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.
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 Ser(10) and Thr(187) 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 Ser(10) and Thr(187) sites on subcellular localization, cell cycle, and proliferation, a plasmid was constructed containing mutations of p27(Kip1) at Ser(10) and Thr(187) (S10A/T187A p27), and transfected into the HepG(2) 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 HepG(2) 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 G(0) 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 Ser(10) and Thr(187) 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 HepG(2) cell line.
Guan
Xiaoxiang
X
Department of Oncology, Jinling Hospital, Nanjing University School of Medicine, China.
Chen
Longbang
L
Wang
Jinghua
J
Geng
Huaicheng
H
Chu
Xiaoyuan
X
Zhang
Qun
Q
Du
Lili
L
De
Wei
W
D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D016193:G1 Phase; D006801:Humans; D009154:Mutation / Q000235:genetics; D010768:Phosphoserine / Q000378:metabolism*; D010769:Phosphothreonine / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016192:Resting Phase, Cell Cycle; D019311:Staurosporine / Q000494:pharmacology
D005231:Fatty Acids, Unsaturated; D011994:Recombinant Proteins; D010769:Phosphothreonine; D050760:Cyclin-Dependent Kinase Inhibitor p27; D010768:Phosphoserine; D019311:Staurosporine; C038753:leptomycin B