Regulation of outside-in signaling in platelets by integrin-associated protein kinase Cβ
Regulation of outside-in signaling in platelets by integrin-associated protein kinase Cβ
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DOI：
10.1074/jbc.m410229200
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发表时间：
2005-01-07
影响因子：
4.8
通讯作者：
Shattil, SJ
中科院分区：
文献类型：
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作者：
Buensuceso, CS;Obergfell, A;Shattil, SJ
抑制剂的研究表明，蛋白激酶C（PKC）参与了血小板中整合素α（IIb）β（3）的粘附功能，但负责的PKC亚型和机制尚不清楚。α（IIb）β（3）直接与酪氨酸激酶c-Src和Syk相互作用。因此，我们想知道α（IIb）β（3）是否也可能与PKC相互作用。在血小板中表达的几种PKC亚型中，只有PKC β与α（IIb）β（3）免疫共沉淀，以响应血小板与可溶性或固定化纤维蛋白原的相互作用。PKC β向α（IIb）β（3）募集伴随着α（IIb）β（3）免疫沉淀物中PKC活性的9倍增加。RACK 1是活化PKC β的细胞内适配器，也与α IIb β 3共免疫沉淀，但在这种情况下，相互作用是组成性的。广谱PKC抑制剂阻断PKC β向α（IIb）β（3）的募集和血小板在纤维蛋白原上的扩散。类似地，尽管正常激动剂诱导的纤维蛋白原结合，但PKC β基因缺陷的小鼠血小板在纤维蛋白原上的扩散很差。在中国仓鼠卵巢细胞模型系统中，与纤维蛋白原的粘附导致绿色荧光蛋白-PKC β I与α（IIb）β（3）结合，并与其共定位在板脂边缘。这些反应，以及中国仓鼠卵巢细胞在纤维蛋白原上的迁移，被β 3胞质尾的缺失或不能结合β 3的RACK 1突变体的共表达所阻断。这些研究表明，α（IIb）β（3）与活化的PKC β的相互作用受整联蛋白占据的调节，并可由RACK 1介导，并且这种相互作用是通过α（IIb）β（3）触发的血小板扩散所必需的。此外，这些研究扩展了α（IIb）β（3）作为调节血小板肌动蛋白细胞骨架的多种蛋白激酶的支架的概念。
Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin alpha(IIb)beta(3) in platelets, but the responsible PKC isoforms and mechanisms are unknown. alpha(IIb)beta(3) interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether alpha(IIb)beta(3) might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKCbeta co-immunoprecipitated with alpha(IIb)beta(3) in response to the interaction of platelets with soluble or immobilized fibrinogen. PKCbeta recruitment to alpha(IIb)beta(3) was accompanied by a 9-fold increase in PKC activity in alpha(IIb)beta(3) immunoprecipitates. RACK1, an intracellular adapter for activated PKCbeta, also co-immunoprecipitated with alphaIIbbeta3, but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKCbeta recruitment to alpha(IIb)beta(3) and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKCbeta 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-PKCbetaI to associate with alpha(IIb)beta(3) 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 beta(3) cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to beta(3). These studies demonstrate that the interaction of alpha(IIb)beta(3) with activated PKCbeta is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through alpha(IIb)beta(3). Furthermore, the studies extend the concept of alpha(IIb)beta(3) as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton.