title: PKC-dependent phosphorylation of p27 at T198 contributes to p27 stabilization and cell cycle arrest.
abstract: In this manuscript, we present experimental evidence that PKCs phosphorylate p27 at T198 in vitro and in vivo, resulting in p27 stabilization and cell cycle arrest in MCF-7 and HeLa cells. Our findings indicate that (1) recombinant PKCα, βII, δ, η and θ isoforms phosphorylate, in in vitro kinase assays, wild-type recombinant p27 protein expressed in E. coli and wild-type p27 protein immunoprecpitated from transfected HEK-293 cells but not the T198A mutant, (2) adoptive expressed PKCα and δ phosphorylate both transfected and endogenous p27 at T198 in HEK-293 cells, (3) T198 phosphorylation of transfected and endogenous p27 is increased by PKC activators [Phorbol 12-myristate 13-acetate (PMA)] and suppressed by PKC inhibitors (Rottlerin A, G06976, Calphostin C), (4) in parallel with increased T198 phosphorylation, PMA induces stabilization of p27 protein in HeLa cells, whereas PKC inhibitors induce a decrease in p27 stability and, finally, (5) PMA-induced p27 upregulation is necessary for growth arrest of HeLa and MCF-7 cells induced by PKC activation by PMA.       Overall, these results suggest that PKC-dependent upregulation of p27 induced by its phosphorylation at T198 represents a mechanism that mediates growth arrest promoted by PMA and provide novel insights on the ability of different PKC isoforms to play a role in controlling cell cycle progression.
authors.affiliation: Biogem S.C.A.R.L., Institute for Genetic Research, Avellino, Italy.
authors.forename: Fernanda
authors.initials: F
authors.lastname: De Vita
authors.forename: Miriam
authors.initials: M
authors.lastname: Riccardi
authors.forename: Donatella
authors.initials: D
authors.lastname: Malanga
authors.forename: Marianna
authors.initials: M
authors.lastname: Scrima
authors.forename: Carmela
authors.initials: C
authors.lastname: De Marco
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Viglietto
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D009281:Naphthalenes / Q000737:chemistry / Q000494:pharmacology; D010766:Phosphorylation; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D014162:Transfection; D015854:Up-Regulation
chemical_list: D009281:Naphthalenes; D020033:Protein Isoforms; D011994:Recombinant Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; C016658:phorbolol myristate acetate; D011493:Protein Kinase C; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate