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 b-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 Ga as well as through other mechanisms. RGS22/2 mice are hypertensive, show increased anxiety, and are prone to heart failure. RGS2 has a very short protein halflife 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 concentrationdependent manner. Similar results were obtained with phorbol 12-myristate 13-acetate as well as activation of the Gqcoupled muscarinic M 3 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 PKCb-selective inhibitor Ruboxastaurin, as well as small interfering RNA-mediated knockdown of PKCb. 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.

Regulator of G Protein Signaling 10 (Rgs10) Expression Is Transcriptionally Silenced in Activated Microglia by Histone Deacetylase Activity

“…Strategies to target regulation of RGS protein expression have focused on post-translational mechanisms that control protein stability (Sjögren et al, 2012;Raveh et al, 2014). In contrast, our data suggest predominantly transcriptional regulation of Rgs10 expression.…”

“…Given the ability of RGS10 to regulate inflammatory signaling, understanding the mechanisms that control RGS10 levels in microglia may reveal new therapeutic strategies to address neuroinflammatory disease. The most commonly described mechanism for regulation of RGS protein abundance involves critical post-translational mechanisms that control protein stability (Sjögren et al, 2012;Raveh et al, 2014). However, our studies in ovarian cancer cells suggest that Rgs10 is transcriptionally regulated by DNA and histonetargeted epigenetic mechanisms (Ali et al, 2013;Cacan et al, 2014).…”

The evolution of regulators of G protein signalling proteins as drug targets – 20 years in the making: IUPHAR Review 21

“…Another member of the R4 family, RGS2, was demonstrated to be phosphorylated by PKC, which inhibited GAP activity of RGS2 in vitro (Cunningham et al, 2001). In contrast, we recently demonstrated that activation of PKC enhances RGS2 protein levels, leading to increased RGS2-mediated suppression of GPCR signalling in HEK-293 cells (Raveh et al, 2014). Although it is not clear whether this effect is due to direct phosphorylation of RGS2 by PKC, it provides a clear example of the importance of the experimental context in which RGS protein function is studied and the complexity of RGS protein biology.…”

“…Several RGS proteins are downregulated during pathological insults so finding ways to increase their expression, and thereby function, is an attractive alternative strategy to achieve this goal. This encouraged us to develop a cell based enzyme complementation assay to screen for small molecule stabilizers of RGS2 (Sjögren et al, 2012;Raveh et al, 2014). As discussed above, RGS2 is one of several RGS proteins that is rapidly degraded through the ubiquitin-proteasomal pathway.…”

R4 Regulator of G Protein Signaling (RGS) Proteins in Inflammation and Immunity

“…Topical or mucosal (e.g., inhalational) administration of such compounds could increase their efficacy in inflammatory diseases of the mucosa (e.g., asthma), while limiting systemic side effects. Finally, a recent cell-based HTS of a microbial-derived natural compound library identified indolactam V as a specific upregulator of RGS2 but not RGS4, through a protein kinase C (PKC)-mediated mechanism, supporting the feasibility of this experimental strategy (117).…”