Skp1 (S-phase kinase-associated protein 1) is a core component of SCF ubiquitin ligases and mediates protein degradation, thereby regulating eukaryotic fundamental processes such as cell cycle progression, transcriptional regulation, and signal transduction. Among the four components of the SCF complexes, Rbx1 and Cullin form a core catalytic complex, an F-box protein acts as a receptor for target proteins, and Skp1 is an adaptor between one of the variable F-box proteins and Cullin. Whereas protists, fungi and some vertebrates have a single SKP1 gene, many animal and plant species possess multiple SKP1 homologs. It has been shown that the same Skp1 homolog can interact with two or more F-box proteins, and different Skp1 homologs from the same species sometimes can interact with the same F-box protein. Multiple Skp1 homologs from the same species have evolved at highly heterogeneous rates. Parametric bootstrap analyses suggest that the differences in evolutionary rate are so large that true phylogenies are not recoverable from the full data set. Only when the original data set is partitioned into sets of genes with slow, medium, and rapid rates of evolution and analyzed separately, are better resolved relationships observed. The slowly evolving Skp1 homologs, which are relatively highly conserved in sequence and expressed widely and/or at high levels, usually have very low dN/dS values, suggesting that they have evolved under functional constraint and serve the most fundamental function(s). In contrast, the rapidly evolving members are structurally more diverse and usually have limited expression patterns and higher dN/dS values, suggesting that they may have evolved under relaxed or altered constraint, or even under positive selection. Some rapidly evolving members may have lost their original function(s) and/or acquired new function(s), or become pseudogenes, as suggested by their expression patterns, dN/dS values, and amino acid changes at key positions. In addition, these analyses revealed several monophyletic groups within the SKP1 gene family, one for each of protists, fungi, animals and plants, as well as nematodes, arthropods and angiosperms, suggesting that the extant SKP1 genes within each of these eukaryote groups shared only one common ancestor (Kong, 2003).
The yeast and human SKP1 genes regulate the mitotic cell cycle but are not yet known to be required for meiosis. Nine Arabidopsis SKP1 homologs have been uncovered and were named ASK1 through ASK9. A male sterile Arabidopsis mutant has been isolated and characterized that was caused by a Ds transposon insertion into the ASK1 gene. In the ask1-1 mutant, abnormal microspores exhibit a range of sizes. Furthermore, during mutant male meiosis, although homologous chromosome pairing appears normal at metaphase I, chromosome segregation at anaphase I is unequal, and some chromosomes are abnormally extended. Therefore, in ask1-1, at least some homologs remain associated after metaphase I. In addition, immunofluorescence microscopy indicates that the mutant spindle morphology at both metaphase I and early anaphase I is normal; thus, the abnormal chromosome segregation is not likely caused by a spindle defect. Because the yeast Skp1p is required for targeting specific proteins for ubiquitin-mediated proteolysis, it is proposed that ASK1 controls homolog separation by degrading or otherwise removing a protein that is required directly or indirectly for homolog association before anaphase I (Yang, 1999).
The infection of plants by Agrobacterium tumefaciens leads to the formation of crown gall tumors due to the transfer of a nucleoprotein complex into plant cells that is mediated by the virulence (vir) region-encoded transport system. In addition, A. tumefaciens secretes the Vir proteins, VirE2 and VirF, directly into plant cells via the same VirB/VirD4 transport system, and both assist there in the transformation of normal cells into tumor cells. The function of the 22 kDa VirF protein is not clear. Deletion of the virF gene in A. tumefaciens leads to diminished virulence and can be complemented by the expression of the virF gene in the host plant. This finding indicates that VirF functions within the plant cell. The VirF protein is the first prokaryotic protein with an F box; it can interact via its F box with plant homologs of the yeast Skp1 protein. The presence of the F box turns out to be essential for the biological function of VirF. F box proteins and Skp1p are both subunits of a class of E3 ubiquitin ligases referred to as SCF complexes. Thus, VirF may be involved in the targeted proteolysis of specific host proteins in early stages of the transformation process (Schrammeijer, 2001).
The yeast Skp1 protein is a component of the SCF complex, an E3 enzyme involved in the specific protein degradation pathway via ubiquitination. Skp1 binds to F-box proteins to trigger specific recognition of proteins targeted for degradation. SKP1-like genes have been found in a variety of eukaryotes including yeast, man, Caenorhabditis elegans and Arabidopsis thaliana. The Arabidopsis genome contains 20 SKP1-like genes called ASK (for Arabidopsis SKP1-like), among which only ASK1 has been characterized in detail. The analysis of the expression pattern of the ASK genes in Arabidopsis should provide key information for the understanding of the biological role of this family in protein degradation and in different cellular mechanisms. The expression profiles are described of 19 ASK promoter-GUS fusions in stable transformants of Arabidopsis, with a special emphasis on floral organ development. Four ASK promoters did not show any detectable expression in either inflorescences or seedlings. These results on the ASK1 expression profile are consistent with previous reports. Several ASK promoters show clear tissue-specific expression (for instance in the connective of anthers or in the embryo). Almost half (9/19) of ASK promoters direct a post-meiotic expression in the male gametophyte. Tight regulation of the expression of this gene family indicates a crucial role of the ubiquitin degradation pathway during development, particularly during male gametophyte development (Marrocco, 2003).
Ubiquitin-mediated proteolysis by the proteasome is a critical regulatory mechanism controlling many biological processes. In particular, SKP1, cullin/CDC53, F-box protein (SCF) complexes play important roles in selecting substrates for proteolysis by facilitating the ligation of ubiquitin to specific proteins. In plants, SCF complexes have been found to regulate auxin responses and jasmonate signaling and may be involved in several other processes, such as flower development, circadian clock, and gibberellin signaling. Although 21 Skp1-related genes, called Arabidopsis-SKP1-like (ASK), have been uncovered in the Arabidopsis genome, ASK1 is the only gene that has been analyzed genetically. As a first step toward understanding their functions, expression of 20 ASK genes was tested using reverse transcription-polymerase chain reaction experiments. Also, the expression patterns of 11 ASK genes were examined by in situ hybridizations. The ASK genes exhibit a spectrum of expression levels and patterns, with a large subset showing expression in the flower and/or fruit. In addition, the ASK genes that have similar sequences tend to have similar expression patterns. On the basis of the expression results, the expression of a few ASK genes was selectively suppressed using RNA interference. Compared with the ask1 mutant, the strong ASK1 RNA interference (RNAi) line exhibits similar or enhanced phenotypes in both vegetative and floral development, whereas ASK11 RNAi plants had normal vegetative growth but mild defects in flower development. The diverse expression patterns and distinct defects observed in RNAi plants suggest that the ASK gene family may collectively perform a range of functions and may regulate different developmental and physiological processes (Zhao, 2003).
Posttranslational modification of a protein by ubiquitin usually results in rapid degradation of the ubiquitinated protein by the proteasome. The transfer of ubiquitin to substrate is a multistep process. Cdc4p is a component of a ubiquitin ligase that tethers the ubiquitin-conjugating enzyme Cdc34p to its substrates. Among the domains of Cdc4p that are crucial for function are the F-box, which links Cdc4p to Cdc53p through Skp1p, and the WD-40 repeats, which are required for binding the substrate for Cdc34p. In addition to Cdc4p, other F-box proteins, including Grr1p and Met30p, may similarly act together with Cdc53p and Skp1p to function as ubiquitin ligase complexes. Because the relative abundance of these complexes, known collectively as SCFs, is important for cell viability, evidence of mechanisms that modulate F-box protein regulation have been sought. The abundance of Cdc4p is subject to control by a peptide segment that has been termed the R-motif (for "reduced abundance"). Binding of Skp1p to the F-box of Cdc4p inhibits R-motif-dependent degradation of Cdc4p. These results suggest a general model for control of SCF activities (Mathias, 1999).
SCF ubiquitin ligases are composed of Skp1, Cdc53, Hrt1 and one member of a large family of substrate receptors known as F-box proteins (FBPs). Using sequential rounds of epitope tagging, affinity purification and mass spectrometry, 16 Skp1 and Cdc53-associated proteins have been identified in budding yeast, including all components of SCF, 9 FBPs, Yjr033 (Rav1) and Ydr202 (Rav2). Rav1, Rav2 and Skp1 form a complex named here 'regulator of the (H+)-ATPase of the vacuolar and endosomal membranes' (RAVE), which associates with the V1 domain of the vacuolar membrane (H+)-ATPase (V-ATPase). V-ATPases are conserved throughout eukaryotes, and have been implicated in tumor metastasis and multidrug resistance; this study shows that RAVE promotes glucose-triggered assembly of the V-ATPase holoenzyme. Previous systematic genome-wide two-hybrid screens yielded 17 proteins that interact with Skp1 and Cdc53, only 3 of which overlap with those reported here. Thus, these results provide a distinct view of the interactions that link proteins into a comprehensive cellular network (Seol, 2001).
Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin-ligases composed of a core complex including Skp1p, Cdc53p, Hrt1p, the E2 enzyme Cdc34p, and one of multiple F-box proteins which are thought to provide substrate specificity to the complex. F-box protein Rcy1p is required for recycling of the v-SNARE Snc1p in Saccharomyces cerevisiae. Rcy1p localizes to areas of polarized growth, and this polarized localization requires its CAAX box and an intact actin cytoskeleton. Rcy1p interacts with Skp1p in vivo in an F-box-dependent manner, and both deletion of its F box and loss of Skp1p function impair recycling. In contrast, cells deficient in Cdc53p, Hrt1p, or Cdc34p do not exhibit recycling defects. Unlike the case for F-box proteins that are known to participate in SCF complexes, degradation of Rcy1p requires neither its F box nor functional 26S proteasomes or other SCF core subunits. Importantly, Skp1p is the only major partner that copurifies with Rcy1p. These results thus suggest that a complex composed of Rcy1p and Skp1p but not other SCF components may play a direct role in recycling of internalized proteins (Galan, 2001).
Binding of CBF3, a protein complex consisting of Ndc10p, Cep3p, Ctf13p, and Skp1p, to the centromere DNA nucleates kinetochore formation in budding yeast. This study investigates how the Ctf13p/Skp1p complex becomes competent to form the CBF3-centromere DNA complex. As revealed by mass spectrometry, Ctf13p and Skp1p carry two and four phosphate groups, respectively. Complete dephosphorylation of Ctf13p and Skp1p does not interfere with the formation of CBF3-centromere DNA complexes in vitro. Furthermore, deletion of corresponding phosphorylation sites results in viable cells. Thus, in contrast to the current view, phosphorylation of Ctf13p and Skp1p is not essential for the formation of CBF3-centromere DNA complexes. Instead, the formation of active Ctf13p/Skp1p requires Hsp90. Several lines of evidence support this conclusion: activation of heterologous Ctf13p/Skp1p by reticulocyte lysate is inhibited by geldanamycin and Hsp90 depletion. skp1 mutants exhibit growth defects on media containing geldanamycin. A skp1 mutation together with Hsp90 mutations exhibits synthetic lethality. An Hsp90 mutant contains decreased levels of active Ctf13p/Skp1p (Stemmann, 2002).
SCF complexes are a conserved family of ubiquitin-ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity. The F-box motif links the F-box protein to the core components via its interaction with Skp1p. In yeast, the SCFMet30p complex contains the Met30p F-box protein and regulates Met4p, a transcription factor that mediates sulfur fixation and methionine biosynthesis. Although a nuclear protein, Met30p lacks a definable nuclear localization sequence. The entire amino terminal half of Met30p is required for its proper nuclear localization. Mutations in the F-box, but not mutations in Skp1p, affect Met30p nuclear localization, indicating that the F-box motif plays an important role in Met30p trafficking independent of its interaction with Skp1p binding. Met30p mutants that poorly localize to the nucleus display increased nuclear to cytoplasmic exchange, indicating that the amino terminus mediates nuclear retention in addition to nuclear import. The Met30p F-box motif, residues 180-225, is necessary and sufficient to bind Skp1p, however, mutations upstream of the Met30p F-box inhibit Skp1p binding. It is proposed that additional factors bind the amino terminal region of Met30p and mediate its nuclear localization and assimilation into an SCF complex (Brunson, 2003).
The SCF ubiquitin-ligase complex targets the ubiquitin-mediated degradation of proteins in multiple dynamic cellular processes. A key SCF component is the Skp1 protein that functions within the complex to link the substrate-recognition subunit to a cullin that in turn binds the ubiquitin-conjugating enzyme. In contrast to yeast and humans, Caenorhabditis elegans contains multiple expressed Skp1-related (skr) genes. The 21 Skp1-related (skr) genes in C. elegans form one phylogenetic clade, suggesting that a single ancestral Skp1 gene underwent independent expansion in C. elegans. The cellular and developmental functions of the 21 C. elegans skr genes were probed by dsRNA-mediated gene inactivation (RNAi). The RNAi phenotypes of the skr genes fall into two classes: (1) the highly similar skr-7, -8, -9, and -10 genes are required for posterior body morphogenesis, embryonic and larval development, and cell proliferation; (2) the related skr-1 and -2 genes are required for the restraint of cell proliferation, progression through the pachytene stage of meiosis, and the formation of bivalent chromosomes at diakinesis. CUL-1 was found to interact with SKR-1, -2, -3, -7, -8, and -10 in the yeast two-hybrid system. Interestingly, SKR-3 could interact with both CUL-1 and its close paralog CUL-6. It is concluded that members of the expanded skr gene family in C. elegans perform critical functions in regulating cell proliferation, meiosis, and morphogenesis. The finding that multiple SKRs are able to bind cullins suggests an extensive set of combinatorial SCF complexes (Nayak, 2002).
The ubiquitin-proteasome pathway of proteolysis controls the abundance of specific regulatory proteins. The SCF complex is a type of ubiquitin-protein ligase (E3) that contributes to this pathway in many biological systems. In yeast and mammals, the SCF complex consists of common components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Whereas only one functional Skp1 gene is present in the human genome, the genome of Caenorhabditis elegans has now been shown to contain at least 21 Skp1-related (skr) genes. The biochemical properties, expression, and function of the C. elegans SKR proteins were examined. Of the 17 SKR proteins examined, eight (SKR-1, -2, -3, -4, -7, -8, -9, and -10) were shown to interact with C. elegans CUL1 by yeast two-hybrid analysis or a coimmunoprecipitation assay in mammalian cells. Furthermore, SKR proteins exhibit diverse binding specificities for C. elegans F-box proteins. The tissue specificity of expression of the CUL1-interacting SKR proteins is also varied. Suppression of skr-1 or skr-2 genes by double-stranded RNA interference results in embryonic death, whereas that of skr-7, -8, -9, or -10 is associated with slow growth and morphological abnormalities. It is concluded that the multiple C. elegans SKR proteins exhibit marked differences in their association with Cullins and F-box proteins, in tissue specificity of expression, and in phenotypes associated with functional suppression by RNAi. At least eight of the SKR proteins may, like F-box proteins, act as variable components of the SCF complex in C. elegans (Yamanaka, 2002).
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 homolog 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).
F-box proteins are members of a large family that regulates the cell cycle, the immune response, signalling cascades and developmental programs by targeting proteins, such as cyclins, cyclin-dependent kinase inhibitors, IkappaBalpha and beta-catenin, for ubiquitination. F-box proteins are the substrate-recognition components of SCF (Skp1-Cullin-F-box protein) ubiquitin-protein ligases. They bind the SCF constant catalytic core by means of the F-box motif interacting with Skp1, and they bind substrates through their variable protein-protein interaction domains. The large number of F-box proteins is thought to allow ubiquitination of numerous, diverse substrates. Most organisms have several Skp1 family members, but the function of these Skp1 homologs and the rules of recognition between different F-box and Skp1 proteins remain unknown. The crystal structure of the human F-box protein Skp2 bound to Skp1 is described. Skp1 recruits the F-box protein through a bipartite interface involving both the F-box and the substrate-recognition domain. The structure raises the possibility that different Skp1 family members evolved to function with different subsets of F-box proteins, and suggests that the F-box protein may not only recruit substrate, but may also position it optimally for the ubiquitination reaction (Schulman, 2000).
Selective protein degradation targeted by members of the F-box protein family plays pivotal roles in cell biology. It is widely accepted that an F-box protein directs substrate ubiquitination within a Skp1.CUL1.F-box protein.ROC1 (SCF-ROC1) E3 ubiquitin ligase complex. This assembly utilizes the CUL1 molecular scaffold, allowing the F-box protein to position its bound substrate for ubiquitination by a ROC1-recruited E2-conjugating enzyme. An alternative mechanism is described for assembling an F-box protein-based E3 complex through a previously uncharacterized cullin, CUL7, identified by mass spectrometry as a ROC1-interacting protein. CUL7 is a large polypeptide containing a cullin domain, which is responsible for ROC1 binding, and a DOC domain, which is also present in the anaphase-promoting complex. Remarkably, CUL7 assembles an SCF-ROC1-like E3 ubiquitin ligase complex consisting of Skp1, CUL7, the Fbx29 F-box protein, and ROC1. In contrast to CUL1 that binds Skp1 by itself, CUL7 interacts with the Skp1.Fbx29 complex, but not with Skp1 alone. Strikingly, CUL7 selectively interacts with Skp1.Fbx29 but not with Skp1.betaTRCP2 or Skp1.Skp2. Thus, CUL7 may define a previously uncharacterized, Fbx29-mediated, and ubiquitin-dependent proteolysis pathway (Dias, 2002).
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 (Zheng, 2002).
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 requires 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).
In Saccharomyces cerevisiae, the initiation of DNA replication and mitotic progression requires SKP1p function. SKP1p is an essential subunit of a newly identified class of E3 ubiquitin protein ligases, the SCF complexes, that catalyze ubiquitin-mediated proteolysis of key cell-cycle-regulatory proteins at distinct times in the cell cycle. SKP1p is also required for proper kinetochore assembly. Little is known about the corresponding human homolog, p19(SKP1), except that it is expressed throughout the cell cycle and that it too is a component of an S-phase-regulating SCF-E3 ligase complex. p19(SKP1) localizes to the centrosomes. Centrosome association occurs throughout the mammalian cell cycle, including all stages of mitosis. These findings suggest that p19(SKP1) is a novel component of the centrosome and the mitotic spindle, which, in turn, implies a physiological role of this protein in the regulation of one or more aspects of the centrosome cycle (Gstaiger, 1999).
Skp1 is involved in a variety of crucial cellular functions, among which the best understood is the formation, together with Cul1, of Skp1-cullin-F-box protein ubiquitin ligases. To investigate the role of Skp1, transgenic (Tg) mice were generated expressing a Cul1 deletion mutant (Cul1-N252) able to sequestrate and inactivate Skp1. In vivo interference with Skp1 function through expression of the Cul1-N252 mutant into the T-cell lineage results in lymphoid organ hypoplasia and reduced proliferation. Nonetheless, after a period of latency, Cul1-N252 Tg mice succumb to T-cell lymphomas with high penetrance (>80%). Both T-cell depletion and the neoplastic phenotype of Cul1-N252 Tg mice are largely rescued in Cul1-N252, Skp1 double-Tg mice, indicating that the effects of Cul1-N252 are due to a sequestration of the endogenous Skp1. Analysis of Cul1-N252 lymphomas demonstrates striking karyotype heterogeneity associated with c-myc amplification and c-Myc overexpression. The in vitro expression of the Cul1-N252 mutant causes a pleiotrophic phenotype, which includes the formation of multinucleated cells, centrosome and mitotic spindle abnormalities, and impaired chromosome segregation. These findings support a crucial role for Skp1 in proper chromosomal segregation, which is required for the maintenance of euploidy and suppression of transformation (Piva, 2002).
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).
beta-catenin plays an essential role in the Wingless/Wnt signaling cascade and is a component of the cadherin cell adhesion complex. Deregulation of beta-catenin accumulation as a result of mutations in adenomatous polyposis coli (APC) tumor suppressor protein is believed to initiate colorectal neoplasia. beta-catenin levels are regulated by the ubiquitin-dependent proteolysis system and beta-catenin ubiquitination is preceded by phosphorylation of its N-terminal region by the glycogen synthase kinase-3beta (GSK-3beta)/Axin kinase complex. FWD1 (the mouse homolog of Slimb/betaTrCP: see Drosophila supernumerary limbs), an F-box/WD40-repeat protein, specifically forms a multi-molecular complex with beta-catenin, Axin, GSK-3beta and APC. Mutations at the signal-induced phosphorylation site of beta-catenin inhibit beta-catenin association with FWD1. FWD1 facilitates ubiquitination and promotes degradation of beta-catenin, resulting in reduced cytoplasmic beta-catenin levels. In contrast, a dominant-negative mutant form of FWD1 inhibits the ubiquitination process and stabilizes beta-catenin. These results suggest that the Skp1/Cullin/F-box protein FWD1 (SCFFWD1)-ubiquitin ligase complex is involved in beta-catenin ubiquitination and that FWD1 serves as an intracellular receptor for phosphorylated beta-catenin. FWD1 also links the phosphorylation machinery to the ubiquitin-proteasome pathway to ensure prompt and efficient proteolysis of beta-catenin in response to external signals. SCFFWD1 may be critical for tumor development and suppression through regulation of beta-catenin protein stability (Kitagawa, 1999).
Destruction of ß-catenin is regulated through phosphorylation-dependent interactions with the F box protein ß-TrCP. A novel pathway for ß-catenin degradation was discovered involving mammalian homologs of Drosophila Sina (Siah), which bind ubiquitin ß-conjugating enzymes, and Ebi, an F box protein that binds ß-catenin independent of the phosphorylation sites recognized by ß-TrCP. A series of protein interactions were identified in which Siah is physically linked to Ebi by association with a novel Sgt1 homolog SIP that binds Skp1, a central component of Skp1-Cullin-F box complexes. Expression of Siah is induced by p53, revealing a way of linking genotoxic injury to destruction of ß-catenin, thus reducing activity of Tcf/LEF transcription factors and contributing to cell cycle arrest (Matsuzawa, 2001).
A pathway linking the RING protein Siah-1 to the F box protein Ebi has been mapped and it has been shown that Ebi can bind ß-catenin. Unlike ß-TrCP, however, which requires GSK3ß-mediated phosphorylation of ß-catenin on serine 33 and serine 37, Ebi interacts with ß-catenin independently of these phosphorylation sites. Also, the Siah binding protein SIP associates with complexes containing Ebi but not ß-TrCP, suggesting differences compared to previously characterized E3 ubiquitin ligase complexes, where E2 enzymes are supplied via Cullin-mediated interactions with RING-containing proteins such as Rbx-1/Roc-1. Recent identification of interactions between Siah-1 and the ß-catenin binding protein APC suggest that this scaffold protein represents a point of common intersection of the Wnt and Siah-1 pathways for ß-catenin degradation (Matsuzawa, 2001).
Two alternative pathways for regulation of ß-catenin levels are presented, involving different F box proteins (Ebi versus ß-TrCP). One pathway is initiated by increases in the expression of Siah-family proteins, which can be induced, for example, by p53 in response to DNA damage, and involves sequential protein interactions with SIP, Skp1, and Ebi. Ebi binds ß-catenin, thus recruiting it to the Siah-1-SIP-Skp1 complex for polyubiquitination and subsequent proteosome-mediated degradation. Siah-1 binds the E2 UbcH5. The other pathway is regulated by Wnt signals (Dsh) and possibly PI3K/Akt. This pathway is phosphorylation dependent and involves GSK3ß-induced phosphorylation of Ser-33 and Ser-37 on ß-catenin, allowing ß-TrCP binding, resulting in recruitment of ß-catenin to Skp1-Cullin-1- ß-TrCP complexes (SCF). Cullin-1, in collaboration with other proteins, supplies this SCF complex with E2s, such as UbcH3. APC is required for both pathways as a scaffold protein, binding ß-catenin via one domain and also binding Siah-1 and GSK3ß (Matsuzawa, 2001).
In the fly, Sina recruits E2s to Phyllopod/Tramtrack complexes, targeting Tramtrack for ubiquitination. The ebi-gene product also binds Tramtrack and promotes its degradation in vitro and when expressed in insect cells in culture. Loss-of-function mutations of ebi cause Tramtrack accumulation and prevent R7 cell differentiation. Similar to ß-TrCP, the ebi gene of Drosophila encodes an F box/WD-40-repeat protein with sequence homology to Cdc4 (yeast), Sel-10 (C. elegans), and Slimb (Drosophila), suggesting that it provides a functional connection between a Sina-regulated pathway and SCF complexes. How this linkage between Sina and SCF complexes is achieved, however, has been unclear (Matsuzawa, 2001).
The finding that SIP functions as a molecular bridge between the human homologs of Sina and the SCF-component Skp1 provides evidence of a physical linkage between components of these two ubiquitin ligase systems, thus corroborating the genetic evidence from Drosophila that these two pathways for targeted protein degradation interact. The Drosophila ortholog of SIP is also capable of bridging the fly Skp1 and Sina proteins in three-hybrid experiments. Thus, an evolutionarily conserved network of protein interactions exists in which Siah-1 (Sina) binds to SIP, which in turn binds to Skp1, which binds Ebi (Matsuzawa, 2001).
p53 can induce expression of Siah-family genes in mammals, establishing p53 as one factor capable of invoking Siah-dependent pathways for protein degradation. Siah-family proteins are normally maintained at a relatively low level through ubiquitination-dependent protein turnover, where human Siah-1 and Siah-2 promote their own degradation through interactions of their RING domains with E2s. This therefore suggests that activation of p53 leads to a burst of Siah-1 mRNA and protein production, triggering the Siah/SIP/Skp1/Ebi pathway for ß-catenin degradation. In contrast to Siah-family proteins, it seems unlikely that SIP, Skp1, or Ebi are limiting components of this pathway, since overexpression of them has little effect on ß-catenin levels (Matsuzawa, 2001).
Though p53-mediated degradation of ß-catenin correlates with cell cycle arrest, it remains to be established whether these events are functionally linked. Activation of Tcf/LEF-family transcription factors by ß-catenin is known to induce expression of cyclin D1, c-myc, and other genes important for cell proliferation, making it plausible that ß-catenin degradation is linked to p53-mediated cell cycle arrest. However, given the role established for the cyclin-dependent kinase inhibitor p21Waf1 in mediating G1 arrest induced by p53, it is unclear whether a parallel pathway for ß-catenin degradation would be required. Circumstances have been described where p53 fails to induce cell cycle arrest despite inducing p21Waf1 expression, raising the question of whether p21Waf1 is necessary but insufficient for p53-mediated G1 arrest. Recently, a genetic interaction between ebi and p21Waf1 has been identified using an assay in Drosophila where flies are engineered to ectopically express human p21Waf1 in the developing eye disc (Boulton, 2000). Specifically, mutant alleles of ebi abrogate inhibition of S phase entry by p21Waf1, implying a need for Ebi in p21-mediated cell cycle arrest. Flies with mutant ebi also display ectopic S phases and overproliferation phenotypes (Boulton, 2000), further implying a role for ebi in growth suppression. Defects in cell cycle arrest in ebi mutants, however, do not necessarily implicate ß-catenin/Armadillo. For example, p53 can induce degradation of c-Myb through a proteosome-dependent mechanism partly mediated by Siah (Tanikawa, 2000). Thus, Ebi may have other targets in addition to ß-catenin that are relevant to mechanisms of p53-mediated cell cycle arrest. Future experiments should explore whether the fly homolog of p53 is linked to an ebi-dependent pathway for cell cycle arrest entailing degradation of Armadillo. In the M1 cell model, p53 induces both G1 arrest and apoptosis. Though Ebi(DeltaF)-expressing M1 cells may exhibit some delay in p53-induced apoptosis, this could result indirectly because of failed G1 arrest. Moreover, Siah-1 often fails to induce apoptosis when overexpressed in cells. However, links of Siah to apoptosis can occur under some circumstances, as demonstrated by the observation that coexpression of Siah-1 with a Siah binding protein Pw1/Peg3 causes apoptosis, whereas neither Siah-1 nor Pw1/Peg3 alone are sufficient. Mutations affecting components of the Wnt-signaling pathway are commonly observed in human cancers, resulting in aberrant accumulation of ß-catenin and activation of Tcf/LEF-target genes. Wnt-family ligands, frizzled-family receptors, and the signaling proteins downstream of these define one mechanism for regulating ß-catenin levels. However, additional inputs into pathways controlling ß-catenin turnover have recently been identified, including a mitogen-activated protein kinase pathway involving a Tak1 homolog and Nemo-like kinases in C. elegans and a cell adhesion-dependent pathway involving integrin-linked kinase. The findings reported here reveal yet another pathway for regulating ß-catenin levels that is linked at least in part to p53-dependent responses to genotoxic injury. It is speculated that loss of p53 or components of the Siah/SIP/Skp/Ebi pathway for ß-catenin destruction may contribute to aberrant ß-catenin accumulation in cancers (Matsuzawa, 2001).
The SCF ubiquitin ligases catalyze protein ubiquitination in diverse cellular processes. SCFs bind substrates through the interchangeable F box protein subunit, with the >70 human F box proteins, allowing the recognition of a wide range of substrates. The F box protein β-TrCP1 recognizes the doubly phosphorylated DpSGφXpS destruction motif, present in β-catenin and IκB, and directs the SCFβ-TrCP1 to ubiquitinate these proteins at specific lysines. The 3.0 Å structure of a β-TrCP1-Skp1-β-catenin complex reveals the basis of substrate recognition by the β-TrCP1 WD40 domain. The structure, together with the previous SCFSkp2 structure, leads to the model of SCF catalyzing ubiquitination by increasing the effective concentration of the substrate lysine at the E2 active site. The model's prediction that the lysine-destruction motif spacing is a determinant of ubiquitination efficiency is confirmed by measuring ubiquitination rates of mutant β-catenin peptides, solidifying the model and also providing a mechanistic basis for lysine selection (Wu, 2003).
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