title: Expression of cyclin E renders cyclin D-CDK4 dispensable for inactivation of the retinoblastoma tumor suppressor protein, activation of E2F, and G1-S phase progression.
abstract: The activation of CDK2-cyclin E in late G1 phase has been shown to play a critical role in retinoblastoma protein (pRb) inactivation and G1-S phase progression of the cell cycle. The phosphatidylinositol 3-OH-kinase inhibitor LY294002 has been shown to block cyclin D1 accumulation, CDK4 activity and, thus, G1 progression in alpha-thrombin-stimulated IIC9 cells (Chinese hamster embryonic fibroblasts). Our previous results show that expression of cyclin E rescues S phase progression in alpha-thrombin-stimulated IIC9 cells treated with LY294002, arguing that cyclin E renders CDK4 activity dispensable for G1 progression. In this work we investigate the ability of alpha-thrombin-induced CDK2-cyclin E activity to inactivate pRb in the absence of prior CDK4-cyclin D1 activity. We report that in the absence of CDK4-cyclin D1 activity, CDK2-cyclin E phosphorylates pRb in vivo on at least one residue and abolishes pRb binding to E2F response elements. We also find that expression of cyclin E rescues E2F activation and cyclin A expression in cyclin D kinase-inhibited, alpha-thrombin-stimulated cells. Furthermore, the rescue of E2F activity, cyclin A expression, and DNA synthesis by expression of E can be blocked by the expression of either CDK2(D145N) or RbDeltaCDK, a constitutively active mutant of pRb. However, restoring four known cyclin E-CDK2 phosphorylation sites to RbDeltaCDK renders it susceptible to inactivation in late G1, as assayed by E2F activation, cyclin A expression, and S phase progression. These data indicate that CDK2-cyclin E, without prior CDK4-cyclin D activity, can phosphorylate and inactivate pRb, activate E2F, and induce DNA synthesis.
authors.affiliation: Department of Pharmacological and Physiological Sciences, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, USA.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Keenan
authors.forename: Nathan H
authors.initials: NH
authors.lastname: Lents
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Baldassare
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D006224:Cricetinae; D019925:Cyclin A / Q000378:metabolism; D056741:Cyclin D; D019927:Cyclin E / Q000494:pharmacology*; D051358:Cyclin-Dependent Kinase 4; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000502:physiology*; D016213:Cyclins / Q000378:metabolism / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D016193:G1 Phase; D005799:Genes, Dominant; D017930:Genes, Reporter; D018922:HL-60 Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism / Q000502:physiology*; D016196:S Phase; D013917:Thrombin / Q000378:metabolism; D013936:Thymidine / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D002867:Chromones; D019925:Cyclin A; D056741:Cyclin D; D019927:Cyclin E; D019941:Cyclin-Dependent Kinase Inhibitor p16; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D004791:Enzyme Inhibitors; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; D014157:Transcription Factors; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004247:DNA; D008156:Luciferases; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D013917:Thrombin; D013936:Thymidine