title: Identification of protein kinase C activation as a novel mechanism for RGS2 protein upregulation through phenotypic screening of natural product extracts.
abstract: Biochemical high-throughput screening is widely used in drug discovery, using a variety of small molecule libraries. However, broader screening strategies may be more beneficial to identify novel biologic mechanisms. In the current study we used a β-galactosidase complementation method to screen a selection of microbial-derived pre-fractionated natural product extracts for those that increase regulator of G protein signaling 2 (RGS2) protein levels. RGS2 is a member of a large family of proteins that all regulate signaling through G protein-coupled receptors (GPCRs) by accelerating GTPase activity on active Gα as well as through other mechanisms. RGS2(-/-) mice are hypertensive, show increased anxiety, and are prone to heart failure. RGS2 has a very short protein half-life due to rapid proteasomal degradation, and we propose that enhancement of RGS2 protein levels could be a beneficial therapeutic strategy. Bioassay-guided fractionation of one of the hit strains yielded a pure compound, Indolactam V, a known protein kinase C (PKC) activator, which selectively increased RGS2 protein levels in a time- and concentration-dependent manner. Similar results were obtained with phorbol 12-myristate 13-acetate as well as activation of the Gq-coupled muscarinic M3 receptor. The effect on RGS2 protein levels was blocked by the nonselective PKC inhibitor Gö6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), the PKCβ-selective inhibitor Ruboxastaurin, as well as small interfering RNA-mediated knockdown of PKCβ. Indolactam V-mediated increases in RGS2 protein levels also had functional effects on GPCR signaling. This study provides important proof-of-concept for our screening strategy and could define a negative feedback mechanism in Gq/Phospholipase C signaling through RGS2 protein upregulation.
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (A.R., P.J.S., D.H.S.); Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan (L.A., R.R.N., B.S.); Department of Pharmacology (C.C.), Department of Medicinal Chemistry (D.H.S.), Department of Microbiology and Immunology (D.H.S.), Department of Chemistry (D.H.S.), Center for Chemical Genomics, University of Michigan, Ann Arbor, Michigan (D.H.S.); Unidad Estrategica de Bioprospección, Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica & CIPRONA, Escuela de Química, Universidad de Costa Rica, San Pedro, Costa Rica (G.T-C.); Harvard Medical School, Boston, Massachusetts (S.C., J.C.); and University of Hawaii Cancer Center, Honolulu, Hawaii (S.C.).
authors.forename: Avi
authors.initials: A
authors.lastname: Raveh
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (A.R., P.J.S., D.H.S.); Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan (L.A., R.R.N., B.S.); Department of Pharmacology (C.C.), Department of Medicinal Chemistry (D.H.S.), Department of Microbiology and Immunology (D.H.S.), Department of Chemistry (D.H.S.), Center for Chemical Genomics, University of Michigan, Ann Arbor, Michigan (D.H.S.); Unidad Estrategica de Bioprospección, Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica & CIPRONA, Escuela de Química, Universidad de Costa Rica, San Pedro, Costa Rica (G.T-C.); Harvard Medical School, Boston, Massachusetts (S.C., J.C.); and University of Hawaii Cancer Center, Honolulu, Hawaii (S.C.).
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Schultz
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (A.R., P.J.S., D.H.S.); Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan (L.A., R.R.N., B.S.); Department of Pharmacology (C.C.), Department of Medicinal Chemistry (D.H.S.), Department of Microbiology and Immunology (D.H.S.), Department of Chemistry (D.H.S.), Center for Chemical Genomics, University of Michigan, Ann Arbor, Michigan (D.H.S.); Unidad Estrategica de Bioprospección, Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica & CIPRONA, Escuela de Química, Universidad de Costa Rica, San Pedro, Costa Rica (G.T-C.); Harvard Medical School, Boston, Massachusetts (S.C., J.C.); and University of Hawaii Cancer Center, Honolulu, Hawaii (S.C.).
authors.forename: Lauren
authors.initials: L
authors.lastname: Aschermann
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (A.R., P.J.S., D.H.S.); Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan (L.A., R.R.N., B.S.); Department of Pharmacology (C.C.), Department of Medicinal Chemistry (D.H.S.), Department of Microbiology and Immunology (D.H.S.), Department of Chemistry (D.H.S.), Center for Chemical Genomics, University of Michigan, Ann Arbor, Michigan (D.H.S.); Unidad Estrategica de Bioprospección, Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica & CIPRONA, Escuela de Química, Universidad de Costa Rica, San Pedro, Costa Rica (G.T-C.); Harvard Medical School, Boston, Massachusetts (S.C., J.C.); and University of Hawaii Cancer Center, Honolulu, Hawaii (S.C.).
authors.forename: Colleen
authors.initials: C
authors.lastname: Carpenter
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (A.R., P.J.S., D.H.S.); Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan (L.A., R.R.N., B.S.); Department of Pharmacology (C.C.), Department of Medicinal Chemistry (D.H.S.), Department of Microbiology and Immunology (D.H.S.), Department of Chemistry (D.H.S.), Center for Chemical Genomics, University of Michigan, Ann Arbor, Michigan (D.H.S.); Unidad Estrategica de Bioprospección, Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica & CIPRONA, Escuela de Química, Universidad de Costa Rica, San Pedro, Costa Rica (G.T-C.); Harvard Medical School, Boston, Massachusetts (S.C., J.C.); and University of Hawaii Cancer Center, Honolulu, Hawaii (S.C.).
authors.forename: Giselle
authors.initials: G
authors.lastname: Tamayo-Castillo
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (A.R., P.J.S., D.H.S.); Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan (L.A., R.R.N., B.S.); Department of Pharmacology (C.C.), Department of Medicinal Chemistry (D.H.S.), Department of Microbiology and Immunology (D.H.S.), Department of Chemistry (D.H.S.), Center for Chemical Genomics, University of Michigan, Ann Arbor, Michigan (D.H.S.); Unidad Estrategica de Bioprospección, Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica & CIPRONA, Escuela de Química, Universidad de Costa Rica, San Pedro, Costa Rica (G.T-C.); Harvard Medical School, Boston, Massachusetts (S.C., J.C.); and University of Hawaii Cancer Center, Honolulu, Hawaii (S.C.).
authors.forename: Shugeng
authors.initials: S
authors.lastname: Cao
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (A.R., P.J.S., D.H.S.); Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan (L.A., R.R.N., B.S.); Department of Pharmacology (C.C.), Department of Medicinal Chemistry (D.H.S.), Department of Microbiology and Immunology (D.H.S.), Department of Chemistry (D.H.S.), Center for Chemical Genomics, University of Michigan, Ann Arbor, Michigan (D.H.S.); Unidad Estrategica de Bioprospección, Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica & CIPRONA, Escuela de Química, Universidad de Costa Rica, San Pedro, Costa Rica (G.T-C.); Harvard Medical School, Boston, Massachusetts (S.C., J.C.); and University of Hawaii Cancer Center, Honolulu, Hawaii (S.C.).
authors.forename: Jon
authors.initials: J
authors.lastname: Clardy
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (A.R., P.J.S., D.H.S.); Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan (L.A., R.R.N., B.S.); Department of Pharmacology (C.C.), Department of Medicinal Chemistry (D.H.S.), Department of Microbiology and Immunology (D.H.S.), Department of Chemistry (D.H.S.), Center for Chemical Genomics, University of Michigan, Ann Arbor, Michigan (D.H.S.); Unidad Estrategica de Bioprospección, Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica & CIPRONA, Escuela de Química, Universidad de Costa Rica, San Pedro, Costa Rica (G.T-C.); Harvard Medical School, Boston, Massachusetts (S.C., J.C.); and University of Hawaii Cancer Center, Honolulu, Hawaii (S.C.).
authors.forename: Richard R
authors.initials: RR
authors.lastname: Neubig
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (A.R., P.J.S., D.H.S.); Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan (L.A., R.R.N., B.S.); Department of Pharmacology (C.C.), Department of Medicinal Chemistry (D.H.S.), Department of Microbiology and Immunology (D.H.S.), Department of Chemistry (D.H.S.), Center for Chemical Genomics, University of Michigan, Ann Arbor, Michigan (D.H.S.); Unidad Estrategica de Bioprospección, Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica & CIPRONA, Escuela de Química, Universidad de Costa Rica, San Pedro, Costa Rica (G.T-C.); Harvard Medical School, Boston, Massachusetts (S.C., J.C.); and University of Hawaii Cancer Center, Honolulu, Hawaii (S.C.).
authors.forename: David H
authors.initials: DH
authors.lastname: Sherman
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (A.R., P.J.S., D.H.S.); Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan (L.A., R.R.N., B.S.); Department of Pharmacology (C.C.), Department of Medicinal Chemistry (D.H.S.), Department of Microbiology and Immunology (D.H.S.), Department of Chemistry (D.H.S.), Center for Chemical Genomics, University of Michigan, Ann Arbor, Michigan (D.H.S.); Unidad Estrategica de Bioprospección, Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica & CIPRONA, Escuela de Química, Universidad de Costa Rica, San Pedro, Costa Rica (G.T-C.); Harvard Medical School, Boston, Massachusetts (S.C., J.C.); and University of Hawaii Cancer Center, Honolulu, Hawaii (S.C.) sjogren1@msu.edu.
authors.forename: Benita
authors.initials: B
authors.lastname: Sjögren
mesh_terms: D039903:Actinobacteria / Q000737:chemistry; D000818:Animals; D001688:Biological Products / Q000494:pharmacology*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D007769:Lactams / Q000494:pharmacology*; D008301:Maleimides / Q000494:pharmacology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects; D010641:Phenotype; D064546:Protein Kinase C beta / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D043587:Receptor, Muscarinic M3 / Q000819:agonists; D054852:Small Molecule Libraries / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015854:Up-Regulation
chemical_list: C465664:2-(1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1H-indol-3-yl)maleimide; D001688:Biological Products; D007211:Indoles; D007769:Lactams; D008301:Maleimides; D047428:Protein Kinase Inhibitors; D020710:RGS Proteins; C087182:RGS2 protein, human; D043587:Receptor, Muscarinic M3; D054852:Small Molecule Libraries; C098799:RGS4 protein; C099154:ruboxistaurin; C043969:indolactam V; D064546:Protein Kinase C beta; D013755:Tetradecanoylphorbol Acetate
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