Originally published In Press as doi:10.1074/jbc.M410229200 on November 9, 2004
J. Biol. Chem., Vol. 280, Issue 1, 644-653, January 7, 2005
Regulation of Outside-in Signaling in Platelets by Integrin-associated Protein Kinase C
*
Charito S. Buensuceso
,
Achim Obergfell,
Alessandra Soriani,
Koji Eto,
William B. Kiosses,
Elena G. Arias-Salgado,
Toshiaki Kawakami¶, and
Sanford J. Shattil||
From the
Hematology-Oncology Division, Department of Medicine, University of California San Diego, La Jolla, California 92093, Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, and the ¶Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
Received for publication, September 7, 2004
, and in revised form, November 8, 2004.
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ABSTRACT
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Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin 
IIb
3 in platelets, but the responsible PKC isoforms and mechanisms are unknown. IIb3 interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether IIb3 might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKC co-immunoprecipitated with IIb3 in response to the interaction of platelets with soluble or immobilized fibrinogen. PKC recruitment to IIb3 was accompanied by a 9-fold increase in PKC activity in IIb3 immunoprecipitates. RACK1, an intracellular adapter for activated PKC, also co-immunoprecipitated with IIb3, but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKC recruitment to IIb3 and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKC spread poorly on fibrinogen, despite normal agonist-induced fibrinogen binding. In a Chinese hamster ovary cell model system, adhesion to fibrinogen caused green fluorescent protein-PKCI to associate with IIb3 and to co-localize with it at lamellipodial edges. These responses, as well as Chinese hamster ovary cell migration on fibrinogen, were blocked by the deletion of the 3 cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to 3. These studies demonstrate that the interaction of IIb3 with activated PKC is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through IIb3. Furthermore, the studies extend the concept of IIb3 as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton.
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INTRODUCTION
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In addition to their roles in cell adhesion, integrins transmit signals in both directions across the plasma membrane to regulate cytoskeletal organization, motility, and other anchorage-dependent cellular responses (1). In platelets, for example, IIb3 responds to "inside-out" signals with an increase in affinity for cognate ligands such as fibrinogen that bridge platelets to each other and mediate platelet adhesion to sites of vascular damage. In turn, ligand binding to IIb3 triggers outside-in signals that promote cytoskeletal changes necessary for full platelet aggregation and spreading (2, 3). Bidirectional IIb3 signaling is controlled, in part, by specific intracellular proteins that interact with the relatively short cytoplasmic tails of IIb or 3. For example, binding of talin to 3 is a final common step in the cellular modulation of IIb3 affinity (4), and binding of c-Src and Syk protein-tyrosine kinases to 3 is required for platelet spreading on fibrinogen (5, 6). Several other intracellular proteins, for example, CIB and 3-endonexin, can also bind to IIb or 3 tails, respectively, and may influence IIb3 functions (7-9). However, the full complement of intracellular proteins that are capable of interacting directly or indirectly with IIb3 is unknown.
The protein kinase C (PKC)1 subfamily of AGC serine/threonine kinases has been implicated in integrin function or dynamics in many cell types (10). In platelets, PKC is thought to regulate IIb3 affinity, based on the stimulatory effects of phorbol esters, which bind to PKC C1 domains, and the blocking effects of broad spectrum PKC inhibitors (3). However, the lack of specificity of these compounds limits data interpretation and does not permit conclusions about the roles of specific PKC isoforms (11). PKCs have been categorized as classical (diacylglycerol- and Ca2+-regulated through C1 and C2 domains, respectively), novel (Ca2+-independent but diacylglycerol-regulated), or atypical (Ca2+- and diacylglycerol-independent) (12, 13). Platelets are reported to contain members of all three classes of PKC isozymes (14-21), as well as the related protein kinase D (22). Recently, experimental tools have become available to study specific PKC isoforms in cells, including overexpression, gene targeting and gene knock-down strategies, and molecular imaging (23-25). Some of these tools are potentially relevant to platelets.
PKC function depends on the maturation of catalytic activity of the enzyme through phosphorylation and PKC binding to membranes or specific proteins. The latter interactions place PKC in proximity to substrates and relieve autoinhibitory restraints imposed by the binding of a pseudosubstrate sequence to the active site (12, 13, 26). One group of PKC targeting proteins has been termed RACK, which binds selectively to activated PKCs (27). The best characterized protein of this group is RACK1, a 36-kDa protein composed of seven WD40 repeats. RACK1 was originally identified based on its interaction with activated PKC and subsequently shown to interact with certain other PKC isoforms and with several other proteins, most notably integrin cytoplasmic tails and c-Src (see Fig. 1A) (28-30).
Given the potential for the cytoplasmic tails of IIb3 to serve as binding sites for signaling molecules and the apparent functional relationships between PKC and IIb3, the present studies were carried out to determine whether specific PKCs associate with IIb3 and if so to determine what the functional relevance of the association is. By using human and mouse platelets and a CHO cell model system, the results show that one particular PKC isoform, PKC, inducibly associates with IIb3 in response to fibrinogen binding to cells. The PKC/IIb3 interaction appears to be mediated by RACK1 and is required for cytoskeletal reorganization and platelet spreading on fibrinogen, but it is dispensable for the affinity modulation of IIb3.
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EXPERIMENTAL PROCEDURES
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Reagents—Monoclonal antibodies to PKC, -, -
, -
, -
, -
, and -
were from Transduction Laboratories (Lexington, KY), and antibodies specific for PKCI, PKCII, or cortactin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The phospho-specific antibody to c-Src, tyrosine 418, was from BIOSOURCE, and anti-phosphotyrosine antibodies 4G10 and PY20 were from Upstate Biotechnology (Lake Placid, NY) and Transduction Laboratories, respectively. Monoclonal antibody 327 to c-Src was from Dr. Joan Brugge (Harvard Medical School), and monoclonal antibody D57 (specific for IIb3) and polyclonal antibody Rb8053 (specific for integrin 3) were from Dr. Mark H. Ginsberg (University of California San Diego, La Jolla, CA). Horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad, and Cy-5- and TRITC-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Rhodamine-phalloidin was from Molecular Probes (Eugene, OR), purified human fibrinogen was from Enzyme Research Laboratories, Inc. (South Bend, IN), and protein A-Sepharose was from Amersham Biosciences. Cell-permeable PKC antagonist peptide p99 was purchased from Stanford University (31). Src kinase inhibitor SU6656 was from SUGEN, Inc. (South San Francisco, CA). Platelet agonists were from Sigma, except for convulxin, which was a gift from Dr. Steve Watson (University of Birmingham, Birmingham, UK).
Cell Culture, Plasmids, and Transfections—A5 CHO cells stably expressing IIb3 were maintained in culture as described previously (32). Transfections were performed at 70-80% confluency using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Twenty-four hours later, the concentration of fetal calf serum was decreased from 10 to 0.5%, and cells were cultured for another 24 h before being used in functional assays. Mammalian expression plasmids included pEGFP-PKCI (a chimera containing GFP fused to the N terminus of full-length PKCI (a gift from Dr. Stephen S. G. Ferguson, Robarts Research Institute, Ontario, Canada)), pRC-CMV/Src (encoding wild-type non-neuronal murine c-Src), and pcDNA/RACK1/WD6/7/HA (a hemagglutinin-tagged chimera containing RACK1 WD repeats 6 and 7). To construct the latter, pTarget/WD6/7 (a gift from Dr. Arnaud Besson, Fred Hutchinson Cancer Research Center, Seattle, WA) was subjected to PCR using Platinum Pfx polymerase to introduce a5'-KpnI site and a hemagglutinin tag-3'-XhoI site. After digestion and ligation into pcDNA3.1, transformed colonies were screened by colony PCR, and coding sequences were verified by DNA sequencing. Plasmids were amplified and purified before use (QIAfilter plasmid Maxi kit, Qiagen, Inc., Chatsworth, CA).
Interaction of Cells with Fibrinogen—Human platelets or mouse platelets from PKC-/-, PKC+/-, and PKC+/+ littermates (33) were obtained from fresh, anticoagulated whole blood and were washed and resuspended to 3 x 108 cells/ml in a platelet incubation buffer (34, 35). The PKC status of mouse platelets was established by genotyping the animals and by Western blotting of platelet lysates. CHO cells were harvested using 0.5 mM EDTA, washed once in Dulbecco's modified Eagle's medium, resuspended to 3 x 106 cells/ml, and incubated for 45 min in the presence 20 µM cycloheximide. Binding of fluorescein isothiocyanate-fibrinogen to mouse platelets was quantified by flow cytometry (35). To test the effects of soluble fibrinogen binding on the interaction of intracellular proteins with IIb3, platelets or CHO cells were incubated for 20 or 30 min, respectively, with 250 µg/ml fibrinogen in the presence or absence of 0.5 mM MnCl2 (to activate IIb3) (36) and in some cases with 2 mM RGDS (to block fibrinogen binding). Cells were collected by centrifugation, washed once with phosphate-buffered saline, and solubilized for 10 min on ice in a lysis buffer containing 0.5% Nonidet P-40, 50 mM NaCl, 50 mM Tris, pH 7.4, and inhibitors (1 mM sodium vanadate, 0.5 mM sodium fluoride, and 1x Complete protease inhibitor (Roche Applied Science)). Lysates were clarified at 4 °C by sedimentation at 10,000 rpm in a microcentrifuge and subjected to immunoprecipitation and Western blotting. To test the effects of cell adhesion to fibrinogen, 100-mm bacterial culture dishes were precoated with 5 mg/ml bovine serum albumin (BSA) or 100 µg/ml fibrinogen. After blocking with heat-denatured BSA, 4.5 x 108 platelets in 1.5 ml or 3 x 106 CHO cells in 2 ml were added to each dish, and incubations were carried out in a CO2 incubator for the indicated periods of time at 37 °C. Non-adherent cells from the BSA plates were sedimented and lysed immediately. Cells adherent to fibrinogen were gently washed twice with phosphate-buffered saline, lysed on the plates, and subjected to immunoprecipitation and Western blotting.
Immunoprecipitation and Western Blotting—Five hundred-microliter aliquots of lysate containing equal amounts of protein (ranging from 500 to 800 µg, depending on the experiment) were incubated with relevant antibodies for 2 h or overnight at 4 °C with gentle agitation. Then, 50 µl of protein A-Sepharose (50% v/v) were added for 2 h at 4 °C. Immune complexes were washed twice with phosphate-buffered saline, SDS-PAGE sample buffer was added, and samples were electrophoresed in 7.5 or 10% SDS-polyacrylamide gels. After electrotransfer to nitrocellulose, Western blotting was carried out using enhanced chemiluminescence for detection (Supersignal West Pico substrate, Pierce).
PKC Activity Measurements—IIb3 was immunoprecipitated from platelet lysates with antibody Rb8053 and protein A-Sepharose. Beads were washed twice with lysis buffer and resuspended in kinase buffer and [32P]ATP, and PKC activity was measured using a substrate peptide (QKRPSQRSKYL) according to the manufacturer's instructions (PKC assay kit, Upstate Biotechnology, Inc., Lake Placid, NY).
Cell Spreading and Migration Assays—Glass coverslips were coated with 100 µg/ml fibrinogen, washed cells were allowed to adhere for 45 min at room temperature, and cell morphology and spreading were assessed by confocal microscopy (6, 34). To study CHO cell migration on fibrinogen, cells were serum-starved overnight in Dulbecco's modified Eagle's medium containing 0.5% fetal calf serum and resuspended in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, and 1-ml aliquots were seeded at 30,000 cells/ml onto fibrinogen-coated coverslips. After 1 h, cells were microinjected with a 50 µg/ml solution of RACK1 WD6/7 and/or GFP-PKCI or GFP. Microinjected cells were monitored in real time in a temperature-controlled environment chamber using a Nikon TE2000U microscope. Images were acquired every 10 min for a 6-h period with a CoolSnap HQ charge-coupled device camera (Roper Scientific, Tucson, AZ) running on a Linux work station using ISee software (ISee Imaging Systems, Raleigh, NC). At the end of the filming period, cells were stained with an antibody to hemagglutinin tag to detect those cells expressing RACK1 WD6/7.
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RESULTS
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Interactions between IIb3 and RACK1 in Platelets—The RACK1 adapter molecule contains binding sites for c-Src, certain isoforms of activated PKC, and integrin cytoplasmic tails (Fig. 1A) (27, 29, 30). In platelets, IIb3 is constitutively associated with c-Src, and fibrinogen binding leads to c-Src activation (5). Therefore, we considered whether RACK1 might also be associated with IIb3. Washed human platelets were allowed to adhere for 30 min to immobilized fibrinogen or incubated in suspension over BSA. During this time, adherent platelets reorganize their actin cytoskeletons and spread to varying degrees (34). After washing and cell lysis in buffer containing Nonidet P-40 detergent, IIb3 immunoprecipitates were probed for the presence of RACK1 by Western blotting. IIb3 associated with RACK1 whether platelets were in suspension or adherent to fibrinogen (Fig. 1B). Therefore, potential relationships among IIb3, RACK1, and PKC were assessed.
Interactions between IIb3 and PKC in Platelets—As reported previously (14-21), two classical PKC isoforms (PKC and PKC) and two novel isoforms (PKC and PKC
) were detected readily in human platelets, whereas other isoforms were less prominent or undetectable with the antibodies used (Fig. 2A). Of the PKC isoforms detected, only PCK was inducibly associated with IIb3 in response to platelet adhesion to fibrinogen (Fig. 2B). In addition, when platelets were incubated in suspension for 10 min with 200 nM phorbol 12-myristate 13-acetate (PMA) to activate PKC, PKC became associated with IIb3 (Fig. 2B). Similar results were obtained if platelets were stimulated instead with 50 µM PAR1 thrombin receptor-activating peptide (SSFLRN) or if murine platelets were used instead of human platelets (data not shown). There are two splice variants of PKC, PCKI and PKCII, in which the sequences differ in the C-terminal V5 region (37). Human platelets are reported to contain both variants (16), and we could detect both in human and mouse platelets with variant-specific antibodies (data not shown). The platelet studies described below used an antibody that recognizes both variants of PKC.
The inducible association of PKC with IIb3 could be a direct consequence of fibrinogen binding and IIb3 clustering (38) or the result of additional signaling events stimulated during platelet spreading caused by co-stimulation with endogenous ADP (3). However, the association of PKC with IIb3 that was induced by platelet adhesion to fibrinogen was not affected by the presence of 2 µM apyrase, an amount of enzyme sufficient to remove any ADP that might be released from the platelets (Fig. 2B). In addition, soluble fibrinogen binding to platelets in suspension was induced with 0.5 mM MnCl2, which activates IIb3 directly. Under these conditions, PKC became associated with IIb3 as early as 30 s after fibrinogen binding, the earliest time point tested, and the association was stable for up to 20 min (Fig. 2C). This association was maintained even if platelets were pretreated with 10 µM latrunculin A to block actin polymerization (data not shown). However, the interaction was blocked by 2 mM RGDS, a competitive inhibitor peptide of fibrinogen binding to IIb3 (Fig. 2C), and it was not observed whether fibrinogen was omitted from the incubation mixture. Thus, the recruitment of PKC to IIb3 is caused by fibrinogen binding and is not the result of additional signaling events induced by ADP co-stimulation.
Factors Regulating PKC Association with IIb3—Recruitment of PKC to plasma membranes often coincides with PKC activation (12, 13). To test PKC activity associated with IIb3, platelets were incubated with MnCl2 with or without fibrinogen for up to 20 min, and PKC activity was quantified in IIb3 immunoprecipitates. Immunoprecipitates from control platelets incubated with MnCl2 or fibrinogen alone displayed relatively low PKC activity. However, IIb3 immunoprecipitates from platelets incubated with MnCl2 and fibrinogen displayed a significant increase in PKC activity, even at 30 s (p 
0.001). At 20 min, this increase was 9-fold over baseline, which was 25-30% of the response observed with maximal PKC activation by PMA, and it could be blocked by RGDS (Fig. 3A). In addition, the fibrinogen-dependent increase in the association of PKC with IIb3 was blocked partially by p99, a cell-permeable pseudosubstrate peptide inhibitor of classical and novel PKCs (31) and by bisindolylmaleimide I, a general PKC inhibitor (Fig. 3B). These results indicated that fibrinogen binding stimulates an increase in PKC activity associated with IIb3 and suggest that the association of PKC with IIb3 requires catalytically active PKC.
The activity and subcellular localization of classical PKCs are influenced by phosphorylation and products of phospholipid hydrolysis, e.g. Ca2+ and diacylglycerol (12, 13). Because fibrinogen binding to platelets leads to both the activation of integrin-associated c-Src (5) and the association of active PKC with IIb3, functional relationships between PKC and c-Src were examined. In fibrinogen-adherent platelets, 2 µM SU6656, a selective inhibitor of Src family kinases, failed to block the interaction of PKC with IIb3 (Fig. 4A), despite the fact that it adequately blocked c-Src activation, as assessed by tyrosine phosphorylation of c-Src activation loop Tyr-418 (Fig. 4B). Furthermore, the inhibition of PKC by 12 µM bisindolylmaleimide I failed to block the activation of integrin-associated c-Src (data not shown).
Platelet and IIb3 Responses Dependent on PKC—Prominent responses following platelet adhesion to fibrinogen include reorganization of the actin cytoskeleton and spreading (34). Forty-five minutes after plating on fibrinogen, normal human platelets were generally well spread and exhibited prominent F-actin cables and a peripheral distribution of phosphotyrosine-containing proteins (Fig. 5B). In contrast, platelets incubated with the cell-permeable p99 peptide to inhibit classical and novel PKCs attached to fibrinogen and displayed filopodia but largely failed to reorganize their cytoskeletons, develop a peripheral distribution of phosphotyrosine-containing proteins, or spread (Fig. 5A). Similar results were obtained with bisindolylmaleimide I (Fig. 5C), whereas platelets incubated with the inactive congener, bisindolylmaleimide V, responded normally (Fig. 5D). The differences in spreading between PKC inhibitor-treated and control platelets were significant, as determined by computerized image analysis of cell surface areas (p < 0.001).
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FIG. 5.
Role of PKC in platelet cytoskeletal reorganization and spreading on fibrinogen. Platelets were preincubated with 12 µM bisindolylmaleimide I (Bis) (C), 12 µM bisindolylmaleimide V (D), 1 µM p99 peptide (A), or buffer (B) for 10 min. After adhesion to fibrinogen for 45 min, cells were fixed, permeabilized, and stained with rhodaminephalloidin to visualize F-actin (red) and anti-phosphotyrosine antibodies (green) by confocal microscopy. Bar, 10 µm.
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Because the inhibition of PKC by p99 or by bisindolylmaleimide I had also blocked the fibrinogen-dependent interaction of PKC with IIb3 (Fig. 3B), these results suggested that an integrin-associated pool of PKC is required for full cytoskeletal and morphological responses of platelets to fibrinogen. However, because neither inhibitor is specific for PKC, this issue was also addressed by studying mouse platelets that were deficient in PKC (PKC-/-). Similar to human platelets incubated with PKC inhibitors, PKC-/- platelets failed to spread on fibrinogen, although platelets from PKC+/+ littermates spread normally (Fig. 6A). In addition, PKC-/- platelets spread less well than PKC+/+ platelets in response to PMA. These differences in spreading between PKC-/- and PKC+/+ platelets were statistically significant (p < 0.001). In contrast, PKC-/- platelets spread as well as PKC+/+ platelets when co-stimulated with agonists to G protein-coupled receptors (PAR4 receptor-activating peptide, ADP) or an agonist to glycoprotein VI (convulxin) (Fig. 6A). Furthermore, PKC-/- platelets bound soluble fibrinogen as well as PKC+/- platelets in response to G protein-coupled receptor agonists (Fig. 6B). Altogether, these results indicated that PKC is required for normal outside-in signaling from IIb3 to the platelet actin cytoskeleton. On the other hand, PKC is dispensable for ADP- or thrombin-induced activation of IIb3. Because small amounts of endogenous platelet ADP are required for full spreading of platelets on fibrinogen (3), we cannot exclude a role for PKC in this type of ADP co-stimulation.
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FIG. 6.
Role of PKC in IIb3 function in mouse platelets. A, platelets from PKC-/- and PKC+/+ mice were plated on fibrinogen for 45 min in the absence or presence of 0.5 mM PAR4 receptor-activating peptide (AYPGKF (59)), 50 µM ADP, 200 nM PMA, or 10 µg/ml convulxin (CVX). Cells were fixed, permeabilized, stained with anti-phosphotyrosine antibodies (green) and rhodaminephalloidin (red), and analyzed by confocal microscopy. Bar, 10 µm. B, washed platelets were stimulated for 20 min as indicated in the presence of 200 µg/ml fluorescein isothiocyanate (FITC)-fibrinogen, and fibrinogen binding was quantified by flow cytometry. Data represent specific fibrinogen binding, defined as binding that is inhibitable by 5 mM EDTA (35), and are the means ± S.E. of three experiments.
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Functional Relationships between RACK1, PKC, and IIb3—A CHO cell model system was used to further explore relationships among RACK1, PKC, and IIb3. A5 CHO cells that express RACK1 endogenously and IIb3 after stable transfection were transiently transfected with GFP-PKCI. Forty-eight hours later, cells were plated on fibrinogen or BSA for 45 min, and the presence of GFP-PKC in IIb3 immunoprecipitates was assessed. As had been observed with platelets, RACK1 was constitutively associated with IIb3 (data not shown), and adhesion to fibrinogen (Fig. 7A) or binding of soluble fibrinogen (Fig. 7B) caused an increase in the amount of GFP-PKCI associated with IIb3. However, no PKC/IIb3 association was observed if cells had been simultaneously transfected with a RACK1 truncation mutant (WD6/7) consisting of WD repeats 6 and 7, which can interact with PKC but not with integrin cytoplasmic tails (39) (Fig. 7, A and B). RACK1 WD6/7 had no effect on the expression of RACK1, GFP-PKCI, or IIb3 (Fig. 7A), nor did it affect the constitutive association of IIb3 with c-Src (data not shown). Because RACK1 WD6/7 may sequester PKC away from a RACK1/IIb3 complex, these results are consistent with the idea that RACK1 mediates the interaction of PKC with IIb3, although it does not mediate the interaction of c-Src with IIb3. If RACK1 were the intermediary between PKC and IIb3, this interaction would be dependent on the 3 cytoplasmic tail. Indeed, when fibrinogen binding was stimulated by MnCl2, GFP-PKCI co-immunoprecipitated with IIb but not with IIb3
724, a carboxylterminal truncation mutant lacking 39 of the 47 residues of the 3 tail (Fig. 7C).
RACKs mediate translocation of activated PKCs to specific subcellular locations (27). To evaluate whether RACK1 is capable of promoting GFP-PKCI localization to IIb3-based adhesion sites, A5 cells transfected with PKCI were plated on fibrinogen-coated coverslips for 45 min and evaluated by confocal microscopy. Adherent cells spread and exhibited prominent lamellipodia containing small peripheral focal complexes and larger somewhat more centralized focal adhesions containing IIb3 (Fig. 8A). GFP-PKCI co-localized strongly with IIb3 in these structures (Fig. 8, B and C). However, when cells were simultaneously transfected with GFP-PKCI and RACK1 WD6/7, GFP-PKCI appeared to be distributed more diffusely throughout the cells and exhibited less co-localization with IIb3 (Fig. 8, A-C). To localize GFP-PKCI more precisely, fibrinogen-adherent cells were co-stained for cortactin, which co-localizes with IIb3 primarily at lamellipodial edges, and paxillin, which localizes predominantly to focal adhesions. PKC1 co-localized with both cortactin and paxillin, although more completely with the former (78.9 ± 1.3% co-localization) than the latter (54.2 ± 2.5% co-localization) (Fig. 8D). Thus, RACK1 promotes the localization of PKCI to IIb3 adhesion sites, especially at lamellipodial edges.
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FIG. 8.
Localization of GFP-PKCI in fibrinogen-adherent IIb3 CHO cells. A, A5 CHO cells were transfected with GFP-PKCI (top) or GFP-PKCI plus RACK1 WD6/7 (bottom). After 24 h, cells were plated on fibrinogen-coated coverslips, and 18 h later localization of GFP-PKCI (green) and IIb3 (red) was determined by confocal microscopy. Cells expressing RACK1 WD6/7 were identified with an antibody to the hemagglutinin epitope tag (insets A, top, middle and D, top and bottom right). Images depicting fluorescence from a single fluorophore are shown in black and white for clarity. Arrowheads (A and D) illustrate co-localization of GFP-PKCI and IIb3. Although not shown, GFP-PKC fluorescence was clearly positive in successfully transfected cells when compared with "control" untransfected cells on the same coverslip. B, localization of GFP-PKCI was scored as lamellipodial or dispersed. Data represent means ± S.E. of three experiments. C, proportion of cells showing co-localization of GFP-PKCI and IIb3. Data represent means ± S.E. of three experiments. D, distribution of GFP-PKCI, IIb3, cortactin, and paxillin in fibrinogen-adherent A5 cells transfected with GFP-PKCI. Co-localization of GFP-PKCI with cortactin (top) or paxillin (bottom) is indicated in yellow in the images labeled Co-localization or Zoom. Zoomed areas are identified by white rectangles. Bars, 10 µm.
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To assess the functional relevance of RACK1-mediated PKCI association with IIb3, the migration of A5 cells on fibrinogen was studied in the presence of fetal calf serum as a source of growth factor. After adhesion to fibrinogen for 1 h, A5 cells were microinjected with either GFP, GFP-PKCI, or GFP-PKCI plus RACK1 WD6/7. Two hours later, individual cell movement was monitored for a period of 6 h (Fig. 9A). Cells expressing GFP-PKCI exhibited rates of migration similar to the control cells injected with GFP. However, cells expressing GFP-PKCI and RACK1 WD6/7 displayed significantly less migration (p < 0.01) (Fig. 9Bi). The effect of RACK1 WD6/7 on the cell migration rate was enhanced over time (Fig. 9Bii) presumably because of a progressive increase in expression of the mutant protein. Thus, RACK1 WD6/7 affects IIb3-dependent cell migration. Although there is little evidence that platelets migrate, these results in CHO cells indicated that RACK1 has the potential to influence IIb3 function by forming a bridge between activated PKC and the integrin.
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DISCUSSION
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Important functional relationships exist between integrins and PKCs in many cell types (10). These relationships include PKC regulation of integrin affinity, avidity, and trafficking (3, 40), PKC involvement in cellular responses that are dependent on integrins (41-43), and the modulation of PKC activity by integrin-mediated cell adhesion (44). In platelets, PKC has been implicated in the agonist regulation of IIb3 affinity and initiation of platelet aggregation (3, 45). This conclusion is based largely on the stimulatory effects of phorbol esters and the inhibitory effects of PKC inhibitors. However, because these compounds can target signaling molecules in addition to PKC, observations derived solely from their use are ambiguous (11). Furthermore, platelets express several PKC isoforms, and with rare exception (46) the relationships of individual isoforms to IIb3 function have not been evaluated. In addition, the role of specific PKC isoforms in outside-in IIb3 signaling is unknown, and the mechanisms by which PKCs and IIb3 regulate each other's functions remain poorly characterized.
Therefore, several experimental systems were used here to better understand the relationship between IIb3 and PKC. The main conclusions are: 1) of all the PKC isoforms expressed in platelets, only PKC becomes associated with IIb3 in response to fibrinogen binding; 2) the interaction of PKC with IIb3 is dependent on the cytoplasmic tail of 3, mediated likely by RACK1, and results in an increase in PKC activity associated with IIb3; 3) the recruitment of PKC to IIb3 localizes the enzyme to lamellipodial edges and focal complexes, precisely the subcellular locations where the early phases of outside-in IIb3 signaling take place (3, 47); 4) PKC is required for cytoskeletal reorganization and spreading of platelets on fibrinogen; and 5) the identification of PKC within IIb3-based signaling complexes provides new support for a scaffold function of IIb3 in adhesive signaling.
Role of RACK1 in Bridging IIb3 and PKC—In cells other than platelets, some PKC isoforms may be able to interact with 1 integrins, perhaps directly (e.g. PKC) (48) or indirectly through tetraspanins (e.g. PKCII) (49) or RACK1 (28). Several observations suggest that RACK1 is physically interposed between IIb3 and activated PKC in platelets. First, RACK1 can bind directly to integrin 1, 2, and 3 tails in vitro through interactions that require conserved, membrane-proximal residues in the integrin tails and the WD5-7 repeats in RACK1 (28, 50). Second, PKC is one of the PKC isoforms that has been shown to bind directly to RACK1 through interactions involving the C2 domain of PKC and repeats WD3 and WD6 of RACK1 (27, 51). Interestingly, the V5 region of PKCII but not PKCI contains a second binding site for RACK1 (52). Third, RACK1 is constitutively associated with IIb3 in platelets, as assessed by co-immunoprecipitation (Fig. 1B), thus placing a pool of this adapter molecule in the proper location to bridge IIb3 and activated PKC. The constitutive association of RACK1 with IIb3 contrasts with the reported PMA-inducible association of recombinant glutathione S-transferase (GST)-RACK1 with 1 integrin in 293T cells (28). Fourth, fibrinogen binding to platelets was associated with a marked increase in PKC activity in IIb3 immunoprecipitates (Fig. 3A). This would be expected if the interaction was mediated by RACK1 because it binds selectively to activated PKCs (27, 51). Finally in IIb3 CHO cells, expression of the RACK1 WD6/7 truncation mutant, which can bind PKC but not integrins (28, 39), abrogated fibrinogen-dependent interaction of GFP-PKC with IIb3 (Figs. 7 and 8) and blocked cell migration (Fig. 9).
Previous work has demonstrated that RACK1 can bind to a yeast two-hybrid bait representing the membrane-proximal 20 amino acid residues of the 2 cytoplasmic tail (28). In the current study, fibrinogen-dependent association of PKC with IIb3 in CHO cells no longer occurred when the 3 cytoplasmic tail was truncated at residue 724 (Fig. 7C). By eliminating 39 carboxyl-terminal residues from 3, this truncation also eliminates many of the conserved residues that correspond to those sufficient for binding of 2 to RACK1 (28). Therefore, the weight of evidence suggests that RACK1 mediates the interaction of PKC with IIb3 in platelets. However, additional modes of PKC interaction with IIb3 are possible.
Role of PKC in IIb3 Signaling—PKC co-immunoprecipitated with IIb3 after fibrinogen binding to platelets, and it localized to lamellipodial edges and nascent adhesion sites in fibrinogen-adherent IIb3 CHO cells. Thus, a stable association of PKC with IIb3 may be required for specific aspects of outside-in signaling. This formulation is supported by the inability of platelets from PKC-/- mice to reorganize their cytoskeletons or to spread on fibrinogen (Fig. 6A) and by the migration defect of IIb3 CHO cells expressing RACK1 WD6/7, which prevented PKC association with IIb3 (Figs. 8 and 9). On the other hand, PKC-/- platelets retained inside-out signaling, as manifested by normal agonist-induced fibrinogen binding (Fig. 6B). Therefore, PKC is not essential for IIb3 activation, either because other PKC isoforms can compensate for the loss of PKC in PKC-/- platelets or because proteins that play key roles in inside-out signaling, such as talin, are substrates for PKC isoforms other than PKC (3, 4). There is precedent in platelets for individual PKC isozymes fulfilling unique roles by virtue of their targeting to specific substrates. For example, PKC interacts specifically with Bruton's tyrosine kinase downstream of glycoprotein Ib-IX-V and glycoprotein VI receptors, resulting in the reciprocal modulation of each other's activity (20). In contrast, PKC selectively interacts with Fyn downstream from these same receptors, resulting in translocation of both kinases to the platelet membrane (21).
Several proteins that are required for normal outside-in signaling are known to bind directly or indirectly to IIb3 and may, therefore, be considered potential substrates for PKC. These include c-Src, Syk, Vav, SLP-76, and PTP-1B (3, 53). Some of these proteins contain potential phosphorylation sites for classical PKCs (motif scan analysis, medium stringency); however, the substrates of PKC that participate in outside-in IIb3 signaling remain to be identified. Interestingly, the 3 cytoplasmic tail becomes phosphorylated on Thr-753, an event proposed to be implicated in outside-in signaling; however, that site is said to be recognized preferentially by PDK1 and Akt/PKB (54, 55). In principle, PKC might phosphorylate relevant substrates directly or influence the action of another protein kinase or phosphatase. In this context, both PKC and c-Src can bind to RACK1, and in NIH 3T3 cells the c-Src interaction is enhanced by PKC, resulting in down-regulation of c-Src signaling, cytoskeletal rearrangements, and cell growth (29, 56, 57). However, in platelets, c-Src can bind directly to the 3 integrin cytoplasmic tail, suggesting that the c-Src interaction with RACK1 or PKC may not be essential in the platelet system (5, 6). In support of this, the inhibition of PKC with bisindolylmaleimide I had no effect on c-Src activation in fibrinogen-adherent platelets, and the inhibition of Src with SU6656 had no effect on IIb3 interaction with RACK1 or PKC. Therefore in platelets, PKC and c-Src may function in parallel rather than by direct cross-talk, at least during the initial phases of outside-in IIb3 signaling.
In the future, advances in proteomics may allow unbiased detection of the range of phospho-proteins associated with IIb3 in platelets, providing a more complete picture of integrin signaling (58). Nonetheless, by uncovering a functionally significant interaction between PKC and IIb3, the present studies provide new support for the idea that the IIb3 cytoplasmic tails function as a scaffold for the assembly of a protein complex that initiates and propagates signaling to the platelet actin cytoskeleton (3). These results also lead to new questions. How does fibrinogen binding recruit PKC to IIb3? Is the catalytic activity of PKC regulated by this interaction? Do PKCI and PKCII have different roles in outside-in signaling? What are the stoichiometries of RACK1 interaction with IIb3 and PKC interaction with RACK1? Is PKC required for normal platelet function during hemostasis?
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FOOTNOTES
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* This work was supported by National Institutes of Health Grants HL56595, HL57900, and AI38348. 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 should be addressed: Hematology-Oncology Div., Dept. of Medicine, University of California San Diego, Leichtag Biomedical Research Bldg., Rm. 180, 9500 Gilman Dr., Mail Code 0726, La Jolla, CA 92093. Tel.: 858-822-6425; Fax: 858-822-6444; E-mail: sshattil{at}ucsd.edu.
1 The abbreviations used are: PKC, protein kinase C; RACK, receptor for activated PKC; CHO, Chinese hamster ovary; GFP, green fluorescent protein; TRITC, tetramethylrhodamine isothiocyanate; BSA, bovine serum albumin; PMA, phorbol 12-myristate 13-acetate.
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ACKNOWLEDGMENTS
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We are grateful to Arnaud Besson, Joan Brugge, Stephen Ferguson, Mark Ginsberg, and Steve Watson for reagents.
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ABSTRACT
INTRODUCTION
60 microinjected cells in at least three separate experiments/condition.