Chandhoke Surjit K, Williams Matthew, Schaefer Erik, Zorn Linda, Blystone Scott D
Department of Cell and Developmental Biology SUNY Upstate Medical University, 750 East Adams St, Syracuse, NY 13210, USA.
J Cell Sci. 2004 Mar 15;117(Pt 8):1431-41. doi: 10.1242/jcs.00987. Epub 2004 Mar 2.
Integrins play a pivotal role in self-regulated hematopoietic adhesion and migration. Leukocyte alpha(V)beta(3) integrin-mediated adhesion to vitronectin requires protein kinase C activation and phosphorylation on tyrosine 747 of the beta(3) cytoplasmic tail. We have previously shown that beta(3) phosphorylation is required for Rho activation. In this study, an antibody specific to phosphorylated beta(3) tyrosine 747 was used to localize phosphorylated alpha(V)beta(3) in vitronectin adhesive structures. Early adhesion contacts containing phosphorylated beta(3) preceded actin stress fiber formation. beta(3) phosphorylation decreased progressively throughout the course of adhesion coincident with the appearance of actin stress fibers. Time-dependent increases in colocalization of beta(3) with tyrosine 402 phosphorylated Pyk2 in similar adhesive structures was observed, providing evidence for downstream signaling complex formation. Surprisingly, Arp3 organized into similar adhesion contacts in cells expressing wild-type beta(3) but not in those expressing a nonphosphorylatable mutant of beta(3), suggesting that beta(3) phosphorylation is required for sequestration of Arp3 to adhesion complexes. Suppression of actin stress fiber formation by an inhibitor to Rho kinase disrupted Arp3 organization while prolonging beta(3) phosphorylation throughout the adhesion time course. These data confirm a requirement for beta(3) phosphorylation in alpha(V)beta(3)-mediated adhesion to vitronectin and suggest that beta(3) phosphorylation permits signaling complex assembly at the adhesion site necessary for actin stress fiber formation in leukocytes.
整合素在自我调节的造血细胞黏附和迁移中起关键作用。白细胞α(V)β(3)整合素介导的与玻连蛋白的黏附需要蛋白激酶C激活以及β(3)细胞质尾部酪氨酸747位点的磷酸化。我们之前已经表明β(3)磷酸化是Rho激活所必需的。在本研究中，使用针对磷酸化β(3)酪氨酸747的特异性抗体来定位玻连蛋白黏附结构中的磷酸化α(V)β(3)。含有磷酸化β(3)的早期黏附接触先于肌动蛋白应力纤维的形成。在黏附过程中，β(3)磷酸化随着肌动蛋白应力纤维的出现而逐渐降低。在类似的黏附结构中观察到β(3)与酪氨酸402磷酸化的Pyk2的共定位随时间增加，这为下游信号复合物的形成提供了证据。令人惊讶的是，在表达野生型β(3)的细胞中Arp3组装成类似的黏附接触，但在表达β(3)非磷酸化突变体的细胞中则不然，这表明β(3)磷酸化是将Arp3隔离到黏附复合物所必需的。用Rho激酶抑制剂抑制肌动蛋白应力纤维的形成会破坏Arp3的组装，同时在整个黏附时间过程中延长β(3)磷酸化。这些数据证实了α(V)β(3)介导的与玻连蛋白黏附中β(3)磷酸化的必要性，并表明β(3)磷酸化允许在黏附位点组装信号复合物，这对于白细胞中肌动蛋白应力纤维的形成是必需的。
