The cysteine‐cluster motif of c‐Src: Its role for the heavy metal‐mediated activation of kinase

Abstract

We have previously reported that c-Src is activated by mercuric chloride (HgCl 2 ). We investigated the mechanism of this activation and found that in vitro activation of c-Src by HgCl 2 did not require tyrosine residues at 416 and 527. Both SH2 and SH3 domains of c-Src were dispensable for the activation by HgCl 2 . In contrast, iodoacetoamide (IAA) that binds to thiol side chain of cysteine blocked the activation of c-Src by HgCl 2 . To obtain more clues, each cysteine residue of c-Src was replaced with alanine. Of six cysteine residues in the kinase domain of c-Src, Cys483 and Cys498 located in the C-terminal portion as a cysteine-cluster (CC) motif were critical for the activation. In addition, other Src family kinases, Yes and Lyn, were activated by treatment with HgCl 2 , and cysteine residues, especially those correspond to Cys498 of Src in the CC motif of these kinases, were also required for the activation of the kinases by HgCl 2 . In addition to these observations, treatment of cells with HgCl 2 dramatically activated the wild-type c-Src, whereas it could not activate the mutant form of Src with a substitution of Cys498. Taken together, our results disclose that cysteine residues in the CC motif of c-Src, Cys483 and Cys498, act as a module for the activation of the kinase by a heavy metal compound, mercuric chloride. c -Src, a cellular counterpart of viral oncogene product, v-Src, is a non-receptor tyrosine kinase distributed widely in tissues.(1) Activation of c-Src is frequently observed in various human carcinomas including those of breast and colon carcinomas and is critical for their metastasis, (2) suggesting the importance of Src signaling in human malignant tumors. Regulation of kinase activity of c-Src has been well characterized. Phosphorylation of tyrosine 527 (Tyr527) located near the Cterminus appears to down-regulate the kinase. Point mutation of Tyr527 renders c-Src active and highly oncogenic, (3)(4)(5) whereas targeted disruption of C-terminal SRC kinase (CSK), (6) the kinase that phosphorylates Tyr527, caused activation of c-Src. (7) Studies of the three-dimensional structure of c-Src revealed that interaction between SH2 and phosphorylated Tyr527 together with SH3 and linker region folded the molecule in a closed, inactive state. (8,9) In addition to the Tyr527-based regulation, increasing evidence suggests that the c-Src kinase has a redox-linked regulatory mechanism.(10) One of the models of the redox-linked regulation, we found, is the activation of c-Src by mercuric chloride (HgCl 2 ).(11) Treatment of purified c-Src with mercuric chloride changed V max and K m of the kinase, suggesting the kinase activation to be a direct one.(11) Mercuric chloridetreatment of cells activates signal pathways leading to tyrosine phosphorylation of cellular proteins, (12) and activation of mitogenactivated protein (MAP) kinases, (13)(14)(15)(16) which in turn induce the expression of cytokines, (17) unregulated cell growth and DNA synthesis. (13,18) Consistently, treatment of c-Src with mercuric ...

S-Nitrosylation at Cysteine 498 of c-Src Tyrosine Kinase Regulates Nitric Oxide-mediated Cell Invasion

“…Mutation of cysteine residues in the CC motif abolished the inactivation of the kinase by these agents (19). On the other hand, mercuric ions that have high affinity for thiol side chain of cysteine residues activate c-Src, which is dependent on the modification of Cys 498 (20). These previous studies and our present study strongly suggest the importance of cysteine modification for the regulation of c-Src for diverse intracellular signaling pathways.…”

“…Our previous studies have indicated the importance of cysteine residues located in the C terminus of the catalytic domain (18,19). Four cysteines, namely Cys 483 , Cys 487 , Cys 496 , and Cys 498 are clustered in the C terminus of c-Src, which we referred to as the cysteine-clustered motif (CC motif) (20). Substitution of these cysteine residues rendered the kinase refractory to the inactivation by SH-alkylating agents and the activation by mercuric ions that have high affinity for thiols of cysteines (19 -21).…”

ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17

“…While the precise mechanism of Src activation by H 2 O 2 is currently unknown, a number of reports indicate that Src and related kinases, such as Lyn and Yes, are subject to redox regulation which involves oxidation of one or more specific conserved cysteine residues within a cysteine-cluster in the C-terminal region [32], [33]. It has been suggested that tyrosine auto-phosphorylation at Y416 and cysteine oxidation within Src are successive events during e.g.…”

“…Another potential mechanism by which H 2 O 2 may mediate EGFR transactivation is by oxidative activation of non-receptor tyrosine kinases of the Src family [27], [28], which promote EGFR phosphorylation at selected residues in a ligand-independent fashion [29], [30]. The activity of Src is tightly controlled by inhibitory tyrosine phosphorylation at Y527 and by auto-phosphorylation at Y416 during activation, but recent evidence indicate that Src family kinases are also regulated by oxidation of conserved cysteine residues with the C-terminal region [31], [32], [33], and such oxidative modification of Src kinases have been implicated in cell adhesion and spreading and in wound responses [31], [34].…”

Hydrogen Peroxide as a Damage Signal in Tissue Injury and Inflammation: Murderer, Mediator, or Messenger?

“…wound responses, the precise oxidative cysteine modification(s) or their direct consequence for SFK activity have not been established. Moreover, contrasting the identification of specific cysteines involved in SFK activation by ROS or by thiol-reactive heavy metals [29, 30] are findings that oxidative mechanisms can also inactivate Src, e.g. through homodimerization by disulfide linkage involving Cys277 [31].…”