An RNAi screen identifies additional members of the Drosophila Integrator complex and a requirement for cyclin C/Cdk8 in snRNA 3′-end formation

Formation of the 3′ end of RNA polymerase II–transcribed snRNAs requires a poorly understood group of proteins called the Integrator complex. Here we used a fluorescence-based read-through reporter that expresses GFP in response to snRNA misprocessing and performed a genome-wide RNAi screen in Drosophila S2 cells to identify novel factors required for snRNA 3′-end formation. In addition to the known Integrator complex members, we identified Asunder and CG4785 as additional Integrator subunits. Functional and biochemical experiments revealed that Asunder and CG4785 are additional core members of the Integrator complex. We also identified a conserved requirement in both fly and human snRNA 3′-end processing for cyclin C and Cdk8 that is distinct from their function in the Mediator Cdk8 module. Moreover, we observed biochemical association between Integrator proteins and cyclin C/Cdk8, and that overexpression of a kinase-dead Cdk8 causes snRNA misprocessing. These data functionally define the Drosophila Integrator complex and demonstrate an additional function for cyclin C/Cdk8 unrelated to its function in Mediator.     

Analysis of phosphotyrosine signaling in glioblastoma identifies STAT5 as a novel downstream target of ΔEGFR

An in-frame deletion mutation in Epidermal Growth Receptor (EGFR), ΔEGFR is a common and potent oncogene in glioblastoma (GBM), promoting growth and survival of cancer cells. This mutated receptor is ligand independent and constitutively active. Its activity is low in intensity and thought to be qualitatively different from acutely ligand stimulated wild-type receptor implying that the preferred downstream targets of ΔEGFR play a significant role in malignancy. To understand the ΔEGFR signal, we compared it to that of a kinase-inactivated mutant of ΔEGFR and wild-type EGFR with shotgun phosphoproteomics using an electron-transfer dissociation (ETD) enabled ion trap mass spectrometer. We identified and quantified 354 phosphopeptides corresponding to 249 proteins. Among the ΔEGFR-associated phosphorylations were the previously described Gab1, c-Met and Mig-6, and also novel phosphorylations including that of STAT5 on Y694/9. We have confirmed the most prominent phosphorylation events in cultured cells and in murine xenograft models of glioblastoma. Pathway analysis of these proteins suggests a preference for an alternative signal transduction pathway by ΔEGFR compared to wild-type EGFR. This understanding will potentially benefit the search for new therapeutic targets for ΔEGFR expressing tumors.     

Quantitative mass spectrometry of diabetic kidney tubules identifies GRAP as a novel regulator of TGF-β signaling

The aim of this study was to define novel mediators of tubule injury in diabetic kidney disease. For this, we used state-of-the-art proteomic methods combined with a label-free quantitative strategy to define protein expression differences in kidney tubules from transgenic OVE26 type 1 diabetic and control mice. The analysis was performed with diabetic samples that displayed a pro-fibrotic phenotype. We have identified 476 differentially expressed proteins. Bioinformatic analysis indicated several clusters of regulated proteins in relevant functional groups such as TGF-β signaling, tight junction maintenance, oxidative stress, and glucose metabolism. Mass spectrometry detected expression changes of four physiologically relevant proteins were confirmed by immunoblot analysis. Of these, the Grb2-related adaptor protein (GRAP) was up-regulated in kidney tubules from diabetic mice and fibrotic kidneys from diabetic patients, and subsequently confirmed as a novel component of TGF-β signaling in cultured human renal tubule cells. Thus, indicating a potential novel role for GRAP in TGF-β-induced tubule injury in diabetic kidney disease. Although we targeted a specific disease, this approach offers a robust, high-sensitivity methodology that can be applied to the discovery of novel mediators for any experimental or disease condition.     

Matrilin-3 as a putative effector of C-type natriuretic peptide signaling during TGF-β induced chondrogenic differentiation of mesenchymal stem cells

C-type natriuretic peptide (CNP) signaling has been implicated as an important regulator of chondrogenic differentiation during endochondral bone development. This preliminary study further investigated the putative effectors and/or targets of CNP signaling in transforming growth factor (TGF)-β induced in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs). Previously characterized human trabecular bone derived MSCs were induced either with only TGF-β1 or with a combination of TGF-β1 and CNP in micromass culture for 10 or 20 days. Genome wide gene expression profile changes in between these two groups were analyzed on day-10 or day-20 of culture. Results revealed that there were only 7 genes, whose expression change was fourfolds or higher in TGF-β1 and CNP fed group in comparison to only TGF-β1 fed group. The up-regulated genes included matrilin-3 (MATN3), engulfment and cell motility 1 (ELMO1), CD24, and DCN1, defective in cullin neddylation 1, domain containing 1 (DCUN1D1). The down-regulated genes, on the other hand, included LIM domain kinase 2 (LIMK2), Ewing sarcoma breakpoint region 1, and guanine nucleotide binding protein (G protein), gamma 12 (GNG12). The up-regulation of MATN3 was confirmed on the basis of RT-PCR. The known literature on both CNP signaling and MATN3 function in chondrogenesis match with each other and suggest MATN3 as a putative effector and/or target of CNP signaling during this process.     

High-throughput chemical screen identifies a novel potent modulator of cellular circadian rhythms and reveals CKIα as a clock regulatory kinase

The circadian clock underlies daily rhythms of diverse physiological processes, and alterations in clock function have been linked to numerous pathologies. To apply chemical biology methods to modulate and dissect the clock mechanism with new chemical probes, we performed a circadian screen of ～120,000 uncharacterized compounds on human cells containing a circadian reporter. The analysis identified a small molecule that potently lengthens the circadian period in a dose-dependent manner. Subsequent analysis showed that the compound also lengthened the period in a variety of cells from different tissues including the mouse suprachiasmatic nucleus, the central clock controlling behavioral rhythms. Based on the prominent period lengthening effect, we named the compound longdaysin. Longdaysin was amenable for chemical modification to perform affinity chromatography coupled with mass spectrometry analysis to identify target proteins. Combined with siRNA-mediated gene knockdown, we identified the protein kinases CKIδ, CKIα, and ERK2 as targets of longdaysin responsible for the observed effect on circadian period. Although individual knockdown of CKIδ, CKIα, and ERK2 had small period effects, their combinatorial knockdown dramatically lengthened the period similar to longdaysin treatment. We characterized the role of CKIα in the clock mechanism and found that CKIα-mediated phosphorylation stimulated degradation of a clock protein PER1, similar to the function of CKIδ. Longdaysin treatment inhibited PER1 degradation, providing insight into the mechanism of longdaysin-dependent period lengthening. Using larval zebrafish, we further demonstrated that longdaysin drastically lengthened circadian period in vivo. Taken together, the chemical biology approach not only revealed CKIα as a clock regulatory kinase but also identified a multiple kinase network conferring robustness to the clock. Longdaysin provides novel possibilities in manipulating clock function due to its ability to simultaneously inhibit several key components of this conserved network across species.     

Suppression of TNF-α and IL-1 signaling identifies a mechanism of homeostatic regulation of macrophages by IL-27

IL-27 is a pleiotropic cytokine with both activating and inhibitory functions on innate and acquired immunity. IL-27 is expressed at sites of inflammation in cytokine-driven autoimmune/inflammatory diseases, such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, and sarcoidosis. However, its role in modulating disease pathogenesis is still unknown. In this study, we found that IL-27 production is induced by TNF-α in human macrophages (MΦ) and investigated the effects of IL-27 on the responses of primary human MΦ to the endogenous inflammatory cytokines TNF-α and IL-1. In striking contrast to IL-27-mediated augmentation of TLR-induced cytokine production, we found that IL-27 suppressed MΦ responses to TNF-α and IL-1β, thus identifying an anti-inflammatory function of IL-27. IL-27 blocked the proximal steps of TNF-α signaling by downregulating cell-surface expression of the signaling receptors p55 and p75. The mechanism of inhibition of IL-1 signaling was downregulation of the ligand-binding IL-1RI concomitant with increased expression of the receptor antagonist IL-1Ra and the decoy receptor IL-1RII. These findings provide a mechanism for suppressive effects of IL-27 on innate immune cells and suggest that IL-27 regulates inflammation by limiting activation of MΦ by inflammatory cytokines while preserving initial steps in host defense by augmenting responses to microbial products.     

A functional genomics screen identifies an Importin-α homolog as a regulator of stem cell function and tissue patterning during planarian regeneration

Background

Planarians are renowned for their regenerative capacity and are an attractive model for the study of adult stem cells and tissue regeneration. In an effort to better understand the molecular mechanisms underlying planarian regeneration, we performed a functional genomics screen aimed at identifying genes involved in this process in Schmidtea mediterranea.

Methods

We used microarrays to detect changes in gene expression in regenerating and non-regenerating tissues in planarians regenerating one side of the head and followed this with high-throughput screening by in situ hybridization and RNAi to characterize the expression patterns and function of the differentially expressed genes.

Results

Along with five previously characterized genes (Smed-cycD, Smed-morf41/mrg-1, Smed-pdss2/dlp1, Smed-slbp, and Smed-tph), we identified 20 additional genes necessary for stem cell maintenance (Smed-sart3, Smed-smarcc-1, Smed-espl1, Smed-rrm2b-1, Smed-rrm2b-2, Smed-dkc1, Smed-emg1, Smed-lig1, Smed-prim2, Smed-mcm7, and a novel sequence) or general regenerative capability (Smed-rbap46/48-2, Smed-mcm2, Smed-ptbp1, and Smed-fen-1) or that caused tissue-specific defects upon knockdown (Smed-ddc, Smed-gas8, Smed-pgbd4, and Smed-b9d2). We also found that a homolog of the nuclear transport factor Importin-α plays a role in stem cell function and tissue patterning, suggesting that controlled nuclear import of proteins is important for regeneration.

Conclusions

Through this work, we described the roles of several previously uncharacterized genes in planarian regeneration and implicated nuclear import in this process. We have additionally created an online database to house our in situ and RNAi data to make it accessible to the planarian research community.

     

��-Actin is a downstream effector of the PI3K/AKT signaling pathway in myeloma cells

Interleukin 6 is the in vivo growth factor of myeloma cells. In response to IL-6 stimulation, the PI3K/AKT signaling pathway is activated in these cells. With comparative proteomic approaches, this study reveals many putative downstream effectors of the PI3K/AKT pathway. Mass spectrometry analysis of excised protein spots from 2-dimensional gel allowed the identification of proteins such as β-Actin, cyclophilin A, E3 SUMO-protein ligase PIAS-NY protein, HSP 27, PML, and transforming growth factor β-2. Among these putative effectors, β-Actin was chosen for further characterization. Phosphorylation of β-Actin by AKT upon IL-6 stimulation was confirmed by western blotting using a phospho-AKT substrate antibody. Interestingly, IL-6 significantly increased cell migration (P < 0.05) and the content of filamentous actin (P < 0.05). Therefore, IL-6 stimulation could have effects on the migration of myeloma cells, and the phosphorylation of β-Actin is probably involved in the process.     

A biotin switch-based proteomics approach identifies 14-3-3ζ as a target of Sirt1 in the metabolic regulation of caspase-2

While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes14-3-3ζ dissociation from caspase-2 in both egg extract and human cultured cells. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation.     

Identification of Integrin α3 as a New Substrate of the Adenovirus E4orf6/E1B 55-Kilodalton E3 Ubiquitin Ligase Complex

The human adenovirus E4orf6 and E1B55K proteins promote viral replication by targeting several cellular proteins for degradation. The E4orf6 product has been shown by our group and others to form an E3 ubiquitin ligase complex that contains elongins B and C and cullin family member Cul5. E1B55K associates with this complex, where it is believed to function primarily to introduce bound substrates for degradation via proteasomes. In addition to p53, its first known substrate, the E4orf6/E1B 55-kDa complex (E4orf6/E1B55K) was shown to promote the degradation of Mre11 and DNA ligase IV; however, additional substrates are believed to exist. This notion is strengthened by the fact that none of these substrates seems likely to be associated with additional functions shown to be mediated by the E4orf6-associated E3 ubiquitin ligase complex, including export of late viral mRNAs and blockage of export of the bulk cellular mRNAs from the nucleus. In an attempt to identify new E4orf6/E1B55K substrates, we undertook a proteomic screen using human p53-null, non-small-cell lung carcinoma H1299 cells expressing either E4orf6 protein alone or in combination with E1B55K through infection by appropriate adenovirus vectors. One cellular protein that appeared to be degraded by E1B55K in combination with the E4orf6 protein was a species of molecular mass ∼130 kDa that was identified as the integrin α3 subunit (i.e., very late activation antigen 3 alpha subunit). Preliminary analyses suggested that degradation of α3 may play a role in promoting release and spread of progeny virions.Viruses are well known to promote replication by inhibiting or enhancing endogenous cellular machinery or, in some cases, by reprogramming key cellular pathways. Human adenoviruses have developed effective ways to modulate the immune response, apoptosis, double-strand break repair, mRNA export, and translation to optimize virus replication and the spreading of progeny virions. The expression of adenovirus E1A proteins stabilizes p53 and induces apoptosis (8, 33); however, this effect is reversed in infected cells by the action of two early products: the E1B 55-kDa (E1B55K) and E4orf6 proteins (35, 36). We and others have shown that these proteins act through the formation of an E3 ubiquitin ligase complex analogous to the SCF and VBC complexes but which contains, in addition to elongins B and C and the RING protein Rbx1, the cullin family member Cul5 (18, 41, 43). This E4orf6-mediated E3 ligase complex blocks p53-induced apoptosis (35, 36) by promoting the ubiquitination of p53, followed by its degradation by proteasomes (41, 43). E4orf6 protein mediates the assembly of the complex by its interaction with elongin C through its three BC boxes (11, 41, 43). E1B55K, which appears to associate with the E4orf6 protein only when present in the ligase complex (4), is thought to function as a substrate recognition factor that brings substrates to the complex because, although both E4orf6 and E1B55K bind p53 independently, interaction of E1B55K with p53 is essential for the efficient degradation of p53 (41, 48). In addition to protecting infected cells from early lysis via p53-induced apoptosis, the E4orf6/E1B55K ligase complex performs other functions essential for virus replication. Two other substrates of the complex have been identified: a member of the MRN DNA repair complex, Mre11, and the central component of the nonhomologous end-joining DNA repair system, DNA ligase IV (2, 56). Degradation of both of these proteins prevents viral genome concatenation, which interferes with the packaging of viral DNA into virions (2, 56). E1B55K binds to p53, Mre11, and DNA ligase IV and has been demonstrated to colocalize with p53 and Mre11 in perinuclear cytoplasmic bodies termed aggresomes (1, 2, 32). More recently, we and others have obtained results that suggest that the E4orf6-associated E3 ligase complex regulates viral and cellular mRNA export (5, 66). The Cul5-based ligase activity was shown to be essential for selective viral mRNA export and the block of cellular mRNA export from the nucleus (66), thus contributing to the shutoff of cellular protein synthesis initiated by L4-100K (20). The actual substrates of the complex responsible for regulating mRNA export are currently unknown.As discussed in detail below, our efforts to identify substrates of the E4orf6/E1B55K complex led us to consider a member of the integrin family as a potential substrate. Integrins are members of a family of surface receptors that function in several ways through the formation of cell-extracellular matrices and cell-cell interactions (reviewed in references 21, 26, and 63). Integrins are typically composed of two transmembrane glycoproteins forming heterodimers of α and β subunits each of approximately 80 to 150 kDa. There are at least 18 α subunits and 8 β subunits in mammals that can dimerize in limited combinations to form more than 20 functionally distinct integrins with different ligand specificities. Integrin heterodimers function as transmembrane receptors that link external factors to intracellular signaling pathways. In addition to roles in cell adhesion, these communication events are implicated in a large range of cellular processes, including proliferation, differentiation, translation, migration, and apoptosis. Some of these processes depend on the intracellular trafficking pathways of the integrins (reviewed in references 9, 24, 40, and 44), including the long-loop recycling pathway in which integrins present in clathrin-coated endosomes move first to the perinuclear recycling center, where some accumulate, including the β1 integrin subunit (31), before returning to the plasma membrane. The integrin α3β1 is a member of the β1 integrin subfamily in which the α3 subunit (VLA-3a) is coupled to the β1 subunit to form the very late activation antigen (VLA-3 or CD49c) (21, 59, 60). α3β1 is expressed in a wide range of tissues in which it binds a variety of extracellular matrix substrates, including fibronectin, collagen, thrombospondin 1, and laminins 1, 5, 8, 10, and 11 (13). These associations allow the integrin α3β1 to fill its primary role in cell adhesion. α3β1 also participates in intercellular adhesion through several protein-protein interactions (10, 27, 53, 55, 58), making it a major contributor in the regulation of cellular adhesion.Human adenovirus type 5 (Ad5) particles interact with cell surface receptors to facilitate internalization into target cells. In the high-affinity interacting model (reviewed in reference 29), the viral fiber knob polypeptide binds the coxsackie adenovirus receptor (CAR) protein on the surface of cells as the primary cell binding event (primary receptor). The penton base polypeptide then binds a cell surface integrin (secondary receptor), leading to entry of the capsid into the cell by a process termed receptor-mediated endocytosis or clathrin-mediated endocytosis. Several types of integrins have been identified as being used by Ad5 to mediate virus internalization: αMβ1, αMβ2, αVβ1, αVβ3, αVβ5, and α5β1 (22, 30, 49, 65). Salone et al. have shown that α3β1 serves as an alternative cellular receptor for adenovirus serotype 5 (49). It promotes entry of the virus into cells, transduction of DNA, and mediates adenovirus infection in both CAR-positive and CAR-negative cell lines. Thus, in addition to functions related to cell adhesion, integrin α3β1 plays an important role in the adenovirus infection cycle.To identify new targets for degradation by the E4orf6/E1B55K ubiquitin ligase, we used a proteomic screen covering most cellular proteins to look for any polypeptide that exhibited a significant decrease in amount following the coexpression from appropriate adenovirus vectors of the E4orf6 protein and E1B55K. This screen revealed several interesting candidates, including integrin α3, a species of 130 kDa that also was found to be reduced in wild-type (wt) virus infection. The degradation of α3 was seen to be dependent on the Cul5-based ligase complex driven by E4orf6 and E1B55K. We also found evidence that the E4orf6/E1B55K ligase complex appears to be involved in cell detachment from the extracellular matrix, a function that could play a role in virus spread.     

PIASxα Ligase Enhances SUMO1 Modification of PTEN Protein as a SUMO E3 Ligase

The tumor suppressor PTEN plays a critical role in the regulation of multiple cellular processes that include survival, cell cycle, proliferation, and apoptosis. PTEN is frequently mutated or deleted in various human cancer cells to promote tumorigenesis. PTEN is regulated by SUMOylation, but the SUMO E3 ligase involved in the SUMOylation of PTEN remains unclear. Here, we demonstrated that PIASxα is a SUMO E3 ligase for PTEN. PIASxα physically interacted with PTEN both in vitro and in vivo. Their interaction depended on the integrity of phosphatase and C2 domains of PTEN and the region of PIASxα comprising residues 134–347. PIASxα enhanced PTEN protein stability by reducing PTEN ubiquitination, whereas the mutation of PTEN SUMO1 conjugation sites neutralized the effect of PIASxα on PTEN protein half-life. Functionally, PIASxα, as a potential tumor suppressor, negatively regulated the PI3K-Akt pathway through stabilizing PTEN protein. Overexpression of PIASxα led to G₀/G₁ cell cycle arrest, thus triggering cell proliferation inhibition and tumor suppression, whereas PIASxα knockdown or deficiency in catalytic activity abolished the inhibition. Together our studies suggest that PIASxα is a novel SUMO E3 ligase for PTEN, and it positively regulates PTEN protein level in tumor suppression.     

A genome wide shRNA screen identifies α/β hydrolase domain containing 4 (ABHD4) as a novel regulator of anoikis resistance

Acquisition of resistance to anchorage dependant cell death, a process termed anoikis, is a requirement for cancer cell metastasis. However, the molecular determinants of anoikis resistance and sensitivity are poorly understood. To better understand resistance to anoikis we conducted a genome wide lentiviral shRNA screen to identify genes whose knockdown render anoikis-sensitive RWPE-1 prostate cells resistant to anoikis. RWPE-1 cells were infected with a pooled lentiviral shRNA library with 54,021 shRNA targeting 11,255 genes. After infection, an anoikis-resistant cell population was selected and shRNA sequences were amplified and sequenced. Thirty-four shRNA sequences reproducibly protected RWPE-1 cells from anoikis after culture under suspension conditions including the top validated hit, α/β hydrolase domain containing 4 (ABHD4). In validation studies, ABHD4 knockdown inhibited anoikis in RWPE-1 cells as well as anoikis sensitive NP69 nasopharyngeal and OVCAR3 ovarian cancer cells, while over-expression of the gene increased sensitivity. Induction of anoikis after ABHD4 knockdown was associated with cleavage of PARP and activation of caspases-3, but was independent in changes of FLIP, FAK and Src expression. Interestingly, induction of anoikis after ABHD4 knockdown was independent of the known role of ABHD4 in the anandamide synthesis pathway and the generation of glycerophospho-N-acyl ethanolamines. Thus, ABHD4 is a novel genetic regulator of anoikis sensitivity.