Phosphorylation of Ser166 in RGS5 by protein kinase C causes loss of RGS function

RGS5 is a member of regulators of G protein signaling (RGS) proteins that attenuate heterotrimeric G protein signaling by functioning as GTPase-activating proteins (GAPs). We investigated phosphorylation of RGS5 and the resulting change of its function. In 293T cells, transiently expressed RGS5 was phosphorylated by endogenous protein kinases in the basal state. The phosphorylation was enhanced by phorbol 12-myristate 13-acetate (PMA) and endothelin-1 (ET-1), and suppressed by protein kinase C (PKC) inhibitors, H7, calphostin C and staurosporine. These results suggest involvement of PKC in phosphorylation of RGS5. In in vitro experiments, PKC phosphorylated recombinant RGS5 protein at serine residues. RGS5 protein phosphorylated by PKC showed much lower binding capacity for and GAP activity toward Galpha subunits than did the unphosphorylated RGS5. In cells expressing RGS5, the inhibitory effect of RGS5 on ET-1-induced Ca(2+) responses was enhanced by staurosporine. Mass spectrometric analysis of the phosphorylated RGS5 revealed that Ser166 was one of the predominant phosphorylation sites. Substitution of Ser166 by aspartic acid abolished the binding capacity to Galpha subunits and the GAP activity, and markedly reduced the inhibitory effect on ET-1-induced Ca(2+) responses. These results indicate that phosphorylation at Ser166 of RGS5 by PKC causes loss of the function of RGS5 in G protein signaling. Since this serine residue is conserved in RGS domains of many RGS proteins, the phosphorylation at Ser166 by PKC might act as a molecular switch and have functional significance.     

Gbetagamma inhibits Galpha GTPase-activating proteins by inhibition of Galpha-GTP binding during stimulation by receptor

Gbetagamma subunits modulate several distinct molecular events involved with G protein signaling. In addition to regulating several effector proteins, Gbetagamma subunits help anchor Galpha subunits to the plasma membrane, promote interaction of Galpha with receptors, stabilize the binding of GDP to Galpha to suppress spurious activation, and provide membrane contact points for G protein-coupled receptor kinases. Gbetagamma subunits have also been shown to inhibit the activities of GTPase-activating proteins (GAPs), both phospholipase C (PLC)-betas and RGS proteins, when assayed in solution under single turnover conditions. We show here that Gbetagamma subunits inhibit G protein GAP activity during receptor-stimulated, steady-state GTPase turnover. GDP/GTP exchange catalyzed by receptor requires Gbetagamma in amounts approximately equimolar to Galpha, but GAP inhibition was observed with superstoichiometric Gbetagamma. The potency of inhibition varied with the GAP and the Galpha subunit, but half-maximal inhibition of the GAP activity of PLC-beta1 was observed with 5-10 nM Gbetagamma, which is at or below the concentrations of Gbetagamma needed for regulation of physiologically relevant effector proteins. The kinetics of GAP inhibition of both receptor-stimulated GTPase activity and single turnover, solution-based GAP assays suggested a competitive mechanism in which Gbetagamma competes with GAPs for binding to the activated, GTP-bound Galpha subunit. An N-terminal truncation mutant of PLC-beta1 that cannot be directly regulated by Gbetagamma remained sensitive to inhibition of its GAP activity, suggesting that the Gbetagamma binding site relevant for GAP inhibition is on the Galpha subunit rather than on the GAP. Using fluorescence resonance energy transfer between cyan or yellow fluorescent protein-labeled G protein subunits and Alexa532-labeled RGS4, we found that Gbetagamma directly competes with RGS4 for high-affinity binding to Galpha(i)-GDP-AlF4.     

Molecular mechanism of the inhibition of phospholipase C beta 3 by protein kinase C

Activation of protein kinase C (PKC) can result from stimulation of the receptor-G protein-phospholipase C (PLCbeta) pathway. In turn, phosphorylation of PLCbeta by PKC may play a role in the regulation of receptor-mediated phosphatidylinositide (PI) turnover and intracellular Ca(2+) release. Activation of endogenous PKC by phorbol 12-myristate 13-acetate inhibited both Galpha(q)-coupled (oxytocin and M1 muscarinic) and Galpha(i)-coupled (formyl-Met-Leu-Phe) receptor-stimulated PI turnover by 50-100% in PHM1, HeLa, COSM6, and RBL-2H3 cells expressing PLCbeta(3). Activation of conventional PKCs with thymeleatoxin similarly inhibited oxytocin or formyl-Met-Leu-Phe receptor-stimulated PI turnover. The PKC inhibitory effect was also observed when PLCbeta(3) was stimulated directly by Galpha(q) or Gbetagamma in overexpression assays. PKC phosphorylated PLCbeta(3) at the same predominant site in vivo and in vitro. Peptide sequencing of in vitro phosphorylated recombinant PLCbeta(3) and site-directed mutagenesis identified Ser(1105) as the predominant phosphorylation site. Ser(1105) is also phosphorylated by protein kinase A (PKA; Yue, C., Dodge, K. L., Weber, G., and Sanborn, B. M. (1998) J. Biol. Chem. 273, 18023-18027). Similar to PKA, the inhibition by PKC of Galpha(q)-stimulated PLCbeta(3) activity was completely abolished by mutation of Ser(1105) to Ala. In contrast, mutation of Ser(1105) or Ser(26), another putative phosphorylation target, to Ala had no effect on inhibition of Gbetagamma-stimulated PLCbeta(3) activity by PKC or PKA. These data indicate that PKC and PKA act similarly in that they inhibit Galpha(q)-stimulated PLCbeta(3) as a result of phosphorylation of Ser(1105). Moreover, PKC and PKA both inhibit Gbetagamma-stimulated activity by mechanisms that do not involve Ser(1105).     

Erk1/2- and p38 MAP kinase-dependent phosphorylation and activation of cPLA2 by m3 and m2 receptors

This study examined the upstream signaling pathways initiated by muscarinic m2 and m3 receptors that mediate sustained ERK1/2- and p38 MAP kinase-dependent phosphorylation and activation of the 85-kDa cytosolic phospholipase (cPL)A(2) in smooth muscle. The pathway initiated by m2 receptors involved sequential activation of Gbetagamma(i3), phosphatidylinositol (PI)3-kinase, Cdc42, and Rac1, p21-activated kinase (PAK1), p38 mitogen-activated protein (MAP) kinase, and cPLA(2), and phosphorylation of cPLA(2) at Ser(505). cPLA(2) activity was inhibited to the same extent (61 +/- 5 to 72 +/- 4%) by the m2 antagonist methoctramine, Gbeta antibody, pertussis toxin, the PI3-kinase inhibitor LY 294002, PAK1 antibody, the p38 MAP kinase inhibitor SB-203580, and a Cdc42/Rac1 GEF (Vav2) antibody and by coexpression of dominant-negative Cdc42 and Rac1 mutants. The pathway initiated by m3 receptors involved sequential activation of Galpha(q), PLC-beta1, PKC, ERK1/2, and cPLA(2), and phosphorylation of cPLA(2) at Ser(505). cPLA(2) activity was inhibited to the same extent (35 +/- 3 to 41 +/- 5%) by the m3 antagonist 4-diphenylacetoxy-N-methylpiperdine (4-DAMP), the phosphoinositide hydrolysis inhibitor U-73122, the PKC inhibitor bisindolylmaleimide, and the ERK1/2 inhibitor PD 98059. cPLA(2) activity was not affected in cells coexpressing dominant-negative RhoA and PLC-delta1 mutants, implying that PKC was not derived from phosphatidylcholine hydrolysis. The effects of ERK1/2 and p38 MAP kinase on cPLA(2) activity were additive and accounted fully for activation and phosphorylation of cPLA(2).     

Mapping of a yeast G protein betagamma signaling interaction.

The mating pathway of Saccharomyces cerevisiae is widely used as a model system for G protein-coupled receptor-mediated signal transduction. Following receptor activation by the binding of mating pheromones, G protein betagamma subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gbeta (Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identify residues in Ste4p required for the interaction with Ste5p. These residues define a new signaling interface close to the Ste20p binding site within the Gbetagamma coiled-coil. Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (Galpha) and Ste18p (Ggamma) but cannot function in signal transduction because cells expressing these mutants are sterile. Ste4 L65S is temperature-sensitive for its interaction with Ste5p, and also for signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong evidence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues. The correlation between disruption of the Ste4p-Ste5p interaction and sterility confirms the importance of this interaction in signal transduction. Identification of the Gbetagamma coiled-coil in Ste5p binding may set a precedent for Gbetagamma-effector interactions in more complex organisms.     

RGS16 function is regulated by epidermal growth factor receptor-mediated tyrosine phosphorylation

Galpha(i)-coupled receptor stimulation results in epidermal growth factor receptor (EGFR) phosphorylation and MAPK activation. Regulators of G protein signaling (RGS proteins) inhibit G protein-dependent signal transduction by accelerating Galpha(i) GTP hydrolysis, shortening the duration of G protein effector stimulation. RGS16 contains two conserved tyrosine residues in the RGS box, Tyr(168) and Tyr(177), which are predicted sites of phosphorylation. RGS16 underwent phosphorylation in response to m2 muscarinic receptor or EGFR stimulation in HEK 293T or COS-7 cells, which required EGFR kinase activity. Mutational analysis suggested that RGS16 was phosphorylated on both tyrosine residues (Tyr(168) Tyr(177)) after EGF stimulation. RGS16 co-immunoprecipitated with EGFR, and the interaction did not require EGFR activation. Purified EGFR phosphorylated only recombinant RGS16 wild-type or Y177F in vitro, implying that EGFR-mediated phosphorylation depended on residue Tyr(168). Phosphorylated RGS16 demonstrated enhanced GTPase accelerating (GAP) activity on Galpha(i). Mutation of Tyr(168) to phenylalanine resulted in a 30% diminution in RGS16 GAP activity but completely eliminated its ability to regulate G(i)-mediated MAPK activation or adenylyl cyclase inhibition in HEK 293T cells. In contrast, mutation of Tyr(177) to phenylalanine had no effect on RGS16 GAP activity but also abolished its regulation of G(i)-mediated signal transduction in these cells. These data suggest that tyrosine phosphorylation regulates RGS16 function and that EGFR may potentially inhibit Galpha(i)-dependent MAPK activation in a feedback loop by enhancing RGS16 activity through tyrosine phosphorylation.     

Inhibition of a Gi-activated potassium channel (GIRK1/4) by the Gq-coupled m1 muscarinic acetylcholine receptor

The G protein-coupled inwardly rectifying K+ channel, GIRK1/GIRK4, can be activated by receptors coupled to the Galpha(i) subunit. An opposing role for Galpha(q) receptor signaling in GIRK regulation has only recently begun to be established. We have studied the effects of m1 muscarinic acetylcholine receptor (mAChR) stimulation, which is known to mobilize calcium and activate protein kinase C (PKC) by a Galpha(q)-dependent mechanism, on whole cell GIRK1/4 currents in Xenopus oocytes. We found that stimulation of the m1 mAChR suppresses both basal and dopamine 2 receptor-activated GIRK 1/4 currents. Overexpression of Gbetagamma subunits attenuates this effect, suggesting that increased binding of Gbetagamma to the GIRK channel can effectively compete with the G(q)-mediated inhibitory signal. This G(q) signal requires the use of second messenger molecules; pharmacology implicates a role for PKC and Ca2+ responses as m1 mAChR-mediated inhibition of GIRK channels is mimicked by PMA and Ca2+ ionophore. We have analyzed a series of mutant and chimeric channels suggesting that the GIRK4 subunit is capable of responding to G(q) signals and that the resulting current inhibition does not occur via phosphorylation of a canonical PKC site on the channel itself.     

Protein kinase C phosphorylates RGS2 and modulates its capacity for negative regulation of Galpha 11 signaling

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 Galpha(q)-mediated inositol lipid signaling. Although purified RGS2 blocks PLC-beta activation by the nonhydrolyzable GTP analog guanosine 5'-O-thiophosphate (GTPgammaS), 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 GTPgammaS-stimulated Galpha(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-betat activity than of GTPgammaS-promoted PLC-betat 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 4beta-phorbol 12beta-myristate 13alpha-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 GTPgammaS-stimulated PLC-betat 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 Galpha(q)betagamma. 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.     

Investigation of the mechanisms of G protein: effector coupling by the human and mouse prostacyclin receptors. Identification of critical species-dependent differences

We recently identified a novel mechanism explaining how the mouse (m) prostacyclin receptor (IP) couples to Galpha(s), Galpha(i), and Galpha(q) (Lawler, O. A., Miggin, S. M., and Kinsella, B. T. (2001) J. Biol. Chem. 276, 33596-33607) whereby mIP coupling to Galpha(i) and Galpha(q) is dependent on its initial coupling to Galpha(s) and subsequent phosphorylation by cAMP-dependent protein kinase A (PKA) on Ser(357). In the current study, the generality of that mechanism was investigated by examining the G protein coupling specificity of the human (h) IP. The hIP efficiently coupled to Galpha(s)/adenylyl cyclase and to Galpha(q)/phospholipase C activation but failed to couple to Galpha(i). Coupling of the hIP to Galpha(q), or indeed to Galpha(s) or Galpha(i), was unaffected by the PKA or protein kinase C (PKC) inhibitors H-89 and GF 109203X, respectively. Thus, mIP and hIP exhibit essential differences in their coupling to Galpha(i) and in their dependence on PKA in regulating their coupling to Galpha(q). Analysis of their primary sequences revealed that the critical PKA phosphorylation site within the mIP, at Ser(357), is replaced by a PKC site within the hIP, at Ser(328). Conversion of the PKC site of the hIP to a PKA site generated hIP(QL325,326RP) that efficiently coupled to Galpha(s) and to Galpha(i) and Galpha(q); coupling of hIP(QL325,326RP) to Galpha(i) but not to Galpha(s) or Galpha(q) was inhibited by H-89. Abolition of the PKC site of the hIP generated hIP(S328A) that efficiently coupled to Galpha(s) and Galpha(q) but failed to couple to Galpha(i). Finally, conversion of the PKA site at Ser(357) within the mIP to a PKC site generated mIP(RP354,355QL) that efficiently coupled to Galpha(s) but not to Galpha(i) or Galpha(q). Collectively, our data highlight critical differences in signaling by the mIP and hIP that are regulated by their differential phosphorylation by PKA and PKC together with contextual sequence differences surrounding those sites.     

RACK1 regulates specific functions of Gbetagamma

We showed previously that Gbetagamma interacts with Receptor for Activated C Kinase 1 (RACK1), a protein that not only binds activated protein kinase C (PKC) but also serves as an adaptor/scaffold for many signaling pathways. Here we report that RACK1 does not interact with Galpha subunits or heterotrimeric G proteins but binds free Gbetagamma subunits released from activated heterotrimeric G proteins following the activation of their cognate receptors in vivo. The association with Gbetagamma promotes the translocation of RACK1 from the cytosol to the membrane. Moreover, binding of RACK1 to Gbetagamma results in inhibition of Gbetagamma-mediated activation of phospholipase C beta2 and adenylyl cyclase II. However, RACK1 has no effect on other functions of Gbetagamma, such as activation of the mitogen-activated protein kinase signaling pathway or chemotaxis of HEK293 cells via the chemokine receptor CXCR2. Similarly, RACK1 does not affect signal transduction through the Galpha subunits of G(i), G(s), or G(q). Collectively, these findings suggest a role of RACK1 in regulating specific functions of Gbetagamma.     

Regulation of G protein-mediated signal transduction by RGS proteins

RGS proteins form a new family of regulatory proteins of G protein signaling. They contain homologous core domains (RGS domains) of about 120 amino acids. RGS domains interact with activated Galpha subunits. Several RGS proteins have been shown biochemically to act as GTPase activating proteins (GAPs) for their interacting Galpha subunits. Other than RGS domains, RGS proteins differ significantly in size, amino acid sequences, and tissue distribution. In addition, many RGS proteins have other protein-protein interaction motifs involved in cell signaling. We have shown that p115RhoGEF, a newly identified GEF(guanine nucleotide exchange factor) for RhoGTPase, has a RGS domain at its N-terminal region and this domain acts as a specific GAP for Galpha12 and Galpha13. Furthermore, binding of activated Galpha13 to this RGS domain stimulated GEF activity of p115RhoGEF. Activated Galpha12 inhibited Galpha13-stimulated GEF activity. Thus p115RhoGEF is a direct link between heterotrimeric G protein and RhoGTPase and it functions as an effector for Galpha12 and Galpha13 in addition to acting as their GAP. We also found that RGS domain at N-terminal regions of G protein receptor kinase 2 (GRK2) specifically interacts with Galphaq/11 and inhibits Galphaq-mediated activation of PLC-beta, apparently through sequestration of activated Galphaq. However, unlike other RGS proteins, this RGS domain did not show significant GAP activity to Galphaq. These results indicate that RGS proteins have far more diverse functions than acting simply as GAPs and the characterization of function of each RGS protein is crucial to understand the G protein signaling network in cells.     

Palmitoylation of a conserved cysteine in the regulator of G protein signaling (RGS) domain modulates the GTPase-activating activity of RGS4 and RGS10

RGS4 and RGS10 expressed in Sf9 cells are palmitoylated at a conserved Cys residue (Cys(95) in RGS4, Cys(66) in RGS10) in the regulator of G protein signaling (RGS) domain that is also autopalmitoylated when the purified proteins are incubated with palmitoyl-CoA. RGS4 also autopalmitoylates at a previously identified cellular palmitoylation site, either Cys(2) or Cys(12). The C2A/C12A mutation essentially eliminates both autopalmitoylation and cellular [(3)H]palmitate labeling of Cys(95). Membrane-bound RGS4 is palmitoylated both at Cys(95) and Cys(2/12), but cytosolic RGS4 is not palmitoylated. RGS4 and RGS10 are GTPase-activating proteins (GAPs) for the G(i) and G(q) families of G proteins. Palmitoylation of Cys(95) on RGS4 or Cys(66) on RGS10 inhibits GAP activity 80-100% toward either Galpha(i) or Galpha(z) in a single-turnover, solution-based assay. In contrast, when GAP activity was assayed as acceleration of steady-state GTPase in receptor-G protein proteoliposomes, palmitoylation of RGS10 potentiated GAP activity >/=20-fold. Palmitoylation near the N terminus of C95V RGS4 did not alter GAP activity toward soluble Galpha(z) and increased G(z) GAP activity about 2-fold in the vesicle-based assay. Dual palmitoylation of wild-type RGS4 remained inhibitory. RGS protein palmitoylation is thus multi-site, complex in its control, and either inhibitory or stimulatory depending on the RGS protein and its sites of palmitoylation.     

G Protein activation without subunit dissociation depends on a G{alpha}(i)-specific region

G proteins transmit a variety of extracellular signals into intracellular responses. The Galpha and Gbetagamma subunits are both known to regulate effectors. Interestingly, the Galpha subunit also determines subtype specificity of Gbetagamma effector interactions. However, in light of the common paradigm that Galpha and Gbetagamma subunits dissociate during activation, a plausible mechanism of how this subtype specificity is generated was lacking. Using a fluorescence resonance energy transfer (FRET)-based assay developed to directly measure mammalian G protein activation in intact cells, we demonstrate that fluorescent Galpha(i1,2,3), Galpha(z), and Gbeta(1)gamma(2) subunits do not dissociate during activation but rather undergo subunit rearrangement as indicated by an activation-induced increase in FRET. In contrast, fluorescent Galpha(o) subunits exhibited an activation-induced decrease in FRET, reflecting subunit dissociation or, alternatively, a distinct subunit rearrangement. The alpha(B/C)-region within the alpha-helical domain, which is much more conserved within Galpha(i1,2,3) and Galpha(z) as compared with that in Galpha(o), was found to be required for exhibition of an activation-induced increase in FRET between fluorescent Galpha and Gbetagamma subunits. However, the alpha(B/C)-region of Galpha(il) alone was not sufficient to transfer the activation pattern of Galpha(i) to the Galpha(o) subunit. Either residues in the first 91 amino acids or in the C-terminal remainder (amino acids 93-354) of Galpha(il) together with the alpha(B/C)-helical region of Galpha(i1) were needed to transform the Galpha(o)-activation pattern into a Galpha(i1)-type of activation. The discovery of subtype-selective mechanisms of G protein activation illustrates that G protein subfamilies have specific mechanisms of activation that may provide a previously unknown basis for G protein signaling specificity.     

RGS17/RGSZ2, a novel regulator of Gi/o, Gz, and Gq signaling

To identify novel regulators of Galpha(o), the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Galpha(o) as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Galpha-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of Galpha(i1-3), Galpha(o), Galpha(z), or Galpha(q) but not Galpha(s) were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Galpha(i2) and Galpha(o) under pre-steady-state conditions, and on M2-muscarinic receptor-activated Galpha(i1), Galpha(i2), Galpha(i3), Galpha(z), and Galpha(o) in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for G(z), RGS17 exhibited limited selectivity for G(o) among G(i)/G(o) proteins. All RZ family members reduced dopamine-D2/Galpha(i)-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Galpha(q)-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G(i/o), G(z), and G(q) over G(s) to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors.     

Distinct roles for Galpha(i)2 and Gbetagamma in signaling to DNA synthesis and Galpha(i)3 in cellular transformation by dopamine D2S receptor activation in BALB/c 3T3 cells

Control of cell proliferation depends on intracellular mediators that determine the cellular response to external cues. In neuroendocrine cells, the dopamine D2 receptor short form (D2S receptor) inhibits cell proliferation, whereas in mesenchymal cells the same receptor enhances cell proliferation. Nontransformed BALB/c 3T3 fibroblast cells were stably transfected with the D2S receptor cDNA to study the G proteins that direct D2S signaling to stimulate cell proliferation. Pertussis toxin inactivates G(i) and G(o) proteins and blocks signaling of the D2S receptor in these cells. D2S receptor signaling was reconstituted by individually transfecting pertussis toxin-resistant Galpha(i/o) subunit mutants and measuring D2-induced responses in pertussis toxin-treated cells. This approach identified Galpha(i)2 and Galpha(i)3 as mediators of the D2S receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity; Galpha(i)2-mediated D2S-induced stimulation of p42 and p44 mitogen-activated kinase (MAPK) and DNA synthesis, whereas Galpha(i)3 was required for formation of transformed foci. Transfection of toxin-resistant Galpha(i)1 cDNA induced abnormal cell growth independent of D2S receptor activation, while Galpha(o) inhibited dopamine-induced transformation. The role of Gbetagamma subunits was assessed by ectopic expression of the carboxyl-terminal domain of G protein receptor kinase to selectively antagonize Gbetagamma activity. Mobilization of Gbetagamma subunits was required for D2S-induced calcium mobilization, MAPK activation, and DNA synthesis. These findings reveal a remarkable and distinct G protein specificity for D2S receptor-mediated signaling to initiate DNA synthesis (Galpha(i)2 and Gbetagamma) and oncogenic transformation (Galpha(i)3), and they indicate that acute activation of MAPK correlates with enhanced DNA synthesis but not with transformation.     

Functional interaction between Galpha(z) and Rap1GAP suggests a novel form of cellular cross-talk

G(z) is a member of the G(i) family of trimeric G proteins whose primary role in cell physiology is still unknown. In an ongoing effort to elucidate the cellular functions of G(z), the yeast two-hybrid system was employed to identify proteins that specifically interact with a mutationally activated form of Galpha(z). One of the molecules uncovered in this screen was Rap1GAP, a previously identified protein that specifically stimulates GTP hydrolytic activity of the monomeric G protein Rap1 and thus is believed to function as a down-regulator of Rap1 signaling. Like G(z), the precise role of Rap1 in cell physiology is poorly understood. Biochemical analysis using purified recombinant proteins revealed that the physical interaction between Galpha(z) and Rap1GAP blocks the ability of RGSs (regulators of G protein signaling) to stimulate GTP hydrolysis of the alpha subunit, and also attenuates the ability of activated Galpha(z) to inhibit adenylyl cyclase. Structure-function analyses indicate that the first 74 amino-terminal residues of Rap1GAP, a region distinct from the catalytic core domain responsible for the GAP activity toward Rap1, is required for this interaction. Co-precipitation assays revealed that Galpha(z), Rap1GAP, and Rap1 can form a stable complex. These data suggest that Rap1GAP acts as a signal integrator to somehow coordinate and/or integrate G(z) signaling and Rap1 signaling in cells.     

Phosphorylation of the MEKK Ste11p by the PAK-like kinase Ste20p is required for MAP kinase signaling in vivo

BACKGROUND: Many signals are transduced from the cell surface to the nucleus through mitogen-activated protein (MAP) kinase cascades. Activation of MAP kinase requires phosphorylation by MEK, which in turn is controlled by Raf, Mos or a group of structurally related kinases termed MEKKs. It is not understood how MEKKs are regulated by extracellular signals. In yeast, the MEKK Ste11p functions in multiple MAP kinase cascades activated in response to pheromones, high osmolarity and nutrient starvation. Genetic evidence suggests that the p21-activated protein kinase (PAK) Ste20p functions upstream of Ste11p, and Ste20p has been shown to phosphorylate Ste11p in vitro. RESULTS: Ste20p phosphorylated Ste11p on Ser302 and/or Ser306 and Thr307 in yeast, residues that are conserved in MEKKs of other organisms. Mutating these sites to non-phosphorylatable residues abolished Ste11p function, whereas changing them to aspartic acid to mimic the phosphorylated form constitutively activated Ste11p in vivo in a Ste20p-independent manner. The amino-terminal regulatory domain of Ste11p interacted with its catalytic domain, and overexpression of a small amino-terminal fragment of Ste11p was able to inhibit signaling in response to pheromones. Mutational analysis suggested that this interaction was regulated by phosphorylation and dependent on Thr596, which is located in the substrate cleft of the catalytic domain. CONCLUSIONS: Our results suggest that, in response to multiple extracellular signals, phosphorylation of Ste11p by Ste20p removes an amino-terminal inhibitory domain, leading to activation of the Ste11 protein kinase. This mechanism may serve as a paradigm for the activation of mammalian MEKKs.     

Prostacyclin receptor-induced STAT3 phosphorylation in human erythroleukemia cells is mediated via Galpha(s) and Galpha(16) hybrid signaling

Human prostacyclin receptor (hIP) stimulates STAT3 via pertussis toxin-insensitive G proteins in human erythroleukemia (HEL) cells. Since hIP can utilize G(s) and G(q) proteins for signal transduction and that both G proteins can induce STAT3 phosphorylation and activation via complex signaling networks, we sought to determine if one of them is predominant in mediating the hIP signal. Stimulation of STAT3 Tyr(705) and Ser(727) phosphorylations by the IP-specific agonist, cicaprost, was sensitive to inhibition of protein kinase A, phospholipase Cbeta, protein kinase C, calmodulin-dependent protein kinase II and Janus kinase 2/3. Unlike Galpha(16)-mediated regulation of STAT3 in the same cells, cicaprost-induced STAT3 Tyr(705) phosphorylation was resistant to inhibition of Src and MEK while STAT3 Ser(727) phosphorylation distinctly required phosphatidylinositol-3 kinase. This unique inhibitor-sensitivity pattern of STAT3 phosphorylation was reproduced in HEL cells by stimulating the G(16)-coupled C5a receptor in the presence of dibutyryl-cAMP, suggesting that the change in inhibitor-sensitivity was due to activation of the G(s) pathway. This postulation was confirmed by expressing constitutively active Galpha(16)QL and Galpha(s)QL in human embryonic kidney 293 cells and the inhibitor-sensitivity of Galpha(16)QL-induced STAT3 phosphorylations could be converted by the mere presence of Galpha(s)QL to resemble that obtained with cicaprost in HEL cells. In addition, the restoration of the Galpha(16)-mediated inhibitor-sensitivity upon cicaprost induction in Galpha(s)-knocked down HEL cells again verified the pivotal role of G(s) signal. Taken together, our observations illustrate that co-stimulation of G(s) and G(q) can result in the fine-tuning of STAT3 activation status, and this may provide the basis for cell type-specific responses following activation of hIP.     

Dopamine-D2S receptor inhibition of calcium influx,adenylyl cyclase,and mitogen-activated protein kinase in pituitary cells: distinct Galpha and Gbetagamma requirements

The G protein specificity of multiple signaling pathways of the dopamine-D2S (short form) receptor was investigated in GH4ZR7 lactotroph cells. Activation of the dopamine-D2S receptor inhibited forskolin-induced cAMP production, reduced BayK8644- activated calcium influx, and blocked TRH-mediated p42/p44 MAPK phosphorylation. These actions were blocked by pretreatment with pertussis toxin (PTX), indicating mediation by G(i/o) proteins. D2S stimulation also decreased TRH-induced MAPK/ERK kinase phosphorylation. TRH induced c-Raf but not B-Raf activation, and the D2S receptor inhibited both TRH-induced c-Raf and basal B-Raf kinase activity. After PTX treatment, D2S receptor signaling was rescued in cells stably transfected with individual PTX-insensitive Galpha mutants. Inhibition of adenylyl cyclase was partly rescued by Galpha(i)2 or Galpha(i)3, but Galpha(o) alone completely reconstituted D2S-mediated inhibition of BayK8644-induced L-type calcium channel activation. Galpha(o) and Galpha(i)3 were the main components involved in D2S-mediated p42/44 MAPK inhibition. In cells transfected with the carboxyl-terminal domain of G protein receptor kinase to inhibit Gbetagamma signaling, only D2S-mediated inhibition of calcium influx was blocked, but not inhibition of adenylyl cyclase or MAPK. These results indicate that the dopamine-D2S receptor couples to distinct G(i/o) proteins, depending on the pathway addressed, and suggest a novel Galpha(i)3/Galpha(o)-dependent inhibition of MAPK mediated by c-Raf and B-Raf-dependent inhibition of MAPK/ERK kinase.