Cytoplasmic Localization of Cyclin Kinase Inhibitor p21 Delays the Progression of Apoptosis

Abstract

The different functions of the cyclin kinase inhibitor, p21, rely on its localization to either the cytoplasm or nucleus. Phosphorylation at Thr-145 and/or Ser-146 was reported to target p21 to the cytoplasm. To clarify the function of cytoplasmic p21, we constructed non-phosphorylatable mutants, Thr-145 to Ala (T145A) and Ser-146 to Ala (S146A), and phosphorylation mimic mutants, Thr-145 to Asp (T145D) and Ser-146 to Asp (S146D), and the cells stably expressing those mutants were identified. The association of all four mutants with either CyclinA or CDK2 was increased by Á-irradiation, indicating that the mutants functioned as cyclin kinase inhibitors. PCNA binding was detected in T145A and S146A, but not in T145D and S146D. In the stably-expressing cells, T145D and S146D binding was observed in the cytoplasm, while T145A and S146A in the nucleus. Further, lactacystin treatment enhanced T145A and S146A, but not T145D and S146D, which is consistent with the degradation of p21 by proteasome in the nucleus. Apoptosis induced by Á-irradiation was delayed in the cells expressing either T145D or S146D. The activities of caspase 3 were not reduced in mutant-expressing cells. These results suggest that the PCNA-unbound form of the full length p21 in the cytoplasm delays apoptosis through the interaction with caspase 3 or downstream components.