Mutations of phosphorylation sites Ser10 and Thr187 of p27Kip1 abolish cytoplasmic redistribution but do not abrogate G0/1 phase arrest in the HepG2 cell line

Abstract

Listen

Translate article

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 is an important regulator of cell cycle progression as it negatively regulates G0/1 progression and plays a major role in controlling the cell cycle. The screening of the p27Kip1 sequence identified many potential phosphorylation sites. Although Ser10 and Thr187 were shown to be important for p27Kip1 function, the effects of a combined deletion of both sites on p27Kip1 function are still unknown. To investigate the effects of the overexpression of exogenous p27Kip1 protein lacking both the Ser10 and Thr187 sites on subcellular localization, cell cycle, and proliferation, a plasmid was constructed containing mutations of p27Kip1 at Ser10 and Thr187 (S10A/T187A p27), and transfected into the HepG2 cell line with Lipofectamine. Wild-type and mutant p27 plasmids S10A and T187A were transfected separately as control groups. As a result, the proliferation of HepG2 cells was greatly inhibited and cell cycle was arrested in G0/1 phase after exogenous p27Kip1 double-mutant expression. All recombinant p27Kip1 constructs were distributed in the nucleus after synchronization in G0 phase by treatment with leptomycin B. The expressed wild-type and T187A p27Kip1 proteins were translocated from the nucleus into cytoplasm when cells were exposed to 20% serum for 8h, whereas the S10A p27Kip1 and S10A/T187A p27Kip1 proteins remained in the nucleus. FACS profiles and cell growth curves indicated that the Ser10 and Thr187 double mutant has no significant effect on the biological activities of cell cycle control and growth inhibition. Our results suggest that expression of the p27Kip1 double-mutant abolishes its cytoplasmic redistribution but does not abrogate G0/1 phase arrest in the HepG2 cell line.