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Cell Cycle, Cell Death, and Senescence

Pim-1 Kinase-Dependent Phosphorylation of p21^Cip1/WAF1 Regulates Its Stability and Cellular Localization in H1299 Cells

School of Molecular Biosciences, Washington State University, Pullman, Washington

Requests for reprints: Nancy S. Magnuson, School of Molecular Biosciences, Washington State University, Pullman, WA 99163. Phone: 509-335-0966; Fax: 509-335-1907. E-mail: magnuson{at}wsu.edu

Previous studies from our laboratory showed that p21^Cip1/WAF1 can be phosphorylated by Pim-1 kinase in vitro, implying that part of the function of Pim-1 might involve influencing the cell cycle. In the present study, site-directed mutagenesis and phosphorylated-specific antibodies were used as tools to identify the sites phosphorylated by Pim-1 and the consequences of this phosphorylation. What we found was that Pim-1 can efficiently phosphorylate p21 on Thr¹⁴⁵ in vitro using recombinant protein and in vivo in intact cells. Unexpectedly, we found that Ser¹⁴⁶ is a second site that is phosphorylated in vivo, but this phosphorylation event seems to be an indirect result of Pim-1 expression. More importantly, the consequences of phosphorylation of either Thr¹⁴⁵ or Ser¹⁴⁶ are distinct. When p21 is phosphorylated on Thr¹⁴⁵, it localizes to the nucleus and results in the disruption of the association between proliferating cell nuclear antigen and p21. Furthermore, phosphorylation of Thr¹⁴⁵ promotes stabilization of p21. On the other hand, when p21 is phosphorylated on Ser¹⁴⁶, it localizes primarily in the cytoplasm and the effect of phosphorylation on stability is minimal. Cotransfection of wild-type Pim-1 with p21 increases the rate of proliferation compared with cotransfection of p21 with kinase-dead Pim-1. Knocking down Pim-1 expression greatly decreases the rate of proliferation of H1299 cells and their ability to grow in soft agar. These data suggest that Pim-1 overexpression may contribute to tumorigenesis in part by influencing the cellular localization and stability of p21 and by promoting cell proliferation. (Mol Cancer Res 2007;5(9):909–22)