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 th e Has GTPase. Because activation of Pas induced by growth factor stimu lation likely requires the localization of mSos1 to the plasma membran e, we have investigated the possibility that the PH domain of mSos1 mi ght mediate an interaction of mSos1 with phospholipid membranes. A glu tathione S-transferase fusion protein containing the pleckstrin homolo gy (PH) domain of mSos1 bound specifically and tightly to phosphatidyl inositol 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 o f Arg(452) within the PH domain with Ala had only a slight effect on b inding to PI(4,5)P-2, whereas substitution of Arg(459) severely compro mised the ability of the mSos1 PH domain to bind to PI(4,5)P-2 contain ing vesicles. Purified full-length mSos1 and mSos1 complexed with Grb2 were also tested for binding to various phosphoinositol derivatives a nd 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 sugges t that the PH domain of mSos1 can interact in vitro with phospholipid vesicles containing PI(4,5)P-2 and that this interaction is facilitate d by the ionic interaction of Arg(459) with the negatively charged hea d group of PI(4,5)P-2. The association of the mSos1 PH domain with pho spholipid may therefore play a role in regulating the function of this enzyme in vivo.