Identification of Distinctive Patterns of USP19-Mediated Growth Regulation in Normal and Malignant Cells

We previously reported that the USP19 deubiquitinating enzyme positively regulates proliferation in fibroblasts by stabilizing KPC1, a ubiquitin ligase for p27Kip1. To explore whether this role of USP19 extends to other cellular systems, we tested the effects of silencing of USP19 in several human prostate and breast models, including carcinoma cell lines. Depletion of USP19 inhibited proliferation in prostate cancer DU145, PC-3 and 22RV1 cells, which was similar to the pattern established in fibroblasts in that it was due to decreased progression from G1 to S phase and associated with a stabilization of the cyclin-dependent kinase inhibitor p27Kip1. However, in contrast to previous findings in fibroblasts, the stabilization of p27Kip1 upon USP19 depletion was not associated with changes in the levels of the KPC1 ligase. USP19 could also regulate the growth of immortalized MCF10A breast epithelial cells through a similar mechanism. This regulatory pattern was lost, though, in breast cancer MCF7 and MDA-MB-231 cells and in prostate carcinoma LNCaP cells. Of interest, the transformation of fibroblasts through overexpression of an oncogenic form of Ras disrupted the USP19-mediated regulation of cell growth and of levels of p27Kip1 and KPC1. Thus, the cell context appears determinant for the ability of USP19 to regulate cell proliferation and p27Kip1 levels. This may occur through both KPC1 dependent and independent mechanisms. Moreover, a complete loss of USP19 function on cell growth may arise as a result of oncogenic transformation of cells

Degradation of p57(Kip2) mediated by SCF(Skp2)-dependent ubiquitylation

The abundance of the cyclin-dependent kinase (CDK) inhibitor p57(Kip2), an important regulator of cell cycle progression, is thought to be controlled by the ubiquitin-proteasome pathway. The Skp1/Cul1/F-box (SCF)-type E3 ubiquitin ligase complex SCF(Skp2) has now been shown to be responsible for regulating the cellular level of p57(Kip2) by targeting it for ubiquitylation and proteolysis. The elimination of p57(Kip2) was impaired in Skp2(-/-) cells, resulting in abnormal accumulation of the protein. Coimmunoprecipitation analysis also revealed that Skp2 interacts with p57(Kip2) in vivo. Overexpression of WT Skp2 promoted degradation of p57(Kip2), whereas expression of a dominant negative mutant of Skp2 prolonged the half-life of p57(Kip2). Mutation of the threonine residue (Thr-310) of human p57(Kip2) that is conserved between the COOH-terminal QT domains of p57(Kip2) and p27(Kip1) prevented the effect of Skp2 on the stability of p57(Kip2), suggesting that phosphorylation at this site is required for SCF(Skp2)-mediated ubiquitylation. Finally, the purified recombinant SCF(Skp2) complex mediated p57(Kip2) ubiquitylation in vitro in a manner dependent on the presence of the cyclin E-CDK2 complex. These observations thus demonstrate that the SCF(Skp2) complex plays an important role in cell-cycle progression by determining the abundance of p57(Kip2) and that of the related CDK inhibitor p27(Kip1)