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

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 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. 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. INTRODUCTIONGuanine nucleotide exchange factors catalyze the conversion of Ras from its inactive GDP state to an active GTP-bound state, leading to the activation of downstream effectors (1McCormick F. Curr. Opin. Genet. & Dev. 1994; 4: 71-76Crossref PubMed Scopus (209) Google Scholar). A Ras-guanine nucleotide exchange factor specifically implicated in tyrosine kinase signaling is Sos, which was first identified in Drosophila (2Simon M.A. Bowtell D.D. Dodson G.S. Laverty T.R. Rubin G.M. Cell. 1991; 67: 701-716Abstract Full Text PDF PubMed Scopus (651) Google Scholar, 3Bonfini L. Karlovich C.A. Dasgupta C. Banerjee U. Science. 1992; 255: 603-606Crossref PubMed Scopus (198) Google Scholar); subsequently two mammalian homologues have been described, mSos1 (murine form of the son-of-sevenless 1) and mSos2 (4Bowtell D. Fu P. Simon M. Senior P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6511-6515Crossref PubMed Scopus (237) Google Scholar). Sos is a large protein (∼150 kDa) that consists of an N-terminal region, with a Dbl homology domain and a pleckstrin homology (PH) 1The abbreviations used are: PHpleckstrin homologyPI(4,5)P2phosphatidylinositol 4,5-bisphosphatePI(4)Pphosphatidylinositol 4-monophosphatePI(3,4,5)P3phosphatidylinositol 3,4,5-triphosphateIns(1,4,5)inositol 1,4,5-triphosphatePIphosphatidylinositolPCphoshpatidylcholinePSphosphatidylserineGSTglutathione S-transferase. domain, a catalytic region that is responsible for the protein's guanine nucleotide exchange activity, and a C-terminal proline-rich tail with binding sites for the SH3 domains of the Grb2 adaptor protein.Because Ras is membrane-associated, activation of Sos may involve a change in Sos subcellular localization from the cytosol to the membrane, where it can gain access to GDP-bound Ras (1McCormick F. Curr. Opin. Genet. & Dev. 1994; 4: 71-76Crossref PubMed Scopus (209) Google Scholar, 4Bowtell D. Fu P. Simon M. Senior P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6511-6515Crossref PubMed Scopus (237) Google Scholar, 5Satoh T. Nakafuku M. Kaziro Y. J. Biol. Chem. 1992; 267: 24149-24152Abstract Full Text PDF PubMed Google Scholar). It has been observed that targeting of mSos1 to the plasma membrane is sufficient for the activation of the Ras signaling pathway, consistent with the notion that mSos1 is active when situated at the membrane (6Aronheim A. Engelberg D. Li N. Al-Alawi N. Schlessinger J. Karin M. Cell. 1994; 78: 949-961Abstract Full Text PDF PubMed Scopus (422) Google Scholar, 7Quilliam L.A. Huff S.Y. Rabun K.M. Wei W. Park W. Broek D. Der C.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8512-8516Crossref PubMed Scopus (127) Google Scholar). One possibility is that the association of mSos1 with the SH3 domains of Grb2 may serve to modify mSos1 localization upon growth factor stimulation through interaction of the Grb2 SH2 domain with the tyrosine phosphate of activated growth factor receptors (8Gale N.W. Kaplan S. Lowenstein E.J. Schlessinger J. Bar-Sagi D. Nature. 1993; 363: 88-92Crossref PubMed Scopus (449) Google Scholar, 9Li N. Batzer A. Daly R. Yajnik V. Skolnik E. Chardin P. Bar-Sagi D. Margolis B. Schlessinger J. Nature. 1993; 363: 85-88Crossref PubMed Scopus (797) Google Scholar, 10Rozakis-Adcock M. Fernley R. Wade J. Pawson T. Bowtell D. Nature. 1993; 363: 83-85Crossref PubMed Scopus (835) Google Scholar) or through an intermediate docking protein, such as Shc or IRS-1 (11Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R. Li W. Batzer A.G. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (825) Google Scholar, 12Baltensperger K. Kozma L.M. Cherniack A.D. Klarlund J.K. Chawla A. Banerjee U. Czech M.P. Science. 1993; 260: 1950-1952Crossref PubMed Scopus (230) Google Scholar, 13Skolnik E.Y. Batzer A. Li N. Lee C.-H. Lowenstein E. Mohammadi M. Margolis B. Schlessinger J. Science. 1993; 260: 1953-1955Crossref PubMed Scopus (499) Google Scholar).Recent evidence has raised the possibility that the interactions of Grb2 SH3 domains with the C-terminal tail of mSos1 proteins may have more complex functions than simple relocalization and conversely has suggested that other regions of mSos1 may be involved in membrane targeting. Deletion of the mSos1 C-terminal region yields an mSos1 variant that transforms Rat1 cells (14Wang W. Fisher E.M.C. Jia Q. Dunn J.M. Porfiri E. Downward J. Egan S.E. Nat. Genet. 1995; 10: 294-299Crossref PubMed Scopus (59) Google Scholar). Similarly, Drosophila Sos constructs lacking the proline-rich region are able to support the development of R7 photoreceptor cells in a genetically sensitized background (15Karlovich C.A. Bonfini L. McCollam L. Rogge R.D. Daga A. Czech M.P. Banerjee U. Science. 1995; 268: 576-579Crossref PubMed Scopus (74) Google Scholar) and to stimulate Ras activation when ectopically expressed in COS-1 monkey cells (16McCollam L. Bonfini L. Karlovich C.A. Conway B.R. Kozma L.M. Banerjee U. Czech M.P. J. Biol. Chem. 1995; 270: 15954-15957Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). These results are consistent with the possibility that a membrane localization region exists in Sos apart from the C-terminal proline-rich domain.A logical candidate for this potential membrane binding region is the PH domain of Sos. Pleckstrin contains N-terminal and C-terminal repeats of about 100 amino acids (17Tyers M. Rachubinski R.A. Stewart M.I. Varrichio A.M. Shoo R.G.L. Haslam R. Harley C.B. Nature. 1988; 333: 470-473Crossref PubMed Scopus (190) Google Scholar) that have been found in a large number of proteins including GTPase-activating proteins, tyrosine or serine/threonine kinases, kinase substrates, structural and regulatory elements of the cytoskeleton, and phospholipases (18Musacchio A. Gibson T. Rice P. J. M. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar). A number of for the PH domains have been including of proteins K. J. G. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) and protein kinase L. Y. T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). The PH domains of proteins and have been to interact in vitro with phospholipid vesicles containing PI(4,5)P2 its soluble head group Nature. 1994; PubMed Scopus Google Scholar, K.M. M.A. Schlessinger J. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, M.A. K.M. R. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google although only the PH domain has a association with this we that the PH domain of mSos1 PI(4,5)P2 with and results are consistent with the that Sos localization may be facilitated by the interaction of its PH domain with the plasma that the PH domain of Sos has a role in (14Wang W. Fisher E.M.C. Jia Q. Dunn J.M. Porfiri E. Downward J. Egan S.E. Nat. Genet. 1995; 10: 294-299Crossref PubMed Scopus (59) Google Scholar, C.A. Bonfini L. McCollam L. Rogge R.D. Daga A. Czech M.P. Banerjee U. Science. 1995; 268: 576-579Crossref PubMed Scopus (74) Google Scholar, L. Bonfini L. Karlovich C.A. Conway B.R. Kozma L.M. Banerjee U. Czech M.P. J. Biol. Chem. 1995; 270: 15954-15957Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and may be involved in membrane association to the ability of this domain to interact with derivatives in this the PH domain of mSos1 was expressed as a fusion protein in containing of have that a charged of PH domains is for binding PI(4,5)P2 1995; PubMed Scopus Google Scholar). the mSos1 PH domain has that might be involved in phospholipid the amino acids and of PH domains of were with the region of the mSos1 PH domain or to be involved in with the and of PI(4,5)P2, were identified in the mSos1 PH of the mSos1 PH to the interaction of the mSos1 PH domain with the PH fusion protein was and with to the PH The was with containing which were to a the and were by as in the of PI(4,5)P2 was at μM, the of the mSos1 PH domain was found in the an association with the phospholipid vesicles. with this the of this a in the of the mSos1 PH domain, in with mSos1 PH domain was with vesicles of the phospholipid or with vesicles containing negatively charged The by which as an in the in and binding to of the These results that the mSos1 PH domain specifically to phospholipid vesicles containing a binding of PH domain of mSos1 to phospholipid vesicles. A of the PH domain of mSos1 from with was and as an The of protein used was in an of The of used was vesicles were in the of the and were more with to interaction of the fusion protein with various phospholipid vesicles as by and by as in the mSos1 PH domain binding to specific we tested the ability of the domain fusion protein to bind phospholipid vesicles containing or in the fusion protein was in phospholipid vesicles containing PI(4,5)P2 and to a in association with vesicles containing mSos1 PH domain fusion protein was in association with vesicles that or These suggest that the mSos1 PH domain a specific interaction with PI(4,5)P2 and to interaction with of the phospholipid vesicles tested of the interaction of the PH domain of mSos1 with phospholipid was as has been for other binding proteins L. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). is a for interactions and is on the of the the of upon association of a protein with a phospholipid The in upon the of the domain to various phospholipid vesicles are in The mSos1 PH domain specifically with phospholipid vesicles containing PI(4,5)P2, as demonstrated by an in of about upon the of the fusion association was observed with vesicles containing other The fusion protein induced an of about which the consistent in observed for other vesicles containing or A was observed for vesicles containing or or that negatively charged a interaction with the PH domain of this interaction was as as that observed for PI(4,5)P2. when with PI(4,5)P2 containing in in to mSos1 binding to vesicles containing PI(4,5)P2 and by the in observed upon the of fusion protein of domain of mSos1 fusion protein to to a of various phospholipid vesicles. protein of upon domain interaction with vesicles and by of of A containing of phospholipid vesicles with PI(4,5)P2 was domain of mSos1 fusion protein at the in A at a of was the in the as the effect on of the fusion protein in to a of phospholipid vesicles. to at about μM, of a interaction the PH domain of mSos1 and PI(4,5)P2. This in be competed away by at a of of of and mSos1 PH to ability of the mSos1 PH domain to bind vesicles containing of PI(4,5)P2 was the also the of two of the mSos1 PH domain, Arg452 and in the of PI(4,5)P2 by domain fusion proteins in which or of these were with The PH domain of mSos1 bound tightly to PI(4,5)P2 containing phospholipid with a Kd of 1.8 ± 0.4 These results suggest that the mSos1 PH domain a for PI(4,5)P2, in with other PH the mSos1 PH domain only a slight in its to PI(4,5)P2, a Kd of ± μM. the a than in binding for PI(4,5)P2, that it has a for PI(4,5)P2, and the a in of and PH domains of mSos1 to is for of the by to the binding ± ± ± ± ± as from binding ± as from binding is for of the by to the binding as from binding in a these had the of the and PH domains was investigated by that the of the domain and the were to that of the protein these structural that the of the PH domain for PI(4,5)P2 is likely to a specific effect on phospholipid binding than a effect on the of the PH of mSos1 and a to from cells Pawson T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google was tested for its ability to bind various phospholipid vesicles. mSos1 demonstrated a specific interaction with vesicles at than that by the domain fusion protein (Kd ± μM, Because mSos1 is found bound to Grb2 in we tested the ability of a complex containing full-length mSos1 and Grb2 to bind The complex demonstrated a specific interaction with vesicles with an to that of mSos1 ± μM, The of a on the Shc that with to the Grb2 SH2 domain binding of the complex to complex was with various phospholipid vesicles and for phospholipid binding by A of the complex with phospholipid vesicles as in The mSos1 and Grb2 are The of used was μM, whereas the phospholipid vesicles were at a of has been that SH2 domains can interact with Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). therefore tested the binding of Grb2 to the various used in this Grb2 from cells a fusion protein expressed in E. interaction with the various and of phospholipid results in this that the PH domain of mSos1 to PI(4,5)P2 with and a number of PH domains have been to interact with phosphatidylinositol derivatives Nature. 1994; PubMed Scopus Google Scholar, K.M. M.A. Schlessinger J. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, M. M. M. M. J. 1995; PubMed Scopus Google Scholar) in the are about the interactions in vivo. It is to the Kd for the mSos1 PH domain with on other pleckstrin the number of binding sites are on the and the of the phospholipid vesicles. the of the mSos1 PH for domain for PI(4,5)P2 to be sufficient to to the localization of the full-length mSos1 to the Kd for binding of the mSos1 PH domain to PI(4,5)P2 is than that for the pleckstrin N-terminal PH domain Nature. 1994; PubMed Scopus Google respectively) and of the of as for the PH domain M.A. K.M. R. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, P. S. S. V. S. 1995; PubMed Scopus Google the mSos1 PH domain the of the PH domain for the soluble inositol it has been suggested that interaction of its PH domain with PI(4,5)P2 at the membrane the enzyme to its in a and that the interaction of the this and catalytic M.A. K.M. R. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, P. S. S. V. S. 1995; PubMed Scopus Google Scholar, L. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, T. Y. S. M. T. S. M. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). this of by is to be involved in the of the mSos1 PH domain from the membrane have been of PH domains binding to PI(4,5)P2 and with Nature. 1994; PubMed Scopus Google Scholar, M.A. K.M. R. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar, P. S. S. V. S. 1995; PubMed Scopus Google Scholar, M. M. M. M. J. 1995; PubMed Scopus Google although various protein suggest that these domains interact with the within the membranes. with that the head group inositol is at an to the to interaction of the negatively charged with the of Nat. Biol. PubMed Scopus Google Scholar). it may be that the inositol head bound to in a membrane than that of the soluble inositol the binding of to the mSos1 PH domain has It may be for to with PI(4,5)P2 for the PH domain, although it that of in cells the to mSos1 P. S. S. V. S. 1995; PubMed Scopus Google of can of or number of have that PH domains have structural Nature. 1994; PubMed Scopus Google Scholar, K.M. M.A. Schlessinger J. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, M. M. M. M. J. 1995; PubMed Scopus Google in the of homology (18Musacchio A. Gibson T. Rice P. J. M. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar). the Shc and IRS-1 binding domains have been demonstrated to the as PH domains A.M. Wade Nature. 1995; PubMed Scopus Google Scholar, S. T. S.E. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The PI(4,5)P2 binding in the N-terminal PH domain of pleckstrin has been identified Nature. 1994; PubMed Scopus Google Scholar) at the of the in the N-terminal of the found to be for this interaction were and 1995; PubMed Scopus Google Scholar). The amino acids within the PH domain of mSos1 are Arg452 and Arg459 (18Musacchio A. Gibson T. Rice P. J. M. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar). have identified Arg459 of mSos1 as for the interaction of this protein's PH domain with PI(4,5)P2 in This is to the of the PH domain (18Musacchio A. Gibson T. Rice P. J. M. Sci. 1993; Full Text PDF PubMed Scopus Google has been demonstrated by its to in P. V. Science. 1993; PubMed Scopus Google Scholar, S. L. M. Science. 1993; PubMed Scopus Google Scholar). The that Arg452 with Ala has only a effect on binding be as that this form an ionic interaction with the negatively charged or that a upon substitution of Arg452 that the weaker interactions of the full-length mSos1 and complex with that of the PH domain are It is to that a factor is to the PH domain of mSos1 to interact with PI(4,5)P2, which may localization and other SH2 domains Cell. 1995; Full Text PDF PubMed Scopus Google Grb2 interaction with the tested in this role of these interactions as for the to of the enzyme to its of also by the for This the of proteins by the of the that the complex mSos1 and Ras on the plasma membrane be a to the of signaling through the pathway, as as a to by its subcellular to phospholipid it has been suggested that function of PH domains is to bind to K. J. G. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). vitro to saturable binding to D. N. J. P. D. C. C. J. T. 1995; PubMed Scopus Google Scholar). it is that as with PH domain K. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google PI(4,5)P2 interaction with the mSos1 PH domain may mediate or with the interaction of this domain with or with as protein we have that is a interaction of the PH domain of mSos1 to PI(4,5)P2 in vitro and that this interaction can be competed away by The complex also bound PI(4,5)P2, more than the PH of within the PH domain of mSos1 has demonstrated that Arg459 a role in this interaction with PI(4,5)P2. INTRODUCTIONGuanine nucleotide exchange factors catalyze the conversion of Ras from its inactive GDP state to an active GTP-bound state, leading to the activation of downstream effectors (1McCormick F. Curr. Opin. Genet. & Dev. 1994; 4: 71-76Crossref PubMed Scopus (209) Google Scholar). A Ras-guanine nucleotide exchange factor specifically implicated in tyrosine kinase signaling is Sos, which was first identified in Drosophila (2Simon M.A. Bowtell D.D. Dodson G.S. Laverty T.R. Rubin G.M. Cell. 1991; 67: 701-716Abstract Full Text PDF PubMed Scopus (651) Google Scholar, 3Bonfini L. Karlovich C.A. Dasgupta C. Banerjee U. Science. 1992; 255: 603-606Crossref PubMed Scopus (198) Google Scholar); subsequently two mammalian homologues have been described, mSos1 (murine form of the son-of-sevenless 1) and mSos2 (4Bowtell D. Fu P. Simon M. Senior P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6511-6515Crossref PubMed Scopus (237) Google Scholar). Sos is a large protein (∼150 kDa) that consists of an N-terminal region, with a Dbl homology domain and a pleckstrin homology (PH) 1The abbreviations used are: PHpleckstrin homologyPI(4,5)P2phosphatidylinositol 4,5-bisphosphatePI(4)Pphosphatidylinositol 4-monophosphatePI(3,4,5)P3phosphatidylinositol 3,4,5-triphosphateIns(1,4,5)inositol 1,4,5-triphosphatePIphosphatidylinositolPCphoshpatidylcholinePSphosphatidylserineGSTglutathione S-transferase. domain, a catalytic region that is responsible for the protein's guanine nucleotide exchange activity, and a C-terminal proline-rich tail with binding sites for the SH3 domains of the Grb2 adaptor protein.Because Ras is membrane-associated, activation of Sos may involve a change in Sos subcellular localization from the cytosol to the membrane, where it can gain access to GDP-bound Ras (1McCormick F. Curr. Opin. Genet. & Dev. 1994; 4: 71-76Crossref PubMed Scopus (209) Google Scholar, 4Bowtell D. Fu P. Simon M. Senior P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6511-6515Crossref PubMed Scopus (237) Google Scholar, 5Satoh T. Nakafuku M. Kaziro Y. J. Biol. Chem. 1992; 267: 24149-24152Abstract Full Text PDF PubMed Google Scholar). It has been observed that targeting of mSos1 to the plasma membrane is sufficient for the activation of the Ras signaling pathway, consistent with the notion that mSos1 is active when situated at the membrane (6Aronheim A. Engelberg D. Li N. Al-Alawi N. Schlessinger J. Karin M. Cell. 1994; 78: 949-961Abstract Full Text PDF PubMed Scopus (422) Google Scholar, 7Quilliam L.A. Huff S.Y. Rabun K.M. Wei W. Park W. Broek D. Der C.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8512-8516Crossref PubMed Scopus (127) Google Scholar). One possibility is that the association of mSos1 with the SH3 domains of Grb2 may serve to modify mSos1 localization upon growth factor stimulation through interaction of the Grb2 SH2 domain with the tyrosine phosphate of activated growth factor receptors (8Gale N.W. Kaplan S. Lowenstein E.J. Schlessinger J. Bar-Sagi D. Nature. 1993; 363: 88-92Crossref PubMed Scopus (449) Google Scholar, 9Li N. Batzer A. Daly R. Yajnik V. Skolnik E. Chardin P. Bar-Sagi D. Margolis B. Schlessinger J. Nature. 1993; 363: 85-88Crossref PubMed Scopus (797) Google Scholar, 10Rozakis-Adcock M. Fernley R. Wade J. Pawson T. Bowtell D. Nature. 1993; 363: 83-85Crossref PubMed Scopus (835) Google Scholar) or through an intermediate docking protein, such as Shc or IRS-1 (11Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R. Li W. Batzer A.G. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (825) Google Scholar, 12Baltensperger K. Kozma L.M. Cherniack A.D. Klarlund J.K. Chawla A. Banerjee U. Czech M.P. Science. 1993; 260: 1950-1952Crossref PubMed Scopus (230) Google Scholar, 13Skolnik E.Y. Batzer A. Li N. Lee C.-H. Lowenstein E. Mohammadi M. Margolis B. Schlessinger J. Science. 1993; 260: 1953-1955Crossref PubMed Scopus (499) Google Scholar).Recent evidence has raised the possibility that the interactions of Grb2 SH3 domains with the C-terminal tail of mSos1 proteins may have more complex functions than simple relocalization and conversely has suggested that other regions of mSos1 may be involved in membrane targeting. Deletion of the mSos1 C-terminal region yields an mSos1 variant that transforms Rat1 cells (14Wang W. Fisher E.M.C. Jia Q. Dunn J.M. Porfiri E. Downward J. Egan S.E. Nat. Genet. 1995; 10: 294-299Crossref PubMed Scopus (59) Google Scholar). Similarly, Drosophila Sos constructs lacking the proline-rich region are able to support the development of R7 photoreceptor cells in a genetically sensitized background (15Karlovich C.A. Bonfini L. McCollam L. Rogge R.D. Daga A. Czech M.P. Banerjee U. Science. 1995; 268: 576-579Crossref PubMed Scopus (74) Google Scholar) and to stimulate Ras activation when ectopically expressed in COS-1 monkey cells (16McCollam L. Bonfini L. Karlovich C.A. Conway B.R. Kozma L.M. Banerjee U. Czech M.P. J. Biol. Chem. 1995; 270: 15954-15957Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). These results are consistent with the possibility that a membrane localization region exists in Sos apart from the C-terminal proline-rich domain.A logical candidate for this potential membrane binding region is the PH domain of Sos. Pleckstrin contains N-terminal and C-terminal repeats of about 100 amino acids (17Tyers M. Rachubinski R.A. Stewart M.I. Varrichio A.M. Shoo R.G.L. Haslam R. Harley C.B. Nature. 1988; 333: 470-473Crossref PubMed Scopus (190) Google Scholar) that have been found in a large number of proteins including GTPase-activating proteins, tyrosine or serine/threonine kinases, kinase substrates, structural and regulatory elements of the cytoskeleton, and phospholipases (18Musacchio A. Gibson T. Rice P. J. M. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar). A number of for the PH domains have been including of proteins K. J. G. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) and protein kinase L. Y. T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). The PH domains of proteins and have been to interact in vitro with phospholipid vesicles containing PI(4,5)P2 its soluble head group Nature. 1994; PubMed Scopus Google Scholar, K.M. M.A. Schlessinger J. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, M.A. K.M. R. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google although only the PH domain has a association with this we that the PH domain of mSos1 PI(4,5)P2 with and results are consistent with the that Sos localization may be facilitated by the interaction of its PH domain with the plasma