Mutations of phosphorylation sites Ser¹⁰ and Thr¹⁸⁷ of p27^Kip1 abolish cytoplasmic redistribution but do not abrogate G_0/1 phase arrest in the HepG₂ cell line

The cyclin-dependent kinase (CDK) inhibitor p27^Kip1 is an important regulator of cell cycle progression as it negatively regulates G_0/1 progression and plays a major role in controlling the cell cycle. The screening of the p27^Kip1 sequence identified many potential phosphorylation sites. Although Ser¹⁰ and Thr¹⁸⁷ were shown to be important for p27^Kip1 function, the effects of a combined deletion of both sites on p27^Kip1 function are still unknown. To investigate the effects of the overexpression of exogenous p27^Kip1 protein lacking both the Ser¹⁰ and Thr¹⁸⁷ sites on subcellular localization, cell cycle, and proliferation, a plasmid was constructed containing mutations of p27^Kip1 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 G_0/1 phase after exogenous p27^Kip1 double-mutant expression. All recombinant p27^Kip1 constructs were distributed in the nucleus after synchronization in G₀ phase by treatment with leptomycin B. The expressed wild-type and T187A p27^Kip1 proteins were translocated from the nucleus into cytoplasm when cells were exposed to 20% serum for 8 h, whereas the S10A p27^Kip1 and S10A/T187A p27^Kip1 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 p27^Kip1 double-mutant abolishes its cytoplasmic redistribution but does not abrogate G_0/1 phase arrest in the HepG₂ cell line.