title: SSeCKS, a major protein kinase C substrate with tumor suppressor activity, regulates G(1)-->S progression by controlling the expression and cellular compartmentalization of cyclin D.
abstract: SSeCKS, first isolated as a G(1)-->S inhibitor that is downregulated in src- and ras-transformed cells, is a major cytoskeleton-associated PKC substrate with tumor suppressor and kinase-scaffolding activities. Previous attempts at constitutive expression resulted in cell variants with truncated ectopic SSeCKS products. Here, we show that tetracycline-regulated SSeCKS expression in NIH 3T3 cells induces G(1) arrest marked by extracellular signal-regulated kinase 2-dependent decreases in cyclin D1 expression and pRb phosphorylation. Unexpectedly, the forced reexpression of cyclin D1 failed to rescue SSeCKS-induced G(1) arrest. Confocal microscopy analysis revealed cytoplasmic colocalization of cyclin D1 with SSeCKS. Because the SSeCKS gene encodes two potential cyclin-binding motifs (CY) flanking major in vivo protein kinase C (PKC) phosphorylation sites (Ser(507/515)), we addressed whether SSeCKS encodes a phosphorylation-dependent cyclin scaffolding function. Bacterially expressed SSeCKS-CY bound cyclins D1 and E, whereas K-->S mutations within either CY motif ablated binding. Activation of PKC in vivo caused a rapid translocation of cyclin D1 to the nucleus. Cell permeable, penetratin-linked peptides encoding wild-type SSeCKS-CY, but not K-->S or phospho-Ser(507/515) variants, released cyclin D1 from its cytoplasmic sequestration and induced higher saturation density in cyclin D1-overexpressor cells or rat embryo fibroblasts. Our data suggest that SSeCKS controls G(1)-->S progression by regulating the expression and localization of cyclin D1. These data suggest that downregulation of SSeCKS in tumor cells removes gating checkpoints for saturation density, an effect that may promote contact independence.
authors.affiliation: Departments of Microbiology, Mount Sinai School of Medicine, New York, New York 10029-6574, USA.
authors.forename: X
authors.initials: X
authors.lastname: Lin
authors.forename: P
authors.initials: P
authors.lastname: Nelson
authors.forename: I H
authors.initials: IH
authors.lastname: Gelman
mesh_terms: D016475:3T3 Cells / Q000166:cytology / Q000378:metabolism; D054758:A Kinase Anchor Proteins; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D042846:CDC2-CDC28 Kinases; D002451:Cell Compartmentation / Q000502:physiology*; D018797:Cell Cycle Proteins; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D003260:Contact Inhibition / Q000502:physiology*; D056741:Cyclin D; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D016193:G1 Phase / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D016147:Genes, Tumor Suppressor; D020935:MAP Kinase Signaling System; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D008934:Mitogens / Q000235:genetics / Q000502:physiology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016196:S Phase / Q000502:physiology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D054758:A Kinase Anchor Proteins; C491161:Akap12 protein, mouse; C102361:Akap12 protein, rat; D018797:Cell Cycle Proteins; D056741:Cyclin D; D016213:Cyclins; D008934:Mitogens; D011993:Recombinant Fusion Proteins; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D019950:Mitogen-Activated Protein Kinase 1