The human F box protein beta-Trcp associates with the Cul1/Skp1 complex and regulates the stability of beta-catenin.
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Ubiquitin-conjugation targets numerous cellular regulators for proteasome-mediated degradation. Thus, the identification of ubiquitin ligases and their physiological substrates is crucially important, especially for those cases in which aberrant levels of regulatory proteins (e.g., beta-catenin, p27) result from a deregulated ubiquitination pathway. In yeast, the proteolysis of several G1 regulators is controlled by ubiquitin ligases (or SCFs) formed by three subunits: Skp1, Cul A (Cdc53), and one of many F-box proteins. Specific F-box proteins (Fbps) recruit different substrates to the SCF. Although many Fbps have been identified in mammals, their specific substrates and the existence of multiple SCFs have not yet been reported. We have found that one human Fbp, beta-Trcp (beta-Transducin repeat containing protein), does indeed form a novel SCF with human Skp1 and Cul1. Consistent with recent reports indicating that Xenopus and Drosophila beta-Trcp homologs act as negative regulators of the Wnt/beta-catenin signaling pathway, we report here that human beta-Trcp interacts with beta-catenin in vivo. Furthermore, beta-catenin is specifically stabilized in vivo by the expression of a dominant negative beta-Trcp. These results indicate that the Cul1/Skp1/beta-Trcp complex forms a ubiquitin ligase that mediates the degradation of beta-catenin. (+info)
Identification of a human HECT family protein with homology to the Drosophila tumor suppressor gene hyperplastic discs.
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Use of the differential display technique to isolate progestin-regulated genes in T-47D human breast cancer cells led to identification of a novel gene, EDD. The cDNA sequence contains a 2799 amino acid open reading frame sharing 40% identity with the predicted 2894 amino acid product of the Drosophila melanogaster tumor suppressor gene hyperplastic discs, while the carboxy-terminal 889 amino acids show 96% identity to a rat 100 kDa HECT domain protein. EDD mRNA was progestin-induced in T-47D cells and was highly abundant in testes and expressed at moderately high levels in other tissues, suggesting a broad role for EDD. Anti-EDD antibodies immunoprecipitated an approximately 300 kDa protein from T-47D cell lysates. HECT family proteins function as E3 ubiquitin-protein ligases, targeting specific proteins for ubiquitin-mediated proteolysis. EDD is likely to function as an E3 as in vitro translated protein bound ubiquitin reversibly through a conserved HECT domain cysteine residue. EDD was localized by FISH to chromosome 8q22, a locus disrupted in a variety of cancers. Given the homology between EDD and the hyperplastic discs protein, which is required for control of imaginal disc growth in Drosophila, EDD potentially has a role in regulation of cell proliferation or differentiation. (+info)
Function of WW domains as phosphoserine- or phosphothreonine-binding modules.
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Protein-interacting modules help determine the specificity of signal transduction events, and protein phosphorylation can modulate the assembly of such modules into specific signaling complexes. Although phosphotyrosine-binding modules have been well-characterized, phosphoserine- or phosphothreonine-binding modules have not been described. WW domains are small protein modules found in various proteins that participate in cell signaling or regulation. WW domains of the essential mitotic prolyl isomerase Pin1 and the ubiquitin ligase Nedd4 bound to phosphoproteins, including physiological substrates of enzymes, in a phosphorylation-dependent manner. The Pin1 WW domain functioned as a phosphoserine- or phosphothreonine-binding module, with properties similar to those of SRC homology 2 domains. Phosphoserine- or phosphothreonine-binding activity was required for Pin1 to interact with its substrates in vitro and to perform its essential function in vivo. (+info)
Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of twitching motility.
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Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important human pathogen. The production of several virulence factors by P. aeruginosa is controlled through two quorum-sensing systems, las and rhl. We have obtained evidence that both the las and rhl quorum-sensing systems are also required for type 4 pilus-dependent twitching motility and infection by the pilus-specific phage D3112cts. Mutants which lack the ability to synthesize PAI-1, PAI-2, or both autoinducers were significantly or greatly impaired in twitching motility and in susceptibility to D3112cts. Twitching motility and phage susceptibility in the autoinducer-deficient mutants were partially restored by exposure to exogenous PAI-1 and PAI-2. Both twitching motility and infection by pilus-specific phage are believed to be dependent on the extension and retraction of polar type 4 pili. Western blot analysis of whole-cell lysates and enzyme-linked immunosorbent assays of intact cells were used to measure the amounts of pilin on the cell surfaces of las and rhl mutants relative to that of the wild type. It appears that PAI-2 plays a crucial role in twitching motility and phage infection by affecting the export and assembly of surface type 4 pili. The ability of P. aeruginosa cells to adhere to human bronchial epithelial cells was also found to be dependent on the rhl quorum-sensing system. Microscopic analysis of twitching motility indicated that mutants which were unable to synthesize PAI-1 were defective in the maintenance of cellular monolayers and migrating packs of cells. Thus, PAI-1 appears to have an essential role in maintaining cell-cell spacing and associations required for effective twitching motility. (+info)
Identification of determinants in E2 ubiquitin-conjugating enzymes required for hect E3 ubiquitin-protein ligase interaction.
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Members of the hect domain protein family are characterized by sequence similarity of their C-terminal regions to the C terminus of E6-AP, an E3 ubiquitin-protein ligase. An essential intermediate step in E6-AP-dependent ubiquitination is the formation of a thioester complex between E6-AP and ubiquitin in the presence of distinct E2 ubiquitin-conjugating enzymes including human UbcH5, a member of the UBC4/UBC5 subfamily of E2s. Similarly, several hect domain proteins, including Saccharomyces cerevisiae RSP5, form ubiquitin thioester complexes, indicating that hect domain proteins in general have E3 activity. We show here, by the use of chimeric E2s generated between UbcH5 and other E2s, that a region of UbcH5 encompassing the catalytic site cysteine residue is critical for its ability to interact with E6-AP and RSP5. Of particular importance is a phenylalanine residue at position 62 of UbcH5 that is conserved among the members of the UBC4/UBC5 subfamily but is not present in any of the other known E2s, whereas the N-terminal 60 amino acids do not contribute significantly to the specificity of these interactions. The conservation of this phenylalanine residue throughout evolution underlines the importance of the ability to interact with hect domain proteins for the cellular function of UBC4/UBC5 subfamily members. (+info)
Histone ubiquitination and chromatin remodeling in mouse spermatogenesis.
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Male infertility in HR6B knockout mice is associated with impairment of spermatogenesis. The HR6B gene is a mammalian, autosomal homolog of the Saccharomyces cerevisiae gene Rad6 encoding a ubiquitin-conjugating enzyme. In addition, X-chromosomal HR6A has been identified, in human and mouse. RAD6 in yeast is required for a variety of cellular functions, including sporulation, DNA repair, and mutagenesis. Since RAD6 and its mammalian homologs can ubiquitinate histones in vitro, we have investigated the pattern of histone ubiquitination in mouse testis. By immunoblot and immunohistochemical analysis of wild-type mouse testis, a high amount of ubiquitinated H2A (uH2A) was detected in pachytene spermatocytes. This signal became undetectable in round spermatids, but then increased again during a relatively short developmental period, in elongating spermatids. No other ubiquitinated histones were observed. In the HR6B knockout mice, we failed to detect an overt defect in the overall pattern of histone ubiquitination. For somatic cell types, it has been shown that histone ubiquitination is associated with destabilization of nucleosomes, in relation to active gene transcription. Unexpectedly, the most intense uH2A signal in pachytene spermatocytes was detected in the sex body, an inactive nuclear structure that contains the heterochromatic X and Y chromosomes. The postmeiotic uH2A immunoexpression in elongating spermatids indicates that nucleosome destabilization induced by histone ubiquitination may play a facilitating role during histone-to-protamine replacement. (+info)
A wide variety of mutations in the parkin gene are responsible for autosomal recessive parkinsonism in Europe. French Parkinson's Disease Genetics Study Group and the European Consortium on Genetic Susceptibility in Parkinson's Disease.
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Autosomal recessive juvenile parkinsonism (AR-JP, PARK2; OMIM 602544), one of the monogenic forms of Parkinson's disease (PD), was initially described in Japan. It is characterized by early onset (before age 40), marked response to levodopa treatment and levodopa-induced dyskinesias. The gene responsible for AR-JP was recently identified and designated parkin. We have analysed the 12 coding exons of the parkin gene in 35 mostly European families with early onset autosomal recessive parkinsonism. In one family, a homozygous deletion of exon 4 could be demonstrated. By direct sequencing of the exons in the index patients of the remaining 34 families, eight previously undescribed point mutations (homozygous or heterozygous) were detected in eight families that included 20 patients. The mutations segregated with the disease in the families and were not detected on 110-166 control chromosomes. Four mutations caused truncation of the parkin protein. Three were frameshifts (202-203delAG, 255delA and 321-322insGT) and one a nonsense mutation (Trp453Stop). The other four were missense mutations (Lys161Asn, Arg256Cys, Arg275Trp and Thr415Asn) that probably affect amino acids that are important for the function of the parkin protein, since they result in the same phenotype as truncating mutations or homozygous exon deletions. Mean age at onset was 38 +/- 12 years, but onset up to age 58 was observed. Mutations in the parkin gene are therefore not invariably associated with early onset parkinsonism. In many patients, the phenotype is indistinguishable from that of idiopathic PD. This study has shown that a wide variety of different mutations in the parkin gene are a common cause of autosomal recessive parkinsonism in Europe and that different types of point mutations seem to be more frequently responsible for the disease phenotype than are deletions. (+info)
Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14.
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BACKGROUND: Exit from mitosis requires inactivation of mitotic cyclin-dependent kinases (CDKs). A key mechanism of CDK inactivation is ubiquitin-mediated cyclin proteolysis, which is triggered by the late mitotic activation of a ubiquitin ligase known as the anaphase-promoting complex (APC). Activation of the APC requires its association with substoichiometric activating subunits termed Cdc20 and Hct1 (also known as Cdh1). Here, we explore the molecular function and regulation of the APC regulatory subunit Hct1 in Saccharomyces cerevisiae. RESULTS: Recombinant Hct1 activated the cyclin-ubiquitin ligase activity of APC isolated from multiple cell cycle stages. APC isolated from cells arrested in G1, or in late mitosis due to the cdc14-1 mutation, was more responsive to Hct1 than APC isolated from other stages. We found that Hct1 was phosphorylated in vivo at multiple CDK consensus sites during cell cycle stages when activity of the cyclin-dependent kinase Cdc28 is high and APC activity is low. Purified Hct1 was phosphorylated in vitro at these sites by purified Cdc28-cyclin complexes, and phosphorylation abolished the ability of Hct1 to activate the APC in vitro. The phosphatase Cdc14, which is known to be required for APC activation in vivo, was able to reverse the effects of Cdc28 by catalyzing Hct1 dephosphorylation and activation. CONCLUSIONS: We conclude that Hct1 phosphorylation is a key regulatory mechanism in the control of cyclin destruction. Phosphorylation of Hct1 provides a mechanism by which Cdc28 blocks its own inactivation during S phase and early mitosis. Following anaphase, dephosphorylation of Hct1 by Cdc14 may help initiate cyclin destruction. (+info)