cDNA cloning and expression analysis of new members of the mammalian F-box protein family

F-box proteins are critical components of the SCF ubiquitin-protein ligase complex and are involved in substrate recognition and recruitment for ubiquitination and consequent degradation by the proteasome. We have isolated cDNAs encoding a further 10 mammalian F-box proteins. Five of them (FBL3 to FBL7) share structural similarities with Skp2 and contain C-terminal leucine-rich repeats. The other 5 proteins have different putative protein-protein interaction motifs. Specifically, FBS and FBWD4 proteins contain Sec7 and WD40-repeat domains, respectively. The C-terminal region of FBA shares similarity with bacterial protein ApaG while FBG2 shows homology with the F-box protein NFB42. The marked differences in F-box gene expression in human tissues suggest their distinct role in ubiquitin-dependent protein degradation.     

Molecular genetic characterization of a Korean split hand/split foot malformation (SHFM)

Split hand/split foot malformation (SHFM; ectrodactyly) is genetically heterogeneous, with mutations identified at five loci (SHFM1 at 7q21.3, SHFM2 at Xq26, SHFM3 at 10q24, SHFM4 at 3q27 and SHFM5 at 2q31). In this study, we attempted to identify and localize the causative allele of a Korean case of SHFM. Pedigree analysis showed that the Korean SHFM was autosomally dominant and its penetrance was high, indicating that it was not caused by SHFM2. Clinical features were variable, but limited to the four limbs unlike SHFM1, SHFM4 and SHFM5. G-banding and FISH failed to identify any chromosomal abnormalities. We also performed mutation screening by SSCP and DNA sequencing, as well as loss of heterozygosity (LOH) analysis, to exclude the possibility that SHFM4 or SHFM5 were involved; these revealed no mutations in gene p63 and no LOH on 2q31, respectively. It therefore appears that the Korean SHFM may be caused by mutation of SHFM3. In fact, linkage analysis using informative microsatellite markers indicated that SHFM3 was linked to D10S577 with a maximum LOD score of 1.15 at recombination fraction zero. Finally, we identified two novel alleles (191 and 211 bp) of D10S577 that have not been found in Western populations.     

Proviral insertions in the zebrafish hagoromo gene, encoding an F-box/WD40-repeat protein, cause stripe pattern anomalies

The zebrafish, Danio rerio, has three types of pigment cells (melanophores, xanthophores and iridophores) and, in adult fish, these cells are organized into a stripe pattern. The mechanisms underlying formation of the stripe pattern are largely unknown. We report here the identification and characterization of a novel dominant zebrafish mutation, hagoromo (hag), which was generated by insertional mutagenesis using a pseudotyped retrovirus. The hag mutation caused disorganized stripe patterns. Two hag mutant alleles were isolated independently and proviruses were located within the fifth intron of a novel gene, which we named hag, encoding an F-box/WD40-repeat protein. The hag gene was mapped to linkage group (LG)13, close to fgf8 and pax2.1. Amino acid sequence similarity, conserved exon-intron boundaries and conserved synteny indicated that zebrafish hag is an ortholog of mouse Dactylin, the gene mutated in the Dactylaplasia (Dac) mouse [1]. The Dac mutation is dominant and causes defects in digit formation in fore- and hindlimbs. This study revealed that the hag locus is important for pattern formation in fish but is involved in distinct morphogenetic events in different vertebrates.     

Targeted degradation of beta-catenin by chimeric F-box fusion proteins

Adenomatous polyposis coli (APC) tumor suppressor protein, together with Axin and glycogen synthase kinase 3beta (GSK-3beta), forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of cytoplasmic beta-catenin by ubiquitin-dependent proteolysis. Degradation of phosphorylated beta-catenin is initiated by interaction through the WD40-repeat of a F-box protein beta-TrCP, a component of SCF ubiquitin ligase complex. Mutations in APC, Axin, and beta-catenin that prevent down-regulation of cytoplasmic beta-catenin are found in various types of cancers. In the search for efficient treatment and prevention of malignancies associated with increased levels of cytoplasmic beta-catenin, we created chimeric F-box fusion proteins by replacing the WD40-repeat of beta-TrCP with the beta-catenin-binding domains of Tcf4 and E-cadherin. Expression of chimeric F-box fusion proteins successfully promotes degradation of beta-catenin independently of GSK-3beta-mediated phosphorylation. More importantly, this degradation does not require intact APC protein (pAPC).     

SEL-10 is an inhibitor of notch signaling that targets notch for ubiquitin-mediated protein degradation

Notch receptors and their ligands play important roles in both normal animal development and pathogenesis. We show here that the F-box/WD40 repeat protein SEL-10 negatively regulates Notch receptor activity by targeting the intracellular domain of Notch receptors for ubiquitin-mediated protein degradation. Blocking of endogenous SEL-10 activity was done by expression of a dominant-negative form containing only the WD40 repeats. In the case of Notch1, this block leads to an increase in Notch signaling stimulated by either an activated form of the Notch1 receptor or Jagged1-induced signaling through Notch1. Expression of dominant-negative SEL-10 leads to stabilization of the intracellular domain of Notch1. The Notch4 intracellular domain bound to SEL-10, but its activity was not increased as a result of dominant-negative SEL-10 expression. SEL-10 bound Notch4 via the WD40 repeats and bound preferentially to a phosphorylated form of Notch4 in cells. We mapped the region of Notch4 essential for SEL-10 binding to the C-terminal region downstream of the ankyrin repeats. When this C-terminal fragment of Notch4 was expressed in cells, it was highly labile but could be stabilized by the expression of dominant-negative SEL-10. Ubiquitination of Notch1 and Notch4 intracellular domains in vitro was dependent on SEL-10. Although SEL-10 interacts with the intracellular domains of both Notch1 and Notch4, these proteins respond differently to interference with SEL-10 function. Thus, SEL-10 functions to promote the ubiquitination of Notch proteins; however, the fates of these proteins may differ.     

泛素连接酶FBW7的肿瘤抑制作用和机制

泛素-蛋白酶体系统是一个主要的蛋白质降解调节通路,在细胞分裂过程中发挥着重要作用,其成员在肿瘤中频繁存在着表达异常现象.FBW7又名AGO、hCDC4、FBXW7和SEL-10,是一种拥有7串联WD40重复结构域的F-box蛋白,可作为SCF型泛素连接酶（E3）复合物的底物识别亚基发挥作用.FBW7是一种肿瘤抑制蛋白,其基因在多种肿瘤包括直肠癌、胃癌、卵巢癌和白血病中存在着基因突变或缺失.FBW7可直接结合和靶向作用多种转录激活因子或原癌基因,如周期蛋白E、c-Myc、c-Jun、Notch、MCL1、KLF5 和mTOR等并对其进行泛素化修饰和随后的26S蛋白酶体降解.肿瘤抑制蛋白FBW7的研究对肿瘤发生机制的理解具有重要意义,同时也为肿瘤的诊断和治疗提供了新的靶点.本文综述了FBW7的特征、肿瘤抑制作用及机制.     

Five human genes encoding F-box proteins: chromosome mapping and analysis in human tumors

Members of the F-box protein (Fbp) family are characterized by an approximately 40 amino acid F-box motif. SCF complexes (formed by Skp1, cullin, and one of many Fbps) act as protein-ubiquitin ligases that control the G(1)/S transition of the eukaryotic cell cycle. The substrate specificity of SCF complexes is determined by the presence of different Fbp subunits that recruit specific substrates for ubiquitination. Unchecked degradation of cellular regulatory proteins has been observed in certain tumors and it is possible that deregulated ubiquitin ligases play a role in the altered degradation of cell cycle regulators. We have recently identified a family of human Fbps. As a first step aimed at determining if FBP genes could be involved in human neoplasia, we have mapped the chromosome positions of 5 FBP genes by fluorescence in situ hybridization (FISH) to 10q24 (BTRC alias beta-TRCP/FBW1a), 9q34 (FBXW2 alias FBW2), 13q22 (FBXL3A alias FBL3a), 5p12 (FBXO4 alias FBX4) and 6q25-->q26 (FBXO5 alias FBX5). Since most of these are chromosomal loci frequently altered in tumors, we have screened 42 human tumor cell lines and 48 human tumor samples by Southern hybridization and FISH. While no gross alterations of the genes encoding beta-Trcp/Fbw1a, Fbw2, Fbx4 and Fbx5 were found, heterozygous deletion of the FBXL3A gene was found in four of 13 small cell carcinoma cell lines. This is the first evaluation of genes encoding Fbps in human tumors.     

A WD40 repeat protein regulates fungal cell differentiation and can be replaced functionally by the mammalian homologue striatin

Fruiting body development in fungi is a complex cellular differentiation process that is controlled by more than 100 developmental genes. Mutants of the filamentous fungus Sordaria macrospora showing defects in fruiting body formation are pertinent sources for the identification of components of this multicellular differentiation process. Here we show that the sterile mutant pro11 carries a defect in the pro11 gene encoding a multimodular WD40 repeat protein. Complementation analysis indicates that the wild-type gene or C-terminally truncated versions of the wild-type protein are able to restore the fertile phenotype in mutant pro11. PRO11 shows significant homology to several vertebrate WD40 proteins, such as striatin and zinedin, which seem to be involved in Ca²⁺-dependent signaling in cells of the central nervous system and are supposed to function as scaffolding proteins linking signaling and eukaryotic endocytosis. Cloning of a mouse cDNA encoding striatin allowed functional substitution of the wild-type protein with restoration of fertility in mutant pro11. Our data strongly suggest that an evolutionarily conserved cellular process controlling eukaryotic cell differentiation may regulate fruiting body formation.     

Proteasomal degradation of the multifunctional regulator YB-1 is mediated by an F-Box protein induced during programmed cell death

F-Box proteins (FBPs) are variable adaptor proteins that earmark protein substrates for ubiquination and destruction by the proteasome. Through their N-terminal F-box motif, they couple specific protein substrates to a catalytic machinery known as SCF (Skp-1/Cul1/F-Box) E3-ubiquitin ligase. Typical FBPs bind the specific substrates in a phosphorylation dependent manner via their C-termini using either leucine rich repeats (LRR) or tryptophan-aspartic acid (WD40) domains for substrate recognition. By using a gene trap strategy that selects for genes induced during programmed cell death, we have isolated the mouse homolog of the hypothetical human F-Box protein 33 (FBX33). Here we identify FBX33 as a component of an SCF E3-ubiquitin ligase that targets the multifunctional regulator Y-box binding protein 1 (YB-1)/dbpB/p50 for polyubiquitination and destruction by the proteasome. By targeting YB-1 for proteasomal degradation, FBX33 negatively interferes with YB-1 mediated functions. In contrast to typical FBPs, FBX33 has no C-terminal LRR or WD40 domains and associates with YB-1 via its N-terminus. The present study confirms the existence of a formerly hypothetical F-Box protein in living cells and describes one of its substrates.     

Roles of F-box proteins in plant hormone responses

The F-box protein is an important component of the E3 ubiquitin ligase Skpl-Cullin-F-box protein complex. It binds specific substrates for ubiquitin-mediated proteolysis. The F-box proteins contain a signature F-box motif at their amino-terminus and some protein-protein interaction motifs at their carboxyterminus, such as Trp-Asp repeats or leucine rich repeats. Many F-box proteins have been identified to be involved in plant hormone response as receptors or important medial components. These breakthrough findings shed light on our current understanding of the structure and function of the various F-box proteins, their related plant hormone signaling pathways, and their roles in regulating plant development.     

Mouse model of split hand/foot malformation type I

Split hand/foot malformation type I (SHFM1) disease locus maps to chromosome 7q21.3-q22, a region that includes the distal-less-related (dll) genes DLX5 and DLX6. However, incomplete penetrance, variable expressivity, segregation distortion, and syndromic association with other anomalies have so far prevented the identification of the SHFM1 gene(s) in man. Here we show that the targeted double inactivation of Dlx5 and Dlx6 in the mouse causes in homozygous mutant animals bilateral ectrodactyly with a severe defect of the central ray of the hindlimbs, a malformation typical of SHFM1. This is the first evidence that the role of dll/Dlx genes in appendage development is conserved from insects to mammals and proves their involvement in SHFM1.     

人类新基因WDR24的研究初报

WD40家族是一类结构保守、功能复杂的蛋白.目前很多研究显示该家族成员通过参与MAPK信号途径调控细胞内信号转导而影响细胞的基本生命活动.为了鉴定参与细胞生命活动的新基因,运用同源基因克隆法,通过PCR技术扩增获得一个新的人类基因WDR24, 其cDNA全长3 302 bp,2 373 bp长的开放阅读框编码由790个氨基酸残基组成的蛋白质.生物信息学分析表明,WDR24蛋白在进化上高度保守,与其他脊椎动物中的同源蛋白组成了一个功能未知的亚家族.蛋白序列分析显示其中有6个WD40重复序列和1个ERK的停泊位点D-domain.RT-PCR分析表明,该基因在所有被检测的人类胚胎组织中表达.     

Structure and expression of the gene encoding mouse F-box protein, Fwd2

A novel class of ubiquitin ligases, termed the SCF complex, consists of invariable components, Skp1 and Cullin, and variable components called F-box proteins, which have a primary role in determining substrate specificity. We have isolated a cDNA encoding the mouse F-box protein Fwd2 (also known as MD6) as a possible constituent of an SCF-type ubiquitin ligase. Fwd2 cDNA contains 1890 bp with a 1362-bp open reading frame and encodes an approximately 51.5-kDa protein. Fwd2 is expressed predominantly in liver and, to a lesser extent, in the testis, lung, heart, and skeletal muscle. Immunofluorescence staining for Fwd2 protein shows a pattern with the cytoplasm. A coimmunoprecipitation assay has revealed the in vivo interaction between Skp1 and Fwd2 through the F-box domain. Fwd2 also interacts with Cul1 through Skp1, suggesting that Skp1, Cul1, and the F-box protein Fwd2 form an SCF complex (SCF(Fwd2)). We have also isolated and determined the nucleotide sequence and genomic organization of the gene that encodes mouse Fwd2. This gene spans approximately 17 kb and consists of six exons and five introns. Our results suggest that Fwd2 is an F-box protein that constitutes an SCF ubiquitin ligase complex and that it plays a critical role in the ubiquitin-dependent degradation of proteins expressed in the liver.     

Fine mapping of the split-hand/split-foot locus (SHFM3) at 10q24: evidence for anticipation and segregation distortion.

Split-hand/split-foot malformation (SHFM, ectrodactyly, or lobster-claw deformity) is a human limb malformation characterized by aberrant development of central digital rays with absence of fingers and toes, a deep median cleft, and fusion of remaining digits. SHFM is clinically heterogeneous, presenting both in an isolated form and in combination with additional abnormalities affecting the tibia and/or other organ systems, including the genitourinary, craniofacial, and ectodermal structures. Three SHFM disease loci have been genetically mapped to chromosomes 7q21 (SHFM1), Xq26 (SHFM2), and 10q24 (SHFM3). We mapped data from a large Turkish family with isolated SHFM to chromosome 10q24 and have narrowed the SHFM3 region from 9 cM to an approximately 2-cM critical interval between genetic markers D10S1147 and D10S1240. In several instances we found evidence for a more severe phenotype in offspring of a mildly affected parent, suggesting anticipation. Finally, data from this family, combined with those from six other pedigrees, mapped to 10q24, demonstrate biased transmission of SHFM3 alleles from affected fathers to offspring. The degree of this segregation distortion is obvious in male offspring and is possibly of the same magnitude for female offspring.     

Isolation and characterization of a novel F-box protein Pof10 in fission yeast

The SCF complex is a type of ubiquitin-protein ligase (E3) that consists of invariable components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Using a yeast two-hybrid system, we isolated six proteins that interact with Schizosaccharomyces pombe Skp1. Among them, Pof10 is a novel F-box protein consisting of 662 amino acids, harboring the F-box domain required for the binding to Skp1 and followed by four WD40 repeats. Overexpression of Pof10 in fission yeast resulted in loss of viability with marked morphological changes that are similar to those in pop1 mutant yeast. Coexpression of Skp1 with Pof10 prevented the lethality, suggesting that the lethality from Pof10 overexpression results from the sequestration of Skp1 from other F-box proteins including Pop1. Whereas most F-box proteins show rapid turnover, Pof10 has a remarkably long half-life in vivo and has been shown to be localized predominantly in cytoplasm. These results suggest that the stable F-box protein Pof10 might target abundant cytoplasmic proteins for degradation in fission yeast.     

F-box proteins: the key to protein degradation

Summary The eukaryotic protein degradation pathway involves the ubiquitin (Ub) modification of substrates targeted for degradation by the 26S proteasome. The addition of Ub, a process called ubiquitination, is mediated by enzymes including the E3 Ub ligases which transfer the Ub to targeted substrates. A major type of E3 Ub ligases, the SCF (Skp–Cullin–F-box) complex, is composed of four major components: Skp1, Cul1/Cdc53, Roc1/Rbx1/Hrt1, and an F-box protein. The F-box component of the SCF machineries is responsible for recognizing different substrates for ubiquitination. Interaction with components of the SCF complex is mediated through the F-box motif of the F-box protein while it associates with phosphorylated substrates through its second protein–protein interaction motif such as Trp–Asp (WD) repeats or leucine-rich repeats (LRRs). By targeting diverse substrates, F-box proteins exert controls over stability of proteins and regulate the mechanisms for a wide-range of cellular processes. Here we discuss the importance of F-box proteins by providing a general overview and examples of how F-box proteins function in various cellular settings such as tissue development, cell proliferation, and cell death, in the modeling organism Drosophila.     

Characterization of a mouse gene (Fbxw6) that encodes a homologue of Caenorhabditis elegans SEL-10.

The SCF complex is a type of ubiquitin ligase that consists of the invariable components SKP1, CUL1, and RBX1 as well as a variable component, known as an F-box protein, that is the main determinant of substrate specificity. The Caenorhabditis elegans F-box- and WD40-repeat-containing protein SEL-10 functionally and physically associates with LIN-12 and SEL-12, orthologues of mammalian Notch and presenilin, respectively. We have now identified a gene (which we call Fbxw6) that encodes a mouse homologue (F-box-WD40 repeat protein 6, or FBW6) of SEL-10 and is expressed mainly in brain, heart, and testis. Co-immunoprecipitation analysis showed that FBW6 interacts with SKP1 and CUL1, indicating that these three proteins form an SCF complex. Comparison of the genomic organization of Fbxw6, which is located on mouse chromosome 3.3E3, with that of mouse Fbxw1, Fbxw2, and Fbxw4 showed only a low level of similarity, indicating that these genes diverged relatively early and thereafter evolved independently.