title: Clustered cysteine residues in the kinase domain of v-Src: critical role for protein stability, cell transformation and sensitivity to herbimycin A.
abstract: We have previously reported the activation of Src by mercuric chloride based on the sulfhydryl modification. To evaluate the significance of cysteine residues in v-Src, we replaced each cysteine to alanine by oligonucleotide-directed mutagenesis and examined its effect on cell transformation. Of ten cysteine residues scattered over v-Src, four cysteines clustered in kinase domain, Cys483, Cys487, Cys496 and Cys498, were important for protein stability and cell transformation, whereas those in SH2 domain were dispensable. A single mutation in Cys498 yielded suppression of kinase activity and a temperature-sensitivity in anchorage independent growth. Double mutation either in Cys483/Cys487 or in Cys496/Cys498 yielded clear temperature-sensitivity in cell transformation and in stability of Src protein. Instability of Src protein was magnified by quadruple mutation in the cysteines, which decreased the half-life of Src to be less than one quarter of that of wild-type. In addition, both Cys483/Cyr487 and Cys496/Cys498 kinases became resistant to in vitro inactivation by herbimycin A, which directly inactivates v-Src in addition to its effect on HSP90. Taken together, our results strongly suggest that the cysteine clustered motif of v-Src are critical for protein stability, cell transformation and in vitro inactivation by herbimycin A.
authors.affiliation: Department of Molecular Pathogenesis, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-Ku, Nagoya 466-8550, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Senga
authors.forename: K
authors.initials: K
authors.lastname: Miyazaki
authors.forename: K
authors.initials: K
authors.lastname: Machida
authors.forename: H
authors.initials: H
authors.lastname: Iwata
authors.forename: S
authors.initials: S
authors.lastname: Matsuda
authors.forename: I
authors.initials: I
authors.lastname: Nakashima
authors.forename: M
authors.initials: M
authors.lastname: Hamaguchi
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D016227:Benzoquinones; D019556:COS Cells; D002460:Cell Line; D002472:Cell Transformation, Viral / Q000235:genetics / Q000502:physiology*; D003545:Cysteine / Q000378:metabolism*; D004351:Drug Resistance / Q000235:genetics; D004795:Enzyme Stability; D006540:Herbicides / Q000494:pharmacology*; D047029:Lactams, Macrocyclic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism / Q000502:physiology*; D011809:Quinones / Q000494:pharmacology*; D051381:Rats; D017828:Rifabutin / Q000031:analogs & derivatives; D013696:Temperature; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D016227:Benzoquinones; D006540:Herbicides; D047029:Lactams, Macrocyclic; D011809:Quinones; D017828:Rifabutin; C020754:herbimycin; D015688:Oncogene Protein pp60(v-src); D019061:src-Family Kinases; D003545:Cysteine