DOCSLIB.ORG

Sperm Proteasome Degrades Egg Envelope Glycoprotein ZP1 During

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Sperm Proteasome Degrades Egg Envelope Glycoprotein ZP1 During REPRODUCTIONRESEARCH Expression profiles of protein tyrosine kinase genes in human embryonic stem cells Mi-Young Son, Janghwan Kim, Hyo-Won Han, Sun-Mi Woo, Yee Sook Cho, Yong-Kook Kang and Yong-Mahn Han1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), Center for Regenerative Medicine, 52 Eoeun- Dong, Yuseong-Gu, Daejeon 305-806, South Korea and 1Department of Biological Sciences and Center for Stem Cell Differentiation, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, South Korea Correspondence should be addressed to Y-K Kang; Email: [email protected] Y-M Han; Email: [email protected] Abstract Complex signaling pathways operate in human embryonic stem cells (hESCs) and are coordinated to maintain self-renewal and stem cell characteristics in them. Protein tyrosine kinases (PTKs) participate in diverse signaling pathways in various types of cells. Because of their functions as key molecules in various cellular processes, PTKs are anticipated to have important roles also in hESCs. In this study, we investigated the roles of PTKs in undifferentiated and differentiated hESCs. To establish comprehensive PTK expression profiles in hESCs, we performed reverse transcriptase PCR using degenerate primers according to the conserved catalytic PTK motifs in both undifferentiated and differentiated hESCs. Here, we identified 42 different kinases in two hESC lines, including 5 non-receptor tyrosine kinases (RTKs), 24 RTKs, and 13 dual and other kinases, and compared the protein kinase expression profiles of hESCs and retinoic acid- treated hESCs. Significantly, up- and downregulated kinases in undifferentiated hESCs were confirmed by real-time PCR and western blotting. MAP3K3, ERBB2, FGFR4, and EPHB2 were predominantly upregulated, while CSF1R, TYRO3, SRC, and GSK3A were consistently downregulated in two hESC lines. Western blot analysis showed that the transcriptional levels of these kinases were consistent with the translational levels. The obstruction of upregulated kinases’ activities using specific inhibitors disturbed the undifferentiated status and induced the differentiation of hESCs. Our results support the dynamic expression of PTKs during hESC maintenance and suggest that specific PTKs that are consistently up- and downregulated play important roles in the maintenance of stemness and the direction of differentiation of hESCs. Reproduction (2008) 136 423–432 Introduction differentiation, and development (Hunter 1987). These proteins are among the key molecules that regulate Human embryonic stem cells (hESCs) have the ability signaling pathways, but only account for less than 10% of to self-renew and differentiate into a variety of cell the total cellular kinases (Hunter 1994). PTKs are types within the three embryonic germ layers classified into receptor tyrosine kinases (RTK) and (Thomson et al. 1998). To identify the key molecules cytoplasmic non-RTKs (NRTK). In humans, 90 PTKs involved in maintaining stemness of hESCs, transcrip- have been identified to date, comprising 58 receptor and tome characterization procedures, such as microarray 32 NRTKs (Robinson et al. 2000). Expression of most (Ramalho-Santos et al. 2002, Sato et al. 2003, Sperger PTKs may be tightly regulated to retain unique features of et al. 2003, Dvash et al. 2004), serial analysis of gene a specific cell type. However, limited information is expression (Richards et al.2004), and expressed available on the molecular behavior of PTKs in hESCs. sequence tags (ESTs; Brandenberger et al. 2004), have Here, we present the PTK-focused expression profiles been employed. Molecules participating in several of hESCs. The transcriptional profiles of protein kinases signaling pathways, including fibroblast growth factor (PKs) from hESCs are compared with those of retinoic (FGF), WNT, NODAL, PI3K/AKT, MAP kinase (MAPK)/ acid (RA)-treated hESCs by an RT-PCR-based cloning ERK, and NF-kB, may be involved in maintaining the technique using degenerate primers corresponding to the stemness of hESCs (Sato et al. 2003, Brandenberger highly conserved catalytic domains of PTKs. We et al. 2004, Armstrong et al. 2006, Rho et al. 2006). analyzed 1298 clones and identified 42 kinase genes in Generally, protein tyrosine kinases (PTKs) play pivotal two genetically independent hESC lines. Our PTK roles in cell proliferation, apoptosis, oncogenesis, profiling data should be valuable in understanding the q 2008 Society for Reproduction and Fertility DOI: 10.1530/REP-08-0080 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/02/2021 08:27:48PM via free access 424 M-Y Son and others molecular mechanisms underlying the stemness, devel- oping the novel biomarkers for the pluripotency, and identifying the potential regulators for differentiation in hESCs. Results Identification of the tyrosine kinase family in hESCs To elucidate the molecular mechanisms of PTKs regulat- ing the stemness of hESCs, differentiated cells were derived from the hESCs used as controls. To induce differentiation, hESCs were treated with RA for 5 days. As shown in Fig. 1A, alkaline phosphatase (AP) activity was considerably weak in RA-treated hESCs, compared with control hESCs. Moreover, transcriptional expression of stem cell marker genes, such as POU5F1 (OCT4) and NANOG, was remarkably reduced as assessed by real- time PCR (Fig. 1B). To assess the degree of diversity in differentiation capacity, we performed the real-time PCR analyses of the lineage markers for endoderm (amylase, albumin), mesoderm (GATA2), and ectoderm (NCAM). The RA-treated hESCs used in this study expressed diverse lineage markers (Supplementary Fig. 1, which can be viewed online at www.reproduction-online.org/supple- mental). Thus, the RA treatment appeared to result in the loss of stemness with no preference for differentiation towards a specific lineage, consistent with a previous report (Draper et al. 2002). To determine the transcrip- tional patterns of PTKs, degenerate RT-PCR was per- formed in both control and RA-treated hESCs. Degenerate primers were designed on the basis of conserved motifs within the catalytic domains of tyrosine kinase, DFG and DVW. The DFG motif is present in all PK types; while DVW is primarily encoded by tyrosine kinases, but a small subset of serine/threonine kinases also contain the motif (Robinson et al. 1996, Lin et al. 1998). In this study, the forward (F1, F2, and F3) and reverse primer (R) are based on DFG and DVW motifs respectively (Supple- mentary Table 1, which can be viewed online at www.reproduction-online.org/supplemental). DNA fragments of 150–170 bp were successfully amplified by degenerate PCR (Fig. 1C). The F1-R combination yielded the least PCR products in two hESC lines. Similar results have been observed with other tissues and cell lines (Lin et al. 1998, Wu et al. 2000). We performed two independent degenerate PCRs for each hESC lines and pooled the amplification products together respectively, Figure 1 Induction of hESC differentiation by retinoic acid (RA). (A) Alkaline phosphatase staining of hESC colonies. (B) Expression of hESC markers, POU5F1 and NANOG, by real-time PCR analysis. Statistical significance of data was assessed using Student’s t-test (*P!0.01). (C) Representative gel electrophoresis of amplified protein tyrosine kinase RT-PCR products (150–170 bp). RT-PCR using a combination of degenerate primers, F1, F2, F3, and R, was performed, as described in the Materials and Methods section. Reproduction (2008) 136 423–432 www.reproduction-online.org Downloaded from Bioscientifica.com at 10/02/2021 08:27:48PM via free access Expression profiles of tyrosine kinases in hESCs 425 for subsequent analysis. The pooled amplified products EPHA2, EPHB3, and MERTK) were commonly up- and were eluted, cloned, and sequenced. BLASTN analyses downregulated in both hESC lines respectively. The revealed that 90.0% (743/826 clones) and 94.3% (445/ cellular functions of the kinase genes identified in this 472 clones) genes from CHA-hES3 and SNUhES3 study are presented in Table 3. These PK profiles encoded human PKs respectively (Table 1). Thus, we obtained using gene frequency analyses provide presented a high fidelity procedure for identifying PKs in valuable information on the dynamic behavior of hESCs. Among the identified genes, there were 36 and 31 these proteins between undifferentiated and differen- non-redundant human kinase genes in CHA-hES3 and tiated hESCs. SNUhES3 respectively. Non-PKs were additionally detected, possibly as a result of PCR-related mispriming. Validation of selected tyrosine kinases by real-time PCR Murine PKs were observed at a low frequency in both and western blotting hESC types, which appear to be derived from feeder cells. Twenty-three human PTK genes and two serine/threonine To validate the differential expression of PK genes kinases were commonly expressed in the two hESCs between normal and differentiated hESCs, further experi- (Table 2). ments were performed on several selected genes. Prior to choosing the genes for ensuing analyses, we needed to set up selection criteria because the two different hESC lines Differential expression of PK genes in hESCs of different genetic backgrounds that we analyzed To identify the candidate genes associated with the showed some variations in gene expression patterns and stemness of hESCs, expression profiles of PK genes in possibly in preferential differentiation potential, as hESCs were compared with those of RA-treated hESCs by reported previously (Abeyta et al. 2004, Skottman et al. analyzing gene frequency (Table 2). In total, 386 clones 2005). We selected the genes using the following criteria: from CHA-hES3 cells and 357 clones from RA-treated (i) The expression of genes should be commonly up- CHA-hES3 cells were assessed. Out of the 36 PKs and downregulated in both hESC lines respectively. (ii) Among commonly up- and downregulated genes, identified in CHA-hES3 cells, 15 were transcriptionally the significant P values of the genes from the gene upregulated and 19 were downregulated, compared frequency analysis on PK expression profile should be with RA-treated CHA-hES3 cells. Two PK genes were lower than 0.01 in at least one of the two hESC lines.
Load more

Recommended publications
  • Failures in Preclinical and Clinical Trials of C-Met Inhibitors: Evaluation of Pathway Activity As a Promising Selection Criterion
    www.oncotarget.com Oncotarget, 2019, Vol. 10, (No. 2), pp: 184-197 Research Paper Failures in preclinical and clinical trials of c-Met inhibitors: evaluation of pathway activity as a promising selection criterion Veronica S. Hughes1 and Dietmar W. Siemann1 1University of Florida, Department of Radiation Oncology, UF Health Cancer Center, Gainesville, FL 32608, USA Correspondence to: Dietmar W. Siemann, email: [email protected] Keywords: cancer; c-Met; small molecule inhibitor; microenvironment; HGF Received: November 01, 2018 Accepted: December 20, 2018 Published: January 04, 2019 Copyright: Hughes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT C-Met is a frequently overexpressed or amplified receptor tyrosine kinase involved in metastatic-related functions, including migration, invasion, cell survival, and angiogenesis. Because of its role in cancer progression and metastasis, many inhibitors have been developed to target this pathway. Unfortunately, most c-Met inhibitor clinical trials have failed to show significant improvement in survival of cancer patients. In these trials tumor type, protein overexpression, or gene amplification are the primary selection criteria for patient inclusion. Our data show that none of these criteria are associated with c-Met pathway activation. Hence, it is conceivable that the majority of c-Met inhibitor clinical trial failures are the consequence of a lack of appropriate patient selection. Further complicating matters, c-Met inhibitors are routinely tested in preclinical studies in the presence of high levels of exogenous Hepatocyte Growth Factor (HGF), its activating ligand.
    [Show full text]
  • Mtor Mutations in Smith-Kingsmore Syndrome: Four Additional Patients and a Review
    Received: 4 July 2017 Revised: 31 August 2017 Accepted: 5 September 2017 DOI: 10.1111/cge.13135 ORIGINAL ARTICLE mTOR mutations in Smith-Kingsmore syndrome: Four additional patients and a review G. Gordo1,2,3 | J. Tenorio1,2 | P. Arias1,2 | F. Santos-Simarro1,4 | S. García-Miñaur1,4 | J.C. Moreno1,2 | J. Nevado1,5 | E. Vallespin1,5 | L. Rodriguez-Laguna1,3 | R. de Mena1,5 | I. Dapia1,2 | M. Palomares-Bralo1,5 | A. del Pozo1,6 | K. Ibañez1,6 | J.C. Silla1,6 | E. Barroso1,2 | V.L. Ruiz-Pérez1,7 | V. Martinez-Glez1,3,4 | P. Lapunzina1,2,4 1Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain 2Molecular Endocrinology Section, Overgrowth Syndromes Laboratory, Instituto de Genética Médica y Molecular (INGEMM), IdiPAZ, Hospital Universitario la Paz, Universidad Autónoma de Madrid (UAM), Madrid, Spain 3Vascular Malformations Section, Instituto de Genética Médica y Molecular (INGEMM), IdiPAZ, Hospital Universitario la Paz, Universidad Autónoma de Madrid (UAM), Madrid, Spain 4Clinical Genetics Section, Instituto de Genética Médica y Molecular (INGEMM), IdiPAZ, Hospital Universitario la Paz, Universidad Autónoma de Madrid (UAM), Madrid, Spain 5Structural and Functional Genomics Section, Instituto de Genética Médica y Molecular (INGEMM), IdiPAZ, Hospital Universitario la Paz, Universidad Autónoma de Madrid (UAM), Madrid, Spain 6Bioinformatics Section, Instituto de Genética Médica y Molecular (INGEMM), IdiPAZ, Hospital Universitario la Paz, Universidad Autónoma de Madrid (UAM), Madrid, Spain 7IIB, Instituto de Investigación “Alberto Sols”, Universidad Autónoma de Madrid (UAM), Madrid, Spain Correspondence Smith-Kingsmore syndrome (SKS) OMIM #616638, also known as MINDS syndrome (ORPHA Pablo Lapunzina, MD, PhD, Instituto de Genética Médica y Molecular (INGEMM), 457485), is a rare autosomal dominant disorder reported so far in 23 patients.
    [Show full text]
  • Emery and Rimoin's Principles and Practice Of
    9 Disorders of the Venous System Pascal Brouillard,1 Nisha Limaye,1 Laurence M. Boon,1,2 Miikka Vikkula1,3 1Human Molecular Genetics, de Duve Institute, Université catholique de Louvain, Brussels, Belgium, 2Center for Vascular Anomalies, Division of Plastic Surgery, Cliniques Universitaires St-Luc, Université catholique de Louvain, Brussels, Belgium, 3Walloon Excellence in Lifesciences and Biotechnology (WELBIO), Université catholique de Louvain, Brussels, Belgium 9.1 INTRODUCTION hemangioma) and more slow-growing vascular malfor- mations. The latter are subcategorized according to the The vasculature is the first organ system to develop during type(s) of vessel(s) altered [5–7] into capillary, venous, embryogenesis, delivering nutrients, growth factors, and arteriovenous, lymphatic, and combined malformations. oxygen to tissues and removing wastes. It is made up of four major types of vessels: arteries, capillaries, veins, and lymphatic vessels, all of which have a single layer of endo- 9.2 THE VENOUS SYSTEM thelial cells (ECs) forming the innermost layer. In blood Veins collect CO2-rich blood from the capillary net- vessels, the endothelial tubes are supported by a layer of work and contain about 75%–80% of the total volume of vascular smooth muscle cells (vSMCs) and/or pericytes blood in the body. They have larger lumens than arteries, (together called mural cells) of variable thickness. A base- with thinner, less muscular walls. Venous flow is passive, ment membrane separates the endothelial and vSMC essentially mediated by physical movements of the body layers, with an extracellular matrix (ECM) of fibrous and and the aspirating effect exerted by the heart. The pres- elastic proteins and carbohydrate polymers forming the ence of valves ensures correct orientation of blood flow.
    [Show full text]
  • Supplementary Table 1. in Vitro Side Effect Profiling Study for LDN/OSU-0212320. Neurotransmitter Related Steroids
    Supplementary Table 1. In vitro side effect profiling study for LDN/OSU-0212320. Percent Inhibition Receptor 10 µM Neurotransmitter Related Adenosine, Non-selective 7.29% Adrenergic, Alpha 1, Non-selective 24.98% Adrenergic, Alpha 2, Non-selective 27.18% Adrenergic, Beta, Non-selective -20.94% Dopamine Transporter 8.69% Dopamine, D1 (h) 8.48% Dopamine, D2s (h) 4.06% GABA A, Agonist Site -16.15% GABA A, BDZ, alpha 1 site 12.73% GABA-B 13.60% Glutamate, AMPA Site (Ionotropic) 12.06% Glutamate, Kainate Site (Ionotropic) -1.03% Glutamate, NMDA Agonist Site (Ionotropic) 0.12% Glutamate, NMDA, Glycine (Stry-insens Site) 9.84% (Ionotropic) Glycine, Strychnine-sensitive 0.99% Histamine, H1 -5.54% Histamine, H2 16.54% Histamine, H3 4.80% Melatonin, Non-selective -5.54% Muscarinic, M1 (hr) -1.88% Muscarinic, M2 (h) 0.82% Muscarinic, Non-selective, Central 29.04% Muscarinic, Non-selective, Peripheral 0.29% Nicotinic, Neuronal (-BnTx insensitive) 7.85% Norepinephrine Transporter 2.87% Opioid, Non-selective -0.09% Opioid, Orphanin, ORL1 (h) 11.55% Serotonin Transporter -3.02% Serotonin, Non-selective 26.33% Sigma, Non-Selective 10.19% Steroids Estrogen 11.16% 1 Percent Inhibition Receptor 10 µM Testosterone (cytosolic) (h) 12.50% Ion Channels Calcium Channel, Type L (Dihydropyridine Site) 43.18% Calcium Channel, Type N 4.15% Potassium Channel, ATP-Sensitive -4.05% Potassium Channel, Ca2+ Act., VI 17.80% Potassium Channel, I(Kr) (hERG) (h) -6.44% Sodium, Site 2 -0.39% Second Messengers Nitric Oxide, NOS (Neuronal-Binding) -17.09% Prostaglandins Leukotriene,
    [Show full text]
  • The Role of C-Abl Kinase in HCC Development
    Loyola University Chicago Loyola eCommons Master's Theses Theses and Dissertations 2016 The Role of c-Abl Kinase in HCC Development Lennox Chitsike Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_theses Part of the Molecular Biology Commons Recommended Citation Chitsike, Lennox, "The Role of c-Abl Kinase in HCC Development" (2016). Master's Theses. 3259. https://ecommons.luc.edu/luc_theses/3259 This Thesis is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Master's Theses by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 2016 Lennox Chitsike LOYOLA UNIVERSITY CHICAGO THE ROLE OF C-ABL KINASE IN HCC DEVELOPMENT A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF MASTER OF SCIENCE PROGRAM IN BIOCHEMISTRY AND MOLECULAR BIOLOGY BY LENNOX CHITSIKE CHICAGO, ILLINOIS AUGUST, 2016 I dedicate this thesis to my mother ACKNOWLEDGEMENTS Firstly, I would like to express my sincere gratitude to my Ph.D advisor and mentor, Dr. Qiu for his tutelage, guidance, and support for the past year I have been in his lab. I could not have achieved half without the assistance. Secondly, I would like to express my sincere gratitude to the members of my thesis committee: Dr. Mitchell Denning and Dr. Zeleznik-Le for their insightful advice and suggestions that gave my study better perspective and new directions.
    [Show full text]
  • The Receptor Tyrosine Kinase Epha2 Promotes Mammary Adenocarcinoma Tumorigenesis and Metastatic Progression in Mice by Amplifying Erbb2 Signaling
    The receptor tyrosine kinase EphA2 promotes mammary adenocarcinoma tumorigenesis and metastatic progression in mice by amplifying ErbB2 signaling Dana M. Brantley-Sieders, … , Rebecca S. Muraoka-Cook, Jin Chen J Clin Invest. 2008;118(1):64-78. https://doi.org/10.1172/JCI33154. Research Article Oncology Overexpression of the receptor tyrosine kinase EPH receptor A2 (EphA2) is commonly observed in aggressive breast cancer and correlates with a poor prognosis. However, while EphA2 has been reported to enhance tumorigenesis, proliferation, and MAPK activation in several model systems, other studies suggest that EphA2 activation diminishes these processes and inhibits the activity of MAPK upon ligand stimulation. In this study, we eliminated EphA2 expression in 2 transgenic mouse models of mammary carcinoma. EphA2 deficiency impaired tumor initiation and metastatic progression in mice overexpressing ErbB2 (also known as Neu) in the mammary epithelium (MMTV-Neu mice), but not in mice overexpressing the polyomavirus middle T antigen in mammary epithelium (MMTV–PyV-mT mice). Histologic and ex vivo analyses of MMTV-Neu mouse mammary epithelium indicated that EphA2 enhanced tumor proliferation and motility. Biochemical analyses revealed that EphA2 formed a complex with ErbB2 in human and murine breast carcinoma cells, resulting in enhanced activation of Ras-MAPK signaling and RhoA GTPase. Additionally, MMTV-Neu, but not MMTV–PyV-mT, tumors were sensitive to therapeutic inhibition of EphA2. These data suggest that EphA2 cooperates with ErbB2 to promote tumor progression in mice and may provide a novel therapeutic target for ErbB2-dependent tumors in humans. Moreover, EphA2 function in tumor progression appeared to depend on oncogene context, an important consideration for the application of therapies targeting EphA2.
    [Show full text]
  • Dual Targeting of TAM Receptors Tyro3, Axl, and Mertk: Role in Tumors and the Tumor Immune Microenvironment Kai‑Hung Wanga, Dah‑Ching Dingb*
    [Downloaded free from http://www.tcmjmed.com on Monday, July 5, 2021, IP: 118.163.42.220] Tzu Chi Medical Journal 2021; 33(3): 250-256 Review Article Dual targeting of TAM receptors Tyro3, Axl, and MerTK: Role in tumors and the tumor immune microenvironment Kai‑Hung Wanga, Dah‑Ching Dingb* aDepartment of Medical Research, Hualien Tzu Chi Abstract Hospital, Buddhist Tzu Chi In both normal and tumor tissues, receptor tyrosine kinases (RTKs) may be pleiotropically Medical Foundation, Hualien, expressed. The RTKs not only regulate ordinary cellular processes, including proliferation, Taiwan, bDepartment of survival, adhesion, and migration, but also have a critical role in the development of Obstetrics and Gynecology, many types of cancer. The Tyro3, Axl, and MerTK (TAM) family of RTKs (Tyro3, Axl, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical and MerTK) plays a pleiotropic role in phagocytosis, inflammation, and normal cellular Foundation and Tzu Chi processes. In this article, we highlight the cellular activities of TAM receptors and discuss University, Hualien, Taiwan their roles in cancer and immune cells. We also discuss cancer therapies that target TAM receptors. Further research is needed to elucidate the function of TAM receptors in immune cells toward the development of new targeted immunotherapies for cancer. Submission : 25-May-2020 Revision : 12-Jun-2020 Keywords: Axl, MerTK, Tyro3, Axl, and MerTK receptors, Tumor immune Acceptance : 02-Jul-2020 Web Publication : 15-Oct-2020 microenvironment, Tyro3 Introduction located on chromosome 19q13.2 and was cloned in 1991 [6]. he Tyro3, Axl, and MerTK (TAM) proteins belong to The next TAM family receptor to be identified was v‑ryk, iso- T the receptor tyrosine kinase (RTK) subclass of protein lated from the avian retrovirus RLP30 [7], followed by cloning kinases.
    [Show full text]
  • Genome-Wide Screen of Gamma-Secretase–Mediated Intramembrane Cleavage of Receptor Tyrosine Kinases
    M BoC | ARTICLE Genome-wide screen of gamma-secretase– mediated intramembrane cleavage of receptor tyrosine kinases Johannes A. M. Merilahtia,b,c, Veera K. Ojalaa, Anna M. Knittlea, Arto T. Pulliainena, and Klaus Eleniusa,b,d,* aDepartment of Medical Biochemistry and Genetics, bMedicity Research Laboratory, and cTurku Doctoral Programme of Molecular Medicine, University of Turku, 20520 Turku, Finland; dDepartment of Oncology, Turku University Hospital, 20520 Turku, Finland ABSTRACT Receptor tyrosine kinases (RTKs) have been demonstrated to signal via regulated Monitoring Editor intramembrane proteolysis, in which ectodomain shedding and subsequent intramembrane Carl-Henrik Heldin cleavage by gamma-secretase leads to release of a soluble intracellular receptor fragment Ludwig Institute for Cancer Research with functional activity. For most RTKs, however, it is unknown whether they can exploit this new signaling mechanism. Here we used a system-wide screen to address the frequency of Received: Apr 27, 2017 susceptibility to gamma-secretase cleavage among human RTKs. The screen covering 45 of Revised: Aug 11, 2017 the 55 human RTKs identified 12 new as well as all nine previously published gamma-secre- Accepted: Sep 6, 2017 tase substrates. We biochemically validated the screen by demonstrating that the release of a soluble intracellular fragment from endogenous AXL was dependent on the sheddase disintegrin and metalloprotease 10 (ADAM10) and the gamma-secretase component prese- nilin-1. Functional analysis of the cleavable RTKs indicated that proliferation promoted by overexpression of the TAM family members AXL or TYRO3 depends on gamma-secretase cleavage. Taken together, these data indicate that gamma-secretase–mediated cleavage provides an additional signaling mechanism for numerous human RTKs.
    [Show full text]
  • Integrated Molecular Characterisation of the MAPK Pathways in Human
    ARTICLE https://doi.org/10.1038/s42003-020-01552-6 OPEN Integrated molecular characterisation of the MAPK pathways in human cancers reveals pharmacologically vulnerable mutations and gene dependencies 1234567890():,; ✉ Musalula Sinkala 1 , Panji Nkhoma 2, Nicola Mulder 1 & Darren Patrick Martin1 The mitogen-activated protein kinase (MAPK) pathways are crucial regulators of the cellular processes that fuel the malignant transformation of normal cells. The molecular aberrations which lead to cancer involve mutations in, and transcription variations of, various MAPK pathway genes. Here, we examine the genome sequences of 40,848 patient-derived tumours representing 101 distinct human cancers to identify cancer-associated mutations in MAPK signalling pathway genes. We show that patients with tumours that have mutations within genes of the ERK-1/2 pathway, the p38 pathways, or multiple MAPK pathway modules, tend to have worse disease outcomes than patients with tumours that have no mutations within the MAPK pathways genes. Furthermore, by integrating information extracted from various large-scale molecular datasets, we expose the relationship between the fitness of cancer cells after CRISPR mediated gene knockout of MAPK pathway genes, and their dose-responses to MAPK pathway inhibitors. Besides providing new insights into MAPK pathways, we unearth vulnerabilities in specific pathway genes that are reflected in the re sponses of cancer cells to MAPK targeting drugs: a revelation with great potential for guiding the development of innovative therapies.
    [Show full text]
  • EPH Receptor A2 Governs a Feedback Loop That Activates Wnt/Β-Catenin
    Peng et al. Cell Death and Disease (2018) 9:1146 DOI 10.1038/s41419-018-1164-y Cell Death & Disease ARTICLE Open Access EPH receptor A2 governs a feedback loop that activates Wnt/β-catenin signaling in gastric cancer Qiu Peng1,2,LingChen3,WeiWu3,JiaWang2, Xiang Zheng2,ZihuaChen3,QinJiang1,JiaqiHan1,LingyuWei2, Lujuan Wang2,JinHuang1 and Jian Ma1,2,4 Abstract The erythropoietin-producing hepatoma (EPH) receptor A2 (EphA2) belongs to the Eph family of receptor tyrosine kinases. EphA2 is highly correlated with the formation of many solid tumors and has been linked to the dysregulation of signaling pathways that promote tumor cell proliferation, migration, and invasion as well as angiogenesis. Deregulation of Wnt signaling is implicated in many forms of human disease including gastric cancer. We previously reported that EphA2 promotes the epithelial–mesenchymal transition through Wnt/β-catenin signaling in gastric cancer. Herein, we present a novel mechanism by which EphA2 regulates Wnt/β-catenin signaling. EphA2 acts as a receptor for Wnt ligands and recruits Axin1 to the plasma membrane by directly binding Dvl2. The EphA2-Dvl2/Axin1 interaction was enhanced by Wnt3a treatment, suggesting that EphA2 acts as a functional receptor for the Wnt/β- catenin pathway and plays a vital role in downstream signaling. We showed that Dvl2 mediates the EphA2-Axin1 interaction by binding to the tyrosine kinase domain of EphA2. We propose that EphA2/Dvl2/Axin1 forms a complex that destabilizes the β-catenin destruction complex and allows β-catenin to translocate to the nucleus and initiate the 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; transcription of c-MYC, the primary Wnt signaling target gene.
    [Show full text]
  • Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy*□S
    crossmark Research Author’s Choice © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy*□S Rolf Turk‡§¶ʈ**, Jordy J. Hsiao¶, Melinda M. Smits¶, Brandon H. Ng¶, Tyler C. Pospisil‡§¶ʈ**, Kayla S. Jones‡§¶ʈ**, Kevin P. Campbell‡§¶ʈ**, and Michael E. Wright¶‡‡ Mutations in genes encoding components of the sar- The muscular dystrophies are hereditary diseases charac- colemmal dystrophin-glycoprotein complex (DGC) are re- terized primarily by the progressive degeneration and weak- sponsible for a large number of muscular dystrophies. As ness of skeletal muscle. Most are caused by deficiencies in such, molecular dissection of the DGC is expected to both proteins associated with the cell membrane (i.e. the sarco- reveal pathological mechanisms, and provides a biologi- lemma in skeletal muscle), and typical features include insta- cal framework for validating new DGC components. Es- bility of the sarcolemma and consequent death of the myofi- tablishment of the molecular composition of plasma- ber (1). membrane protein complexes has been hampered by a One class of muscular dystrophies is caused by mutations lack of suitable biochemical approaches. Here we present in genes that encode components of the sarcolemmal dys- an analytical workflow based upon the principles of pro- tein correlation profiling that has enabled us to model the trophin-glycoprotein complex (DGC). In differentiated skeletal molecular composition of the DGC in mouse skeletal mus- muscle, this structure links the extracellular matrix to the cle. We also report our analysis of protein complexes in intracellular cytoskeleton.
    [Show full text]
  • PRODUCTS and SERVICES Target List
    PRODUCTS AND SERVICES Target list Kinase Products P.1-11 Kinase Products Biochemical Assays P.12 "QuickScout Screening Assist™ Kits" Kinase Protein Assay Kits P.13 "QuickScout Custom Profiling & Panel Profiling Series" Targets P.14 "QuickScout Custom Profiling Series" Preincubation Targets Cell-Based Assays P.15 NanoBRET™ TE Intracellular Kinase Cell-Based Assay Service Targets P.16 Tyrosine Kinase Ba/F3 Cell-Based Assay Service Targets P.17 Kinase HEK293 Cell-Based Assay Service ～ClariCELL™ ～ Targets P.18 Detection of Protein-Protein Interactions ～ProbeX™～ Stable Cell Lines Crystallization Services P.19 FastLane™ Structures ～Premium～ P.20-21 FastLane™ Structures ～Standard～ Kinase Products For details of products, please see "PRODUCTS AND SERVICES" on page 1～3. Tyrosine Kinases Note: Please contact us for availability or further information. Information may be changed without notice. Expression Protein Kinase Tag Carna Product Name Catalog No. Construct Sequence Accession Number Tag Location System HIS ABL(ABL1) 08-001 Full-length 2-1130 NP_005148.2 N-terminal His Insect (sf21) ABL(ABL1) BTN BTN-ABL(ABL1) 08-401-20N Full-length 2-1130 NP_005148.2 N-terminal DYKDDDDK Insect (sf21) ABL(ABL1) [E255K] HIS ABL(ABL1)[E255K] 08-094 Full-length 2-1130 NP_005148.2 N-terminal His Insect (sf21) HIS ABL(ABL1)[T315I] 08-093 Full-length 2-1130 NP_005148.2 N-terminal His Insect (sf21) ABL(ABL1) [T315I] BTN BTN-ABL(ABL1)[T315I] 08-493-20N Full-length 2-1130 NP_005148.2 N-terminal DYKDDDDK Insect (sf21) ACK(TNK2) GST ACK(TNK2) 08-196 Catalytic domain
    [Show full text]