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Rpn13 is a proteasome ubiquitin receptor that has emerged as a therapeutic target for human cancers. Its ubiquitin-binding activity is confined to an N-terminal Pru (pleckstrin-like receptor for ubiquitin) domain that also docks it into the proteasome, while its C-terminal DEUBAD (DEUBiquitinase ADaptor) domain recruits deubiquitinating enzyme Uch37 to the proteasome. Bis-benzylidine piperidone derivatives that were found to bind covalently to Rpn13 C88 caused the accumulation of polyubiquitinated proteins as well as ER stress-related apoptosis in various cancer cell lines, including bortezomib-resistant multiple myeloma lines. We find that a 38-amino acid peptide derived from the C-terminus of proteasome PC repeat protein hRpn2/PSMD1 binds to hRpn13 Pru domain with 12 nM affinity. By using NMR, we identify the hRpn13-interacting amino acids in this hRpn2 fragment, some of which are conserved among eukaryotes. Importantly, we find the hRpn2-derived peptide to immunoprecipitate endogenous Rpn13 from 293T cells, and to displace it from the proteasome. These findings indicate that this region of hRpn2 is the primary binding site for hRpn13 in the proteasome. Moreover, the hRpn2-derived peptide was no longer able to interact with endogenous hRpn13 when a strictly conserved phenylalanine (F948 in humans) was replaced with arginine or a stop codon, or when Y950 and I951 were substituted with aspartic acid. Finally, over-expression of the hRpn2-derived peptide leads to an increased presence of ubiquitinated proteins in 293T cells. We propose that this hRpn2-derived peptide could be used to develop peptide-based strategies that specifically target hRpn13 function in the proteasome. [ABSTRACT FROM AUTHOR]

The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases. [ABSTRACT FROM AUTHOR]

The Hippo pathway regulates cell proliferation and apoptosis through the Yes-associated protein (YAP) transcriptional activator. YAP has a well-described role in promoting cell proliferation and survival, but the precise mechanisms and transcriptional targets that underlie these properties are still unclear and likely context-dependent. We found, using siRNA-mediated knockdown, that YAP is required for proliferation in endothelial cells but not HeLa cells. Specifically, YAP is required for S-phase entry and its absence causes cells to accumulate in G1. Microarray analysis suggests that YAP mediates this effect by regulating the transcription of genes involved in the assembly and/or firing of replication origins and homologous recombination of DNA. These findings thus provide insight into the molecular mechanisms by which YAP regulates cell cycle progression. [ABSTRACT FROM AUTHOR]

Rap1GAP is a GTPase-activating protein (GAP) that specifically stimulates the GTP hydrolysis of Rap1 GTPase. Although Rap1GAP is recognized as a tumor suppressor gene and downregulated in various cancers, little is known regarding the regulation of Rap1GAP ubiquitination and degradation under physiological conditions. Here, we demonstrated that Rap1GAP is ubiquitinated and degraded through proteasome pathway in mitosis. Proteolysis of Rap1GAP requires the PLK1 kinase and β-TrCP ubiquitin ligase complex. We revealed that PLK1 interacts with Rap1GAP in vivo through recognition of an SSP motif within Rap1GAP. PLK1 phosphorylates Ser525 in conserved 524DSGHVS529 degron of Rap1GAP and promotes its interaction with β-TrCP. We also showed that Rap1GAP was a cell cycle regulator and that tight regulation of the Rap1GAP degradation in mitosis is required for cell proliferation. [ABSTRACT FROM AUTHOR]

Temperature-sensitive (ts) CHO-K1 mutant tsTM3 exhibits chromosomal instability and cell-cycle arrest in the S to G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. Previously, complementation tests with other mutants showed that tsTM3 harbors a genetic defect in the ubiquitin-activating enzyme Uba1. Sequence comparison of the Uba1 gene between wild-type and mutant cells in this study revealed that the mutant phenotype is caused by a G-to-A transition that yields a Met-to-Ile substitution at position 256 in hamster Uba1. The ts defects in tsTM3 were complemented by expression of the wild-type Uba1 tagged with green fluorescent protein. Expression of the Uba1 primarily in the nucleus appeared to rescue tsTM3 cells. Incubation at 39°C resulted in a decrease of nuclear Uba1 in tsTM3 cells, suggesting that loss of Uba1 in the nucleus may lead to the ts defects. Analyses with the fluorescent ubiquitination-based cell cycle indicator revealed that loss of function of Uba1 leads to failure of the ubiquitin system in the nucleus. Incubation at 39°C caused an increase in endogenous geminin in tsTM3 cells. A ts mutation of Uba1 found in tsTM3 cells appears to be a novel mutation reflecting the important roles of Uba1 in nucleus. [ABSTRACT FROM AUTHOR]

Eukaryotic cells invoke mechanisms to promote survival when confronted with cellular stress or damage to the genome. The protein kinase Chk1 is an integral and conserved component of the DNA damage response pathway. Mutation or inhibition of Chk1 results in mitotic death when cells are exposed to DNA damage. Oxidative stress activates a pathway that results in nuclear accumulation of the bZIP transcription factor Pap1. We report the novel finding that fission yeast Pap1 confers resistance to drug- and non-drug-induced DNA damage even when the DNA damage checkpoint is compromised. Multi-copy expression of Pap1 restores growth to chk1-deficient cells exposed to camptothecin or hydroxyurea. Unexpectedly, increased Pap1 expression also promotes survival of chk1-deficient cells with mutations in genes encoding DNA ligase (cdc17) or DNA polymerase δ (cdc6), but not DNA replication initiation mutants. The ability of Pap1 to confer resistance to DNA damage was not specific to chk1 mutants, as it also improved survival of rad1- and rad9-deficient cells in the presence of CPT. To confer resistance to DNA damage Pap1 must localize to the nucleus and be transcriptionally active. [ABSTRACT FROM AUTHOR]

The yeast HECT-family E3 ubiquitin ligase Rsp5 has been implicated in diverse cell functions. Previously, we and others [1], [2] reported the physical and functional interaction of Rsp5 with the deubiquitinating enzyme Ubp2, and the ubiquitin associated (UBA) domain-containing cofactor Rup1. To investigate the mechanism and significance of the Rsp5-Rup1-Ubp2 complex, we examined Rsp5 ubiquitination status in the presence or absence of these cofactors. We found that, similar to its mammalian homologues, Rsp5 is auto-ubiquitinated in vivo. Association with a substrate or Rup1 increased Rsp5 self-ubiquitination, whereas Ubp2 efficiently deubiquitinates Rsp5 in vivo and in vitro. The data reported here imply an auto-modulatory mechanism of Rsp5 regulation common to other E3 ligases. [ABSTRACT FROM AUTHOR]

Identification of novel proteins that can potentially contribute to carcinogenesis is a requisite venture. Herein, we report the first biochemical characterization of the novel F-box and WD40 containing protein, FBXW4. We have identified interacting protein partners and demonstrated that FBXW4 is part of a ubiquitin ligase complex. Furthermore, the Fbxw4 locus is a common site of proviral insertion in a variety of retroviral insertional mutagenesis murine cancer models and Fbxw4 mRNA is highly expressed in the involuting murine mammary gland. To begin to characterize the biochemical function of Fbxw4, we used proteomic analysis to demonstrate that Fbxw4 interacts with Skp1 (SKP1), Cullin1 (CUL1), Ring-box1 (RBX1) and all components of the COP9 signalosome. All of these interactions are dependent on an intact F-box domain of Fbxw4. Furthermore, Fbxw4 is capable of interacting with ubiquitinated proteins within cells in an F-box dependent manner. Finally, we demonstrate that FBXW4 is mutated, lost and under-expressed in a variety of human cancer cell lines and clinical patient samples. Importantly, expression of FBXW4 correlates with survival of patients with non-small cell lung cancer. Taken together, we suggest that FBXW4 may be a novel tumor suppressor that regulates important cellular processes. [ABSTRACT FROM AUTHOR]