Phosphorylation of the integrin α4 cytoplasmic domain regulates paxillin binding

alpha4 integrins are essential for embryogenesis, hematopoiesis, inflammation, and immune response possibly because alpha (4) integrins have distinct signaling properties from other integrins. Specifically, the alpha (4) cytoplasmic domain binds tightly to paxillin, a signaling adaptor protein, leading to increased cell migration and an altered cytoskeletal organization that results in reduced cell spreading. The alpha (4) tail contains potential phosphorylation sites clustered in its core paxillin binding region. We now report that the alpha (4) tail is phosphorylated in vitro and in viva. Furthermore, Ser(988) is a major phosphorylation site. Using antibodies specific for Ser(988)- phosphorylated alpha (4), we found the stoichiometry of a4 phosphorylation varied in different cells. However, > 60% of a4 was phosphorylated in Jurkat T cells. Phosphorylation at Ser(988) blocked paxillin binding to the alpha (4) tail. A phosphorylation-mimicking mutant of alpha (4) (alpha (4)S988D) blocked paxillin binding and reversed the inhibitory effect of alpha (4) on cell spreading. Consequently, alpha (4) phosphorylation is a biochemical mechanism to modulate paxillin binding to alpha (4) integrins with consequent regulation of alpha (4) integrin-dependent cellular functions.