Actin Cytoskeletal Association of Cytohesin-1 Is Regulated by Specific Phosphorylation of Its Carboxyl-terminal Polybasic Domain

Henning Dierks,Johanna Kolanus,Waldemar Kolanus

doi:10.1074/jbc.m101502200

Abstract

Cell adhesion mediated by integrin receptors is controlled by intracellular signal transduction cascades. Cytohesin-1 is an integrin-binding protein and guanine nucleotide exchange factor that activates binding of the leukocyte integrin leukocyte function antigen-1 to its ligand, intercellular adhesion molecule 1. Cytohesin-1 bears a carboxyl-terminal pleckstrin homology domain that aids in reversible membrane recruitment and functional regulation of the protein. Although phosphoinositide-dependent membrane attachment of cytohesin-1 is mediated primarily by the pleckstrin homology domain, this function is further strengthened by a short carboxyl-terminal polybasic amino acid sequence. We show here that a serine/threonine motif within the short polybasic stretch of cytohesin-1 is phosphorylated by purified protein kinase C delta in vitro. Furthermore, the respective residues are also found to be phosphorylated after phorbol ester stimulation in vivo. Biochemical and functional analyses show that phosphorylated cytohesin-1 is able to tightly associate with the actin cytoskeleton, and we further demonstrate that phosphorylation of the protein is required for maximal leukocyte function antigen-1-mediated adhesion of Jurkat cells to intercellular adhesion molecule 1. These data suggest that both phosphatidylinositol 3-kinase and protein kinase C-dependent intracellular pathways that stimulate beta(2)-integrin-mediated adhesion of T lymphocytes converge on cytohesin-1 as functional integrator.

Highlights

Integrins are a diverse family of heterodimeric transmembrane adhesion receptors that are present on most vertebrate cell types
Biochemical and functional analyses show that phosphorylated cytohesin-1 is able to tightly associate with the actin cytoskeleton, and we further demonstrate that phosphorylation of the protein is required for maximal leukocyte function antigen-1-mediated adhesion of Jurkat cells to intercellular adhesion molecule 1
The pleckstrin homology (PH) domain was supported in this function by the carboxyl-terminal polybasic region of the protein, which proved to be necessary for membrane association and stabilized PIP3 binding in vitro [22]

Summary

Introduction

Integrins are a diverse family of heterodimeric transmembrane adhesion receptors that are present on most vertebrate cell types They are known to play important roles in either development or somatic functions such as wound healing and the regulation of complex cell-cell or cell-matrix interactions within the immune system [1]. A number of PH domains, including the PH domain of cytohesin-1, were shown to bind phosphatidylinositol 3,4,5-trisphosphate (PIP3) in vitro with high affinity [12, 15,16,17,18] These findings implicated the involvement of phosphatidylinositol 3-kinase in the LFA-1 activation pathway, and we have subsequently shown that a constitutively active version of phosphatidylinositol 3-kinase suffices to activate the ␣L␤2 adhesion pathway in a T-cell line [17]. We have very recently shown that a mutation in the Sec domain of cytohesin-1 that blocks GTP binding to ARF proteins in vitro interferes with cell spreading and adhesion in Jurkat cells and peripheral blood mononuclear cells [25]

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