title: Collective inhibition of pRB family proteins by phosphorylation in cells with p16INK4a loss or cyclin E overexpression.
abstract: The activity of the retinoblastoma protein pRB is regulated by phosphorylation that is mediated by G(1) cyclin-associated cyclin-dependent kinases (CDKs). Since the pRB-related pocket proteins p107 and p130 share general structures and biological functions with pRB, their activity is also considered to be regulated by phosphorylation. In this work, we generated phosphorylation-resistant p107 and p130 molecules by replacing potential cyclin-CDK phosphorylation sites with non-phosphorylatable alanine residues. These phosphorylation-resistant mutants retained the ability to bind E2F and cyclin. Upon introduction into p16(INK4a)-deficient U2-OS osteosarcoma cells, in which cyclin D-CDK4/6 is dysregulated, the phosphorylation-resistant mutants, but not wild-type p107 or p130, were capable of inhibiting cell proliferation. Furthermore, when ectopically expressed in pRB-deficient SAOS-2 osteosarcoma cells, the wild-type as well as the phosphorylation-resistant pRB family proteins were capable of inducing large flat cells. The flat cell-inducing activity of the wild-type proteins, but not that of the phosphorylation-resistant mutants, was abolished by coexpressing cyclin E. Our results indicate that the elevated cyclin D- or cyclin E-associated kinase leads to systemic inactivation of the pRB family proteins and suggest that dysregulation of the pRB kinase provokes an aberrant cell cycle in a broader range of cell types than those induced by genetic inactivation of the RB gene.
authors.affiliation: Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University, Kita-ku, Sapporo 060-0815, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Ashizawa
authors.forename: H
authors.initials: H
authors.lastname: Nishizawa
authors.forename: M
authors.initials: M
authors.lastname: Yamada
authors.forename: H
authors.initials: H
authors.lastname: Higashi
authors.forename: T
authors.initials: T
authors.lastname: Kondo
authors.forename: H
authors.initials: H
authors.lastname: Ozawa
authors.forename: A
authors.initials: A
authors.lastname: Kakita
authors.forename: M
authors.initials: M
authors.lastname: Hatakeyama
mesh_terms: D019927:Cyclin E / Q000096:biosynthesis* / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016160:Retinoblastoma Protein / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D019927:Cyclin E; D019941:Cyclin-Dependent Kinase Inhibitor p16; D016160:Retinoblastoma Protein