Mammalian Ras interacts directly with the serine/threonine kinase raf

We have identified proteins that interact with H-Ras using a two hybrid system screen of a mouse cDNA library. Approximately 50% of the clones identified encoded portions of the c-Raf and A-Raf serine/threonine kinases. Overlaps among these clones define a conserved 81 residue region of the N-terminus of Raf as the Ras interaction region. We show that Raf interacts with wild-type and activated Ras, but not with an effector domain mutant of Ras or with a dominant-interfering Ras mutant. Using purified bacterially expressed fusion proteins, we show, furthermore, that Ras and the N-terminal region of Raf associate directly in vitro and that this interaction is dependent on GTP bound to Ras.

[1]  N. Perrimon,et al.  Requirement of the Drosophila raf homologue for torso function , 1989, Nature.

[2]  Jonathan A. Cooper,et al.  Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit. , 1991, Cell regulation.

[3]  Mark R. Smith,et al.  Requirement for c-ras proteins during viral oncogene transformation , 1986, Nature.

[4]  Morrison Dk,et al.  The Raf-1 kinase as a transducer of mitogenic signals. , 1990 .

[5]  J. Bishop,et al.  Torso, a receptor tyrosine kinase required for embryonic pattern formation, shares substrates with the sevenless and EGF-R pathways in Drosophila. , 1993, Genes & development.

[6]  Jonathan A. Cooper,et al.  Identification and characterization of a cDNA encoding mouse CAP: a homolog of the yeast adenylyl cyclase associated protein. , 1993, Journal of cell science.

[7]  S. Fields,et al.  A novel genetic system to detect protein–protein interactions , 1989, Nature.

[8]  G. Rubin,et al.  Signalling by the sevenless protein tyrosine kinase is mimicked by Rasl activation , 1992, Nature.

[9]  G. Yu,et al.  Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins , 1992, Molecular and cellular biology.

[10]  W. Kolch,et al.  Raf-1 protein kinase is required for growth of induced NIH/3T3 cells , 1991, Nature.

[11]  T. Pawson,et al.  Mutational activation of c-raf-1 and definition of the minimal transforming sequence , 1990, Molecular and cellular biology.

[12]  P. Fürst,et al.  The Saccharomyces cerevisiae CDC25 gene product binds specifically to catalytically inactive ras proteins in vivo , 1992, Molecular and cellular biology.

[13]  M. Ptashne,et al.  Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae , 1984, Molecular and cellular biology.

[14]  T. Haystead,et al.  Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro. , 1992, Science.

[15]  M. Wigler,et al.  RAS proteins can induce meiosis in xenopus oocytes , 1985, Cell.

[16]  Anthony J. Muslin,et al.  Raf-1 kinase is essential for early Xenopus development and mediates the induction of mesoderm by FGF , 1993, Cell.

[17]  C. Marshall,et al.  The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product , 1988, Nature.

[18]  C. Marshall,et al.  MAP kinase regulation--the oncogene connection. , 1992, Trends in cell biology.

[19]  M. Wigler,et al.  Functional homology of mammalian and yeast RAS genes , 1985, Cell.

[20]  Frank McCormick,et al.  The GTPase superfamily: a conserved switch for diverse cell functions , 1990, Nature.

[21]  James R. Broach,et al.  SRV2, a gene required for RAS activation of adenylate cyclase in yeast , 1990, Cell.

[22]  E. Scolnick,et al.  Identification of effector residues and a neutralizing epitope of Ha-ras-encoded p21. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[23]  V. Stanton,et al.  Definition of the human raf amino-terminal regulatory region by deletion mutagenesis , 1989, Molecular and cellular biology.

[24]  D. Morrison,et al.  Signal transduction from membrane to cytoplasm: growth factors and membrane-bound oncogene products increase Raf-1 phosphorylation and associated protein kinase activity. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[25]  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.

[26]  F. McCormick,et al.  Role of GTPase activating protein in mitogenic signalling through phosphatidylcholine‐hydrolysing phospholipase C. , 1991, The EMBO journal.

[27]  U. Rapp,et al.  Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. , 1992, Genes & development.

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

[29]  M. Liyanage,et al.  Activation of the c-Raf protein kinase by protein kinase C phosphorylation. , 1992, Oncogene.

[30]  V. Zakian,et al.  Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA , 1982, Molecular and cellular biology.

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

[32]  P. Sternberg,et al.  let-60, a gene that specifies cell fates during C. elegans vulval induction, encodes a ras protein , 1990, Cell.

[33]  M. Wigler,et al.  Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein , 1990, Cell.

[34]  David L. Brautigan,et al.  Raf-1 activates MAP kinase-kinase , 1992, Nature.

[35]  John Calvin Reed,et al.  Differential regulation of the p72-74 RAF-1 kinase in 3T3 fibroblasts expressing ras or src oncogenes. , 1991, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[36]  T. Roberts,et al.  Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Frank McCormick,et al.  The GTPase superfamily: conserved structure and molecular mechanism , 1991, Nature.

[38]  J. Blenis,et al.  ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK , 1992, Cell.

[39]  R. Brent,et al.  Mxi1, a protein that specifically interacts with Max to bind Myc-Max recognition sites , 1993, Cell.

[40]  F. Clerc,et al.  Identification of the SH3 domain of GAP as an essential sequence for Ras-GAP-mediated signaling. , 1993, Science.

[41]  D. Lowy,et al.  Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. , 1988, Science.

[42]  M. Wigler,et al.  Dominant yeast and mammalian RAS mutants that interfere with the CDC25-dependent activation of wild-type RAS in Saccharomyces cerevisiae , 1989, Molecular and cellular biology.

[43]  L. Feig.  The many roads that lead to Ras. , 1993, Science.

[44]  D. Lowy,et al.  Suppression of c-ras transformation by GTPase-activating protein , 1990, Nature.

[45]  E. Hafen,et al.  Raf functions downstream of Rasl in the Sevenless signal transduction pathway , 1992, Nature.

[46]  N. Perrimon,et al.  Control of cell fate determination by p21ras/Ras1, an essential component of torso signaling in Drosophila. , 1993, Genes & development.

[47]  M. Yamamoto,et al.  Role of a ras homolog in the life cycle of schizosaccharomyces pombe , 1986, Cell.

[48]  F. McCormick,et al.  A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. , 1987, Science.

[49]  M. Wigler,et al.  The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway , 1987, Cell.

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

[51]  D. Beach,et al.  Involvement of ras in sexual differentiation but not in growth control in fission yeast , 1986, The EMBO journal.

[52]  T. Bonner,et al.  Characterization of murine A-raf, a new oncogene related to the v-raf oncogene , 1986, Molecular and cellular biology.

[53]  S. Hattori,et al.  The ras oncogene product p21 is not a regulatory component of adenylate cyclase , 1985, Nature.

[54]  N. Copeland,et al.  Sequence homology shared by neurofibromatosis type-1 gene and IRA-1 and IRA-2 negative regulators of the RAS cyclic AMP pathway , 1990, Nature.

[55]  D. Lowy,et al.  Mutational analysis of a ras catalytic domain , 1986, Molecular and cellular biology.

[56]  J. Cleveland,et al.  Expression of raf family proto-oncogenes in normal mouse tissues. , 1990, Oncogene.

[57]  R. Treisman,et al.  Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element , 1992, Cell.

[58]  H. Horvitz,et al.  Caenorhabditis elegans ras gene let-60 acts as a switch in the pathway of vulval induction , 1990, Nature.

[59]  N. Perrimon,et al.  Developmental and molecular characterization of mutations in the Drosophila-raf serine/threonine protein kinase. , 1993, Development.

[60]  Margaret Robertson,et al.  The neurofibromatosis type 1 gene encodes a protein related to GAP , 1990, Cell.

[61]  G. Rubin,et al.  A putative Ras GTPase activating protein acts as a negative regulator of signaling by the Sevenless receptor tyrosine kinase , 1992, Cell.

[62]  T. Roberts,et al.  The cytoplasmic raf oncogene induces a neuronal phenotype in PC12 cells: a potential role for cellular raf kinases in neuronal growth factor signal transduction. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[63]  M. Wigler,et al.  Mutations of the adenylyl cyclase gene that block RAS function in Saccharomyces cerevisiae. , 1990, Science.

[64]  M. Boguski,et al.  Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins , 1993, Molecular and cellular biology.

[65]  L. Breeden,et al.  Regulation of the yeast HO gene. , 1985, Cold Spring Harbor symposia on quantitative biology.

[66]  D. E. Griffiths,et al.  DMSO-enhanced whole cell yeast transformation. , 1991, Nucleic acids research.

[67]  Paul W. Sternberg,et al.  C. elegans lin-45 raf gene participates in let-60 ras-stimulated vulval differentiation , 1993, Nature.

[68]  D. Melton,et al.  Involvement of p21ras in Xenopus mesoderm induction , 1992, Nature.

[69]  A. Brown,et al.  ras p21 and GAP inhibit coupling of muscarinic receptors to atrial K+ channels , 1990, Cell.