Protein Kinase C Phosphorylates RGS2 and Modulates Its Capacity for Negative Regulation of Gα11 Signaling

Abstract

RGS proteins (regulators of G protein signaling) attenuate heterotrimeric G protein signaling by functioning as both GTPase-activating proteins (GAPs) and inhibitors of G protein/effector interaction. RGS2 has been shown to regulate G␣ q -mediated inositol lipid signaling. Although purified RGS2 blocks PLC-␤ activation by the nonhydrolyzable GTP analog guanosine 5-O-thiophosphate (GTP␥S), its capacity to regulate inositol lipid signaling under conditions where GTPase-promoted hydrolysis of GTP is operative has not been fully explored. Utilizing the turkey erythrocyte membrane model of inositol lipid signaling, we investigated regulation by RGS2 of both GTP and GTP␥S-stimulated G␣ 11 signaling. Different inhibitory potencies of RGS2 were observed under conditions assessing its activity as a GAP versus as an effector antagonist; i.e. RGS2 was a 10 -20-fold more potent inhibitor of aluminum fluoride and GTP-stimulated PLC-␤t activity than of GTP␥S-promoted PLC-␤t activity. We also examined whether RGS2 was regulated by downstream components of the inositol lipid signaling pathway. RGS2 was phosphorylated by PKC in vitro to a stoichiometry of approximately unity by both a mixture of PKC isozymes and individual calcium and phospholipid-dependent PKC isoforms. Moreover, RGS2 was phosphorylated in intact COS7 cells in response to PKC activation by 4␤-phorbol 12␤-myristate 13␣-acetate and, to a lesser extent, by the P2Y 2 receptor agonist UTP. In vitro phosphorylation of RGS2 by PKC decreased its capacity to attenuate both GTP and GTP␥S-stimulated PLC-␤t activation, with the extent of attenuation correlating with the level of RGS2 phosphorylation. A phosphorylation-dependent inhibition of RGS2 GAP activity was also observed in proteoliposomes reconstituted with purified P2Y 1 receptor and G␣ q ␤␥. These results identify for the first time a phosphorylation-induced change in the activity of an RGS protein and suggest a mechanism for potentiation of inositol lipid signaling by PKC.A variety of hormone and neurotransmitter receptors transduce signals through heterotrimeric G proteins. In their inactive state, G proteins exist as heterotrimers consisting of ␣, ␤, and ␥ subunits with GDP bound to G␣. Upon agonist occupation, the receptor promotes GDP/GTP exchange, and the active GTP-bound G␣ subunit and G␤␥ dissociate to interact with target effector proteins. Signaling is terminated by the hydrolysis of GTP to GDP and the subsequent formation of the heterotrimer. Therefore, the magnitude and duration of signaling is determined by the length of time G␣ remains in the active GTP-bound conformation.A recently identified family of proteins termed RGS (regulators of G protein signaling) proteins interact directly with G␣ subunits to decrease the lifetime of the active GTP-bound complex (1-4). RGS proteins attenuate heterotrimeric G protein 1 signaling by functioning as both GTPase-activating proteins (GAPs) (5, 6) and inhibitors of G protein/effector interaction (6, 7). In vitro studies illustrate that RGS2 interacts with and fu...

Differential Contribution of GTPase Activation and Effector Antagonism to the Inhibitory Effect of RGS Proteins on Gq-mediated Signaling in Vivo

“…effector antagonism) are sufficient to mediate their inhibitory effects. RGS2 was previously reported to be 10 -20-fold more potent as a GAP than effector antagonist in vitro (36). The stoichiometry of signaling components cannot be easily controlled in vivo, partly because protein expression can be altered upon co-transfection, as seen in the present study (see above).…”

“…Because the N terminus of R4 RGS proteins is believed to serve as a scaffold for receptors and signaling proteins (46), it is conceivable that structural differences between RGS2/3 and RGS5/16 contribute to their differential effects. Phosphorylation of RGS proteins may also play a role, but so far little is known about its regulation and functional implications (36,49,50). Changes in post-translational modifications on G␣ subunits, such as an increase in palmitate turnover in constitutively active G␣ q * (51), also have the potential to differentially affect RGS function (52,53).…”

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

“…Phosphorylation of RGS14 by PKA at Thr 494 , adjacent to the GoLoco motif, enhances its guanine nucleotide dissociation inhibitory activity towards Gα i , while having no effect on GAP activity (Hollinger et al, 2003).In contrast, GPCR ligand-dependent phosphorylation of RGS16 at Ser 53 has been shown to inhibit GAP activity (Chen et al, 2001), while Src-mediated phosphorylation at Tyr 168 protects RGS16 from degradation leading to enhanced GAP activity in cells (Derrien and Druey, 2001;Derrien et al, 2003). 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).…”

R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1

“…Other RGS proteins in general show highly varied distribution patterns (19)(20)(21)(22)(23)(24)(25)(26). Variable subcellular targeting of RGS proteins may be a mechanism for regulation of their functions (18,(27)(28)(29).…”