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Research ArticleArticle

Identification of Protein Kinase C Activation as a Novel Mechanism for RGS2 Protein Upregulation through Phenotypic Screening of Natural Product Extracts

Avi Raveh, Pamela J. Schultz, Lauren Aschermann, Colleen Carpenter, Giselle Tamayo-Castillo, Shugeng Cao, Jon Clardy, Richard R. Neubig, David H. Sherman and Benita Sjögren
Molecular Pharmacology October 2014, 86 (4) 406-416; DOI: https://doi.org/10.1124/mol.114.092403
Avi Raveh
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.)
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Pamela J. Schultz
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.)
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Lauren Aschermann
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.)
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Colleen Carpenter
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.)
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Giselle Tamayo-Castillo
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.)
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Shugeng Cao
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.)
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Jon Clardy
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.)
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Richard R. Neubig
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.)
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David H. Sherman
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.)
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Benita Sjögren
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.)
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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.

Footnotes

    • Received February 21, 2014.
    • Accepted July 31, 2014.
  • The project described was supported by the International Cooperative Biodiversity Groups initiative from the Fogarty International Center [U01TW007404], the H. W. Vahlteich Professorship, and the Swedish Heart and Lung Foundation.

  • dx.doi.org/10.1124/mol.114.092403

  • ↵Embedded ImageThis article has supplemental material available at mol.aspetjournals.org.

  • Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 86 (4)
Molecular Pharmacology
Vol. 86, Issue 4
1 Oct 2014
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Research ArticleArticle

PKC Activation Increases RGS2 Protein Levels

Avi Raveh, Pamela J. Schultz, Lauren Aschermann, Colleen Carpenter, Giselle Tamayo-Castillo, Shugeng Cao, Jon Clardy, Richard R. Neubig, David H. Sherman and Benita Sjögren
Molecular Pharmacology October 1, 2014, 86 (4) 406-416; DOI: https://doi.org/10.1124/mol.114.092403

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Research ArticleArticle

PKC Activation Increases RGS2 Protein Levels

Avi Raveh, Pamela J. Schultz, Lauren Aschermann, Colleen Carpenter, Giselle Tamayo-Castillo, Shugeng Cao, Jon Clardy, Richard R. Neubig, David H. Sherman and Benita Sjögren
Molecular Pharmacology October 1, 2014, 86 (4) 406-416; DOI: https://doi.org/10.1124/mol.114.092403
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