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 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.

Phosphoinositide 3-OH kinase activates the beta2 integrin adhesion pathway and induces membrane recruitment of cytohesin-1.

Signal transduction through phosphoinositide 3-OH kinase (PI 3-kinase) has been implicated in the regulation of lymphocyte adhesion mediated by integrin receptors. Cellular phosphorylation products of PI 3-kinases interact with a subset of pleckstrin homology (PH) domains, a module that has been shown to recruit proteins to cellular membranes. We have recently identified cytohesin-1, a cytoplasmic regulator of beta2 integrin adhesion to intercellular adhesion molecule 1. We describe here that expression of a constitutively active PI 3-kinase is sufficient for the activation of Jurkat cell adhesion to intercellular adhesion molecule 1, and for enhanced membrane association of cytohesin-1. Up-regulation of cell adhesion by PI 3-kinase and membrane association of endogenous cytohesin-1 is abrogated by overexpression of the isolated cytohesin-1 PH domain, but not by a mutant of the PH domain which fails to associate with the plasma membrane. The PH domain of Bruton's tyrosine kinase (Btk), although strongly associated with the plasma membrane, had no effect on either membrane recruitment of cytohesin-1 or on induction of adhesion by PI 3-kinase. Having delineated the critical steps of the beta2 integrin activation pathway by biochemical and functional analyses, we conclude that PI 3-kinase activates inside-out signaling of beta2 integrins at least partially through cytohesin-1.

Restricted activation of general amino acid control under conditions of glutamine limitation in Neurospora crassa.

In Neurospora crassa limitation for single amino acids normally results in increased formation of enzymes required for amino acid synthesis via 'general amino acid control'. Glutamine limitation, however, led to comparatively low and delayed derepression of enzyme synthesis. Nitrate reductase activity increased steeply under these conditions confirming that de novo protein synthesis could occur. Derepression levels were unaffected by addition of glutamine-derived metabolites. Only small and delayed increases in mRNA levels occurred for the anabolic enzyme genes arg-12, his-3 and trp-1 under conditions of glutamine limitation in contrast to the immediate and far larger increase found on histidine limitation. The trans-acting regulatory gene of general amino acid control in Neurospora, cpc-1, responded with a significant increase in mRNA level to histidine and to glutamine limitation. The restricted response of the amino acid synthesis genes could imply a post-transcriptional block to the positive regulatory function of cpc-1 under condition of glutamine limitation. The results suggest that the expression of general amino acid control is restricted under conditions of inadequate nitrogen supply.

The range of amino acids whose limitation activates general amino-acid control in Neurospora crassa.

Several amino-acid synthetic enzymes, belonging to arginine, glutamine, leucine, lysine and phenylalanine biosynthesis, respectively, were investigated under conditions of reduced availability of any one of 16 out of the 20 amino acids represented in proteins. The enzymes showed simultaneous derepression under each condition, albeit to different degrees. Derepression was abolished and the remaining basal enzyme levels reduced by mutations at the cpc-1 locus which governs general amino-acid control in Neurospora. Glutamine synthetase was shown to be under cpc-1 and additional controls. The evidence emphasizes the global nature of general amino-acid control.