High affinity binding of the pleckstrin homology domain of mSos1 to phosphatidylinositol (4,5)-bisphosphate

mSos1 has been implicated in coupling mammalian tyrosine kinases to the Has GTPase. Because activation of Pas induced by growth factor stimulation likely requires the localization of mSos1 to the plasma membrane, we have investigated the possibility that the PH domain of mSos1 might mediate an interaction of mSos1 with phospholipid membranes. A glutathione S-transferase fusion protein containing the pleckstrin homology (PH) domain of mSos1 bound specifically and tightly to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) with a K-d of 1.8 +/- 0.4 mu M. This interaction was saturable and was competed away with the soluble head group of PI(4,5)P-2, inositol 1,4,5-triphosphate. Substitution of Arg(452) within the PH domain with Ala had only a slight effect on binding to PI(4,5)P-2, whereas substitution of Arg(459) severely compromised the ability of the mSos1 PH domain to bind to PI(4,5)P-2 containing vesicles. Purified full-length mSos1 and mSos1 complexed with Grb2 were also tested for binding to various phosphoinositol derivatives and demonstrated a specific interaction with PI(4,5)P-2, although these interactions were weaker (K-d = similar to 53 and similar to 69 mu M, respectively) than that of the PH domain alone. These findings suggest that the PH domain of mSos1 can interact in vitro with phospholipid vesicles containing PI(4,5)P-2 and that this interaction is facilitated by the ionic interaction of Arg(459) with the negatively charged head group of PI(4,5)P-2. The association of the mSos1 PH domain with phospholipid may therefore play a role in regulating the function of this enzyme in vivo.