DOCSLIB.ORG

Local Protein Kinase a Action Proceeds Through Intact Holoenzymes F

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Local Protein Kinase a Action Proceeds Through Intact Holoenzymes F RESEARCH SIGNAL TRANSDUCTION observed between mass/charge ratios of 5700 and 6300. These species preferentially contained two, but up to four, molecules of cAMP (fig. S1). When analysis was done at higher concentrations of cAMP Local protein kinase A action (5 mM), the predominant species exhibited cyclic nucleotide occupancy of 4 mol per RII dimer (fig. proceeds through intact holoenzymes S1 and table S3). Notably, the C subunit remained attached under these conditions (fig. S1). Thus, F. Donelson Smith,1 Jessica L. Esseltine,1* Patrick J. Nygren,1 David Veesler,2 we can conclude that a substantial proportion Dominic P. Byrne,3 Matthias Vonderach,3 Ilya Strashnov,4 Claire E. Eyers,3 of the PKA C subunit remained associated with 297–427 Patrick A. Eyers,3 Lorene K. Langeberg,1 John D. Scott1† the AKAP79 -RII dimer when cAMP-binding sites were occupied. Hormones can transmit signals through adenosine 3ʹ,5ʹ-monophosphate (cAMP) to For a more stringent in situ test, we monitored precise intracellular locations. The fidelity of these responses relies on the activation of the integrity of cellular AKAP-PKA complexes in localized protein kinase A (PKA) holoenzymes. Association of PKA regulatory type II (RII) response to ligand activation. AKAP79 or AKAP18g subunits with A-kinase–anchoring proteins (AKAPs) confers location, and catalytic (C) complexes were immunoprecipitated from cell subunits phosphorylate substrates. Single-particle electron microscopy demonstrated that lysates prepared after stimulation of cells with AKAP79 constrains RII-C subassemblies within 150 to 250 angstroms of its targets. the b-adrenergic agonist isoproterenol (Iso, 1 mM). Native mass spectrometry established that these macromolecular assemblies Immunoblot analysis detected equivalent amounts incorporated stoichiometric amounts of cAMP. Chemical-biology– and live cell–imaging of C subunit in samples from cells stimulated with techniques revealed that catalytically active PKA holoenzymes remained intact within the isoproterenol or vehicle control (Fig. 2, A and B, Downloaded from cytoplasm. These findings indicate that the parameters of anchored PKA holoenzyme top, lane 2). Local cAMP flux is controlled through action are much more restricted than originally anticipated. a balance of second-messenger production by adenylyl cyclases and degradation by phospho- diesterases (PDEs) (10). Cells were stimulated with lassical in vitro biochemistry and elegant with the AKAP (Fig. 1F) or in proximity to the isoproterenol in the presence of the general PDE structural studies have led to a commonly peripheral lobes of the extended PKA holoenzyme inhibitor 3-isobutyl-1-methylxanthine (IBMX), the held view that the active protein kinase A (Fig. 1G). selective PDE3 inhibitor milrinone, or the PDE4- http://science.sciencemag.org/ C (PKA) catalytic (C) subunit dissociates from An implication of our structural model is that, specific inhibitor rolipram (11, 12). The composition the regulatory subunit dimer in the pres- although local cAMP production stimulates ki- of AKAP79-PKA complexes was assessed by im- ence of excess adenosine 3′,5′-monophosphate nase activity, AKAP79:2RII:2C assemblies can munoblot. Intact complexes were detected in con- (cAMP) (1–3). Yet the utility of this rudimentary remain intact. Native mass spectrometry (MS) trol and Iso-treated samples (Fig. 2C, lanes 1 and mechanism inside cells remains unclear (4). Single- allows the measurement of molecular mass and 2). Inclusion of rolipram promoted full dissocia- particle negative-stain electron microscopy (EM) is used to determine the stoichiometry of intact tion of the C subunit (Fig. 2C, lane 3). Likewise, analysis of A-kinase–anchoring protein 79 with protein complexes in the gas phase (7–9). In the application of IBMX induced release of the C sub- PKA (AKAP79:PKA) holoenzyme complexes of presence of cAMP, which copurifies with RII, we unit from AKAP complexes (Fig. 2C, lane 5). In AKAP79, the regulatory II (RII) subunit, and the observed higher-order complexes, including the contrast, inclusion of milrinone had little effect, C subunit (AKAP79:2RII:2C) detected a range of intact 2RII:2C PKA holoenzyme and the pen- indicating that PDE3 is nonfunctional in these conformationally distinct species (Fig. 1, A to D, tameric AKAP79:2RII:2C assembly (Fig. 1H, fig. S1, AKAP79-PKA microdomains (Fig. 2C, lane 4). Ki- on June 25, 2017 and fig. S1). A set of ~14,000 particles was sub- and tables S1 and S2). From these experiments, nase activation was monitored by immunoblot jected to reference-free two-dimensional (2D) clas- we infer a minimal disruption of the AKAP-PKA detection of phospho-PKA substrates (Fig. 2C, sification using RELION (5). Poorly aligned classes architecture even in the presence of cAMP. bottom panel). Similar findings were obtained were discarded over multiple iterations to reveal To address the relative stability of the pentame- upon analysis of AKAP18g complexes and when an ensemble of three-lobed assemblies in which ric AKAP79:2RII:2C assembly in the presence of prostaglandin E1 (PGE1) or epinephrine was used the peripheral densities are observed at different elevated cAMP, we performed pull-down experi- as an agonist (fig. S2). Treatment with rolipram distances from each other. These range from 150 Å ments with a fragment of the anchoring protein alonedidnotactivatePKAorreleaseCsubunits in the compact configuration to 250 Å in the ex- AKAP79297–427. Retention of anchored C subunits (Fig.2C,lane6).Thus,nativeproductionofcAMP tended conformation (Fig. 1E). The tandem pe- in the presence of increasing concentrations of in response to physiological effectors of GPCR ripheral lobes represent the C subunit in complex cAMP was assessed by quantification of Coomassie signaling appears not to promote C subunit release with the cAMP-binding domains of RII (6), whereas blue–stained protein (Fig. 1I and inset). At physi- from anchored PKA holoenzymes. the central density represents the AKAP-RII di- ological concentrations of cAMP (1 to 2 mM) most Förster resonance energy transfer (FRET) was mer interface (Fig. 1E). Thus, AKAP79 constrains of the C subunit (~70 to 80%) remained associated used to investigate PKA holoenzyme composition each subassembly of regulatory and C subunits with the AKAP79297–427-RII complex (Fig. 1I and in real time (13). Initially, we used RII conjugated to within 250 Å of substrates. We surmise that inset). Substantial release of the C subunit was to cyan fluorescent protein (RII-CFP) and the C the restricted movement of the anchored C sub- only evident at supraphysiological levels of cAMP subunit conjugated to yellow fluorescent pro- unit in this configuration augments phosphoryl- (Fig. 1I; 10 to 90 mM), and no change in binding tein (C-YFP) as intermolecular FRET probes to ation of local substrates that are either interacting of RII to AKAP79297–427 was observed. In control monitor the integrity of the PKA holoenzyme af- experiments, 5′-AMP (100 mM), a degradation ter agonist stimulation of cells (Fig. 2D). Isopro- product of cAMP, did not alter anchored holo- terenol promoted minimal change in the CFP/ 1Howard Hughes Medical Institute, Department of enzyme composition (Fig. 1I). Thus, physiological YFP FRET ratio over time courses of 400 s (Fig. Pharmacology, University of Washington, Seattle, WA 98195, amounts of cAMP promote minimal release of 2, E and H, black), consistent with negligible USA. 2Department of Biochemistry, University of Washington, Seattle, WA 98195, USA. 3Department of Biochemistry, the C subunit from the anchored holoenzyme dissociation of the PKA holoenzyme. In contrast, Institute of Integrative Biology, University of Liverpool, in vitro. preincubation of cells with rolipram before stim- Liverpool L69 7ZB, UK. 4School of Chemistry, The University Additional studies used high-resolution native ulation with b-adrenergic agonist triggered a of Manchester, Manchester M13 9PL, UK. MS to evaluate cAMP occupancy in PKA holo- pronounced and time-dependent reduction in *Present address: Schulich School of Medicine and Dentistry, Western University, London, Ontario N6A 5C1, Canada. enzyme complexes. At basal cAMP concentrations, the FRET ratio (Fig. 2, F and H, red). Thus, dis- †Corresponding author. Email: [email protected] multiple charge states of 2RII:C subcomplexes were sociation of the PKA holoenzyme only occurred Smith et al., Science 356, 1288–1293 (2017) 23 June 2017 1of6 RESEARCH | REPORT if cAMP accumulated to supraphysiological con- lize cAMP signaling promote very little dissocia- binding protein domain–tagged RII dimers (RII- centrations. Pretreatment of cells with the PDE3 tion of native PKA holoenzymes inside cells. FKBP) in the context of the AKAP18 complex. In inhibitor milrinone had little effect on the FRET To test whether covalent coupling of RII to the this system, the small molecule rapamycin bridges response(Fig.2,GandH,gray).ComparableFRET C subunit would alter PKA action inside cells, we the tagged proteins to maintain a stable complex recordings were obtained when PGE1 was used generated a construct that encodes RIIa and Ca (Fig.3D).ImmunoblotanalysesofAKAP18im- as the agonist (fig. S2). in one polypeptide, creating a nondissociable PKA mune complexes from cells expressing RII-FKBP The ICUE3 FRET biosensor detects agonist- fusion enzyme designated “R2C2” (Fig. 3A). To and C-FRB revealed that both proteins were asso- responsive accumulation of cAMP (Fig. 2I) (14). exploit this tool in a simplified genetic background, ciated after
Load more

Recommended publications
  • Gpr161 Anchoring of PKA Consolidates GPCR and Camp Signaling
    Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling Verena A. Bachmanna,1, Johanna E. Mayrhofera,1, Ronit Ilouzb, Philipp Tschaiknerc, Philipp Raffeinera, Ruth Röcka, Mathieu Courcellesd,e, Federico Apeltf, Tsan-Wen Lub,g, George S. Baillieh, Pierre Thibaultd,i, Pia Aanstadc, Ulrich Stelzlf,j, Susan S. Taylorb,g,2, and Eduard Stefana,2 aInstitute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, 6020 Innsbruck, Austria; bDepartment of Chemistry and Biochemistry, University of California, San Diego, CA 92093; cInstitute of Molecular Biology, University of Innsbruck, 6020 Innsbruck, Austria; dInstitute for Research in Immunology and Cancer, Université de Montréal, Montreal, QC, Canada H3C 3J7; eDépartement de Biochimie, Université de Montréal, Montreal, QC, Canada H3C 3J7; fOtto-Warburg Laboratory, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany; gDepartment of Pharmacology, University of California, San Diego, CA 92093; hInstitute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, G12 8QQ, United Kingdom; iDepartment of Chemistry, Université de Montréal, Montreal, QC, Canada H3C 3J7; and jInstitute of Pharmaceutical Sciences, Pharmaceutical Chemistry, University of Graz, 8010 Graz, Austria Contributed by Susan S. Taylor, May 24, 2016 (sent for review February 18, 2016; reviewed by John J. G. Tesmer and Mark von Zastrow) Scaffolding proteins organize the information flow from activated G accounts for nanomolar binding affinities to PKA R subunit dimers protein-coupled receptors (GPCRs) to intracellular effector cascades (12, 13). Moreover, additional components of the cAMP signaling both spatially and temporally. By this means, signaling scaffolds, such machinery, such as GPCRs, adenylyl cyclases, and phosphodiester- as A-kinase anchoring proteins (AKAPs), compartmentalize kinase ases, physically interact with AKAPs (1, 5, 11, 14).
    [Show full text]
  • 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]
  • Identification and Characterization of RHOA-Interacting Proteins in Bovine Spermatozoa1
    BIOLOGY OF REPRODUCTION 78, 184–192 (2008) Published online before print 10 October 2007. DOI 10.1095/biolreprod.107.062943 Identification and Characterization of RHOA-Interacting Proteins in Bovine Spermatozoa1 Sarah E. Fiedler, Malini Bajpai, and Daniel W. Carr2 Department of Medicine, Oregon Health & Sciences University and Veterans Affairs Medical Center, Portland, Oregon 97239 ABSTRACT Guanine nucleotide exchange factors (GEFs) catalyze the GDP for GTP exchange [2]. Activation is negatively regulated by In somatic cells, RHOA mediates actin dynamics through a both guanine nucleotide dissociation inhibitors (RHO GDIs) GNA13-mediated signaling cascade involving RHO kinase and GTPase-activating proteins (GAPs) [1, 2]. Endogenous (ROCK), LIM kinase (LIMK), and cofilin. RHOA can be RHO can be inactivated via C3 exoenzyme ADP-ribosylation, negatively regulated by protein kinase A (PRKA), and it and studies have demonstrated RHO involvement in actin-based interacts with members of the A-kinase anchoring (AKAP) cytoskeletal response to extracellular signals, including lyso- family via intermediary proteins. In spermatozoa, actin poly- merization precedes the acrosome reaction, which is necessary phosphatidic acid (LPA) [2–4]. LPA is known to signal through for normal fertility. The present study was undertaken to G-protein-coupled receptors (GPCRs) [4, 5]; specifically, LPA- determine whether the GNA13-mediated RHOA signaling activated GNA13 (formerly Ga13) promotes RHO activation pathway may be involved in acrosome reaction in bovine through GEFs [4, 6]. Activated RHO-GTP then signals RHO caudal sperm, and whether AKAPs may be involved in its kinase (ROCK), resulting in the phosphorylation and activation targeting and regulation. GNA13, RHOA, ROCK2, LIMK2, and of LIM-kinase (LIMK), which in turn phosphorylates and cofilin were all detected by Western blot in bovine caudal inactivates cofilin, an actin depolymerizer, the end result being sperm.
    [Show full text]
  • A SARS-Cov-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing
    A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing Supplementary Information Supplementary Discussion All SARS-CoV-2 protein and gene functions described in the subnetwork appendices, including the text below and the text found in the individual bait subnetworks, are based on the functions of homologous genes from other coronavirus species. These are mainly from SARS-CoV and MERS-CoV, but when available and applicable other related viruses were used to provide insight into function. The SARS-CoV-2 proteins and genes listed here were designed and researched based on the gene alignments provided by Chan et. al. 1 2020 .​ Though we are reasonably sure the genes here are well annotated, we want to note that not every ​ protein has been verified to be expressed or functional during SARS-CoV-2 infections, either in vitro or in vivo. ​ ​ In an effort to be as comprehensive and transparent as possible, we are reporting the sub-networks of these functionally unverified proteins along with the other SARS-CoV-2 proteins. In such cases, we have made notes within the text below, and on the corresponding subnetwork figures, and would advise that more caution be taken when examining these proteins and their molecular interactions. Due to practical limits in our sample preparation and data collection process, we were unable to generate data for proteins corresponding to Nsp3, Orf7b, and Nsp16. Therefore these three genes have been left out of the following literature review of the SARS-CoV-2 proteins and the protein-protein interactions (PPIs) identified in this study.
    [Show full text]
  • (P -Value<0.05, Fold Change≥1.4), 4 Vs. 0 Gy Irradiation
    Table S1: Significant differentially expressed genes (P -Value<0.05, Fold Change≥1.4), 4 vs. 0 Gy irradiation Genbank Fold Change P -Value Gene Symbol Description Accession Q9F8M7_CARHY (Q9F8M7) DTDP-glucose 4,6-dehydratase (Fragment), partial (9%) 6.70 0.017399678 THC2699065 [THC2719287] 5.53 0.003379195 BC013657 BC013657 Homo sapiens cDNA clone IMAGE:4152983, partial cds. [BC013657] 5.10 0.024641735 THC2750781 Ciliary dynein heavy chain 5 (Axonemal beta dynein heavy chain 5) (HL1). 4.07 0.04353262 DNAH5 [Source:Uniprot/SWISSPROT;Acc:Q8TE73] [ENST00000382416] 3.81 0.002855909 NM_145263 SPATA18 Homo sapiens spermatogenesis associated 18 homolog (rat) (SPATA18), mRNA [NM_145263] AA418814 zw01a02.s1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:767978 3', 3.69 0.03203913 AA418814 AA418814 mRNA sequence [AA418814] AL356953 leucine-rich repeat-containing G protein-coupled receptor 6 {Homo sapiens} (exp=0; 3.63 0.0277936 THC2705989 wgp=1; cg=0), partial (4%) [THC2752981] AA484677 ne64a07.s1 NCI_CGAP_Alv1 Homo sapiens cDNA clone IMAGE:909012, mRNA 3.63 0.027098073 AA484677 AA484677 sequence [AA484677] oe06h09.s1 NCI_CGAP_Ov2 Homo sapiens cDNA clone IMAGE:1385153, mRNA sequence 3.48 0.04468495 AA837799 AA837799 [AA837799] Homo sapiens hypothetical protein LOC340109, mRNA (cDNA clone IMAGE:5578073), partial 3.27 0.031178378 BC039509 LOC643401 cds. [BC039509] Homo sapiens Fas (TNF receptor superfamily, member 6) (FAS), transcript variant 1, mRNA 3.24 0.022156298 NM_000043 FAS [NM_000043] 3.20 0.021043295 A_32_P125056 BF803942 CM2-CI0135-021100-477-g08 CI0135 Homo sapiens cDNA, mRNA sequence 3.04 0.043389246 BF803942 BF803942 [BF803942] 3.03 0.002430239 NM_015920 RPS27L Homo sapiens ribosomal protein S27-like (RPS27L), mRNA [NM_015920] Homo sapiens tumor necrosis factor receptor superfamily, member 10c, decoy without an 2.98 0.021202829 NM_003841 TNFRSF10C intracellular domain (TNFRSF10C), mRNA [NM_003841] 2.97 0.03243901 AB002384 C6orf32 Homo sapiens mRNA for KIAA0386 gene, partial cds.
    [Show full text]
  • S41467-019-10037-Y.Pdf
    ARTICLE https://doi.org/10.1038/s41467-019-10037-y OPEN Feedback inhibition of cAMP effector signaling by a chaperone-assisted ubiquitin system Laura Rinaldi1, Rossella Delle Donne1, Bruno Catalanotti 2, Omar Torres-Quesada3, Florian Enzler3, Federica Moraca 4, Robert Nisticò5, Francesco Chiuso1, Sonia Piccinin5, Verena Bachmann3, Herbert H Lindner6, Corrado Garbi1, Antonella Scorziello7, Nicola Antonino Russo8, Matthis Synofzik9, Ulrich Stelzl 10, Lucio Annunziato11, Eduard Stefan 3 & Antonio Feliciello1 1234567890():,; Activation of G-protein coupled receptors elevates cAMP levels promoting dissociation of protein kinase A (PKA) holoenzymes and release of catalytic subunits (PKAc). This results in PKAc-mediated phosphorylation of compartmentalized substrates that control central aspects of cell physiology. The mechanism of PKAc activation and signaling have been largely characterized. However, the modes of PKAc inactivation by regulated proteolysis were unknown. Here, we identify a regulatory mechanism that precisely tunes PKAc stability and downstream signaling. Following agonist stimulation, the recruitment of the chaperone- bound E3 ligase CHIP promotes ubiquitylation and proteolysis of PKAc, thus attenuating cAMP signaling. Genetic inactivation of CHIP or pharmacological inhibition of HSP70 enhances PKAc signaling and sustains hippocampal long-term potentiation. Interestingly, primary fibroblasts from autosomal recessive spinocerebellar ataxia 16 (SCAR16) patients carrying germline inactivating mutations of CHIP show a dramatic dysregulation of PKA signaling. This suggests the existence of a negative feedback mechanism for restricting hormonally controlled PKA activities. 1 Department of Molecular Medicine and Medical Biotechnologies, University Federico II, 80131 Naples, Italy. 2 Department of Pharmacy, University Federico II, 80131 Naples, Italy. 3 Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.
    [Show full text]
  • Whole Transcriptomic Expression Differences in EBV Immortalized Versus Primary B-Cells
    W&M ScholarWorks Undergraduate Honors Theses Theses, Dissertations, & Master Projects 12-2010 Whole Transcriptomic Expression Differences in EBV Immortalized versus Primary B-Cells Dolores Huberts College of William and Mary Follow this and additional works at: https://scholarworks.wm.edu/honorstheses Part of the Biology Commons Recommended Citation Huberts, Dolores, "Whole Transcriptomic Expression Differences in EBV Immortalized versus Primary B- Cells" (2010). Undergraduate Honors Theses. Paper 347. https://scholarworks.wm.edu/honorstheses/347 This Honors Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Undergraduate Honors Theses by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. Whole Transcriptomic Expression Differences in EBV Immortalized versus Primary B-Cells A thesis submitted in partial fulfillment of the requirement for the degree of Bachelor of Science with Honors in Biology from the College of William and Mary in Virginia By Dolores Huberts Accepted for Honors ________________________________________ Lizabeth A. Allison, Director ________________________________________ Matthew Wawersik ________________________________________ Drew LaMar ________________________________________ Beverly Sher Williamsburg, Virginia December 17, 2010 ABSTRACT The Epstein–Barr Virus (EBV) is a human gamma herpes virus that infects more than 90% of the human population worldwide. It is commonly known in the US as the cause of Infectious Mononucleosis, and around the world as the cause of nasopharyngeal carcinoma and malignant lymphomas such as non-Hodgkin lymphoma, endemic Burkett’s lymphoma and Hodgkin lymphoma. Additionally, the EBV is used to immortalize cells to create cell lines for in-vitro studies.
    [Show full text]
  • A Cytoplasmic COMPASS Is Necessary for Cell Survival and Triple-Negative Breast Cancer Pathogenesis by Regulating Metabolism
    Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press A cytoplasmic COMPASS is necessary for cell survival and triple-negative breast cancer pathogenesis by regulating metabolism Lu Wang,1 Clayton K. Collings,1 Zibo Zhao,1 Kira Alia Cozzolino,1,2 Quanhong Ma,3 Kaiwei Liang,1 Stacy A. Marshall,1 Christie C. Sze,1 Rintaro Hashizume,2 Jeffrey Nicholas Savas,2 and Ali Shilatifard1,4 1Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA; 2Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA; 3Department of Neurosurgery, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA; 4Robert H. Lurie National Cancer Institute Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA Mutations and translocations within the COMPASS (complex of proteins associated with Set1) family of histone lysine methyltransferases are associated with a large number of human diseases, including cancer. Here we report that SET1B/COMPASS, which is essential for cell survival, surprisingly has a cytoplasmic variant. SET1B, but not its SET domain, is critical for maintaining cell viability, indicating a novel catalytic-independent role of SET1B/ COMPASS. Loss of SET1B or its unique cytoplasmic-interacting protein, BOD1, leads to up-regulation of expression of numerous genes modulating fatty acid metabolism, including ADIPOR1 (adiponectin receptor 1), COX7C, SDC4, and COQ7. Our detailed molecular studies identify ADIPOR1 signaling, which is inactivated in both obesity and human cancers, as a key target of SET1B/COMPASS. Collectively, our study reveals a cytoplasmic function for a member of the COMPASS family, which could be harnessed for therapeutic regulation of signaling in human dis- eases, including cancer.
    [Show full text]
  • Anti-PRKAR2A / PKR2 Antibody [Hs-36] (ARG42439)
    Product datasheet [email protected] ARG42439 Package: 100 μg anti-PRKAR2A / PKR2 antibody [Hs-36] Store at: -20°C Summary Product Description Mouse Monoclonal antibody [Hs-36] recognizes PRKAR2A / PKR2 Tested Reactivity Hu Tested Application ICC/IF, WB Specificity The antibody Hs-36 reacts with PRKAR2A (protein kinase A regulatory type II alpha subunit), an intra- acrosomal protein. Host Mouse Clonality Monoclonal Clone Hs-36 Isotype IgM Target Name PRKAR2A / PKR2 Species Human Immunogen Freshly ejaculated Human sperms were washed in PBS and extracted in 3% acetic acid, 10% glycerol, 30 mM benzaminidine. The acid extract was dialyzed against 0.2% acetic acid and subsequently used for immunization. Conjugation Un-conjugated Alternate Names cAMP-dependent protein kinase type II-alpha regulatory subunit; PRKAR2; PKR2 Application Instructions Application table Application Dilution ICC/IF 10 µg/ml WB Assay-dependent Application Note * The dilutions indicate recommended starting dilutions and the optimal dilutions or concentrations should be determined by the scientist. Properties Form Liquid Purification Precipitation and chromatography. Buffer TBS and 15 mM Sodium azide. Preservative 15 mM Sodium azide Concentration 1 mg/ml Storage instruction For continuous use, store undiluted antibody at 2-8°C for up to a week. For long-term storage, aliquot and store at -20°C or below. Storage in frost free freezers is not recommended. Avoid repeated freeze/thaw cycles. Suggest spin the vial prior to opening. The antibody solution should be gently mixed www.arigobio.com 1/2 before use. Note For laboratory research only, not for drug, diagnostic or other use. Bioinformation Gene Symbol PRKAR2A Gene Full Name protein kinase, cAMP-dependent, regulatory, type II, alpha Background cAMP is a signaling molecule important for a variety of cellular functions.
    [Show full text]
  • A SARS-Cov-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug- Repurposing
    bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.002386; this version posted March 23, 2020. 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 4.0 International license. A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug- Repurposing David E. Gordon1,2,3,4, Gwendolyn M. Jang1,2,3,4, Mehdi Bouhaddou1,2,3,4, Jiewei Xu1,2,3,4, Kirsten Obernier1,2,3,4, Matthew J. O'Meara5, Jeffrey Z. Guo1,2,3,4, Danielle L. Swaney1,2,3,4, Tia A. Tummino1,2,6, Ruth Huettenhain1,2,3,4, Robyn Kaake1,2,3,4, Alicia L. Richards1,2,3,4, Beril Tutuncuoglu1,2,3,4, Helene Foussard1,2,3,4, Jyoti Batra1,2,3,4, Kelsey Haas1,2,3,4, Maya Modak1,2,3,4, Minkyu Kim1,2,3,4, Paige Haas1,2,3,4, Benjamin J. Polacco1,2,3,4, Hannes Braberg1,2,3,4, Jacqueline M. Fabius1,2,3,4, Manon Eckhardt1,2,3,4, Margaret Soucheray1,2,3,4, Melanie J. Bennett1,2,3,4, Merve Cakir1,2,3,4, Michael J. McGregor1,2,3,4, Qiongyu Li1,2,3,4, Zun Zar Chi Naing1,2,3,4, Yuan Zhou1,2,3,4, Shiming Peng1,2,6, Ilsa T. Kirby1,4,7, James E. Melnyk1,4,7, John S. Chorba1,4,7, Kevin Lou1,4,7, ShiZhong A. Dai1,4,7, Wenqi Shen1,4,7, Ying Shi1,4,7, Ziyang Zhang1,4,7, Inigo Barrio-HernandeZ8, Danish Memon8, Claudia Hernandez-Armenta8, Christopher J.P.
    [Show full text]
  • A SARS-Cov-2 Protein Interaction Map Reveals Targets for Drug Repurposing
    Article A SARS-CoV-2 protein interaction map reveals targets for drug repurposing https://doi.org/10.1038/s41586-020-2286-9 A list of authors and affiliations appears at the end of the paper Received: 23 March 2020 Accepted: 22 April 2020 A newly described coronavirus named severe acute respiratory syndrome Published online: 30 April 2020 coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than Check for updates 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efcacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and eforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identifed the human proteins that physically associated with each of the SARS-CoV-2 proteins using afnity-purifcation mass spectrometry, identifying 332 high-confdence protein–protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors.
    [Show full text]
  • Research Article Complex and Multidimensional Lipid Raft Alterations in a Murine Model of Alzheimer’S Disease
    SAGE-Hindawi Access to Research International Journal of Alzheimer’s Disease Volume 2010, Article ID 604792, 56 pages doi:10.4061/2010/604792 Research Article Complex and Multidimensional Lipid Raft Alterations in a Murine Model of Alzheimer’s Disease Wayne Chadwick, 1 Randall Brenneman,1, 2 Bronwen Martin,3 and Stuart Maudsley1 1 Receptor Pharmacology Unit, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224, USA 2 Miller School of Medicine, University of Miami, Miami, FL 33124, USA 3 Metabolism Unit, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224, USA Correspondence should be addressed to Stuart Maudsley, [email protected] Received 17 May 2010; Accepted 27 July 2010 Academic Editor: Gemma Casadesus Copyright © 2010 Wayne Chadwick et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Various animal models of Alzheimer’s disease (AD) have been created to assist our appreciation of AD pathophysiology, as well as aid development of novel therapeutic strategies. Despite the discovery of mutated proteins that predict the development of AD, there are likely to be many other proteins also involved in this disorder. Complex physiological processes are mediated by coherent interactions of clusters of functionally related proteins. Synaptic dysfunction is one of the hallmarks of AD. Synaptic proteins are organized into multiprotein complexes in high-density membrane structures, known as lipid rafts. These microdomains enable coherent clustering of synergistic signaling proteins.
    [Show full text]