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

Terry J. Kubiseski, Yuh Min Chook, Wendy E. Parris, Maria Rozakis-Adcock, Tony Pawson

Research output: Contribution to journalArticlepeer-review

63 Scopus citations

Abstract

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 hind 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.

Original languageEnglish (US)
Pages (from-to)1799-1804
Number of pages6
JournalJournal of Biological Chemistry
Volume272
Issue number3
DOIs
StatePublished - 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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