Identification of protein kinase C activation as a novel mechanism for RGS2 protein upregulation through phenotypic screening of natural product extracts

Mol Pharmacol. 2014 Oct;86(4):406-16. doi: 10.1124/mol.114.092403. Epub 2014 Aug 1.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actinobacteria / chemistry
  • Animals
  • Biological Products / pharmacology*
  • HEK293 Cells
  • High-Throughput Screening Assays
  • Humans
  • Indoles / pharmacology*
  • Lactams / pharmacology*
  • Maleimides / pharmacology
  • Myocytes, Smooth Muscle / drug effects
  • Phenotype
  • Protein Kinase C beta / antagonists & inhibitors
  • Protein Kinase C beta / drug effects*
  • Protein Kinase C beta / metabolism
  • Protein Kinase Inhibitors / pharmacology
  • RGS Proteins / genetics
  • RGS Proteins / metabolism*
  • Rats
  • Receptor, Muscarinic M3 / agonists
  • Small Molecule Libraries / pharmacology*
  • Tetradecanoylphorbol Acetate / pharmacology
  • Up-Regulation*


  • 2-(1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1H-indol-3-yl)maleimide
  • Biological Products
  • Indoles
  • Lactams
  • Maleimides
  • Protein Kinase Inhibitors
  • RGS Proteins
  • RGS2 protein, human
  • Receptor, Muscarinic M3
  • Small Molecule Libraries
  • RGS4 protein
  • ruboxistaurin
  • indolactam V
  • Protein Kinase C beta
  • Tetradecanoylphorbol Acetate