Negative regulation of SCFSkp2 ubiquitin ligase by TGF-β signaling

Abstract

TGF-β is a multifunctional growth factor whose best-known function is to inhibit cell growth and suppress tumor formation. TGF-β causes cells to accumulate in mid-to-late G1 phase by blocking the transition from G1 to S. It has been shown that TGF-β inhibits Cdk2-cyclin E kinase activity by promoting the binding of cell cycle inhibitor p27Kip1 to the kinase complexes. Here, we show that TGF-β treatment leads to stabilization of p27Kip1 during G1 to S transition. We found that TGF-β negatively regulates components of the SCF complex, which degrades the p27 Kip1 during the G1 to S transition, through two distinct mechanisms. Using a pulse-chase analysis, we demonstrated that the stability of Skp2 decreases in the presence of TGF-β. Destabilization of Skp2 by ubiquitin-mediated proteolysis was also demonstrated that in an in vitro degradation system, using cell extracts prepared from TGF-β-treated cultured cells. In addition, TGF-β treatment decreases the levels of Cks1 mRNA. The deficiency of Cksl in TGF-β-treated cells likely contributes to the stabilization of p27Kip1 and destabilization of Skp2, because in the absence of Cks1, SCFSkp2 cannot ubiquitinate p27Kip1; instead, self-ubiquitination of Skp2 occurs. Thus, stabilization of the cell cycle inhibitor p27Kip1 and cell growth inhibition in response to TGF-β occur in part through limiting the threshold of the SCF Skp2 ubiquitin ligase by transcriptional and post-transcriptional mechanisms.