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1.  Multiple Regulation of Rad51-Mediated Homologous Recombination by Fission Yeast Fbh1 
PLoS Genetics  2014;10(8):e1004542.
Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCFFbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated.
Author Summary
Homologous recombination is required for repairing DNA double-strand breaks (DSBs), which are induced by exogenous factors such as DNA damaging agents or by endogenous factors such as collapse of DNA replication fork in mitotic cells. If improperly processed, DSBs could lead to chromosome rearrangement, cell death, or tumorigenesis in mammals, and thus HR is strictly controlled at several steps, including Rad51 recombinase-driven DNA strand exchange reaction. Specifically, DNA helicases have been shown to be important for suppression of inappropriate recombination events. In this study, we analyzed one such DNA helicase, fission yeast Fbh1. We used an in vivo single-DSB repair assay to show that Fbh1 suppresses crossover formation between homologous chromosomes. Next, we obtained in vitro evidence that Fbh1 acts as an inhibitor of the strand-exchange reaction in the absence of Swi5-Sfr1, but stimulates the reaction after it starts. Furthermore, we found that SCFFbh1 has ubiquitin-ligase activity toward Rad51 in vitro and that Fbh1 regulates the protein level of Rad51 in the stationary phase. These results suggest Fbh1 regulates Rad51-mediated homologous recombination by its seemingly-unrelated two activities, DNA helicase/translocase and ubiquitin ligase.
PMCID: PMC4148199  PMID: 25165823
2.  The Human F-Box DNA Helicase FBH1 Faces Saccharomyces cerevisiae Srs2 and Postreplication Repair Pathway Roles▿  
Molecular and Cellular Biology  2007;27(21):7439-7450.
The Saccharomyces cerevisiae Srs2 UvrD DNA helicase controls genome integrity by preventing unscheduled recombination events. While Srs2 orthologues have been identified in prokaryotic and lower eukaryotic organisms, human orthologues of Srs2 have not been described so far. We found that the human F-box DNA helicase hFBH1 suppresses specific recombination defects of S. cerevisiae srs2 mutants, consistent with the finding that the helicase domain of hFBH1 is highly conserved with that of Srs2. Surprisingly, hFBH1 in the absence of SRS2 also suppresses the DNA damage sensitivity caused by inactivation of postreplication repair-dependent functions leading to PCNA ubiquitylation. The F-box domain of hFBH1, which is not present in Srs2, is crucial for hFBH1 functions in substituting for Srs2 and postreplication repair factors. Furthermore, our findings indicate that an intact F-box domain, acting as an SCF ubiquitin ligase, is required for the DNA damage-induced degradation of hFBH1 itself. Overall, our findings suggest that the hFBH1 helicase is a functional human orthologue of budding yeast Srs2 that also possesses self-regulation properties necessary to execute its recombination functions.
PMCID: PMC2169053  PMID: 17724085
3.  Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities 
The Journal of Cell Biology  2009;186(5):655-663.
Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities.
Homologous recombination (HR) is essential for faithful repair of DNA lesions yet must be kept in check, as unrestrained HR may compromise genome integrity and lead to premature aging or cancer. To limit unscheduled HR, cells possess DNA helicases capable of preventing excessive recombination. In this study, we show that the human Fbh1 (hFbh1) helicase accumulates at sites of DNA damage or replication stress in a manner dependent fully on its helicase activity and partially on its conserved F box. hFbh1 interacted with single-stranded DNA (ssDNA), the formation of which was required for hFbh1 recruitment to DNA lesions. Conversely, depletion of endogenous Fbh1 or ectopic expression of helicase-deficient hFbh1 attenuated ssDNA production after replication block. Although elevated levels of hFbh1 impaired Rad51 recruitment to ssDNA and suppressed HR, its small interfering RNA–mediated depletion increased the levels of chromatin-associated Rad51 and caused unscheduled sister chromatid exchange. Thus, by possessing both pro- and anti-recombinogenic potential, hFbh1 may cooperate with other DNA helicases in tightly controlling cellular HR activity.
PMCID: PMC2742184  PMID: 19736316
4.  Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair 
DNA double-strand breaks (DSBs) are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51)-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage.
To determine the functional roles of the highly conserved F-box and helicase domains, we have characterized fbh1 mutants carrying specific mutations in these domains. We show that the F-box mutation fbh1-fb disturbs the nuclear localization of Fbh1, conferring an fbh1 null-like phenotype. Moreover, nuclear foci do not form in fbh1-fb cells with DNA damage even if Fbh1-fb is targeted to the nucleus by fusion to a nuclear localization signal sequence. In contrast, the helicase mutation fbh1-hl causes the accumulation of Fbh1 foci irrespective of the presence of DNA damage and confers damage sensitivity greater than that conferred by the null allele. Additional mutation of the F-box alleviates the hypermorphic phenotype of the fbh1-hl mutant.
These results suggest that the F-box and DNA helicase domains play indispensable but distinct roles in Fbh1 function. Assembly of the SCFFbh1 complex is required for both the nuclear localization and DNA damage-induced focus formation of Fbh1 and is therefore prerequisite for the Fbh1 recombination function.
PMCID: PMC2294136  PMID: 18312697
5.  Mammalian Fbh1 is important to restore normal mitotic progression following decatenation stress 
DNA repair  2010;9(6):708-717.
We have addressed the role of the F-box helicase 1 (Fbh1) protein during genome maintenance in mammalian cells. For this, we generated two mouse embryonic stem cell lines deficient for Fbh1: one with a homozygous deletion of the N-terminal F-box domain (Fbh1f/f), and the other with a homozygous disruption (Fbh1−/−). Consistent with previous reports of Fbh1-deficiency in vertebrate cells, we found that Fbh1−/− cells show a moderate increase in Rad51 localization to DNA damage, but no clear defect in chromosome break repair. In contrast, we found that Fbh1f/f cells show a decrease in Rad51 localization to DNA damage and increased cytoplasmic localization of Rad51. However, these Fbh1f/f cells show no clear defects in chromosome break repair. Since some Rad51 partners and F-box-associated proteins (Skp1-Cul1) have been implicated in progression through mitosis, we considered whether Fbh1 might play a role in this process. To test this hypothesis, we disrupted mitosis using catalytic topoisomerase II inhibitors (bisdioxopiperazines), which inhibit chromosome decatenation. We found that both Fbh1f/f and Fbh1−/− cells show hypersensitivity to topoisomerase II catalytic inhibitors, even though the degree of decatenation stress was not affected. Furthermore, following topoisomerase II catalytic inhibition, both Fbh1-deficient cell lines show substantial defects in anaphase separation of chromosomes. These results indicate that Fbh1 is important for restoration of normal mitotic progression following decatenation stress.
PMCID: PMC2883650  PMID: 20457012
Fbh1; Rad51; decatenation; bisdioxopiperazine; topoisomerase; mitosis
6.  FBH1 promotes DNA double-strand breakage and apoptosis in response to DNA replication stress 
The Journal of Cell Biology  2013;200(2):141-149.
Enzymatic activity of the UvrD DNA helicase FBH1 is required for the efficient induction of DSBs and apoptosis specifically in response to DNA replication stress.
Proper resolution of stalled replication forks is essential for genome stability. Purification of FBH1, a UvrD DNA helicase, identified a physical interaction with replication protein A (RPA), the major cellular single-stranded DNA (ssDNA)–binding protein complex. Compared with control cells, FBH1-depleted cells responded to replication stress with considerably fewer double-strand breaks (DSBs), a dramatic reduction in the activation of ATM and DNA-PK and phosphorylation of RPA2 and p53, and a significantly increased rate of survival. A minor decrease in ssDNA levels was also observed. All these phenotypes were rescued by wild-type FBH1, but not a FBH1 mutant lacking helicase activity. FBH1 depletion had no effect on other forms of genotoxic stress in which DSBs form by means that do not require ssDNA intermediates. In response to catastrophic genotoxic stress, apoptosis prevents the persistence and propagation of DNA lesions. Our findings show that FBH1 helicase activity is required for the efficient induction of DSBs and apoptosis specifically in response to DNA replication stress.
PMCID: PMC3549964  PMID: 23319600
7.  The F-Box DNA Helicase Fbh1 Prevents Rhp51-Dependent Recombination without Mediator Proteins 
Molecular and Cellular Biology  2005;25(18):8084-8096.
A key step in homologous recombination is the loading of Rad51 onto single-stranded DNA to form a nucleoprotein filament that promotes homologous DNA pairing and strand exchange. Mediator proteins, such as Rad52 and Rad55-Rad57, are thought to aid filament assembly by overcoming an inhibitory effect of the single-stranded-DNA-binding protein replication protein A. Here we show that mediator proteins are also required to enable fission yeast Rad51 (called Rhp51) to function in the presence of the F-box DNA helicase Fbh1. In particular, we show that the critical function of Rad22 (an orthologue of Rad52) in promoting Rhp51-dependent recombination and DNA repair can be mostly circumvented by deleting fbh1. Similarly, the reduced growth/viability and DNA damage sensitivity of an fbh1− mutant are variously suppressed by deletion of any one of the mediators Rad22, Rhp55, and Swi5. From these data we propose that Rhp51 action is controlled through an interplay between Fbh1 and the mediator proteins. Colocalization of Fbh1 with Rhp51 damage-induced foci suggests that this interplay occurs at the sites of nucleoprotein filament assembly. Furthermore, analysis of different fbh1 mutant alleles suggests that both the F-box and helicase activities of Fbh1 contribute to controlling Rhp51.
PMCID: PMC1234329  PMID: 16135800
8.  Cooperative Roles of Vertebrate Fbh1 and Blm DNA Helicases in Avoidance of Crossovers during Recombination Initiated by Replication Fork Collapse▿ †  
Molecular and Cellular Biology  2007;27(8):2812-2820.
Fbh1 (F-box DNA helicase 1) orthologues are conserved from Schizosaccharomyces pombe to chickens and humans. Here, we report the disruption of the FBH1 gene in DT40 cells. Although the yeast fbh1 mutant shows an increase in sensitivity to DNA damaging agents, FBH1−/− DT40 clones show no prominent sensitivity, suggesting that the loss of FBH1 might be compensated by other genes. However, FBH1−/− cells exhibit increases in both sister chromatid exchange and the formation of radial structures between homologous chromosomes without showing a defect in homologous recombination. This phenotype is reminiscent of BLM−/− cells and suggests that Fbh1 may be involved in preventing extensive strand exchange during homologous recombination. In addition, disruption of RAD54, a major homologous recombination factor in FBH1−/− cells, results in a marked increase in chromosome-type breaks (breaks on both sister chromatids at the same place) following replication fork arrest. Further, FBH1BLM cells showed additive increases in both sister chromatid exchange and the formation of radial chromosomes. These data suggest that Fbh1 acts in parallel with Bloom helicase to control recombination-mediated double-strand-break repair at replication blocks and to reduce the frequency of crossover.
PMCID: PMC1899948  PMID: 17283053
9.  SCF Ensures Meiotic Chromosome Segregation Through a Resolution of Meiotic Recombination Intermediates 
PLoS ONE  2012;7(1):e30622.
The SCF (Skp1-Cul1-F-box) complex contributes to a variety of cellular events including meiotic cell cycle control, but its function during meiosis is not understood well. Here we describe a novel function of SCF/Skp1 in meiotic recombination and subsequent chromosome segregation. The skp1 temperature-sensitive mutant exhibited abnormal distribution of spindle microtubules in meiosis II, which turned out to originate from abnormal bending of the spindle in meiosis I. Bent spindles were reported in mitosis of this mutant, but it remained unknown how SCF could affect spindle morphology. We found that the meiotic bent spindle in skp1 cells was due to a hypertension generated by chromosome entanglement. The spindle bending was suppressed by inhibiting double strand break (DSB) formation, indicating that the entanglement was generated by the meiotic recombination machinery. Consistently, Rhp51/Rad51-Rad22/Rad52 foci persisted until meiosis I in skp1 cells, proving accumulation of recombination intermediates. Intriguingly bent spindles were also observed in the mutant of Fbh1, an F-box protein containing the DNA helicase domain, which is involved in meiotic recombination. Genetic evidence suggested its cooperation with SCF/Skp1. Thus, SCF/Skp1 together with Fbh1 is likely to function in the resolution of meiotic recombination intermediates, thereby ensuring proper chromosome segregation.
PMCID: PMC3264600  PMID: 22292001
10.  An SCF complex containing Fbxl12 mediates DNA damage-induced Ku80 ubiquitylation 
Cell Cycle  2013;12(4):587-595.
The Ku heterodimer, composed of Ku70 and Ku80, is the initiating factor of the nonhomologous end joining (NHEJ) double-strand break (DSB) repair pathway. Ku is also thought to impede the homologous recombination (HR) repair pathway via inhibition of DNA end resection. Using the cell-free Xenopus laevis egg extract system, we had previously discovered that Ku80 becomes polyubiquitylated upon binding to DSBs, leading to its removal from DNA and subsequent proteasomal degradation. Here we show that the Skp1-Cul1-F box (SCF) E3 ubiquitin ligase complex is required for Ku80 ubiquitylation and removal from DNA. A screen for DSB-binding F box proteins revealed that the F box protein Fbxl12 was recruited to DNA in a DSB- and Ku-sensitive manner. Immunodepletion of Fbxl12 prevented Cul1 and Skp1 binding to DSBs and Ku80 ubiquitylation, indicating that Fbxl12 is the F box protein responsible for Ku80 substrate recognition. Unlike typical F box proteins, the F box of Fbxl12 was essential for binding to both Skp1 and its substrate Ku80. Besides Fbxl12, six other chromatin-binding F box proteins were identified in our screen of a subset of Xenopus F box proteins: β-TrCP, Fbh1, Fbxl19, Fbxo24, Fbxo28 and Kdm2b. Our study unveils a novel function for the SCF ubiquitin ligase in regulating the dynamic interaction between DNA repair machineries and DSBs.
PMCID: PMC3594259  PMID: 23324393
Ku80; Ku86; Ku70; SCF; DNA damage; double-strand break; nonhomologous end joining; Fbxl12; Fbl12; ubiquitin
11.  Single-molecule sorting reveals how ubiquitylation affects substrate recognition and activities of FBH1 helicase 
Nucleic Acids Research  2013;41(6):3576-3587.
DNA repair helicases function in the cell to separate DNA duplexes or remodel nucleoprotein complexes. These functions are influenced by sensing and signaling; the cellular pool of a DNA helicase may contain subpopulations of enzymes carrying different post-translational modifications and performing distinct biochemical functions. Here, we report a novel experimental strategy, single-molecule sorting, which overcomes difficulties associated with comprehensive analysis of heterologously modified pool of proteins. This methodology was applied to visualize human DNA helicase F-box–containing DNA helicase (FBH1) acting on the DNA structures resembling a stalled or collapsed replication fork and its interactions with RAD51 nucleoprotein filament. Individual helicase molecules isolated from human cells with their native post-translational modifications were analyzed using total internal reflection fluorescence microscopy. Separation of the activity trajectories originated from ubiquitylated and non-ubiquitylated FBH1 molecules revealed that ubiquitylation affects FBH1 interaction with the RAD51 nucleoprotein filament, but not its translocase and helicase activities.
PMCID: PMC3616717  PMID: 23393192
12.  Fbh1 Limits Rad51-Dependent Recombination at Blocked Replication Forks▿ ‡  
Molecular and Cellular Biology  2009;29(17):4742-4756.
Controlling the loading of Rad51 onto DNA is important for governing when and how homologous recombination is used. Here we use a combination of genetic assays and indirect immunofluorescence to show that the F-box DNA helicase (Fbh1) functions in direct opposition to the Rad52 orthologue Rad22 to curb Rad51 loading onto DNA in fission yeast. Surprisingly, this activity is unnecessary for limiting spontaneous direct-repeat recombination. Instead it appears to play an important role in preventing recombination when replication forks are blocked and/or broken. When overexpressed, Fbh1 specifically reduces replication fork block-induced recombination, as well as the number of Rad51 nuclear foci that are induced by replicative stress. These abilities are dependent on its DNA helicase/translocase activity, suggesting that Fbh1 exerts its control on recombination by acting as a Rad51 disruptase. In accord with this, overexpression of Fbh1 also suppresses the high levels of recombinant formation and Rad51 accumulation at a site-specific replication fork barrier in a strain lacking the Rad51 disruptase Srs2. Similarly overexpression of Srs2 suppresses replication fork block-induced gene conversion events in an fbh1Δ mutant, although an inability to suppress deletion events suggests that Fbh1 has a distinct functionality, which is not readily substituted by Srs2.
PMCID: PMC2725720  PMID: 19546232
13.  Role of the Schizosaccharomyces pombe F-Box DNA Helicase in Processing Recombination Intermediates 
Molecular and Cellular Biology  2005;25(18):8074-8083.
In an effort to identify novel genes involved in recombination repair, we isolated fission yeast Schizosaccharomyces pombe mutants sensitive to methyl methanesulfonate (MMS) and a synthetic lethal with rad2. A gene that complements such mutations was isolated from the S. pombe genomic library, and subsequent analysis identified it as the fbh1 gene encoding the F-box DNA helicase, which is conserved in mammals but not conserved in Saccharomyces cerevisiae. An fbh1 deletion mutant is moderately sensitive to UV, MMS, and γ rays. The rhp51 (RAD51 ortholog) mutation is epistatic to fbh1. fbh1 is essential for viability in stationary-phase cells and in the absence of either Srs2 or Rqh1 DNA helicase. In each case, lethality is suppressed by deletion of the recombination gene rhp57. These results suggested that fbh1 acts downstream of rhp51 and rhp57. Following UV irradiation or entry into the stationary phase, nuclear chromosomal domains of the fbh1Δ mutant shrank, and accumulation of some recombination intermediates was suggested by pulsed-field gel electrophoresis. Focus formation of Fbh1 protein was induced by treatment that damages DNA. Thus, the F-box DNA helicase appears to process toxic recombination intermediates, the formation of which is dependent on the function of Rhp51.
PMCID: PMC1234317  PMID: 16135799
14.  The helicase FBH1 is tightly regulated by PCNA via CRL4(Cdt2)-mediated proteolysis in human cells 
Nucleic Acids Research  2013;41(13):6501-6513.
During replication, DNA damage can challenge replication fork progression and cell viability. Homologous Recombination (HR) and Translesion Synthesis (TLS) pathways appear as major players involved in the resumption and completion of DNA replication. How both pathways are coordinated in human cells to maintain genome stability is unclear. Numerous helicases are involved in HR regulation. Among them, the helicase FBH1 accumulates at sites of DNA damage and potentially constrains HR via its anti-recombinase activity. However, little is known about its regulation in vivo. Here, we report a mechanism that controls the degradation of FBH1 after DNA damage. Firstly, we found that the sliding clamp Proliferating Cell Nuclear Antigen (PCNA) is critical for FBH1 recruitment to replication factories or DNA damage sites. We then showed the anti-recombinase activity of FBH1 is partially dependent on its interaction with PCNA. Intriguingly, after its re-localization, FBH1 is targeted for degradation by the Cullin-ring ligase 4-Cdt2 (CRL4Cdt2)–PCNA pathway via a PCNA-interacting peptide (PIP) degron. Importantly, expression of non-degradable FBH1 mutant impairs the recruitment of the TLS polymerase eta to chromatin in UV-irradiated cells. Thus, we propose that after DNA damage, FBH1 might be required to restrict HR and then degraded by the Cdt2–proteasome pathway to facilitate TLS pathway.
PMCID: PMC3711418  PMID: 23677613
15.  The SCFHOS/β-TRCP-ROC1 E3 Ubiquitin Ligase Utilizes Two Distinct Domains within CUL1 for Substrate Targeting and Ubiquitin Ligation 
Molecular and Cellular Biology  2000;20(4):1382-1393.
We describe a purified ubiquitination system capable of rapidly catalyzing the covalent linkage of polyubiquitin chains onto a model substrate, phosphorylated IκBα. The initial ubiquitin transfer and subsequent polymerization steps of this reaction require the coordinated action of Cdc34 and the SCFHOS/β-TRCP-ROC1 E3 ligase complex, comprised of four subunits (Skp1, cullin 1 [CUL1], HOS/β-TRCP, and ROC1). Deletion analysis reveals that the N terminus of CUL1 is both necessary and sufficient for binding Skp1 but is devoid of ROC1-binding activity and, hence, is inactive in catalyzing ubiquitin ligation. Consistent with this, introduction of the N-terminal CUL1 polypeptide into cells blocks the tumor necrosis factor alpha-induced and SCF-mediated degradation of IκB by forming catalytically inactive complexes lacking ROC1. In contrast, the C terminus of CUL1 alone interacts with ROC1 through a region containing the cullin consensus domain, to form a complex fully active in supporting ubiquitin polymerization. These results suggest the mode of action of SCF-ROC1, where CUL1 serves as a dual-function molecule that recruits an F-box protein for substrate targeting through Skp1 at its N terminus, while the C terminus of CUL1 binds ROC1 to assemble a core ubiquitin ligase.
PMCID: PMC85290  PMID: 10648623
16.  FBH1 protects melanocytes from transformation and is deregulated in melanomas 
Cell Cycle  2013;12(7):1128-1132.
FBH1 is a member of the UvrD family of DNA helicases and plays a crucial role in the response to DNA replication stress. In particular, upon DNA replication stress, FBH1 promotes double-strand breakage and activation of the DNA-PK and ATM signaling cascades in a helicase-dependent manner. In the present manuscript, we show that FBH1 is often deleted or mutated in melanoma cells, which results in their increased survival in response to replicative stress. Accordingly, FBH1 depletion promotes UV-mediated transformation of human melanocytes. Thus, FBH1 inactivation appears to contribute to oncogenic transformation by allowing survival of cells undergoing replicative stress due to external factors such as UV irradiation.
PMCID: PMC3646868  PMID: 23466708
FBH1; F-box protein; helicase; DNA replication stress; melanoma
17.  A failure of meiotic chromosome segregation in a fbh1Δ mutant correlates with persistent Rad51-DNA associations 
Nucleic Acids Research  2010;39(5):1718-1731.
The F-box DNA helicase Fbh1 constrains homologous recombination in vegetative cells, most likely through an ability to displace the Rad51 recombinase from DNA. Here, we provide the first evidence that Fbh1 also serves a vital meiotic role in fission yeast to promote normal chromosome segregation. In the absence of Fbh1, chromosomes remain entangled or segregate unevenly during meiosis, and genetic and cytological data suggest that this results in part from a failure to efficiently dismantle Rad51 nucleofilaments that form during meiotic double-strand break repair.
PMCID: PMC3061084  PMID: 21149262
18.  Phosphorylation of Ser72 is dispensable for Skp2 assembly into an active SCF ubiquitin ligase and its subcellular localization 
Cell cycle (Georgetown, Tex.)  2010;9(5):971-974.
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 G0 and during the G1 phase of the cell cycle, Skp2 is degraded via the APC/CCdh1 ubiquitin ligase to allow stabilization of p27 and inhibition of CDKs, facilitating the maintenance of the G0/G1 state. APC/CCdh1 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 SCFSkp2 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 SCFSkp2 ubiquitin ligase activity, and does not affect the subcellular localization of Skp2.
PMCID: PMC3827631  PMID: 20160477
Skp2; Akt; SCF; ubiquitin
19.  Calcium-sensing receptor mutations in familial benign hypercalcemia and neonatal hyperparathyroidism. 
Journal of Clinical Investigation  1995;96(6):2683-2692.
Familial benign hypercalcemia (FBH) and neonatal hyperparathyroidism (NHPT) are disorders of calcium homeostasis that are associated with missense mutations of the calcium-sensing receptor (CaR). We have undertaken studies to characterize such CaR mutations in FBH and NHPT and to explore methods for their more rapid detection. Nine unrelated kindreds (39 affected, 32 unaffected members) with FBH and three unrelated children with sporadic NHPT were investigated for mutations in the 3,234-bp coding region of the CaR gene by DNA sequencing. Six novel heterozygous (one nonsense and five missense) mutations were identified in six of the nine FBH kindreds, and two de novo heterozygous missense mutations and one homozygous frame-shift mutation were identified in the three children with NHPT. Our results expand the phenotypes associated with CaR mutations to include sporadic NHPT. Single-stranded conformational polymorphism analysis was found to be a sensitive and specific mutational screening method that detected > 85% of these CaR gene mutations. The single-stranded conformational polymorphism identification of CaR mutations may help in the distinction of FBH from mild primary hyperparathyroidism which can be clinically difficult. Thus, the results of our study will help to supplement the clinical evaluation of some hypercalcemic patients and to elucidate further the structure-function relationships of the CaR.
PMCID: PMC185975  PMID: 8675635
20.  The Papillomavirus E7 Oncoprotein Is Ubiquitinated by UbcH7 and Cullin 1- and Skp2-Containing E3 Ligase 
Journal of Virology  2004;78(10):5338-5346.
Recurrent infections with high-risk human papillomaviruses (HPVs) are associated with human cervical cancers. All HPV-associated cancer tissues express the viral oncoproteins E6 and E7, which stimulate cell growth. The expression of E7 is crucial for both the initiation and the maintenance of HPV-associated cancer. Recent studies showed that the level of E7 in cancer cells is regulated by ubiquitin-dependent proteolysis through the 26S proteasome. In this study, we characterized the enzymes involved in the ubiquitin-dependent proteolysis of E7. We show that UbcH7, an E2 ubiquitin-conjugating enzyme, is specifically involved in the ubiquitination of E7. Furthermore, we show that E7 interacts with the SCF (Skp-Cullin-F box) ubiquitin ligase complex containing Cullin 1 (Cul1) and Skp2 and can be ubiquitinated by the Cul1-containing ubiquitin ligase in vitro. Coimmunoprecipitation analyses revealed that E7 interacts with Skp2 and Cul1 in vivo. Finally, the half-life of E7 was found to be significantly longer in Skp2−/− mouse embryo fibroblasts (MEFs) than in wild-type MEFs. Taken together, these results suggest that the Cul1- and Skp2-containing ubiquitin ligase plays a role in the ubiquitination and proteolysis of E7. In HPV type 16-containing cervical carcinoma cell line Caski, E7 localizes to both the cytoplasm and the nucleus. Brief treatment of Caski cells with MG132 (a proteasome inhibitor) causes the accumulation of E7 in discrete nuclear bodies. These nuclear bodies are detergent insoluble and contain polyubiquitinated E7. We suggest that E7 relocates to specific nuclear bodies for proteolysis in HPV-containing epithelial cells.
PMCID: PMC400333  PMID: 15113913
21.  Deconjugation of Nedd8 from Cul1 Is Directly Regulated by Skp1-F-box and Substrate, and the COP9 Signalosome Inhibits Deneddylated SCF by a Noncatalytic Mechanism* 
The Journal of Biological Chemistry  2012;287(35):29679-29689.
Background: A detailed description of the kinetics of deneddylation of cullin by CSN has been lacking.
Results: Selected factors and SCF subunits are able to inhibit deneddylation to varying degrees. CSN interferes with SCF-mediated ubiquitination through a noncatalytic mechanism.
Conclusion: Deneddylation of Cul1 by CSN is regulated by F-box protein, substrate, and other factors.
Significance: Our work reported here could facilitate the development of directed therapies.
COP9 signalosome (CSN) mediates deconjugation of the ubiquitin-like protein Nedd8 from the cullin subunits of SCF and other cullin-RING ubiquitin ligases (CRLs). This process is essential to maintain the proper activity of CRLs in cells. Here, we report a detailed kinetic characterization of CSN-mediated deconjugation of Nedd8 from SCF. CSN is an efficient enzyme, with a kcat of ∼1 s−1 and Kmfor neddylated Cul1-Rbx1 of ∼200 nm, yielding a kcat/Km near the anticipated diffusion-controlled limit. Assembly with an F-box-Skp1 complex markedly inhibited deneddylation, although the magnitude varied considerably, with Fbw7-Skp1 inhibiting by ∼5-fold but Skp2-Cks1-Skp1 by only ∼15%. Deneddylation of both SCFFbw7 and SCFSkp2-Cks1 was further inhibited ∼2.5-fold by the addition of substrate. Combined, the inhibition by Fbw7-Skp1 plus its substrate cyclin E was greater than 10-fold. Unexpectedly, our results also uncover significant product inhibition by deconjugated Cul1, which results from the ability of Cul1 to bind tightly to CSN. Reciprocally, CSN inhibits the ubiquitin ligase activity of deneddylated Cul1. We propose a model in which assembled CRL complexes engaged with substrate are normally refractory to deneddylation. Upon consumption of substrate and subsequent deneddylation, CSN can remain stably bound to the CRL and hold it in low state of reduced activity.
PMCID: PMC3436198  PMID: 22767593
Analytical Biochemistry; Enzyme Kinetics; Protein Degradation; Protein-Protein Interactions; Ubiquitin Ligase; CSN; Cop9; Cul1; Nedd8; Deneddylation
22.  The Steady-State Repertoire of Human SCF Ubiquitin Ligase Complexes Does Not Require Ongoing Nedd8 Conjugation* 
Molecular & Cellular Proteomics : MCP  2010;10(5):M110.006460.
The human genome encodes 69 different F-box proteins (FBPs), each of which can potentially assemble with Skp1-Cul1-RING to serve as the substrate specificity subunit of an SCF ubiquitin ligase complex. SCF activity is switched on by conjugation of the ubiquitin-like protein Nedd8 to Cul1. Cycles of Nedd8 conjugation and deconjugation acting in conjunction with the Cul1-sequestering factor Cand1 are thought to control dynamic cycles of SCF assembly and disassembly, which would enable a dynamic equilibrium between the Cul1-RING catalytic core of SCF and the cellular repertoire of FBPs. To test this hypothesis, we determined the cellular composition of SCF complexes and evaluated the impact of Nedd8 conjugation on this steady-state. At least 42 FBPs assembled with Cul1 in HEK 293 cells, and the levels of Cul1-bound FBPs varied by over two orders of magnitude. Unexpectedly, quantitative mass spectrometry revealed that blockade of Nedd8 conjugation led to a modest increase, rather than a decrease, in the overall level of most SCF complexes. We suggest that multiple mechanisms including FBP dissociation and turnover cooperate to maintain the cellular pool of SCF ubiquitin ligases.
PMCID: PMC3098594  PMID: 21169563
23.  Mechanisms of mono- and poly-ubiquitination: Ubiquitination specificity depends on compatibility between the E2 catalytic core and amino acid residues proximal to the lysine 
Cell Division  2010;5:19.
Ubiquitination involves the attachment of ubiquitin to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin attachment to different lysine residues can generate diverse substrate-ubiquitin structures, targeting proteins to different fates. The mechanisms of lysine selection are not well understood. Ubiquitination by the largest group of E3 ligases, the RING-family E3 s, is catalyzed through co-operation between the non-catalytic ubiquitin-ligase (E3) and the ubiquitin-conjugating enzyme (E2), where the RING E3 binds the substrate and the E2 catalyzes ubiquitin transfer. Previous studies suggest that ubiquitination sites are selected by E3-mediated positioning of the lysine toward the E2 active site. Ultimately, at a catalytic level, ubiquitination of lysine residues within the substrate or ubiquitin occurs by nucleophilic attack of the lysine residue on the thioester bond linking the E2 catalytic cysteine to ubiquitin. One of the best studied RING E3/E2 complexes is the Skp1/Cul1/F box protein complex, SCFCdc4, and its cognate E2, Cdc34, which target the CDK inhibitor Sic1 for K48-linked polyubiquitination, leading to its proteasomal degradation. Our recent studies of this model system demonstrated that residues surrounding Sic1 lysines or lysine 48 in ubiquitin are critical for ubiquitination. This sequence-dependence is linked to evolutionarily conserved key residues in the catalytic region of Cdc34 and can determine if Sic1 is mono- or poly-ubiquitinated. Our studies indicate that amino acid determinants in the Cdc34 catalytic region and their compatibility to those surrounding acceptor lysine residues play important roles in lysine selection. This may represent a general mechanism in directing the mode of ubiquitination in E2 s.
PMCID: PMC2927562  PMID: 20704751
24.  Regulation of Cullin-RING ubiquitin ligase 1 by Spliceosome-associated protein 130 (SAP130) 
Biology Open  2013;2(8):838-844.
Cullin-RING ubiquitin ligases (CRLs) mediate the ubiquitination of numerous protein substrates and target them for proteasomal degradation. The function of CRLs is under tight regulation by Cullin-binding proteins. It has been reported that the Spliceosome-associated protein 130 (SAP130/SF3b-3) binds to several Cullin proteins, yet it remains unknown whether SAP130 plays any role in regulating the function of CRLs. Here, we report that SAP130 overexpression reduces the binding of adaptor protein Skp1 and substrate receptor Skp2 to Cul1, whereas it has no effect on CAND1 binding to Cul1. Overexpression of SAP130 decreases the degradation rate of p27, a protein substrate of the SCFSkp2 ligase. Interestingly, silencing of SAP130 also inhibits the degradation of p27, suggesting a dual role for SAP130 in the regulation of SCF activity. We hypothesized that the regulatory role of SAP130 could extend to other CRLs; however, overexpression of SAP130 is unable to affect the protein stability of the Cul2 and Cul3 substrates, HIF-1 and NRF-2. SAP130 binds to Cul1, Cul2 and Cul4 with similar affinity, and it binds to Cul3 more strongly. SAP130 localizes in both the nucleus and the cytoplasm. Hence, the inability of SAP130 to regulate Cul2 and Cul3 CRLs cannot be explained by low binding affinity of SAP130 to these cullins or by subcellular sequestration of SAP130. We propose a novel role for SAP130 in the regulation of SCF, whereby SAP130 physically competes with the adaptor protein/F-box protein for Cul1 binding and interferes with the assembly of a functional SCF ligase.
PMCID: PMC3744076  PMID: 23951410
Cullin-RING ligase 1; SAP130; p27
25.  Signal-induced disassembly of the SCF ubiquitin ligase complex by Cdc48/p97 
Molecular cell  2012;48(2):288-297.
A large group of E3 ubiquitin ligases is formed by the multisubunit SCF complex, whose core complex (Rbx1/Cul1-Cdc53/Skp1) binds one of many substrate recruiting F-box proteins to form an array of SCF ligases with diverse substrate specificities. It has long been thought that ubiquitylation by SCF ligases is regulated at the level of substrate binding. Here we describe an alternative mechanism of SCF regulation by active dissociation of the F-box subunit. We show that cadmium stress induces selective recruitment of the AAA+ ATPase Cdc48/p97 to catalyze dissociation of the F-box subunit from the yeast SCFMet30 ligase to block substrate ubiquitylation and trigger downstream events. Our results not only provide an additional layer of ubiquitin ligase regulation but also suggest that targeted, signal-dependent dissociation of multisubunit enzyme complexes is an important mechanism in control of enzyme function.
PMCID: PMC3483439  PMID: 23000173

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