SSeCKS, a Major Protein Kinase C Substrate with Tumor Suppressor Activity, Regulates G₁→S Progression by Controlling the Expression and Cellular Compartmentalization of Cyclin D

Abstract

SSeCKS, first isolated as a G 1 3S 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 K3S 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 K3S 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 3S 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.Cell cycle progression from the first gap (G 1 ) to the DNA synthetic (S) phase is mainly controlled by the activity of complexes containing cyclin D-cyclin-dependent kinase 4 (CDK4) or CDK6 and cyclin E-CDK2 (53). In contrast to cycling cells in which regulation of these activities has been well studied, little is known regarding the control of cyclin-dependent complexes when cells become contact inhibited or how these controls are subverted in cancer or diseases marked by cell hyperplasia.Cyclin protein levels are largely controlled at the transcriptional level and by ubiquitin-mediated degradation. Except for cyclin D, whose expression increases rapidly in G 1 and stays high until the end of G 2 /M, the remaining cyclins are expressed in discrete phases (20,49). In contrast, the levels of CDKs are stable throughout the cell cycle; however, their phosphorylation status is cell cycle regulated (48). Current models suggest that cyclin D-CDK4 or -CDK6 complexes are responsible for G 1 progression whereas cyclin E-CDK2 complexes are responsible for G 1 3S transition. The primary role of cyclin D in cell cycle regulation is most likely to phosphorylate pRb, because microinjection of antibodies to cyclin D1 has no effect in Rb Ϫ/Ϫ cells (54). Phosphorylation of pRb by cyclin D-CDK4 or -CDK6, which occurs in mid-G 1 , leads to th...

Lack of Relation of AKAP12 with p53 and Bcl-2 in Colorectal Carcinoma

“…It causes separation of isoenzymes of PKC from the plasma membrane. By this way cytoplasmic levels and intracellular localization of cyclin D1 changes (Lin et al, 2000). Cyclin D1 is synthesized during transition from G1 to S phase and it breaks down rapidly when the cell enters S phase (Vermeulen et al, 2003).…”

Progressive and Inhibitory Cell Cycle Proteins Act Simultaneously to Regulate Neurotrophin-mediated Proliferation and Maturation of Neuronal Precursors

“…Regulation of cyclin D1 protein levels occurs through a variety of post-translational mechanisms, including alterations in proteolysis, [50][51][52][53][54][55][56] and sequestration. 65 BDNF may act through either mechanism. We hypothesize that BDNF is able to elicit a rapid post-translational increase in cyclin D1 protein since cyclin D1 mRNA and protein have already been synthesized during prior exposure of earlier precursors to NGF.…”

SSeCKS sequesters cyclin D1 in glomerular parietal epithelial cells and influences proliferative injury in the glomerulus

“…11,12 SSeCKS is unique among AKAPs by containing protein kinase C (PKC) phosphorylation sites that flank cyclin-binding motifs, which bind cyclin D1. 13,14 In response to cell-cell contact inhibition of proliferation, 15 SSeCKS expression is induced, resulting in the sequestration of existing cyclin D1 protein in the cytoplasm and the downregulation of further cyclin D1 expression through extracellular signal-regulated kinase-dependent pathways. This leads to growth arrest by inhibiting G 1 -to-S cell-cycle progression.…”