Phosphorylation of the cell cycle inhibitor p21Cip1/WAF1 by Pim-1 kinase

Biochim Biophys Acta. 2002 Dec 16;1593(1):45-55. doi: 10.1016/s0167-4889(02)00347-6.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Cycle / physiology*
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins / genetics
  • Cyclins / physiology*
  • Humans
  • In Vitro Techniques
  • Leukemia, Monocytic, Acute
  • Mutation
  • Phosphorylation
  • Precipitin Tests
  • Protein-Serine-Threonine Kinases / physiology*
  • Proto-Oncogene Proteins / physiology*
  • Proto-Oncogene Proteins c-pim-1
  • Substrate Specificity

Substances

  • CDKN1A protein, human
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins
  • Proto-Oncogene Proteins
  • PIM1 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-pim-1