Negative regulation of EGFR-Vav2 signaling axis by Cbl ubiquitin ligase controls EGF receptor-mediated epithelial cell adherens junction dynamics and cell migration

The E3 ubiquitin ligase Casitas B lymphoma protein (Cbl) controls the ubiquitin-dependent degradation of EGF receptor (EGFR), but its role in regulating downstream signaling elements with which it associates and its impact on biological outcomes of EGFR signaling are less clear. Here, we demonstrate that stimulation of EGFR on human mammary epithelial cells disrupts adherens junctions (AJs) through Vav2 and Rac1/Cdc42 activation. In EGF-stimulated cells, Cbl regulates the levels of phosphorylated Vav2 thereby attenuating Rac1/Cdc42 activity. Knockdown of Cbl and Cbl-b enhanced the EGF-induced disruption of AJs and cell motility. Overexpression of constitutively active Vav2 activated Rac1/Cdc42 and reorganized junctional actin cytoskeleton; these effects were suppressed by WT Cbl and enhanced by a ubiquitin ligase-deficient Cbl mutant. Cbl forms a complex with phospho-EGFR and phospho-Vav2 and facilitates phospho-Vav2 ubiquitinylation. Cbl can also interact with Vav2 directly in a Cbl Tyr-700-dependent manner. A ubiquitin ligase-deficient Cbl mutant enhanced the morphological transformation of mammary epithelial cells induced by constitutively active Vav2; this effect requires an intact Cbl Tyr-700. These results indicate that Cbl ubiquitin ligase plays a critical role in the maintenance of AJs and suppression of cell migration through down-regulation of EGFR-Vav2 signaling.     

Uncleaved BAP31 in association with A4 protein at the endoplasmic reticulum is an inhibitor of Fas-initiated release of cytochrome c from mitochondria

BAP31 is a polytopic integral protein of the endoplasmic reticulum membrane and, like BID, is a preferred substrate of caspase-8. Upon Fas/CD95 stimulation, BAP31 is cleaved within its cytosolic domain, generating proapoptotic p20 BAP31. In human KB epithelial cells expressing the caspase-resistant mutant crBAP31, Fas stimulation resulted in cleavage of BID and insertion of BAX into mitochondrial membrane, but subsequent oligomerization of BAX and BAK, egress of cytochrome c to the cytosol, and apoptosis were impaired. Bap31-null mouse cells expressing crBAP31 cannot generate the endogenous p20 BAP31 cleavage product, yet crBAP31 conferred resistance to cellular condensation and cytochrome c release in response to activation of ectopic FKBPcasp8 by FK1012z. Full-length BAP31, therefore, is a direct inhibitor of these caspase-8-initiated events, acting independently of its ability to sequester p20, with which it interacts. Employing a novel split ubiquitin yeast two-hybrid screen for BAP31-interacting membrane proteins, the putative ion channel protein of the endoplasmic reticulum, A4, was detected and identified as a constitutive binding partner of BAP31 in human cells. Ectopic A4 that was introduced into A4-deficient cells cooperated with crBAP31 to resist Fas-induced egress of cytochrome c from mitochondria and cytoplasmic apoptosis.     

Detection of MAPK signal transduction proteins in an ischemia/reperfusion model of mouse intestine using in vivo cryotechnique

Intestinal ischemia and ischemia–reperfusion rapidly progress to tissue destruction and reconstruction of functional organs. To date, precise immunolocalizations and the timing of appearance of cell signaling components under such conditions have not been well visualized. Mitogen-activated protein kinase (MAPK) signal transduction pathways have been reported to be activated under various types of cell damage, and cyclic AMP response element-binding protein (CREB) was directly phosphorylated with various cellular stimuli. In this study, both the expression and the immunolocalization of ERK1/2, a member of the MAPK family, were examined in mouse intestinal tissues by in vivo cryotechnique, which is useful to retain soluble molecules including cell signaling molecules. Under normal conditions, although ERK was widely immunolocalized in the cytoplasm of epithelial cells, phosphorylated (p) ERK1/2 was slightly detected in a small amount of epithelial cells in crypt and top parts of the villi. In 5 min ischemia, more pERK1/2 immunolocalization was detected in epithelial cells of the crypt part. Up to 60 min, the pERK1/2 immunoreactivity was remarkably increased in wide areas of epithelial cells. In the 20 and 60 min ischemia groups, phosphorylated CREB was also immunostained in the nuclei of the same epithelial cell areas of pERK1/2. In 20 min ischemia with 60 min reperfusion experiments, pERK1/2 immunointensity was reduced in the crypt areas. In 60 min ischemia with 60 min reperfusion, however, it was still strongly immunolocalized in epithelial cells of the crypts. Thus, rapidly changing ERK1/2 phosphorylation was visualized in the intestinal epithelial stem cells of mouse small intestine.     

Overexpression of Smurf1 negatively regulates mouse embryonic lung branching morphogenesis by specifically reducing Smad1 and Smad5 proteins

Early embryonic lung branching morphogenesis is regulated by many growth factor-mediated pathways. Bone morphogenetic protein 4 (BMP4) is one of the morphogens that stimulate epithelial branching in mouse embryonic lung explant culture. To further understand the molecular mechanisms of BMP4-regulated lung development, we studied the biological role of Smad-ubiquitin regulatory factor 1 (Smurf1), an ubiquitin ligase specific for BMP receptor-regulated Smads, during mouse lung development. The temporo-spatial expression pattern of Smurf1 in mouse embryonic lung was first determined by quantitative real-time PCR and immunohistochemistry. Overexpression of Smurf1 in airway epithelial cells by intratracheal introduction of recombinant adenoviral vector dramatically inhibited embryonic day (E) 11.5 lung explant growth in vitro. This inhibition of lung epithelial branching was restored by coexpression of Smad1 or by addition of soluble BMP4 ligand into the culture medium. Studies at the cellular level show that overexpression of Smurf1 reduced epithelial cell proliferation and differentiation, as documented by reduced PCNA-positive cell index and by reduced mRNA levels for surfactant protein C and Clara cell protein 10 expression. Further studies found that overexpression of Smurf1 reduced BMP-specific Smad1 and Smad5, but not Smad8, protein levels. Thus overexpression of Smurf1 specifically promotes Smad1 and Smad5 ubiquitination and degradation in embryonic lung epithelium, thereby modulating the effects of BMP4 on embryonic lung growth.     

The Insect Peptide CopA3 Increases Colonic Epithelial Cell Proliferation and Mucosal Barrier Function to Prevent Inflammatory Responses in the Gut

The epithelial cells of the gut form a physical barrier against the luminal contents. The collapse of this barrier causes inflammation, and its therapeutic restoration can protect the gut against inflammation. EGF enhances mucosal barrier function and increases colonocyte proliferation, thereby ameliorating inflammatory responses in the gut. Based on our previous finding that the insect peptide CopA3 promotes neuronal growth, we herein tested whether CopA3 could increase the cell proliferation of colonocytes, enhance mucosal barrier function, and ameliorate gut inflammation. Our results revealed that CopA3 significantly increased epithelial cell proliferation in mouse colonic crypts and also enhanced colonic epithelial barrier function. Moreover, CopA3 treatment ameliorated Clostridium difficile toxin As-induced inflammation responses in the mouse small intestine (acute enteritis) and completely blocked inflammatory responses and subsequent lethality in the dextran sulfate sodium-induced mouse model of chronic colitis. The marked CopA3-induced increase of colonocyte proliferation was found to require rapid protein degradation of p21^Cip1/Waf1, and an in vitro ubiquitination assay revealed that CopA3 directly facilitated ubiquitin ligase activity against p21^Cip1/Waf1. Taken together, our findings indicate that the insect peptide CopA3 prevents gut inflammation by increasing epithelial cell proliferation and mucosal barrier function.     

Identification of components of the murine histone deacetylase 6 complex: link between acetylation and ubiquitination signaling pathways.

The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.     

Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2

Insulin and insulin-like growth factor-1 (IGF-1) regulate metabolism and body growth through homologous receptor tyrosine kinases that phosphorylate the insulin receptor substrate (IRS) proteins. IRS-2 is an important IRS protein, as it mediates peripheral insulin action and beta-cell survival. In this study, we show that insulin, IGF-1, or osmotic stress promoted ubiquitin/proteasome-mediated degradation of IRS-2 in 3T3-L1 cells, Fao hepatoma, cells and mouse embryo fibroblasts; however, insulin/IGF-1 did not promote degradation of IRS-1 in 3T3-L1 preadipocytes or mouse embryo fibroblasts. MG132 or lactacystin, specific inhibitors of 26S proteasome, blocked insulin/IGF-1-induced degradation of IRS-2 and enhanced the detection of ubiquitinated IRS-2. Insulin/IGF1-induced ubiquitination and degradation of IRS-2 was blocked by inhibitors of phosphatidylinositol 3-kinase (wortmannin or LY294002) or mTOR (rapamycin). Chronic insulin or IGF-1 treatment of IRS-1-deficient mouse embryo fibroblasts inhibited IRS-2-mediated activation of Akt and ERK1/2, which was reversed by lactacystin pretreatment. By contrast, IRS-1 activation of Akt and ERK1/2 was not inhibited by chronic insulin/IGF-1 stimulation in IRS-2-deficient mouse embryo fibroblasts. Thus, we identified a novel negative feedback mechanism by which the ubiquitin/proteasome-mediated degradation of IRS-2 limits the magnitude and duration of the response to insulin or IGF-1.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of (3H] uridine incorporation into RNA and [3H] leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10-21 M). Insulin stimulated the rate of [3H] thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100-1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H] thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of 3H- uridine, [3H] thymidine and [3H] leucine into their respective precursor pools is not responsible for the apparent stimulation of RNA, DNA and protein synthesis.     

Ezrin ubiquitylation by the E3 ubiquitin ligase, WWP1, and consequent regulation of hepatocyte growth factor receptor activity

The membrane cytoskeleton linker ezrin participates in several functions downstream of the receptor Met in response to Hepatocyte Growth Factor (HGF) stimulation. Here we report a novel interaction of ezrin with a HECT E3 ubiquitin ligase, WWP1/Aip5/Tiul1, a potential oncogene that undergoes genomic amplification and overexpression in human breast and prostate cancers. We show that ezrin binds to the WW domains of WWP1 via the consensus motif PPVY(477) present in ezrin's C-terminus. This association results in the ubiquitylation of ezrin, a process that requires an intact PPVY(477) motif. Interestingly ezrin ubiquitylation does not target the protein for degradation by the proteasome. We find that ezrin ubiquitylation by WWP1 in epithelial cells leads to the upregulation of Met level in absence of HGF stimulation and increases the response of Met to HGF stimulation as measured by the ability of the cells to heal a wound. Interestingly this effect requires ubiquitylated ezrin since it can be rescued, after depletion of endogenous ezrin, by wild type ezrin but not by a mutant of ezrin that cannot be ubiquitylated. Taken together our data reveal a new role for ezrin in Met receptor stability and activity through its association with the E3 ubiquitin ligase WWP1. Given the role of Met in cell proliferation and tumorigenesis, our results may provide a mechanistic basis for understanding the role of ezrin in tumor progression.     

The E3 ubiquitin ligase activity of Trip12 is essential for mouse embryogenesis

Protein ubiquitination is a post-translational protein modification that regulates many biological conditions. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12(mt/mt)) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12(mt/mt) embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16. In contrast, Trip12(mt/mt) ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12(mt/mt) ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.     

The eye lens has an active ubiquitin-protein conjugation system

Using exogenous 125I-ubiquitin, ubiquitin-lens protein conjugation was observed with supernatants of cultured rabbit lens epithelial cells and lens cortex tissue. Conjugation was ATP-dependent with the greatest variety and amount of conjugates larger than 150 kDa. In vivo production of ubiquitin-protein conjugates in cultured rabbit and beef lens epithelial cells and rabbit lens tissues of different developmental age was established using immunological detection. There were limited similarities between conjugates found in youngest as opposed to oldest tissue. Cultured rabbit cells contained 27 pmol/mg free ubiquitin and 18 pmol/mg conjugated ubiquitin. Levels of free ubiquitin in lens tissue epithelium, cortex, and core were 36, 5, and 5 pmol/mg, respectively. There were only 2 pmol/mg conjugated ubiquitin in each of these tissues. Hydrolysis of 125I-ubiquitin was catalyzed by supernatants of cultured lens cells, beef and human lens tissues, and reticulocytes. Degradation was greatest in epithelial tissues, and least in core. This corroborates studies which show that proteolytic capabilities are attenuated in older tissue. Decreased initiation of proteolysis by ubiquitination as well as diminished proteolysis in older lens tissue may be related to the accumulation of damaged proteins in aging lens tissue.     

Mechanism of extracellular ubiquitination in the mammalian epididymis

Posttranslational modification by ubiquitination marks defective or outlived intracellular proteins for proteolytic degradation by the 26S proteasome. The ATP-dependent, covalent ligation and formation of polyubiquitin chains on substrate proteins requires the presence and activity of a set of ubiquitin activating and conjugating enzymes. While protein ubiquitination typically occurs in the cell cytosol or nucleus, defective mammalian spermatozoa become ubiquitinated on their surface during post-testicular sperm maturation in the epididymis, suggesting an active molecular mechanism for sperm quality control. Consequently, we hypothesized that the bioactive constituents of ubiquitin-proteasome pathway were secreted in the mammalian epididymal fluid (EF) and capable of ubiquitinating extrinsic substrates. Western blotting indeed detected the presence of the ubiquitin-activating enzyme E1 and presumed E1-ubiquitin thiol-ester intermediates, ubiquitin-carrier enzyme E2 and presumed E2-ubiquitin thiol-ester intermediates and the ubiquitin C-terminal hydrolase PGP 9.5/UCHL1 in the isolated bovine EF. Thiol-ester assays utilizing recombinant ubiquitin-activating and ubiquitin-conjugating enzymes, biotinylated substrates, and isolated bovine EF confirmed the activity of the ubiquitin activating and conjugating enzymes within EF. Ubiquitinated proteins were found to be enriched in the defective bull sperm fraction and appropriate proteasomal deubiquitinating and proteolytic activities were measured in the isolated EF by specific fluorescent substrates. The apocrine secretion of cytosolic proteins was visualized in transgenic mice and rats expressing the enhanced green fluorescent protein (eGFP) under the direction of ubiquitin-C promoter. Accumulation of eGFP, ubiquitin and proteasomes was detected in the apical blebs, the apocrine secretion sites of the caput epididymal epithelia of both the rat and mouse epididymal epithelium, although region-specific differences exist. Secretion of eGFP and proteasomes continued during the prolonged culture of the isolated rat epididymal epithelial cells in vitro. This study provides evidence that the activity of the ubiquitin system is not limited to the intracellular environment, contributing to a greater understanding of the sperm maturation process during epididymal passage.