title: Successive phosphorylation of p27(KIP1) protein at serine-10 and C terminus crucially controls its potency to inactivate Cdk2.
abstract: During the G(1)-S transition, the activity of Cdk2 is regulated by its association with p27(KIP1), which in rodent fibroblasts undergoes phosphorylation mainly at serine 10, threonine 187, and C-terminal threonine 197 by KIS, Cdk2, and Pim or ROCK, respectively. Recently Cdc6 the AAA+ ATPase, identified initially to assemble pre-replicative complexes on origins of replication and later to activate p21(CIP1)-inactivated Cdk2, was found also to activate p27-bound Cdk2 but only after the bound p27 is C-terminally phosphorylated. On the other hand, the biological significance of the serine 10 phosphorylation remains elusive aside from its involvement in the stability of p27 itself. We report here that serine 10 phosphorylation is required for efficient C-terminal phosphorylation of its own by PIM and ROCK kinases and critically controls the potency of p27 as a Cdk2 inhibitor. In vitro, PIM1 and active ROCK1 efficiently phosphorylated free as well as Cdk2-bound p27 but only when the p27 was phosphorylated at Ser-10 in advance. Consistently, a Ser-10 nonphosphorylatable mutant p27 protein was not phosphorylated at the C terminus in vivo. Furthermore, when double-phosphorylated, free p27 was no longer a potent inhibitor of Cdk2, and Cdk2-bound p27 could be removed by Cdc6 to reactivate the Cdk2. Thus, phosphorylation at these two sites crucially controls the potency of this CDK inhibitor in two distinct modes.
authors.affiliation: Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors.forename: Atish R
authors.initials: AR
authors.lastname: Mohanty
authors.forename: Qiuming
authors.initials: Q
authors.lastname: Kan
authors.forename: Saumya
authors.initials: S
authors.lastname: Srivastava
authors.forename: Baasanjav
authors.initials: B
authors.lastname: Uranbileg
authors.forename: Shiho
authors.initials: S
authors.lastname: Arakawa-Takeuchi
authors.forename: Naoya
authors.initials: N
authors.lastname: Fujita
authors.forename: Hiroto
authors.initials: H
authors.lastname: Okayama
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry; D002460:Cell Line; D049109:Cell Proliferation; D051357:Cyclin-Dependent Kinase 2 / Q000737:chemistry*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D009687:Nuclear Proteins / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry*
chemical_list: C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D012694:Serine; D006639:Histidine; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2