Clustered cysteine residues in the kinase domain of v-Src: critical role for protein stability, cell transformation and sensitivity to herbimycin A.
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.
Senga
T
T
Department of Molecular Pathogenesis, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-Ku, Nagoya 466-8550, Japan.
Miyazaki
K
K
Machida
K
K
Iwata
H
H
Matsuda
S
S
Nakashima
I
I
Hamaguchi
M
M
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*
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