Mutational activation of c-raf-1 and definition of the minimal transforming sequence

A series of wild-type and mutant raf genes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine- and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and CR2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.

[1]  B. Wasylyk,et al.  Expression of raf oncogenes activates the PEA1 transcription factor motif , 1989, Molecular and cellular biology.

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

[3]  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.

[4]  T. Sugimura,et al.  Identification of a transforming activity suppressing sequence in the c-raf oncogene. , 1988, Oncogene.

[5]  Y. Ueyama,et al.  B-raf, a new member of the raf family, is activated by DNA rearrangement , 1988, Molecular and cellular biology.

[6]  R. Evans,et al.  The steroid and thyroid hormone receptor superfamily. , 1988, Science.

[7]  Y. Nishizuka,et al.  Proliferation of both somatic and germ cells is affected in the Drosophila mutants of raf proto‐oncogene. , 1988, The EMBO journal.

[8]  W. Kolch,et al.  Preparation of raf-oncogene-specific antiserum with raf protein produced in E. coli. , 1988, Biochimica et biophysica acta.

[9]  U. Rapp,et al.  Identification and characterization of c-raf phosphoproteins in transformed murine cells. , 1988, Oncogene.

[10]  F. Galibert,et al.  Replacement of lys 622 in the ATP binding domain of P100gag‐mil abolishes the in vitro autophosphorylation of the protein and the biological properties of the v‐mil oncogene of MH2 virus. , 1988, The EMBO journal.

[11]  V. Stanton,et al.  Activation of human raf transforming genes by deletion of normal amino-terminal coding sequences , 1987, Molecular and cellular biology.

[12]  T. Bonner,et al.  The complete coding sequence of the human A-raf-1 oncogene and transforming activity of a human A-raf carrying retrovirus. , 1987, Nucleic acids research.

[13]  A. Ullrich,et al.  The complete primary structure of protein kinase C--the major phorbol ester receptor. , 1986, Science.

[14]  R. Kriz,et al.  Cloning and expression of multiple protein kinase C cDNAs , 1986, Cell.

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

[16]  T. Bonner,et al.  Actively transcribed genes in the raf oncogene group, located on the X chromosome in mouse and human. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[17]  T. Sugimura,et al.  Activation of rat c-raf during transfection of hepatocellular carcinoma DNA. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[18]  J. Berg,et al.  Potential metal-binding domains in nucleic acid binding proteins. , 1986, Science.

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

[20]  M. Kozak.  Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes , 1986, Cell.

[21]  P. Seeburg,et al.  The complete coding sequence of the human raf oncogene and the corresponding structure of the c-raf-1 gene. , 1986, Nucleic acids research.

[22]  U. Rapp,et al.  Detection of the myristylated gag-raf transforming protein with raf-specific antipeptide sera. , 1985, Virology.

[23]  T. Sugimura,et al.  Molecular cloning of an activated human oncogene, homologous to v-raf, from primary stomach cancer. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[24]  T. Yamamoto,et al.  Detection of a raf-related and two other transforming DNA sequences in human tumors maintained in nude mice. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[25]  D. Baltimore,et al.  Specific transforming potential of oncogenes encoding protein‐tyrosine kinases. , 1985, The EMBO journal.

[26]  T. Bonner,et al.  Integration of transfected LTR sequences into the c‐raf proto‐oncogene: activation by promoter insertion. , 1985, The EMBO journal.

[27]  H. Hanafusa,et al.  Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein. , 1985, Science.

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

[29]  B. Sefton,et al.  Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog , 1985, Journal of virology.

[30]  K. Moelling,et al.  Serine- and threonine-specific protein kinase activities of purified gag–mil and gag–raf proteins , 1984, Nature.

[31]  Susan S. Taylor,et al.  Direct evidence that oncogenic tyrosine kinases and cyclic AMP-dependent protein kinase have homologous ATP-binding sites , 1984, Nature.

[32]  S. Hughes,et al.  Mutagenesis of the region between env and src of the SR-A strain of Rous sarcoma virus for the purpose of constructing helper-independent vectors. , 1984, Virology.

[33]  T. Pawson,et al.  Identification of functional regions in the transforming protein of Fujinami sarcoma virus by in-phase insertion mutagenesis , 1984, Cell.

[34]  U. Rapp,et al.  Primary structure of v-raf: relatedness to the src family of oncogenes. , 1984, Science.

[35]  J. Devereux,et al.  A comprehensive set of sequence analysis programs for the VAX , 1984, Nucleic Acids Res..

[36]  D. Baltimore,et al.  Sequences of the A-MuLV protein needed for fibroblast and lymphoid cell transformation , 1983, Cell.

[37]  F H Reynolds,et al.  Structure and biological activity of v-raf, a unique oncogene transduced by a retrovirus. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[38]  Wim G. J. Hol,et al.  Predicted nucleotide-binding properties of p21 protein and its cancer-associated variant , 1983, Nature.

[39]  J. Stephenson,et al.  New mammalian transforming retrovirus: demonstration of a polyprotein gene product , 1983, Journal of virology.

[40]  M. Smith,et al.  Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. , 1982, Nucleic acids research.

[41]  M. O. Dayhoff,et al.  Viral src gene products are related to the catalytic chain of mammalian cAMP-dependent protein kinase. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[42]  E. Krebs,et al.  Amino acid sequence at the ATP-binding site of cGMP-dependent protein kinase. , 1982, The Journal of biological chemistry.

[43]  S. Taylor,et al.  Affinity labeling of cAMP-dependent protein kinase with p-fluorosulfonylbenzoyl adenosine. Covalent modification of lysine 71. , 1981, The Journal of biological chemistry.

[44]  R. Lerner,et al.  Nucleotide sequence of Moloney murine leukaemia virus , 1981, Nature.

[45]  A. van der Eb,et al.  A new technique for the assay of infectivity of human adenovirus 5 DNA. , 1973, Virology.

[46]  Gary Smith.  Genes and Signal Transduction in Multistage Carcinogenesis , 1990 .

[47]  T. Pawson,et al.  raf family serine/threonine protein kinases in mitogen signal transduction. , 1988, Cold Spring Harbor symposia on quantitative biology.

[48]  T. Pawson,et al.  Role of raf oncogenes in lung carcinogenesis , 1988 .

[49]  J. Cleveland,et al.  Transformation by raf and myc oncogenes. , 1986, Princess Takamatsu symposia.

[50]  F. Cross,et al.  An N-terminal peptide from p60src can direct myristylation and plasma membrane localization when fused to heterologous proteins , 1985, Nature.

[51]  H. Krutzsch,et al.  Myristyl amino-terminal acylation of murine retrovirus proteins: an unusual post-translational proteins modification. , 1983, Proceedings of the National Academy of Sciences of the United States of America.