USP19 Deubiquitinating Enzyme Supports Cell Proliferation by Stabilizing KPC1, a Ubiquitin Ligase for p27Kip1

p27^Kip1 is a cyclin-dependent kinase inhibitor that regulates the G₁/S transition. Increased degradation of p27^Kip1 is associated with cellular transformation. Previous work demonstrated that the ubiquitin ligases KPC1/KPC2 and SCF^Skp2 ubiquitinate p27^Kip1 in G₁ and early S, respectively. The regulation of these ligases remains unclear. We report here that the USP19 deubiquitinating enzyme interacts with and stabilizes KPC1, thereby modulating p27^Kip1 levels and cell proliferation. Cells depleted of USP19 by RNA interference exhibited an inhibition of cell proliferation, progressing more slowly from G₀/G1 to S phase, and accumulated p27^Kip1. This increase in p27^Kip1 was associated with normal levels of Skp2 but reduced levels of KPC1. The overexpression of KPC1 or the use of p27^−/− cells inhibited significantly the growth defect observed upon USP19 depletion. KPC1 was ubiquitinated in vivo and stabilized by proteasome inhibitors and by overexpression of USP19, and it also coimmunoprecipitated with USP19. Our results identify USP19 as the first deubiquitinating enzyme that regulates the stability of a cyclin-dependent kinase inhibitor and demonstrate that progression through G₁ to S phase is, like the metaphase-anaphase transition, controlled in a hierarchical, multilayered fashion.The ubiquitin proteasome pathway plays essential roles in regulating the cell cycle. The best-defined functions of this pathway in cell cycle regulation are those mediated by the multisubunit ubiquitin protein ligases SKP1-CUL1-F-box (SCF) and the anaphase-promoting complex/cyclosome (APC) (reviewed in reference 25). The functions of the APC in the cell cycle are predominant at the mitosis-anaphase transition, while the activities of SCF-type ubiquitin protein ligase complexes are involved at various steps of the cycle (reviewed in references 21 and 25). One of the best-defined functions of the SCF is mediated by SCF^skp2, which plays a vital role in regulating the G₁-S transition by ubiquitinating the cyclin-dependent kinase inhibitor p27^Kip1, thereby targeting it for degradation by the proteasome (3, 31, 32).The central role of p27^Kip1 in restricting cell proliferation is demonstrated by the fact that mice lacking the p27^Kip1 gene manifest increased body and organ weights and develop pituitary adenomas (6, 13, 18). In addition, the results of clinical studies suggest that low p27^Kip1 levels are associated with increased aggressivity of tumors (1, 28). Unlike the case with the p53 or Rb tumor suppressors, mutation or deletion of p27^Kip1 in tumors is rare. Rather, its deregulation in human tumors is due mainly to reduced protein levels, mediated in large part by increased proteolysis (reviewed in reference 21). In support of this, the low p27^Kip1 levels seen in tumors are associated with increased levels of Skp2, the substrate recognition subunit of the SCF^skp2 ligase. The loss of Skp2 in mice results in p27^Kip1 accumulation, and cells from Skp2^−/− animals contain enlarged nuclei with polyploidy and multiple centrosomes. They also show a reduced growth rate and increased apoptosis (19). Many of the cellular phenotypes observed in Skp2^−/− mice disappear in Skp2^−/− p27^−/− double-mutant mice (14, 20). Thus, the oncogenic nature of Skp2 is largely due to its ability to mediate p27^Kip1 degradation.In spite of the clear role of SCF^skp2 in mediating the ubiquitination and degradation of p27^Kip1, the downregulation of this cyclin-dependent kinase inhibitor proceeds normally in lymphocytes isolated from Skp2^−/− mice (8, 21). In addition, in the normal cell cycle, p27^Kip1 is also degraded in G₁, before the expression of Skp2, which occurs in early S phase (8, 9, 36). Also, p27^Kip1 is exported from the nucleus to the cytoplasm in G₁, whereas Skp2 is localized in the nucleus (9, 26). These observations suggest the existence of another pathway for the degradation of p27^Kip1. Indeed, KPC (Kip1 ubiquitination-promoting complex) was subsequently identified as a novel cytoplasmic ligase complex that interacts with and ubiquitinates p27^Kip1 (10). KPC consists of two subunits: KPC1, a 140-kDa RING-finger domain-containing protein, and KPC2, a 50-kDa protein containing a ubiquitin-like domain and two ubiquitin-associated domains.Although the role of ubiquitin protein ligases in the cell cycle has received considerable attention, fewer data are available regarding the roles of deubiquitinating enzymes in the cell cycle (22). We recently described USP19 as a deubiquitinating enzyme that is induced in skeletal muscle atrophying in response to numerous catabolic stimuli. To study its function, we used RNA interference to explore the consequences of depletion of this enzyme in cultured muscle cells. Our early studies indicated that the loss of USP19 interfered with the growth of L6 myoblasts. We have observed similar effects in FR3T3 fibroblasts and have explored the underlying mechanisms of this growth defect.