Phosphorylation of the integrin alpha(4) cytoplasmic domain regulates paxillin binding

alpha4 integrins are essential for embryogenesis, hematopoiesis, inflammati on, and immune response possibly because alpha (4) integrins have distinct signaling properties from other integrins. Specifically, the alpha (4) cyto plasmic domain binds tightly to paxillin, a signaling adaptor protein, lead ing to increased cell migration and an altered cytoskeletal organization th at results in reduced cell spreading. The alpha (4) tail contains potential phosphorylation sites clustered in its core paxillin binding region. We no w report that the alpha (4) tail is phosphorylated in vitro and in viva. Fu rthermore, Ser(988) is a major phosphorylation site. Using antibodies speci fic for Ser(988)- phosphorylated alpha (4), we found the stoichiometry of a 4 phosphorylation varied in different cells. However, > 60% of a4 was phosp horylated in Jurkat T cells. Phosphorylation at Ser(988) blocked paxillin b inding to the alpha (4) tail. A phosphorylation-mimicking mutant of alpha ( 4) (alpha (4)S988D) blocked paxillin binding and reversed the inhibitory ef fect of alpha (4) on cell spreading. Consequently, alpha (4) phosphorylatio n is a biochemical mechanism to modulate paxillin binding to alpha (4) inte grins with consequent regulation of alpha (4) integrin-dependent cellular f unctions.