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Volume 272, Number 3, Issue of January 17, 1997 pp. 1799-1804
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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High Affinity Binding of the Pleckstrin Homology Domain of mSos1 to Phosphatidylinositol (4,5)-Bisphosphate

(Received for publication, November 7, 1996)

From the Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada

mSos1 has been implicated in coupling mammalian tyrosine kinases to the Ras GTPase. Because activation of Ras 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)P₂) with a K_d of 1.8 ± 0.4 µM. This interaction was saturable and was competed away with the soluble head group of PI(4,5)P₂, inositol 1,4,5-triphosphate. Substitution of Arg⁴⁵² within the PH domain with Ala had only a slight effect on binding to PI(4,5)P₂, whereas substitution of Arg⁴⁵⁹ severely compromised the ability of the mSos1 PH domain to bind to PI(4,5)P₂ 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)P₂, although these interactions were weaker (K_d = ~53 and ~69 µM, 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)P₂ and that this interaction is facilitated by the ionic interaction of Arg⁴⁵⁹ with the negatively charged head group of PI(4,5)P₂. The association of the mSos1 PH domain with phospholipid may therefore play a role in regulating the function of this enzyme in vivo.

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