Title

Cysteine residues in the C-terminal lobe of Src: their role in the suppression of the Src kinase

Document Type

Article

Publication Date

3-2009

Abstract

To evaluate the function of cysteine residues of the Src kinase, we constructed a series of Src mutants in which some of cyteines were replaced to alanines. With these mutants, we studied the effect of SH-alkylating agents, N-[p-(2-benzimidazolyl)phenyl] maleimide (BIPM) and N-(9-acridinyl) maleimide (NAM), on their kinase activity. Of 10 cysteine residues scattered over v-Src, either a single mutation at Cys520 or multiple mutations at the four clustered cyteines, Cys483, Cys487, Cys496 and Cys498, yielded clear resistance to the treatment with 10 μ M BIPM or 1 μ M NAM. In contrast, other cysteines including those in the SH2 domain and those in the catalytic cleft of the kinase domain were dispensable for the inactivation by BIPM and NAM. Similarly, deletion of SH2 and SH3 did not confer the resistance to v-Src, suggesting the inactivation by the SH-alkylating agents is SH2/SH3-independent. Although Cys520-mutated v-Src was resistant to 1 μ M NAM, it was inactivated by 5 μ M NAM. However, combined mutation including all of Cys483, Cys487, Cys496, Cys498 and Cys520 yielded clear resistance to 5 μ M NAM. Among these mutants, those with double mutations in the four clustered cysteines yielded a temperature sensitive phenotype in the transfected cells, whereas Cys520 did not, suggesting that Cys520 has, at least in part, a discrete function. In contrast to v-Src, c-Src, which lacks cysteine at position 520, was resistant to 1 μ M NAM but sensitive to 5 μ M NAM. While replacement of Phe520 of c-Src to cysteine made it sensitive to 1 μ M NAM, double mutation in clustered cysteines again yielded resistance to 5 μ M NAM. Taken together, our results strongly suggest that the multiple cysteine residues clustered at the end of the C-terminal lobe are critical for the inhibition by the SH-alkylating agents and, thereby, have an allosteric repressor effect on the catalytic activity of Src in a SH2-phosphoTyr527 independent manner.

Comments

Copyright © 2003 Nature Publishing Group All rights reserved.

Share

COinS