Faculty Bibliography

Ubiquitin-specific proteases (USPs) are a subclass of cysteine proteases that catalyze the removal of ubiquitin (either monomeric or chains) from substrates, thus counteracting the activity of E3 ubiquitin ligases. Although the importance of USPs in a multitude of processes, from hereditary cancer to neurodegeneration, is well established, our knowledge on their mode of regulation, substrate specificity and biological function is quite limited. In this study we identify USP47 as a novel interactor of the E3 ubiquitin ligase, Skp1/Cul1/F-box protein beta-transducin repeat-containing protein (SCF(beta-Trcp)). We found that both beta-Trcp1 and beta-Trcp2 bind specifically to USP47, and point mutations in the beta-Trcp WD-repeat region completely abolished USP47 binding, indicating an E3-substrate-type interaction. However, unlike canonical beta-Trcp substrates, USP47 protein levels were neither affected by silencing of beta-Trcp nor modulated in a variety of processes, such as cell-cycle progression, DNA damage checkpoint responses or tumor necrosis factor (TNF) pathway activation. Notably, genetic or siRNA-mediated depletion of USP47 induced accumulation of Cdc25A, decreased cell survival and augmented the cytotoxic effects of anticancer drugs. In conclusion, we showed that USP47, a novel beta-Trcp interactor, regulates cell growth and survival, potentially providing a novel target for anticancer therapies.

Androgen receptor (AR), a member of the steroid receptor family, is a transcription factor that has an important role in the regulation of both prostate cell proliferation and growth suppression. AR coactivators may influence the transition between cell growth and growth suppression. We have shown previously that the internally spliced ARA70 isoform, ARA70beta, promotes prostate cancer cell growth and invasion. Here we report that the full length ARA70alpha, in contrast, represses prostate cancer cell proliferation and anchorage-independent growth in vitro and inhibits tumor growth in nude mice xenograft experiments in vivo. Further, the growth inhibition by ARA70alpha is AR-dependent and mediated through induction of apoptosis rather than cell cycle arrest. Interestingly, AR with T877A mutation in LNCaP cells decreased its physical and functional interaction with ARA70alpha, facilitating the growth of LNCaP cells. This is consistent with our previous findings that ARA70alpha expression is decreased in prostate cancer cells compared with benign prostate. ARA70alpha also reduced the invasion ability of LNCaP cells. Although growth inhibition by ARA70alpha is AR-dependent, the inhibition of cell invasion is an androgen-independent process. These results strongly suggest that ARA70alpha functions as a tumor suppressor gene

Generally, F-box proteins are the substrate recognition subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes, which mediate the timely proteolysis of important eukaryotic regulatory proteins. Mammalian genomes encode roughly 70 F-box proteins, but only a handful have established functions. The F-box protein family obtained its name from Cyclin F (also called Fbxo1), in which the F-box motif (the approximately 40-amino-acid domain required for binding to Skp1) was first described. Cyclin F, which is encoded by an essential gene, also contains a cyclin box domain, but in contrast to most cyclins, it does not bind or activate any cyclin-dependent kinases (CDKs). However, like other cyclins, Cyclin F oscillates during the cell cycle, with protein levels peaking in G2. Despite its essential nature and status as the founding member of the F-box protein family, Cyclin F remains an orphan protein, whose functions are unknown. Starting from an unbiased screen, we identified CP110, a protein that is essential for centrosome duplication, as an interactor and substrate of Cyclin F. Using a mode of substrate binding distinct from other F-box protein-substrate pairs, CP110 and Cyclin F physically associate on the centrioles during the G2 phase of the cell cycle, and CP110 is ubiquitylated by the SCF(Cyclin F) ubiquitin ligase complex, leading to its degradation. siRNA-mediated depletion of Cyclin F in G2 induces centrosomal and mitotic abnormalities, such as multipolar spindles and asymmetric, bipolar spindles with lagging chromosomes. These phenotypes were reverted by co-silencing CP110 and were recapitulated by expressing a stable mutant of CP110 that cannot bind Cyclin F. Finally, expression of a stable CP110 mutant in cultured cells also promotes the formation of micronuclei, a hallmark of chromosome instability. We propose that SCF(Cyclin F)-mediated degradation of CP110 is required for the fidelity of mitosis and genome integrity

F-box proteins are the substrate recognition subunits of SCF (Skp1, Cul1, F-box protein) ubiquitin ligase complexes. Skp2 is a nuclear F-box protein that targets the CDK inhibitor p27 for ubiquitin- and proteasome-dependent degradation. In G(0) and during the G(1) phase of the cell cycle, Skp2 is degraded via the APC/C(Cdh1) ubiquitin ligase to allow stabilization of p27 and inhibition of CDKs, facilitating the maintenance of the G(0)/G(1) state. APC/C(Cdh1) binds Skp2 through an N-terminal domain (amino acids 46-94 in human Skp2). It has been shown that phosphorylation of Ser64 and Ser72 in this domain dissociates Skp2 from APC/C. More recently, it has instead been proposed that phosphorylation of Skp2 on Ser72 by Akt/PKB allows Skp2 binding to Skp1, promoting the assembly of an active SCF(Skp2) ubiquitin ligase, and Skp2 relocalization/retention into the cytoplasm, promoting cell migration via an unknown mechanism. According to these reports, a Skp2 mutant in which Ser72 is substituted with Ala is unable to promote cell proliferation and loses its oncogenic potential. Given the contrasting reports, we revisited these results and conclude that phosphorylation of Skp2 on Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCF(Skp2) ubiquitin ligase activity, and does not affect the subcellular localization of Skp2

Maintenance of genomic integrity is one of the fundamental biological properties shared by all living organisms. To counterbalance deleterious and potentially mutagenic effects of omnipresent DNA damaging assaults, organisms have developed a network of genome surveillance and maintenance pathways known as the DNA damage response. In eukaryotes, the orchestration of cell-cycle checkpoints, DNA damage repair, and apoptosis in response to DNA damage relies on posttranslational modifications of key regulatory proteins. Although the role of phosphorylation in these pathways is relatively well established, the significance of ubiquitylation has only recently emerged. In this review, we survey current research on the ubiquitin-proteasome system, focusing on the DNA damage response in the G2 phase of the cell cycle and two prominent classes of ubiquitin ligases, the SCF- and APC/C complexes. These ubiquitin ligases are reviewed with regard to their function in activating, maintaining, and terminating the checkpoint and in light of increasing evidence that suggests a dynamic balance of substrate ubiquitylation and deubiquitylation. We further discuss the impact of defective G2 checkpoint signaling on genomic stability and cancer risk, highlighting strategies for targeted antitumor drug discovery

Background: Skp2, a known oncogene, is overexpressed in several types of tumors and is associated with worse recurrence rate and overall survival in primary melanoma patients. Moreover, the anti-proliferative effects of Skp2 siRNA on various tumor cell lines have prompted the preclinical testing of Skp2 small molecule inhibitors. In this study, we assessed the clinical relevance and molecular mechanism(s) underlying Skp2 overexpression in metastatic melanoma patients. Methods: Skp2 protein levels were measured in 122 metastatic melanoma specimens using immunohistochemistry (IHC), and the association between Skp2 overexpression and post-recurrence survival was examined. Moreover, 22 cell lines (2 normal primary melanocytes, 2 primary immortal melanocytes, 4 primary melanoma cell lines, and 18 metastatic melanoma cell lines) were evaluated for Skp2 genomic amplification using Single Nucleotide Polymorphism (SNP) arrays (Affymetrix 6.0) and Skp2 gene expression using mRNA arrays (Affymetrix U133A 2.0) and quantitative RT-PCR. We also screened 18 cell lines for Skp2 mutation by sequencing. Results: Skp2 overexpression, defined as >25% tumor cells, was associated with shorter 3-yr post-recurrence survival (37%) compared to Skp2 expression <= 25% (55%) (HR=1.89, 95%, CI= 1.04, 3.42, p=0.04). Skp2 overexpression was significantly associated with the site of melanoma metastasis: visceral (n= 12; 89%), lymph node (n=49; 36%), brain (n=15; 14%), and soft-tissue (n=36; 6%) (p<0.001). SNP array revealed genomic amplification at the Skp2 locus in 6 (33%) metastatic cell lines and one primary melanoma cell line. Skp2 genomic amplification was associated with increased transcript expression. No Skp2 mutations were identified. Conclusions: Skp2 protein overexpression is associated with worse prognosis in metastasis in melanoma. Our results also support that gene amplification, rather than a Skp2 gene mutation, may be the major mechanism responsible for Skp2 aberrant expression in metastatic melanoma

The transcription factor p73, a member of the p53 family, mediates cell-cycle arrest and apoptosis in response to DNA damage-induced cellular stress, acting thus as a proapoptotic gene. Similar to p53, p73 activity is regulated by post-translational modification, including phosphorylation, acetylation and ubiquitylation. In C. elegans, the F-box protein FSN-1 controls germline apoptosis by regulating CEP-1, the single ancestral p53 family member. Here we report that FBXO45, the human ortholog of FSN-1, binds specifically to p73 triggering its proteasome-dependent degradation. Importantly, SCF(FBXO45) ubiquitylates p73 both in vivo and in vitro. Moreover, siRNA-mediated depletion of FBXO45 stabilizes p73 and concomitantly induces cell death in a p53-independent manner. All together, these results show that the orphan F-box protein FBXO45 regulates the stability of p73, highlighting a conserved pathway evolved from nematode to human by which the p53 members are regulated by an SCF-dependent mechanism.

Cyclin A accumulates at the onset of S phase, remains high during G(2) and early mitosis and is degraded at prometaphase. Here, we report that the acetyltransferase P/CAF directly interacts with cyclin A that as a consequence becomes acetylated at lysines 54, 68, 95 and 112. Maximal acetylation occurs simultaneously to ubiquitylation at mitosis, indicating importance of acetylation on cyclin A stability. This was further confirmed by the observation that the pseudoacetylated cyclin A mutant can be ubiquitylated whereas the nonacetylatable mutant cannot. The nonacetylatable mutant is more stable than cyclin A WT (cycA WT) and arrests cell cycle at mitosis. Moreover, in cells treated with histone deacetylase inhibitors cyclin A acetylation increases and its stability decreases, thus supporting the function of acetylation on cyclin A degradation. Although the nonacetylatable mutant cannot be ubiquitylated, it interacts with the proteins needed for its degradation (cdks, Cks, Cdc20, Cdh1 and APC/C). In fact, its association with cdks is increased and its complexes with these kinases display higher activity than control cycA WT-cdk complexes. All these results indicate that cyclin A acetylation at specific lysines is crucial for cyclin A stability and also has a function in the regulation of cycA-cdk activity.