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ABSTRACT: TEAD proteins are transcription factors that are crucial for development, but also play a role in cancers. Several developmentally and pathologically important genes are upregulated by TEADs. TEADs have a TEA domain that enables them to bind specific DNA elements and a transactivation domain that enables them to interact with coactivators. TEADs on their own are unable to activate transcription and they require the help of coactivators. Several TEAD-interacting coactivators are known and they can be classified into three groups: (1) YAP and its paralog TAZ; (2) Vgll proteins; and (3) p160s. Accordingly, these coactivators also play a role in development and cancers. Recent studies have shown that TEADs and their coactivators aid in the progression of various cancers, including the difficult to treat glioblastoma, liver and ovarian cancers. They facilitate cancer progression through expression of proliferation promoting genes such as c-myc, survivin, Axl, CTGF and Cyr61. There is also a good correlation between high TEAD or its coactivator expression and poor prognosis in various cancers. Given the fact that TEADs and their coactivators need to work together for a functional outcome, disrupting the interaction between them appears to be a viable option for cancer therapy. Structures of TEAD-coactivator complexes have been elucidated and will facilitate drug design and development.
Cancer biology & therapy 02/2013; 14(5). · 2.64 Impact Factor
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ABSTRACT: The Hippo pathway restricts the activity of transcriptional coactivators TAZ (WWTR1) and YAP. TAZ and YAP are reported to be overexpressed in various cancers, however, their prognostic significance in colorectal cancers remains unstudied. The expression levels of TAZ and YAP, and their downstream transcriptional targets, AXL and CTGF, were extracted from two independent colon cancer patient datasets available in the Gene Expression Omnibus database, totaling 522 patients. We found that mRNA expressions of both TAZ and YAP were positively correlated with those of AXL and CTGF (p<0.05). High level mRNA expression of TAZ, AXL or CTGF significantly correlated with shorter survival. Importantly, patients co-overexpressing all 3 genes had a significantly shorter survival time, and combinatorial expression of these 3 genes was an independent predictor for survival. The downstream target genes for TAZ-AXL-CTGF overexpression were identified by Java application MyStats. Interestingly, genes that are associated with colon cancer progression (ANTXR1, EFEMP2, SULF1, TAGLN, VCAN, ZEB1 and ZEB2) were upregulated in patients co-overexpressing TAZ-AXL-CTGF. This TAZ-AXL-CTGF gene expression signature (GES) was then applied to Connectivity Map to identify small molecules that could potentially be utilized to reverse this GES. Of the top 20 small molecules identified by connectivity map, amiloride (a potassium sparing diuretic,) and tretinoin (all-trans retinoic acid) have shown therapeutic promise in inhibition of colon cancer cell growth. Using MyStats, we found that low level expression of either ANO1 or SQLE were associated with a better prognosis in patients who co-overexpressed TAZ-AXL-CTGF, and that ANO1 was an independent predictor of survival together with TAZ-AXL-CTGF. Finally, we confirmed that TAZ regulates Axl, and plays an important role in clonogenicity and non-adherent growth in vitro and tumor formation in vivo. These data suggest that TAZ could be a therapeutic target for the treatment of colon cancer.
PLoS ONE 01/2013; 8(1):e54211. · 4.09 Impact Factor
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ABSTRACT: Conventional Western blot based pull-down methods involve lengthy and laborious work and the results are generally not quantitative. Here, we report the imaging beads-retained prey (IBRP) assay that is rapid and quantitative in studying protein-protein interactions. In this assay, the bait is immobilized onto beads and the prey is fused with a fluorescence protein. The assay takes advantage of the fluorescence of prey and directly quantifies the amount of prey binding to the immobilized bait under a microscope. We validated the assay using previously well studied interactions and found that the amount of prey retained on beads could have a relative linear relationship to both the inputs of bait and prey. IBRP assay provides a universal, fast, quantitative and economical method to study protein interactions and it could be developed to a medium- or high-throughput compatible method. With the availability of fluorescence tagged whole genome ORFs in several organisms, we predict IBRP assay should have wide applications.
PLoS ONE 01/2013; 8(3):e59727. · 4.09 Impact Factor
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ABSTRACT: The Hippo signaling pathway was initially defined by genetic studies in Drosophila to regulate tissue growth and organ size [1,2]. This pathway is highly conserved in mammals and dysregulation of the Hippo pathway has been implicated in human cancer. Although the exact extracellular signal that controls the Hippo pathway is currently unknown, compelling evidence supports a critical role of the Hippo pathway in cell contact inhibition, which is a property commonly lost in cancer cells. Many molecules, such as the merlin tumor suppressor protein, have been identified as regulating the activity of the core Hippo pathway components [1,2]. Acting downstream are two key transcription co-activators, YAP and TAZ, which mediate the major gene regulation and biological functions of the Hippo pathway. This article will focus on the physiological function and molecular regulation of YAP/TAZ and its Drosophila homolog Yki.
Seminars in Cell and Developmental Biology 05/2012; 23(7):785-93. · 6.65 Impact Factor
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ABSTRACT: The structure of the complex between the transcription cofactor Vgll1 and the transcription factor TEAD4, the mammalian equivalent of the Drosophila Vestigial and Scalloped, respectively, is determined in this study. Remarkably, Vgll1 interacts with TEAD in a manner similar to the transcription coactivators, as well as oncogenes YAP and TAZ, despite having a varied primary sequence. Vgll1-TEAD complex upregulates the expression of IGFBP-5, a proliferation-promoting gene, and facilitates anchorage-independent cell proliferation. The YAP/TAZ-TEAD complex also upregulates several other proliferation-promoting genes and also promotes anchorage-independent cell proliferation. Given its structural and functional similarity to YAP/TAZ, Vgll1 has the potential to promote cancer progression.
Structure 05/2012; 20(7):1135-40. · 6.35 Impact Factor
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ABSTRACT: Rab7 plays an important role in regulating endocytic traffic. In view of an emerging role of membrane traffic in signaling and diseases, we have examined the possible role of Rab7 in oncogenesis. The role of Rab7 was investigated using shRNA-mediated knockdown in A431 and MCF7 cancer cells. To our surprise, Rab7 knockdown effectively suppressed anchorage-independent growth of cancer cells in soft agar. Anoikis (matrix-detachment triggered apoptosis) was enhanced, while the level of phosphorylated (active) Akt (which is a key survival factor) was significantly reduced. Also intriguing was the observation that EGFR and Her2 levels were significantly reduced when Rab7 was knocked-down. More robust reduction of EGFR and Her2 levels was observed when knocked-down cells were treated with HSP90 inhibitor geldanamycin (GA). Low concentration of GA (50-100 nm)-induced apoptosis of the Rab7 knocked-down cells but not control cells, suggesting that Rab7 and HSP90 together contribute to the optimal stability of EGFR and Her2 as well as to protect cancer cells from apoptosis. Rab7 seems to protect EGFR and Her2 from proteosome-mediated degradation. These results suggest that Rab7 is likely involved in protecting EGFR and Her2 from being degraded by the proteosome and in maintaining optimal Akt survival signal (especially during cell detachment or when HSP90 is inhibited). Rab7 is potentially a novel target for combinatory therapy with Hsp90 inhibitors.
Journal of Cellular Physiology 09/2011; 227(6):2788-97. · 3.87 Impact Factor
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ABSTRACT: The Hippo pathway restricts the activity of transcriptional co-activators TAZ and YAP by phosphorylating them for cytoplasmic
sequestration or degradation. In this report, we describe an independent mechanism for the cell to restrict the activity of
TAZ and YAP through interaction with angiomotin (Amot) and angiomotin-like 1 (AmotL1). Amot and AmotL1 were robustly co-immunoprecipitated
with FLAG-tagged TAZ, and their interaction is dependent on the WW domain of TAZ and the PPXY motif in the N terminus of Amot. Amot and AmotL1 also interact with YAP via the first WW domain of YAP. Overexpression of
Amot and AmotL1 caused cytoplasmic retention of TAZ and suppressed its transcriptional outcome such as the expression of CTGF and Cyr61. Hippo refractory TAZ mutant (S89A) is also negatively regulated by Amot and AmotL1. HEK293 cells express the highest level
of Amot and AmotL1 among nine cell lines examined, and silencing the expression of endogenous Amot increased the expression
of CTGF and Cyr61 either at basal levels or upon overexpression of exogenous S89A. These results reveal a novel mechanism to restrict the activity
of TAZ and YAP through physical interaction with Amot and AmotL1.
Journal of Biological Chemistry 03/2011; 286(9):7018-7026. · 4.77 Impact Factor
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ABSTRACT: Protein-protein interaction cascades are crucial for cellular signaling pathways and cell morphogenesis. Membrane traffic along the secretory and endocytic pathways is similarly governed by regulated protein-protein interactions of diverse machineries, which are inter-regulated, assembled and disassembled sequentially to drive membrane budding, vesicle transport, membrane fission and fusion. Rab7, the key regulator in endo-lysosomal trafficking investigated extensively in the past decades, is emerging to govern early-to-late endosomal maturation, microtubule minus-end as well as plus-end directed endosomal migration and positioning, and endosome-lysosome transport through different protein-protein interaction cascades. We summarize here the key protein interaction cascades of Rab7 by focusing on endo-lysosomal trafficking regulated by its interaction with HOPs, RILP, ORP1L, FYCO1 and Mon1/Sand1-CCZ1 complex.
Cellular signalling 03/2011; 23(3):516-21. · 4.09 Impact Factor
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ABSTRACT: Mutations in the Conserved Oligomeric Golgi (COG) complex give rise to type II congenital disorders of glycosylation (CDG). Thus far, mutations have been identified in 6 of the 8 COG subunits. Here we present data identifying a previously reported CDG-IIx case from Singapore as a new COG4 patient with 2 novel mutations leading to p.E233X and p.L773R; with p.E233X being a de novo mutation. As a result, COG4 protein expression was dramatically reduced, while expression of the other subunits remained unaffected. Analysis of serum N-glycans revealed deficiencies in both sialylation and galactosylation. Furthermore, patient fibroblasts have impaired O-glycosylation. Importantly, patient fibroblasts exhibited a delay in Brefeldin A (BFA) induced retrograde transport, a common characteristic seen in COG deficiencies.
Molecular Genetics and Metabolism 03/2011; 102(3):364-7. · 3.19 Impact Factor
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ABSTRACT: Phox (PX) domain-containing sorting nexins (SNXs) are emerging as important regulators of endocytic trafficking. Sorting nexin 27 (SNX27) is unique, as it contains a PDZ (Psd-95/Dlg/ZO1) domain. We show here that SNX27 is primarily targeted to the early endosome by interaction of its PX domain with PtdIns(3)P. Although targeted ablation of the SNX27 gene in mice did not significantly affect growth and survival during embryonic development, SNX27 plays an essential role in postnatal growth and survival. N-Methyl-d-aspartate (NMDA) receptor 2C (NR2C) was identified as a novel SNX27-interacting protein, and this interaction is mediated by the PDZ domain of SNX27 and the C-terminal PDZ-binding motif of NR2C. Increased NR2C expression levels, together with impaired NR2C endocytosis in SNX27(-/-) neurons, indicate that SNX27 may function to regulate endocytosis and/or endosomal sorting of NR2C. This is consistent with a role of SNX27 as a general regulator for sorting of membrane proteins containing a PDZ-binding motif, and its absence may alter the trafficking of these proteins, leading to growth and survival defects.
Molecular and cellular biology 02/2011; 31(8):1734-47. · 6.06 Impact Factor
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ABSTRACT: The Hippo pathway restricts the activity of transcriptional co-activators TAZ and YAP by phosphorylating them for cytoplasmic sequestration or degradation. In this report, we describe an independent mechanism for the cell to restrict the activity of TAZ and YAP through interaction with angiomotin (Amot) and angiomotin-like 1 (AmotL1). Amot and AmotL1 were robustly co-immunoprecipitated with FLAG-tagged TAZ, and their interaction is dependent on the WW domain of TAZ and the PPXY motif in the N terminus of Amot. Amot and AmotL1 also interact with YAP via the first WW domain of YAP. Overexpression of Amot and AmotL1 caused cytoplasmic retention of TAZ and suppressed its transcriptional outcome such as the expression of CTGF and Cyr61. Hippo refractory TAZ mutant (S89A) is also negatively regulated by Amot and AmotL1. HEK293 cells express the highest level of Amot and AmotL1 among nine cell lines examined, and silencing the expression of endogenous Amot increased the expression of CTGF and Cyr61 either at basal levels or upon overexpression of exogenous S89A. These results reveal a novel mechanism to restrict the activity of TAZ and YAP through physical interaction with Amot and AmotL1.
Journal of Biological Chemistry 01/2011; 286(9):7018-26. · 4.77 Impact Factor
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ABSTRACT: Breast cancer, the most common malignancy in women, still holds many secrets. The causes for non-hereditary breast cancer are still unknown. To elucidate any role for circulating naturally secreted proteins, a screen of secreted proteins' influence of MCF10A cell anchorage independent growth was set up.
To systematically screen secreted proteins for their capacity to transform mammalian breast epithelial cells, a soft agar screen of MCF10A cells was performed using a library of ~ 470 secreted proteins. A high concentration of infecting viral particles was used to obtain multiple infections in individual cells to specifically study the combined effect of multiple secreted proteins.
Several known breast cancer factors, such as Wnt, FGF and IL were retained, as well as factors that were previously unknown to have a role in breast cancer, such as paraoxonase 1 and fibroblast growth factor binding protein 2. Additionally, a combinatory role of Interleukin 6 with other factors in MCF10A anchorage-independent growth is demonstrated.
The transforming effect of combinations of IL6 with other secreted proteins allows studying the transformation of mammary epithelial cells in vitro, and may also have implications in in vivo studies where secreted proteins are upregulated or overexpressed.
Cell & bioscience. 01/2011; 1(1):13.
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ABSTRACT: Oxysterol-binding protein (OSBP) and its related proteins (ORPs) constitute a large and evolutionarily conserved family of lipid-binding proteins that target organelle membranes to mediate sterol signaling and/or transport. Here we characterize ORP5, a tail-anchored ORP protein that localizes to the endoplasmic reticulum. Knocking down ORP5 causes cholesterol accumulation in late endosomes and lysosomes, which is reminiscent of the cholesterol trafficking defect in Niemann Pick C (NPC) fibroblasts. Cholesterol appears to accumulate in the limiting membranes of endosomal compartments in ORP5-depleted cells, whereas depletion of NPC1 or both ORP5 and NPC1 results in luminal accumulation of cholesterol. Moreover, trans-Golgi resident proteins mislocalize to endosomal compartments upon ORP5 depletion, which depends on a functional NPC1. Our results establish the first link between NPC1 and a cytoplasmic sterol carrier, and suggest that ORP5 may cooperate with NPC1 to mediate the exit of cholesterol from endosomes/lysosomes.
The Journal of Cell Biology 01/2011; 192(1):121-35. · 10.26 Impact Factor
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ABSTRACT: EHD1 is an EH (Eps15 homology) domain-containing protein involved in endosomal recycling. Our yeast two hybrid screening experiments showed that EHD1 interacts with a synaptic protein, snapin, and the present study was carried out to further elucidate the functional significance of this interaction. Immunoreactivity to EHD1 is observed in the cerebral cortex, hippocampus and striatum, in the rat brain. The protein is colocalized with the axon terminal marker synaptophysin in cultured neurons. EHD1 binds to the C terminus of snapin via its C terminus EH domain. It negatively affects the binding of a SNARE complex protein, SNAP-25, to snapin, probably due to the competition for overlapping binding sites on the C terminus of snapin. EHD1 affects the coupling of synaptotagmin-1 to the SNARE complex, and could be a negative regulator of exocytosis. This is supported by electrophysiological findings that PC-12 cells which overexpress EHD1 show reduced depolarization-induced exocytosis compared to controls, but the reduced exocytosis is not observed in cells which overexpress the N terminus of EHD1 that is unable to bind snapin. Together, the above results indicate that EHD1 is a synaptic protein that negatively affects exocytosis through binding to snapin.
Molecular and Cellular Neuroscience 12/2010; 45(4):418-29. · 3.66 Impact Factor
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Chen-Ying Liu,
Zheng-Yu Zha,
Xin Zhou,
Heng Zhang,
Wei Huang,
Di Zhao,
Tingting Li,
Siew Wee Chan,
Chun Jye Lim, Wanjin Hong,
Shimin Zhao,
Yue Xiong,
Qun-Ying Lei,
Kun-Liang Guan
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ABSTRACT: The TAZ transcription co-activator promotes cell proliferation and epithelial-mesenchymal transition. TAZ is inhibited by the Hippo tumor suppressor pathway, which promotes TAZ cytoplasmic localization by phosphorylation. We report here that TAZ protein stability is controlled by a phosphodegron recognized by the F-box protein β-TrCP and ubiquitylated by the SCF/CRL1(β-TrCP) E3 ligase. The interaction between TAZ and β-TrCP is regulated by the Hippo pathway. Phosphorylation of a phosphodegron in TAZ by LATS primes it for further phosphorylation by CK1ε and subsequent binding by β-TrCP. Therefore, the Hippo pathway negatively regulates TAZ function by both limiting its nuclear accumulation and promoting its degradation. The phosphodegron-mediated TAZ degradation plays an important role in negatively regulating TAZ biological functions.
Journal of Biological Chemistry 11/2010; 285(48):37159-69. · 4.77 Impact Factor
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Chen-Ying Liu,
Zheng-Yu Zha,
Xin Zhou,
Heng Zhang,
Wei Huang,
Di Zhao,
Tingting Li,
Siew Wee Chan,
Chun Jye Lim, Wanjin Hong,
Shimin Zhao,
Yue Xiong,
Qun-Ying Lei,
Kun-Liang Guan
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ABSTRACT: The TAZ transcription co-activator promotes cell proliferation and epithelial-mesenchymal transition. TAZ is inhibited by
the Hippo tumor suppressor pathway, which promotes TAZ cytoplasmic localization by phosphorylation. We report here that TAZ
protein stability is controlled by a phosphodegron recognized by the F-box protein β-TrCP and ubiquitylated by the SCF/CRL1β-TrCP E3 ligase. The interaction between TAZ and β-TrCP is regulated by the Hippo pathway. Phosphorylation of a phosphodegron in
TAZ by LATS primes it for further phosphorylation by CK1ϵ and subsequent binding by β-TrCP. Therefore, the Hippo pathway negatively
regulates TAZ function by both limiting its nuclear accumulation and promoting its degradation. The phosphodegron-mediated
TAZ degradation plays an important role in negatively regulating TAZ biological functions.
Journal of Biological Chemistry 11/2010; 285(48):37159-37169. · 4.77 Impact Factor
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ABSTRACT: The Hippo pathway is an evolutionally conserved protein kinase cascade involved in regulating organ size in vivo and cell contact inhibition in vitro by governing cell proliferation and apoptosis. Deregulation of the Hippo pathway is linked to cancer development. Its first core kinase Warts was identified in Drosophila more than 15 years ago, but it gained much attention when other core components of the pathway were identified 8 years later. Major discoveries of the pathway were made during past several years. The core kinase components Hippo, Salvador, Warts, and Mats in the fly and Mst1/2, WW45, Lats1/2, and Mob1 in mammals phosphorylate and inactivate downstream transcriptional co-activators Yorkie in the fly, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) in mammals, respectively. Phosphorylated Yorkie, YAP, and TAZ are sequestered in the cytoplasm by interaction with 14-3-3 proteins. Here we review recent progresses of this pathway by focusing on how these proteins communicate with each other and how loss of regulation results in cancers.
Journal of Cellular Physiology 10/2010; 226(4):928-39. · 3.87 Impact Factor
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ABSTRACT: The maintenance of cellular homeostasis and execution of regulatory mechanisms to dynamically govern various cellular processes require the correct delivery of proteins to their target subcellular compartments. It is estimated that over 30% of the proteins encoded by the human genome, projected to encode about 25 000 proteins and other macromolecules, are delivered to the secretory and endocytic pathways where movement of proteins between various compartments is primarily mediated by vesicles/carriers budding from one compartment for delivery to another. Sorting of cargo proteins into budding vesicles/carriers is mediated by adaptors that link the cargo proteins to the coat proteins. The adaptor function of VHS domain proteins, GGA proteins, STAM proteins and Hrs is well-established and is evolutionarily conserved from yeast to humans. Recent studies suggest that Tom1, Tom1L1 and Tom1L2 subfamily of VHS domain proteins, which do not exist in yeast, are emerging as novel regulators for post-Golgi trafficking and signaling.
Traffic 09/2010; 11(9):1119-28. · 4.92 Impact Factor
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ABSTRACT: Coat protein II (COPII)-mediated export from the endoplasmic reticulum (ER) involves sequential recruitment of COPII complex components, including the Sar1 GTPase, the Sec23/Sec24 subcomplex, and the Sec13/Sec31 subcomplex. p125A was originally identified as a Sec23A-interacting protein. Here we demonstrate that p125A also interacts with the C-terminal region of Sec31A. The Sec31A-interacting domain of p125A is between residues 260-600, and is therefore a distinct domain from that required for interaction with Sec23A. Gel filtration and immunodepletion studies suggest that the majority of cytosolic p125A exists as a ternary complex with the Sec13/Sec31A subcomplex, suggesting that Sec 13, Sec31A, and p125A exist in the cytosol primarily as preassembled Sec13/Sec31A/p125A heterohexamers. Golgi morphology and protein export from the ER were affected in p125A-silenced cells. Our results suggest that p125A is part of the Sec13/Sec31A subcomplex and facilitates ER export in mammalian cells.
The Journal of Cell Biology 08/2010; 190(3):331-45. · 10.26 Impact Factor
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ABSTRACT: The Hippo signaling pathway controls cell growth, proliferation, and apoptosis by regulating the expression of target genes that execute these processes. Acting downstream from this pathway is the YAP transcriptional coactivator, whose biological function is mediated by the conserved TEAD family transcription factors. The interaction of YAP with TEADs is critical to regulate Hippo pathway-responsive genes. Here, we describe the crystal structure of the YAP-interacting C-terminal domain of TEAD4 in complex with the TEAD-interacting N-terminal domain of YAP. The structure reveals that the N-terminal region of YAP is folded into two short helices with an extended loop containing the PXXPhiP motif in between, while the C-terminal domain of TEAD4 has an immunoglobulin-like fold. YAP interacts with TEAD4 mainly through the two short helices. Point mutations of TEAD4 indicate that the residues important for YAP interaction are required for its transforming activity. Mutagenesis reveals that the PXXPhiP motif of YAP, although making few contacts with TEAD4, is important for TEAD4 interaction as well as for the transforming activity.
Genes & development 02/2010; 24(3):290-300. · 12.08 Impact Factor
