Inferring Protein Function in an Emerging Virus: Detection of the Nucleoprotein in Tilapia Lake Virus.

Emerging viruses impose global threats to animal and human populations and may bear novel genes with limited homology to known sequences, necessitating the development of novel approaches to infer and test protein functions. This challenge is dramatically evident in tilapia lake virus (TiLV), an emerging "orthomyxo-like" virus that threatens the global tilapia aquaculture and food security of millions of people. The majority of TiLV proteins have no homology to known sequences, impeding functionality assessments. Using a novel bioinformatics approach, we predicted that TiLV's Protein 4 encodes the nucleoprotein, a factor essential for viral RNA replication. Multiple methodologies revealed the expected properties of orthomyxoviral nucleoproteins. A modified yeast three-hybrid assay detected Protein 4-RNA interactions, which were independent of the RNA sequence, and identified specific positively charged residues involved. Protein 4-RNA interactions were uncovered by R-DeeP and XRNAX methodologies. Immunoelectron microscopy found that multiple Protein 4 copies localized along enriched ribonucleoproteins. TiLV RNA from cells and virions coimmunoprecipitated with Protein 4. Immunofluorescence microscopy detected Protein 4 in the cytoplasm and nuclei, and nuclear Protein 4 increased upon CRM1 inhibition, suggesting CRM1-dependent nuclear export of TiLV RNA. Together, these data reveal TiLV's nucleoprotein and highlight the ability to infer protein functionality, including novel RNA-binding proteins, in emerging pathogens. These are important in light of the expected discovery of many unknown viruses and the zoonotic potential of such pathogens. IMPORTANCE Tilapia is an important source of dietary protein, especially in developing countries. Massive losses of tilapia were identified worldwide, risking the food security of millions of people. Tilapia lake virus (TiLV) is an emerging pathogen responsible for these disease outbreaks. TiLV's genome encodes 10 major proteins, 9 of which show no homology to other known viral or cellular proteins, hindering functionality assessment of these proteins. Here, we describe a novel bioinformatics approach to infer the functionality of TiLV proteins, which predicted Protein 4 as the nucleoprotein, a factor essential for viral RNA replication. We provided experimental support for this prediction by applying multiple molecular, biochemical, and imaging approaches. Overall, we illustrate a strategy for functional analyses in viral discovery. The strategy is important in light of the expected discovery of many unknown viruses and the zoonotic potential of such pathogens.

In vivo anti-MUC1+ tumor activity and sequences of high-affinity anti-MUC1-SEA antibodies.

Cleavage of the MUC1 glycoprotein yields two subunits, an extracellular alpha-subunit bound to a smaller transmembrane beta-subunit. Monoclonal antibodies (mAbs) directed against the MUC1 alpha-beta junction comprising the SEA domain, a stable cell-surface moiety, were generated. Sequencing of all seven anti-SEA domain mAbs showed that they clustered into four groups and sequences of all groups are presented here. mAb DMB5F3 with picomolar affinity for the MUC1 SEA target was selected for further evaluation. Immunohistochemical staining of a series of malignancies with DMB5F3 including lung, prostate, breast, colon, and pancreatic carcinomas revealed qualitative and qualitative differences between MUC1 expression on normal versus malignant cells: DMB5F3 strongly stained malignant cells in a near-circumferential pattern, whereas MUC1 in normal pancreatic and breast tissue showed only weak apical positivity of ductal/acinar cells. Humanized chimeric DMB5F3 linked to ZZ-PE38 (ZZ IgG-binding protein fused to Pseudomonas exotoxin) induced vigorous cytotoxicity of MUC1+ malignant cells in vitro. The intensity of cell killing correlated with the level of MUC1 expression by the target cell, suggesting a MUC1 expression threshold for cell killing. MUC1+ Colo357 pancreatic cancer cells xenotransplanted into nude and SCID mice models were treated with the chDMB5F3:ZZ-PE38 immunocomplex. In both transplant models, chDMB5F3:ZZ-PE38 exhibited significant in vivo anti-tumor activity, suppressing up to 90% of tumor volume in the SCID model compared with concomitant controls. The efficacy of chDMB5F3:ZZ-PE38 immunotoxin in mediating tumor killing both in vitro and in vivo strongly suggests a clinical role for anti-MUC1 SEA antibody in the treatment of MUC1-expressing malignancies.

Requirement of ATM-dependent monoubiquitylation of histone H2B for timely repair of DNA double-strand breaks.

The cellular response to DNA double-strand breaks (DSBs) is mobilized by the protein kinase ATM, which phosphorylates key players in the DNA damage response (DDR) network. A major question is how ATM controls DSB repair. Optimal repair requires chromatin relaxation at damaged sites. Chromatin reorganization is coupled to dynamic alterations in histone posttranslational modifications. Here, we show that in human cells, DSBs induce monoubiquitylation of histone H2B, a modification that is associated in undamaged cells with transcription elongation. We find that this process relies on recruitment to DSB sites and ATM-dependent phosphorylation of the responsible E3 ubiquitin ligase: the RNF20-RNF40 heterodimer. H2B monoubiquitylation is required for timely recruitment of players in the two major DSB repair pathways-nonhomologous end-joining and homologous recombination repair-and optimal repair via both pathways. Our data and previous data suggest a two-stage model for chromatin decondensation that facilitates DSB repair.

Differential molecular regulation of processing and membrane expression of Type-I BMP receptors: implications for signaling.

The Type-I bone morphogenetic protein receptors (BMPRs), BMPR1A and BMPR1B, present the highest sequence homology among BMPRs, suggestive of functional similitude. However, sequence elements within their extracellular domain, such as signal sequence or N-glycosylation motifs, may result in differential regulation of biosynthetic processing and trafficking and in alterations to receptor function. We show that (i) BMPR1A and the ubiquitous isoform of BMPR1B differed in mode of translocation into the endoplasmic reticulum; and (ii) BMPR1A was N-glycosylated while BMPR1B was not, resulting in greater efficiency of processing and plasma membrane expression of BMPR1A. We further demonstrated the importance of BMPR1A expression and glycosylation in ES-2 ovarian cancer cells, where (i) CRISPR/Cas9-mediated knockout of BMPR1A abrogated BMP2-induced Smad1/5/8 phosphorylation and reduced proliferation of ES-2 cells and (ii) inhibition of N-glycosylation by site-directed mutagenesis, or by tunicamycin or 2-deoxy-D-glucose treatments, reduced biosynthetic processing and plasma membrane expression of BMPR1A and BMP2-induced Smad1/5/8 phosphorylation.

Constitutive negative regulation in the processing of the anti-Müllerian hormone receptor II.

The levels and intracellular localization of wild-type transforming growth factor ß superfamily (TGFß-SF) receptors are tightly regulated by endocytic trafficking, shedding and degradation. In contrast, a main regulatory mechanism of mutation-bearing receptors involves their intracellular retention. Anti-Müllerian hormone receptor II (AMHRII, also known as AMHR2) is the type-II receptor for anti-Müllerian hormone (AMH), a TGFß-SF ligand that mediates Müllerian duct regression in males. Here, we studied AMHRII processing and identified novel mechanisms of its constitutive negative regulation. Immunoblot analysis revealed that a significant portion of AMHRII was missing most of its extracellular domain (ECD) and, although glycosylated, was unfolded and retained in the endoplasmic reticulum. Exogenous expression of AMHRII, but not of type-II TGF-ß receptor (TßRII, also known as TGFR2), resulted in its disulfide-bond-mediated homo-oligomerization and intracellular retention, and in a decrease in its AMH-binding capacity. At the plasma membrane, AMHRII differed from TßRII, forming high levels of non-covalent homomeric complexes, which exhibited a clustered distribution and restricted lateral mobility. This study identifies novel mechanisms of negative regulation of a type-II TGFß-SF receptor through cleavage, intracellular retention and/or promiscuous disulfide-bond mediated homo-oligomerization.

FIE, a nuclear PRC2 protein, forms cytoplasmic complexes in Arabidopsis thaliana.

Polycomb group (PcG) proteins are evolutionarily conserved chromatin modifiers that regulate developmental pathways in plants. PcGs form nuclear multi-subunit Polycomb Repressive Complexes (PRCs). The PRC2 complex mediates gene repression via methylation of lysine 27 on histone H3, which consequently leads to chromatin condensation. In Arabidopsis thaliana, several PRC2 complexes with different compositions were identified, each controlling a particular developmental program.The core subunit FIE is crucial for PRC2 function throughout the plant life cycle, yet accurate information on its spatial and temporal localization was absent. This study focused on identifying FIE accumulation patterns, using microscopy and biochemical approaches. Analysing endogenous FIE and transgenic gFIE-green fluorescent protein fusion protein (gFIE-GFP) showed that FIE accumulates in the nuclei of every cell type examined. Interestingly, gFIE-GFP, as well as the endogenous FIE, also localized to the cytoplasm in all examined tissues. In both vegetative and reproductive organs, FIE formed cytoplasmic high-molecular-mass complexes, in parallel to the nuclear PRC2 complexes. Moreover, size-exclusion chromatography and bimolecular fluorescence complementation assays indicated that in inflorescences FIE formed a cytoplasmic complex with MEA, a PRC2 histone methyltransferase subunit. In contrast, CLF and SWN histone methyltransferases were strictly nuclear. Presence of PRC2 subunits in cytoplasmic complexes has not been previously described in plants. Our findings are in agreement with accumulating evidence demonstrating cytoplasmic localization and function of PcGs in metazoa. The cytosolic accumulation of PRC2 components in plants supports the model that PcGs have alternative non-nuclear functions that go beyond chromatin methylation.

Epizootic hemorrhagic disease virus induces and benefits from cell stress, autophagy, and apoptosis.

The mode and timing of virally induced cell death hold the potential of regulating viral yield, viral transmission, and the severity of virally induced disease. Orbiviruses such as the epizootic hemorrhagic disease virus (EHDV) are nonenveloped and cytolytic. To date, the death of cells infected with EHDV, the signal transduction pathways involved in this process, and the consequence of their inhibition have yet to be characterized. Here, we report that the Ibaraki strain of EHDV2 (EHDV2-IBA) induces apoptosis, autophagy, a decrease in cellular protein synthesis, the activation of c-Jun N-terminal kinase (JNK), and the phosphorylation of the JNK substrate c-Jun. The production of infectious virions decreased upon inhibition of apoptosis with the pan-caspase inhibitor Q-VD-OPH (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone), upon inhibition of autophagy with 3-methyladenine or via the knockout of the autophagy regulator Atg5, or upon treatment of infected cells with the JNK inhibitor SP600125 or the cyclin-dependent kinase (CDK) inhibitor roscovitine, which also inhibited c-Jun phosphorylation. Moreover, Q-VD-OPH, SP600125, and roscovitine partially reduced EHDV2-IBA-induced cell death, and roscovitine diminished the induction of autophagy by EHDV2-IBA. Taken together, our results imply that EHDV induces and benefits from the activation of signaling pathways involved in cell stress and death.

Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA.

We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection.

Negative regulation of the endocytic adaptor disabled-2 (Dab2) in mitosis.

Mitotic cells undergo extensive changes in shape and size through the altered regulation and function of their membrane trafficking machinery. Disabled 2 (Dab2), a multidomain cargo-specific endocytic adaptor and a mediator of signal transduction, is a potential integrator of trafficking and signaling. Dab2 binds effectors of signaling and trafficking that localize to different intracellular compartments. Thus, differential localization is a putative regulatory mechanism of Dab2 function. Furthermore, Dab2 is phosphorylated in mitosis and is thus regulated in the cell cycle. However, a detailed description of the intracellular localization of Dab2 in the different phases of mitosis and an understanding of the functional consequences of its phosphorylation are lacking. Here, we show that Dab2 is progressively displaced from the membrane in mitosis. This phenomenon is paralleled by a loss of co-localization with clathrin. Both phenomena culminate in metaphase/anaphase and undergo partial recovery in cytokinesis. Treatment with 2-methoxyestradiol, which arrests cells at the spindle assembly checkpoint, induces the same effects observed in metaphase cells. Moreover, 2-methoxyestradiol also induced Dab2 phosphorylation and reduced Dab2/clathrin interactions, endocytic vesicle motility, clathrin exchange dynamics, and the internalization of a receptor endowed with an NPXY endocytic signal. Serine/threonine to alanine mutations, of residues localized to the central region of Dab2, attenuated its phosphorylation, reduced its membrane displacement, and maintained its endocytic abilities in mitosis. We propose that the negative regulation of Dab2 is part of an accommodation of the cell to the altered physicochemical conditions prevalent in mitosis, aimed at allowing endocytic activity throughout the cell cycle.

LY6S, a New IFN-Inducible Human Member of the Ly6a Subfamily Expressed by Spleen Cells and Associated with Inflammation and Viral Resistance.

Syntenic genomic loci on human chromosome 8 and mouse chromosome 15 (mChr15) code for LY6/Ly6 (lymphocyte Ag 6) family proteins. The 23 murine Ly6 family genes include eight genes that are flanked by the murine Ly6e and Ly6l genes and form an Ly6 subgroup referred to in this article as the Ly6a subfamily gene cluster. Ly6a, also known as Stem Cell Ag-1 and T cell-activating protein, is a member of the Ly6a subfamily gene cluster. No LY6 genes have been annotated within the syntenic LY6E to LY6L human locus. We report in this article on LY6S, a solitary human LY6 gene that is syntenic with the murine Ly6a subfamily gene cluster, and with which it shares a common ancestry. LY6S codes for the IFN-inducible GPI-linked LY6S-iso1 protein that contains only 9 of the 10 consensus LY6 cysteine residues and is most highly expressed in a nonclassical spleen cell population. Its expression leads to distinct shifts in patterns of gene expression, particularly of genes coding for inflammatory and immune response proteins, and LY6S-iso1-expressing cells show increased resistance to viral infection. Our findings reveal the presence of a previously unannotated human IFN-stimulated gene, LY6S, which has a 1:8 ortholog relationship with the genes of the Ly6a subfamily gene cluster, is most highly expressed in spleen cells of a nonclassical cell lineage, and whose expression induces viral resistance and is associated with an inflammatory phenotype and with the activation of genes that regulate immune responses.

ERK and PI3K regulate different aspects of the epithelial to mesenchymal transition of mammary tumor cells induced by truncated MUC1.

Epithelial to mesenchymal transition (EMT) integrates changes to cell morphology and signaling pathways resulting from modifications to the cell's transcriptional response. Different combinations of stimuli ignite this process in the contexts of development or tumor progression. The human MUC1 gene encodes multiple alternatively spliced forms of a polymorphic oncoprotein that is aberrantly expressed in epithelial malignancies. MUC1 is endowed with various signaling modules and has the potential to mediate proliferative and morphological changes characteristic of the progression of epithelial tumors. The tyrosine-rich cytoplasmic domain and the heavily glycosylated extracellular domain both play a role in MUC1-mediated signal transduction. However, the attribution of function to specific domains of MUC1 is difficult due to the concomitant presence of multiple forms of the protein, which stem from alternative splicing and proteolytic cleavage. Here we show that DA3 mouse mammary tumor cells stably transfected with a truncated genomic fragment of human MUC1 undergo EMT. In their EMT, these cells demonstrate altered [i] morphology, [ii] signaling pathways and [iii] expression of epithelial and mesenchymal markers. Similarly to well characterized human breast cancer cell lines, cells transfected with truncated MUC1 show an ERK-dependent increased spreading on fibronectin, and a PI3K-dependent enhancement of their proliferative rate.

The MUC1 oncoprotein as a functional target: immunotoxin binding to alpha/beta junction mediates cell killing.

MUC1, a heavily glycosylated mucin, has generated considerable interest as a target for tumor killing because of its overexpression in malignancies. Full-length MUC1 (MUC1/TM) is proteolytically cleaved after synthesis generating alpha and beta subunits, which specifically bind in a noncovalent interaction. Although the beta chain remains on the cell surface, the alpha chain binds in an on-and-off interaction. Most anti-MUC1 antibodies (Abs) described to date recognize epitopes within the highly immunogenic alpha-chain tandem repeat. Because the alpha-chain is shed, such Abs are sequestered and fail to reach MUC1-expressing cells. Immunizing with cDNA encoding MUC1/TM and the spliced MUC1/X isoform from which the tandem repeat has been deleted yielded antibodies to the MUC1 alpha/beta junction. Pseudomonas toxin PE38 linked to polyclonal anti-MUC1 alpha/beta junction Abs both bound and killed MUC1-positive malignant cells. Monoclonal DMC209 binds the MUC1 alpha/beta junction in both MUC1/X and MUC1/TM. When injected into SCID mice xenotransplanted with human breast cancer MDA-MB-231, monoclonal DMC209 showed significant in vivo tumor-suppressive activity. The MUC1/X alpha/beta junction presents a biologically-significant target in MUC1-expressing malignancies because (i) antibodies directed against cell-bound alpha/beta junction epitopes reach the intended cellular target, (ii) antibodies to junction epitope are internalized into cells, (iii) anti alpha/beta junction antibodies can effectively kill high MUC1-expressing cancer cells as antibody-toxin conjugates and (iv) antibodies targeting the MUC1 cell-bound alpha/beta junction results in tumor suppression in vivo. Our results indicate that cell-bound MUC1 alpha/beta junction, unlike shed alpha chain, represents a highly effective moiety for targeting and killing MUC1-expressing malignancies.

Tumor-microenvironment interactions: the fucose-generating FX enzyme controls adhesive properties of colorectal cancer cells.

Extravasation of tumor cells is a pivotal step in metastasis formation. This step is initiated by an interaction of extravasating tumor cells with endothelial cells. Among the molecules mediating tumor-endothelium interactions are selectins and their fucosylated ligands. In a previous study, we demonstrated that the fucose-generating FX enzyme regulates the expression of selectin ligands by B and T lymphocytes and by head and neck squamous cell carcinoma cells. It was also shown that the FX enzyme regulated important interaction parameters between these cancer cells and endothelial cells. The present study was aimed to determine whether the FX enzyme controls adhesive interactions between colorectal cancer cells and endothelial cells. The results clearly indicate that this is indeed the case. Overexpressing the FX enzyme by the transfer of FX cDNA to low FX-expressing colorectal cancer cells resulted in an increased adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. Down-regulating FX levels in colorectal cancer cells expressing high levels of endogenous FX by transfection with small-interfering RNA resulted in a down-regulated expression of the selectin ligand sialyl Lewis-a and a decrease in the adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. These transfection experiments also indicated that manipulating the levels of the FX enzyme affected global cellular fucosylation and altered the interaction of colorectal cancer cells with some extracellular matrix components such as fibronectin. We also found that highly metastatic colorectal cancer variants express higher levels of FX and of sialyl Lewis-a than low metastatic variants originating in the same tumors. These results lead us to hypothesize that the FX enzyme controls the capacity of colorectal cancer to extravasate and form metastasis. If this hypothesis will be confirmed the FX enzyme could become a target molecule for metastasis prevention.

Active immunization against vasoactive intestinal polypeptide decreases neuronal recruitment and inhibits reproduction in zebra finches.

Neurogenesis and neuronal recruitment occur in adult brains of many vertebrates, and the hypothesis is that these phenomena contribute to the brain plasticity that enables organisms to adjust to environmental changes. In mammals, vasoactive intestinal polypeptide (VIP) is known to have many neuroprotective properties, but in the avian brain, although widely distributed, its role in neuronal recruitment is not yet understood. In the present study we actively immunized adult zebra finches against VIP conjugated to KLH and compared neuronal recruitment in their brains, with brains of control birds, which were immunized against KLH. We looked at two forebrain regions: the nidopallium caudale (NC), which plays a role in vocal communication, and the hippocampus (HC), which is involved in the processing of spatial information. Our data demonstrate that active immunization against VIP reduces neuronal recruitment, inhibits reproduction, and induces molting, with no change in plasma prolactin levels. Thus, our observations suggest that VIP has a direct positive role in neuronal recruitment and reproduction in birds. J. Comp. Neurol. 524:2516-2528, 2016. © 2016 Wiley Periodicals, Inc.

MUC1 immunotherapy against a metastatic mammary adenocarcinoma model: Importance of IFN-gamma.

Immunotherapy using mucin 1 (MUC1) linked to oxidised mannan (MFP) was investigated in an aggressive MUC1+ metastatic tumour, DA3-MUC1 because, unlike many MUC1+ tumour models, DA3-MUC1 is not spontaneously rejected in mice making it an alternative model for immunotherapy studies. Further, DA3-MUC1 cells are resistant to lysis by anti-MUC1 cytotoxic T cells (CTLs). The inability of DA3-MUC1 tumours to be rejected in naïve mice as well as vaccination to MUC1 was attributed to a deficiency of expression of MHC class I molecules on the tumour cell surface. In vitro and in vivo analysis of subcutaneous tumours and lung metastases demonstrated that DA3-MUC1 tumour cells have a low expression (< 6%) of MHC class I which can be upregulated (> 90%) following culturing with IFN-γ. Results from flow cytometry analysis and immunoperoxidase staining indicated that the in vitro up-regulation of MHC class I could be maintained for up to seven days in vivo, without affecting the expression levels of MUC1 antigen. Interestingly, MUC1-specific CTL that lyse DA3-MUC1 targets in vitro were induced in MFP immunised mice but failed to protect mice from a DA3-MUC1 tumour challenge. These results highlight the importance of MHC class I molecules in the induction of anti-tumour immunity and the MFP immune response.

MUC1-ARF-A Novel MUC1 Protein That Resides in the Nucleus and Is Expressed by Alternate Reading Frame Translation of MUC1 mRNA.

Translation of mRNA in alternate reading frames (ARF) is a naturally occurring process heretofore underappreciated as a generator of protein diversity. The MUC1 gene encodes MUC1-TM, a signal-transducing trans-membrane protein highly expressed in human malignancies. Here we show that an AUG codon downstream to the MUC1-TM initiation codon initiates an alternate reading frame thereby generating a novel protein, MUC1-ARF. MUC1-ARF, like its MUC1-TM 'parent' protein, contains a tandem repeat (VNTR) domain. However, the amino acid sequence of the MUC1-ARF tandem repeat as well as N- and C- sequences flanking it differ entirely from those of MUC1-TM. In vitro protein synthesis assays and extensive immunohistochemical as well as western blot analyses with MUC1-ARF specific monoclonal antibodies confirmed MUC1-ARF expression. Rather than being expressed at the cell membrane like MUC1-TM, immunostaining showed that MUC1-ARF protein localizes mainly in the nucleus: Immunohistochemical analyses of MUC1-expressing tissues demonstrated MUC1-ARF expression in the nuclei of secretory luminal epithelial cells. MUC1-ARF expression varies in different malignancies. While the malignant epithelial cells of pancreatic cancer show limited expression, in breast cancer tissue MUC1-ARF demonstrates strong nuclear expression. Proinflammatory cytokines upregulate expression of MUC1-ARF protein and co-immunoprecipitation analyses demonstrate association of MUC1-ARF with SH3 domain-containing proteins. Mass spectrometry performed on proteins coprecipitating with MUC1-ARF demonstrated Glucose-6-phosphate 1-dehydrogenase (G6PD) and Dynamin 2 (DNM2). These studies not only reveal that the MUC1 gene generates a previously unidentified MUC1-ARF protein, they also show that just like its 'parent' MUC1-TM protein, MUC1-ARF is apparently linked to signaling and malignancy, yet a definitive link to these processes and the roles it plays awaits a precise identification of its molecular functions. Comprising at least 524 amino acids, MUC1-ARF is, furthermore, the longest ARF protein heretofore described.

ATM-dependent activation of the gene encoding MAP kinase phosphatase 5 by radiomimetic DNA damage.

Cellular responses to DNA damage are mediated by an extensive network of signaling pathways. The ATM protein kinase is a master regulator of the response to double-strand breaks (DSBs), the most cytotoxic DNA lesion caused by ionizing radiation. ATM is the protein missing or inactive in patients with the pleiotropic genetic disorder ataxia-telangiectasia (A-T). A major response to DNA damage is altered expression of numerous genes. While studying gene expression in control and A-T cells following treatment with the radiomimetic chemical neocarzinostatin (NCS), we identified an expressed sequence tag that represented a gene that was induced by DSBs in an ATM-dependent manner. The corresponding cDNA encoded a dual specificity phosphatase of the MAP kinase phosphatase family, MKP-5. MKP-5 dephosphorylates and inactivates the stress-activated MAP kinases JNK and p38. The phosphorylation-dephosphorylation cycle of JNK and p38 by NCS was attenuated in A-T cells. Thus, ATM modulates this cycle in response to DSBs. These results further highlight ATM as a link between the DNA damage response and major signaling pathways involved in proliferative and apoptotic processes.

Generation of ligand-receptor alliances by "SEA" module-mediated cleavage of membrane-associated mucin proteins.

A mechanism is described whereby one and the same gene can encode both a receptor protein as well as its specific ligand. Generation of this receptor-ligand partnership is effected by proteolytic cleavage within a specific module located in a membrane resident protein. It is postulated here that the "SEA" module, found in a number of heavily O-linked glycosylated membrane-associated proteins, serves as a site for proteolytic cleavage. The subunits generated by proteolytic cleavage of the SEA module reassociate, and can subsequently elicit a signaling cascade. We hypothesize that all membrane resident proteins containing such a "SEA" module will undergo cleavage, thereby generating a receptor-ligand alliance. This requires that the protein subunits resulting from the proteolytic cleavage reassociate with each other in a highly specific fashion. The same SEA module that serves as the site for proteolytic cleavage, probably also contains the binding sites for reassociation of the resultant two subunits. More than one type of module can function as a site for proteolytic cleavage; this can occur not only in one-pass membrane proteins but also in 7-transmembrane proteins and other membrane-associated proteins. The proposal presented here is likely to have significant practical consequences. It could well lead to the rational design and identification of molecules that, by binding to one of the cleaved partners, will act either as agonists or antagonists, alter signal transduction and, hence, cellular behavior.

Transmembrane and truncated (SEC) isoforms of MUC1 in the human endometrium and Fallopian tube.

The cell surface mucin MUC1 is expressed by endometrial epithelial cells with increased abundance in the secretory phase of the menstrual cycle, when it is found both at the apical cell surface and in secretions. This suggests the presence of a maternal cell surface glycoprotein barrier to embryo implantation, arising from the anti-adhesive property of MUC1. In previous work, we demonstrated alternatively spliced MUC1 variant forms in tumour cells. The variant MUC1/SEC lacks the transmembrane and cytoplasmic sequences found in the full-length variant. We now show that MUC1/SEC mRNA is present in endometrial carcinoma cell lines, endometrial tissue and primary cultured endometrial epithelial cells. The protein can be detected using isoform-specific antibodies in uterine flushings, suggesting release from endometrium in vivo. However, on the basis of immunolocalisation studies, MUC1/SEC also remains associated with the apical epithelial surface both in tissue and in cultured cells. Transmembrane MUC1 and MUC1/SEC are both strikingly localised to the apical surface of tubal epithelium. Thus MUC1 may contribute to the anti-adhesive character of the tubal surface, inhibiting ectopic implantation. The mechanism by which this barrier is overcome in endometrium at implantation is the subject of ongoing investigation.

Dab2 inhibits the cholesterol-dependent activation of JNK by TGF-ß.

Transforming growth factor-ß (TGF-ß) ligands activate Smad-mediated and noncanonical signaling pathways in a cell context-dependent manner. Localization of signaling receptors to distinct membrane domains is a potential source of signaling output diversity. The tumor suppressor/endocytic adaptor protein disabled-2 (Dab2) was proposed as a modulator of TGF-ß signaling. However, the molecular mechanism(s) involved in the regulation of TGF-ß signaling by Dab2 were not known. Here we investigate these issues by combining biophysical studies of the lateral mobility and endocytosis of the type I TGF-ß receptor (TßRI) with TGF-ß phosphoprotein signaling assays. Our findings demonstrate that Dab2 interacts with TßRI to restrict its lateral diffusion at the plasma membrane and enhance its clathrin-mediated endocytosis. Small interfering RNA-mediated knockdown of Dab2 or Dab2 overexpression shows that Dab2 negatively regulates TGF-ß-induced c-Jun N-terminal kinase (JNK) activation, whereas activation of the Smad pathway is unaffected. Moreover, activation of JNK by TGF-ß in the absence of Dab2 is disrupted by cholesterol depletion. These data support a model in which Dab2 regulates the domain localization of TßRI in the membrane, balancing TGF-ß signaling via the Smad and JNK pathways.