Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway

Activation of growth factor receptors results in tyrosine autophosphorylation and recruitment of SH2 domain-containing effectors, including Grb2. Grb2 recruitment mediates activation of the Ras nucleotide exchanger Sos by an unknown mechanism. To examine the role of membrane recruitment, we prepared Sos derivatives containing either myristoylation or farnesylation signals. This resulted in plasma membrane targeting of Sos and stimulation of the Ras signaling pathway, including ERK and AP-1 activities leading to oncogenic transformation. Sos derivatives with nonfunctional myristoylation or farnesylation sequences were inactive. Farnesylation of Sos also activated Ras signaling in yeast. In both mammalian cells and yeast, membrane-targeted Sos derivatives lacking the C-terminal region were considerably more active. Therefore, targeting of Sos to the plasma membrane in the vicinity of Ras appears to be the primary mechanism leading to activation of the Ras pathway. A secondary mechanism could involve relief of the inhibitory effect of the Sos C-terminal region.

[1]  F. McCormick.  How receptors turn Ras on , 1993, Nature.

[2]  M. Weber,et al.  Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase. , 1993, Science.

[3]  B. Tocqué,et al.  Implication of GAP in Ras-dependent transactivation of a polyoma enhancer sequence. , 1992, Science.

[4]  Joseph Schlessinger,et al.  Signal transduction by receptors with tyrosine kinase activity , 1990, Cell.

[5]  C. Marshall,et al.  All ras proteins are polyisoprenylated but only some are palmitoylated , 1989, Cell.

[6]  M. Karin.  Signal transduction from the cell surface to the nucleus through the phosphorylation of transcription factors. , 1994, Current opinion in cell biology.

[7]  A. Ullrich,et al.  The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling , 1992, Cell.

[8]  A. Zilberstein,et al.  PDGF stimulation of inositol phospholipid hydrolysis requires PLC-γ1 phosphorylation on tyrosine residues 783 and 1254 , 1991, Cell.

[9]  R. Weinberg,et al.  Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation , 1993, Nature.

[10]  Nanxin Li,et al.  Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling , 1993, Nature.

[11]  R. Treisman,et al.  The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain , 1993, Cell.

[12]  G. Carpenter,et al.  Stimulation of phospholipase C-gamma 1 membrane association by epidermal growth factor. , 1990, Science.

[13]  J Downward,et al.  The GRB2/Sem‐5 adaptor protein , 1994, FEBS letters.

[14]  Jonathan A. Cooper,et al.  Mammalian Ras interacts directly with the serine/threonine kinase raf , 1993, Cell.

[15]  T Pawson,et al.  SH2 and SH3 domains , 1993, Current Biology.

[16]  Julian Downward,et al.  Epidermal growth factor regulates p21 ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor , 1993, Cell.

[17]  L. Cantley,et al.  Oncogenes and signal transduction , 1991, Cell.

[18]  A. Levitzki,et al.  In vitro reconstitution of cdc25 regulated S. cerevisiae adenylyl cyclase and its kinetic properties. , 1990, The EMBO journal.

[19]  M. Jacquet,et al.  Characterization, cloning and sequence analysis of the CDC25 gene which controls the cyclic AMP level of Saccharomyces cerevisiae. , 1986, The EMBO journal.

[20]  M. Wigler,et al.  In yeast, RAS proteins are controlling elements of adenylate cyclase , 1985, Cell.

[21]  J. Schlessinger.  SH2/SH3 signaling proteins. , 1994, Current opinion in genetics & development.

[22]  M. Karin,et al.  The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. , 1991, Biochimica et biophysica acta.

[23]  M. Wigler,et al.  Purification and characterization of human H-ras proteins expressed in Escherichia coli , 1985, Molecular and cellular biology.

[24]  G. Rubin,et al.  Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase , 1991, Cell.

[25]  K. Robbins,et al.  Association of p60fyn with middle tumor antigen in murine polyomavirus-transformed rat cells , 1989, Journal of virology.

[26]  A. Schönthal,et al.  Autoregulation of fos: the dyad symmetry element as the major target of repression. , 1989, The EMBO journal.

[27]  R. Davis,et al.  Serum-induced translocation of mitogen-activated protein kinase to the cell surface ruffling membrane and the nucleus , 1993, The Journal of cell biology.

[28]  M. Šuša,et al.  Platelet-derived growth factor activates membrane-associated phosphatidylinositol 3-kinase and mediates its translocation from the cytosol. Detection of enzyme activity in detergent-solubilized cell extracts. , 1992, The Journal of biological chemistry.

[29]  B. Sefton,et al.  Mutation of NH2-terminal glycine of p60src prevents both myristoylation and morphological transformation. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Mark R. Smith,et al.  Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells , 1985, Nature.

[31]  M. Wigler,et al.  Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. , 1993, Science.

[32]  A. Cherniack,et al.  Phosphorylation of the Ras nucleotide exchange factor son of sevenless by mitogen-activated protein kinase. , 1994, The Journal of biological chemistry.

[33]  F. Tamanoi,et al.  Mutants of Saccharomyces cerevisiae defective in the farnesylation of Ras proteins. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[34]  R. Weinberg,et al.  Isolation of a transforming sequence from a human bladder carcinoma cell line , 1982, Cell.

[35]  J. Schlessinger,et al.  Signaling by Receptor Tyrosine Kinases , 1993 .

[36]  C. Der,et al.  The six amino-terminal amino acids of p60src are sufficient to cause myristylation of p21v-ras , 1988, Molecular and cellular biology.

[37]  F. Hilger,et al.  Comparison of thermosensitive alleles of the CDC25 gene involved in the cAMP metabolism of Saccharomyces cerevisiae. , 1990, Genetics.

[38]  E. Martegani,et al.  Cloning by functional complementation of a mouse cDNA encoding a homologue of CDC25, a Saccharomyces cerevisiae RAS activator. , 1992, The EMBO journal.

[39]  L. Hokin,et al.  Role of phosphoinositides in transmembrane signaling. , 1990, Physiological reviews.

[40]  Dan R. Littman,et al.  Signal transduction by lymphocyte antigen receptors , 1994, Cell.

[41]  G. Cooper,et al.  Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins , 1988, Molecular and cellular biology.

[42]  F. Shibasaki,et al.  Insulin treatment stimulates the tyrosine phosphorylation of the alpha-type 85-kDa subunit of phosphatidylinositol 3-kinase in vivo. , 1992, The Journal of biological chemistry.

[43]  C J Marshall,et al.  A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins. , 1991, The EMBO journal.

[44]  S. Elledge,et al.  Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1 , 1993, Nature.

[45]  M. Karin,et al.  c-Jun N-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinases , 1994, Molecular and cellular biology.

[46]  F. McCormick,et al.  Ras p21: effects and regulation. , 1991, Biochimica et biophysica acta.

[47]  A. Kazlauskas,et al.  Disruption of PDGF receptor trafficking by mutation of its PI-3 kinase binding sites. , 1994, Science.

[48]  R. Weinberg,et al.  Behavior of myc and ras oncogenes in transformation of rat embryo fibroblasts , 1986, Molecular and cellular biology.

[49]  A. Ullrich,et al.  Growth factor signaling by receptor tyrosine kinases , 1992, Neuron.

[50]  W. Fantl,et al.  Signalling by receptor tyrosine kinases. , 1993, Annual review of biochemistry.

[51]  A. Aronheim,et al.  The E2A gene product contains two separable and functionally distinct transcription activation domains. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[52]  M. Jacquet,et al.  The Saccharomyces cerevisiae CDC25 gene product is a 180 kDa polypeptide and is associated with a membrane fraction , 1990, FEBS letters.

[53]  J. Hancock,et al.  Activation of Raf as a result of recruitment to the plasma membrane. , 1994, Science.

[54]  I Gout,et al.  PI 3‐kinase is a dual specificity enzyme: autoregulation by an intrinsic protein‐serine kinase activity. , 1994, The EMBO journal.

[55]  A. Levitzki,et al.  Phosphorylation of the S. cerevisiae Cdc25 in response to glucose results in its dissociation from Ras , 1992, Nature.

[56]  E. Nishida,et al.  The MAP kinase cascade is essential for diverse signal transduction pathways. , 1993, Trends in biochemical sciences.

[57]  M. Wigler,et al.  DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[58]  R. Lerner,et al.  Generation of protein-reactive antibodies by short peptides is an event of high frequency: implications for the structural basis of immune recognition. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[59]  D. Bar-Sagi,et al.  Grb2 mediates the EGF-dependent activation of guanine nucleotide exchange on Ras , 1993, Nature.

[60]  E. Scolnick,et al.  Identification of guanine nucleotides bound to ras-encoded proteins in growing yeast cells. , 1987, The Journal of biological chemistry.

[61]  M. Alava,et al.  Microfilament assembly modulates phospholipase C-mediated signal transduction by the TCR/CD3 in murine T helper lymphocytes. , 1992, Journal of immunology.

[62]  C. Crews,et al.  Raf-1 forms a stable complex with Mek1 and activates Mek1 by serine phosphorylation. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[63]  Sally J. Leevers,et al.  Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane , 1994, Nature.

[64]  M. Wigler,et al.  Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase. , 1989, Proceedings of the National Academy of Sciences of the United States of America.