SCF-mediated Cdh1 degradation defines a negative feedback system that coordinates cell-cycle progression.
ABSTRACT: Proper cell-cycle transitions are driven by waves of ubiquitin-dependent degradation of key regulators by the anaphase-promoting complex (APC) and Skp1-Cullin1-F-box (SCF) E3 ubiquitin ligase complexes. But precisely how APC and SCF activities are coordinated to regulate cell-cycle progression remains largely unclear. We previously showed that APC/Cdh1 earmarks the SCF component Skp2 for degradation. Here, we continue to report that SCF(?-TRCP) reciprocally controls APC/Cdh1 activity by governing Cdh1 ubiquitination and subsequent degradation. Furthermore, we define both cyclin A and Plk1, two well-known Cdh1 substrates, as upstream modifying enzymes that promote Cdh1 phosphorylation to trigger Cdh1 ubiquitination and subsequent degradation by SCF(?-TRCP). Thus, our work reveals a negative repression mechanism for SCF to control APC, thereby illustrating an elegant dual repression system between these two E3 ligase complexes to create the ordered cascade of APC and SCF activities governing timely cell-cycle transitions.
Project description:The Set8/PR-Set7/KMT5a methyltransferase plays critical roles in governing transcriptional regulation, cell cycle progression and tumorigenesis. Although CRL4(Cdt2) was reported to regulate Set8 stability, deleting the PIP motif only led to partial resistance to ultraviolet-induced degradation of Set8, indicating the existence of additional E3 ligase(s) controlling Set8 stability. Furthermore, it remains largely undefined how DNA damage-induced kinase cascades trigger the timely destruction of Set8 to govern tumorigenesis. Here, we report that SCF(?-TRCP) earmarks Set8 for ubiquitination and degradation in a casein kinase I-dependent manner, which is activated by DNA-damaging agents. Biologically, both CRL4(Cdt2) and SCF(?-TRCP)-mediated pathways contribute to ultraviolet-induced Set8 degradation to control cell cycle progression, governing the onset of DNA damage-induced checkpoints. Therefore, like many critical cell cycle regulators including p21 and Cdt1, we uncover a tight regulatory network to accurately control Set8 abundance. Our studies further suggest that aberrancies in this delicate degradation pathway might contribute to aberrant elevation of Set8 in human tumours.
Project description:NIPA (Nuclear Interaction Partner of Alk kinase) is an F-box like protein that targets nuclear Cyclin B1 for degradation. Integrity and therefore activity of the SCF(NIPA) E3 ligase is regulated by cell-cycle-dependent phosphorylation of NIPA, restricting substrate ubiquitination to interphase. Here we show that phosphorylated NIPA is degraded in late mitosis in an APC/C(Cdh1)-dependent manner. Binding of the unphosphorylated form of NIPA to Skp1 interferes with binding to the APC/C-adaptor protein Cdh1 and therefore protects unphosphorylated NIPA from degradation in interphase. Our data thus define a novel mode of regulating APC/C-mediated ubiquitination.
Project description:The HECT domain-containing ubiquitin E3 ligase NEDD4 is widely expressed in mammalian tissues and plays a crucial role in governing a wide spectrum of cellular processes including cell growth, tissue development and homeostasis. Recent reports have indicated that NEDD4 might facilitate tumorigenesis through targeted degradation of multiple tumor suppressor proteins including PTEN. However, the molecular mechanism by which NEDD4 stability is regulated has not been fully elucidated. Here we report that SCF(?-TRCP) governs NEDD4 protein stability by targeting it for ubiquitination and subsequent degradation in a Casein Kinase-I (CKI) phosphorylation-dependent manner. Specifically, depletion of ?-TRCP, or inactivation of CKI, stabilized NEDD4, leading to down-regulation of its ubiquitin target PTEN and subsequent activation of the mTOR/Akt oncogenic pathway. Furthermore, we found that CKI?-mediated phosphorylation of Ser347 and Ser348 on NEDD4 promoted its interaction with SCF(?-TRCP) for subsequent ubiquitination and degradation. As a result, compared to ectopic expression of wild-type NEDD4, introducing a non-degradable NEDD4 (S347A/S348A-NEDD4) promoted cancer cell growth and migration. Hence, our findings revealed the CKI/SCF(?-TRCP) signaling axis as the upstream negative regulator of NEDD4, and further suggested that enhancing NEDD4 degradation, presumably with CKI or SCF(?-TRCP) agonists, could be a promising strategy for treating human cancers.
Project description:During cell division, the activation of glycolysis is tightly regulated by the action of two ubiquitin ligases, anaphase-promoting complex/cyclosome-Cdh1 (APC/C-Cdh1) and SKP1/CUL-1/F-box protein-?-transducin repeat-containing protein (SCF-?-TrCP), which control the transient appearance and metabolic activity of the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3). We now demonstrate that the breakdown of PFKFB3 during S phase occurs specifically via a distinct residue (S(273)) within the conserved recognition site for SCF-?-TrCP. Glutaminase 1 (GLS1), the first enzyme in glutaminolysis, is also targeted for destruction by APC/C-Cdh1 and, like PFKFB3, accumulates after the activity of this ubiquitin ligase decreases in mid-to-late G1. However, our results show that GLS1 differs from PFKFB3 in that its recognition by APC/C-Cdh1 requires the presence of both a Lys-Glu-Asn box (KEN box) and a destruction box (D box) rather than a KEN box alone. Furthermore, GLS1 is not a substrate for SCF-?-TrCP and is not degraded until cells progress from S to G2/M. The presence of PFKFB3 and GLS1 coincides with increases in generation of lactate and in utilization of glutamine, respectively. The contrasting posttranslational regulation of PFKFB3 and GLS1, which we have verified by studies of ubiquitination and protein stability, suggests the different roles of glucose and glutamine at distinct stages in the cell cycle. Indeed, experiments in which synchronized cells were deprived of either of these substrates show that both glucose and glutamine are required for progression through the restriction point in mid-to-late G1, whereas glutamine is the only substrate essential for the progression through S phase into cell division.
Project description:?-transducin repeat-containing protein (?-TrCP), one of the best-characterized substrate recognition components of the SKP1-CUL1-F-box (SCF) E3 ligase, has two distinct paralogs, ?-TrCP1 and ?-TrCP2, expressed in mammals. Through governing the ubiquitination and degradation of numerous key regulators, ?-TrCP1/2 regulates various cellular physiological and pathological processes. However, whether and how these two proteins cross talk and whether they regulate cell autophagy and proliferation in different manners is completely unknown. Herein, we report that ?-TrCP1 and ?-TrCP2 are the physiological substrates of SCF E3 ligase and target each other for degradation that is dependent on their ?-TrCP degron sequences. Furthermore, glucose deprivation activates AMPK kinase to phosphorylate ?-TrCP1 and promotes the subsequent ubiquitination and degradation of ?-TrCP1 by ?-TrCP2, but does not promote ?-TrCP2 degradation by ?-TrCP1. Finally, we found that ?-TrCP2, not ?-TrCP1, preferentially degrades DEPTOR and REDD1, the inhibitors of mTORC1, to activate mTORC1, leading to autophagy inhibition and cell growth. Thus, our study demonstrates that ?-TrCP1 and ?-TrCP2 mutually target each other for degradation and that ?-TrCP2 acts as a dominant paralog in the regulation of cell autophagy and growth, which might be a promising anticancer target.
Project description:The levels of proteins that control the cell cycle are regulated by ubiquitin-mediated degradation via the ubiquitin-proteasome system (UPS) by substrate-specific E3 ubiquitin ligases. The cyclin-dependent kinase inhibitor, p27kip1 (p27), that blocks the cell cycle in G1, is ubiquitylated by the E3 ligase SCF-Skp2/Cks1 for degradation by the UPS. In turn, Skp2 and Cks1 are ubiquitylated by the E3 ligase complex APC/Cdh1 for destruction thereby maintaining abundant levels of nuclear p27. We previously showed that perpetual proteasomal degradation of p27 is an early event in Type I endometrial carcinogenesis (ECA), an estrogen (E2)-induced cancer. The present studies demonstrate that E2 stimulates growth of ECA cell lines and normal primary endometrial epithelial cells (EECs) and induces MAPK-ERK1/2-dependent phosphorylation of p27 on Thr187, a prerequisite for p27 ubiquitylation by nuclear SCF-Skp2/Cks1 and subsequent degradation. In addition, E2 decreases the E3 ligase [APC]Cdh1 leaving Skp2 and Cks1 intact to cause p27 degradation. Furthermore, knocking-down Skp2 prevents E2-induced p27 degradation and growth stimulation suggesting that the pathogenesis of E2-induced ECA is dependent on Skp2-mediated degradation of p27. Conversely, progesterone (Pg) as an inhibitor of endometrial proliferation increases nuclear p27 and Cdh1 in primary EECs and ECA cells. Pg, also increases Cdh1 binding to APC to form the active E3ligase. Knocking-down Cdh1 obviates Pg-induced stabilization of p27 and growth inhibition. Notably, neither E2 nor Pg affected transcription of Cdh1, Skp2, Cks1 nor p27. These studies provide new insights into hormone regulation of cell proliferation through the UPS. The data implicates that preventing nuclear p27 degradation by blocking Skp2/Cks1-mediated degradation of p27 or increasing Cdh1 to mediate degradation of Skp2-Cks1 are potential strategies for the prevention and treatment of ECA.
Project description:The HBx oncoprotein of hepatitis B Virus has been accredited as one of the protagonists in driving hepatocarcinogenesis. HBx exerts its influence over the cell cycle progression by potentiating the activity of cyclin A/E-CDK2 complex, the Cyclin A partner of which is a well-known target of cellular deubiquitinase USP37. In the present study, we observed the intracellular accumulation of cyclin A and USP37 proteins under the HBx microenvironment. Flow cytometry analysis of the HBx-expressing cells showed deregulation of cell cycle apparently due to the enhanced gene expression and stabilization of USP37 protein and deubiquitination of Cyclin A by USP37. Our co-immunoprecipitation and confocal microscopic studies suggested a direct interaction between USP37 and HBx. This interaction promoted the translocation of USP37 outside the nucleus and prevented its association and ubiquitination by E3 ubiquitin ligases - APC/CDH1 and SCF/?-TrCP. Thus, HBx seems to control the cell cycle progression via the cyclin A-CDK2 complex by regulating the intracellular distribution and stability of deubiquitinase USP37.
Project description:DEPTOR, an inhibitor of mTORC1 and mTORC2, is degraded via ubiquitin-proteasome pathway by an unknown E3 ubiquitin ligase. Here we report that DEPTOR is a physiological substrate of SCF(?TrCP) E3 ligase for targeted degradation. Upon growth factor stimulation, RSK1 and S6K1 kinases are activated to phosphorylate DEPTOR, which is then recognized by the F box protein, ?TrCP, via its degron sequence for subsequent ubiquitination and degradation by SCF E3. Endogenous DEPTOR levels are negatively regulated by ?TrCP. DEPTOR half-life is shortened by ?TrCP but extended by a dominant-negative mutant of ?TrCP, by RSK1/S6K1 inhibition, and by ?TrCP degron site mutations. Biologically, DEPTOR accumulation upon ?TrCP knockdown inactivates mTORC1 and activates AKT in cancer cells to confer resistance to rapamycin and paclitaxel. Furthermore, DEPTOR accumulates upon glucose deprivation and mTOR inhibition to induce autophagy. Thus, ?TrCP-DEPTOR-mTOR intertwine to regulate cell survival and autophagy.
Project description:Wnt signaling has emerged as a major regulator of tissue development by governing the self-renewal and maintenance of stem cells in most tissue types. As a key upstream regulator of the Wnt pathway, the transmembrane E3 ligase ZNRF3 has recently been established to play a role in negative regulation of Wnt signaling by targeting Frizzled (FZD) receptor for ubiquitination and degradation. However, the upstream regulation of ZNRF3, in particular the turnover of ZNRF3, is still unclear. Here we report that ZNRF3 is accumulated in the presence of proteasome inhibitor treatment independent of its E3-ubiquitin ligase activity. Furthermore, the Cullin 1-specific SCF complex containing ?-TRCP has been identified to directly interact with and ubiquitinate ZNRF3 thereby regulating its protein stability. Similar with the degradation of ?-catenin by ?-TRCP, ZNRF3 is ubiquitinated by ?-TRCP in both CKI-phosphorylation- and degron-dependent manners. Thus, our findings not only identify a novel substrate for ?-TRCP oncogenic regulation, but also highlight the dual regulation of Wnt signaling by ?-TRCP in a context-dependent manner where ?-TRCP negatively regulates Wnt signaling by targeting ?-catenin, and positively regulates Wnt signaling by targeting ZNRF3.
Project description:The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase and core component of the cell-cycle oscillator. During G1 phase, APC/C binds to its substrate receptor Cdh1 and APC/C(Cdh1) plays an important role in restricting S-phase entry and maintaining genome integrity. We describe a reciprocal feedback circuit between APC/C and a second ubiquitin ligase, the SCF (Skp1-Cul1-F box). We show that cyclin F, a cell-cycle-regulated substrate receptor (F-box protein) for the SCF, is targeted for degradation by APC/C. Furthermore, we establish that Cdh1 is itself a substrate of SCF(cyclin F). Cyclin F loss impairs Cdh1 degradation and delays S-phase entry, and this delay is reversed by simultaneous removal of Cdh1. These data indicate that the coordinated, temporal ordering of cyclin F and Cdh1 degradation, organized in a double-negative feedback loop, represents a fundamental aspect of cell-cycle control. This mutual antagonism could be a feature of other oscillating systems.