Mutagenic analysis of functional domains of the mos proto-oncogene and identification of the sites important for MAPK activation and DNA binding.
We constructed in-frame deletion/replacement mutations in the Xenopus mos proto-oncogene that lie within conserved Mos-specific codons, but outside of the regions that are conserved among the src kinase family of genes. All gene products were assayed in vitro for kinase activity and in vivo for their ability to induce oocyte maturation, embryonic cleavage arrest and cellular transformation. Most mutations in Mos eliminated both kinase and biological activity. However, a mutation in Mos that removed two basic amino acid residues (R94 and K97) downstream from the lysine at the ATP binding site (K90) markedly enhanced autophosphorylation activity. Moreover, this mutant displayed markedly reduced biological activity, lacked transforming activity, and failed to activate mitogen activated protein kinase (MAPK). A second mutant Mos product, lacking amino acids R45-A54, displayed a five-fold increase in cellular transforming activity. This Mos mutant specifically localized to the cytoplasm; in contrast to wild-type (wt) Mos that localized to both the nucleus and the cytoplasm. These data indicate that Mos transforming activity is mediated via signalling exerted in the cytoplasm, presumably through MAPK, and that nuclear localization of the oncogene product interferes with transforming activity. We also show that amino acids R45-A54 are important for Mos DNA binding activity.
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002471:Cell Transformation, Neoplastic; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D019084:Fluorescent Antibody Technique, Indirect; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D017231:Proto-Oncogene Proteins c-mos / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D017931:DNA Primers; D004268:DNA-Binding Proteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017231:Proto-Oncogene Proteins c-mos; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1