Role of Threonine Phosphorylation in Integrin Function

苏氨酸磷酸化在整合素功能中的作用

• 批准号: 9816832
• 负责人: Kenneth Lerea
• 金额: $ 34.5万
• 依托单位: New York Medical College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-15 至 2002-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9816832&HistoricalAwards=false
• 关键词: Role; Threonine; Phosphorylation; Integrin; Function

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.

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。 整合素功能研究最充分的模型系统之一是血小板，它响应整合素激活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。 第一个目标是检查血小板特异性整合素 αIIb/β3 的 β 亚基的 Thr 磷酸化。 血小板扩散和血小板聚集诱导的其他反应是该整合素激活的关键步骤。 蛋白质 Ser/Thr 磷酸酶抑制剂可阻止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 β3 亚基的羧基末端片段具有包含酪氨酸 (Tyr) 和 Thr 磷酸化位点的序列。 正常情况下，纤维蛋白原激活αIIb/β3整合素会导致Tyr残基磷酸化，进而促进β3羧基末端信号复合物的组装。 相反，抑制血小板 Ser/Thr 磷酸酶会促进羧基末端 Thr 残基的磷酸化。 假设这种磷酸化调节 alphaIIb/beta3 活性。 第一个具体目标是测试 beta3 的 Thr 磷酸化是否影响 Tyr 磷酸化或该亚基形成信号复合物的能力。 这将使用完整的 beta3 和源自其细胞质结构域的肽（带有或不带有目标 Thr 残基）作为 Tyr 激酶底物来完成。 第二个具体目标是表征磷酸化 β3 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 β3 整合素，或者还影响具有其他 β 亚基的整合素。 具体来说，将测试花萼蛋白 A 改变 β1 整合素在粘着斑上的定位的能力。 这些研究对于了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具有重要意义。