Thiol deprivation induced premature enhancement of both enzymatic activity and phosphorylation of CDK6 and CDK2. (A) NK3.3 cells were stimulated with IL-2 (200 U/mL) in CYS(+) or CYS(−) medium for the indicated time, and equivalent protein amounts in each lysate were immunoprecipitated with antibodies to CDK6 or CDK2. In vitro kinase assays were performed on the immunoprecipitates using truncated RB (56 kD) or H1 histone as substrates for CDK6 or CDK2, respectively. (B) NK3.3 cells were labeled with [³²P]-orthophosphate and stimulated with IL-2 (200 U/mL) in CYS(+) or CYS(-) media for the indicated times, and lysates were immunoprecipitated with antibodies to CDK6 or CDK2. Equivalent protein content in each lane and identity of each band were verified by Western blot analysis with antibody to CDK6 or CDK2, respectively. (C) Dephosphorylation of CDK6 was assessed in cells that had been labeled with [³²P]-orthophosphate in CYS(−) medium in the presence of IL-2 (200 U/mL), and split into two cultures, one continued in CYS(−) medium, and one placed in CYS(+), each with IL-2, for the indicated times. CDK6 protein amount in each lane was verified by Western blot analysis in one of two independent experiments (not shown). Each set of data is representative of two independent experiments.