Phosphorylation of the Integrin α4 Cytoplasmic Domain Regulates Paxillin Binding

Abstract α4 integrins are essential for embryogenesis, hematopoiesis, inflammation, and immune response possibly because α4 integrins have distinct signaling properties from other integrins. Specifically, the α4cytoplasmic 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 α4 tail contains potential phosphorylation sites clustered in its core paxillin binding region. We now report that the α4 tail is phosphorylated in vitro andin vivo. Furthermore, Ser988 is a major phosphorylation site. Using antibodies specific for Ser988-phosphorylated α4, we found the stoichiometry of α4 phosphorylation varied in different cells. However, >60% of α4 was phosphorylated in Jurkat T cells. Phosphorylation at Ser988 blocked paxillin binding to the α4 tail. A phosphorylation-mimicking mutant of α4 (α4S988D) blocked paxillin binding and reversed the inhibitory effect of α4 on cell spreading. Consequently, α4 phosphorylation is a biochemical mechanism to modulate paxillin binding to α4 integrins with consequent regulation of α4integrin-dependent cellular functions.