GSK-3? regulates cell growth, migration, and angiogenesis via Fbw7 and USP28-dependent degradation of HIF-1?.
ABSTRACT: The hypoxia-inducible transcription factor-1? (HIF-1?) is a major regulator of angiogenesis, carcinogenesis, and various processes by which cells adapt to hypoxic conditions. Therefore, the identification of critical players regulating HIF-1? is not only important for the understanding of angiogenesis and different cancer phenotypes, but also for unraveling new therapeutic options. We report a novel mechanism by which HIF-1? is degraded after glycogen synthase kinase-3 (GSK-3)-induced phosphorylation and recruitment of the ubiquitin ligase and tumor suppressor F-box and WD protein Fbw7. Further, experiments with GSK-3? and Fbw7-deficient cells revealed that GSK-3? and Fbw7-dependent HIF-1? degradation can be antagonized by ubiquitin-specific protease 28 (USP28). In agreement with this, Fbw7 and USP28 reciprocally regulated cell migration and angiogenesis in an HIF-1?-dependent manner. Therefore, we have identified a new pathway that could be targeted at the level of GSK-3, Fbw7, or USP28 to influence HIF-1?-dependent processes like angiogenesis and metastasis.
Project description:The stability of several oncoproteins, including c-Myc, is regulated by ubiquitin-dependent degradation mediated by the SCF(Fbw7) ubiquitin ligase. This activity is antagonized by the deubiquitinase Usp28, which is highly expressed in murine and human intestinal cancers. Usp28 was previously shown to interact with its substrates via a "piggyback" interaction with Fbw7, which suggested that Fbw7 is required for Usp28 activity. Unexpectedly, we found that genetic deletion of Usp28 rescued the lethality of Fbw7-deficient primary fibroblasts. Moreover, Usp28 inactivation in the intestine (Usp28(?IEC)) ameliorated the hyperproliferation and the impaired goblet and Paneth cell differentiation observed in Fbw7(?IEC) mice. The aggressive intestinal tumor formation of APC(Min/+); Fbw7(?IEC) mice was restrained when Usp28 was inactivated concomitantly. In both fibroblasts and intestinal cells, Usp28 deficiency corrected the accumulation of SCF(Fbw7) substrate proteins, including NICD1, c-Jun, and c-Myc. These findings suggested that Usp28 function does not depend on the presence of Fbw7, but instead independently recognizes and deubiquitylates the same substrates as SCF(Fbw7). Fbw7 binds to a phosphorylated motif termed the phosphodegron and we found that Usp28 also interacted with this same motif, but only when it is unphosphorylated, offering a mechanistic explanation for identical substrate selection by Fbw7 and Usp28. Our results indicate an unusually direct antagonism between an E3 ligase and a deubiquitinase, Fbw7 and Usp28, in modulating intestinal homeostasis and cancer.
Project description:Fbw7, the substrate recognition subunit of SCF(Fbw7) ubiquitin ligase, mediates turnover of multiple proto-oncoproteins and promotes its own degradation. Fbw7-mediated substrate degradation is antagonized by the Usp28 deubiquitinase. We now show, using knockout mice, that Usp28 preferentially deubiquitinates and stabilizes Fbw7. Monoallelic deletion of Usp28 maintains stable Fbw7 but destabilizes Fbw7 substrates. In contrast, complete knockout of Usp28 promotes Pin1-dependent autocatalytic turnover of Fbw7, accumulation of Fbw7 substrates and oncogenic transformation. Overexpression of Usp28 stabilizes both Fbw7 and its substrates and similarly enhances transformation. We propose that dual regulation of Fbw7 activity by Usp28 maintains physiological levels of Fbw7 substrates, and that both loss and overexpression of Usp28 in human cancer promote Fbw7 substrate accumulation. RNAseq experiments of E13.5 murine embryonic fibroblasts (MEFs) derived from animals in which Usp28 was either deleted (-/-), wildtype (+/+) or heterozygous (+/-). In a first set of experiments immortalized MEFs of all three genotypes were analysed in biological triplicates. In a second set of experiments immortalized and Ras transformed MEFs of all three genotypes and MEFs which overexpress USP28 (+/+/+) where sequenced in duplicates.
Project description:The Usp28 deubiquitinase antagonizes Fbw7-mediated turnover of multiple oncoproteins, including Myc, Jun, and Notch, and promotes tumorigenesis in the intestine. Our recent study reveals that Usp28 also counteracts autocatalytic ubiquitination of Fbw7, suggesting a complex role for Usp28 in the regulation of Fbw7 activity and tumor development.
Project description:Hypoxia inducible factor-1? (HIF-1?) stimulates expression of genes associated with angiogenesis and is associated with poor outcomes in ovarian and other cancers. In normoxia, HIF-1? is ubiquitinated and degraded through the E3 ubiquitin ligase, von Hippel-Lindau; however, little is known about the regulation of HIF-1? in hypoxic conditions. FBW7 is an E3 ubiquitin ligase that recognizes proteins phosphorylated by glycogen synthase kinase 3? (GSK3?) and targets them for destruction. This study used an ovarian cancer cell model to test the hypothesis that HIF-1? phosphorylation by GSK3? in hypoxia leads to interaction with FBW7 and ubiquitin-dependent degradation. Expression of constitutively active GSK3? reduced HIF-1? protein and transcriptional activity and increased ubiquitination of HIF-1? in hypoxia, whereas pharmacologic inhibition of GSK3 or expression of siGSK3? promoted HIF-1? stabilization and activity. A mechanism through FBW7 was supported by the observed decrease in HIF-1? stabilization when FBW7 was overexpressed and both the elevation of HIF-1? levels and decrease in ubiquitinated HIF-1? when FBW7 was suppressed. Furthermore, HIF-1? associated with FBW7? by co-immunoprecipitation, and the interaction was weakened by inhibition of GSK3 or mutation of GSK3? phosphorylation sites. The relevance of this pathway to angiogenic signaling was supported by the finding that endothelial cell tube maturation was increased by conditioned media from hypoxic SK-OV-3 cell lines expressing suppressed GSK3? or FBW7. These data introduce a new mechanism for regulation of HIF-1? during hypoxia that utilizes phosphorylation to target HIF-1? for ubiquitin-dependent degradation through FBW7 and may identify new targets in the regulation of angiogenesis.
Project description:F-box and WD repeat domain-containing 7 (FBW7), the substrate-binding subunit of E3 ubiquitin ligase SCF(FBW7) (a complex of SKP1, cullin-1 and FBW7), plays important roles in various physiological and pathological processes. Although FBW7 is required for vascular development, its function in the endothelium remains to be investigated. In this study, we show that FBW7 is an important regulator of endothelial functions, including angiogenesis, leukocyte adhesion and the endothelial barrier integrity. Using RNA interference, we found that the depletion of FBW7 markedly impairs angiogenesis in vitro and in vivo. We identified the zinc finger transcription factor Krüppel-like factor 2 (KLF2) as a physiological target of FBW7 in endothelial cells. Knockdown of FBW7 expression resulted in the accumulation of endogenous KLF2 protein in endothelial cells. FBW7-mediated KLF2 destruction was shown to depend on the phosphorylation of KLF2 via glycogen synthase kinase-3 (GSK3) at two conserved phosphodegrons. Mutating these phosphodegron motifs abolished the FBW7-mediated degradation and ubiquitination of KLF2. The siRNA-mediated knockdown of FBW7 showed that KLF2 is an essential target of FBW7 in the regulation of endothelial functions. Moreover, FBW7-mediated KLF2 degradation was shown to be critical for angiogenesis in teratomas and in zebrafish development. Taken together, our study suggests a role for FBW7 in the processes of endothelial cell migration, angiogenesis, inflammation and barrier integrity, and provides novel insights into the regulation of KLF2 stability in vivo.
Project description:Cyclin E1, an activator of cyclin-dependent kinase 2 (Cdk2) that promotes replicative functions, is normally expressed periodically within the mammalian cell cycle, peaking at the G(1)-S-phase transition. This periodicity is achieved by E2F-dependent transcription in late G(1) and early S phases and by ubiquitin-mediated proteolysis. The ubiquitin ligase that targets phosphorylated cyclin E is SCF(Fbw7) (also known as SCF(Cdc4)), a member of the cullin ring ligase (CRL) family. Fbw7, a substrate adaptor subunit, is expressed as three splice-variant isoforms with different subcellular distributions: Fbw7? is nucleoplasmic but excluded from the nucleolus, Fbw7? is cytoplasmic, and Fbw7? is nucleolar. Degradation of cyclin E in vivo requires SCF complexes containing Fbw7? and Fbw7?, respectively. In vitro reconstitution showed that the role of SCF(Fbw7?) in cyclin E degradation, rather than ubiquitylation, is to serve as a cofactor of the prolyl cis-trans isomerase Pin1 in the isomerization of a noncanonical proline-proline bond in the cyclin E phosphodegron. This isomerization is required for subsequent binding and ubiquitylation by SCF(Fbw7?). Here we show that Pin1-mediated isomerization of the cyclin E phosphodegron and subsequent binding to Fbw7? drive nucleolar localization of cyclin E, where it is ubiquitylated by SCF(Fbw7?) prior to its degradation by the proteasome. It is possible that this constitutes a mechanism for rapid inactivation of phosphorylated cyclin E by nucleolar sequestration prior to its multiubiquitylation and degradation.
Project description:Colorectal cancer is the third most common cancer worldwide. Although the transcription factor c-MYC is misregulated in the majority of colorectal tumors, it is difficult to target directly. The deubiquitinase USP28 stabilizes oncogenic factors, including c-MYC; however, the contribution of USP28 in tumorigenesis, particularly in the intestine, is unknown. Here, using murine genetic models, we determined that USP28 antagonizes the ubiquitin-dependent degradation of c-MYC, a known USP28 substrate, as well as 2 additional oncogenic factors, c-JUN and NOTCH1, in the intestine. Mice lacking Usp28 had no apparent adverse phenotypes, but exhibited reduced intestinal proliferation and impaired differentiation of secretory lineage cells. In a murine model of colorectal cancer, Usp28 deletion resulted in fewer intestinal tumors, and importantly, in established tumors, Usp28 deletion reduced tumor size and dramatically increased lifespan. Moreover, we identified Usp28 as a c-MYC target gene highly expressed in murine and human intestinal cancers, which indicates that USP28 and c-MYC form a positive feedback loop that maintains high c-MYC protein levels in tumors. Usp28 deficiency promoted tumor cell differentiation accompanied by decreased proliferation, which suggests that USP28 acts similarly in intestinal homeostasis and colorectal cancer models. Hence, inhibition of the enzymatic activity of USP28 may be a potential target for cancer therapy.
Project description:USP28 (ubiquitin-specific protease 28) is a deubiquitinating enzyme that has been implicated in the DNA damage response, the regulation of Myc signaling, and cancer progression. The half-life stability of major regulators of critical cellular pathways depends on the activities of specific ubiquitin E3 ligases that target them for proteosomal degradation and deubiquitinating enzymes that promote their stabilization. One function of the post-translational small ubiquitin modifier (SUMO) is the regulation of enzymatic activity of protein targets. In this work, we demonstrate that the SUMO modification of the N-terminal domain of USP28 negatively regulates its deubiquitinating activity, revealing a role for the N-terminal region as a regulatory module in the control of USP28 activity. Despite the presence of ubiquitin-binding domains in the N-terminal domain, its truncation does not impair deubiquitinating activity on diubiquitin or polyubiquitin chain substrates. In contrast to other characterized USP deubiquitinases, our results indicate that USP28 has a chain preference activity for Lys(11), Lys(48), and Lys(63) diubiquitin linkages.
Project description:The SCF(Fbw7) ubiquitin ligase complex plays important roles in cell growth, survival, and differentiation via the ubiquitin-proteasome-mediated regulation of protein stability. Fbw7 (also known as Fbxw7, Sel-10, hCdc4, or hAgo), a substrate recognition subunit of SCF(Fbw7) ubiquitin ligase, facilitates the degradation of several proto-oncogene products by the proteasome. Given that mutations in Fbw7 are found in various types of human cancers, Fbw7 is considered to be a potent tumor suppressor. In the present study, we show that E1A, an oncogene product derived from adenovirus, interferes with the activity of the SCF(Fbw7) ubiquitin ligase. E1A interacted with SCF(Fbw7) and attenuated the ubiquitylation of its target proteins in vivo. Furthermore, using in vitro purified SCF(Fbw7) component proteins, we found that E1A directly bound to Roc1/Rbx1 and CUL1 and that E1A inhibited the ubiquitin ligase activity of the Roc1/Rbx1-CUL1 complex but not that of another RING-type ubiquitin ligase, Mdm2. Ectopically expressed E1A interacted with cellular endogenous Roc1/Rbx1 and CUL1 and decelerated the degradation of several protooncogene products that were degraded by SCF(Fbw7) ubiquitin ligase. Moreover, after wild-type adenovirus infection, adenovirus-derived E1A interacted with endogenous Roc1/Rbx1 and decelerated degradation of the endogenous target protein of SCF(Fbw7). These observations demonstrated that E1A perturbs protein turnover regulated by SCF(Fbw7) through the inhibition of SCF(Fbw7) ubiquitin ligase. Our findings may help to explain the mechanism whereby adenovirus infection induces unregulated proliferation.
Project description:The E3 ubiquitin ligase and tumor suppressor SCF(Fbw7) exists as three isoforms that govern the degradation of a host of critical cell regulators, including c-Myc, cyclin E, and PGC-1?. Peroxisome proliferator activated receptor-gamma coactivator 1? (PGC-1?) is a transcriptional coactivator with broad effects on cellular energy metabolism. Cellular PGC-1? levels are tightly controlled in a dynamic state by the balance of synthesis and rapid degradation via the ubiquitin-proteasome system. Isoform-specific functions of SCF(Fbw7) are yet to be determined. Here, we show that the E3 ubiquitin ligase, SCF(Fbw7), regulates cellular PGC-1? levels via two independent, isoform-specific, mechanisms. The cytoplasmic isoform (SCF(Fbw7?)) reduces cellular PGC-1? levels via accelerated ubiquitin-proteasome degradation. In contrast, the nuclear isoform (SCF(Fbw7?)) increases cellular PGC-1? levels and protein stability via inhibition of ubiquitin-proteasomal degradation. When nuclear Fbw7? proteins are redirected to the cytoplasm, cellular PGC-1? protein levels are reduced through accelerated ubiquitin-proteasomal degradation. We find that SCF(Fbw7?) catalyzes high molecular weight PGC-1?-ubiquitin conjugation, whereas SCF(Fbw7?) produces low molecular weight PGC-1?-ubiquitin conjugates that are not effective degradation signals. Thus, selective ubiquitination by specific Fbw7 isoforms represents a novel mechanism that tightly regulates cellular PGC-1? levels. Fbw7 isoforms mediate degradation of a host of regulatory proteins. The E3 ubiquitin ligase, Fbw7, mediates PGC-1? levels via selective isoform-specific ubiquitination. Fbw7? reduces cellular PGC-1? via ubiquitin-mediated degradation, whereas Fbw7? increases cellular PGC-1? via ubiquitin-mediated stabilization.