Clustered cysteine residues in the kinase domain of v-Src: critical role for protein stability, cell transformation and sensitivity to herbimycin A

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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.