DOCSLIB.ORG

CCM3/PDCD10 Heterodimerizes With

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
CCM3/PDCD10 Heterodimerizes With THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 28, pp. 25056–25064, July 15, 2011 © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. CCM3/PDCD10 Heterodimerizes with Germinal Center Kinase III (GCKIII) Proteins Using a Mechanism Analogous to CCM3 Homodimerization*ࡗ Received for publication, December 17, 2010, and in revised form, April 26, 2011 Published, JBC Papers in Press, May 11, 2011, DOI 10.1074/jbc.M110.213777 Derek F. Ceccarelli‡1, Rob C. Laister§1, Vikram Khipple Mulligan¶, Michelle J. Kean‡ʈ2, Marilyn Goudreault‡, Ian C. Scottʈ**, W. Brent Derryʈ**, Avijit Chakrabartty¶‡‡, Anne-Claude Gingras‡ʈ3, and Frank Sicheri‡ʈ4 From the ‡Centre for Systems Biology, Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto, Ontario M5G 1X5, the §Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, Toronto, Ontario M5G 1L7, the ¶Campbell Family Institute for Cancer Research, Ontario Cancer Institute/University Health Network, Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1L7, the ʈDepartment of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, the **Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, and the ‡‡Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7, Canada CCM3 mutations give rise to cerebral cavernous malforma- three distinct genes, denoted CCM1–3, that are causative for tions (CCMs) of the vasculature through a mechanism that the formation of most familial CCM lesions (1, 2). CCM3, also remains unclear. Interaction of CCM3 with the germinal center named PDCD10, is a 212 amino acid protein conserved among Downloaded from kinase III (GCKIII) subfamily of Sterile 20 protein kinases, metazoans (3, 4). Knockdown of CCM3 in zebrafish causes an MST4, STK24, and STK25, has been implicated in cardiovascu- enlarged heart phenotype (5), whereas targeted deletion of lar development in the zebrafish, raising the possibility that dys- CCM3 in the mouse results in defects of early angiogenesis and regulated GCKIII function may contribute to the etiology of early embryonic lethality, a phenotype also observed following CCM disease. Here, we show that the amino-terminal region of tissue-specific deletion in the vascular endothelium (6). A non- http://www.jbc.org/ CCM3 is necessary and sufficient to bind directly to the C-ter- cell autonomous role for CCM3 in neuroglia on the vasculature minal tail region of GCKIII proteins. This same region of CCM3 has also been uncovered in mouse recently, indicating that was shown previously to mediate homodimerization through CCMs may arise in the central nervous system by the loss of the formation of an interdigitated ␣-helical domain. Sequence CCM3 signaling in neural as well as endothelial populations (7). conservation and binding studies suggest that CCM3 may CCM3 has been detected in complex with CCM1 and CCM2 by guest on May 21, 2015 preferentially heterodimerize with GCKIII proteins through proteins, suggesting that the three proteins may share a com- a manner structurally analogous to that employed for CCM3 mon biochemical function (8–10). Yeast two-hybrid, co-im- homodimerization. munoprecipitation, and GST pulldown experiments from cell lysates demonstrated that CCM3 also readily interacts with MST4, STK24, and STK25, a grouping of protein kinases Cerebral cavernous malformations (CCMs)5 are vascular termed the germinal center kinase class III (GCKIII) family abnormalities in the brain characterized by focal dilations of (10–13). CCM3 and the GCKIII proteins have also been cranial vasculature that can progress to hemorrhages and detected as part of a large multiprotein complex termed STRI- stroke (OMIM ID 116860). Mutations have been identified in PAK (striatin-interacting phosphatase and kinase; see Refs. 14, 15). The knockdown of GCKIII proteins in zebrafish gives rise to the same cardiovascular defects as CCM3 knockdown, sug- * This work was supported in part by Canadian Institutes of Health Research gesting the CCM3-GCKIII protein interaction is important for Grants MOP-36399 (to F. S.) and MOP-84314 (to A.-C. G.). 6 ࡗ This article was selected as a Paper of the Week. proper CCM3 function (5). 1 Both authors contributed equally to this work. The GCKIII proteins (STK24, STK25, and MST4) are mem- 2 Supported by the Canadian Institutes of Health Research through a Banting bers of the larger Sterile 20 kinase family and are characterized and Best Canada graduate scholarship. 3 Canada Research chair in Functional Proteomics and the Lea Reichmann by highly conserved catalytic domains and a 100–120 residue chair in Cancer Proteomics. To whom correspondence may be addressed: carboxyl-terminal tail, whose function is not currently known. Samuel Lunenfeld Research Institute at Mount Sinai Hospital, 600 Univer- The closely related GCKII proteins MST1 and MST2 possess sity Ave., Toronto, ON M5G1X5, Canada. Tel.: 416-586-5027; Fax: 416-586- 8869; E-mail: [email protected]. completely distinct C-terminal tails that mediate homotypic 4 Canada Research chair in Structural Principles of Signal Transduction. To and heterotypic interactions (16), raising the possibility that an whom correspondence may be addressed: Samuel Lunenfeld Research analogous function might be served by the tail region of GCKIII Institute at Mount Sinai Hospital, 600 University Ave., Toronto, ON M5G1X5, Canada. Tel.: 416-586-8471; Fax: 416-586-8869; E-mail: sicheri@ proteins, albeit through an unrelated structural mechanism. lunenfeld.ca. Crystal structures of the CCM3 protein revealed an architec- 5 The abbreviations used are: CCM, cerebral cavernous malformation; GCKIII, ture consisting of two distinct structural domains (17, 18). The germinal center kinase group III; STRIPAK, striatin-interacting phosphatase N-terminal helical domain of CCM3 mediates homodimeriza- and kinase; TEV, tobacco etch virus; SEC-MALS, size exclusion chromatog- raphy-multiangle light scattering; FAT, focal adhesion targeting; PP4C, cat- alytic subunit of the serine/threonine protein phosphatase 4; mAU, milli absorbance units. 6 B. Yoruk, B. S. Gillers, N. C. Chi, and Ian C. Scott, submitted for publication. 25056 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 28•JULY 15, 2011 CCM3 Interacts Directly with GCKIII Proteins tion. The C-terminal four-helix bundle, termed the focal adhe- S-75 size exclusion column (GE Healthcare) in 20 mM HEPES, sion targeting (FAT) homology domain (17), functions as a lin- pH 7.5, 100 mM NaCl, and ␤-mercaptoethanol. Fractions con- ear peptide binding module that mediates direct interactions taining purified protein were concentrated, flash frozen, and with CCM2, paxillin, and the striatin component of STRIPAK stored at Ϫ80 °C. Bacterial pellets of GST-tagged proteins were 7 (17). Of note, the N-terminal region of CCM3 has also been lysed in 20 mM HEPES, pH 7.5, 400 mM NaCl, and 5 mM ␤-mer- implicated in the interaction with GCKIII proteins in cells and captoethanol, and the supernatant was applied to glutathione- model organisms (5, 13, 19). Given the critical role for CCM3 Sepharose resin (GE Healthcare). The protein of interest was and GCKIII proteins in maintaining vascular integrity, we have separated from the GST affinity tag following overnight incu- probed the basis for their interaction in close detail. The results bation with TEV protease and further purified by size exclusion presented here demonstrate that the amino terminus of CCM3 chromatography as described above. The heterodimeric CCM3 interacts directly with the C-terminal regions of GCKIII pro- and MST4 complex was obtained by mixing bacterial lysates teins. Based on sequence similarity between the interacting expressing GST-MST4 and His-CCM3 proteins. Protein eluted regions of CCM3 and GCKIII proteins, we propose that het- from a nickel-chelating column was applied directly to gluta- erodimerization of the two proteins is achieved through an thione Sepharose resin and subsequently purified as described analogous structural mechanism to that reported for the above for GST-tagged proteins. homodimerization for CCM3 and present data indicating that In Vitro Binding Studies—Bacterial or SF9 cell lysates con- heterodimerization may be favored over homodimerization. taining GST fusion proteins were prepared as above, however EXPERIMENTAL PROCEDURES the supernatant was applied to glutathione-Sepharose resin (GE Healthcare) in GST-loading buffer containing 20 mM Cloning of Expression Constructs—PDCD10/CCM3, Downloaded from HEPES, pH 7.5, 400 mM NaCl, and 5 mM ␤-mercaptoethanol STK25/SOK1, MST4, STK4/MST1, and STRN3 were ampli- and washed extensively. The protein-bound affinity resin was fied by PCR and cloned into the modified bacterial expres- equilibrated with interaction buffer (20 mM HEPES, pH 7.5, 150 sion vectors pGEX-TEV and/or ProEX-TEV. CCM3 point mM NaCl, and 5 mM ␤-mercaptoethanol). Previously purified mutations were generated by PCR using standard tech- binding partner proteins were added to the protein-bound niques, and all clones were sequence verified. The N- http://www.jbc.org/ affinity resin and incubated on ice with gentle mixing for 20 terminal mutant of CCM3 (LAIL-4D) comprises L44D, min. The affinity resin was then washed three times with 500 ␮l A47D,I66D,L67D and the C-terminal mutant (K4A) comprises of interaction buffer and aliquots of the binding reactions were K132A,K139A, K172A,K179A. CCM3 point mutants were sub- separated by SDS-PAGE with proteins visualized following cloned into the pcDNA5-FRT-GFP vector (20) for expression Coomassie staining. in mammalian cells. pcDNA3-FLAG-MST4 was reported pre- Light Scattering—Size exclusion chromatography-multi- by guest on May 21, 2015 viously (14). ␮ Protein Expression and Purification—BL21-Codonϩ cells angle light scattering (SEC-MALS) was performed with 200 M (Agilent Technologies) were transformed and grown to A of protein samples injected onto a 24-ml S200 Superdex column 600 and measured using a three-angle (45, 90, and 135°) miniDawn 0.8 and induced by addition of 0.25 mM isopropyl 1-thio-␤-D- galactopyranoside for 12–18 h at 18 °C.
Load more

Recommended publications
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Downloaded from [266]
    Patterns of DNA methylation on the human X chromosome and use in analyzing X-chromosome inactivation by Allison Marie Cotton B.Sc., The University of Guelph, 2005 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Medical Genetics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) January 2012 © Allison Marie Cotton, 2012 Abstract The process of X-chromosome inactivation achieves dosage compensation between mammalian males and females. In females one X chromosome is transcriptionally silenced through a variety of epigenetic modifications including DNA methylation. Most X-linked genes are subject to X-chromosome inactivation and only expressed from the active X chromosome. On the inactive X chromosome, the CpG island promoters of genes subject to X-chromosome inactivation are methylated in their promoter regions, while genes which escape from X- chromosome inactivation have unmethylated CpG island promoters on both the active and inactive X chromosomes. The first objective of this thesis was to determine if the DNA methylation of CpG island promoters could be used to accurately predict X chromosome inactivation status. The second objective was to use DNA methylation to predict X-chromosome inactivation status in a variety of tissues. A comparison of blood, muscle, kidney and neural tissues revealed tissue-specific X-chromosome inactivation, in which 12% of genes escaped from X-chromosome inactivation in some, but not all, tissues. X-linked DNA methylation analysis of placental tissues predicted four times higher escape from X-chromosome inactivation than in any other tissue. Despite the hypomethylation of repetitive elements on both the X chromosome and the autosomes, no changes were detected in the frequency or intensity of placental Cot-1 holes.
    [Show full text]
  • Genome-Wide DNA Methylation Analysis of KRAS Mutant Cell Lines Ben Yi Tew1,5, Joel K
    www.nature.com/scientificreports OPEN Genome-wide DNA methylation analysis of KRAS mutant cell lines Ben Yi Tew1,5, Joel K. Durand2,5, Kirsten L. Bryant2, Tikvah K. Hayes2, Sen Peng3, Nhan L. Tran4, Gerald C. Gooden1, David N. Buckley1, Channing J. Der2, Albert S. Baldwin2 ✉ & Bodour Salhia1 ✉ Oncogenic RAS mutations are associated with DNA methylation changes that alter gene expression to drive cancer. Recent studies suggest that DNA methylation changes may be stochastic in nature, while other groups propose distinct signaling pathways responsible for aberrant methylation. Better understanding of DNA methylation events associated with oncogenic KRAS expression could enhance therapeutic approaches. Here we analyzed the basal CpG methylation of 11 KRAS-mutant and dependent pancreatic cancer cell lines and observed strikingly similar methylation patterns. KRAS knockdown resulted in unique methylation changes with limited overlap between each cell line. In KRAS-mutant Pa16C pancreatic cancer cells, while KRAS knockdown resulted in over 8,000 diferentially methylated (DM) CpGs, treatment with the ERK1/2-selective inhibitor SCH772984 showed less than 40 DM CpGs, suggesting that ERK is not a broadly active driver of KRAS-associated DNA methylation. KRAS G12V overexpression in an isogenic lung model reveals >50,600 DM CpGs compared to non-transformed controls. In lung and pancreatic cells, gene ontology analyses of DM promoters show an enrichment for genes involved in diferentiation and development. Taken all together, KRAS-mediated DNA methylation are stochastic and independent of canonical downstream efector signaling. These epigenetically altered genes associated with KRAS expression could represent potential therapeutic targets in KRAS-driven cancer. Activating KRAS mutations can be found in nearly 25 percent of all cancers1.
    [Show full text]
  • Structure-Based Screen Identification of a Mammalian Ste20-Like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors
    Published OnlineFirst December 31, 2015; DOI: 10.1158/1535-7163.MCT-15-0703 Small Molecule Therapeutics Molecular Cancer Therapeutics Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors Weipeng Xiong1,3, Christopher J. Matheson2, Mei Xu1,3, Donald S. Backos2, Taylor S. Mills1, Smita Salian-Mehta1, Katja Kiseljak-Vassiliades1,3, Philip Reigan2, and Margaret E. Wierman1,3 Abstract Pituitary tumors of the gonadotrope lineage are often large described as an Aurora kinase inhibitor, exhibited potent inhi- and invasive, resulting in hypopituitarism. No medical treat- bition of the MST4 kinase at nanomolar concentrations. The ments are currently available. Using a combined genetic and LbT2 gonadotrope pituitary cell hypoxic model was used to test genomic screen of individual human gonadotrope pituitary the ability of this inhibitor to antagonize MST4 actions. Under tumor samples, we recently identified the mammalian ster- short-term severe hypoxia (1% O2), MST4 protection from ile-20 like kinase 4 (MST4) as a protumorigenic effector, driving hypoxia-induced apoptosis was abrogated in the presence of increased pituitary cell proliferation and survival in response to hesperadin. Similarly, under chronic hypoxia (5%), hesperadin a hypoxic microenvironment. To identify novel inhibitors of blocked the proliferative and colony-forming actions of MST4 the MST4 kinase for potential future clinical use, computation- as well as the ability to activate specific downstream signaling al-based virtual library screening was used to dock the Sell- and hypoxia-inducible factor-1 effectors. Together, these data eckChem kinase inhibitor library into the ATP-binding site of identify hesperadin as the first potent, selective inhibitor of the the MST4 crystal structure.
    [Show full text]
  • A Semantic Approach for Knowledge Capture of Microrna-Target Gene Interactions
    2015 IEEE International Conference on Bioinformatics and Biomedicine (BIBM) A semantic approach for knowledge capture of microRNA-target gene interactions Jingshan Huang* Fernando Gutierrez Dejing Dou School of Computing, University of South Alabama CIS Dept., University of Oregon CIS Dept., University of Oregon Mobile, Alabama 36688, U.S.A. Eugene, Oregon 97403, U.S.A. Eugene, Oregon 97403, U.S.A. Judith A. Blake Karen Eilbeck Darren A. Natale The Jackson Laboratory School of Medicine, University of Utah Georgetown University Medical Center Bar Harbor, Maine 04609, U.S.A. Salt Lake City, Utah 84112, U.S.A. Washington D.C. 20007, U.S.A. Barry Smith Yu Lin Xiaowei Wang Dept. Philosophy, University at Buffalo University of Miami Washington University School of Medicine Buffalo, New York 14260, U.S.A. Miami, Florida 33146, U.S.A. St. Louis, Missouri 63110, U.S.A. Zixing Liu Ming Tan Alan Ruttenberg MCI, University of South Alabama MCI, University of South Alabama Dental Medicine, University at Buffalo Mobile, Alabama 36604, U.S.A. Mobile, Alabama 36604, U.S.A. Buffalo, New York 14214, U.S.A. Abstract—Research has indicated that microRNAs (miRNAs), Conventionally, data end users (that is, biologists, bioin- a special class of non-coding RNAs (ncRNAs), can perform formaticians, and clinical investigators) need to search for important roles in different biological and pathological processes. (1) biologically validated miRNA targets (for example, by miRNAs’ functions are realized by regulating their respective querying the PubMed database [3]) and (2) computationally target genes (targets). It is thus critical to identify and analyze putative miRNA targets (for example, by initiating inquiries miRNA-target interactions for a better understanding and de- on various prediction databases or websites such as miRDB lineation of miRNAs’ functions.
    [Show full text]
  • STRIPAK Complexes in Cell Signaling and Cancer
    Oncogene (2016), 1–9 © 2016 Macmillan Publishers Limited All rights reserved 0950-9232/16 www.nature.com/onc REVIEW STRIPAK complexes in cell signaling and cancer Z Shi1,2, S Jiao1 and Z Zhou1,3 Striatin-interacting phosphatase and kinase (STRIPAK) complexes are striatin-centered multicomponent supramolecular structures containing both kinases and phosphatases. STRIPAK complexes are evolutionarily conserved and have critical roles in protein (de) phosphorylation. Recent studies indicate that STRIPAK complexes are emerging mediators and regulators of multiple vital signaling pathways including Hippo, MAPK (mitogen-activated protein kinase), nuclear receptor and cytoskeleton remodeling. Different types of STRIPAK complexes are extensively involved in a variety of fundamental biological processes ranging from cell growth, differentiation, proliferation and apoptosis to metabolism, immune regulation and tumorigenesis. Growing evidence correlates dysregulation of STRIPAK complexes with human diseases including cancer. In this review, we summarize the current understanding of the assembly and functions of STRIPAK complexes, with a special focus on cell signaling and cancer. Oncogene advance online publication, 15 February 2016; doi:10.1038/onc.2016.9 INTRODUCTION in the central nervous system and STRN4 is mostly abundant in Recent proteomic studies identified a group of novel multi- the brain and lung, whereas STRN3 is ubiquitously expressed in 5–9 component complexes named striatin (STRN)-interacting phos- almost all tissues. STRNs share a
    [Show full text]
  • Structure-Based Screen Identification of a Mammalian Ste20-Like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors
    Published OnlineFirst December 31, 2015; DOI: 10.1158/1535-7163.MCT-15-0703 Small Molecule Therapeutics Molecular Cancer Therapeutics Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors Weipeng Xiong1,3, Christopher J. Matheson2, Mei Xu1,3, Donald S. Backos2, Taylor S. Mills1, Smita Salian-Mehta1, Katja Kiseljak-Vassiliades1,3, Philip Reigan2, and Margaret E. Wierman1,3 Abstract Pituitary tumors of the gonadotrope lineage are often large described as an Aurora kinase inhibitor, exhibited potent inhi- and invasive, resulting in hypopituitarism. No medical treat- bition of the MST4 kinase at nanomolar concentrations. The ments are currently available. Using a combined genetic and LbT2 gonadotrope pituitary cell hypoxic model was used to test genomic screen of individual human gonadotrope pituitary the ability of this inhibitor to antagonize MST4 actions. Under tumor samples, we recently identified the mammalian ster- short-term severe hypoxia (1% O2), MST4 protection from ile-20 like kinase 4 (MST4) as a protumorigenic effector, driving hypoxia-induced apoptosis was abrogated in the presence of increased pituitary cell proliferation and survival in response to hesperadin. Similarly, under chronic hypoxia (5%), hesperadin a hypoxic microenvironment. To identify novel inhibitors of blocked the proliferative and colony-forming actions of MST4 the MST4 kinase for potential future clinical use, computation- as well as the ability to activate specific downstream signaling al-based virtual library screening was used to dock the Sell- and hypoxia-inducible factor-1 effectors. Together, these data eckChem kinase inhibitor library into the ATP-binding site of identify hesperadin as the first potent, selective inhibitor of the the MST4 crystal structure.
    [Show full text]
  • MST4 Regulates Epithelial–Mesenchymal Transition of Choriocarcinoma by Mediating TGF- Β1 Expression
    OncoTargets and Therapy Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH MST4 Regulates Epithelial–Mesenchymal Transition of Choriocarcinoma by Mediating TGF- β1 Expression This article was published in the following Dove Press journal: OncoTargets and Therapy Hanxi Yu 1,* Background: Mammalian Ste20-like kinase 4 (MST4), also known as serine/threonine Weichen Zhang2,* kinase 26 (STK26), promotes development of several cancers and is found to be highly Peilin Han1 expressed in the placenta. However, in choriocarcinoma that originated from the placenta, the Beng Yang2 expression of MST4 was undetermined and its mechanism was unknown. In this study, the Xiaode Feng 2 expression of MST4 in choriocarcinoma as well as the underlying mechanism was explored. Purpose: To detect the expression of MST4 in patient samples and mechanism of mediating Ping Zhou1 EMT by MST4 in choriocarcinoma. Xiaoxu Zhu1 3 Patients and Methods: The metastatic lesions of choriocarcinoma (n=17) and volunteer Bingqian Zhou villus (n=17) were collected to determine MST4 expression using immunohistochemistry and 4 Wei Chen H&E staining. We use siRNA and lentiviral vector to knockdown MST4 and use plasmid to 1 Jianhua Qian overexpress MST4 in choriocarcinoma. Then, we apply real-time polymerase chain reaction 2 Jun Yu (RT-PCR), Western blot assay and immunofluorescence assay to detect target protein expres­ 1Department of Gynecology, The First sions. Cell invasion and migration and cell proliferation were detected by transwell assay and Affiliated Hospital of Zhejiang University, wound healing assay and CCK-8 and cell colony formation. School of Medicine, Hangzhou 310006, Results: MST4 is lowly expressed in the metastatic lesions of choriocarcinoma patients when People’s Republic of China; 2Division of Hepatobiliary and Pancreatic Surgery, compared with normal villus.
    [Show full text]
  • STRIPAK, a Highly Conserved Signaling Complex, Controls Multiple
    Biol. Chem. 2019; 400(8): 1005–1022 Review Ulrich Kück*, Daria Radchenko and Ines Teichert STRIPAK, a highly conserved signaling complex, controls multiple eukaryotic cellular and developmental processes and is linked with human diseases https://doi.org/10.1515/hsz-2019-0173 networks are now known to underlie the transmission and Received February 27, 2019; accepted March 28, 2019; previously modulation of such signals. These networks, controlled published online May 1, 2019 by diverse regulators at different cellular levels, are highly conserved across eukaryotic organisms. Abstract: The striatin-interacting phosphatases and Among the signaling components that have received kinases (STRIPAK) complex is evolutionary highly con- increased attention in the last decade is the STRIPAK served and has been structurally and functionally complex, which was initially identified in mammals by described in diverse lower and higher eukaryotes. In recent mass spectrometry (MS) analysis (Goudreault et al., 2009). years, this complex has been biochemically characterized Here we will provide a summary of the key studies leading better and further analyses in different model systems have to the discovery of striatin, the major regulatory phos- shown that it is also involved in numerous cellular and phatase subunit of the STRIPAK complex. Further, the developmental processes in eukaryotic organisms. Fur- components and architecture as well as the assembly and ther recent results have shown that the STRIPAK complex structural insights of the STRIPAK complex will be com- functions as a macromolecular assembly communicating prehensively reviewed. Another focus will be the control through physical interaction with other conserved signal- of developmental processes as a result of bi-directional ing protein complexes to constitute larger dynamic protein interaction of STRIPAK with other components involved networks.
    [Show full text]
  • MOB (Mps One Binder) Proteins in the Hippo Pathway and Cancer
    Review MOB (Mps one Binder) Proteins in the Hippo Pathway and Cancer Ramazan Gundogdu 1 and Alexander Hergovich 2,* 1 Vocational School of Health Services, Bingol University, 12000 Bingol, Turkey; [email protected] 2 UCL Cancer Institute, University College London, WC1E 6BT, London, United Kingdom * Correspondence: [email protected]; Tel.: +44 20 7679 2000; Fax: +44 20 7679 6817 Received: 1 May 2019; Accepted: 4 June 2019; Published: 10 June 2019 Abstract: The family of MOBs (monopolar spindle-one-binder proteins) is highly conserved in the eukaryotic kingdom. MOBs represent globular scaffold proteins without any known enzymatic activities. They can act as signal transducers in essential intracellular pathways. MOBs have diverse cancer-associated cellular functions through regulatory interactions with members of the NDR/LATS kinase family. By forming additional complexes with serine/threonine protein kinases of the germinal centre kinase families, other enzymes and scaffolding factors, MOBs appear to be linked to an even broader disease spectrum. Here, we review our current understanding of this emerging protein family, with emphases on post-translational modifications, protein-protein interactions, and cellular processes that are possibly linked to cancer and other diseases. In particular, we summarise the roles of MOBs as core components of the Hippo tissue growth and regeneration pathway. Keywords: Mps one binder; Hippo pathway; protein kinase; signal transduction; phosphorylation; protein-protein interactions; structure biology; STK38; NDR; LATS; MST; STRIPAK 1. Introduction The family of MOBs (monopolar spindle-one-binder proteins) is highly conserved in eukaryotes [1–4]. To our knowledge, at least two different MOBs have been found in every eukaryote analysed so far.
    [Show full text]
  • MST4 Antibody A
    Revision 1 C 0 2 - t MST4 Antibody a e r o t S Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) 2 2 Web: [email protected] 8 www.cellsignal.com 3 # 3 Trask Lane Danvers Massachusetts 01923 USA For Research Use Only. Not For Use In Diagnostic Procedures. Applications: Reactivity: Sensitivity: MW (kDa): Source: UniProt ID: Entrez-Gene Id: WB H M R Mk B Endogenous 52 Rabbit Q9P289 51765 Product Usage Information 4. Zhao, B. et al. (2011) Nat Cell Biol 13, 877-83. 5. Dan, I. et al. (2002) J Biol Chem 277, 5929-39. Application Dilution 6. Sung, V. et al. (2003) Cancer Res 63, 3356-63. 7. Preisinger, C. et al. (2004) J Cell Biol 164, 1009-20. Western Blotting 1:1000 Storage Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody. Specificity / Sensitivity MST4 Antibody recognizes endogenous levels of total MST4 protein independent of phosphorylation. The antibody does not cross-react with MST1-3 proteins. Species Reactivity: Human, Mouse, Rat, Monkey, Bovine Source / Purification Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the amino-terminal residues of human MST4 protein. Antibodies are purified by protein A and peptide affinity chromatography. Background Mammalian sterile-20-like (MST) kinases are upstream regulators of mitogen-activated protein kinase (MAPK) signaling pathways that regulate multiple biological processes, including apoptosis, morphogenesis, cell migration, and cytoskeletal rearrangements (1). This group of serine/threonine kinases includes a pair of closely related proteins (MST1, MST2) that are functionally distinct from the more distantly related MST3 and MST4 kinases.
    [Show full text]
  • A Novel, Non-Canonical Mechanism of Regulation of MST3 (Mammalian Sterile20- Related Kinase 3)
    A novel, non-canonical mechanism of regulation of MST3 (mammalian Sterile20- related kinase 3) Article Published Version Fuller, S. J., McGuffin, L. J., Marshall, A. K., Giraldo, A., Pikkarainen, S., Clerk, A. and Sugden, P. (2012) A novel, non- canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3). Biochemical Journal, 442 (3). pp. 595-610. ISSN 0264-6021 doi: https://doi.org/10.1042/BJ20112000 Available at http://centaur.reading.ac.uk/26012/ It is advisable to refer to the publisher’s version if you intend to cite from the work. See Guidance on citing . Published version at: http://dx.doi.org/10.1042/BJ20112000 To link to this article DOI: http://dx.doi.org/10.1042/BJ20112000 Publisher: Portland Press Limited Publisher statement: The final version of record is available at http://www.biochemj.org/bj All outputs in CentAUR are protected by Intellectual Property Rights law, including copyright law. Copyright and IPR is retained by the creators or other copyright holders. Terms and conditions for use of this material are defined in the End User Agreement . www.reading.ac.uk/centaur CentAUR Central Archive at the University of Reading Reading’s research outputs online www.biochemj.org Biochem. J. (2012) 442, 595–610 (Printed in Great Britain) doi:10.1042/BJ20112000 595 A novel non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3) Stephen J. FULLER*, Liam J. MCGUFFIN*, Andrew K. MARSHALL*, Alejandro GIRALDO*, Sampsa PIKKARAINEN†, Angela CLERK* and Peter H. SUGDEN*1 *School of Biological
    [Show full text]