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Zhang Z, Mei X, Wang H, Gong H, Chen R, Liu B, Wei Y, Gan Y, Yuan T, Wu Y, Shao G, Xiong Q, Zhang C, Feng Z. Long non-coding RNA MMTP mediates necroptosis in alveolar macrophages during Mycoplasma hyopneumoniae infection by enhancing TNF-α transcription. Int J Biol Macromol 2025; 288:138649. [PMID: 39674476 DOI: 10.1016/j.ijbiomac.2024.138649] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2024] [Revised: 12/01/2024] [Accepted: 12/09/2024] [Indexed: 12/16/2024]
Abstract
Mycoplasma hyopneumoniae (M. hyo), a major respiratory pathogen in swine, causes chronic respiratory diseases characterized by severe lung inflammation. Alveolar macrophages, which serve as the first line of defense in the respiratory immune system, undergo necroptosis in response to M. hyo infection. This form of programmed cell death amplifies pulmonary inflammation and leads to impaired lung function, yet the precise molecular mechanisms remain poorly understood. Long non-coding RNAs (lncRNAs), known for their regulatory roles in transcriptional and epigenetic processes, have been linked to various inflammatory and infectious diseases. In this study, we identified a novel lncRNA, lncRNA-MMTP, as a critical regulator of necroptosis during M. hyo infection. Mechanistically, lncRNA-MMTP interacts with the transcription factor TFII-I to enhance c-Fos promoter activity, leading to increased transcription of TNF-α and activation of the RIPK1/RIPK3/MLKL necroptotic pathway. Importantly, knockdown of lncRNA-MMTP or inhibition of TFII-I significantly reduced TNF-α levels and necroptosis in alveolar macrophages. These findings not only elucidate a new molecular pathway underlying M. hyo-induced lung inflammation but also suggest potential therapeutic targets for managing pathogen-induced inflammatory responses in the respiratory system.
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Affiliation(s)
- Zhenzhen Zhang
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China; School of Animal Medicine, Nanjing Agricultural University, Nanjing 210095, China.
| | - Xiuzhen Mei
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China; School of Animal Medicine, Nanjing Agricultural University, Nanjing 210095, China
| | - Hui Wang
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China; Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Marine Science and Engineering, Nanjing Normal University, Nanjing 210023, China
| | - Hanfei Gong
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China; School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Rong Chen
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China
| | - Beibei Liu
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China
| | - Yanna Wei
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China
| | - Yuan Gan
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China
| | - Ting Yuan
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China
| | - Yuzi Wu
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China
| | - Guoqing Shao
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China
| | - Qiyan Xiong
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China
| | - Chao Zhang
- School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, China.
| | - Zhixin Feng
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China; School of Animal Medicine, Nanjing Agricultural University, Nanjing 210095, China.
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De D, Thapliyal N, Prakash Tiwari V, Toyama Y, Flemming Hansen D, Kay LE, Vallurupalli P. Mapping the FF domain folding pathway via structures of transiently populated folding intermediates. Proc Natl Acad Sci U S A 2024; 121:e2416682121. [PMID: 39630857 DOI: 10.1073/pnas.2416682121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2024] [Accepted: 11/04/2024] [Indexed: 12/07/2024] Open
Abstract
Despite the tremendous accomplishments of AlpaFold2/3 in predicting biomolecular structure, the protein folding problem remains unsolved in the sense that accurate atomistic models of how protein molecules fold into their native conformations from an unfolded ensemble are still elusive. Here, using chemical exchange saturation transfer (CEST) NMR experiments and a comprehensive four-state kinetic model of the folding trajectory of a 71 residue four-helix bundle FF domain from human HYPA/FBP11 we present an atomic resolution structure of a transiently formed intermediate, I2, that along with the structure of a second intermediate, I1, provides a description of the FF domain folding trajectory. By recording CEST profiles as a function of urea concentration the extent of compaction along the folding pathway is evaluated. Our data establish that unlike the partially disordered I1 state, the I2 intermediate that is also formed before the rate-limiting folding barrier is well ordered and compact like the native conformer, while retaining nonnative interactions similar to those found in I1. The slow-interconversion from I2 to F, involving changes in secondary structure and the breaking of nonnative interactions, proceeds via a compact transition-state. Interestingly, the native state of the FF1 domain from human p190-A Rho GAP resembles the I2 conformation, suggesting that well-ordered folding intermediates can be repurposed by nature in structurally related proteins to assume functional roles. It is anticipated that the strategy for elucidation of sparsely populated and transiently formed structures of intermediates along kinetic pathways described here will be of use in other studies of protein dynamics.
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Affiliation(s)
- Debajyoti De
- Tata Institute of Fundamental Research Hyderabad, Ranga Reddy District, Hyderabad 500046, India
| | - Nemika Thapliyal
- Tata Institute of Fundamental Research Hyderabad, Ranga Reddy District, Hyderabad 500046, India
| | - Ved Prakash Tiwari
- Tata Institute of Fundamental Research Hyderabad, Ranga Reddy District, Hyderabad 500046, India
| | - Yuki Toyama
- Department of Molecular Genetics, University of Toronto, Toronto M5S 1A8, Canada
- Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6, Canada
- Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada
- Center for Biosystems Dynamics Research, RIKEN, Kanagawa 230-0045, Japan
| | - D Flemming Hansen
- Department of Structural and Molecular Biology, Division of Biosciences, University College London, London WC1E 6BT, United Kingdom
- The Francis Crick Institute, London NW1 1AT, United Kingdom
| | - Lewis E Kay
- Department of Molecular Genetics, University of Toronto, Toronto M5S 1A8, Canada
- Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6, Canada
- Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada
| | - Pramodh Vallurupalli
- Tata Institute of Fundamental Research Hyderabad, Ranga Reddy District, Hyderabad 500046, India
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3
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Kannan A, Chaurasiya DK, Naganathan AN. Conflicting Interfacial Electrostatic Interactions as a Design Principle to Modulate Long-Range Interdomain Communication. ACS BIO & MED CHEM AU 2024; 4:53-67. [PMID: 38404745 PMCID: PMC10885104 DOI: 10.1021/acsbiomedchemau.3c00047] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/17/2023] [Revised: 10/19/2023] [Accepted: 10/23/2023] [Indexed: 02/27/2024]
Abstract
The extent and molecular basis of interdomain communication in multidomain proteins, central to understanding allostery and function, is an open question. One simple evolutionary strategy could involve the selection of either conflicting or favorable electrostatic interactions across the interface of two closely spaced domains to tune the magnitude of interdomain connectivity. Here, we study a bilobed domain FF34 from the eukaryotic p190A RhoGAP protein to explore one such design principle that mediates interdomain communication. We find that while the individual structural units in wild-type FF34 are marginally coupled, they exhibit distinct intrinsic stabilities and low cooperativity, manifesting as slow folding. The FF3-FF4 interface harbors a frustrated network of highly conserved electrostatic interactions-a charge troika-that promotes the population of multiple, decoupled, and non-native structural modes on a rugged native landscape. Perturbing this network via a charge-reversal mutation not only enhances stability and cooperativity but also dampens the fluctuations globally and speeds up the folding rate by at least an order of magnitude. Our work highlights how a conserved but nonoptimal network of interfacial electrostatic interactions shapes the native ensemble of a bilobed protein, a feature that could be exploited in designing molecular systems with long-range connectivity and enhanced cooperativity.
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Affiliation(s)
- Adithi Kannan
- Department of Biotechnology,
Bhupat & Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India
| | - Dhruv Kumar Chaurasiya
- Department of Biotechnology,
Bhupat & Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India
| | - Athi N. Naganathan
- Department of Biotechnology,
Bhupat & Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India
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4
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Ouyang H, Wu S, Li W, Grey MJ, Wu W, Hansen SH. p120 RasGAP and ZO-2 are essential for Hippo signaling and tumor-suppressor function mediated by p190A RhoGAP. Cell Rep 2023; 42:113486. [PMID: 37995182 PMCID: PMC10809936 DOI: 10.1016/j.celrep.2023.113486] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2022] [Revised: 10/19/2023] [Accepted: 11/08/2023] [Indexed: 11/25/2023] Open
Abstract
ARHGAP35, which encodes p190A RhoGAP (p190A), is a major cancer gene. p190A is a tumor suppressor that activates the Hippo pathway. p190A was originally cloned via direct binding to p120 RasGAP (RasGAP). Here, we determine that interaction of p190A with the tight-junction-associated protein ZO-2 is dependent on RasGAP. We establish that both RasGAP and ZO-2 are necessary for p190A to activate large tumor-suppressor (LATS) kinases, elicit mesenchymal-to-epithelial transition, promote contact inhibition of cell proliferation, and suppress tumorigenesis. Moreover, RasGAP and ZO-2 are required for transcriptional modulation by p190A. Finally, we demonstrate that low ARHGAP35 expression is associated with shorter survival in patients with high, but not low, transcript levels of TJP2 encoding ZO-2. Hence, we define a tumor-suppressor interactome of p190A that includes ZO-2, an established constituent of the Hippo pathway, and RasGAP, which, despite strong association with Ras signaling, is essential for p190A to activate LATS kinases.
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Affiliation(s)
- Hanyue Ouyang
- GI Cell Biology Laboratory, Boston Children's Hospital, Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA; State Key Laboratory of Oncology in South China, Guangdong Provincial Clinical Research Center for Cancer, Sun Yat-sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, P.R. China
| | - Shuang Wu
- Department of Radiation Oncology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, P.R. China
| | - Wangji Li
- GI Cell Biology Laboratory, Boston Children's Hospital, Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
| | - Michael J Grey
- GI Cell Biology Laboratory, Boston Children's Hospital, Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
| | - Wenchao Wu
- State Key Laboratory of Oncology in South China, Guangdong Provincial Clinical Research Center for Cancer, Sun Yat-sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, P.R. China
| | - Steen H Hansen
- GI Cell Biology Laboratory, Boston Children's Hospital, Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
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5
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Ouyang H, Li W, Hansen SH. p120 RasGAP and ZO-2 are essential for Hippo signaling and tumor suppressor function mediated by p190A RhoGAP. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.05.22.541483. [PMID: 37292741 PMCID: PMC10245842 DOI: 10.1101/2023.05.22.541483] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/10/2023]
Abstract
ARHGAP35 , which encodes p190A RhoGAP (p190A), is a major cancer gene. p190A is a tumor suppressor that activates the Hippo pathway. p190A was originally cloned via direct binding to p120 RasGAP (RasGAP). Here, we determine that a novel interaction of p190A with the tight junction-associated protein ZO-2 is dependent on RasGAP. We establish that both RasGAP and ZO-2 are necessary for p190A to activate LATS kinases, elicit mesenchymal-to-epithelial transition, promote contact inhibition of cell proliferation and suppress tumorigenesis. Moreover, RasGAP and ZO-2 are required for transcriptional modulation by p190A. Finally, we demonstrate that low ARHGAP35 expression is associated with shorter survival in patients with high, but not low, transcript levels of TJP2 encoding ZO-2. Hence, we define a tumor suppressor interactome of p190A that includes ZO-2, an established constituent of the Hippo pathway, and RasGAP, which despite strong association with Ras signaling, is essential for p190A to activate LATS kinases.
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6
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Golla H, Kannan A, Gopi S, Murugan S, Perumalsamy LR, Naganathan AN. Structural-Energetic Basis for Coupling between Equilibrium Fluctuations and Phosphorylation in a Protein Native Ensemble. ACS CENTRAL SCIENCE 2022; 8:282-293. [PMID: 35233459 PMCID: PMC8880421 DOI: 10.1021/acscentsci.1c01548] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/16/2021] [Indexed: 06/14/2023]
Abstract
The functioning of proteins is intimately tied to their fluctuations in the native ensemble. The structural-energetic features that determine fluctuation amplitudes and hence the shape of the underlying landscape, which in turn determine the magnitude of the functional output, are often confounded by multiple variables. Here, we employ the FF1 domain from human p190A RhoGAP protein as a model system to uncover the molecular basis for phosphorylation of a buried tyrosine, which is crucial to the transcriptional activity associated with transcription factor TFII-I. Combining spectroscopy, calorimetry, statistical-mechanical modeling, molecular simulations, and in vitro phosphorylation assays, we show that the FF1 domain samples a diverse array of conformations in its native ensemble, some of which are phosphorylation-competent. Upon eliminating unfavorable charge-charge interactions through a single charge-reversal (K53E) or charge-neutralizing (K53Q) mutation, we observe proportionately lower phosphorylation extents due to the altered structural coupling, damped equilibrium fluctuations, and a more compact native ensemble. We thus establish a conformational selection mechanism for phosphorylation in the FF1 domain with K53 acting as a "gatekeeper", modulating the solvent exposure of the buried tyrosine. Our work demonstrates the role of unfavorable charge-charge interactions in governing functional events through the modulation of native ensemble characteristics, a feature that could be prevalent in ordered protein domains.
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Affiliation(s)
- Hemashree Golla
- Department
of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India
| | - Adithi Kannan
- Department
of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India
| | - Soundhararajan Gopi
- Department
of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India
| | - Sowmiya Murugan
- Department
of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India
| | - Lakshmi R Perumalsamy
- Department
of Biomedical Sciences, Sri Ramachandra
Institute of Higher Education and Research, Chennai 600116, India
| | - Athi N. Naganathan
- Department
of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India
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7
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Li Y, Chen B, Zhao J, Li Q, Chen S, Guo T, Li Y, Lai H, Chen Z, Meng Z, Guo W, He X, Huang S. HNRNPL Circularizes ARHGAP35 to Produce an Oncogenic Protein. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2021; 8:2001701. [PMID: 34258149 PMCID: PMC8261482 DOI: 10.1002/advs.202001701] [Citation(s) in RCA: 63] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/08/2020] [Revised: 02/25/2021] [Indexed: 05/27/2023]
Abstract
Circular RNAs (circRNAs) are an intriguing class of widely prevalent endogenous RNAs, the vast majority of which have not been characterized functionally. Here, we identified a novel oncogenic circRNA originating from the back-splicing of Exon2 and Exon3 of a tumor suppressor gene, ARHGAP35 (also known as P190-A), termed as circARHGAP35. have observe that circARHGAP35 and linear ARHGAP35 have antithetical expression and functions. Interestingly, circARHGAP35 contains a 3867 nt long ORF with an m6A-modified start codon and encodes a truncated protein comprising four FF domains and lacking the Rho GAP domain. Mechanistically, circARHGAP35 protein promotes cancer cell progression by interacting with TFII-I protein in the nucleus. The RNA binding protein, HNRNPL, facilitates the formation of circARHGAP35. Clinically, circARHGAP35 is associated with poor survival in cancer patients. Our findings characterize an oncogenic circRNA and demonstrate a novel mechanism of oncogene activation in cancer by circRNA through the production of a truncated protein.
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Affiliation(s)
- Yan Li
- Department of Integrative OncologyFudan University Shanghai Cancer CenterShanghai Key Laboratory of Medical EpigeneticsInstitutes of Biomedical SciencesShanghai Medical CollegeFudan UniversityShanghai200032China
| | - Bing Chen
- Department of Integrative OncologyFudan University Shanghai Cancer CenterShanghai Key Laboratory of Medical EpigeneticsInstitutes of Biomedical SciencesShanghai Medical CollegeFudan UniversityShanghai200032China
| | - Jingjing Zhao
- Department of Integrative OncologyFudan University Shanghai Cancer CenterShanghai Key Laboratory of Medical EpigeneticsInstitutes of Biomedical SciencesShanghai Medical CollegeFudan UniversityShanghai200032China
| | - Qin Li
- Department of Integrative OncologyFudan University Shanghai Cancer CenterShanghai Key Laboratory of Medical EpigeneticsInstitutes of Biomedical SciencesShanghai Medical CollegeFudan UniversityShanghai200032China
| | - Siyuan Chen
- Department of Medical OncologyFudan University Shanghai Cancer CenterShanghai Medical CollegeFudan UniversityShanghai200032China
| | - Tianan Guo
- Department of Colorectal SurgeryFudan University Shanghai Cancer CenterShanghai Medical CollegeFudan UniversityShanghai200032China
| | - Yuchen Li
- Department of Integrative OncologyFudan University Shanghai Cancer CenterShanghai Key Laboratory of Medical EpigeneticsInstitutes of Biomedical SciencesShanghai Medical CollegeFudan UniversityShanghai200032China
| | - Hongyan Lai
- Department of Integrative OncologyFudan University Shanghai Cancer CenterShanghai Key Laboratory of Medical EpigeneticsInstitutes of Biomedical SciencesShanghai Medical CollegeFudan UniversityShanghai200032China
| | - Zhiao Chen
- Fudan University Shanghai Cancer Center and Institutes of Biomedical SciencesShanghai Key Laboratory of Radiation OncologyShanghai Medical CollegeFudan UniversityShanghai200032China
| | - Zhiqiang Meng
- Department of Integrative OncologyFudan University Shanghai Cancer CenterShanghai Key Laboratory of Medical EpigeneticsInstitutes of Biomedical SciencesShanghai Medical CollegeFudan UniversityShanghai200032China
| | - Weijie Guo
- Fudan University Shanghai Cancer Center and Institutes of Biomedical SciencesShanghai Key Laboratory of Radiation OncologyShanghai Medical CollegeFudan UniversityShanghai200032China
| | - Xianghuo He
- Fudan University Shanghai Cancer Center and Institutes of Biomedical SciencesShanghai Key Laboratory of Radiation OncologyShanghai Medical CollegeFudan UniversityShanghai200032China
| | - Shenglin Huang
- Department of Integrative OncologyFudan University Shanghai Cancer CenterShanghai Key Laboratory of Medical EpigeneticsInstitutes of Biomedical SciencesShanghai Medical CollegeFudan UniversityShanghai200032China
- Fudan University Shanghai Cancer Center and Institutes of Biomedical SciencesShanghai Key Laboratory of Radiation OncologyShanghai Medical CollegeFudan UniversityShanghai200032China
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8
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Linzer N, Trumbull A, Nar R, Gibbons MD, Yu DT, Strouboulis J, Bungert J. Regulation of RNA Polymerase II Transcription Initiation and Elongation by Transcription Factor TFII-I. Front Mol Biosci 2021; 8:681550. [PMID: 34055891 PMCID: PMC8155576 DOI: 10.3389/fmolb.2021.681550] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Accepted: 04/20/2021] [Indexed: 11/13/2022] Open
Abstract
Transcription by RNA polymerase II (Pol II) is regulated by different processes, including alterations in chromatin structure, interactions between distal regulatory elements and promoters, formation of transcription domains enriched for Pol II and co-regulators, and mechanisms involved in the initiation, elongation, and termination steps of transcription. Transcription factor TFII-I, originally identified as an initiator (INR)-binding protein, contains multiple protein–protein interaction domains and plays diverse roles in the regulation of transcription. Genome-wide analysis revealed that TFII-I associates with expressed as well as repressed genes. Consistently, TFII-I interacts with co-regulators that either positively or negatively regulate the transcription. Furthermore, TFII-I has been shown to regulate transcription pausing by interacting with proteins that promote or inhibit the elongation step of transcription. Changes in TFII-I expression in humans are associated with neurological and immunological diseases as well as cancer. Furthermore, TFII-I is essential for the development of mice and represents a barrier for the induction of pluripotency. Here, we review the known functions of TFII-I related to the regulation of Pol II transcription at the stages of initiation and elongation.
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Affiliation(s)
- Niko Linzer
- Department of Biochemistry and Molecular Biology, College of Medicine, UF Health Cancer Center, Genetics Institute, Powell Gene Therapy Center, University of Florida, Gainesville, FL, United States
| | - Alexis Trumbull
- Department of Biochemistry and Molecular Biology, College of Medicine, UF Health Cancer Center, Genetics Institute, Powell Gene Therapy Center, University of Florida, Gainesville, FL, United States
| | - Rukiye Nar
- Department of Biochemistry and Molecular Biology, College of Medicine, UF Health Cancer Center, Genetics Institute, Powell Gene Therapy Center, University of Florida, Gainesville, FL, United States
| | - Matthew D Gibbons
- Department of Biochemistry and Molecular Biology, College of Medicine, UF Health Cancer Center, Genetics Institute, Powell Gene Therapy Center, University of Florida, Gainesville, FL, United States
| | - David T Yu
- Department of Biochemistry and Molecular Biology, College of Medicine, UF Health Cancer Center, Genetics Institute, Powell Gene Therapy Center, University of Florida, Gainesville, FL, United States
| | - John Strouboulis
- Comprehensive Cancer Center, School of Cancer and Pharmaceutical Sciences, King's College London, United Kingdom
| | - Jörg Bungert
- Department of Biochemistry and Molecular Biology, College of Medicine, UF Health Cancer Center, Genetics Institute, Powell Gene Therapy Center, University of Florida, Gainesville, FL, United States
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9
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Sheng J, Olrichs NK, Gadella BM, Kaloyanova DV, Helms JB. Regulation of Functional Protein Aggregation by Multiple Factors: Implications for the Amyloidogenic Behavior of the CAP Superfamily Proteins. Int J Mol Sci 2020; 21:E6530. [PMID: 32906672 PMCID: PMC7554809 DOI: 10.3390/ijms21186530] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2020] [Revised: 09/01/2020] [Accepted: 09/03/2020] [Indexed: 12/13/2022] Open
Abstract
The idea that amyloid fibrils and other types of protein aggregates are toxic for cells has been challenged by the discovery of a variety of functional aggregates. However, an identification of crucial differences between pathological and functional aggregation remains to be explored. Functional protein aggregation is often reversible by nature in order to respond properly to changing physiological conditions of the cell. In addition, increasing evidence indicates that fast fibril growth is a feature of functional amyloids, providing protection against the long-term existence of potentially toxic oligomeric intermediates. It is becoming clear that functional protein aggregation is a complexly organized process that can be mediated by a multitude of biomolecular factors. In this overview, we discuss the roles of diverse biomolecules, such as lipids/membranes, glycosaminoglycans, nucleic acids and metal ions, in regulating functional protein aggregation. Our studies on the protein GAPR-1 revealed that several of these factors influence the amyloidogenic properties of this protein. These observations suggest that GAPR-1, as well as the cysteine-rich secretory proteins, antigen 5 and pathogenesis-related proteins group 1 (CAP) superfamily of proteins that it belongs to, require the assembly into an amyloid state to exert several of their functions. A better understanding of functional aggregate formation may also help in the prevention and treatment of amyloid-related diseases.
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Affiliation(s)
| | | | | | | | - J. Bernd Helms
- Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands; (J.S.); (N.K.O.); (B.M.G.); (D.V.K.)
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10
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Ouyang H, Luong P, Frödin M, Hansen SH. p190A RhoGAP induces CDH1 expression and cooperates with E-cadherin to activate LATS kinases and suppress tumor cell growth. Oncogene 2020; 39:5570-5587. [PMID: 32641858 PMCID: PMC7426264 DOI: 10.1038/s41388-020-1385-2] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2020] [Revised: 06/09/2020] [Accepted: 06/29/2020] [Indexed: 01/06/2023]
Abstract
The ARHGAP35 gene encoding p190A RhoGAP (p190A) is significantly altered by both mutation and allelic deletion in human cancer, but the functional implications of such alterations are not known. Here, we demonstrate for the first time that p190A is a tumor suppressor using a xenograft mouse model with carcinoma cells harboring defined ARHGAP35 alterations. In vitro, restoration of p190A expression in carcinoma cells promotes contact inhibition of proliferation (CIP) through activation of LATS kinases and phosphorylation of the proto-oncogenic transcriptional co-activator YAP. In contrast, p190A forms harboring recurrent cancer mutations exhibit loss of function in modulating the Hippo pathway, inducing CIP, as well as attenuated suppression of tumor growth in mice. We determine that p190A promotes mesenchymal to epithelial transition (MET) and elicits expression of a cassette of epithelial adherens junction-associated genes in a cell density-dependent manner. This cassette includes CDH1 encoding E-cadherin, which amplifies p190A-mediated LATS activation and is necessary for CIP. Oppositely, we establish that p190A is obligatory for E-cadherin to activate LATS kinases and induce CIP. Collectively, this work defines a novel mechanism by which p190A and E-cadherin cooperate in modulating Hippo signaling to suppress tumor cell growth.
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Affiliation(s)
- Hanyue Ouyang
- GI Cell Biology Laboratory, Boston Children's Hospital and Harvard Medical School, Boston, MA, 02115, USA
| | - Phi Luong
- GI Cell Biology Laboratory, Boston Children's Hospital and Harvard Medical School, Boston, MA, 02115, USA
| | - Morten Frödin
- Biotech Research & Innovation Centre (BRIC), University of Copenhagen, 2200, Copenhagen N, Denmark
| | - Steen H Hansen
- GI Cell Biology Laboratory, Boston Children's Hospital and Harvard Medical School, Boston, MA, 02115, USA.
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11
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Héraud C, Pinault M, Lagrée V, Moreau V. p190RhoGAPs, the ARHGAP35- and ARHGAP5-Encoded Proteins, in Health and Disease. Cells 2019; 8:cells8040351. [PMID: 31013840 PMCID: PMC6523970 DOI: 10.3390/cells8040351] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2019] [Revised: 04/05/2019] [Accepted: 04/09/2019] [Indexed: 12/30/2022] Open
Abstract
Small guanosine triphosphatases (GTPases) gathered in the Rat sarcoma (Ras) superfamily represent a large family of proteins involved in several key cellular mechanisms. Within the Ras superfamily, the Ras homolog (Rho) family is specialized in the regulation of actin cytoskeleton-based mechanisms. These proteins switch between an active and an inactive state, resulting in subsequent inhibiting or activating downstream signals, leading finally to regulation of actin-based processes. The On/Off status of Rho GTPases implicates two subsets of regulators: GEFs (guanine nucleotide exchange factors), which favor the active GTP (guanosine triphosphate) status of the GTPase and GAPs (GTPase activating proteins), which inhibit the GTPase by enhancing the GTP hydrolysis. In humans, the 20 identified Rho GTPases are regulated by over 70 GAP proteins suggesting a complex, but well-defined, spatio-temporal implication of these GAPs. Among the quite large number of RhoGAPs, we focus on p190RhoGAP, which is known as the main negative regulator of RhoA, but not exclusively. Two isoforms, p190A and p190B, are encoded by ARHGAP35 and ARHGAP5 genes, respectively. We describe here the function of each of these isoforms in physiological processes and sum up findings on their role in pathological conditions such as neurological disorders and cancers.
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Affiliation(s)
- Capucine Héraud
- INSERM, UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, F-33000 Bordeaux, France.
- University of Bordeaux, UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, Bordeaux F-33000, France.
- Equipe Labellisée Fondation pour la Recherche Médicale (FRM) 2018, 75007 Paris, France.
| | - Mathilde Pinault
- INSERM, UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, F-33000 Bordeaux, France.
- University of Bordeaux, UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, Bordeaux F-33000, France.
- Equipe Labellisée Fondation pour la Recherche Médicale (FRM) 2018, 75007 Paris, France.
| | - Valérie Lagrée
- INSERM, UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, F-33000 Bordeaux, France.
- University of Bordeaux, UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, Bordeaux F-33000, France.
- Equipe Labellisée Fondation pour la Recherche Médicale (FRM) 2018, 75007 Paris, France.
| | - Violaine Moreau
- INSERM, UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, F-33000 Bordeaux, France.
- University of Bordeaux, UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, Bordeaux F-33000, France.
- Equipe Labellisée Fondation pour la Recherche Médicale (FRM) 2018, 75007 Paris, France.
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12
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Frank SR, Köllmann CP, Luong P, Galli GG, Zou L, Bernards A, Getz G, Calogero RA, Frödin M, Hansen SH. p190 RhoGAP promotes contact inhibition in epithelial cells by repressing YAP activity. J Cell Biol 2018; 217:3183-3201. [PMID: 29934311 PMCID: PMC6122998 DOI: 10.1083/jcb.201710058] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2017] [Revised: 04/06/2018] [Accepted: 05/29/2018] [Indexed: 12/14/2022] Open
Abstract
ARHGAP35 encoding p190A RhoGAP is a cancer-associated gene with a mutation spectrum suggestive of a tumor-suppressor function. In this study, we demonstrate that loss of heterozygosity for ARHGAP35 occurs in human tumors. We sought to identify tumor-suppressor capacities for p190A RhoGAP (p190A) and its paralog p190B in epithelial cells. We reveal an essential role for p190A and p190B to promote contact inhibition of cell proliferation (CIP), a function that relies on RhoGAP activity. Unbiased mRNA sequencing analyses establish that p190A and p190B modulate expression of genes associated with the Hippo pathway. Accordingly, we determine that p190A and p190B induce CIP by repressing YAP-TEAD-regulated gene transcription through activation of LATS kinases and inhibition of the Rho-ROCK pathway. Finally, we demonstrate that loss of a single p190 paralog is sufficient to elicit nuclear translocation of YAP and perturb CIP in epithelial cells cultured in Matrigel. Collectively, our data reveal a novel mechanism consistent with a tumor-suppressor function for ARHGAP35.
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Affiliation(s)
- Scott R Frank
- GI Cell Biology Research Laboratory, Boston Children's Hospital and Harvard Medical School, Boston, MA
| | - Clemens P Köllmann
- GI Cell Biology Research Laboratory, Boston Children's Hospital and Harvard Medical School, Boston, MA
| | - Phi Luong
- GI Cell Biology Research Laboratory, Boston Children's Hospital and Harvard Medical School, Boston, MA
| | - Giorgio G Galli
- Stem Cell Program, Boston Children's Hospital and Harvard Stem Cell Institute, Boston, MA
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA
| | - Lihua Zou
- The Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA
| | - André Bernards
- The Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA
- Massachusetts General Hospital Center for Cancer Research and Harvard Medical School, Charlestown, MA
| | - Gad Getz
- The Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA
- Massachusetts General Hospital Center for Cancer Research and Harvard Medical School, Charlestown, MA
| | - Raffaele A Calogero
- University of Torino, Department of Molecular Biotechnology and Health Sciences, Torino, Italy
| | - Morten Frödin
- Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark
| | - Steen H Hansen
- GI Cell Biology Research Laboratory, Boston Children's Hospital and Harvard Medical School, Boston, MA
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13
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Marinelli P, Navarro S, Baño-Polo M, Morel B, Graña-Montes R, Sabe A, Canals F, Fernandez MR, Conejero-Lara F, Ventura S. Global Protein Stabilization Does Not Suffice to Prevent Amyloid Fibril Formation. ACS Chem Biol 2018; 13:2094-2105. [PMID: 29966079 DOI: 10.1021/acschembio.8b00607] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Mutations or cellular conditions that destabilize the native protein conformation promote the population of partially unfolded conformations, which in many cases assemble into insoluble amyloid fibrils, a process associated with multiple human pathologies. Therefore, stabilization of protein structures is seen as an efficient way to prevent misfolding and subsequent aggregation. This has been suggested to be the underlying reason why proteins living in harsh environments, such as the extracellular space, have evolved disulfide bonds. The effect of protein disulfides on the thermodynamics and kinetics of folding has been extensively studied, but much less is known on its effect on aggregation reactions. Here, we designed a single point mutation that introduces a disulfide bond in the all-α FF domain, a protein that, despite being devoid of preformed β-sheets, forms β-sheet-rich amyloid fibrils. The novel and unique covalent bond in the FF domain dramatically increases its thermodynamic stability and folding speed. Nevertheless, these optimized properties cannot counteract the inherent aggregation propensity of the protein, thus indicating that a high global protein stabilization does not suffice to prevent amyloid formation unless it contributes to hide from exposure the specific regions that nucleate the aggregation reaction.
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Affiliation(s)
- Patrizia Marinelli
- Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain
| | - Susanna Navarro
- Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain
| | - Manuel Baño-Polo
- Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain
| | - Bertrand Morel
- Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain
| | - Ricardo Graña-Montes
- Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain
| | - Anna Sabe
- Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron University Hospital, 08135 Barcelona, Spain
| | - Francesc Canals
- Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron University Hospital, 08135 Barcelona, Spain
| | - Maria Rosario Fernandez
- Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain
| | - Francisco Conejero-Lara
- Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain
| | - Salvador Ventura
- Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain
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14
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A single cysteine post-translational oxidation suffices to compromise globular proteins kinetic stability and promote amyloid formation. Redox Biol 2017; 14:566-575. [PMID: 29132128 PMCID: PMC5684091 DOI: 10.1016/j.redox.2017.10.022] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2017] [Revised: 10/24/2017] [Accepted: 10/29/2017] [Indexed: 12/22/2022] Open
Abstract
Oxidatively modified forms of proteins accumulate during aging. Oxidized protein conformers might act as intermediates in the formation of amyloids in age-related disorders. However, it is not known whether this amyloidogenic conversion requires an extensive protein oxidative damage or it can be promoted just by a discrete, localized post-translational modification of certain residues. Here, we demonstrate that the irreversible oxidation of a single free Cys suffices to severely perturb the folding energy landscape of a stable globular protein, compromise its kinetic stability, and lead to the formation of amyloids under physiological conditions. Experiments and simulations converge to indicate that this specific oxidation-promoted protein aggregation requires only local unfolding. Indeed, a large scale analysis indicates that many cellular proteins are at risk of undergoing this kind of deleterious transition; explaining how oxidative stress can impact cell proteostasis and subsequently lead to the onset of pathological states.
The population of aggregation-prone states by natural proteins does not require their extensive oxidation. A single residue irreversible oxidation suffices to promote the formation of amyloid fibrils. Under oxidative stress, many cellular proteins are at risk of aggregating into toxic species.
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15
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Bidaud-Meynard A, Binamé F, Lagrée V, Moreau V. Regulation of Rho GTPase activity at the leading edge of migrating cells by p190RhoGAP. Small GTPases 2017; 10:99-110. [PMID: 28287334 DOI: 10.1080/21541248.2017.1280584] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Cell migration, a key feature of embryonic development, immunity, angiogenesis, and tumor metastasis, is based on the coordinated regulation of actin dynamics and integrin-mediated adhesion. Rho GTPases play a major role in this phenomenon by regulating the onset and maintenance of actin-based protruding structures at cell leading edges (i.e. lamellipodia and filopodia) and contractile structures (i.e., stress fibers) at their trailing edge. While spatio-temporal analysis demonstrated the tight regulation of Rho GTPases at the migration front during cell locomotion, little is known about how the main regulators of Rho GTPase activity, such as GAPs, GEFs and GDIs, play a role in this process. In this review, we focus on a major negative regulator of RhoA, p190RhoGAP-A and its close isoform p190RhoGAP-B, which are necessary for efficient cell migration. Recent studies, including our, demonstrated that p190RhoGAP-A localization and activity undergo a complex regulatory mechanism, accounting for the tight regulation of RhoA, but also other members of the Rho GTPase family, at the cell periphery.
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Affiliation(s)
- Aurélien Bidaud-Meynard
- a Institut National de la Santé et de la Recherche Médicale , Bordeaux , France.,b Université de Bordeaux, Unité Mixte de Recherche 1053 Bordeaux Research In Translational Oncology , Bordeaux , France
| | - Fabien Binamé
- a Institut National de la Santé et de la Recherche Médicale , Bordeaux , France.,b Université de Bordeaux, Unité Mixte de Recherche 1053 Bordeaux Research In Translational Oncology , Bordeaux , France
| | - Valérie Lagrée
- a Institut National de la Santé et de la Recherche Médicale , Bordeaux , France.,b Université de Bordeaux, Unité Mixte de Recherche 1053 Bordeaux Research In Translational Oncology , Bordeaux , France
| | - Violaine Moreau
- a Institut National de la Santé et de la Recherche Médicale , Bordeaux , France.,b Université de Bordeaux, Unité Mixte de Recherche 1053 Bordeaux Research In Translational Oncology , Bordeaux , France
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16
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Parasuraman P, Mulligan P, Walker JA, Li B, Boukhali M, Haas W, Bernards A. Interaction of p190A RhoGAP with eIF3A and Other Translation Preinitiation Factors Suggests a Role in Protein Biosynthesis. J Biol Chem 2016; 292:2679-2689. [PMID: 28007963 DOI: 10.1074/jbc.m116.769216] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2016] [Indexed: 11/06/2022] Open
Abstract
The negative regulator of Rho family GTPases, p190A RhoGAP, is one of six mammalian proteins harboring so-called FF motifs. To explore the function of these and other p190A segments, we identified interacting proteins by tandem mass spectrometry. Here we report that endogenous human p190A, but not its 50% identical p190B paralog, associates with all 13 eIF3 subunits and several other translational preinitiation factors. The interaction involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A, is enhanced by serum stimulation and reduced by phosphatase treatment. The p190A/eIF3A interaction is unaffected by mutating phosphorylated p190A-Tyr308, but disrupted by a S296A mutation, targeting the only other known phosphorylated residue in the first FF domain. The p190A-eIF3 complex is distinct from eIF3 complexes containing S6K1 or mammalian target of rapamycin (mTOR), and appears to represent an incomplete preinitiation complex lacking several subunits. Based on these findings we propose that p190A may affect protein translation by controlling the assembly of functional preinitiation complexes. Whether such a role helps to explain why, unique among the large family of RhoGAPs, p190A exhibits a significantly increased mutation rate in cancer remains to be determined.
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Affiliation(s)
- Prasanna Parasuraman
- From the Massachusetts General Hospital Center for Cancer Research and Harvard Medical School, Charlestown, Massachusetts 02129
| | - Peter Mulligan
- From the Massachusetts General Hospital Center for Cancer Research and Harvard Medical School, Charlestown, Massachusetts 02129
| | - James A Walker
- From the Massachusetts General Hospital Center for Cancer Research and Harvard Medical School, Charlestown, Massachusetts 02129
| | - Bihua Li
- From the Massachusetts General Hospital Center for Cancer Research and Harvard Medical School, Charlestown, Massachusetts 02129
| | - Myriam Boukhali
- From the Massachusetts General Hospital Center for Cancer Research and Harvard Medical School, Charlestown, Massachusetts 02129
| | - Wilhelm Haas
- From the Massachusetts General Hospital Center for Cancer Research and Harvard Medical School, Charlestown, Massachusetts 02129
| | - Andre Bernards
- From the Massachusetts General Hospital Center for Cancer Research and Harvard Medical School, Charlestown, Massachusetts 02129
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17
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Herath SCB, Sharghi-Namini S, Du Y, Wang D, Ge R, Wang QG, Asada H, Chen PCY. A Magneto-Microfluidic System for Investigating the Influence of an Externally Induced Force Gradient in a Collagen Type I ECM on HMVEC Sprouting. SLAS Technol 2016; 22:413-424. [DOI: 10.1177/2211068216680078] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Advances in mechanobiology have suggested that physiological and pathological angiogenesis may be differentiated based on the ways in which the cells interact with the extracellular matrix (ECM) that exhibits partially different mechanical properties. This warrants investigating the regulation of ECM stiffness on cell behavior using angiogenesis assays. In this article, we report the application of the technique of active manipulation of ECM stiffness to study in vitro angiogenic sprouting of human microvascular endothelial cells (HMVECs) in a microfluidic device. Magnetic beads were embedded in the ECM through bioconjugation (between the streptavidin-coated beads and collagen fibers) in order to create a pretension in the ECM when under the influence of an external magnetic field. The advantage of using this magneto-microfluidic system is that the resulting change in the local deformability of the collagen fibers is only apparent to a cell at the pericellular level near the site of an embedded bead, while the global intrinsic material properties of the ECM remain unchanged. The results demonstrate that this system represents an effective tool for inducing noninvasively an external force on cells through the ECM, and suggest the possibility of creating desired stiffness gradients in the ECM for manipulating cell behavior in vitro.
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Affiliation(s)
- Sahan C. B. Herath
- Department of Mechanical Engineering, National University of Singapore, Singapore
- Biosystem and Micromechanics Interdisciplinary Research Group, Singapore–MIT Alliance for Research and Technology Program, Singapore
| | - Soheila Sharghi-Namini
- Biosystem and Micromechanics Interdisciplinary Research Group, Singapore–MIT Alliance for Research and Technology Program, Singapore
| | - Yue Du
- Department of Mechanical Engineering, National University of Singapore, Singapore
- Biosystem and Micromechanics Interdisciplinary Research Group, Singapore–MIT Alliance for Research and Technology Program, Singapore
| | - Dongan Wang
- Division of Bioengineering, Nanyang Technological University, Singapore
| | - Ruowen Ge
- Department of Biological Sciences, National University of Singapore, Singapore
| | - Qing-Guo Wang
- Institute for Intelligent Systems, University of Johannesburg, Johannesburg, South Africa
| | - Harry Asada
- Biosystem and Micromechanics Interdisciplinary Research Group, Singapore–MIT Alliance for Research and Technology Program, Singapore
- Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Peter C. Y. Chen
- Department of Mechanical Engineering, National University of Singapore, Singapore
- Biosystem and Micromechanics Interdisciplinary Research Group, Singapore–MIT Alliance for Research and Technology Program, Singapore
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18
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Abstract
Rho GTPases regulate cytoskeletal and cell adhesion dynamics and thereby coordinate a wide range of cellular processes, including cell migration, cell polarity and cell cycle progression. Most Rho GTPases cycle between a GTP-bound active conformation and a GDP-bound inactive conformation to regulate their ability to activate effector proteins and to elicit cellular responses. However, it has become apparent that Rho GTPases are regulated by post-translational modifications and the formation of specific protein complexes, in addition to GTP-GDP cycling. The canonical regulators of Rho GTPases - guanine nucleotide exchange factors, GTPase-activating proteins and guanine nucleotide dissociation inhibitors - are regulated similarly, creating a complex network of interactions to determine the precise spatiotemporal activation of Rho GTPases.
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Affiliation(s)
- Richard G Hodge
- Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK
| | - Anne J Ridley
- Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK
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19
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Bai X, Dee R, Mangum KD, Mack CP, Taylor JM. RhoA signaling and blood pressure: The consequence of failing to “Tone it Down”. World J Hypertens 2016; 6:18-35. [DOI: 10.5494/wjh.v6.i1.18] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2015] [Revised: 11/24/2015] [Accepted: 01/22/2016] [Indexed: 02/06/2023] Open
Abstract
Uncontrolled high blood pressure is a major risk factor for heart attack, stroke, and kidney failure and contributes to an estimated 25% of deaths worldwide. Despite numerous treatment options, estimates project that reasonable blood pressure (BP) control is achieved in only about half of hypertensive patients. Improvements in the detection and management of hypertension will undoubtedly be accomplished through a better understanding of the complex etiology of this disease and a more comprehensive inventory of the genes and genetic variants that influence BP regulation. Recent studies (primarily in pre-clinical models) indicate that the small GTPase RhoA and its downstream target, Rho kinase, play an important role in regulating BP homeostasis. Herein, we summarize the underlying mechanisms and highlight signaling pathways and regulators that impart tight spatial-temporal control of RhoA activity. We also discuss known allelic variations in the RhoA pathway and consider how these polymorphisms may affect genetic risk for hypertension and its clinical manifestations. Finally, we summarize the current (albeit limited) clinical data on the efficacy of targeting the RhoA pathway in hypertensive patients.
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20
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Carmona-Mora P, Widagdo J, Tomasetig F, Canales CP, Cha Y, Lee W, Alshawaf A, Dottori M, Whan RM, Hardeman EC, Palmer SJ. The nuclear localization pattern and interaction partners of GTF2IRD1 demonstrate a role in chromatin regulation. Hum Genet 2015; 134:1099-115. [PMID: 26275350 DOI: 10.1007/s00439-015-1591-0] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2015] [Accepted: 08/04/2015] [Indexed: 12/11/2022]
Abstract
GTF2IRD1 is one of the three members of the GTF2I gene family, clustered on chromosome 7 within a 1.8 Mb region that is prone to duplications and deletions in humans. Hemizygous deletions cause Williams-Beuren syndrome (WBS) and duplications cause WBS duplication syndrome. These copy number variations disturb a variety of developmental systems and neurological functions. Human mapping data and analyses of knockout mice show that GTF2IRD1 and GTF2I underpin the craniofacial abnormalities, mental retardation, visuospatial deficits and hypersociability of WBS. However, the cellular role of the GTF2IRD1 protein is poorly understood due to its very low abundance and a paucity of reagents. Here, for the first time, we show that endogenous GTF2IRD1 has a punctate pattern in the nuclei of cultured human cell lines and neurons. To probe the functional relationships of GTF2IRD1 in an unbiased manner, yeast two-hybrid libraries were screened, isolating 38 novel interaction partners, which were validated in mammalian cell lines. These relationships illustrate GTF2IRD1 function, as the isolated partners are mostly involved in chromatin modification and transcriptional regulation, whilst others indicate an unexpected role in connection with the primary cilium. Mapping of the sites of protein interaction also indicates key features regarding the evolution of the GTF2IRD1 protein. These data provide a visual and molecular basis for GTF2IRD1 nuclear function that will lead to an understanding of its role in brain, behaviour and human disease.
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Affiliation(s)
- Paulina Carmona-Mora
- Cellular and Genetic Medicine Unit, School of Medical Sciences, UNSW Australia, Sydney, NSW, 2052, Australia
| | - Jocelyn Widagdo
- Queensland Brain Institute, The University of Queensland, Brisbane, QLD, 4072, Australia
| | - Florence Tomasetig
- Cellular and Genetic Medicine Unit, School of Medical Sciences, UNSW Australia, Sydney, NSW, 2052, Australia
| | - Cesar P Canales
- Cellular and Genetic Medicine Unit, School of Medical Sciences, UNSW Australia, Sydney, NSW, 2052, Australia
| | - Yeojoon Cha
- Cellular and Genetic Medicine Unit, School of Medical Sciences, UNSW Australia, Sydney, NSW, 2052, Australia
| | - Wei Lee
- Cellular and Genetic Medicine Unit, School of Medical Sciences, UNSW Australia, Sydney, NSW, 2052, Australia
| | - Abdullah Alshawaf
- Centre for Neural Engineering, The University of Melbourne, Melbourne, VIC, 3010, Australia
| | - Mirella Dottori
- Centre for Neural Engineering, The University of Melbourne, Melbourne, VIC, 3010, Australia
| | - Renee M Whan
- Biomedical Imaging Facility, Mark Wainwright Analytical Centre, UNSW Australia, Sydney, NSW, 2052, Australia
| | - Edna C Hardeman
- Cellular and Genetic Medicine Unit, School of Medical Sciences, UNSW Australia, Sydney, NSW, 2052, Australia
| | - Stephen J Palmer
- Cellular and Genetic Medicine Unit, School of Medical Sciences, UNSW Australia, Sydney, NSW, 2052, Australia.
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21
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Fan AX, Papadopoulos GL, Hossain MA, Lin IJ, Hu J, Tang TM, Kilberg MS, Renne R, Strouboulis J, Bungert J. Genomic and proteomic analysis of transcription factor TFII-I reveals insight into the response to cellular stress. Nucleic Acids Res 2014; 42:7625-41. [PMID: 24875474 PMCID: PMC4081084 DOI: 10.1093/nar/gku467] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The ubiquitously expressed transcription factor TFII-I exerts both positive and negative effects on transcription. Using biotinylation tagging technology and high-throughput sequencing, we determined sites of chromatin interactions for TFII-I in the human erythroleukemia cell line K562. This analysis revealed that TFII-I binds upstream of the transcription start site of expressed genes, both upstream and downstream of the transcription start site of repressed genes, and downstream of RNA polymerase II peaks at the ATF3 and other stress responsive genes. At the ATF3 gene, TFII-I binds immediately downstream of a Pol II peak located 5 kb upstream of exon 1. Induction of ATF3 expression increases transcription throughout the ATF3 gene locus which requires TFII-I and correlates with increased association of Pol II and Elongin A. Pull-down assays demonstrated that TFII-I interacts with Elongin A. Partial depletion of TFII-I expression caused a reduction in the association of Elongin A with and transcription of the DNMT1 and EFR3A genes without a decrease in Pol II recruitment. The data reveal different interaction patterns of TFII-I at active, repressed, or inducible genes, identify novel TFII-I interacting proteins, implicate TFII-I in the regulation of transcription elongation and provide insight into the role of TFII-I during the response to cellular stress.
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Affiliation(s)
- Alex Xiucheng Fan
- Department of Biochemistry and Molecular Biology, Center for Epigenetics, Genetics Institute, Powell Gene Therapy Center, Gainesville, Florida, USA
| | - Giorgio L Papadopoulos
- Departmentof Biology, University of Crete, GR1409 Heraklion, Greece Divisionof Molecular Oncology, Biomedical Sciences Research Center "Alexander Fleming", Vari GR 16672, Greece
| | - Mir A Hossain
- Department of Biochemistry and Molecular Biology, Center for Epigenetics, Genetics Institute, Powell Gene Therapy Center, Gainesville, Florida, USA
| | - I-Ju Lin
- Department of Biochemistry and Molecular Biology, Center for Epigenetics, Genetics Institute, Powell Gene Therapy Center, Gainesville, Florida, USA
| | - Jianhong Hu
- Departmentof Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, 32610, USA
| | - Tommy Ming Tang
- Department of Biochemistry and Molecular Biology, Center for Epigenetics, Genetics Institute, Powell Gene Therapy Center, Gainesville, Florida, USA
| | - Michael S Kilberg
- Department of Biochemistry and Molecular Biology, Center for Epigenetics, Genetics Institute, Powell Gene Therapy Center, Gainesville, Florida, USA
| | - Rolf Renne
- Divisionof Molecular Oncology, Biomedical Sciences Research Center "Alexander Fleming", Vari GR 16672, Greece
| | - John Strouboulis
- Department of Biochemistry and Molecular Biology, Center for Epigenetics, Genetics Institute, Powell Gene Therapy Center, Gainesville, Florida, USA Departmentof Biology, University of Crete, GR1409 Heraklion, Greece Divisionof Molecular Oncology, Biomedical Sciences Research Center "Alexander Fleming", Vari GR 16672, Greece Departmentof Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, 32610, USA
| | - Jörg Bungert
- Department of Biochemistry and Molecular Biology, Center for Epigenetics, Genetics Institute, Powell Gene Therapy Center, Gainesville, Florida, USA
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22
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The human papillomavirus E7 proteins associate with p190RhoGAP and alter its function. J Virol 2014; 88:3653-63. [PMID: 24403595 DOI: 10.1128/jvi.03263-13] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
UNLABELLED Using mass spectrometry, we identified p190RhoGAP (p190) as a binding partner of human papillomavirus 16 (HPV16) E7. p190 belongs to the GTPase activating protein (GAP) family and is one of the primary GAPs for RhoA. GAPs stimulate the intrinsic GTPase activity of the Rho proteins, leading to Rho inactivation and influencing numerous biological processes. RhoA is one of the best-characterized Rho proteins and is specifically involved in formation of focal adhesions and stress fibers, thereby regulating cell migration and cell spreading. Since this is the first report that E7 associates with p190, we carried out detailed interaction studies. We show that E7 proteins from other HPV types also bind p190. Furthermore, we found that conserved region 3 (CR3) of E7 and the middle domain of p190 are important for this interaction. More specifically, we identified two residues in CR3 of E7 that are necessary for p190 binding and used mutants of E7 with mutations of these residues to determine the biological consequences of the E7-p190 interaction. Our data suggest that the interaction of E7 with p190 dysregulates this GAP and alters the actin cytoskeleton. We also found that this interaction negatively regulates cell spreading on a fibronectin substrate and therefore likely contributes to important aspects of the HPV life cycle or HPV-induced tumorigenesis. IMPORTANCE This study identifies p190RhoGAP as a novel cellular binding partner for the human papillomavirus (HPV) E7 protein. Our study shows that a large number of different HPV E7 proteins bind p190RhoGAP, and it identifies regions in both E7 and p190RhoGAP which are important for the interaction to occur. This study also highlights the likelihood that the E7-p190RhoGAP interaction may have important biological consequences related to actin organization in the infected cell. These changes could be an important contributor to the viral life cycle and during progression to cancer in HPV-infected cells. Importantly, this work also emphasizes the need for further study in a field which has largely been unexplored as it relates to the HPV life cycle and HPV-induced transformation.
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23
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Zebda N, Tian Y, Tian X, Gawlak G, Higginbotham K, Reynolds AB, Birukova AA, Birukov KG. Interaction of p190RhoGAP with C-terminal domain of p120-catenin modulates endothelial cytoskeleton and permeability. J Biol Chem 2013; 288:18290-9. [PMID: 23653363 DOI: 10.1074/jbc.m112.432757] [Citation(s) in RCA: 61] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
p120-catenin is a multidomain intracellular protein, which mediates a number of cellular functions, including stabilization of cell-cell transmembrane cadherin complexes as well as regulation of actin dynamics associated with barrier function, lamellipodia formation, and cell migration via modulation of the activities of small GTPAses. One mechanism involves p120 catenin interaction with Rho GTPase activating protein (p190RhoGAP), leading to p190RhoGAP recruitment to cell periphery and local inhibition of Rho activity. In this study, we have identified a stretch of 23 amino acids within the C-terminal domain of p120 catenin as the minimal sequence responsible for the recruitment of p190RhoGAP (herein referred to as CRAD; catenin-RhoGAP association domain). Expression of the p120-catenin truncated mutant lacking the CRAD in endothelial cells attenuated effects of barrier protective oxidized phospholipid, OxPAPC. This effect was accompanied by inhibition of membrane translocation of p190RhoGAP, increased Rho signaling, as well as suppressed activation of Rac1 and its cytoskeletal effectors PAK1 (p21-activated kinase 1) and cortactin. Expression of p120 catenin-truncated mutant lacking CRAD also delayed the recovery process after thrombin-induced endothelial barrier disruption. Concomitantly, RhoA activation and downstream signaling were sustained for a longer period of time, whereas Rac signaling was inhibited. These data demonstrate a critical role for p120-catenin (amino acids 820-843) domain in the p120-catenin·p190RhoGAP signaling complex assembly, membrane targeting, and stimulation of p190RhoGAP activity toward inhibition of the Rho pathway and reciprocal up-regulation of Rac signaling critical for endothelial barrier regulation.
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Affiliation(s)
- Noureddine Zebda
- Lung Injury Center, Section of Pulmonary and Critical Medicine, Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA
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24
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Ponik SM, Trier SM, Wozniak MA, Eliceiri KW, Keely PJ. RhoA is down-regulated at cell-cell contacts via p190RhoGAP-B in response to tensional homeostasis. Mol Biol Cell 2013; 24:1688-99, S1-3. [PMID: 23552690 PMCID: PMC3667722 DOI: 10.1091/mbc.e12-05-0386] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
p190RhoGAP-B mediates Rho activity and ductal morphogenesis in response to 3D collagen stiffness. p190B associates with p120-catenin at cell–cell contacts, where RhoA activity is decreased compared to cell–ECM adhesions. This suggests that Rho is in an inactive pool at cell–cell contacts and is recruited to cell–ECM contacts in stiff matrices. Breast epithelial cells cultured in three-dimensional (3D) collagen gels undergo ductal morphogenesis when the gel is compliant and they can achieve tensional homeostasis. We previously showed that this process requires down-regulation of Rho in compliant collagen gels, but the mechanism remains undefined. In this study, we find that p190RhoGAP-B, but not p190RhoGAP-A, mediates down-regulation of RhoA activity and ductal morphogenesis in T47D cells cultured in compliant 3D collagen gels. In addition, both RhoA and p190RhoGAP-B colocalize with p120-catenin at sites of cell–cell contact. The association between p190RhoGAP-B and p120-catenin is regulated by matrix compliance such that it increases in compliant vs. rigid collagen gels. Furthermore, knockdown of p120-catenin disrupts ductal morphogenesis, disregulates RhoA activity, and results in loss of p190B at cell–cell contacts. Consistent with these findings, using a RhoA-specific FRET biosensor (RhoA-FLARE.sc), we determined spatial RhoA activity to be significantly decreased at cell–cell contacts versus cell–ECM adhesions, and, of importance, spatial RhoA activity is regulated by p190B. This finding suggests that RhoA exists as an inactive pool at cell–cell contacts and is recruited to cell–ECM contacts within stiff matrices. Overall, these results demonstrate that RhoA is down-regulated at cell–cell contacts through p190RhoGAP-B, which is localized to cell–cell contacts by association with p120-catenin that is regulated by tensional homeostasis.
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Affiliation(s)
- Suzanne M Ponik
- Department of Cellular and Regenerative Biology, University of Wisconsin-Madison, Madison, WI 53706, USA.
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25
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Lévay M, Bartos B, Ligeti E. p190RhoGAP has cellular RacGAP activity regulated by a polybasic region. Cell Signal 2013; 25:1388-94. [PMID: 23499677 DOI: 10.1016/j.cellsig.2013.03.004] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2012] [Revised: 02/19/2013] [Accepted: 03/06/2013] [Indexed: 01/09/2023]
Abstract
p190RhoGAP is a GTPase-activating protein (GAP) known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of the intrinsic GTPase activity of Rho. Although the GAP domain of p190RhoGAP stimulates the intrinsic' GTPase activity of several Rho family members (Rho, Rac, Cdc42) under in vitro conditions, p190RhoGAP is generally regarded as a GAP for RhoA in the cell. The cellular RacGAP activity of the protein has not been proven directly. We have previously shown that the in vitro RacGAP and RhoGAP activity of p190RhoGAP was inversely regulated through a polybasic region of the protein. Here we provide evidence that p190RhoGAP shows remarkable GAP activity toward Rac also in the cell. The cellular RacGAP activity of p190RhoGAP requires an intact polybasic region adjacent to the GAP domain whereas the RhoGAP activity is inhibited by the same domain. Our data indicate that through its alternating RacGAP and RhoGAP activity, p190RhoGAP plays a more complex role in the Rac-Rho antagonism than it was realized earlier.
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Affiliation(s)
- Magdolna Lévay
- Department of Physiology, Semmelweis University, Budapest, Hungary
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26
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The N-terminal helix controls the transition between the soluble and amyloid states of an FF domain. PLoS One 2013; 8:e58297. [PMID: 23505482 PMCID: PMC3591442 DOI: 10.1371/journal.pone.0058297] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2012] [Accepted: 02/01/2013] [Indexed: 02/03/2023] Open
Abstract
Background Protein aggregation is linked to the onset of an increasing number of human nonneuropathic (either localized or systemic) and neurodegenerative disorders. In particular, misfolding of native α-helical structures and their self-assembly into nonnative intermolecular β-sheets has been proposed to trigger amyloid fibril formation in Alzheimer’s and Parkinson’s diseases. Methods Here, we use a battery of biophysical techniques to elucidate the conformational conversion of native α-helices into amyloid fibrils using an all-α FF domain as a model system. Results We show that under mild denaturing conditions at low pH this FF domain self-assembles into amyloid fibrils. Theoretical and experimental dissection of the secondary structure elements in this domain indicates that the helix 1 at the N-terminus has both the highest α-helical and amyloid propensities, controlling the transition between soluble and aggregated states of the protein. Conclusions The data illustrates the overlap between the propensity to form native α-helices and amyloid structures in protein segments. Significance The results presented contribute to explain why proteins cannot avoid the presence of aggregation-prone regions and indeed use stable α-helices as a strategy to neutralize such potentially deleterious stretches.
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27
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Palmer SJ, Taylor KM, Santucci N, Widagdo J, Chan YKA, Yeo JL, Adams M, Gunning PW, Hardeman EC. GTF2IRD2 from the Williams-Beuren critical region encodes a mobile-element-derived fusion protein that antagonizes the action of its related family members. J Cell Sci 2012; 125:5040-50. [PMID: 22899722 DOI: 10.1242/jcs.102798] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
GTF2IRD2 belongs to a family of transcriptional regulators (including TFII-I and GTF2IRD1) that are responsible for many of the key features of Williams-Beuren syndrome (WBS). Sequence evidence suggests that GTF2IRD2 arose in eutherian mammals by duplication and divergence from the gene encoding TFII-I. However, in GTF2IRD2, most of the C-terminal domain has been lost and replaced by the domesticated remnant of an in-frame hAT-transposon mobile element. In this first experimental analysis of function, we show that transgenic expression of each of the three family members in skeletal muscle causes significant fiber type shifts, but the GTF2IRD2 protein causes an extreme shift in the opposite direction to the two other family members. Mating of GTF2IRD1 and GTF2IRD2 mice restores the fiber type balance, indicating an antagonistic relationship between these two paralogs. In cells, GTF2IRD2 localizes to cytoplasmic microtubules and discrete speckles in the nuclear periphery. We show that it can interact directly with TFII-Iβ and GTF2IRD1, and upon co-transfection changes the normal distribution of these two proteins into a punctate nuclear pattern typical of GTF2IRD2. These data suggest that GTF2IRD2 has evolved as a regulator of GTF2IRD1 and TFII-I; inhibiting their function by direct interaction and sequestration into inactive nuclear zones.
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Affiliation(s)
- Stephen J Palmer
- Neuromuscular and Regenerative Medicine Unit, School of Medical Sciences, The University of New South Wales, Sydney 2052, Australia.
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28
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Goh LL, Manser E. The GTPase-deficient Rnd proteins are stabilized by their effectors. J Biol Chem 2012; 287:31311-20. [PMID: 22807448 DOI: 10.1074/jbc.m111.327056] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Rnd proteins are Rho family GTP-binding proteins with cellular functions that antagonize RhoA signaling. We recently described a new Rnd3 effector Syx, also named PLEKHG5, that interacts with Rnds via a Raf1-like "Ras-binding domain." Syx is a multidomain RhoGEF that participates in early zebrafish development. Here we demonstrated that Rnd1, Rnd2, and Rnd3 stability is acutely dependent on interaction with their effectors such as Syx or p190 RhoGAP. Although Rnd3 turnover is blocked by treatment of cells with MG132, we provide evidence that such turnover is mediated indirectly by effects on the Rnd3 effectors, rather than on Rnd3 itself, which is not significantly ubiquitinated. The minimal regions of Syx and p190 RhoGAP that bind Rnd3 are not sequence-related but have similar effects. We have identified features that allow for Rnd3 turnover including a conserved Lys-45 close to the switch I region and the C-terminal membrane-binding domain of Rnd3, which cannot be substituted by the equivalent Cdc42 CAAX sequence. By contrast, an effector binding-defective mutant of Rnd3 when overexpressed undergoes turnover at normal rates. Interestingly the activity of the RhoA-regulated kinase ROCK stimulates Rnd3 turnover. This study suggests that Rnd proteins are regulated through feedback mechanisms in cells where the level of effectors and RhoA activity influence the stability of Rnd proteins. This effector feedback behavior is analogous to the ability of ACK1 and PAK1 to prolong the lifetime of the active GTP-bound state of Cdc42 and Rac1.
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Affiliation(s)
- Liuh Ling Goh
- Rho GTPases Signaling Group, Institute of Medical Biology, 8A Biomedical Grove, 06-06 Immunos Building 138648, Singapore
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29
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Abstract
Regulatory proteins such as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) determine the activity of small GTPases. In the Rho/Rac family, the number of GEFs and GAPs largely exceeds the number of small GTPases, raising the question of specific or overlapping functions. In our recent study we investigated the first time ARHGAP25 at the protein level, determined its activity as RacGAP and showed its involvement in phagocytosis. With the discovery of ARHGAP25, the number of RacGAPs described in phagocytes is increased to six. We provide data that indicate the specific functions of selected Rho/RacGAPs and we show an example of differential regulation of a Rho/Rac family GAP by different kinases. We propose that the abundance of Rho/Rac family GAPs is an important element of the fine spatiotemporal regulation of diverse cellular functions.
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30
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Grinnell KL, Harrington EO. Interplay between FAK, PKCδ, and p190RhoGAP in the regulation of endothelial barrier function. Microvasc Res 2012; 83:12-21. [PMID: 21549132 PMCID: PMC3175025 DOI: 10.1016/j.mvr.2011.04.005] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2011] [Revised: 04/15/2011] [Accepted: 04/16/2011] [Indexed: 11/16/2022]
Abstract
Disruption of either intercellular or extracellular junctions involved in maintaining endothelial barrier function can result in increased endothelial permeability. Increased endothelial permeability, in turn, allows for the unregulated movement of fluid and solutes out of the vasculature and into the surrounding connective tissue, contributing to a number of disease states, including stroke and pulmonary edema (Ermert et al., 1995; Lee and Slutsky, 2010; van Hinsbergh, 1997; Waller et al., 1996; Warboys et al., 2010). Thus, a better understanding of the molecular mechanisms by which endothelial cell junction integrity is controlled is necessary for development of therapies aimed at treating such conditions. In this review, we will discuss the functions of three signaling molecules known to be involved in regulation of endothelial permeability: focal adhesion kinase (FAK), protein kinase C delta (PKCδ), and p190RhoGAP (p190). We will discuss the independent functions of each protein, as well as the interplay that exists between them and the effects of such interactions on endothelial function.
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Affiliation(s)
- Katie L Grinnell
- Vascular Research Laboratory, Providence VA Medical Center, Department of Medicine, Warren Alpert Medical School of Brown University, Providence, RI 02908, USA
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31
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Roy AL. Biochemistry and biology of the inducible multifunctional transcription factor TFII-I: 10 years later. Gene 2011; 492:32-41. [PMID: 22037610 DOI: 10.1016/j.gene.2011.10.030] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2011] [Revised: 10/08/2011] [Accepted: 10/11/2011] [Indexed: 12/12/2022]
Abstract
Exactly twenty years ago TFII-I was discovered as a biochemical entity that was able to bind to and function via a core promoter element called the Initiator (Inr). Since then several different properties of this signal-induced multifunctional factor were discovered. Here I update these ever expanding functions of TFII-I--focusing primarily on the last ten years since the first review appeared in this journal.
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Affiliation(s)
- Ananda L Roy
- Department of Pathology, Sackler School of Biomedical Sciences, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA.
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32
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Heckman-Stoddard BM, Vargo-Gogola T, Herrick MP, Visbal AP, Lewis MT, Settleman J, Rosen JM. P190A RhoGAP is required for mammary gland development. Dev Biol 2011; 360:1-10. [PMID: 21945077 DOI: 10.1016/j.ydbio.2011.09.006] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2010] [Revised: 08/17/2011] [Accepted: 09/05/2011] [Indexed: 11/19/2022]
Abstract
P190A and p190B Rho GTPase activating proteins (GAPs) are essential genes that have distinct, but overlapping roles in the developing nervous system. Previous studies from our laboratory demonstrated that p190B is required for mammary gland morphogenesis, and we hypothesized that p190A might have a distinct role in the developing mammary gland. To test this hypothesis, we examined mammary gland development in p190A-deficient mice. P190A expression was detected by in situ hybridization in the developing E14.5day embryonic mammary bud and within the ducts, terminal end buds (TEBs), and surrounding stroma of the developing virgin mammary gland. In contrast to previous results with p190B, examination of p190A heterozygous mammary glands demonstrated that p190A deficiency disrupted TEB morphology, but did not significantly delay ductal outgrowth indicating haploinsufficiency for TEB development. To examine the effects of homozygous deletion of p190A, embryonic mammary buds were rescued by transplantation into the cleared fat pads of SCID/Beige mice. Complete loss of p190A function inhibited ductal outgrowth in comparison to wildtype transplants (51% vs. 94% fat pad filled). In addition, the transplantation take rate of p190A deficient whole gland transplants from E18.5 embryos was significantly reduced compared to wildtype transplants (31% vs. 90%, respectively). These results suggest that p190A function in both the epithelium and stroma is required for mammary gland development. Immunostaining for p63 demonstrated that the myoepithelial cell layer is disrupted in the p190A deficient glands, which may result from the defective cell adhesion between the cap and body cell layers detected in the TEBs. The number of estrogen- and progesterone receptor-positive cells, as well as the expression levels of these receptors was increased in p190A deficient outgrowths. These data suggest that p190A is required in both the epithelial and stromal compartments for ductal outgrowth and that it may play a role in mammary epithelial cell differentiation.
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Affiliation(s)
- B M Heckman-Stoddard
- Cancer Prevention Fellowship Program, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD 20892, USA.
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33
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Lu M, Yang J, Ren Z, Sabui S, Espejo A, Bedford MT, Jacobson RH, Jeruzalmi D, McMurray JS, Chen X. Crystal structure of the three tandem FF domains of the transcription elongation regulator CA150. J Mol Biol 2009; 393:397-408. [PMID: 19660470 PMCID: PMC3319151 DOI: 10.1016/j.jmb.2009.07.086] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2009] [Revised: 07/27/2009] [Accepted: 07/29/2009] [Indexed: 10/20/2022]
Abstract
FF domains are small protein-protein interaction modules that have two flanking conserved phenylalanine residues. They are present in proteins involved in transcription, RNA splicing, and signal transduction, and often exist in tandem arrays. Although several individual FF domain structures have been determined by NMR, the tandem nature of most FF domains has not been revealed. Here we report the 2.7-A-resolution crystal structure of the first three FF domains of the human transcription elongation factor CA150. Each FF domain is composed of three alpha-helices and a 3(10) helix between alpha-helix 2 and alpha-helix 3. The most striking feature of the structure is that an FF domain is connected to the next by an alpha-helix that continues from helix 3 to helix 1 of the next. The consequent elongated arrangement allows exposure of many charged residues within the region that can be engaged in interaction with other molecules. Binding studies using a peptide ligand suggest that a specific conformation of the FF domains might be required to achieve higher-affinity binding. Additionally, we explore potential DNA binding of the FF construct used in this study. Overall, we provide the first crystal structure of an FF domain and insights into the tandem nature of the FF domains and suggest that, in addition to protein binding, FF domains might be involved in DNA binding.
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Affiliation(s)
- Ming Lu
- Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030
- Graduate School of Biomedical Sciences, University of Texas Health Science Center, Houston, TX 77030
| | - Jun Yang
- Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030
- Graduate School of Biomedical Sciences, University of Texas Health Science Center, Houston, TX 77030
| | - Zhiyong Ren
- Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030
| | - Subir Sabui
- Department of Experimental Therapeutics, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030
| | - Alexsandra Espejo
- Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center Science Park, Smithville, TX 78957
| | - Mark T. Bedford
- Graduate School of Biomedical Sciences, University of Texas Health Science Center, Houston, TX 77030
- Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center Science Park, Smithville, TX 78957
| | - Raymond H. Jacobson
- Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030
- Graduate School of Biomedical Sciences, University of Texas Health Science Center, Houston, TX 77030
| | - David Jeruzalmi
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
| | - John S. McMurray
- Graduate School of Biomedical Sciences, University of Texas Health Science Center, Houston, TX 77030
- Department of Experimental Therapeutics, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030
| | - Xiaomin Chen
- Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030
- Graduate School of Biomedical Sciences, University of Texas Health Science Center, Houston, TX 77030
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34
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Murphy JM, Hansen DF, Wiesner S, Muhandiram DR, Borg M, Smith MJ, Sicheri F, Kay LE, Forman-Kay JD, Pawson T. Structural Studies of FF Domains of the Transcription Factor CA150 Provide Insights into the Organization of FF Domain Tandem Arrays. J Mol Biol 2009; 393:409-24. [DOI: 10.1016/j.jmb.2009.08.049] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2009] [Revised: 08/06/2009] [Accepted: 08/20/2009] [Indexed: 11/26/2022]
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35
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Fordjour AK, Harrington EO. PKCdelta influences p190 phosphorylation and activity: events independent of PKCdelta-mediated regulation of endothelial cell stress fiber and focal adhesion formation and barrier function. BIOCHIMICA ET BIOPHYSICA ACTA 2009; 1790:1179-90. [PMID: 19632305 PMCID: PMC2759355 DOI: 10.1016/j.bbagen.2009.07.012] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 11/18/2008] [Revised: 07/14/2009] [Accepted: 07/16/2009] [Indexed: 12/11/2022]
Abstract
BACKGROUND We have shown that protein kinase Cdelta (PKCdelta) inhibition results in increased endothelial cell (EC) permeability and decreased RhoA activity; which correlated with diminished stress fibers (SF) and focal adhesions (FA). We have also shown co-precipitation of p190RhoGAP (p190) with PKCdelta. Here, we investigated if PKCdelta regulates p190 and whether PKCdelta-mediated changes in SF and FA or permeability were dependent upon p190. METHODS Protein-protein interaction and activity analyses were performed using co-precipitation assays. Analysis of p190 phosphorylation was performed using in vitro kinase assays. SF and FA were analyzed by immunofluorescence analyses. EC monolayer permeability was measured using electrical cell impedance sensor (ECIS) technique. RESULTS Inhibition of PKCdelta increased p190 activity, while PKCdelta overexpression diminished p190 activity. PKCdelta bound to and phosphorylated both p190FF and p190GTPase domains. p190 protein overexpression diminished SF and FA formation and RhoA activity. Disruption of SF and FA or increased permeability induced upon PKCdelta inhibition, were not attenuated in EC in which the p190 isoforms were suppressed individually or concurrently. GENERAL SIGNIFICANCE Our findings suggest that while PKCdelta can regulate p190 activity, possibly at the FF and/or GTPase domains, the effect of PKCdelta inhibition on SF and FA and barrier dysfunction occurs through a pathway independent of p190.
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Affiliation(s)
- Akua K Fordjour
- Vascular Research Laboratory, Providence Veterans Affairs Medical Center, Department of Medicine, Alpert Medical School of Brown University, Providence, RI 02908, USA
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36
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Bonet R, Ruiz L, Aragón E, Martín-Malpartida P, Macias MJ. NMR structural studies on human p190-A RhoGAPFF1 revealed that domain phosphorylation by the PDGF-receptor alpha requires its previous unfolding. J Mol Biol 2009; 389:230-7. [PMID: 19393245 DOI: 10.1016/j.jmb.2009.04.035] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2009] [Revised: 04/17/2009] [Accepted: 04/17/2009] [Indexed: 11/15/2022]
Abstract
p190-A and -B Rho GAPs (guanosine triphosphatase activating proteins) are the only cytoplasmatic proteins containing FF domains. In p190-A Rho GAP, the region containing the FF domains has been implicated in binding to the transcription factor TFII-I. Moreover, phosphorylation of Tyr308 within the first FF domain inhibits this interaction. Because the structural determinants governing this mechanism remain unknown, we sought to solve the structure of the first FF domain of p190-A Rho GAP (RhoGAPFF1) and to study the potential impact of phosphorylation on the structure. We found that RhoGAPFF1 does not fold with the typical (alpha1-alpha2-3(10)-alpha 3) arrangement of other FF domains. Instead, the NMR data obtained at 285 K show an alpha1-alpha2-alpha 3-alpha 4 topology. In addition, we observed that specific contacts between residues in the first loop and the fourth helix are indispensable for the correct folding and stability of this domain. The structure also revealed that Tyr308 contributes to the domain hydrophobic core. Furthermore, the residues that compose the target motif of the platelet-derived growth factor receptor alpha kinase form part of the alpha 3 helix. We observed that the phosphorylation reaction requires a previous step including domain unfolding, a process that occurs at 310 K. In the absence of phosphorylation, the temperature-dependent RhoGAPFF1 folding/unfolding process is reversible. However, phosphorylation causes an irreversible destabilization of the RhoGAPFF1 structure, which probably accounts for the inhibitory effect that it exerts on the TFII-I interaction. Our results link the ability of a protein domain to be phosphorylated with conformational changes in its three-dimensional structure.
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Affiliation(s)
- R Bonet
- The Institute for Research in Biomedicine, Barcelona (Protein NMR Group), Passeig Lluis Companys 23, Baldiri Reixac 10-13, E-08028 Barcelona, Spain
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37
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Mammoto A, Connor KM, Mammoto T, Yung CW, Huh D, Aderman CM, Mostoslavsky G, Smith LEH, Ingber DE. A mechanosensitive transcriptional mechanism that controls angiogenesis. Nature 2009; 457:1103-8. [PMID: 19242469 PMCID: PMC2708674 DOI: 10.1038/nature07765] [Citation(s) in RCA: 400] [Impact Index Per Article: 25.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2008] [Accepted: 12/31/2008] [Indexed: 01/13/2023]
Abstract
Angiogenesis is controlled by physical interactions between cells and extracellular matrix as well as soluble angiogenic factors, such as VEGF. However, the mechanism by which mechanical signals integrate with other microenvironmental cues to regulate neovascularization remains unknown. Here we show that the Rho inhibitor, p190RhoGAP (also known as GRLF1), controls capillary network formation in vitro in human microvascular endothelial cells and retinal angiogenesis in vivo by modulating the balance of activities between two antagonistic transcription factors, TFII-I (also known as GTF2I) and GATA2, that govern gene expression of the VEGF receptor VEGFR2 (also known as KDR). Moreover, this new angiogenesis signalling pathway is sensitive to extracellular matrix elasticity as well as soluble VEGF. This is, to our knowledge, the first known functional cross-antagonism between transcription factors that controls tissue morphogenesis, and that responds to both mechanical and chemical cues.
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Affiliation(s)
- Akiko Mammoto
- Vascular Biology Program, Departments of Pathology & Surgery, Children's Hospital and Harvard Medical School, Boston, MA 02115
| | - Kip M. Connor
- Department of Ophthalmology, Children's Hospital and Harvard Medical School, Boston, MA 02115
| | - Tadanori Mammoto
- Vascular Biology Program, Departments of Pathology & Surgery, Children's Hospital and Harvard Medical School, Boston, MA 02115
| | - Chong Wing Yung
- Vascular Biology Program, Departments of Pathology & Surgery, Children's Hospital and Harvard Medical School, Boston, MA 02115
| | - Dongeun Huh
- Vascular Biology Program, Departments of Pathology & Surgery, Children's Hospital and Harvard Medical School, Boston, MA 02115
| | - Christopher M. Aderman
- Department of Ophthalmology, Children's Hospital and Harvard Medical School, Boston, MA 02115
| | - Gustavo Mostoslavsky
- Department of Genetics, Harvard Medical School, Harvard Institute of Medicine, Boston, MA 02215, USA
| | - Lois E. H. Smith
- Department of Ophthalmology, Children's Hospital and Harvard Medical School, Boston, MA 02115
| | - Donald E. Ingber
- Vascular Biology Program, Departments of Pathology & Surgery, Children's Hospital and Harvard Medical School, Boston, MA 02115
- Wyss Institute for Biologically Inspired Engineering and Harvard School of Engineering and Applied Sciences, Cambridge, MA 02139
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Ester C, Uetz P. The FF domains of yeast U1 snRNP protein Prp40 mediate interactions with Luc7 and Snu71. BMC BIOCHEMISTRY 2008; 9:29. [PMID: 19014439 PMCID: PMC2613882 DOI: 10.1186/1471-2091-9-29] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/04/2008] [Accepted: 11/11/2008] [Indexed: 11/20/2022]
Abstract
Background The FF domain is conserved across all eukaryotes and usually acts as an adaptor module in RNA metabolism and transcription. Saccharomyces cerevisiae encodes two FF domain proteins, Prp40, a component of the U1 snRNP, and Ypr152c, a protein of unknown function. The structure of Prp40, its relationship to other proteins within the U1 snRNP, and its precise function remain little understood. Results Here we have investigated the essentiality and interaction properties of the FF domains of yeast Prp40. We show that the C-terminal two FF domains of Prp40 are dispensable. Deletion of additional FF domains is lethal. The first FF domain of Prp40 binds to U1 protein Luc7 in yeast two-hybrid and GST pulldown experiments. FF domains 2 and 3 bind to Snu71, another known U1 protein. Peptide array screens identified binding sites for FF1-2 within Snu71 (NDVHY) and for FF1 within Luc7 (ϕ[FHL] × [KR] × [GHL] with ϕ being a hydrophobic amino acid). Conclusion Prp40, Luc7, and Snu71 appear to form a subcomplex within the yeast U1snRNP. Our data suggests that the N-terminal FF domains are critical for these interactions. Crystallization of Prp40, Luc7, and Snu71 have failed so far but co-crystallization of pairs or the whole tri-complex may facilitate crystallographic and further functional analysis.
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Affiliation(s)
- Claudia Ester
- Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, P, O, Box 3640, D-76021 Karlsruhe, Germany.
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Bonet R, Ramirez-Espain X, Macias MJ. Solution structure of the yeast URN1 splicing factor FF domain: Comparative analysis of charge distributions in FF domain structures-FFs and SURPs, two domains with a similar fold. Proteins 2008; 73:1001-9. [DOI: 10.1002/prot.22127] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
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Guegan F, Tatin F, Leste-Lasserre T, Drutel G, Genot E, Moreau V. p190B RhoGAP regulates endothelial-cell-associated proteolysis through MT1-MMP and MMP2. J Cell Sci 2008; 121:2054-61. [PMID: 18505793 DOI: 10.1242/jcs.025817] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The two isoforms of p190 RhoGAP (p190A and p190B) are important regulators of RhoGTPase activity in mammalian cells. Both proteins are ubiquitously expressed, are involved in the same signalling pathways and interact with the same identified binding partners. In search of isoform functional specificity, we knocked down the expression of each p190 protein using siRNA and examined the resulting phenotypic changes in human umbilical vein endothelial cells (HUVECs). We provide evidence that p190B plays a crucial role in the regulation of MT1-MMP expression and cell-surface presentation, as well as subsequent MMP2 activation. p190B is involved in both local extracellular matrix degradation at podosomes and endothelial cell assembly into tube-like structures in Matrigel. In addition, whereas p190B knockdown does not affect podosome formation, p190A knockdown increases the number of cells showing podosome structures in HUVECs. We conclude that the two p190 RhoGAP isoforms play distinct roles in endothelial cells. In addition, our data reveal an unsuspected role for p190B in the expression of the two collaborative proteases MT1-MMP and MMP2, thereby affecting matrix remodelling and angiogenesis.
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Affiliation(s)
- Fabien Guegan
- Institut Européen de Chimie-Biologie, Pessac, France
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41
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Roy AL. Signal-induced functions of the transcription factor TFII-I. BIOCHIMICA ET BIOPHYSICA ACTA 2007; 1769:613-21. [PMID: 17976384 PMCID: PMC2140948 DOI: 10.1016/j.bbaexp.2007.10.002] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 06/18/2007] [Revised: 09/27/2007] [Accepted: 10/01/2007] [Indexed: 12/21/2022]
Abstract
We have learned a great deal over the last several years about the molecular mechanisms that govern cell growth, cell division and cell death. Normal cells pass through cell cycle (growth) and divide in response to mitogenic signals that are transduced through their cognate cell surface receptors to the nucleus. Despite the fact that cellular growth and division are mechanistically distinct steps, they are usually coordinately regulated, which is critical for normal cellular proliferation. The precise mechanistic basis for this coordinated regulation is unclear. TFII-I is a unique, signal-induced multifunctional transcription factor that is activated upon a variety of signaling pathways and appears to participate in distinct phases of cell growth. For instance, TFII-I is required for growth factor-induced transcriptional activation of the c-fos gene, which is essential for cell cycle entry. Two alternatively spliced isoforms of TFII-I exhibit opposing but necessary functions for mitogen-induced transcriptional activation of c-fos. Besides transcriptional activation of the c-fos proto-oncogene and eventual entry into cell cycle, TFII-I also appears to have a role in later phases of the cell cycle and cell division. Here we discuss how a multitude of signaling inputs target TFII-I isoforms, which may exert their functions in distinct phases of the cell cycle and play a key role in the coordinated regulation of cellular proliferation.
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Affiliation(s)
- Ananda L Roy
- Department of Pathology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA.
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Abstract
The Rho GTPases are implicated in almost every fundamental cellular process. They act as molecular switches that cycle between an active GTP-bound and an inactive GDP-bound state. Their slow intrinsic GTPase activity is greatly enhanced by RhoGAPs (Rho GTPase-activating proteins), thus causing their inactivation. To date, more than 70 RhoGAPs have been identified in eukaryotes, ranging from yeast to human, and based on sequence homology of their RhoGAP domain, we have grouped them into subfamilies. In the present Review, we discuss their regulation, biological functions and implication in human diseases.
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Affiliation(s)
- Joseph Tcherkezian
- Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada H3A 2B2
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43
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Heckman BM, Chakravarty G, Vargo-Gogola T, Gonzales-Rimbau M, Hadsell DL, Lee AV, Settleman J, Rosen JM. Crosstalk between the p190-B RhoGAP and IGF signaling pathways is required for embryonic mammary bud development. Dev Biol 2007; 309:137-49. [PMID: 17662267 PMCID: PMC4011021 DOI: 10.1016/j.ydbio.2007.07.002] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2006] [Revised: 06/25/2007] [Accepted: 07/03/2007] [Indexed: 01/12/2023]
Abstract
P190-B RhoGAP (p190-B, also known as ARHGAP5) has been shown to play an essential role in invasion of the terminal end buds (TEBs) into the surrounding fat pad during mammary gland ductal morphogenesis. Here we report that embryos with a homozygous p190-B gene deletion exhibit major defects in embryonic mammary bud development. Overall, p190-B-deficient buds were smaller in size, contained fewer cells, and displayed characteristics of impaired mesenchymal proliferation and differentiation. Consistent with the reported effects of p190-B deletion on IGF-1R signaling, IGF-1R-deficient embryos also displayed a similar small mammary bud phenotype. However, unlike the p190-B-deficient embryos, the IGF-1R-deficient embryos exhibited decreased epithelial proliferation and did not display mesenchymal defects. Because both IGF and p190-B signaling affect IRS-1/2, we examined IRS-1/2 double knockout embryonic mammary buds. These embryos displayed major defects similar to the p190-B-deficient embryos including smaller bud size. Importantly, like the p190-B-deficient buds, proliferation of the IRS-1/2-deficient mesenchyme was impaired. These results indicate that IGF signaling through p190-B and IRS proteins is critical for mammary bud formation and ensuing epithelial-mesenchymal interactions necessary to sustain mammary bud morphogenesis.
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Affiliation(s)
- Brandy M Heckman
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
| | - Geetika Chakravarty
- Department of Molecular & Cellular Oncology, MD Anderson Cancer Center, Houston, TX 77030
| | - Tracy Vargo-Gogola
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
| | - Maria Gonzales-Rimbau
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
| | - Darryl L Hadsell
- U.S. Department of Agriculture/Agricultural Research Service Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030, USA
| | - Adrian V Lee
- The Breast Center, Department of Medicine, Baylor College of Medicine, Houston, TX 77030
| | - Jeffrey Settleman
- Massachusetts General Hospital Cancer Center and Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA
| | - Jeffrey M Rosen
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
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Ashworth T, Roy AL. Cutting Edge: TFII-I controls B cell proliferation via regulating NF-kappaB. THE JOURNAL OF IMMUNOLOGY 2007; 178:2631-5. [PMID: 17312101 DOI: 10.4049/jimmunol.178.5.2631] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The multifunctional transcription factor TFII-I physically and functionally interacts with Bruton's tyrosine kinase in murine B cells. However, the downstream functions of TFII-I in B cells are unknown. Toward achieving this goal, we established stable posttranscriptional silencing of TFII-I in WEHI-231 immature murine B cells, which undergoes growth arrest and apoptosis either upon anti-IgM or TGF-beta signaling. In this study, we show that TFII-I promotes growth arrest of cells in a signal-dependent manner. Unlike control cells, B cells exhibiting loss of TFII-I function fail to undergo arrest upon signaling due to up-regulation of c-Myc expression and concomitant down-regulation of both p21 and p27. Loss of TFII-I is also associated with simultaneous increase in nuclear c-rel and decrease in p50 homodimer binding. Thus, besides controlling c-myc transcription, TFII-I controls B cell proliferation by regulating both nuclear translocation of c-rel and DNA-binding activity of p50 NF-kappaB.
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Affiliation(s)
- Todd Ashworth
- Program in Immmunology, Department of Pathology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA
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45
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Mammoto A, Huang S, Ingber DE. Filamin links cell shape and cytoskeletal structure to Rho regulation by controlling accumulation of p190RhoGAP in lipid rafts. J Cell Sci 2007; 120:456-67. [PMID: 17227794 DOI: 10.1242/jcs.03353] [Citation(s) in RCA: 80] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Cytoskeleton-dependent changes in the activity of the small GTPase Rho mediate the effects of cell shape on cell function; however, little is known about how cell spreading and related distortion of the cytoskeleton regulate Rho activity. Here we show that rearrangements of the actin cytoskeleton associated with early phases of cell spreading in human microvascular endothelial (HMVE) cells suppress Rho activity by promoting accumulation of p190RhoGAP in lipid rafts where it exerts its Rho inhibitory activity. p190RhoGAP is excluded from lipid rafts and Rho activity increases when cell rounding is induced or the actin cytoskeleton is disrupted, and p190RhoGAP knockdown using siRNA prevents Rho inactivation by cell spreading. Importantly, cell rounding fails to prevent accumulation of p190RhoGAP in lipid rafts and to increase Rho activity in cells that lack the cytoskeletal protein filamin. Moreover, filamin is degraded in spread cells and cells that express a calpain-resistant form of filamin exhibit high Rho activity even when spread. Filamin may therefore represent the missing link that connects cytoskeleton-dependent changes of cell shape to Rho inactivation during the earliest phases of cell spreading by virtue of its ability to promote accumulation of p190RhoGAP in lipid rafts.
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Affiliation(s)
- Akiko Mammoto
- Vascular Biology Program, Department of Pathology, Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA
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Santolin L, Meisterernst M. TFII-IDelta and TFII-Ibeta: unequal brothers fostering cellular proliferation. Mol Cell 2006; 24:169-71. [PMID: 17052451 DOI: 10.1016/j.molcel.2006.10.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
Abstract
Activation of the immediate-early gene c-fos through MAP kinases is a hallmark of growth factor signaling. In this issue of Molecular Cell, Roy and colleagues (Hakre et al., 2006) show that TFII-I isoforms play differential roles in this process.
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Affiliation(s)
- Lisa Santolin
- Gene Expression, GSF-National Research Center for Environment and Health, Institute for Molecular Immunology, Marchionistrasse 25, D-81377 München, Germany
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Hakre S, Tussie-Luna MI, Ashworth T, Novina CD, Settleman J, Sharp PA, Roy AL. Opposing functions of TFII-I spliced isoforms in growth factor-induced gene expression. Mol Cell 2006; 24:301-8. [PMID: 17052463 DOI: 10.1016/j.molcel.2006.09.005] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2006] [Revised: 07/18/2006] [Accepted: 09/11/2006] [Indexed: 11/12/2022]
Abstract
Multifunctional transcription factor TFII-I has two spliced isoforms (Delta and beta) in murine fibroblasts. Here we show that these isoforms have distinct subcellular localization and mutually exclusive transcription functions in the context of growth factor signaling. In the absence of signaling, TFII-Ibeta is nuclear and recruited to the c-fos promoter in vivo. But upon growth factor stimulation, the promoter recruitment is abolished and it is exported out of the nucleus. Moreover, isoform-specific silencing of TFII-Ibeta results in transcriptional activation of the c-fos gene. In contrast, TFII-IDelta is largely cytoplasmic in the resting state but translocates to the nucleus upon growth factor signaling, undergoes signal-induced recruitment to the same site on the c-fos promoter, and activates the gene. Importantly, activated TFII-IDelta interacts with Erk1/2 (MAPK) kinase in the cell cytoplasm and imports the Erk1/2 to the nucleus, thereby transducing growth factor signaling. Our results identify a unique growth factor signaling pathway controlled by opposing activities of two TFII-I spliced isoforms.
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Affiliation(s)
- Shweta Hakre
- Program in Immunology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, Massachusetts 02111, USA
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48
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Abstract
In response to extracellular ligands, surface receptor tyrosine kinases and G-protein-coupled receptors activate isoforms of phospholipase C (PLC) and initiate calcium signaling. PLC can activate expression of surface transient receptor potential channels (TRPC) such as TRPC3, which modulate calcium entry through the plasma membrane. A recent paper shows that competitive binding of cytoplasmic TFII-I, a transcription factor, to PLC-gamma results in inhibition of TRPC3-mediated agonist-induced Ca(2+) entry. These results establish a novel cytoplasmic function for TFII-I.
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Affiliation(s)
- Ananda L Roy
- Department of Pathology, Programs in Genetics and Immunology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, Massachusetts 02111, USA.
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49
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Verdier V, Johndrow JE, Betson M, Chen GC, Hughes DA, Parkhurst SM, Settleman J. Drosophila Rho-kinase (DRok) is required for tissue morphogenesis in diverse compartments of the egg chamber during oogenesis. Dev Biol 2006; 297:417-32. [PMID: 16887114 PMCID: PMC2504748 DOI: 10.1016/j.ydbio.2006.05.016] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2005] [Revised: 05/11/2006] [Accepted: 05/15/2006] [Indexed: 11/16/2022]
Abstract
The Rho-kinases are widely utilized downstream targets of the activated Rho GTPase that have been directly implicated in many aspects of Rho-dependent effects on F-actin assembly, acto-myosin contractility, and microtubule stability, and consequently play an essential role in regulating cell shape, migration, polarity, and division. We have determined that the single closely related Drosophila Rho-kinase ortholog, DRok, is required for several aspects of oogenesis, including maintaining the integrity of the oocyte cortex, actin-mediated tethering of nurse cell nuclei, "dumping" of nurse cell contents into the oocyte, establishment of oocyte polarity, and the trafficking of oocyte yolk granules. These defects are associated with abnormalities in DRok-dependent actin dynamics and appear to be mediated by multiple downstream effectors of activated DRok that have previously been implicated in oogenesis. DRok regulates at least one of these targets, the membrane cytoskeletal cross-linker DMoesin, via a direct phosphorylation that is required to promote localization of DMoesin to the oocyte cortex. The collective oogenesis defects associated with DRok deficiency reveal its essential role in multiple aspects of proper oocyte formation and suggest that DRok defines a novel class of oogenesis determinants that function as key regulators of several distinct actin-dependent processes required for proper tissue morphogenesis.
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Affiliation(s)
- Valerie Verdier
- Massachusetts General Hospital Cancer Center and Harvard Medical School, 149 13 Street, Charlestown, MA 02129, USA
| | - James E. Johndrow
- Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A1-162, PO Box 19024, Seattle, WA 98109-1024, USA
| | | | - Guang-Chao Chen
- Massachusetts General Hospital Cancer Center and Harvard Medical School, 149 13 Street, Charlestown, MA 02129, USA
| | - David A. Hughes
- The Faculty of Life Sciences, The University of Manchester, Sackville Street, Manchester, United Kingdom
| | - Susan M. Parkhurst
- Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A1-162, PO Box 19024, Seattle, WA 98109-1024, USA
| | - Jeffrey Settleman
- Massachusetts General Hospital Cancer Center and Harvard Medical School, 149 13 Street, Charlestown, MA 02129, USA
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50
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Desgranges ZP, Ahn J, Lazebnik MB, Ashworth T, Lee C, Pestell RC, Rosenberg N, Prives C, Roy AL. Inhibition of TFII-I-dependent cell cycle regulation by p53. Mol Cell Biol 2005; 25:10940-52. [PMID: 16314517 PMCID: PMC1316948 DOI: 10.1128/mcb.25.24.10940-10952.2005] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2005] [Revised: 07/13/2005] [Accepted: 09/21/2005] [Indexed: 01/27/2023] Open
Abstract
The multifunctional transcription factor TFII-I is tyrosine phosphorylated in response to extracellular growth signals and transcriptionally activates growth-promoting genes. However, whether activation of TFII-I also directly affects the cell cycle profile is unknown. Here we show that under normal growth conditions, TFII-I is recruited to the cyclin D1 promoter and transcriptionally activates this gene. Most strikingly, upon cell cycle arrest resulting from genotoxic stress and p53 activation, TFII-I is ubiquitinated and targeted for proteasomal degradation in a p53- and ATM (ataxia telangiectasia mutated)-dependent manner. Consistent with a direct role of TFII-I in cell cycle regulation and cellular proliferation, stable and ectopic expression of wild-type TFII-I increases cyclin D1 levels, resulting in accelerated entry to and exit from S phase, and overcomes p53-mediated cell cycle arrest, despite radiation. We further show that the transcriptional regulation of cyclin D1 and cell cycle control by TFII-I are dependent on its tyrosine phosphorylation at positions 248 and 611, sites required for its growth signal-mediated transcriptional activity. Taken together, our data define TFII-I as a growth signal-dependent transcriptional activator that is critical for cell cycle control and proliferation and further reveal that genotoxic stress-induced degradation of TFII-I results in cell cycle arrest.
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Affiliation(s)
- Zana P Desgranges
- Program in Immunology, Sackler School of Graduate Biomedical Sciences, Department of Pathology, Tufts University School of Medicine, Boston, MA 02111, USA
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