SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. The crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex is presented; Cul1 is shown to be an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1-F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1-F boxSkp2 and Rbx1 subunits, holding them over 100 Å apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold (N. Zheng, 2002).
SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins copurifying with Cdc53 identified the RING-H2 finger protein Hrt1 as a subunit of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex. Conditional inactivation of hrt1(ts) results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S. Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1Delta, and human HRT2 binds CUL-1. It is concluded that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases (Seol, 1999).
The SCF complex containing Skp1, Cul1, and the F-box protein FWD1 (the mouse homolog of Drosophila Slimb and Xenopus beta-TrCP) functions as the ubiquitin ligase for IkappaBalpha. FWD1 associates with Skp1 through the F-box domain and also recognizes the conserved DSGXXS motif of IkappaBalpha. The structural requirements for the interactions of FWD1 with IkappaBalpha and with Skp1 have now been investigated further. The D31A mutation (but not the G33A mutation) in the DSGXXS motif of IkappaBalpha abolishes the binding of IkappaBalpha to FWD1 and its subsequent ubiquitination without affecting the phosphorylation of IkappaBalpha. The IkappaBalpha mutant D31E still exhibits binding to FWD1 and undergoes ubiquitination. These results suggest that, in addition to site-specific phosphorylation at Ser(32) and Ser(36), an acidic amino acid at position 31 is required for FWD1-mediated ubiquitination of IkappaBalpha. Deletion analysis of Skp1 reveals that residues 61-143 of this protein are required for binding to FWD1. In contrast, the highly conserved residues Pro(149), Ile(160), and Leu(164) in the F-box domain of FWD1 are dispensable for binding to Skp1. Together, these data delineate the structural requirements for the interactions among IkappaBalpha, FWD1, and Skp1 that underlie substrate recognition by the SCF ubiquitin ligase complex (Hattori, 1999).
Ubiquitin-conjugation targets numerous cellular regulators for proteasome-mediated degradation. Thus, the identification of ubiquitin ligases and their physiological substrates is crucially important, especially for those cases in which aberrant levels of regulatory proteins (e.g., beta-catenin, p27) result from a deregulated ubiquitination pathway. In yeast, the proteolysis of several G1 regulators is controlled by ubiquitin ligases (or SCFs) formed by three subunits: Skp1, Cul A (Cdc53), and one of many F-box proteins. Specific F-box proteins (Fbps) recruit different substrates to the SCF. Although many Fbps have been identified in mammals, their specific substrates and the existence of multiple SCFs have not yet been reported. One human Fbp, beta-Trcp (beta-Transducin repeat containing protein), does indeed form a novel SCF with human Skp1 and Cul1. Consistent with recent reports indicating that Xenopus and Drosophila beta-Trcp homologs act as negative regulators of the Wnt/beta-catenin signaling pathway, human beta-Trcp interacts with beta-catenin in vivo. Furthermore, beta-catenin is specifically stabilized in vivo by the expression of a dominant negative beta-Trcp. These results indicate that the Cul1/Skp1/beta-Trcp complex forms a ubiquitin ligase that mediates the degradation of beta-catenin (Latres, 1999).
Cyclin E binds and activates the cyclin-dependent kinase Cdk2 and catalyzes the transition from the G1 phase to the S phase of the cell cycle. The amount of cyclin E protein present in the cell is tightly controlled by ubiquitin-mediated proteolysis. The ubiquitin ligase responsible for cyclin E ubiquitination has been identified as SCFFbw7; it is functionally conserved in yeast, flies, and mammals. Fbw7 associates specifically with phosphorylated cyclin E, and SCFFbw7 catalyzes cyclin E ubiquitination in vitro. Depletion of Fbw7 leads to accumulation and stabilization of cyclin E in vivo in human and Drosophila melanogaster cells. Multiple F-box proteins contribute to cyclin E stability in yeast, suggesting an overlap in SCF E3 ligase specificity that allows combinatorial control of cyclin E degradation (Koepp, 2001).
The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by catalyzing ubiquitination of the S phase cyclin-dependent kinase inhibitor SIC1. SCF is composed of three proteins -- ySKP1, CDC53 (Cullin), and the F-box protein CDC4 -- that are conserved from yeast to humans. As part of an effort to identify components and substrates of a putative human SCF complex, hSKP1 was isolated in a two-hybrid screen with hCUL1, the closest human homologue of CDC53. hCUL1 associates with hSKP1 in vivo and directly interacts with both hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-like particle. Moreover, hCUL1 complements the growth defect of yeast cdc53(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. Taken together, these data suggest that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. Further application of biochemical assays similar to those described here can now be used to identify regulators/components of hCUL1-based SCF complexes, to determine whether the hCUL2-hCUL5 proteins also are components of ubiquitin ligase complexes in human cells, and to screen for chemical compounds that modulate the activities of the hSKP1 and hCUL1 proteins (Lyapina, 1998).
Activation of the transcription factor NF-kappaB in response to proinflammatory stimuli requires the phosphorylation-triggered and ubiquitin-dependent degradation of the NF-kappaB inhibitor, IkappaB alpha. The phosphorylation-dependent ubiquitination of IkappaB alpha has been reconstituted in vitro with purified components. ROC1, a novel SCF-associated protein, is recruited by cullin 1 to form a quatemary SCFHOS-ROC1 holenzyme (with Skp1 and the beta-TRCP homolog HOS). SCFHOS-ROC1 binds IKK beta-phosphorylated IkappaB alpha and catalyzes its ubiquitination in the presence of ubiquitin, E1, and Cdc34. ROC1 plays a unique role in the ubiquitination reaction by heterodimerizing with cullin 1 to catalyze ubiquitin polymerization (Tan, 1999).
ROC1 is a common component of a large family of ubiquitin E3 ligases that regulate cell cycle progression and signal transduction pathways. Evidence is presented suggesting that a conserved RING-H2 structure within ROC1 is critical for its ubiquitin ligation function. Mercury-containing sulfhydryl modification agents (rho-hydroxymercuribenzoate and mercuric chloride) irreversibly inhibit the ROC1-CUL1 ubiquitin ligase activity without disrupting the complex. Consistent with this, these reagents also eliminate the ability of the Skp1-CUL1-HOS-ROC1 E3 ligase complex to support the ubiquitination of IkappaBalpha. Site-directed mutagenesis analysis identifies RING-H2 finger residues Cys(42), Cys(45), Cys(75), His(77), His(80), Cys(83), Cys(94), and Asp(97) as being essential for the ROC1-dependent ubiquitin ligase activity. Furthermore, C42S/C45S and H80A mutations reduce the ability of ROC1 to interact with CUL1 in transfected cells and diminish the capacity of ROC1-CUL1 to form a stable complex with Cdc34 in vitro. However, C75S, H77A, C94S, and D97A substitutions have no detectable effect on ROC1 binding activities. Thus, the ROC1 RING-H2 finger may possess multiple biochemical properties that include stabilizing an interaction with CUL1 and recruiting Cdc34. A possible role of the RING finger in facilitating the Ub transfer reaction is discussed (Chen, 2000).
A purified ubiquitination system is described capable of rapidly catalyzing the covalent linkage of polyubiquitin chains onto phosphorylated IkappaBalpha. The initial ubiquitin transfer and subsequent polymerization steps of this reaction require the coordinated action of Cdc34 and the SCF(HOS/beta-TRCP)-ROC1 E3 ligase complex, comprised of four subunits (Skp1, cullin 1 [CUL1], HOS/beta-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 IkappaB 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 (Wu, 2000a).
The sequential timing of cell-cycle transitions is primarily governed by the availability and activity of key cell-cycle proteins. Recent studies in yeast have identified a class of ubiquitin ligases (E3 enzymes) called SCF complexes, which regulate the abundance of proteins that promote and inhibit cell-cycle progression at the G1-S phase transition. SCF complexes consist of three invariable components -- Skp1, Cul-1 (Cdc53 in yeast) and Rbx1 -- and a variable F-box protein that recruits a specific cellular protein to the ubquitin pathway for degradation. To study the role of Cul-1 in mammalian development and cell-cycle regulation, mice deficient for Cul1 were generated and null embryos and heterozygous cell lines were analyzed. Cul1 is shown to be required for early mouse development and Cul1 mutants fail to regulate the abundance of the G1 cyclin, cyclin E (encoded by Ccne), during embryogenesis (Dealy, 1999).
The stability of many proteins is controlled by the ubiquitin proteolytic system, which recognizes specific substrates through the action of E3 ubiquitin ligases. The SCFs are a class of ubiquitin ligase that target a number of cell cycle regulators and other proteins for degradation in both yeast and mammalian cells. Each SCF complex is composed of the core protein subunits Skp1, Rbx1 and Cul1 (known as Cdc53 in yeast), and substrate-specific adaptor subunits called F-box proteins. To understand the physiological role of SCF complexes in mammalian cells, mice carrying a deletion in the Cul1 gene were generated. Cul1(-/-) embryos arrest around embryonic day 6.5 (E6.5) before the onset of gastrulation. In all cells of the mutant embryos, cyclin E protein, but not mRNA, is highly elevated. Outgrowths of Cul1(-/-) blastocysts have limited proliferative capacity in vitro and accumulate cyclin E in all cells. Within Cul1(-/-) blastocyst cultures, trophoblast giant cells continue to endocycle despite the elevated cyclin E levels. These results suggest that cyclin E abundance is controlled by SCF activity, possibly through SCF-dependent degradation of cyclin E (Wang, 1999).
The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through critical cell cycle transitions. Evidence is provided that c-Myc promotes ubiquitin-dependent proteolysis by directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCF(SKP2). The cell cycle inhibitor p27(kip1) is a known target of the SCF(SKP2) complex, and Myc-induced Cul1 expression matches well with the kinetics of declining p27(kip1) protein. Enforced Cul1 expression or antisense neutralization of p27(kip1) is capable of overcoming the slow-growth phenotype of c-Myc null primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone is able to restore p27(kip1) ubiquitination and degradation in lysates derived from c-myc(-/-) MEFs or density-arrested human fibroblasts. These functional and biochemical data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G(1) exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27 (O'Hagan, 2000).
Deregulation of cell proliferation is a hallmark of cancer. In many transformed cells, the cyclin A/CDK2 complex that contains S-phase kinase associated proteins 1 and 2 (SKP1 and SKP2) is highly induced. To determine the roles of this complex in the cell cycle regulation and transformation, the composition of this complex was analyzed. This complex contains an additional protein, human CUL-1, a member of the cullin/CDC53 family. The identification of CUL-1 as a member of the complex raises the possibility that the p19(SKP1)/p45(SKP2)/CUL-1 complex may function as the yeast SKP1-CDC53-F-box (SCF) protein complex that acts as a ubiquitin E3 ligase to regulate the G1/S transition. In mammalian cells, cyclin D, p21(CIP1/WAF1), and p27(KIP1) are short-lived proteins that are controlled by ubiquitin-dependent proteolysis. To determine the potential in vivo targets of the p19(SKP1)/p45(SKP2)/CUL-1 complex, the specific antisense oligodeoxynucleotides were used against either SKP1, SKP2, or CUL-1 RNA to inhibit their expression. Treatment of cells with these oligonucleotides causes the selective accumulation of p21 and cyclin D proteins. The protein level of p27 is not affected. These data suggest that the human p19(SKP1)/p45(SKP2)/CUL-1 complex is likely to function as an E3 ligase to selectively target cyclin D and p21 for the ubiquitin-dependent protein degradation. Aberrant expression of human p19(SKP1)/p45(SKP2)/CUL-1 complex thus may contribute to tumorigenesis by regulating the protein levels of G1 cell cycle regulators (Yu, 1998).
Centrosomes organize the mitotic spindle to ensure accurate segregation of the chromosomes in mitosis. The mechanism that ensures accurate duplication and separation of the centrosomes underlies the fidelity of chromosome segregation, but remains unknown. In Saccharomyces cerevisiae, entry into S phase and separation of spindle pole bodies each require CDC4 and CDC34, which encode components of an SCF (Skp1-cullin-F-box) ubiquitin ligase, but a direct (SCF) connection to the spindle pole body is unknown. Using immunofluorescence microscopy, it has been shown that in mammalian cells the Skp1 protein and the cullin Cul1 are localized to interphase and mitotic centrosomes and to the cytoplasm and nucleus. Deconvolution and immunoelectron microscopy suggest that Skp1 forms an extended pericentriolar structure that may function to organize the centrosome. Purified centrosomes also contain Skp1, and Cul1 modified by the ubiquitin-like molecule NEDD8, suggesting a role for NEDD8 in targeting. Using an in vitro assay for centriole separation in Xenopus extracts, antibodies to Skp1 or Cul1 block separation. Proteasome inhibitors block both centriole separation in vitro and centrosome duplication in Xenopus embryos. Candidate centrosomal F-box proteins have been identified, suggesting that distinct SCF complexes may direct proteolysis of factors mediating multiple steps in the centrosome cycle (Freed, 1999).
The periodic expression of cell cycle proteins is important for the regulation of cell cycle progression. The amount of CDK inhibitor, p27(kip1), one such protein, seems to be regulated by the ubiquitin-proteasome system. The ubiquitin ligase (E3) toward p27(kip1) is thought to be SCF(skp2). The activity of SCF(skp2) is increased by the addition of Roc1 protein to the complex. Furthermore, the ubiquitination of p27(kip1) seems to be dependent on the phosphorylation of T187 of p27(kip1) because the mutant T187A is not ubiquitinated at all in an in vitro ubiquitination system. Cullin-1, a component of SCF, is modified by ubiquitin-like protein Nedd8. The modification site of cullin-1 was shown to be K696; the K696R mutant is not modified. When the effect of the Nedd8 modification on the SCF(skp2) activity toward p27(kip1) was investigated, the activity was markedly decreased by using the Nedd8-unmodified mutant cullin-1 (K696R), indicating that the modification may play an important role on the SCF(skp2) activity toward p27(kip1) (Morimoto, 2001).
The Cdc25 dual-specificity phosphatases control progression through the eukaryotic cell division cycle by activating cyclin-dependent kinases. Cdc25A regulates entry into S-phase by dephosphorylating Cdk2, it cooperates with activated oncogenes in inducing transformation and is overexpressed in several human tumors. DNA damage or DNA replication blocks induce phosphorylation of Cdc25A and its subsequent degradation via the ubiquitin-proteasome pathway. The regulation of Cdc25 A in the cell cycle has been investigated. Cdc25A degradation during mitotic exit and in early G(1) is found to be mediated by the anaphase-promoting complex or cyclosome (APC/C)(Cdh1) ligase; a KEN-box motif in the N-terminus of the protein is required for its targeted degradation. Interestingly, the KEN-box mutated protein remains unstable in interphase and upon ionizing radiation exposure. Moreover, SCF (Skp1/Cullin/F-box) inactivation using an interfering Cul1 mutant accumulates and stabilizes Cdc25A. The presence of Cul1 and Skp1 in Cdc25A immunocomplexes suggests a direct involvement of SCF in Cdc25A degradation during interphase. A dual mechanism of regulated degradation is proposed that allows for fine tuning of Cdc25A abundance in response to cell environment (Donzelli, 2002).
The anaphase-promoting complex or cyclosome (APC) is an unusually complicated ubiquitin ligase, composed of 13 core subunits and either of two loosely associated regulatory subunits, Cdc20 and Cdh1. The architecture of the APC was analyzed using a recently constructed budding yeast strain that is viable in the absence of normally essential APC subunits. The largest subunit, Apc1, serves as a scaffold that associates independently with two separable subcomplexes, one that contains Apc2 (Cullin), Apc11 (RING), and Doc1/Apc10, and another that contains the three TPR subunits (Cdc27, Cdc16, and Cdc23). The three TPR subunits display a sequential binding dependency, with Cdc27 the most peripheral, Cdc23 the most internal, and Cdc16 between. Apc4, Apc5, Cdc23, and Apc1 associate interdependently, such that loss of any one subunit greatly reduces binding between the remaining three. Intriguingly, the cullin and TPR subunits both contribute to the binding of Cdh1 to the APC. Enzymatic assays performed with APC purified from strains lacking each of the essential subunits revealed that only cdc27Δ complexes retain detectable activity in the presence of Cdh1. This residual activity depends on the C-box domain of Cdh1, but not on the C-terminal IR domain, suggesting that the C-box mediates a productive interaction with an APC subunit other than Cdc27. The IR domain of Cdc20 is dispensable for viability, suggesting that Cdc20 can activate the APC through another domain. This study has provided an updated model for the subunit architecture of the APC (Thornton, 2006).
Genomic integrity is maintained by checkpoints that guard against undesired replication after DNA damage. CDT1, a licensing factor of the pre-replication complex (preRC), is rapidly proteolysed after UV- or gamma-irradiation. The preRC assembles on replication origins at the end of mitosis and during G1 to license DNA for replication in S phase. Once the origin recognition complex (ORC) binds to origins, CDC6 and CDT1 associate with ORC and promote loading of the MCM2-7 proteins onto chromatin, generating the preRC. Radiation-mediated CDT1 proteolysis is independent of ATM and CHK2 and can occur in G1-phase cells. Loss of the COP9-signalosome (CSN) or CUL4-ROC1 complexes completely suppresses CDT1 proteolysis. CDT1 is specifically polyubiquitinated by CUL4 complexes and the interaction between CDT1 and CUL4 is regulated in part by gamma-irradiation. This study reveals an evolutionarily conserved and uncharacterized G1 checkpoint that induces CDT1 proteolysis by the CUL4-ROC1 ubiquitin E3 ligase and CSN complexes in response to DNA damage (Higa, 2003).
Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. With SKP1-CUL1, the DDB1-CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically express DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevents UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. It is suggested that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4A(DDB1), in response to UV irradiation (Hu, 2004).
Regulation of NF-kappaB occurs through phosphorylation-dependent ubiquitination of IkappaBalpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkappaBalpha is carried out by a ubiquitin-ligase enzyme complex called SCF[beta(TrCP)] . Nedd8 modification of the Cul-1 component of SCF[beta(TrCP)] is important for function of SCF[beta(TrCP)] in ubiquitination of IkappaBalpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF[beta(TrCP)] , phosphorylated IkappaBalpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCF[beta(TrCP)] in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed betaTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkappaBalpha requires Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, since SCF[beta(TrCP)] containing a K720R mutant of Cul-1 only weakly supports IkappaBalpha ubiquitination compared to SCF[beta(TrCP)] containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF[beta(TrCP)] . These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkappaBalpha (Read, 2000).
p130 is a tumor suppressor of the pocket protein family whose expression is posttranscriptionally regulated and largely G0 restricted. The mechanism of down-regulation of p130 expression in proliferating cells was investigated. The decline of p130 expression as G0 cells reenter the cell cycle is due to a decrease in protein stability. The enhancement of p130 turnover in late G1 and S phase compared with G0 and early G1 phase is dependent on Cdk4/6-specific phosphorylation of p130 on Serine 672, and is independent of Cdk2 activity. The activity of the ubiquitin ligase complex Skp1-Cul1/Cdc53-F-box protein Skp2 [SCF(Skp2)] and the proteasome are necessary for p130 degradation. In vitro, recombinant Skp2 is able to bind hyperphosphorylated but not dephosphorylated p130. Furthermore, in vitro polyubiquitination of p130 by SCF(Skp2) is specifically dependent on phosphorylation of p130 on Serine 672. Thus, like the Cdk inhibitor p27(Kip1), p130 turnover is regulated by Cdk-dependent G1 phosphorylation leading to ubiquitin-dependent proteolysis (Tedesco, 2002).
Cullins (CULs) are subunits of a prominent class of RING ubiquitin ligases. Whereas the subunits and substrates of CUL1-associated SCF complexes and CUL2 ubiquitin ligases are well established, they are largely unknown for other cullin family members. S. pombe CUL3 (Pcu3p) forms a complex with the RING protein Pip1p and all three BTB/POZ domain proteins encoded in the fission yeast genome. The integrity of the BTB/POZ domain, which shows similarity to the cullin binding proteins SKP1 and elongin C, is required for this interaction. Whereas Btb1p and Btb2p are stable proteins, Btb3p is ubiquitylated and degraded in a Pcu3p-dependent manner. Btb3p degradation requires its binding to a conserved N-terminal region of Pcu3p that precisely maps to the equivalent SKP1/F box adaptor binding domain of CUL1. It is proposed that the BTB/POZ domain defines a recognition motif for the assembly of substrate-specific RING/cullin 3/BTB ubiquitin ligase complexes (Geyer, 2003).
These results identified BTB/POZ proteins as components of Pcu3p/Pip1p ubiquitin ligase complexes. Four pieces of evidence suggest that BTB/POZ domain proteins are functionally equivalent to the SKP1/F box adaptor dimers determining the substrate specificity of CUL1-associate SCF complexes: (1) all three BTB/POZ proteins present in the fission yeast genome interact with Pcu3p/Pip1p complexes; (2) BTB/POZ domains are structurally related to SKP1; (3) N-terminal residues invariably conserved in all CUL3 homologs, including Pcu3p, cluster in the same region of CUL1 that mediates its interaction with SKP1/F box adaptor dimers. Both the Btb3p/Pcu3p interaction and Pcu3p-dependent Btb3p degradation depend on the integrity of this conserved N-terminal region. (4) Btb3p is ubiquitylated in vitro in a Pcu3p-dependent manner, a finding reminiscent of CUL1-dependent ubiquitylation and degradation of F box proteins. Taken together, these findings strongly suggest that the BTB/POZ domain proteins ubiquitously present in eukaryotes define a family of substrate-specific adaptors for CUL3. Since fission yeast encodes three different BTB/POZ domain proteins, all of which interact with Pcu3p and Pip1p, it may form a minimum of three distinct RING/cullin 3/BTB complexes (Geyer, 2003).
The faithful segregation of chromosomes during meiosis is vital for sexual reproduction. Currently, little is known about the molecular mechanisms regulating the initiation and completion of meiotic anaphase. Inactivation of CUL-2, a member of the cullin family of ubiquitin ligases, delays or abolishes meiotic anaphase II with no effect on anaphase I, indicating differential regulation during the two meiotic stages. In cul-2 mutants, the cohesin REC-8 is removed from chromosomes normally during meiosis II and sister chromatids separate, suggesting that the failure to complete anaphase results from a defect in chromosome movement rather than from a failure to sever chromosome attachments. CUL-2 is required for the degradation of cyclin B1 in meiosis and inactivation of cyclin B1 partially rescued the meiotic delay in cul-2 mutants. In cul-2 mutants, the failure to degrade cyclin B1 precedes the metaphase II arrest. CUL-2 is also required for at least two aspects of embryonic polarity. The extended meiosis II in cul-2 mutants induces polarity reversals that include reversed orientation of polarity proteins, P granules, pronuclei migration and asymmetric cell division. Independent of its role in meiotic progression, CUL-2 is required to limit the initiation/spread of the polarity protein PAR-2 in regions distant from microtubule organizing centers. Inactivation of the leucine-rich repeat protein ZYG-11 (Drosophila homolog CG12084) produces meiotic and polarity reversal defects similar to those observed in cul-2 mutants, suggesting that the two proteins function in the same pathways (Liu, 2004).
The mechanisms that ensure coupling between meiotic cell cycle progression and subsequent developmental events, including specification of embryonic axes, are poorly understood. Here, zyg-11 and the cullin cul-2 promote the metaphase-to-anaphase transition and M phase exit at meiosis II in C. elegans. ZYG-11 acts with a CUL-2-based E3 ligase that is essential at meiosis II and that functions redundantly with the anaphase-promoting complex/cyclosome at meiosis I. The data also indicate that delayed M phase exit in zyg-11(RNAi) embryos is due to accumulation of the B type cyclin CYB-3. PAR proteins and P granules become polarized in an inverted manner during the meiosis II delay resulting from zyg-11 or cul-2 inactivation, and zyg-11 and cul-2 can regulate polarity establishment independent of a role in cell cycle progression. Furthermore, microtubules appear dispensable for ectopic polarity during the meiosis II delay in zyg-11(RNAi) embryos, as well as for AP polarity during the first mitotic cell cycle in wild-type embryos. These findings suggest a model in which a CUL-2-based E3 ligase promotes cell cycle progression and prevents polarity establishment during meiosis II, and in which the centrosome acts as a cue to polarize the embryo along the AP axis after exit from the meiotic cell cycle (Sonneville, 2004).
Members of the cullin and RING finger ROC protein families form heterodimeric complexes to constitute a potentially large number of distinct E3 ubiquitin ligases. The highly conserved C-terminal sequence in CUL1 is dually required, both for nuclear localization and for modification by NEDD8. Disruption of ROC1 binding impairs nuclear accumulation of CUL1 and decreases NEDD8 modification in vivo but has no effect on NEDD8 modification of CUL1 in vitro, suggesting that ROC1 promotes CUL1 nuclear accumulation to facilitate its NEDD8 modification. Disruption of NEDD8 binding has no effect on ROC1 binding, nor does it affect nuclear localization of CUL1, suggesting that nuclear localization and NEDD8 modification of CUL1 are two separable steps, with nuclear import preceding and required for NEDD8 modification. Disrupting NEDD8 modification diminishes the IkappaBalpha ubiquitin ligase activity of CUL1. These results identify a pathway for regulation of CUL1 activity-ROC1 and the CUL1 C-terminal sequence collaboratively mediate nuclear accumulation and NEDD8 modification, facilitating assembly of active CUL1 ubiquitin ligase. This pathway may be commonly utilized for the assembly of other cullin ligases (Furukawa, 2000).
The SCF-ROC1 ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase complex targets the ubiquitination and subsequent degradation of protein substrates required for the regulation of cell cycle progression and signal transduction pathways. ROC1-CUL1 is a core subassembly within the SCF-ROC1 complex, capable of supporting the polymerization of ubiquitin. The CUL1 subunit of the bacterially expressed, unmodified ROC1-CUL1 complex is conjugated with Nedd8 at Lys-720 by HeLa cell extracts or by a purified Nedd8 conjugation system (consisting of APP-BP1/Uba3, Ubc12, and Nedd8). This covalent linkage of Nedd8 to CUL1 is both necessary and sufficient to markedly enhance the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization. A mutation of Lys-720 to arginine in CUL1 eliminates the Nedd8 modification, abolishes the activation of the ROC1-CUL1 ubiquitin ligase complex, and significantly reduces the ability of SCF(HOS/beta)(-TRCP)-ROC1 to support the ubiquitination of phosphorylated IkappaBalpha. Thus, although regulation of the SCF-ROC1 action has been shown to preside at the level of recognition of a phosphorylated substrate, this study demonstrates that Nedd8 is a novel regulator of the efficiency of polyubiquitin chain synthesis and, hence, promotes rapid turnover of protein substrates (Wu, 2000b).
COP9 signalosome (CSN) cleaves the ubiquitin-like protein Nedd8 from the Cul1 subunit of SCF ubiquitin ligases. The Jab1/MPN domain metalloenzyme (JAMM) motif in the Jab1/Csn5 subunit (see Drosophila Csn5) was found to underlie CSN's Nedd8 isopeptidase activity. JAMM is found in proteins from archaea, bacteria, and eukaryotes, including the Rpn11 subunit of the 26S proteasome. Metal chelators and point mutations within JAMM abolished CSN-dependent cleavage of Nedd8 from Cul1, yet have little effect on CSN complex assembly. Optimal SCF activity in yeast and both viability and proper photoreceptor cell (R cell) development in Drosophila melanogaster required an intact Csn5 JAMM domain. It is proposed that JAMM isopeptidases play important roles in a variety of physiological pathways (Cope, 2002).
Cullin proteins assemble a large number of RING E3 ubiquitin ligases and regulate various physiological processes. Covalent modification of cullins by the ubiquitin-like protein NEDD8 activates cullin ligases through an as yet undefined mechanism. p120CAND1 selectively binds to unneddylated CUL1 and is dissociated by CUL1 neddylation. CAND1 forms a ternary complex with CUL1 and ROC1. CAND1 dissociates SKP1 from CUL1 and inhibited SCF ligase activity in vitro. Suppression of CAND1 in vivo increases the level of the CUL1-SKP1 complex. It is suggested that by restricting SKP1-CUL1 interaction, CAND1 regulates the assembly of productive SCF ubiquitin ligases, allowing a common CUL1-ROC core to be utilized by a large number of SKP1-F box-substrate subcomplexes (Liu, 2002).
With approximately 50 genes encoding F box proteins in mammals, the incorporation of individual F box proteins into the active CUL1 ligase complex has to be tightly controlled. CUL1 is expressed at a much lower concentration than SKP1 and F box proteins, indicating that it is a rate-limiting factor in the assembly of a large number of SCF ligases. While there is a clear necessity for tight control over the dynamic and destructive ubiquitin ligase complexes, all the previously characterized subunits of the SCF-ROC ligases (CUL1, SKP1, F box, and ROC) are involved in either catalytic activity or substrate recruitment. NEDD8 modification of CUL1 was the only factor known to play a role in regulating cullin ligases, but its mechanism remains elusive. CAND1 functions as a negative regulator of SCF activity and is regulated by neddylation on CUL1. By regulating the assembly of SCF ubiquitin ligases, CAND1 allows a large number of SKP1-F box-substrate subcomplexes to be utilized by the same CUL1-ROC core. CAND1 may also interact with and similarly regulate other cullins (Liu, 2002).
The SCF ubiquitin E3 ligase regulates ubiquitin-dependent proteolysis of many regulatory proteins such as p27(Kip1), IkappaB, and beta-catenin. A CUL1 binding protein, p120(CAND1), has been isolated. The majority of CUL1 is in a complex with CAND1 and ROC1 independent of SKP1 and F box protein SKP2. Both in vivo and in vitro, CAND1 prevents the binding of SKP1 and SKP2 to CUL1 while dissociation of CAND1 from CUL1 promotes the reverse reaction. Neddylation of CUL1 or the presence of SKP1 and ATP causes CAND1 dissociation. These data suggest that CAND1 regulates the formation of the SCF complex, and its dissociation from CUL1 is coupled with the incorporation of F box proteins into the SCF complex, causing their destabilization (J. Zheng, 2002).
Cullin family proteins organize ubiquitin ligase (E3) complexes to target numerous cellular proteins for proteasomal degradation. Neddylation, the process that conjugates the ubiquitin-like polypeptide Nedd8 to the conserved lysines of cullins, is essential for in vivo cullin-organized E3 activities. Deneddylation, which removes the Nedd8 moiety, requires the isopeptidase activity of the COP9 signalosome (CSN). This study shows that in cells deficient for CSN activity, cullin1 (Cul1) and cullin3 (Cul3) proteins are unstable, and that to preserve their normal cellular levels, CSN isopeptidase activity is required. It is further shown that neddylated Cul1 and Cul3 are unstable - as suggested by the evidence that Nedd8 promotes the instability of both cullins - and that the unneddylatable forms of cullins are stable. The protein stability of Nedd8 is also subject to CSN regulation and this regulation depends on its cullin-conjugating ability, suggesting that Nedd8-conjugated cullins are degraded en bloc. It is proposed that while Nedd8 promotes cullin activation through neddylation, neddylation also renders cullins unstable. Thus, CSN deneddylation recycles the unstable, neddylated cullins into stable, unneddylated ones, and promotes cullin-organized E3 activity in vivo (Wu, 2005).
The SCF ubiquitin ligase complex regulates diverse cellular functions by ubiquitinating numerous protein substrates. Cand1 (Cullin-associated and neddylation-dissociated protein: Drosophila homolog: CG5366), a 120 kDa HEAT repeat protein, forms a tight complex with the Cul1-Roc1 SCF catalytic core, inhibiting the assembly of the multisubunit E3 complex. The crystal structure of the Cand1-Cul1-Roc1 complex shows that Cand1 adopts a highly sinuous superhelical structure, clamping around the elongated SCF scaffold protein Cul1. At one end, a Cand1 ß hairpin protrusion partially occupies the adaptor binding site on Cul1, inhibiting its interactions with the Skp1 adaptor and the substrate-recruiting F box protein subunits. At the other end, two Cand1 HEAT repeats pack against a conserved Cul1 surface cleft and bury a Cul1 lysine residue, whose modification by the ubiquitin-like protein, Nedd8, is able to block Cand1-Cul1 association. Together with biochemical evidence, these structural results elucidate the mechanisms by which Cand1 and Nedd8 regulate the assembly-disassembly cycles of SCF and other cullin-dependent E3 complexes (Goldenberg, 2004).
The COP9/signalosome (CSN) is known to remove the stimulatory NEDD8 modification from cullins. The activity of the fission yeast cullins Pcu1p and Pcu3p is dramatically stimulated when retrieved from csn mutants but inhibited by purified CSN. This inhibition is independent of cullin deneddylation but mediated by the CSN-associated deubiquitylating enzyme Ubp12p, which forms a complex with Pcu3p in a CSN-dependent manner. In ubp12 mutants, as in csn mutants, Pcu3p activity is stimulated. CSN is required for efficient targeting of Ubp12p to the nucleus, where both cullins reside. Finally, the CSN/Ubp12p pathway maintains the stability of the Pcu1p-associated substrate-specific adaptor protein Pop1p. It is proposed that CSN/Ubp12p-mediated deubiquitylation creates an environment for the safe de novo assembly of cullin complexes by counteracting the autocatalytic destruction of adaptor proteins (Zhou, 2003).
The COP9 signalosome (CSN) is an evolutionarily conserved multiprotein complex that mediates the repression of photomorphogenesis in the dark in Arabidopsis through the degradation of transcription factors such as HY5 and HYH. CSN-mediated HY5 and HYH degradation also requires the activity of the putative E3 ubiquitin ligase (E3) component COP1 and the E2-conjugating enzyme variant COP10. CSN is also required for auxin responses mediated by the SCF-type E3 SCF(TIR1). To determine whether Arabidopsis CSN is required for E3-mediated processes in a more general manner, plants were generated with reduced E3 function by suppressing AtRBX1, an essential core subunit of SCF-type E3s. AtRBX1 transgenic plants share multiple phenotypes with CSN reduced-function plants, such as morphological defects and reduced responses to auxin, jasmonic acid, and cold stress, suggesting that CSN is required for multiple AtRBX1-mediated processes. Furthermore, mutants with defects in AXR1, a protein that had been described only as a regulator of SCF(TIR1) function, also is required for other E3-mediated processes and for the COP1/COP10/CSN-mediated repression of photomorphogenesis in the dark. It is concluded that CSN and AXR1 are of general importance for different pathways that are controlled by E3-mediated protein degradation (Schweichheimer, 2002).
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