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

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␦ 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 ␤ 2 -integrin-mediated adhesion of T lymphocytes converge on cytohesin-1 as functional integrator.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).The avidity of integrins for their ligands is dependent on the activation state of the cell they are expressed on (1-3). This type of regulation of cell adhesion has been termed inside-out signaling because intracellular signaling pathways triggered by protein tyrosine kinase or G-protein-coupled receptors have been shown to contribute to integrin-mediated adhesiveness. The mechanisms by which cytoplasmic signals are transmitted across the plasma membrane through integrin receptors are just emerging, but compelling evidence suggests that the intracellular domains of both ␣ and ␤ chains participate in this process (4 -8).Previous studies have attempted to elucidate these signaling pathways. Recently, candidate cytoplasmic regulatory factors of integrin activation have been identified, either by biochemical methods or with the help of the two-hybrid system (7). One of them, cytohesin-1, is a 47-kDa intracellular protein that interacts specifically in several systems with the cytoplasmic domain of the leukocyte integrin ␣ L ␤ 2 (CD11a/18, LFA-1) (9). Cytohesin-1 bears a short amino-terminal domain that may aid in oligomerization, an extended central homology region that is similar to that of the yeast Sec7 protein, a...

Cross-Talk between CD14 and Complement Receptor 3 Promotes Phagocytosis of Mycobacteria: Regulation by Phosphatidylinositol 3-Kinase and Cytohesin-1

“…We found several lines of evidence to support a role for cytohesin-1 in CR3-dependent phagocytosis of BCG: 1) the uptake of serum-but not LBP-opsonized BCG was significantly attenuated in THP-1wt cells with knockdown cytohesin-1, 2) intracellular staining showed a CD14-and PI3K-dependent colocalization of cytohesin-1 with CR3 in LAM-stimulated cells, and 3) pull-down experiments detected the physical association between cytohesin-1 and either PtdIns-3,4,5-P 3 and CR3 in LAM-stimulated cells. This role for cytohesin-1 is also consistent with the observation that activated cytohesin-1 is able to tightly associate with the actin cytoskeleton (14).…”

“…Pioneering studies by Kolanus et al (12) led to the discovery of an adaptor protein cytohesin-1, which interacts with the cytoplasmic tail of CD18 leading to changes in the functional properties of ␤ 2 integrins. Cytohesin-1 contains a pleckstrin homology domain that binds the PI3K metabolite, phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P 3 ), leading to changes in properties of the protein (13,14). These findings have suggested a role for PI3K in regulating the activity of ␤ 2 integrins (15,16), which possibly includes CR3.…”

Cytohesin-1 Regulates the Arf6-Phospholipase D Signaling Axis in Human Neutrophils: Impact on Superoxide Anion Production and Secretion

“…The molecular mechanism by which cytohesin-1 regulates granule secretion remains to be studied, but a potential explanation may be provided by inhibition of the production of fusogenic lipids, such as PLD-derived PA (77) and/or the remodeling of the actin cytoskeleton (61). Indeed, the actin cytoskeleton regulates the secretion of several PMN granules (78), and a direct association of cytohesin-1 with the actin cytoskeleton has already been reported (54).…”

“…Targeting of cytohesin-1 and ARNO diglycine constructs to membranes requires the PH domain and is regulated by the C-terminal polybasic region (50,(52)(53)(54). We previously reported that cytohesin-1 triglycine is the major splice variant expressed in differentiated HL60 cells and that fMLF-induced translocation of cytohesin-1 to membranes, but not that of ARNO, was insensitive to inhibition by inhibitors of PI3K (10).…”

ARL4D Recruits Cytohesin-2/ARNO to Modulate Actin Remodeling

“…PH domains of cytohesins bind the lipid second messengers phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) or phosphatidylinositol bisphosphate in vitro. The cytohesins seem to be involved in ARF6-mediated membrane trafficking and cytoskeletal reorganization Dierks et al, 2001;Santy and Casanova, 2001;Moss and Vaughan, 2002). In cells stimulated with an appropriate agonist, cytohesins can translocate from cytosol to plasma membrane to activate ARF6 in a phosphoinositide 3-kinase (PI3K)-dependent manner that requires both the PH and Sec7 domains of the cytohesin (Hemmings, 1997;Nagel et al, 1998b;Venkateswarlu et al, 1998;Venkateswarlu et al, 1999;Cullen and Chardin, 2000;Venkateswarlu, 2003).…”