DOCSLIB.ORG

Phosphorylation of Rabphilin-3A by Ca2+/Calmodulin- and CAMP-Dependent Protein Kinases in Vitro

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Phosphorylation of Rabphilin-3A by Ca2+/Calmodulin- and CAMP-Dependent Protein Kinases in Vitro The Journal of Neuroscience, March 1995, 15(3): 2385-2395 Phosphorylation of Rabphilin-3A by Ca2+/Calmodulin- and CAMP-Dependent Protein Kinases in vitro Else Marie Fykse,” Cai Li, and Thomas C. Siidhof Department of Molecular Genetics and Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, Texas 75235 Regulation of neurotransmitter release is thought to in- dependent protein kinase II (a-CaMK II) exhibit dramatic volve modulation of the release probability by protein changesin short-term synaptic plasticity even in the heterozy- phosphorylation. In order to identify novel targets for such gous state, which presumably only lowers the levels of enzyme regulatory processes, we have studied the phosphorylation by half (Silva et al., 1992; A. Silva, personalcommunication). of rabphilin9A in vitro. Rabphilin-3A is a synaptic vesicle LTP in mossy fiber synapses,a known presynapticform of syn- protein that interacts with rab3A in a GTP-dependent man- aptic plasticity (Zalutsky and Nicoll, 1990), is related to CAMP- ner and binds Ca*+ in a phospholipid-dependent manner. dependentprotein kinase (PKA) activation (Frey et al., 1993). Here we show that rabphilin-3A is an efficient substrate for Pharmacologicalexperiments using a variety of protein kinase Ca*+/calmodulin-dependent protein kinase II, which phos- inhibitors and stimulatorsalso suggesta role for protein kinases phorylates rat rabphilin-3A at residue 234 and 274, and for in synaptic plasticity (Zhuo et al., 1994; reviewed in Stevens, CAMP-dependent protein kinase, which phosphorylates rat 1993). Thus, there is significant evidence for a major role of rabphilin-3A at residue 234. This identifies the middle re- protein phosphorylation events in at least some forms of regu- gion of rabphilin4A situated between the N-terminal rab3A- lation of neurotransmitterrelease. binding sequences and the C-terminal &-domains involved At least two possiblemechanisms can be envisionedby which in Ca2+/phospholipid binding as a regulatory domain. Thus, protein phosphorylation could regulate neurotransmitterrelease. rabphilin9A is a second phosphoprotein on synaptic ves- First, protein kinasescould phosphorylate ion channelsand their icles that, similar to synapsin I, may integrate phosphory- associatedproteins. As a regulatory endpoint, such phosphory- lation signals from multiple protein kinase signaling path- lation events could lead to an increaseor decreasein the intra- ways in the cell. cellular Ca2+levels achieved after an action potential and there- [Key words: synaptic vesicles, synaptic plasticity, exo- by regulate release(for a review, see Nicoll et al., 1990). Sec- cytosis, protein kinases, Ca2+, rab proteins, phosphoryla- ond, protein kinasescould phosphorylate proteins involved in tion] the synaptic vesicle traffic. Thereby, they could modulate the efficiency of synaptic vesicle trafficking as a function of Ca2+. In nerve terminals, neurotransmittersare stored in synaptic ves- Although there is evidence in support of both mechanisms(Tak- icles and secretedby synaptic vesicle exocytosis. Neurotrans- euchi and Takeuchi, 1966; Man-Son-Hing et al., 1989; Dale and mitter secretionis acutely regulated by Ca2+,which triggers exo- Kandel, 1990; Scholz and Miller, 1992), the molecularbasis for cytosis in a rapid (<l msec) and cooperative manner.On a lon- these processesis largely obscure. In particular, for the synaptic ger time scale,neurotransmitter release is modulatedby a variety releaseapparatus, the mechanismsunderlying the modulationof of regulatory processes,such as occur during the different forms releaseand the relevant phosphorylation targets are mostly un- of short-term synaptic plasticity (Zucker, 1989), modulation by known. presynaptic receptors (Takeuchi and Takeuchi, 1966; Silinsky, 1984; Scholz and Miller, 1992; Weisskopf et al., 1993), and pos- Several protein kinase substratesin the nerve terminal have sibly long-term potentiation (LTP) and long-term depression been extensively studied. The synapsinsconstitute a family of (LTD) (Bekkers and Stevens, 1990; Malinow and Tsien, 1990; phosphoproteinsthat are associatedwith synaptic vesicles (De Bolshakov and Siegelbaum,1994). Theseregulatory phenomena Camilli et al., 1983a,b; Stidhof et al., 1989) and are substrates are thought to involve covalent modificationsof proteins, in par- for at least four different protein kinases,CaMK I and II, PKA, ticular, by protein phosphorylation. In support of this notion, and proline-directed kinase (reviewed in Jahn and Stidhof, mice carrying a mutant form of the gene for a-Caz+/calmodulin- 1994). The abundanceand regulatedphosphorylation of the syn- apsinssuggest that they function in regulating neurotransmitter release, but their exact functions are unknown except for the Received July 15, 1994; revised Oct. 3, 1994; accepted Oct. 5, 1994. C-terminal phosphorylation site for CaMK II in synapsin I, We thank I. Leznicki, A. Roth, and E. Borowicz for excellent technical assistance, Dr. H. Schulman (Stanford University), Dr. M. Kennedy (California which appearsto act as a negative regulator during paired-pulse Institute of Technology), and Dr. R. Jahn (Yale University) for supplying us facilitation (Rosahlet al., 1993). GAP-43 (also called B-50) rep- with purified enzymes and with antibodies; and Drs. H. McMahon and B. Davletov for advice. resentsan abundantphosphoprotein of the nerve terminal plas- Correspondence should be addressed to Thomas C. Stidhof at the above mamembrane,with a possiblerole in exocytosis but no currently address. known phosphorylation-dependent function (Dekker et al., “Present address: Norwegian Defence Research Establishment, Division of Environmental Toxicology, P.B. 25, N-2007 Kjeller, Norway. 1989). Dynamin I is a synaptic phosphoproteinwith the inter- Copyright 0 1995 Society for Neuroscience 0270-6474/95/152385-l 1$05,00/O esting property of quantitative dephosphorylationduring nerve 2386 Fykse et al. l Phosphotylation of Rabphilin-3A Anti body MS Anti-Rabphilin Cytosol + - + + + + Activator Ca - - cAMPcGMP Ca . .:i ./ ,...;..;. - 85 kDa Figure 1. Phosphorylation of rabphilin-3A by CAMP- and Ca*+-dependent protein kinases. Immunoprecipitates of rat brain proteins with preim- mune serum (PZS, lej? lane) or with polyclonal antibodies to rabphilin3A (Anti-Rabphilin) were incubated with or without cytosol as a source of kinases and with Ca*+, CAMP, and cGMP as activators. Samples were analyzed by SDS-PAGE, blotted onto nitrocellulose membranes, and exposed to film for 3 hr at -70°C with a screen. Antibody PIS Anti-Rabphilin Kinase CaMKiI - pm CaMKII Activator - Ca - - - Ca - 85 kDa - 85 kDa I - IgG Figure 2. Phosphorylation of rabphilin-3A by the catalytic subunit of CAMP-dependent protein kinase (PKA) and by Ca*+, calmodulin-dependent protein kinase II (C&X II). Rabphilin and control immunoprecipitates (see legend to Fig. 1) were incubated with the indicated protein kinases as described in Experimental Procedures. Samples were subjected to SDS-PAGE, blotted onto nitrocellulose membranes, and analyzed by autoradiog- raphy (iop panel; exposure: 5 hr at -70°C with screen) and by immunoblotting with rabphilin antibodies (bottom panel) to control for the amount of rabphilin-3A immunoprecipitated. The additional phosphoprotein bands observed in the samples incubated with CaMK II and Ca2+ are probably due to autophosphorylated CaMK II (see also Fig. 7). The Journal of Neuroscience, March 1995, f5(3) 2387 PP Rabphilin-3A NH2 COOH domain structure rab3A binding C2domains Residue Numbers GST 0 GST-RaphA 1-181 B 67-226 C 199-361 D 250-361 E 380-684 Figure 3. Schematic overview of the bacterial glutathione S-transferase (GS7’) fusion proteins used to map the F 380-531 phosphorylation sites in rabphilin3A. The diagram olt top depicts the domain structure of rabphilin-3A. The_ I__-_stmc- G 524-684 tures of GST ana of 10 GST-rabphilin PP fusion proteins used in the current study are shown in the middle, and the H 1-361 legend is shown on the bottom. The lo- cations of the phosphorylation sites 1-361 that were identified in the current study I are indicated by “P.” GST-RaphH contains the entire N-terminal half of J 1-361 rabphilin-3A. Serine-to-alanine muta- tions were introduced into serine234 (the putative PK.4 phosphorylation site) in GST-RaphI, and into serine234 and ser- Legend: ine274 (the putative CaMK II phosphor- ylation site) in GST-RaphJ. A mutant Point mutation * in which only the putative CaMK II Fhosphoryiafion site P phosphorylation site (serine*“) was 400 amino acids gggg substituted was also made but the ex- GST pressed protein was unstable in bacteria N-terrnlnal domain / and, therefore, not analyzed. However. First C2 domain m the corresponding mutant was also ex- pressed as a holo-protein in COS cells Second C2 domain (see below). terminal depolarization that probably plays a role in triggering (Frey et al., 1993; Chavez-Noriega and Stevens, 1994). Again, synaptic vesicle endocytosis (Robinson et al., 1993). the only currently known synaptic PKA substrates are synapsins These and other synaptic phosphoproteins (e.g., AP180, syn- I and II, which do not appear to account for all of the regulatory aptotagmin, and others) are candidates for mediating some reg- phenomena observed with PKA. Therefore, it would be of great ulatory functions in the synaptic vesicle cycle. However, the interest to define additional protein kinase substrates in the nerve currently known protein kinase targets at the synapse are clearly terminal
Load more

Recommended publications
  • Rab3a As a Modulator of Homeostatic Synaptic Plasticity
    Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2014 Rab3A as a Modulator of Homeostatic Synaptic Plasticity Andrew G. Koesters Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Biomedical Engineering and Bioengineering Commons Repository Citation Koesters, Andrew G., "Rab3A as a Modulator of Homeostatic Synaptic Plasticity" (2014). Browse all Theses and Dissertations. 1246. https://corescholar.libraries.wright.edu/etd_all/1246 This Dissertation is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. RAB3A AS A MODULATOR OF HOMEOSTATIC SYNAPTIC PLASTICITY A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy By ANDREW G. KOESTERS B.A., Miami University, 2004 2014 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL August 22, 2014 I HEREBY RECOMMEND THAT THE DISSERTATION PREPARED UNDER MY SUPERVISION BY Andrew G. Koesters ENTITLED Rab3A as a Modulator of Homeostatic Synaptic Plasticity BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy. Kathrin Engisch, Ph.D. Dissertation Director Mill W. Miller, Ph.D. Director, Biomedical Sciences Ph.D. Program Committee on Final Examination Robert E. W. Fyffe, Ph.D. Vice President for Research Dean of the Graduate School Mark Rich, M.D./Ph.D. David Ladle, Ph.D. F. Javier Alvarez-Leefmans, M.D./Ph.D. Lynn Hartzler, Ph.D.
    [Show full text]
  • Glioma Cell Secretion: a Driver of Tumor Progression and a Potential Therapeutic Target Damian A
    Published OnlineFirst October 17, 2018; DOI: 10.1158/0008-5472.CAN-18-0345 Cancer Review Research Glioma Cell Secretion: A Driver of Tumor Progression and a Potential Therapeutic Target Damian A. Almiron Bonnin1,2, Matthew C. Havrda1,2, and Mark A. Israel1,2,3 Abstract Cellular secretion is an important mediator of cancer progres- ple oncogenic pathologies. In this review, we describe tumor cell sion. Secreted molecules in glioma are key components of secretion in high-grade glioma and highlight potential novel complex autocrine and paracrine pathways that mediate multi- therapeutic opportunities. Cancer Res; 78(21); 6031–9. Ó2018 AACR. Introduction Glioma-Secreted Molecules Impact Disease Glial cells in the central nervous system (CNS) provide trophic Progression support for neurons (1). In glial tumors, this trophic support is Glioma cells modify their microenvironment by introducing dysregulated creating a pro-oncogenic microenvironment medi- diverse molecules into the extracellular space (Table 1). To exem- ated by a heterogeneous array of molecules secreted into the plify the pro-oncogenic role that secreted molecules can have on – extracellular space (2 15). The glioma secretome includes pro- glioma pathology, we review the functional impact of specific teins, nucleic acids, and metabolites that are often overexpressed cytokines, metabolites, and nucleic acids on glioma biology. By in malignant tissue and contribute to virtually every aspect of describing some of the potent antitumorigenic effects observed in – cancer pathology (Table 1; Fig. 1; refs. 2 15), providing a strong preclinical therapeutic studies targeting tumor cell secretion, we – rationale to target the cancer cell secretory mechanisms. also highlight how blocking secreted molecules might be of fi Although the speci c mechanisms regulating secretion in therapeutic impact in gliomas, as well as other tumors.
    [Show full text]
  • Ubiquitylome Profiling of Parkin-Null Brain Reveals Dysregulation Of
    Neurobiology of Disease 127 (2019) 114–130 Contents lists available at ScienceDirect Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi Ubiquitylome profiling of Parkin-null brain reveals dysregulation of calcium T homeostasis factors ATP1A2, Hippocalcin and GNA11, reflected by altered firing of noradrenergic neurons Key J.a,1, Mueller A.K.b,1, Gispert S.a, Matschke L.b, Wittig I.c, Corti O.d,e,f,g, Münch C.h, ⁎ ⁎ Decher N.b, , Auburger G.a, a Exp. Neurology, Goethe University Medical School, 60590 Frankfurt am Main, Germany b Institute for Physiology and Pathophysiology, Vegetative Physiology and Marburg Center for Mind, Brain and Behavior - MCMBB; Clinic for Neurology, Philipps-University Marburg, 35037 Marburg, Germany c Functional Proteomics, SFB 815 Core Unit, Goethe University Medical School, 60590 Frankfurt am Main, Germany d Institut du Cerveau et de la Moelle épinière, ICM, Paris, F-75013, France e Inserm, U1127, Paris, F-75013, France f CNRS, UMR 7225, Paris, F-75013, France g Sorbonne Universités, Paris, F-75013, France h Institute of Biochemistry II, Goethe University Medical School, 60590 Frankfurt am Main, Germany ARTICLE INFO ABSTRACT Keywords: Parkinson's disease (PD) is the second most frequent neurodegenerative disorder in the old population. Among Parkinson's disease its monogenic variants, a frequent cause is a mutation in the Parkin gene (Prkn). Deficient function of Parkin Mitochondria triggers ubiquitous mitochondrial dysfunction and inflammation in the brain, but it remains unclear howse- Parkin lective neural circuits become vulnerable and finally undergo atrophy. Ubiquitin We attempted to go beyond previous work, mostly done in peripheral tumor cells, which identified protein Calcium targets of Parkin activity, an ubiquitin E3 ligase.
    [Show full text]
  • Ras-Associated, Small GTP-Binding Protein Mouse, Recombinant, E. Coli
    Rab3AGST-His Ras-associated, small GTP-binding protein mouse, recombinant, E. coli Cat. No. Amount Description: N-terminal tagged Rab3A is a small GTPase that belongs to the Ras PR-115 50 µg superfamily. Rab proteins play an important role in various aspects of membrane traffic, including cargo selection, vesicle budding, vesicle motility, tethering, docking, and fusion. Rab3A, a member of For in vitro use only! the Rab small G protein family, is involved in the process of Ca2+- dependent neurotransmitter release. Rab3A activity is regulated by Shipping: shipped on dry ice a GDP/GTP exchange protein (Rab3 GEP), a Rab GDP dissociation inhibitor (Rab GDI), and a GTPaseactivating protein (Rab3 GAP). Storage Conditions: store at -80 °C The Rab3A recycling is coupled with synaptic vesicle trafficking as follows: (i) GDP-Rab3A forms an inactive complex with Rab GDI and Additional Storage Conditions: avoid freeze/thaw cycles stays in the cytosol of nerve terminals. (ii) GDPRab3A released from Shelf Life: 12 months Rab GDI is converted to GTPRab3A by Rab3 GEP. (iii) GTP-Rab3A binds effector molecules, Rabphilin-3 and Rim, localized at synaptic Molecular Weight: 54 kDa vesicles and the active zone, respectively. These complexes facilitate Accession number: NM_009001 translocation and docking of the synaptic vesicles to the active zone. The GST-Tag facilitates the protein‘s application in typical GST Purity: > 90 % (SDS-PAGE) pull-down assays. Form: liquid (Supplied in 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM Activity: MgCl2 and 2 mM beta-mercaptoethanol) 100 pmol of protein can bind > 80 pmol of GDP.
    [Show full text]
  • 140503 IPF Signatures Supplement Withfigs Thorax
    Supplementary material for Heterogeneous gene expression signatures correspond to distinct lung pathologies and biomarkers of disease severity in idiopathic pulmonary fibrosis Daryle J. DePianto1*, Sanjay Chandriani1⌘*, Alexander R. Abbas1, Guiquan Jia1, Elsa N. N’Diaye1, Patrick Caplazi1, Steven E. Kauder1, Sabyasachi Biswas1, Satyajit K. Karnik1#, Connie Ha1, Zora Modrusan1, Michael A. Matthay2, Jasleen Kukreja3, Harold R. Collard2, Jackson G. Egen1, Paul J. Wolters2§, and Joseph R. Arron1§ 1Genentech Research and Early Development, South San Francisco, CA 2Department of Medicine, University of California, San Francisco, CA 3Department of Surgery, University of California, San Francisco, CA ⌘Current address: Novartis Institutes for Biomedical Research, Emeryville, CA. #Current address: Gilead Sciences, Foster City, CA. *DJD and SC contributed equally to this manuscript §PJW and JRA co-directed this project Address correspondence to Paul J. Wolters, MD University of California, San Francisco Department of Medicine Box 0111 San Francisco, CA 94143-0111 [email protected] or Joseph R. Arron, MD, PhD Genentech, Inc. MS 231C 1 DNA Way South San Francisco, CA 94080 [email protected] 1 METHODS Human lung tissue samples Tissues were obtained at UCSF from clinical samples from IPF patients at the time of biopsy or lung transplantation. All patients were seen at UCSF and the diagnosis of IPF was established through multidisciplinary review of clinical, radiological, and pathological data according to criteria established by the consensus classification of the American Thoracic Society (ATS) and European Respiratory Society (ERS), Japanese Respiratory Society (JRS), and the Latin American Thoracic Association (ALAT) (ref. 5 in main text). Non-diseased normal lung tissues were procured from lungs not used by the Northern California Transplant Donor Network.
    [Show full text]
  • APP Anterograde Transport Requires Rab3a Gtpase Activity for Assembly of the Transport Vesicle
    14534 • The Journal of Neuroscience, November 18, 2009 • 29(46):14534–14544 Neurobiology of Disease APP Anterograde Transport Requires Rab3A GTPase Activity for Assembly of the Transport Vesicle Anita Szodorai,2 Yung-Hui Kuan,2,9 Silke Hunzelmann,1,2 Ulrike Engel,4 Ayuko Sakane,6 Takuya Sasaki,6 Yoshimi Takai,7 Joachim Kirsch,3 Ulrike Mu¨ller,5 Konrad Beyreuther,2,10 Scott Brady,8 Gerardo Morfini,8 and Stefan Kins1,2 1Technical University of Kaiserslautern, Department of Human Biology and Human Genetics, D-67663 Kaiserslautern, Germany, 2Centre for Molecular Biology, 3Department of Anatomy and Cell Biology, 4Nikon Imaging Center, Bioquant, and 5Institute for Pharmacia and Molecular Biotechnology, University of Heidelberg, D-69120 Heidelberg, Germany, 6Department of Biochemistry, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan, 7Division of Molecular and Cellular Biology and Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine/Faculty of Medicine, Kobe 650-0017, Japan, 8Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, Illinois 60612, 9Max Planck Institute for Brain Research, D-60528 Frankfurt, Germany, and 10Network Aging Research, D-69115 Heidelberg, Germany The amyloid precursor protein (APP) is anterogradely transported by conventional kinesin in a distinct transport vesicle, but both the biochem- ical composition of such a vesicle and the specific kinesin-1 motor responsible for transport are poorly defined. APP may be sequentially cleaved by ␤- and ␥-secretases leading to accumulation of ␤-amyloid (A␤) peptides in brains of Alzheimer’s disease patients, whereas cleavage of APP by ␣-secretases prevents A␤ generation.
    [Show full text]
  • Identification of Key Genes and Pathways for Alzheimer's Disease
    Biophys Rep 2019, 5(2):98–109 https://doi.org/10.1007/s41048-019-0086-2 Biophysics Reports RESEARCH ARTICLE Identification of key genes and pathways for Alzheimer’s disease via combined analysis of genome-wide expression profiling in the hippocampus Mengsi Wu1,2, Kechi Fang1, Weixiao Wang1,2, Wei Lin1,2, Liyuan Guo1,2&, Jing Wang1,2& 1 CAS Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, Beijing 100101, China 2 Department of Psychology, University of Chinese Academy of Sciences, Beijing 10049, China Received: 8 August 2018 / Accepted: 17 January 2019 / Published online: 20 April 2019 Abstract In this study, combined analysis of expression profiling in the hippocampus of 76 patients with Alz- heimer’s disease (AD) and 40 healthy controls was performed. The effects of covariates (including age, gender, postmortem interval, and batch effect) were controlled, and differentially expressed genes (DEGs) were identified using a linear mixed-effects model. To explore the biological processes, func- tional pathway enrichment and protein–protein interaction (PPI) network analyses were performed on the DEGs. The extended genes with PPI to the DEGs were obtained. Finally, the DEGs and the extended genes were ranked using the convergent functional genomics method. Eighty DEGs with q \ 0.1, including 67 downregulated and 13 upregulated genes, were identified. In the pathway enrichment analysis, the 80 DEGs were significantly enriched in one Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, GABAergic synapses, and 22 Gene Ontology terms. These genes were mainly involved in neuron, synaptic signaling and transmission, and vesicle metabolism. These processes are all linked to the pathological features of AD, demonstrating that the GABAergic system, neurons, and synaptic function might be affected in AD.
    [Show full text]
  • A Novel Rab11-Rab3a Cascade Required for Lysosome Exocytosis
    bioRxiv preprint doi: https://doi.org/10.1101/2021.03.06.434066; this version posted March 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. A novel Rab11-Rab3a cascade required for lysosome exocytosis Cristina Escrevente1,*, Liliana Bento-Lopes1*, José S Ramalho1, Duarte C Barral1,† 1 iNOVA4Health, CDOC, NOVA Medical School, NMS, Universidade NOVA de Lisboa, 1169-056 Lisboa, Portugal. * These authors contributed equally to this work. † Correspondence should be sent to: Duarte C Barral, CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Campo dos Mártires da Pátria 130, 1169-056, Lisboa, Portugal, Tel: +351 218 803 102, Fax: +351 218 803 006, [email protected]. (ORCID 0000-0001-8867-2407). Abbreviations used in this paper: FIP, Rab11-family of interacting protein; GEF, guanine nucleotide exchange factor; LE, late endosomes; LRO, lysosome-related organelle; NMIIA, non-muscle myosin heavy chain IIA; Slp-4a, synaptotagmin-like protein 4a. 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.06.434066; this version posted March 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract Lysosomes are dynamic organelles, capable of undergoing exocytosis. This process is crucial for several cellular functions, namely plasma membrane repair.
    [Show full text]
  • Supplemental Table 1A. Differential Gene Expression Profile of Adehcd40l and Adehnull Treated Cells Vs Untreated Cells
    Supplemental Table 1a. Differential Gene Expression Profile of AdEHCD40L and AdEHNull treated cells vs Untreated Cells Fold change Regulation Fold change Regulation ([AdEHCD40L] vs ([AdEHCD40L] ([AdEHNull] vs ([AdEHNull] vs Probe Set ID [Untreated]) vs [Untreated]) [Untreated]) [Untreated]) Gene Symbol Gene Title RefSeq Transcript ID NM_001039468 /// NM_001039469 /// NM_004954 /// 203942_s_at 2.02 down 1.00 down MARK2 MAP/microtubule affinity-regulating kinase 2 NM_017490 217985_s_at 2.09 down 1.00 down BAZ1A fibroblastbromodomain growth adjacent factor receptorto zinc finger 2 (bacteria-expressed domain, 1A kinase, keratinocyte NM_013448 /// NM_182648 growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer 203638_s_at 2.10 down 1.01 down FGFR2 syndrome, Jackson-Weiss syndrome) NM_000141 /// NM_022970 1570445_a_at 2.07 down 1.01 down LOC643201 hypothetical protein LOC643201 XM_001716444 /// XM_001717933 /// XM_932161 231763_at 3.05 down 1.02 down POLR3A polymerase (RNA) III (DNA directed) polypeptide A, 155kDa NM_007055 1555368_x_at 2.08 down 1.04 down ZNF479 zinc finger protein 479 NM_033273 /// XM_001714591 /// XM_001719979 241627_x_at 2.15 down 1.05 down FLJ10357 hypothetical protein FLJ10357 NM_018071 223208_at 2.17 down 1.06 down KCTD10 potassium channel tetramerisation domain containing 10 NM_031954 219923_at 2.09 down 1.07 down TRIM45 tripartite motif-containing 45 NM_025188 242772_x_at 2.03 down 1.07 down Transcribed locus 233019_at 2.19 down 1.08 down CNOT7 CCR4-NOT transcription complex, subunit 7 NM_013354
    [Show full text]
  • Supplementary Figure 1 Standardization of Gene Expression
    Supplementary Figure 1 Standardization of gene expression Notes: (A) Standardization of GSE86544, (B) standardization of GSE103479, (C) standardization of GSE102238, (D) Standardization of GSE7055. The blue bar represents the data before normalization, and the red bar represents the data after normalization. Supplementary Figure 2 Correlation between module eigengenes and clinical traits especially PNI in GSE103479 and GSE102238 datasets. Notes: (A, B) Module-trait relationships in GSE103479 and GSE102238 datasets. The correlation coefficients and corresponding P-values in the brackets are contained in each cell. The table is color- coded by correlation between eigengenes and traits according to the color legend on the right side. The modules with the most significant differences are displayed in brackets. Abbreviations: PNI, perineural invasion. Supplementary Figure 3 The expression values of CCNB2 in pancreatic cancer (GSE102238) and colon cancer (GSE103479). Notes: (A, B) CCNB2 expression values were detected in GSE102238 and GSE103479. Abbreviations: CCNB2, cyclin B2 Supplementary Table 1 Results of top 20 pathway enrichment analysis of GSE7055 Term Category Description Count Log10(P) Genes GO:0000280 GO Biological nuclear division 33 -23.4 BIRC5,BUB1B,CCNB1,CCNE1,CDC20, Processes CKS2,KIF11,MAD2L1,MYBL2,SPAST, TOP2A,TTK,PRC1,PKMYT1,PTTG1,T RIP13,DLGAP5,TACC3,SMC2,SPAG5, UBE2C,ZWINT,TPX2,FBXO5,RACGA P1,NUSAP1,SPDL1,CDCA8,CEP55,ND C1,NSFL1C,KIF18B,ASPM GO:1902850 GO Biological microtubule 15 -12.89 BIRC5,CCNB1,CDC20,KIF11,MAD2L1 Processes
    [Show full text]
  • Small Rab Gtpases in Intracellular Vesicle Trafficking:The Case Of
    International Journal of Molecular Sciences Review Small Rab GTPases in Intracellular Vesicle Trafficking: The Case of Rab3A/Raphillin-3A Complex in the Kidney Olga Martinez-Arroyo 1,† , Estela Selma-Soriano 2,†, Ana Ortega 1,* , Raquel Cortes 1,‡ and Josep Redon 1,3,*,‡ 1 Cardiometabolic and Renal Risk Research Group, INCLIVA Biomedical Research Institute, 46010 Valencia, Spain; [email protected] (O.M.-A.); [email protected] (R.C.) 2 Physiopathology of Cellular and Organic Oxidative Stress Group, University of Valencia, 46100 Valencia, Spain; [email protected] 3 CIBERObn, Carlos III Institute, 28029 Madrid, Spain * Correspondence: [email protected] (A.O.); [email protected] (J.R.); Tel.: +34-658-909-676 (A.O. & J.R.) † These authors contributed equally as first authors to this work. ‡ These authors contributed equally as senior authors to this work. Abstract: Small Rab GTPases, the largest group of small monomeric GTPases, regulate vesicle traf- ficking in cells, which are integral to many cellular processes. Their role in neurological diseases, such as cancer and inflammation have been extensively studied, but their implication in kidney disease has not been researched in depth. Rab3a and its effector Rabphillin-3A (Rph3A) expression have been demonstrated to be present in the podocytes of normal kidneys of mice rats and humans, around vesicles contained in the foot processes, and they are overexpressed in diseases with proteinuria. In addition, the Rab3A knockout mice model induced profound cytoskeletal changes in podocytes of high glucose fed animals. Likewise, RphA interference in the Drosophila model produced structural Citation: Martinez-Arroyo, O.; and functional damage in nephrocytes with reduction in filtration capacities and nephrocyte number.
    [Show full text]
  • Supporting Information
    Supporting Information Hunter et al. 10.1073/pnas.1404639111 SI Methods and no additional magnesium. SML was added to a concentra- Protein Expression and Purification. A construct encoding codon- tion of 150 μM, and GTP and GDP were added to a concentra- optimized N-terminal His-tobacco etch virus (TEV)-G12C V-Ki- tion of 1.5 mM each in the respective samples. Samples were ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras) in the incubated at 37 °C for the indicated length of time. For each time pJExpressvector(DNA2.0)wassynthesized and used to transform point, 27 μL of sample was mixed with 3 μL of 7-diethylamino- BL21(DE3) cells. Cells were grown in Luria broth (LB) to OD 600 3-(4-maleimidophenyl)-4-methylcoumarin dissolved in DMSO 0.7 and induced with 250 mM isopropyl β-D-1-thiogalactopyrano- (100 μM final concentration) in a black 384-well plate and side (IPTG) for 16 h at 16 °C. Cells were pelleted and resuspended fluorescence was read at 384/470 nm. The assay was run in in lysis buffer [20 mM sodium phosphate (pH 8.0), 500 mM NaCl, triplicate, and normalized data were fit to a one-phase decay 10 mM imidazole, 1 mM 2-mercaptoethanol (BME), 5% (vol/vol) nonlinear regression curve in GraphPad Prism. glycerol] containing PMSF, benzamidine, and 1 mg/mL lysozyme. Lysates were flash-frozen and stored at −80 °C until use. Sequence Conservation Analysis. AminoacidsequencesofRas Protein was purified over an IMAC cartridge (BioRad) fol- family proteins were obtained from the National Center for lowing standard Ni-affinity protocols and desalted into 1× crys- Biotechnology Information Protein Data Bank (PDB) and tallization buffer [20 mM Hepes (pH 8.0), 150 mM NaCl, 5 mM aligned using the multiple alignment server, Clustal-Omega (v.1.2.0).
    [Show full text]