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Pim‐1 phosphorylation of p21Cip1 causes its stabilization
Author(s) -
zhang yandong,
Wang Zeping,
Magnuson Nancy S
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb64
Subject(s) - phosphorylation , microbiology and biotechnology , cell cycle , hela , transfection , mutant , protein phosphorylation , biology , carcinogenesis , cytoplasm , cell , in vitro , chemistry , cell culture , biochemistry , protein kinase a , gene , genetics
Accumulating evidence suggests that Pim‐1's oncogenecity might be a result from its contribution to cell survival, however, the underlying mechanism for this is poorly understood. Our laboratory previously showed that p21 Cip1/WAF1 could be phosphorylated by Pim‐1 protein in vitro , implying that part of Pim‐1's function might involve influencing the cell cycle. In the present study we utilized site‐directed mutagenesis and phosphospecific antibodies as tools to identify the sites phosphorylated by Pim‐1 and studied the consequences of this phosphorylation accordingly. We found that Pim‐1 can efficiently phosphorylate p21 on Thr145 both in vitro (using recombinant protein) and in intact cells. Using transient transfection to overexpress Pim‐1 in intact cells, we found that phosphorylation of p21 by Pim‐1 increases p21's half‐life in H1299 and HeLa cells, which was verified with p21 phosphomimic mutants at residues Thr145 and Ser146. In addition, with phosphomimic mutants of p21 at these two sites, we found that phosphorylation on Thr145 causes translocation of p21 from the nucleus to the cytoplasm in the H1299 and NIH3T3 cells. Finally, when p21 is phosphorylated primarily at Thr145, the interaction between PCNA and p21 alters. These findings suggest that Pim‐1 could contribute to cell survival and tumorigenesis through influencing p21's stability and cellular localization, as well as DNA synthesis. Grant support: NIH‐ CA104470