β3 integrin phosphorylation is essential for Arp3 organization into leukocyte αVβ3-vitronectin adhesion contacts

Abstract

Integrins play a pivotal role in self-regulated hematopoietic adhesion and migration. Leukocyte αVβ3 integrin-mediated adhesion to vitronectin requires protein kinase C activation and phosphorylation on tyrosine 747 of the β3 cytoplasmic tail. We have previously shown that β3 phosphorylation is required for Rho activation. In this study, an antibody specific to phosphorylated β3 tyrosine 747 was used to localize phosphorylated αVβ3 in vitronectin adhesive structures. Early adhesion contacts containing phosphorylated β3 preceded actin stress fiber formation. β3 phosphorylation decreased progressively throughout the course of adhesion coincident with the appearance of actin stress fibers. Time-dependent increases in colocalization of β3 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 β3 but not in those expressing a nonphosphorylatable mutant of β3, suggesting that β3 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 β3 phosphorylation throughout the adhesion time course. These data confirm a requirement for β3 phosphorylation in αVβ3-mediated adhesion to vitronectin and suggest that β3 phosphorylation permits signaling complex assembly at the adhesion site necessary for actin stress fiber formation in leukocytes.