RT Journal Article
SR Electronic
T1 Identification of PKC Activation as a Novel Mechanism for RGS2 Protein Up-regulation Through Phenotypic Screening of Natural Product Extracts
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.114.092403
DO 10.1124/mol.114.092403
A1 Avi Raveh
A1 Pamela J. Schultz
A1 Lauren Aschermann
A1 Colleen Carpenter
A1 Giselle Tamayo-Castillo
A1 Shugeng Cao
A1 Jon Clardy
A1 Richard R Neubig
A1 David H Sherman
A1 Benita Sjogren
YR 2014
UL http://molpharm.aspetjournals.org/content/early/2014/08/01/mol.114.092403.abstract
AB 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 biological mechanisms. In the current study we utilized 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 (PMA) as well as activation of the Gq-coupled muscarinic M3 receptor. The effect on RGS2 protein levels was blocked by the non-selective PKC inhibitor Go6983, the PKCβ-selective inhibitor Ruboxastaurin, as well as siRNA-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/PLC signaling through RGS2 protein up-regulation.