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 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 inter...

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Veröffentlicht in:The Journal of biological chemistry 1997-01, Vol.272 (3), p.1799-1804
Hauptverfasser: Kubiseski, Terry J., Chook, Yuh Min, Parris, Wendy E., Rozakis-Adcock, Maria, Pawson, Tony
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container_end_page 1804
container_issue 3
container_start_page 1799
container_title The Journal of biological chemistry
container_volume 272
creator Kubiseski, Terry J.
Chook, Yuh Min
Parris, Wendy E.
Rozakis-Adcock, Maria
Pawson, Tony
description 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 Kd 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 (Kd = ∼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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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 (Kd = ∼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. 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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 (Kd = ∼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.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>8999863</pmid><doi>10.1074/jbc.272.3.1799</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record>
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subjects Amino Acid Sequence
Blood Proteins - chemistry
Blood Proteins - metabolism
Cell Membrane - metabolism
Fungal Proteins - genetics
Fungal Proteins - metabolism
Glutathione Transferase - genetics
Light
Molecular Sequence Data
Phosphatidylinositol 4,5-Diphosphate - metabolism
Phosphoproteins
Protein Binding
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Repressor Proteins - genetics
Repressor Proteins - metabolism
Scattering, Radiation
Sequence Homology, Amino Acid
SOS1 Protein
title High Affinity Binding of the Pleckstrin Homology Domain of mSos1 to Phosphatidylinositol (4,5)-Bisphosphate
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