binding region of PKC⑀ (C2 domain) can bind the calponin repeats, which have been suggested to be a secondary actin binding region of calponin (10). Thus it is possible that either synergy or competition could exist in actin binding. Mixtures of F-actin, calponin, and full-length GSTregPKC⑀ were subjected to high speed sedimentation (Fig. 7). Neither calponin (1st set of lanes), GSTreg-PKC⑀ (2nd set of lanes), nor a combination thereof (3rd set of lanes) sedimented in the absence of actin. In the presence of actin, binding of calponin was readily detected in the pellet (6th and 9th set of lanes) as well as a small amount of GSTregPKC⑀ (5th and 7th set of lanes). Calponin, added to GSTregPKC⑀ and actin, neither enhanced nor inhibited the binding of GSTreg-PKC⑀ to actin (8th and 10th set of lanes). Thus calponin does not enhance the targeting of PKC⑀ to actin. Conversely, it also does not inhibit the binding of PKC⑀ to actin, indicating that a ternary complex, for example in a signaling scaffold, is possible. DISCUSSION The main findings of the present study are that the actinbinding protein calponin binds the regulatory domains of PKC⑀ and PKC␣ and that calponin activates PKC, as measured by its autophosphorylation and its ability to phosphorylate exogenous substrates. These interactions between an actin-binding protein and a kinase are of some interest because of emerging evidence that cytoskeletal proteins may play a regulatory role in signal transduction.
With respect to the finding that calponin binds the regulatory domain of PKC, the question arises as to whether calponin might function similar to a receptor for activated protein kinase C (RACK) (25). Calponin and PKC have been found to translocate with indistinguishable time courses in ferret portal vein cells and to co-immunoprecipitate from ferret aorta homogenate(s) (3,5). RACK1 was first characterized as a protein that bound activated PKC␤ (25). The portion of RACK1 responsible for binding PKC contains WD40 repeats and binds a region of PKC contained in but not necessarily confined to C2. The coatamer protein ␤-COP was subsequently also found to be a receptor for activated PKC⑀ (26) and hence a type of RACK. ␤-COP also contains WD40 repeats. Calponin has regions similar to WD40 repeats in the region we have shown to bind PKC. Like the ␤-propeller structures formed by WD40 repeats, calponin repeats are predicted to be composed entirely of ␤-sheet and ␤-turn (27). Like calponin, RACK1 has been observed to co-translocate with PKC in agonist-stimulated Chinese hamster ovary cells (28). It was suggested that RACK1 functions as a PKC␤II-shuttling protein targeting active PKC to such targets as ␤-integrin and pleckstrin homology domain-containing proteins. RACKs, by definition, bind only to the active PKC molecule. We have shown here that calponin also binds the inactive PKC molecule; so, in this respect it cannot, strictly speaking, be considered a RACK; however, it is of interest that binding of RACKs to active conformations of PKC has been reported to enhance substrate phosphorylation (29). On the other hand, in some regards calponin might be considered as a possible candidate for a "STICK" (30), a PKC substrate protein that interacts with PKC and is implicated in PKC targeting. However,thebindingofSTICKStoPKCisgenerallyphospholipiddependent, and this does not seem to be the case for calponin.
Additionally, it occurs that basic regions of calponin may be presented to regPKC to cause activation in a manner similar to that described previously for the basic peptide protamine (14). In a survey of the basic peptides protamine, polyarginine, histone, and polylysine, the ability to induce autophosphorylation correlated roughly with the ability to bind a mixture of conventional PKCs (14), suggesting that preference for arginine in the activation of PKC exists simply because arginine-rich peptides bind better to PKC. Calponin does not have any long argininerich stretches like protamine, although it is highly basic with short arginine-rich regions. It has been speculated that the ability of arginine-rich proteins to interact with PKC in the absence of activators allows PKC activity to be regulated by accessibility to arginine-rich protein domains in the cell and distal from cell membranes (14). Interestingly, calcium and diacylglycerol were shown to have little influence on the V max and V max /K m for PKC␣ phosphorylation of the highly argininerich ⑀ peptide, whereas substitution of lysine for arginine rendered phosphorylation dependent on the lipid agonist (22). How the arginines in calponin are presented to PKC cannot reliably be predicted. It might be that the same agonists that cause calponin to translocate (3,4) also cause it to be presented to PKC in such a way as to activate PKC or hold it in the activated state in conjunction with more traditional activators.
In the present study, autophosphorylation was used as an assay of PKC activation. The phosphorylations required for PKC activation have been best described for the ␤II conventional isoform. Transphosphorylation of PKC␤II occurs initially at Thr-500 in the activation loop by a kinase suggested to be PDK (31). This is followed by phosphorylation of Thr-641 and Ser-660 (32). The phosphorylation at Ser-660 was originally thought to be an autophosphorylation but recently has been shown to require extrinsic cofactors (33). The question arises as to whether calponin could function as such a cofactor in smooth muscle cells. These phosphorylation sites are conserved among all isoforms except and (32). Phosphorylation of PKC is essential for optimal catalytic activity (31,34), and dephosphorylation has been suggested to prime PKC for degradation (35). There is also evidence that autophosphorylation influences PKC localization (36). Whether PKC autophosphorylation is regulated or is constitutive after synthesis in smooth muscle cells is not yet clear.
In the present study we have also shown that the addition of calponin to PKC in the absence of lipid cofactors leads to increased phosphorylation of peptide substrates. It is of interest that previous studies have shown that highly basic peptide substrates such as Ser-PKC19-31 also can increase PKC kinase activity in the absence of lipid cofactors (22). However, calponin differs from Ser-PKC19-31 by being a physiologically relevant protein that is present in high concentrations in differentiated smooth muscle cells, co-translocates with PKC, and is co-immunoprecipitated with PKC (5). Furthermore, neither Ser-PKC19-31 nor histone enhanced phosphorylation of PKC itself but calponin did.
Since calponin and PKC co-translocate in at least two cell types (3,5), the question arises as to whether the binding of calponin to PKC helps to target PKC. Because calponin does not enhance binding of regPKC⑀ to actin, it is unlikely that calponin helps target PKC⑀ to actin. On the other hand, calponin does enhance binding of the regulatory domain of PKC⑀ to PS vesicles, suggesting that an event that would cause calponin to translocate to the cell membrane might also result in PKC binding with higher affinity to the cell membrane perhaps even in the absence of diacylglycerol.
However, other cortical targets such as the caveoli cannot be ruled out at this point. Caveolin appears to be a receptor for PKC in COS cells. Peptides corresponding to the scaffolding domain of caveolin inhibit autophosphorylation, peptide phosphorylation, and phorbol ester binding of COS cells (37). Active PKC␣ and PKC⑀ have also been found in the caveolar fraction of cardiac myocytes in response to the phorbol ester phorbol 12-myristate 13-acetate and endothelin but not in response to the inactive phorbol ester 4␣-phorbol 12-myristate 13-acetate (38). It is interesting that a 36-kDa protein was found in the caveolar fraction of cardiac myocytes that might be neutral calponin, the cardiac isoform of calponin (38). This raises the possibility that calponin might serve to target to caveolin.
Calponin may also target substrate to PKC. Perhaps it is possible that the N-terminal half of calponin is recruiting substrate to the catalytic cleft of PKC. It should be further noted that ERK, which is downstream of PKC in the phenylephrineactivated signaling cascade in smooth muscle (13), binds to the CH domain in the N-terminal half of calponin. PKC is known to be capable of directly phosphorylating Raf, an upstream activator of mitogen-activated protein kinase/ERK kinase. Thus, calponin may facilitate the formation of signaling complexes of Raf, mitogen-activated protein kinase/ERK kinase, mitogenactivated protein kinase, and PKC.
We found that the C2 and C1B domains of regPKC bind the calponin repeats. It is hard to make rigorous predictions as to how calponin may control the activity of PKC by these interactions when essentially no information is available on the structure of the calponin repeats. Calponin has been reported to contain between 17 (39) and 32% ␤-structure (40), yet the CH domain is largely composed of ␣-helix, see for example Ref. 41. Thus, the repeats probably contain a fair amount of ␤-structure. Calponin can exist as an extended rod or a collapsed coil (40). A proposed diagrammatic model is shown in Fig. 8. Binding of calponin to PKC␣ C2 and C1B (but not the whole regulatory domain construct) as well as the ability of calponin to activate whole PKC␣ suggests that C2 and C1B may interact with each other in the GSTregPKC␣ construct. The V8 peptide calponin-(160 -291) may be able to interact marginally with GSTregPKC␣, whereas whole calponin does not simply because there are more steric constraints in whole calponin. The interaction of calponin with the PKC regulatory domain may not only hold PKC in the active conformation but also influence the interaction of substrates with the catalytic cleft.
In summary, 1) calponin at residues 160 -291 binds to the regulatory domains of conventional and novel PKC isoforms ␣ and ⑀, respectively. Comparatively better calponin binding to the C2 and C1B domains of PKC␣ than to the whole regulatory domain suggests that these adjacent domains may interact in the intact regulatory domain. 2) Calponin increases PKC autophosphorylation as well as the incorporation of phosphate into exogenous substrates. 3) Calponin, a protein that co-localizes with PKC and is present in high concentrations in differentiated cells, has the potential to facilitate PKC activation and signaling.