supernumerary limbs

EVOLUTIONARY HOMOLOGS part 1/2 | part 2/2

Slimb homologs and protein degradation in yeast

In budding yeast, ubiquitination of the cyclin-dependent kinase (Cdk) inhibitor Sic1 is catalyzed by the E2 ubiquitin conjugating enzyme Cdc34 in conjunction with an E3 ubiquitin ligase complex composed of Skp1, Cdc53 and the F-box protein, Cdc4 (the SCFCdc4 complex). Skp1 binds a motif called the F-box; in turn, F-box proteins appear to recruit specific substrates for ubiquitination. Skp1 has been shown to interact with Cdc53 in vivo, and Skp1 bridges Cdc53 to three different F-box proteins: Cdc4, Met30, and Grr1. Cdc53 contains independent binding sites for Cdc34 and Skp1, suggesting it functions as a scaffold protein within an E2/E3 core complex. F-box proteins show remarkable functional specificity in vivo: Cdc4 is specific for degradation of Sic1; Grr1 is specific for degradation of the G1 cyclin Cln2, and Met30 is specific for repression of methionine biosynthesis genes. In contrast, the Cdc34-Cdc53-Skp1 E2/E3 core complex is required for all three functions. Combinatorial control of SCF complexes may provide a basis for the regulation of diverse cellular processes (Patton, 1998).

Ubiquitin-dependent degradation of regulatory proteins controls many cellular processes, including cell cycle progression, morphogenesis, and signal transduction. Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin ligases composed of a core complex, including Skp1p, Cdc53p, one of multiple F-box proteins that are thought to provide substrate specificity to the complex, and the ubiquitin-conjugating enzyme, Cdc34p. It is not understood how SCF complexes are regulated and how physiological conditions alter their levels. Three F-box proteins, Grr1p, Cdc4p, and Met30p, are unstable components of the SCF, and are themselves degraded in a ubiquitin- and proteasome-dependent manner in vivo. Ubiquitination requires all the core components of the SCF and an intact F-box, suggesting that ubiquitination occurs within the SCF complex by an autocatalytic mechanism. Cdc4p and Grr1p are intrinsically unstable, and their steady-state levels do not fluctuate through the cell cycle. Taken together, these results suggest that ubiquitin-dependent degradation of F-box proteins allows rapid switching among multiple SCF complexes, thereby enabling cells to adapt quickly to changing physiological conditions and progression through different phases of the cell cycle (Galan, 1999).

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 has 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 has been 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).

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: this suggests 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).

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 is 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).

C. elegans Slimb homologs

In multicellular eukaryotes, a complex program of developmental signals regulates cell growth and division by controlling the synthesis, activation and degradation of G1 cell cycle regulators. The lin-23 gene of C. elegans is required to restrain cell proliferation in response to developmental cues. In lin-23 null mutants, all postembryonic blast cells undergo extra divisions, creating supernumerary cells that can differentiate and function normally. In contrast to the inability to regulate the extent of blast cell division in lin-23 mutants, the timing of initial cell cycle entry of blast cells is not affected. lin-23 encodes an F-box/WD-repeat protein that is orthologous to the Saccharomyces cerevisiae gene MET30, the Drosophila gene slmb, and the human gene ßTRCP, all of which function as components of SCF ubiquitin-ligase complexes. Loss of function of the Drosophila slmb gene causes the growth of ectopic appendages in a non-cell autonomous manner. In contrast, lin-23 functions cell autonomously to negatively regulate cell cycle progression, thereby allowing cell cycle exit in response to developmental signals (Kipreos, 2000).

In lin-23 mutants, cells appear to require normal signals to start cell division. Only blast cells and their descendants divide in lin-23 larval stages, while postmitotic non-blast cells do not divide. Blast cells do not divide precociously, but rather wait until the appropriate developmental stage to divide. Further, among the VPCs, those closest to the AC, which secretes an inductive/proliferative signal, divide more than those further from the AC, as is true for wild type. Final differentiation states of lin-23 cells appear normal. Distinct neuronal, hypodermal, somatic gonad, muscle, and intestine phenotypes are readily observed by DIC microscopy (Kipreos, 2000).

Morphogenesis is disorganized in several larval tissues in lin-23 mutants relative to wild type, e.g., spermathecae and uterus, and in embryos lacking lin-23 maternal product. lin-23 may be required for morphogenic processes: alternatively, the disorganization may be the byproduct of excess cell proliferation. In both cyclin-dependent kinase inhibitor cki-1 mutants and cul-1 loss-of-function animals, which also exhibit hyperplasia, there is a similar disorganization of larval tissues and lack of clear morphogenesis in embryos. In contrast to mitotic cell cycles, lin-23 is not required for restraining purely endoreplicative cycles. The ploidy of intestine cells, which endoreplicate in wild type to achieve 32n, does not increase beyond 32n in lin-23 mutants. The first endoreplication for most wild-type intestine cells is preceded by a nuclear division (segregation of chromosomes but no cytokinesis). Interestingly, the first endoreplication after the nuclear division is converted to a second nuclear division in lin-23 mutants, suggesting that lin-23 is required to allow these cells to bypass mitosis. Later endoreplications are not converted to nuclear divisions in lin-23 mutants, suggesting that mitotic bypass is maintained by a lin-23-independent mechanism, potentially via the downregulation of mitotic cyclin, as is observed in Drosophila endoreplicative cycles (Kipreos, 2000).

lin-23 is expressed at its highest levels in embryos and the adult germline, but has some expression in all developmental stages. The complete S. cerevisiae genome has just two F-box/WD-repeat proteins, Cdc4p and Met30p. Both Cdc4p and Met30p function as components of an SCF E3 complex to facilitate the recognition of substrates by a ubiquitin-conjugating enzyme (E2) for ubiquitin-mediated proteolysis. Met30p and Cdc4p function as the substrate recognition subunits of different SCF complexes, SCF Met30 and SCF Cdc4 . Each complex also includes Skp1p, the cullin Cdc53p, and Rbx1p/Roc1p/Hrt1p, and interacts with the ubiquitin-conjugating enzyme Cdc34p. Both Cdc4p and Met30p are involved in the degradation of cell cycle regulators. Cdc4p is required for the degradation of G1 cyclins (Cln1p and Cln2p), cyclin-dependent kinase inhibitors (Sic1p and Far1p), and the DNA replication protein Cdc6p. Met30p is required for the degradation of the CDK inhibitory kinase Swe1p, and for the repression of the sulfur network genes by inactivating the transcription factor Met4p. Increased activity of Met4p in met30 mutants produces a G1 arrest with increased turnover of the mRNA for the G1 cyclins CLN1, CLN2 and PCL2 (Kipreos, 2000).

Parsimony analysis suggests that lin-23 is more closely related to Met30p, while another C. elegans protein SEL-10 is more closely related to Cdc4p. SEL-10 functions to negatively regulate LIN-12 activity and has been found to physically interact with the intracellular domain of LIN-12, suggesting that SEL-10 functions in the turnover of LIN-12. Surprisingly, a null allele of sel-10 has only minor, impenetrant phenotypic consequences in a wild-type lin-12 genetic background. sel-10 is the apparent ortholog of the yeast cell cycle regulator CDC4. While the sel-10 mutant phenotype does not indicate a role in cell cycle regulation, it is possible that another gene functions redundantly with sel-10 to effect cell cycle regulation. However, a double mutant of lin-23 and sel-10 fail to uncover synthetic cell cycle phenotypes, suggesting that lin-23 does not share critical functions with sel-10. Finally, the dissimilar mutant phenotypes of lin-23 (defective cell cycle exit) and met30 (cell cycle arrest) further suggests that the cellular functions of SCF complexes have not been conserved between yeast and metazoa. lin-23 has a mutant hyperplasia phenotype similar to that of the cul-1 gene, which also encodes an SCF component. Orthologs of lin-23 and cul-1 function together in SCF complexes in both yeast and humans, making it likely that lin-23 and CUL-1 also form an SCF complex in C. elegans. The phenotypes of cul-1 and lin-23, while similar, do show differences. Whereas lin-23 maternal products perdure only through embryogenesis, cul-1 maternal products can suffice through the L1 stage. Despite the later onset of the cul-1 mutant phenotype, cul-1 larvae arrest development earlier with a more severe hyperplasia, e.g., cul-1 mutants exhibit on average twice as much vulval hyperplasia as lin-23 mutants (Kipreos, 2000).

Hyperplasia in lin-23 mutants does not appear to be due to morphogen secretion, as the hyperplasia occurs throughout development in all tissue types, and mosaic analysis indicates that lin-23 mutant hyperplasia occurs through a cell autonomous mechanism. It is currently unclear why the two orthologs have such different modes of action in Drosophila and C. elegans. It is tempting to speculate that both genes evolved to negatively regulate cell proliferation, however, differences in the way cell proliferation is regulated in Drosophila, via the action of morphogens to pattern surrounding cells, compared to C. elegans, where cell intrinsic decisions are much more important, have shaped the mechanism of action of the two gene products (Kipreos, 2000).

Cdc25 phosphatases are key positive cell cycle regulators that coordinate cell divisions with growth and morphogenesis in many organisms. Intriguingly in C. elegans, two cdc-25.1(gf) mutations induce tissue-specific and temporally restricted hyperplasia in the embryonic intestinal lineage, despite stabilization of the mutant CDC-25.1 protein in every blastomere. This study investigated the molecular basis underlying the CDC-25.1(gf) stabilization and its associated tissue-specific phenotype. Both mutations were found affect a canonical β-TrCP phosphodegron motif, while the F-box protein LIN-23, the β-TrCP orthologue, is required for the timely degradation of CDC-25.1. Accordingly, depletion of lin-23 in wild-type embryos stabilizes CDC-25.1 and triggers intestinal hyperplasia, which is, at least in part, cdc-25.1 dependent. lin-23(RNAi) causes embryonic lethality owing to cell fate transformations that convert blastomeres to an intestinal fate, sensitizing them to increased levels of CDC-25.1. This characterization of a novel destabilizing cdc-25.1(lf) intragenic suppressor that acts independently of lin-23 indicates that additional cues impinge on different motifs of the CDC-25.1 phosphatase during early embryogenesis to control its stability and turnover, in order to ensure the timely divisions of intestinal cells and coordinate them with the formation of the developing gut (Hebeisen, 2008).

The early cell divisions of the C. elegans embryo are precisely controlled by gene products that are provided from the maternal germ line. The isolation of two gain-of-function alleles of CDC-25.1 that demonstrate a strict maternal effect indicates that this regulation can be perturbed, resulting in supernumerary cell divisions specifically within the E lineage. How these mutations give rise to the extra divisions is unclear, although CDC-25.1(rr31) is more stable than its wild-type counterpart. By developing a GFP-based transgenic assay to assess the dynamics of protein degradation during early embryogenesis, this study shows that the stabilization of CDC-25.1 is mediated by a point mutation within a conserved DSGX4S β-TrCP-like phosphodegron in both cdc-25.1(gf) mutants. This stabilizes the protein, resulting in the abnormal presence of CDC-25.1 during a short, yet crucial, window during early development, which is presumably the cause of the observed cell cycle defect (Hebeisen, 2008).

Mammalian Slimb homologs

Ubiquitin-mediated destruction of regulatory proteins is a frequent means of controlling progression through signaling pathways. F-box proteins are components of modular E3 ubiquitin protein ligases called SCFs, which function in phosphorylation-dependent ubiquitination. F-box proteins contain a carboxy-terminal domain that interacts with substrates and a 42-48 amino-acid F-box motif, which binds to the protein Skp1. Skp1 binding links the F-box protein with a core ubiquitin ligase composed of the proteins Cdc53/Cul1, Rbx1 (also called Hrt1 and Roc1) and the E2 ubiquitin-conjugating enzyme Cdc34. The genomes of the budding yeast Saccharomyces cerevisiae and the nematode worm C. elegans contain, respectively, 16 and more than 60 F-box proteins; in S. cerevisiae, the F-box proteins Cdc4, Grr1 and Met30 target cyclin-dependent kinase inhibitors, G1 cyclins and transcriptional regulators for ubiquitination. Only four mammalian F-box proteins (Cyclin F, Skp1, beta-TRCP and NFB42) have been identified so far. Here, the identification of a family of 33 novel mammalian F-box proteins is reported. The large number of these proteins in mammals suggests that the SCF system controls a correspondingly large number of regulatory pathways in vertebrates. Four of these proteins contain a novel conserved motif: the F-box-associated (FBA) domain, which may represent a new protein-protein interaction motif. The identification of these genes will help uncover pathways controlled by ubiquitin-mediated proteolysis in mammals (Winston, 1999b).

In normal and transformed cells, the F-box protein p45SKP2 is required for S phase and forms stable complexes with p19SKP1 and cyclin A-cyclin-dependent kinase (CDK)2. Human CUL-1, a member of the cullin family, and the ubiquitin-conjugating enzyme CDC34 are identified as additional partners of p45SKP2 in vivo. CUL-1 also associates with cyclin A and p19SKP1 in vivo and, with p45SKP2, they assemble into a large multiprotein complex. In Saccharomyces cerevisiae, a complex of similar molecular composition (an F-box protein, a member of the cullin family and a homolog of p19SKP1) forms a functional E3 ubiquitin protein ligase complex, designated SCFCDC4, that facilitates ubiquitination of a CDK inhibitor by CDC34. The data presented here imply that the p45SKP2-CUL-1-p19SKP1 complex may be a human representative of an SCF-type E3 ubiquitin protein ligase. It is proposed that all eukaryotic cells may use a common ubiquitin conjugation apparatus to promote S phase. Multiprotein complex formation involving p45SKP2-CUL-1 and p19SKP1 is governed, in part, by periodic, S phase-specific accumulation of the p45SKP2 subunit and by the p45SKP2-bound cyclin A-CDK2. The dependency of p45SKP2-p19SKP1 complex formation on cyclin A-CDK2 may ensure tight coordination of the activities of the cell cycle clock with those of a potential ubiquitin conjugation pathway (Lisztwan, 1999).

HOS, a human homolog of Slimb, forms an SCF complex with Skp1 and Cullin1 and targets the phosphorylation-dependent degradation of IkappaB and beta-catenin. SCF E3 ubiquitin ligases mediate ubiquitination and proteasome-dependent degradation of phosphorylated substrates. A human F-box/WD40 repeats protein (HOS), has been identified that is homologous to Slimb/h betaTrCP. Being a part of SCF complex with Skp1 and Cullin1, HOS specifically interacts with the phosphorylated IkappaB and beta-catenin, targeting these proteins for proteasome-dependent degradation in vivo. This targeting requires Cullin1 because expression of a mutant Cullin1 abrogates the degradation of IkappaB and of beta-catenin. Mutant HOS, which lacks the F-box, blocks TNF alpha-induced degradation of IkappaB as well as GSK3beta-mediated degradation of beta-catenin. This mutant also inhibits NF-kappaB transactivation and increases the beta-catenin-dependent transcription activity of Tcf. These results demonstrate that SCF(HOS) E3 ubiquitin ligase regulate both NF-kappaB and beta-catenin signaling pathways (Fuchs, 1999).

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 has been 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; it 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).

HIV-1 Vpu interacts with CD4 in the endoplasmic reticulum and triggers CD4 degradation, presumably by proteasomes. Human beta TrCP identified by interaction with Vpu connects CD4 to this proteolytic machinery; CD4-Vpu-beta TrCP ternary complexes have been detected by coimmunoprecipitation. beta TrCP binding to Vpu and its recruitment to membranes require two phosphoserine residues in Vpu essential for CD4 degradation. In beta TrCP, WD repeats at the C terminus mediate binding to Vpu, and an F box near the N terminus is involved in interaction with Skp1p, a targeting factor for ubiquitin-mediated proteolysis. An F-box deletion mutant of beta TrCP has a dominant-negative effect on Vpu-mediated CD4 degradation. These data suggest that beta TrCP and Skp1p represent components of a novel ER-associated protein degradation pathway that mediates CD4 proteolysis (Margottin, 1998).

Mammalian Slimb homologs: Roles in Wnt signaling

Defects in beta-catenin regulation contribute to the neoplastic transformation of mammalian cells. Dysregulation of beta-catenin (Drosophila homolog: Armadillo) can result from missense mutations that affect critical sites of phosphorylation by glycogen synthase kinase 3beta (GSK3beta). Given that phosphorylation can regulate targeted degradation of beta-catenin by the proteasome, beta-catenin might interact with an E3 ubiquitin ligase complex containing an F-box protein, as is the case for certain cell cycle regulators. Accordingly, disruption of the Drosophila F-box protein Slimb upregulates the beta-catenin homolog Armadillo. It was reasoned that the human homologs of Slimb (beta-TrCP and its isoform beta-TrCP2 [KIAA0696]) might interact with beta-catenin. The binding of beta-TrCP to beta-catenin is direct and dependent upon the WD40 repeat sequences in beta-TrCP and on phosphorylation of the GSK3beta sites in beta-catenin. Endogenous beta-catenin and beta-TrCP can be coimmunoprecipitated from mammalian cells. Overexpression of wild-type beta-TrCP in mammalian cells promotes the downregulation of beta-catenin, whereas overexpression of a dominant-negative deletion mutant upregulates beta-catenin protein levels and activates signaling dependent on the transcription factor Tcf. In contrast, beta-TrCP2 does not associate with beta-catenin. It is concluded that beta-TrCP is a component of an E3 ubiquitin ligase that is responsible for the targeted degradation of phosphorylated beta-catenin (Hart, 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), 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).

Regulation of beta-catenin stability is essential for Wnt signal transduction during development and tumorigenesis. It is well known that serine-phosphorylation of beta-catenin by the Axin-glycogen synthase kinase (GSK)-3beta complex targets beta-catenin for ubiquitination-degradation, and mutations at critical phosphoserine residues stabilize beta-catenin and cause human cancers. Phosphorylated beta-catenin is specifically recognized by beta-Trcp, an F-box/WD40-repeat protein that also associates with Skp1, an essential component of the ubiquitination apparatus. beta-Trcp is a homolog of Drosophila Slimb. beta-catenin harboring mutations at the critical phosphoserine residues escapes recognition by beta-Trcp, thus providing a molecular explanation for why these mutations cause beta-catenin accumulation that leads to cancer. Inhibition of endogenous beta-Trcp function by a dominant negative mutant stabilizes beta-catenin, activates Wnt/beta-catenin signaling, and induces axis formation in Xenopus embryos. Therefore, beta-Trcp plays a central role in recruiting phosphorylated beta-catenin for degradation and in dorsoventral patterning of the Xenopus embryo (Liu, 1999).

Mammalian Slimb homologs: Roles in NF-kappaB signaling

NF-kappaB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules: IkappaBs (Homologs of Drosophila Cactus). Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin-proteasome system to eliminate inhibitory regulators. The pIkappaBalpha-ubiquitin ligase (pIkappaBalpha-E3) has been isolated. This ligase attaches ubiquitin, a small protein that marks other proteins for degradation by the proteasome system, to the phosphorylated NF-kappaB inhibitor pIkappaBalpha. Taking advantage of its high affinity to pIkappaBalpha, this ligase has been isolated from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, the specific component of the ligase that recognizes the pIkappaBalpha degradation motif has been identified as an F-box/WD-domain protein belonging to a recently distinguished family of beta-TrCP/Slimb proteins. This component, which is denoted E3RSIkappaB (pIkappaBalpha-E3 receptor subunit), binds specifically to pIkappaBalpha and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(IkappaB), which tightly binds pIkappaBalpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIkappaBalpha and consequently NF-kappaB activation. E3RS(IkappaB) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation (Yaron, 1998).

FWD1 (the mouse homolog of Drosophila Slimb and Xenopus betaTrCP, a member of the F-box- and WD40 repeat-containing family of proteins, and a component of the SCF ubiquitin ligase complex) interacts with IkappaBalpha and thereby promotes IkappaBalpha ubiquitination and degradation. FWD1 also binds to IkappaBbeta and IkappaBepsilon and induces their ubiquitination and proteolysis. FWD1 recognizes the conserved DSGPsiXS motif (where Psi represents the hydrophobic residue) present in the NH(2)-terminal regions of these three IkappaB proteins only when the component serine residues are phosphorylated. However, in contrast to IkappaBalpha and IkappaBbeta, the recognition site in IkappaBepsilon for FWD1 is not restricted to the DSGPsiXS motif; FWD1 also interacts with other sites in the NH(2)-terminal region of IkappaBepsilon. Substitution of the critical serine residues in the NH(2)-terminal regions of IkappaBalpha, IkappaBbeta, and IkappaBepsilon with alanines also markedly reduces the extent of FWD1-mediated ubiquitination of these proteins and increases their stability. These data indicate that the three IkappaB proteins, despite their substantial structural and functional differences, all undergo ubiquitination mediated by the SCF(FWD1) complex. FWD1 may thus play an important role in NF-kappaB signal transduction through regulation of the stability of multiple IkappaB proteins (Shirane, 1999).

Ubiquitin-mediated proteolysis has a central role in controlling the intracellular levels of several important regulatory molecules, such as cyclins, CKIs, p53, and IkappaBalpha. Many diverse proinflammatory signals lead to the specific phosphorylation and subsequent ubiquitin-mediated destruction of the NF-kappaB inhibitor protein IkappaBalpha. Substrate specificity in ubiquitination reactions is, in large part, mediated by the specific association of the E3-ubiquitin ligases with their substrates. One class of E3 ligases is defined by the recently described SCF complexes, the archetype of which was first described in budding yeast and contains Skp1, Cdc53, and the F-box protein Cdc4. These complexes recognize their substrates through modular F-box proteins in a phosphorylation-dependent manner. A biochemical dissection is described of a novel mammalian SCF complex, SCFbeta-TRCP, that specifically recognizes a 19-amino-acid destruction motif in IkappaBalpha (residues 21-41) in a phosphorylation-dependent manner. This SCF complex also recognizes a conserved destruction motif in beta-catenin, a protein with levels also regulated by phosphorylation-dependent ubiquitination. Endogenous IkappaBalpha-ubiquitin ligase activity cofractionates with SCFbeta-TRCP. Furthermore, recombinant SCFbeta-TRCP assembled in mammalian cells contains phospho-IkappaBalpha-specific ubiquitin ligase activity. These results suggest that an SCFbeta-TRCP complex functions in multiple transcriptional programs by activating the NF-kappaB pathway and inhibiting the beta-catenin pathway (Winston, 1999a).

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by the proteolysis of its inhibitory subunit (IkappaB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IkappaBalpha by a large multisubunit complex containing IkappaB kinases is a prerequisite for ubiquitination. Here, FWD1 (a mouse homolog of Slimb/betaTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IkappaBalpha only when IkappaBalpha is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IkappaBalpha in concert with IkappaB kinases, resulting in nuclear translocation of NF-kappaB. In addition, FWD1 strikingly evokes the ubiquitination of IkappaBalpha in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IkappaBalpha. These results suggest that (1) the substrate-specific degradation of IkappaBalpha is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex, and (2) that FWD1 serves as an intracellular receptor for phosphorylated IkappaBalpha. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-kappaB through control of IkappaB protein stability (Hatakeyama, 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).

Homologue of Slimb (HOS)/beta-transducin repeats-containing proteins up-regulate nuclear factor kappaB activity by targeting its inhibitor (IkappaB) for ubiquitination and subsequent degradation. Whether inhibition of HOS function may modulate apoptosis in human melanoma cells has been investigated. Forced expression of the dominant negative HOSdeltaF construct inhibits IkappaB degradation and leads to sensitization of melanoma cells to apoptosis induced by tumor necrosis factor alpha with cycloheximide, as well as by cisplatin and ionizing and UV irradiation. These data indicate that HOS plays an important role in controlling the IkappaB-dependent apoptotic pathways in human melanoma (Soldatenkov, 1999).

Mammalian Slimb homologs: Roles in Hedgehog signaling

Drosophila Suppressor of fused [Su(fu)] encodes a novel 468-amino-acid cytoplasmic protein which, by genetic analysis, functions as a negative regulator of the Hedgehog segment polarity pathway. Described here is the primary structure, tissue distribution, biochemical and functional analyses of a human Su(fu): [hSu(fu)]. Two alternatively spliced isoforms of hSu(fu) were identified, predicting proteins of 433 and 484 amino acids, with a calculated molecular mass of 48 and 54 kDa, respectively. The two proteins differ only by the inclusion or exclusion of a 52-amino-acid extension at the carboxy terminus. Both isoforms are expressed in multiple embryonic and adult tissues, and exhibit a developmental profile consistent with a role in Hedgehog signaling. The hSu(fu) contains a high-scoring PEST-domain, and exhibits an overall 37% sequence identity (63% similarity) with the Drosophila protein and 97% sequence identity with the mouse Su(fu). The hSu(fu) locus maps to chromosome 10q24-q25, a region that is deleted in glioblastomas, prostate cancer, malignant melanoma and endometrial cancer. HSu(fu) represses activity of the zinc-finger transcription factor Gli, which mediates Hedgehog signaling in vertebrates, and physically interacts with Gli, Gli2 and Gli3 as well as with Slimb, an F-box containing protein which, in the fly, suppresses the Hedgehog response, in part by stimulating the degradation of the fly Gli homolog. Coexpression of Slimb with Su(fu) potentiates the Su(fu)-mediated repression of Gli. Taken together, these data provide biochemical and functional evidence for the hypothesis that Su(fu) is a key negative regulator in the vertebrate Hedgehog signaling pathway. The data further suggest that Su(fu) can act by binding to Gli and inhibiting Gli-mediated transactivation as well as by serving as an adaptor protein, which links Gli to the Slimb-dependent proteasomal degradation pathway (Stone, 1999).

Hedgehog-regulated processing of the transcription factor Cubitus interruptus (Ci) in Drosophila depends on phosphorylation of the C-terminal region of Ci by cAMP-dependent protein kinase and subsequently by Casein kinase 1 and Glycogen synthase kinase 3. Ci processing also requires Slimb, an F-box protein of SCF (Skp1/Cullin/F-box proteins) complex, and the proteasome, but the interplay between phosphorylation and the activity of Slimb and the proteasome remains unclear. This study shows that processing of the Gli3 protein, a homolog of Ci, also depends on phosphorylation of a set of four cAMP-dependent protein kinase sites that primes subsequent phosphorylation of adjacent casein kinase 1 and glycogen synthase kinase 3. Gain- and loss-of-function analyses in cultured cells further reveal that ßTrCP, the vertebrate homolog of Slimb, is required for Gli3 processing, and ßTrCP can bind phosphorylated Gli3 both in vitro and in vivo. Gli3 protein is polyubiquitinated in the cell, and its processing depends on proteasome activity. These findings provide evidence for a direct link between phosphorylation of Gli3/Ci proteins and ßTrCP/Slimb action, thus supporting the hypothesis that the processing of Gli3/Ci is affected by the proteasome (Wang, 2006).

Mammalian Slimb homologs and Presenilin degradation

Mutations in either of two human presenilin genes (PS1 and PS2) cause Alzheimer's disease (see Drosophila Presenilin). Genetic and physical interactions between Caenorhabditis elegans SEL-10, a member of the Cdc4p family of proteins, and SEL-12, a C. elegans presenilin are described. Loss of sel-10 activity can suppress the egg-laying defective phenotype associated with reducing sel-12 activity, and SEL-10 can physically complex with SEL-12. Proteins of the Cdc4p family have been shown to target proteins for ubiquitin-mediated turnover. The functional and physical interaction between sel-10 and sel-12 therefore offers an approach to understanding how presenilin levels are normally regulated (Wu, 1998).

Developmental roles for Slimb homologs

supernumerary limbs:Evolutionary homologs part 2/2

supernumerary limbs: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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