Beta 3 integrin phosphorylation is essential for Arp3 organization into leukocyte alpha V beta 3-vitronectin adhesion contacts.
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.
Department of Cell and Developmental Biology SUNY Upstate Medical University, 750 East Adams St, Syracuse, NY 13210, USA.
Surjit K
SK
Chandhoke
Matthew
M
Williams
Erik
E
Schaefer
Linda
L
Zorn
Scott D
SD
Blystone
D051378:Actin-Related Protein 3; D002448:Cell Adhesion; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D039661:Integrin beta3 / Q000235:genetics / Q000378:metabolism*; D020014:K562 Cells; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D019096:Vitronectin / Q000378:metabolism*
C495928:ACTR3 protein, human; D051378:Actin-Related Protein 3; D003598:Cytoskeletal Proteins; D039302:Integrin alphaVbeta3; D039661:Integrin beta3; D019096:Vitronectin