SH2 mutants of c-src that are host dependent for transformation are trans-dominant inhibitors of mouse cell transformation by activated c-src.

The c-src gene encodes a membrane-associated protein-tyrosine kinase, pp60c-src, whose substrates and regulators have not been identified. In an effort to obtain mutants that might assist the search for proteins that interact with pp60c-src, we have generated by site-directed mutagenesis two alleles of chicken c-src that are host dependent for transformation and inhibit transformation of mouse cells by activated c-src in a trans-dominant manner. These alleles, named M6 and M9, encode nonconservative changes within the highly conserved FLVRES sequence in the src homology-2 (SH2) region of pp60c-src, as well as an activating change, Y527F, near the carboxyl terminus. M6 and M9 transform chicken embryo fibroblasts (CEF) more efficiently than the parental allele (Y527F c-src), but fail to transform mouse NIH-3T3 cells. The product of M6-src is less stable than activated pp60c-src in NIH-3T3 cells and shows decreased protein tyrosine kinase activity on all tested substrates; the product of M9-src, however, is stable, has a novel pattern of substrate preference for tyrosine phosphorylation in vitro, and induces a pattern of phosphotyrosine-containing proteins in mouse cells that is similar to that induced by Y527F c-src, even though it fails to transform these cells. Both M6 and M9 inhibit transformation of NIH-3T3 cells by activated c-src in a dose-dependent manner, as assayed by resistance of M6- or M9-expressing cells to transformation by Y527F c-src or by morphological reversion of cells previously transformed by activated c-src following introduction of M6 or M9. When reversion occurs, the concentration of protein encoded by the active allele declines, without change in level of src mRNA or rate of src protein synthesis, implying that the products of M6 and M9, when present at adequate levels, can destabilize transformation-competent src protein. These alleles offer new opportunities for interfering with the actions of src-related genes and for isolating cellular factors required for the functions of those genes.

[1]  H. Hanafusa,et al.  Activation of the proto-oncogene p60c-src by point mutations in the SH2 domain , 1990, Molecular and cellular biology.

[2]  H. Varmus,et al.  Site-directed mutagenesis of the SH2- and SH3-coding domains of c-src produces varied phenotypes, including oncogenic activation of p60c-src , 1990, Molecular and cellular biology.

[3]  A. Reith,et al.  W mutant mice with mild or severe developmental defects contain distinct point mutations in the kinase domain of the c-kit receptor. , 1990, Genes & development.

[4]  H. Varmus,et al.  The src protein contains multiple domains for specific attachment to membranes , 1990, Molecular and cellular biology.

[5]  M. Resh.  Specific and saturable binding of pp60v-src to plasma membranes: Evidence for a myristyl-src receptor , 1989, Cell.

[6]  A. Levine,et al.  The p53 proto-oncogene can act as a suppressor of transformation , 1989, Cell.

[7]  Klaus Damm,et al.  Protein encoded by v-erbA functions as a thyroid-hormone receptor antagonist , 1989, Nature.

[8]  J. Maller,et al.  Purified maturation promoting factor phosphorylates pp60c-src at the sites phosphorylated during fibroblast mitosis , 1989, Cell.

[9]  D. Baltimore,et al.  N‐terminal mutations activate the leukemogenic potential of the myristoylated form of c‐abl. , 1989, The EMBO journal.

[10]  G. Martin,et al.  Linker insertion-deletion mutagenesis of the v-src gene: isolation of host- and temperature-dependent mutants , 1989, Journal of virology.

[11]  H. Kitayama,et al.  A ras-related gene with transformation suppressor activity , 1989, Cell.

[12]  H. Varmus,et al.  A mutation in v-src that removes a single conserved residue in the SH-2 domain of pp60v-src restricts transformation in a host-dependent manner , 1989, Journal of virology.

[13]  J. Wang,et al.  Deletion of an N‐terminal regulatory domain of the c‐abl tyrosine kinase activates its oncogenic potential. , 1989, The EMBO journal.

[14]  J. Parsons,et al.  Deletions and insertions within an amino-terminal domain of pp60v-src inactivate transformation and modulate membrane stability , 1989, Journal of virology.

[15]  T. Pawson,et al.  Non-catalytic domains of cytoplasmic protein-tyrosine kinases: regulatory elements in signal transduction. , 1988, Oncogene.

[16]  Irving S. Sigal,et al.  Cloning of bovine GAP and its interaction with oncogenic ras p21 , 1988, Nature.

[17]  B. Mayer,et al.  A novel viral oncogene with structural similarity to phospholipase C , 1988, Nature.

[18]  R. Kriz,et al.  Sequence similarity of phospholipase C with the non-catalytic region of src , 1988, Nature.

[19]  Mark D. Johnson,et al.  Overproduction of protein kinase C causesdisordered growth control in rat fibroblasts , 1988, Cell.

[20]  T. Pawson,et al.  A conserved domain regulates interactions of the v-fps protein-tyrosine kinase with the host cell. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[21]  S. Hughes,et al.  Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors , 1987, Journal of virology.

[22]  I. Herskowitz.  Functional inactivation of genes by dominant negative mutations , 1987, Nature.

[23]  D. Baltimore,et al.  Neuronal pp60c-src contains a six-amino acid insertion relative to its non-neuronal counterpart. , 1987, Science.

[24]  H. Varmus,et al.  Identification of a human gene (HCK) that encodes a protein-tyrosine kinase and is expressed in hemopoietic cells , 1987, Molecular and cellular biology.

[25]  R. Perlmutter,et al.  Novel protein-tyrosine kinase gene (hck) preferentially expressed in cells of hematopoietic origin , 1987, Molecular and cellular biology.

[26]  J. Wyke,et al.  Genetic analysis of the form and function of the viral src oncogene product. , 1987, Biochimica et biophysica acta.

[27]  T. Kmiecik,et al.  Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation , 1987, Cell.

[28]  K. Semba,et al.  Characterization of cDNA clones for the human c-yes gene , 1987, Molecular and cellular biology.

[29]  K. Semba,et al.  The yes-related cellular gene lyn encodes a possible tyrosine kinase similar to p56lck , 1987, Molecular and cellular biology.

[30]  R. Jove,et al.  Cell transformation by the viral src oncogene. , 1987, Annual review of cell biology.

[31]  A. Barnekow,et al.  An src-related tyrosine kinase activity in the hydroid, Hydractinia , 1986 .

[32]  T Pawson,et al.  A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps , 1986, Molecular and cellular biology.

[33]  M. Yoshida,et al.  yes-related protooncogene, syn, belongs to the protein-tyrosine kinase family. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[34]  M. Yoshida,et al.  Structure, expression, and chromosomal location of the human c-fgr gene , 1986, Molecular and cellular biology.

[35]  J. Brugge,et al.  Interaction of the Rous sarcoma virus protein pp60src with the cellular proteins pp50 and pp90. , 1986, Current topics in microbiology and immunology.

[36]  E. Krebs,et al.  A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA , 1985, Cell.

[37]  A. V. D. van den Ouweland,et al.  The structure of the human c‐fes/fps proto‐oncogene. , 1985, The EMBO journal.

[38]  T. Kornberg,et al.  The nucleotide sequence and the tissue-specific expression of Drosophila c-src , 1985, Cell.

[39]  F. Cross,et al.  N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene , 1985, Molecular and cellular biology.

[40]  H. Kung,et al.  Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src , 1985, Molecular and cellular biology.

[41]  Thomas A. Kunkel,et al.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[42]  G. Church,et al.  Genomic sequencing. , 1993, Methods in molecular biology.

[43]  Rosalind C. Lee,et al.  The mouse c-abl locus: Molecular cloning and characterization , 1984, Cell.

[44]  H. Hanafusa,et al.  Structure and sequence of the cellular gene homologous to the RSV src gene and the mechanism for generating the transforming virus , 1983, Cell.

[45]  J. Parsons,et al.  Site-directed mutagenesis of the src gene of Rous sarcoma virus: construction and characterization of a deletion mutant temperature sensitive for transformation , 1982, Journal of virology.

[46]  P. Sharp,et al.  Construction of a modular dihydrofolate reductase cDNA gene: analysis of signals utilized for efficient expression , 1982, Molecular and cellular biology.

[47]  H. Varmus,et al.  Two cellular proteins that immunoprecipitate with the transforming protein of Rous sarcoma virus. , 1981, Virology.

[48]  H. Varmus,et al.  Revertants of an ASV-transformed rat cell line have lost the complete provius or sustained mutations in src. , 1981, Virology.

[49]  H. Varmus,et al.  The purified product of the transforming gene of avian sarcoma virus phosphorylates tyrosine. , 1980, The Journal of biological chemistry.