Actin cytoskeletal association of cytohesin-1 is regulated by specific phosphorylation of its carboxyl-terminal polybasic domain

Cell adhesion mediated by integrin receptors is controlled by intracellular signal transduction cascades. Cytohesin-1 is an integrin-binding protein a nd guanine nucleotide exchange factor that activates binding of the leukocy te integrin leukocyte function antigen-1 to its ligand, intercellular adhes ion molecule 1. Cytohesin-1 bears a carboxyl-terminal pleckstrin homology d omain that aids in reversible membrane recruitment and functional regulatio n of the protein. Although phosphoinositide-dependent membrane attachment o f cytohesin-1 is mediated primarily by the pleckstrin homology domain, this function is further strengthened by a short carboxyl-terminal polybasic am ino acid sequence. We show here that a serine/threonine motif within the sh ort polybasic stretch of cytohesin-1 is phosphorylated by purified protein kinase CS in vitro. Furthermore the respective residues are also found to b e phosphorylated after phorbol ester stimulation in vivo. Biochemical and f unctional analyses show that phosphorylated cytohesin-1 is able to tightly associate with the actin cytoskeleton, and we further demonstrate that phos phorylation of the protein is required for maximal leukocyte function antig en-1-mediated adhesion of Jurkat cells to intercellular adhesion molecule 1 . These data suggest that both phosphatidylinositol 3-kinase and protein ki nase C-dependent intracellular pathways that stimulate beta (2)-integrin-me diated adhesion of T lymphocytes converge on cytohesin-1 as functional inte grator.