USP4 is regulated by Akt phosphorylation and deubiquitylates TGF-beta type I receptor

Stability and membrane localization of Transforming growth factor-β (TGF-β) type I receptor (TβRI) is essential for controlling TGF-β signaling. TβRI is targeted for ubiquitination-mediated degradation by Smad7/Smurf2 complex. However, it is unclear whether polyubiquitin modified TβRI can be reversed. Here we performed a genome-wide gain of function screen and identified ubiquitin-specific protease (USP) 4 as a strong inducer of TGF-β signaling. Putative oncogenic USP4 was found to interact with TβRI as deubiquitinating enzyme thus maintains TβR1 levels at the plasma membrane. Depletion of USP4 mitigates TGF-β-induced breast cancer cell migration, epithelial to mesenchymal transition and metastasis. Importantly, Akt/Protein kinase B (PKB), which has been associated with poor prognosis in breast cancer, associates with and phosphorylates USP4. Akt mediated phosphorylation relocates USP4 to cytoplasm and membrane and is required for maintaining its protein stability. Moreover, Akt-induced breast cancer cell migration was inhibited by USP4 depletion and TβRI kinase inhibition. Our results identified USP4 as an important determinant for crosstalk between TGF-β and Akt, which provides new opportunities for cancer treatment

Whole genome functional analysis identifies novel components required for mitotic spindle integrity in human cells

A loss-of-function screen for siRNAs that arrest human cells in metaphase reveals genes involved in mitotic spindle integrity

Identification of Novel Genes and Pathways Regulating SREBP Transcriptional Activity

BACKGROUND: Lipid metabolism in mammals is orchestrated by a family of transcription factors called sterol regulatory element-binding proteins (SREBPs) that control the expression of genes required for the uptake and synthesis of cholesterol, fatty acids, and triglycerides. SREBPs are thus essential for insulin-induced lipogenesis and for cellular membrane homeostasis and biogenesis. Although multiple players have been identified that control the expression and activation of SREBPs, gaps remain in our understanding of how SREBPs are coordinated with other physiological pathways. METHODOLOGY: To identify novel regulators of SREBPs, we performed a genome-wide cDNA over-expression screen to identify proteins that might modulate the transcription of a luciferase gene driven from an SREBP-specific promoter. The results were verified through secondary biological assays and expression data were analyzed by a novel application of the Gene Set Enrichment Analysis (GSEA) method. CONCLUSIONS/SIGNIFICANCE: We screened 10,000 different cDNAs and identified a number of genes and pathways that have previously not been implicated in SREBP control and cellular cholesterol homeostasis. These findings further our understanding of lipid biology and should lead to new insights into lipid associated disorders

Activation of Yap Directed Transcription by Knock-down of Conserved Cellular Functions

The Yap-Hippo pathway has a significant role in regulating cell proliferation and growth, thus controlling organ size and regeneration. The Hippo pathway regulates two highly conserved, transcription co-activators YAP and TAZ. The upstream regulators of the Yap-Hippo pathway have not been fully characterized. The aim of this study was to use a siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that allow activation of the YAP transcription co-activator at high cell density. Activation of the Yap transcription co-activator was monitored using a high content, image based assay that measured the intracellular localization of native YAP protein. Active siRNAs were identified and further validated by quantification of CYR61 mRNA levels (a known Yap target gene). The effect of compounds targeting the putative gene targets identified as hits was also used for further validation. A number of validated hits reveal basic aspects of Yap-Hippo biology; such as components of the nuclear pore, by which YAP cytoplasmic/nuclear shuttling occurs, or how proteasomal degradation regulates intracellular YAP concentrations, which then alter YAP localization and transcription. Such results highlight how targeting conserved cellular functions can lead to validated activity in phenotypic assays

The SIAH E3 ubiquitin ligases promote Wnt/β-catenin signaling through mediating Wnt-induced Axin degradation

The Wnt/β-catenin signaling pathway plays essential roles in embryonic development and adult tissue homeostasis. Axin is a concentration-limiting factor responsible for the formation of the β-catenin destruction complex. Wnt signaling itself promotes the degradation of Axin. However, the underlying molecular mechanism and biological relevance of this targeting of Axin have not been elucidated. Here, we identify SIAH1/2 (SIAH) as the E3 ligase mediating Wnt-induced Axin degradation. SIAH proteins promote the ubiquitination and proteasomal degradation of Axin through interacting with a VxP motif in the GSK3-binding domain of Axin, and this function of SIAH is counteracted by GSK3 binding to Axin. Structural analysis reveals that the Axin segment responsible for SIAH binding is also involved in GSK3 binding but adopts distinct conformations in Axin/SIAH and Axin/GSK3 complexes. Knockout of SIAH1 blocks Wnt-induced Axin ubiquitination and attenuates Wnt-induced β-catenin stabilization. Our data suggest that Wnt-induced dissociation of the Axin/GSK3 complex allows SIAH to interact with Axin not associated with GSK3 and promote its degradation and that SIAH-mediated Axin degradation represents an important feed-forward mechanism to achieve sustained Wnt/β-catenin signaling

Activation of Yap Directed Transcription by Knock-down of Conserved Cellular Functions

The Yap-Hippo pathway has been shown to have a significant role in regulating cell proliferation and growth and hence controlling organ size and regeneration, as well as the differentiation of stem cells. These cell fate decisions are regulated by the Hippo pathway through the action of two, highly conserved, transcription co-activators YAP and TAZ. However, the upstream regulators of the Yap-Hippo pathway have not been fully characterized and may vary in different cell types and organs. The aim of this study was to use a siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that when knocked-down allow activation of the YAP transcription co-activator at high cell density. This project used a commercially available library of siRNAs purchased from Qiagen that consisted of four siRNAs targeting seven thousand genes of the “druggable” genome. The library was screened using a reverse transfection protocol with a biliary derived cell line which was grown to high cell density. Activation of the Yap transcription co-activator was monitored using a high content, image based assay that measured the intracellular localization of native YAP protein. Active siRNAs were identified and further validated by quantification of CYR61 mRNA levels (a known Yap target gene) following knock-down and the effect of compounds targeting the putative gene targets identified as hits was also used for further validation. A number of validated hits reveal basic aspects of Yap-Hippo biology; such as components of the nuclear pore, by which YAP cytoplasmic/nuclear shuttling occurs, or how proteasomal degradation regulates intracellular YAP concentrations, which then alter YAP localization and transcription. Such results highlight how targeting conserved cellular functions can lead to validated activity in phenotypic assays

RNF12 Controls Embryonic Stem Cell Fate and Morphogenesis in Zebrafish Embryos by Targeting Smad7 for Degradation

TGF-b members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-b family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-b signaling. Depletion of RNF12 dramatically reduced TGF-b/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activininduced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-b family signaling

Jenkins-CI, an open source continuous integration system, as a scientific data and image processing platform.

Image based screening assays generate large volumes of heterogeneous data, requiring a plethora of computational tools for processing and analysis. Building robust, scalable workflows for such applications is a major undertaking; requiring collaboration between scientists and engineers with their own specialized area of expertise. Jenkins-CI, is an open source continuous integration system, which has been used to build an analysis platform for processing high content screening results (HCS). The availability of over 800 plugins for Jenkins-CI enables numerous standard computing tasks and allows the robust integration of specialized applications designed high throughput screening. CellProfiler is an open source resource that has become one of the preferred tools for the analysis and quantification of biological images. Jenkins-CI has made it possible to configure CellProfiler for high performance, parallel processing on Linux clusters deployed at two distant locations. This ability to deploy, at scale, and to parallelize the use of CellProfiler greatly facilitates its use in high throughput screening applications. The Jenkins-CI platform described here is accessible via an internet web interface facilitating sharing, annotating and reuse of protocols and results. Image processing pipelines developed using the CellProfiler desktop client can be deployed and shared through a centralized Jenkins repository. Centralized monitoring of the system allows operational issues to be quickly identified, facilitating user support. Limitations in the user interface of Jenkins were addressed using plugins from the Jenkins-CI community. Examples showing how Jenkins-CI can be used to run CellProfiler for the analysis of HCS assays is presented