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 G₁→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 G₁ 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 G₁ 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 G₁→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.

ACKNOWLEDGMENTS

We thank Jean Wang for providing Abl-specific reagents, David Schatz for providing S2-6 cells, Gary Nolan for providing φNX ecotropic packaging cells, J. W. Harper for providing baculovirus-synthesized cyclin D1-CDK4 enzyme, Robert Krauss for stocks of ecotropic viruses encoding pLJ/cyclin D1, and Hermann Bujard for components of the TET-regulated expression system. We thank Lily Ossowski and Ed Johnson for critical review of the manuscript and Scott Henderson for help with confocal microscopy.

This work was supported by NCI grant R29-CA65787 to I.H.G.