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央识别元件。研究得最好的整合素功能模型系统之一是血小板，它在整合素激活后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质丝氨酸/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。第一个目标是检测血小板特异性整合素αIIb/β3的β亚基的苏氨酸磷酸化。血小板聚集引起的血小板扩散和其他反应是该整合素激活的关键步骤。蛋白丝氨酸/苏氨酸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转导。因此，这些反应显然是由磷酸酶活性调节的。整合素的β3亚单位的羧基末端片段包含酪氨酸(Tyr)和苏氨酸(Thr)磷酸化位点。在正常情况下，纤维蛋白原激活αIIb/β3整合素导致Tyr残基的磷酸化，进而促进信号复合体在Beta3的羧基末端组装。相反，抑制血小板丝氨酸/苏氨酸磷酸酶可促进羧基末端苏氨酸残基的磷酸化。假说是这种磷酸化调节alphaIIb/beta3的活性。第一个特定目的是测试Beta3的Thr磷酸化是否影响Tyr的磷酸化或该亚基形成信号复合体的能力。这将使用完整的Beta3和来自其细胞质区域的多肽(带有和不带有靶向Thr残基)作为Tyr激酶底物来实现。第二个特殊目的是表征使β3亚单位磷酸化的蛋白激酶，再次使用适当的合成多肽来测定激酶的活性。第三个具体目的是评估磷酸酶抑制剂Calyculin A是否只影响β3整合素，或者另外影响与其他β亚基的整合素。具体地说，将测试Calyculin A改变β1整合素定位为局部粘连的能力。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.