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

SSeCKS, first isolated as a G(1)-->S inhibitor that is downregulated in src - and ras-transformed tells, is a major cytoskeleton-associated PKC substra te with tumor suppressor and kinase-scaffolding activities. Previous attemp ts at constitutive expression resulted in cell variants with truncated ecto pic SSeCKS products. Here, we show that tetracycline-regulated SSeCKS expre ssion in NIH 3T3 cells induces G(1) arrest marked by extracellular signal-r egulated kinase 2-dependent decreases in cyclin D1 expression and pRb phosp horylation. Unexpectedly, the forced reexpression of cyclin D1 failed to re scue SSeCKS-induced G(1) arrest. Confocal microscopy analysis revealed cyto plasmic colocalization of cyclin D1 with SSeCKS. Because the SSeCHS gene en codes two potential cyclin-binding motifs (CY) flanking major in vivo prote in kinase C (PKC) phosphorylation sites (Ser(507/515)), we addressed whethe r SSeCKS encodes a phosphorylation-dependent cyclin scaffolding function. B acterially expressed SSeCKS-CY bound cyclins D1 and E, whereas K-->S mutati ons within either CY motif ablated binding. Activation of PKC in vivo cause d a rapid translocation of cyclin D1 to the nucleus. Cell permeable, penetr atin-linked peptides encoding wild-type SSeCKS-CY, but not K-->S or phospho -Ser(507/515) variants, released cyclin D1 from its cytoplasmic sequestrati on and induced higher saturation density in cyclin D1-overexpressor cells o r rat embryo fibroblasts. Our data suggest that SSeCKS controls G(1)-->S pr ogression by regulating the expression and localization of cyclin D1. These data suggest that downregulation of SSeCKS in tumor cells removes gating c heckpoints for saturation density, an effect that may promote contact indep endence.