Regulation of outside-in signaling in platelets by integrin-associated protein kinase C beta

Autor: Achim Obergfell, William B. Kiosses, Charito S. Buensuceso, Toshiaki Kawakami, Koji Eto, Elena García Arias-Salgado, Sanford J. Shattil, Alessandra Soriani
Rok vydání: 2004
Předmět:
Zdroj: Digital.CSIC. Repositorio Institucional del CSIC
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ISSN: 0021-9258
Popis: 10 páginas, 9 figuras -- PAGS nros. 644-653
Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin αIIbβ3 in platelets, but the responsible PKC isoforms and mechanisms are unknown. αIIbβ3 interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether αIIbβ3 might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKCβ co-immunoprecipitated with αIIbβ3 in response to the interaction of platelets with soluble or immobilized fibrinogen. PKCβ recruitment to αIIbβ3 was accompanied by a 9-fold increase in PKC activity in αIIbβ3 immunoprecipitates. RACK1, an intracellular adapter for activated PKCβ, also co-immunoprecipitated with αIIbβ3, but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKCβ recruitment to αIIbβ3 and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKCβ 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-PKCβI to associate with αIIbβ3 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 β3 cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to β3. These studies demonstrate that the interaction of αIIbβ3 with activated PKCβ is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through αIIbβ3. Furthermore, the studies extend the concept of αIIbβ3 as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton.
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, αIIbβ3 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 αIIbβ3 triggers outside-in signals that promote cytoskeletal changes necessary for full platelet aggregation and spreading (2, 3). Bidirectional αIIbβ3 signaling is controlled, in part, by specific intracellular proteins that interact with the relatively short cytoplasmic tails of αIIb or β3. For example, binding of talin to β3 is a final common step in the cellular modulation of αIIbβ3 affinity (4), and binding of c-Src and Syk protein-tyrosine kinases to β3 is required for platelet spreading on fibrinogen (5, 6). Several other intracellular proteins, for example, CIB and β3-endonexin, can also bind to αIIb or β3 tails, respectively, and may influence αIIbβ3 functions (7-9). However, the full complement of intracellular proteins that are capable of interacting directly or indirectly with αIIbβ3 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 αIIbβ3 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 PKCβ and subsequently shown to interact with certain other PKC isoforms and with several other proteins, most notably integrin β cytoplasmic tails and c-Src (see Fig. 1A) (28-30).
Given the potential for the cytoplasmic tails of αIIbβ3 to serve as binding sites for signaling molecules and the apparent functional relationships between PKC and αIIbβ3, the present studies were carried out to determine whether specific PKCs associate with αIIbβ3 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, PKCβ, inducibly associates with αIIbβ3 in response to fibrinogen binding to cells. The PKCβ/αIIbβ3 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 αIIbβ3
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.
Databáze: OpenAIRE
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