Mutations of phosphorylation sites Ser{sup 1} and Thr{sup 187} of p27{sup Kip1} abolish cytoplasmic redistribution but do not abrogate G{sub 0/1} phase arrest in the HepG{sub 2} cell line
- Department of Oncology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, 210002 (China)
- Department of Biochemistry, Nanjing Medical University, Nanjing, 210029 (China)
The cyclin-dependent kinase (CDK) inhibitor p27{sup Kip1} is an important regulator of cell cycle progression as it negatively regulates G{sub 0/1} progression and plays a major role in controlling the cell cycle. The screening of the p27{sup Kip1} sequence identified many potential phosphorylation sites. Although Ser{sup 1} and Thr{sup 187} were shown to be important for p27{sup Kip1} function, the effects of a combined deletion of both sites on p27{sup Kip1} function are still unknown. To investigate the effects of the overexpression of exogenous p27{sup Kip1} protein lacking both the Ser{sup 1} and Thr{sup 187} sites on subcellular localization, cell cycle, and proliferation, a plasmid was constructed containing mutations of p27{sup Kip1} at Ser{sup 1} and Thr{sup 187} (S10A/T187A p27), and transfected into the HepG{sub 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{sub 2} cells was greatly inhibited and cell cycle was arrested in G{sub 0/1} phase after exogenous p27{sup Kip1} double-mutant expression. All recombinant p27{sup Kip1} constructs were distributed in the nucleus after synchronization in G phase by treatment with leptomycin B. The expressed wild-type and T187A p27{sup Kip1} proteins were translocated from the nucleus into cytoplasm when cells were exposed to 20% serum for 8 h, whereas the S10A p27{sup Kip1} and S10A/T187A p27{sup Kip1} proteins remained in the nucleus. FACS profiles and cell growth curves indicated that the Ser{sup 1} and Thr{sup 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{sup Kip1} double-mutant abolishes its cytoplasmic redistribution but does not abrogate G{sub 0/1} phase arrest in the HepG{sub 2} cell line.
- OSTI ID:
- 20854434
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 347, Issue 3; Other Information: DOI: 10.1016/j.bbrc.2006.06.114; PII: S0006-291X(06)01419-7; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
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