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Clustered cysteine residues in the kinase domain of v-Src: critical role for protein stability, cell transformation and sensitivity to herbimycin A.
- Source :
-
Oncogene [Oncogene] 2000 Jan 13; Vol. 19 (2), pp. 273-9. - Publication Year :
- 2000
-
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.
- Subjects :
- Amino Acid Sequence
Amino Acid Substitution genetics
Animals
Benzoquinones
COS Cells
Cell Line
Cell Transformation, Viral genetics
Drug Resistance genetics
Enzyme Stability
Lactams, Macrocyclic
Molecular Sequence Data
Mutagenesis, Site-Directed
Oncogene Protein pp60(v-src) metabolism
Rats
Rifabutin analogs & derivatives
Temperature
src Homology Domains genetics
src-Family Kinases genetics
src-Family Kinases metabolism
Cell Transformation, Viral physiology
Cysteine metabolism
Herbicides pharmacology
Oncogene Protein pp60(v-src) physiology
Quinones pharmacology
src Homology Domains physiology
src-Family Kinases physiology
Subjects
Details
- Language :
- English
- ISSN :
- 0950-9232
- Volume :
- 19
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Oncogene
- Publication Type :
- Academic Journal
- Accession number :
- 10645006
- Full Text :
- https://doi.org/10.1038/sj.onc.1203296