Role of Threonine Phosphorylation in Integrin Function

细胞质膜上的整合素是将信号从细胞外基质传递到细胞内部的中心识别元件。 整联蛋白功能的最佳研究模型系统之一是血小板，其响应于整联蛋白活化而经历复杂的生化和结构变化。 该项目的总体目标是确定蛋白丝氨酸/苏氨酸（Ser/Thr）磷酸酶控制与整合素相关的血小板反应的分子机制。 第一个目的是检查血小板特异性整合素α IIb/β 3 β亚基的Thr磷酸化。 血小板聚集诱导的血小板扩散和其他反应是这种整合素激活的关键步骤。 蛋白质Ser/Thr磷酸酶抑制剂阻断纤维蛋白原基质上的扩散和聚集诱导的信号传导。 因此，这些反应显然受到磷酸酶活性的调节。 整联蛋白β 3亚基的羧基末端区段具有包括酪氨酸（Tyr）和Thr磷酸化位点的序列。 在正常条件下，纤维蛋白原激活α IIb/β 3整联蛋白会导致Tyr残基磷酸化，进而促进β 3羧基端信号复合物的组装。 相反，血小板Ser/Thr磷酸酶的抑制促进羧基末端内Thr残基的磷酸化。 假设是这种磷酸化调节α IIb/β 3活性。 第一个具体目的是测试β 3的Thr磷酸化是否影响Tyr磷酸化或该亚基形成信号传导复合物的能力。 这将使用完整的β 3和衍生自其胞质结构域的肽（有和没有靶Thr残基）作为Tyr激酶底物来实现。 第二个具体目标是表征磷酸化β 3亚基的蛋白激酶，再次使用适当的合成肽来测定激酶活性。 第三个具体目标是评估磷酸酶抑制剂calyculin A是否仅影响β 3整联蛋白，或者另外影响具有其他β亚基的整联蛋白。 具体而言，将测试calyculin A改变β 1整联蛋白定位于局灶性粘连的能力。 这些研究对于理解血小板中特定的整合素信号传导以及其他细胞类型中整合素调节的更一般机制都具有重要意义。

Integrins on the plasma membrane of cells are central recognition elements for conveying signals from the extracellular matrix to the cell interior. One of the best studied model systems for integrin function is the platelet, which undergoes complex biochemical and structural changes in response to integrin activation. The overall goal of this project is to define the molecular mechanism by which protein serine/threonine (Ser/Thr) phosphatases control platelet responses associated with integrins. The first objective is to examine Thr phosphorylation of the beta subunit of the platelet-specific integrin alphaIIb/beta3. Platelet spreading and other responses induced by platelet aggregation are critical steps in the activation by this integrin. Inhibitors of protein Ser/Thr phosphatases block spreading on fibrinogen matrices and aggregation-induced signaling. Thus, these responses are apparently regulated by phosphatase activity. The carboxy-terminal segment of the beta3 subunit of the integrin has a sequence that includes sites for both tyrosine (Tyr) and Thr phosphorylation. Under normal conditions, activation of alphaIIb/beta3 integrin by fibrinogen causes phosphorylation of Tyr residues, which in turn promotes the assembly of signaling complexes on the carboxy-terminus of beta3. In contrast, inhibition of platelet Ser/Thr phosphatases promotes phosphorylation of Thr residues within the carboxy-terminus. The hypothesis is that this phosphorylation modulates alphaIIb/beta3 activity. The first specific aim is to test whether Thr phosphorylation of beta3 affects the Tyr phosphorylation or the ability of this subunit to form signaling complexes. This will be accomplished using intact beta3 and peptides derived from its cytoplasmic domain, with and without target Thr residues, as Tyr kinase substrates. The second specific aim is to characterize the protein kinase that phosphorylates the beta3 subunit, again using an appropriate synthetic peptide to assay kinase activity. The third specific aim is to assess whether the phosphatase inhibitor calyculin A affects just beta3 integrins or, in addition, integrins with other beta subunits. Specifically, the ability of calyculin A to alter the localization of beta1 integrins to focal adhesions will be tested. The studies are significant for understanding both specific integrin signaling in platelets as well as more general mechanisms of integrin regulation in other cell types.