THE PH DOMAIN AND THE POLYBASIC-C DOMAIN OF CYTOHESIN-1 COOPERATE SPECIFICALLY IN PLASMA-MEMBRANE ASSOCIATION AND CELLULAR FUNCTION

Recruitment of intracellular proteins to the plasma membrane is a comm only found requirement for the initiation of signal transduction event s. The recently discovered pleckstrin homology (PH) domain, a structur ally conserved element found in similar to 100 signaling proteins, has been implicated in this function, because some PH domains have been d escribed to be involved in plasma membrane association. Furthermore, s everal PH domains bind to the phosphoinositides phosphatidylinositol-( 4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphatein vit ro, however, mostly with low affinity. It is unclear how such weak int eractions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the s tructural and functional requirements for membrane association of cyto hesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-termi nal polybasic sequence of cytohesin-1 (c domain) are necessary for pla sma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosen sor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosph ate binds to the PH domain and c domain together with high affinity (1 00 nM), whereas the isolated PH domain has a substantially lower affin ity (2-3 mu M). The cooperativity of both elements appears specific, b ecause a chimeric protein, consisting of the c domain of cytohesin-1 a nd the PH domain of the P-adrenergic receptor kinase does not associat e with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation-isoprenylation mo tif partially restored the biological function, but the specific targe ting to the plasma membrane was not retained. Thus we conclude that tw o elements of cytohesin-1, the PH domain and the c domain, are require d and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we obs erved a similar functional cooperativity of the PH domain of Bruton's tyrosine kinase with the adjacent Bruton's tyrosine kinase motif, a no vel zinc-containing fold.