WWP1 Research Articles

The R436Q missense mutation in WWP1 disrupts autoinhibition of its E3 ubiquitin ligase activity, leading to self-degradation and loss of function.

Muscular dystrophy in the NH-413 chicken is caused by a missense mutation in the WWP1 gene. WWP1 is a HECT-type E3 ubiquitin ligase containing four tandem WW domains that interact with proline-rich peptide motifs of target proteins, and a short region connecting the second and third WW domains is crucial for the E3 ligase to maintain an autoinhibitory state. A mutation of the arginine in the WW2-WW3 linker to glutamine is thought to affect WWP1 function, but there is little information on this mutation to date. In this study, we generated a transgenic (Tg) mouse model expressing the WWP1 transgene with the R436Q mutation, which corresponds to the missense mutation found in the NH-413 chicken. Tg mice showed marked degradation of mutant WWP1 proteins in various tissues, particularly in striated muscle. Immunoprecipitation analysis using a WWP1-specific antibody demonstrated that the mutant WWP1 proteins lacked the C-terminal catalytic cysteine residue that is required for their binding to the E2-substrate complex during their degradation. In vitro analysis using the R436Q mutant of WWP1 lacking this catalytic cysteine residue showed no autodegradation, indicating that the loss-of-function degradation of this protein is caused by self-ubiquitination. Tg mice expressing R436Q WWP1 did not show stunted growth or premature death. Furthermore, histological analysis did not reveal any obvious changes. These observations suggested that the R436Q mutant WWP1 protein, which is released from autoinhibitory mode by its missense mutation, does not have abnormally activated enzyme function to substrates before its self-degradation and loss of enzyme function.

Caveolin-3: A Causative Process of Chicken Muscular Dystrophy.

The etiology of chicken muscular dystrophy is the synthesis of aberrant WW domain containing E3 ubiquitin-protein ligase 1 (WWP1) protein made by a missense mutation of WWP1 gene. The β-dystroglycan that confers stability to sarcolemma was identified as a substrate of WWP protein, which induces the next molecular collapse. The aberrant WWP1 increases the ubiquitin ligase-mediated ubiquitination following severe degradation of sarcolemmal and cytoplasmic β-dystroglycan, and an erased β-dystroglycan in dystrophic αW fibers will lead to molecular imperfection of the dystrophin-glycoprotein complex (DGC). The DGC is a core protein of costamere that is an essential part of force transduction and protects the muscle fibers from contraction-induced damage. Caveolin-3 (Cav-3) and dystrophin bind competitively to the same site of β-dystroglycan, and excessive Cav-3 on sarcolemma will block the interaction of dystrophin with β-dystroglycan, which is another reason for the disruption of the DGC. It is known that fast-twitch glycolytic fibers are more sensitive and vulnerable to contraction-induced small tears than slow-twitch oxidative fibers under a variety of diseased conditions. Accordingly, the fast glycolytic αW fibers must be easy with rapid damage of sarcolemma corruption seen in chicken muscular dystrophy, but the slow oxidative fibers are able to escape from these damages.

The muscular dystrophic chicken is hypernatremic

ABSTRACT1. The E3 ubiquitin protein ligase 1 (WWP1) gene, the mutation of which causes muscular dystrophy in chickens, is expressed not only in the pectoral muscle, but also in a number of tissues such as the kidney. Therefore, this study examined some parameters related to kidney function in muscular dystrophic (MD) chickens.2. Plasma osmolality, Na+ and K+ concentrations, aldosterone levels, and the expression of aquaporin (AQP) 2, AQP3, and α subunits of the amiloride-sensitive epithelial sodium channel (αENaC) were analysed in the kidneys of 5-week-old MD chickens and White Leghorn (WL) chickens under physiological conditions or after one day of water deprivation.3. Plasma osmolality, Na+ concentrations, and plasma aldosterone levels were significantly higher in MD chickens than in WL chickens. αENaC mRNA expression levels were lower in MD chickens than in WL chickens. AQP2 and AQP3 mRNA expression levels were similar in the two strains of chickens.4. Plasma osmolality correlated with aldosterone levels and AQP2 and αENaC mRNA levels in WL chickens. In MD chickens, plasma osmolality correlated with AQP2 mRNA levels, but not with plasma aldosterone or αENaC mRNA levels.5. These results suggest that neither water reabsorption nor the expression of AQP2 and AQP3 is impaired in MD chickens and that a WWP1 gene mutation may or may not directly induce an abnormality in Na+-reabsorption in the kidneys of MD chickens, potentially through αENaC.

PTEN Regulation by WWP2

WWP2 is a member of NEDD4 family HECT E3 ubiquitin ligases. The architecture of the NEDD4 family proteins include an N‐terminal C2 domain, two to four WW domains, and a C‐terminal HECT catalytic domain. The NEDD4 family E3 ligases play important regulatory roles in development and differentiation, immunological function, and cancer development, and their activity needs to be tightly regulated to prevent pathogenesis. WWP2 is the E3 ligase that catalyzes Lys ubiquitination of numerous proteins including key transcription factors such as OCT4 and signaling molecules such as PTEN. Our lab has been interested in studying how PTEN C‐tail phosphorylation (residues Ser380, Thr382, Thr383, and Ser385) regulates its ubiquitination by WWP2. Using expressed protein ligation, we generated semisynthetic non‐phosphorylated and tetra‐phosphorylated PTEN, and found that the non‐phosphorylated PTEN is more efficiently ubiquitinated by WWP2 than the phosphorylated PTEN, indicating that C‐tail phosphorylation of PTEN could inhibit its ubiquitination by WWP2. In the course of our studies, we have found that WWP2 itself is regulated by autoinhibition. We have dissected aspects of WWP2 that contribute to its autoinhibition and will discuss how the protein may be regulated to ensure proper catalytic tone.

Knockdown of WWP1 inhibits growth and induces apoptosis in hepatoma carcinoma cells through the activation of caspase3 and p53

The activation of oncogenes and the loss of tumor suppressor genes are believed to play critical roles in the pathogenesis of human hepatocellular carcinoma (HCC). The human WW domain containing E3 ubiquitin protein ligase 1 (WWP1) gene is frequently amplified in prostate and breast cancers, however, its role in cancer has not yet been extensively studied. Especially, the role of WWP1 in HCC has not yet been studied. Firstly, we analyzed the expression of WWP1 in HCC samples. We found that protein levels of WWP1 are higher in most HCC cancerous tissues as compared with their matched adjacent non-tumor tissues. Additionally, the WWP1 mRNA was also amplified in all 7 HCC tissues. Knockdown of the endogenous WWP1 using small interfering RNA further showed that deficiency of WWP1 suppressed cell growth and caused apoptosis in HCC cells. Knocking down WWP1 promoted cleaved caspase3 protein and p53 expression in HCC cells, and caspase3 inhibition could prevent cell apoptosis induced by the knockdown of WWP1. All together these results indicate that protein levels of WWP1 in most HCC tissues are higher than non-tumor tissues, and knockdown of WWP1 inhibits growth and induces apoptosis in HCC cells through the activation of caspase3 and p53. Therefore, WWP1 gene might be a potential molecular target of HCC.

MP58-14 DECISION ANALYSIS MODEL TO FACILITATE TREATMENT DECISION-MAKING FOR LOCALIZED PROSTATE CANCER: RESULTS OF A RANDOMIZED TRIAL

andwhether thepatientwasseenbya urologist only or both a urologist and radiation oncologist. Univariate and multivariable logistic regression was used to identify predictors of initial treatment choice within 6 months of diagnosis. RESULTS: Common treatment choices were RP (48%), XRT (21%), and WW (15%). In both univariate and multivariable analyses, men seen by a urologist only were more likely to choose WW (p1⁄40.03 and p1⁄40.02, respectively); employed men were less likely to choose WW (p1⁄40.01 and p1⁄40.02, respectively). In multivariable analysis, married/partnered men were more likely to choose surgery (p1⁄40.03); anxiety was not statistically significantly associated with any treatment choice. CONCLUSIONS: Our findings suggest that treatment choice is related to social infrastructure and type of specialist seen. These findings should guide counseling of patients with newly diagnosed localized CaP. Further studies related to the role of sociodemographic characteristics and access to specialty care in initial treatment choice are warranted.

WWP1 gene is a potential molecular target of human oral cancer

This study investigates the oncogenic role of WWP1, an ubiquitin ligase linked to tumor promotion, in oral cancer. An array-based comparative genomic hybridization was used to detect chromosomal changes in 10 oral cancer specimens. An additional 59 specimens and 6 cultured oral cancer cells were further examined to evaluate changes in the DNA copy number and messenger RNA (mRNA) and protein expression of WWP1. The copy number of the WWP1 gene and its mRNA levels were significantly increased in the oral cancer specimens. An elevated WWP1 gene expression was observed in 6 cultured oral cancer cell lines. Knockdown of the endogenous WWP1using small hairpin RNA further showed that deficiency of WWP1 suppressed cell growth and caused apoptosis in oral cancer cells. Our results reveal that WWP1 might play an oncogenic role in oral cancer cells.

Identification of the Gene Responsible for Chicken Muscular Dystrophy

By a series of positional cloning, we successfully narrowed down the AM candidate region to approximately 1.2 Mbp on GGA2q including 7 functional genes. Subsequently, we identified WWP1 gene as the most likely AM candidate by sequence comparison. The amino acid sequence around the candidate mutation was highly conserved among tetrapods, suggesting that WWP1 is the causative gene of chicken muscular dystrophy. Transfection of mutated WWP1 gene into C₂C₁₂ myoblasts disrupted muscle differentiation process. The abnormal muscle differentiation is a characteristic of chicken muscular dystrophy, so we could demonstrate a part of phenotype of the disease. Furthermore, western blotting revealed that accumulation of caveolin-3 protein is limited in damaged muscle of muscular dystrophic chicken, suggesting caveolin-3 may be associated with the pathological change of the disease. We could conclude that WWP1 gene is the responsible one for chicken muscular dystrophy from these results, but the mechanism leading the onset should be clarified in the future. The information will contribute to the study of chicken muscular dystrophy and the corresponding human dystrophies.

The WWP1 ubiquitin E3 ligase increases TRAIL resistance in breast cancer

WW domain containing E3 ubiquitin protein ligase 1 (WWP1) is an HECT domain-containing E3 ligase regulating apoptosis. The WWP1 gene is frequently amplified and overexpressed in estrogen receptor α (ERα)-positive breast cancer. Inhibition of WWP1 by siRNA induced apoptosis in MCF7 and HCC1500. In our study, we demonstrate that WWP1 depletion by siRNA activated the extrinsic apoptotic pathway. WWP1 depletion-induced apoptosis was rescued by the overexpression of the wild-type WWP1 but not the E3 ligase inactive WWP1-C890A mutant in MCF7 cells. In contrast, WWP1-C890A enhanced apoptosis, suggesting that the E3 ligase activity is required for WWP1 to promote cell survival. The expression levels of WWP1 in four breast cancer cell lines were specifically correlated with the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance, but not TNFα and doxorubicin resistance. Both WWP1 depletion and dominant negative WWP1 overexpression increased the TRAIL-induced caspase-8 recruitment and apoptosis although WWP1 did not regulate FLIP and death receptor levels. Depletion of the initial caspase-8 blocked WWP1 inhibition-induced apoptosis in MCF7. These findings suggest that inhibition of WWP1 may be combined with TRAIL to suppress ERα-positive breast cancer cell survival.

Suppression of WWP1 Gene Via RNAi Induced the Reduction of Proliferation Rate of C2C12 Myoblasts

The WW domain containing E3 ubiquitin protein ligase 1 (WWP1), which plays an important role in ubiquitin-proteasome pathway to degrade unneeded or damaged proteins, was recently identified as the responsible for chicken muscular dystrophy. Despite intensive studies on oncogenic characters, the role of WWP1 to muscular diseases has not yet been fully understood. Previously, we transfected either of wild and mutated types of WWP1 gene into C2C12 mouse myoblasts to monitor the expression pattern of muscle-differentiation markers, so that excessive WWP1 expression enhanced the expression of the myosin heavy chain (MyHC) Ia gene but lowered the expression of the MyHC IIb gene, while mutated WWP1 gene transfected into myoblasts was distinct from these cases in that the MyHC gene or genes expression inhibited the normal myoblast differentiation. However, the mechanism for the mutation to inhibit muscle differentiation remains elucidated. The current study attempted to suppress the WWP1 expression by RNAi technique and to observe its effect on C2C12 cells. The effect of WWP1 suppression was clearly different from that of the R441Q missense mutation in the WWP1 gene. The WWP1 suppression reduced the proliferation rate of C2C12 myoblasts, while clear difference was not observed in the proliferation rate by the transfection of the mutated WWP1 gene into the cells. Our RT-PCR analysis indicated that the reduction of the WWP1 expression is the specific effect mediated by RNAi and that the reduction of proliferation rate observed in this study is largely attributed to the suppression of the WWP1 expression. These data indicated that the mutation responsible for chicken muscular dystrophy does not eliminate the enzymatic activity and provides some new function for the gene.

Expression Pattern of WWP1 in Muscular Dystrophic and Normal Chickens

The WW domain containing E3 ubiquitin protein ligase 1 (WWP1) is classified into one of ubiquitin ligases which play an important role in ubiquitin-proteasome pathway. Previously, we identified the WWP1 gene as a candidate gene of chicken muscular dystrophy by linkage analysis and sequence comparison. However, the mechanism causing pathological changes and underlaying gene function remains elucidated. In the present study, we analyzed the WWP1 gene expression in various muscles and tissues of normal chickens, and compared with those from muscular dystrophic chickens. Two mRNA isoforms were detected in all tissues examined and revealed almost equal expression level. The WWP1 expression of dystrophic chickens was decreased in almost all skeletal muscles including unaffected muscles. These data indicate that there might not be a causal relationship between the alteration of WWP1 expression level and the severity of muscular dystrophy.

The amplified WWP1 gene is a potential molecular target in breast cancer

The amplification of the q21 band of chromosome 8 (8q21) occurs in a large percentage of breast cancers. WWP1, an HECT domain-containing ubiquitin E3 ligase located in the 8q21 region, negatively regulates the TGF-beta tumor suppressor pathway. To characterize the role of WWP1 in breast cancer, we analyzed WWP1 gene dosage and expression level as well as WWP1's function. A copy number gain of WWP1 was found in 51% (18/35) of breast cancer cell lines and in 41% (17/41) of primary breast tumors. Expression of WWP1 mRNA was analyzed with real-time RT-PCR, Northern blot, and Western blot. WWP1 mRNA is up-regulated in 58% (19/33) of breast cancer cell lines, and overexpression of WWP1 is significantly correlated with a gene copy number gain. In a panel of cDNA from primary breast tumors and normal tissues, expression of WWP1 in tumors is significantly higher than that in normal tissues. Functionally, RNAi-mediated WWP1 knockdown significantly induced cell growth arrest and apoptosis in the MCF7 and HCC1500 breast cancer cell lines. Consistently, WWP1 inhibition activated caspases. Forced overexpression of WWP1 by the lentiviral system in 2 immortalized breast epithelial cell lines MCF10A and 184B5 promoted cell proliferation. These results suggest that genomic aberrations of WWP1 may contribute to the pathogenesis of breast cancer.

Proteasomal degradation of the KLF5 transcription factor through a ubiquitin-independent pathway

KLF5 is a Kruppel-like zinc finger transcription factor modulating cell proliferation, differentiation, cell cycle, apoptosis, and angiogenesis. The KLF5 protein undergoes multiple posttranslational modifications including phosphorylation, acetylation and ubiquitination. We have demonstrated that the KLF5 protein can be ubiquitinated by the WWP1 E3 ubiquitin ligase and degraded by the proteasome. In this study, we found that KLF5 protein degradation is blocked by an N-terminal FLAG tag or a small N-terminal deletion without reducing ubiquitination and degradation mediated by WWP1. Interestingly, the N-terminal fragments of KLF5 containing the first 237 or 171 amino acids are as unstable as the full length KLF5 protein. The N-terminal FLAG tag or 19 amino acid deletion also delayed the degradation of the C-terminal truncated KLF5 proteins. To further understand the mechanism, we generated a lysine-less mutant KLF5(1–171). This mutant is efficiently degraded by the proteasome without ubiquitination in vitro and in vivo. These findings suggest that KLF5 protein degradation by the proteasome could be regulated in a ubiquitin-independent manner.

Ubiquitin E3 ligase WWP1 as an oncogenic factor in human prostate cancer

The gene for E3 ubiquitin ligase WWP1 is located at 8q21, a region frequently amplified in human cancers, including prostate cancer. Recent studies have shown that WWP1 negatively regulates the TGFbeta tumor suppressor pathway by inactivating its molecular components, including Smad2, Smad4 and TbetaR1. These findings suggest an oncogenic role of WWP1 in carcinogenesis, but direct supporting evidence has been lacking. In this study, we examined WWP1 for gene dosage, mRNA expression, mutation and functions in a number of human prostate cancer samples. We found that the WWP1 gene had copy number gain in 15 of 34 (44%) xenografts and cell lines from prostate cancer and 15 of 49 (31%) clinical prostate cancer samples. Consistently, WWP1 was overexpressed in 60% of xenografts and cell lines from prostate cancer. Mutation of WWP1 occurred infrequently in prostate cancer. Functionally, WWP1 overexpression promoted colony formation in the 22Rv1 prostate cancer cell line. In PC-3 prostate cancer cells, WWP1 knockdown significantly suppressed cell proliferation and enhanced TGFbeta-mediated growth inhibition. These findings suggest that WWP1 is an oncogene that undergoes genomic amplification at 8q21 in human prostate cancer, and WWP1 overexpression is a common mechanism involved in the inactivation of TGFbeta function in human cancer.