MKK6, a specific upstream activator for the p38 MAPK, contains six cysteine residues. In vitro, MKK6 is inactive in the absence of DTT, a reducing agent that could prevent formation of disulphide bonds between cysteines. Addition of DTT restores the kinase activity of MKK6 towards the p38 MAPK. Similar phenomenon was also observed in cells. Site-directed mutagenesis was performed to identify cysteines that may form disulphide bonds resulting in inactivation of MKK6. Four out of six cysteines (i.e. Cys109, Cys128, Cys196 and Cys294) were implicated in such a process. Interestingly, we found that these cysteine residues have addition roles in either maintaining the proper conformation or participating in enzymatic function of MKK6, as mutation at or modification of these residues signific...[ Read more ]

MKK6, a specific upstream activator for the p38 MAPK, contains six cysteine residues. In vitro, MKK6 is inactive in the absence of DTT, a reducing agent that could prevent formation of disulphide bonds between cysteines. Addition of DTT restores the kinase activity of MKK6 towards the p38 MAPK. Similar phenomenon was also observed in cells. Site-directed mutagenesis was performed to identify cysteines that may form disulphide bonds resulting in inactivation of MKK6. Four out of six cysteines (i.e. Cys109, Cys128, Cys196 and Cys294) were implicated in such a process. Interestingly, we found that these cysteine residues have addition roles in either maintaining the proper conformation or participating in enzymatic function of MKK6, as mutation at or modification of these residues significantly reduced the kinase activity of MKK6. We further demonstrated that, while the ability of the oxidized (i.e., inactive) MKK6 to bind the p38 MAPK was not compromised, its ability to bind ATP was significantly reduced. Thus, our findings provide a clear mechanism for regulation of MKK6 activity by the redox state of its cysteines and suggest that MKK6 may serve as a redox sensor in cells.