Phosphorylation of the cell cycle inhibitor p21Cip1/WAF1 by Pim-1 kinase.
The serine/threonine kinase, Pim-1, appears to be involved in regulating proliferation, differentiation and cell survival of lymphoid and myeloid cells. In this study, we have found that amino acid residues 140-147 (RKRRQTSM) at the C-terminal end of p21(Cip1/WAF1), a cyclin-dependent kinase (CDK) inhibitor, constitute an ideal phosphorylation consensus sequence for Pim-1. We demonstrate that Pim-1 efficiently phosphorylates this peptide sequence as well as the p21 protein in vitro. We also demonstrate by pull-down assay and by immunoprecipitation that Pim-1 associates with p21. During phorbol ester-induced differentiation of U937 cells, both Pim-1 and p21 expression levels increase with Pim-1 levels increasing in both the nucleus and cytoplasm while p21 remains primarily cytoplasmic. Co-transfection of wild type p21 with wild type Pim-1 results in cytoplasmic localization of p21 while co-transfection of wild type p21 with kinase dead Pim-1 results in nuclear localization of p21. Consistent with the results from the phosphoamino acid assay, Pim-1 phosphorylates transfected p21 only on Thr(145) in p21-deficient human fibroblasts and this phosphorylation event results in the cytoplasmic localization of p21. These findings demonstrate that Pim-1 associates with and phosphorylates p21 in vivo, which influences the subcellular localization of p21.
Wang
Zeping
Z
School of Molecular Biosciences and the Cancer Prevention and Research Center, Washington State University, PO Box 644234, Pullman, WA 99164-4234, USA.
Bhattacharya
Nandini
N
Mixter
Philip F
PF
Wei
Wenyi
W
Sedivy
John
J
Magnuson
Nancy S
NS
D002453:Cell Cycle / Q000502:physiology*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D007948:Leukemia, Monocytic, Acute; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051573:Proto-Oncogene Proteins c-pim-1; D013379:Substrate Specificity
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D011518:Proto-Oncogene Proteins; C496634:PIM1 protein, human; D017346:Protein Serine-Threonine Kinases; D051573:Proto-Oncogene Proteins c-pim-1