Molecular basis for interactions of G protein betagamma subunits with effectors

Both the alpha and betagamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) communicate signals from receptors to effectors. Gbetagamma subunits can regulate a diverse array of effectors, including ion channels and enzymes. Galpha subunits bound to guanine diphosphate (Galpha-GDP) inhibit signal transduction through Gbetagamma subunits, suggesting a common interface on Gbetagamma subunits for Galpha binding and effector interaction. The molecular basis for interaction of Gbetagamma with effectors was characterized by mutational analysis of Gbeta residues that make contact with Galpha-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-beta2, and beta-adrenergic receptor kinase revealed the Gbeta residues required for activation of each effector and provides evidence for partially overlapping domains on Gbeta for regulation of these effectors. This organization of interaction regions on Gbeta for different effectors and Galpha explains why subunit dissociation is crucial for signal transmission through Gbetagamma subunits.     

Substitutions in the pheromone-responsive Gbeta protein of Saccharomyces cerevisiae confer a defect in recovery from pheromone treatment

The pheromone-responsive Galpha protein of Saccharomyces cerevisiae, Gpa1p, stimulates an adaptive mechanism that downregulates the mating signal. In a genetic screen designed to identify signaling elements required for Gpa1p-mediated adaptation, a large collection of adaptive-defective (Adp-) mutants were recovered. Of the 49 mutants characterized thus far, approximately three-quarters exhibit a dominant defect in the negative regulation of the pheromone response. Eight of the dominant Adp- mutations showed tight linkage to the gene encoding the pheromone-responsive Gbeta, STE4. Sequence analysis of the STE4 locus in the relevant mutant strains revealed seven novel STE4 alleles, each of which was shown to disrupt proper regulation of the pheromone response. Although the STE4 mutations had only minor effects on basal mating pathway activity, the mutant forms of Gbeta dramatically affected the ability of the cell to turn off the mating response after exposure to pheromone. Moreover, the signaling activity of the aberrant Gbetagamma subunits was suppressed by G322E, a mutant form of Gpa1p that blocks the pheromone response by sequestering Gbetagamma, but not by E364K, a hyperadaptive form of Gpa1p. On the basis of these observations, we propose that Gpa1p-mediated adaptation involves the binding of an unknown negative regulator to Gbetagamma.     

Functional binding between Gbeta and the LIM domain of Ste5 is required to activate the MEKK Ste11

BACKGROUND: In the budding yeast Saccharomyces cerevisiae, the pheromones that induce haploid cells of opposite cell types to mate activate the Gbeta and Ggamma subunits of a heterotrimeric G protein. These subunits signal through the PAK kinase Ste20 to activate a mitogen-activated protein (MAP) kinase cascade comprising the MEKK Ste11, the MEK Ste7 and two MAP kinases, Fus3 and Kss1. The pathway requires Ste5, a scaffold protein that tethers the MAP kinase cascade enzymes into a high molecular weight complex. Ste5 is thought to associate with Gbeta in a pheromone-independent manner, but it is not known if this interaction affects signaling. RESULTS: A ste5C180A mutant - which expresses Ste5 disrupted in the LIM domain, a putative metal-binding motif that has been proposed to be essential for Ste5 oligomerization - could not transmit the pheromone signal from Gbeta through Ste20 to Ste11. The Ste5C180A protein was impaired in binding Gbeta, although it could oligomerize, bind Ste11, Ste7 and Fus3, facilitate the basal activation of Ste11, and relay the Ste11 signal to MAP kinases. Ste5 bound to Gbeta in a pheromone-dependent manner and preferentially associated with a phosphorylated form of Gbeta in wild-type and ste20Delta, but not in ste5C180A, strains. CONCLUSIONS: Pheromone induces binding of Gbeta to Ste5 through its LIM domain. This binding is essential for activation of Ste11 and is distinct from the ability of Ste5 to oligomerize or to serve as a scaffold and relay the signal from Ste11 to the MAP kinases. Pheromone also induces Ste5-dependent phosphorylation of Gbeta.     

Interaction of a G-protein beta-subunit with a conserved sequence in Ste20/PAK family protein kinases

Serine/threonine protein kinases of the Ste20/PAK family have been implicated in the signalling from heterotrimeric G proteins to mitogen-activated protein (MAP) kinase cascades. In the yeast Saccharomyces cerevisiae, Ste20 is involved in transmitting the mating-pheromone signal from the betagamma-subunits (encoded by the STE4 and STE18 genes, respectively) of a heterotrimeric G protein to a downstream MAP kinase cascade. We have identified a binding site for the G-protein beta-subunit (Gbeta) in the non-catalytic carboxy-terminal regions of Ste20 and its mammalian homologues, the p21-activated protein kinases (PAKs). Association of Gbeta with this site in Ste20 was regulated by binding of pheromone to the receptor. Mutations in Gbeta and Ste20 that prevented this association blocked activation of the MAP kinase cascade. Considering the high degree of structural and functional conservation of Ste20/PAK family members and G-protein subunits, our results provide a possible model for a role of these kinases in Gbetagamma-mediated signal transduction in organisms ranging from yeast to mammals.     

Membrane recruitment of the kinase cascade scaffold protein Ste5 by the Gbetagamma complex underlies activation of the yeast pheromone response pathway

In the Saccharomyces cerevisiae pheromone response pathway, the Gbetagamma complex activates downstream responses by an unknown mechanism involving a MAP kinase cascade, the PAK-like kinase Ste20, and a Rho family GTPase, Cdc42. Here we show that Gbetagamma must remain membrane-associated after release from Galpha to activate the downstream pathway. We also show that pheromone stimulates translocation of the kinase cascade scaffold protein Ste5 to the cell surface. This recruitment requires Gbetagamma function and the Gbetagamma-binding domain of Ste5, but not the kinases downstream of Gbetagamma, suggesting that it is mediated by Gbetagamma itself. Furthermore, this event has functional significance, as artificial targeting of Ste5 to the plasma membrane, but not intracellular membranes, activates the pathway in the absence of pheromone or Gbetagamma. Remarkably, although independent of Gbetagamma, activation by membrane-targeted Ste5 requires Ste20, Cdc42, and Cdc24, indicating that their participation in this pathway does not require them to be activated by Gbetagamma. Thus, membrane recruitment of Ste5 defines a molecular activity for Gbetagamma. Moreover, our results suggest that this event promotes kinase cascade activation by delivering the Ste5-associated kinases to the cell surface kinase Ste20, whose function may depend on Cdc42 and Cdc24.     

Ste5 RING-H2 domain: role in Ste4-promoted oligomerization for yeast pheromone signaling

Ste5 is a scaffold for the mitogen-activated protein kinase (MAPK) cascade components in a yeast pheromone response pathway. Ste5 also associates with Ste4, the beta subunit of a heterotrimeric guanine nucleotide-binding protein, potentially linking receptor activation to stimulation of the MAPK cascade. A RING-H2 motif at the Ste5 amino terminus is apparently essential for function because Ste5(C177S) and Ste5(C177A C180A) mutants did not rescue the mating defect of a ste5Delta cell. In vitro Ste5(C177A C180A) bound each component of the MAPK cascade, but not Ste4. Unlike wild-type Ste5, the mutant did not appear to oligomerize; however, when fused to a heterologous dimerization domain (glutathione S-transferase), the chimeric protein restored mating in an ste5Delta cell and an ste4Delta ste5Delta double mutant. Thus, the RING-H2 domain mediates Ste4-Ste5 interaction, which is a prerequisite for Ste5-Ste5 self-association and signaling.     

Dimerization of Ste5, a mitogen-activated protein kinase cascade scaffold protein, is required for signal transduction

The mitogen-activated protein kinase cascade of the Saccharomyces cerevisiae pheromone response pathway is organized on the Ste5 protein, which binds each of the kinases of the cascade prior to signaling. In this study, a structure-function analysis of Ste5 deletion mutants uncovered new functional domains of the Ste5 protein and revealed that Ste5 dimerizes during the course of normal signal transduction. Dimerization, mediated by two regions in the N-terminal half of Ste5, was first suggested by intragenic complementation between pairs of nonfunctional Ste5 mutants and was confirmed by using the two-hybrid system. Coimmunoprecipitation of differently tagged forms of Ste5 from cells in which the pathway has been activated by Ste5 overexpression further confirmed dimerization. A precise correlation between the biological activity of various Ste5 fragments and dimerization suggests that dimerization is essential for Ste5 function.     

Phosphorylation of the pheromone-responsive Gbeta protein of Saccharomyces cerevisiae does not affect its mating-specific signaling function

The pheromone-responsive Gbeta subunit of Saccharomyces cerevisiae (encoded by STE4) is rapidly phosphorylated at multiple sites when yeast cells are exposed to mating pheromone. It has been shown that a mutant form of Ste4 lacking residues 310-346, ste4delta310-346, cannot be phosphorylated, and that its expression leads to defects in recovery from pheromone stimulation. Based on these observations, it was proposed that phosphorylation of Ste4 is associated with an adaptive response to mating pheromone. In this study we used site-directed mutagenesis to create two phosphorylation null (Pho-) alleles of STE4: ste4-T320A/S335A and ste4-T322A/S335A. When expressed in yeast, these mutant forms of Ste4 remained unphosphorylated upon pheromone stimulation. The elimination of Ste4 phosphorylation has no discernible effect on either signaling or adaptation. In addition, disruption of the FUS3 gene, which encodes a pheromone-specific MAP kinase, leads to partial loss of pheromone-induced Ste4 phosphorylation. Two-hybrid analysis suggests that the ste4delta310-346 deletion mutant is impaired in its interaction with Gpa1, the pheromone-responsive Galpha of yeast, whereas the Ste4-T320A/S335A mutant has normal affinity for Gpa1. Taken together, these results indicate that pheromone-induced phosphorylation of Ste4 is not an adaptive mechanism, and that the adaptive defect exhibited by the 310-346 deletion mutant is likely to be due to disruption of the interaction between Ste4 and Gpa1.     

Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbetagamma subunit-mediated activation of mitogen-activated protein kinases

Several G protein-coupled receptors that interact with pertussis toxin-sensitive heterotrimeric G proteins mediate Ras-dependent activation of mitogen-activated protein (MAP) kinases. The mechanism involves Gbetagamma subunit-mediated increases in tyrosine phosphorylation of the Shc adapter protein, Shc*Grb2 complex formation, and recruitment of Ras guanine nucleotide exchange factor activity. We have investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src in activation of the MAP kinase pathway via endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or by transient expression of Gbetagamma subunits in COS-7 cells. In vitro kinase assays of Shc immunoprecipitates following LPA stimulation demonstrated rapid, transient recruitment of tyrosine kinase activity into Shc immune complexes. Recruitment of tyrosine kinase activity was pertussis toxin-sensitive and mimicked by cellular expression of Gbetagamma subunits. Immunoblots for coprecipitated proteins in Shc immunoprecipitates revealed a transient association of Shc and c-Src following LPA stimulation, which coincided with increases in Shc-associated tyrosine kinase activity and Shc tyrosine phosphorylation. LPA stimulation or expression of Gbetagamma subunits resulted in c-Src activation, as assessed by increased c-Src autophosphorylation. Overexpression of wild-type or constitutively active mutant c-Src, but not kinase inactive mutant c-Src, lead to increased tyrosine kinase activity in Shc immunoprecipitates, increased Shc tyrosine phosphorylation, and Shc.Grb2 complex formation. MAP kinase activation resulting from LPA receptor stimulation, expression of Gbetagamma subunits, or expression of c-Src was sensitive to dominant negatives of mSos, Ras, and Raf. Coexpression of Csk, which inactivates Src family kinases by phosphorylating the regulatory C-terminal tyrosine residue, inhibited LPA stimulation of Shc tyrosine phosphorylation, Shc.Grb2 complex formation, and MAP kinase activation. These data suggest that Gbetagamma subunit-mediated formation of Shc.c-Src complexes and c-Src kinase activation are early events in Ras-dependent activation of MAP kinase via pertussis toxin-sensitive G protein-coupled receptors.     

Phosphorylation and localization of Kss1, a MAP kinase of the Saccharomyces cerevisiae pheromone response pathway

Kss1 protein kinase, and the homologous Fus3 kinase, are required for pheromone signal transduction in Saccharomyces cerevisiae. In MATa haploids exposed to alpha-factor, Kss1 was rapidly phosphorylated on both Thr183 and Tyr185, and both sites were required for Kss1 function in vivo. De novo protein synthesis was required for sustained pheromone-induced phosphorylation of Kss1. Catalytically inactive Kss1 mutants displayed alpha-factor-induced phosphorylation on both residues, even in kss1 delta cells; hence, autophosphorylation is not obligatory for these modifications. In kss1 delta fus3 delta double mutants, Kss1 phosphorylation was elevated even in the absence of pheromone; thus, cross-phosphorylation by Fus3 is not responsible for Kss1 activation. In contrast, pheromone-induced Kss1 phosphorylation was eliminated in mutants deficient in two other protein kinases, Ste11 and Ste7. A dominant hyperactive allele of STE11 caused a dramatic increase in the phosphorylation of Kss1, even in the absence of pheromone stimulation, but required Ste7 for this effect, suggesting an order of function: Ste11-->Ste7-->Kss1. When overproduced, Kss1 stimulated recovery from pheromone-imposed G1 arrest. Catalytic activity was essential for Kss1 function in signal transmission, but not for its recovery-promoting activity. Kss1 was found almost exclusively in the particulate material and its subcellular fractionation was unaffected by pheromone treatment. Indirect immunofluorescence demonstrated that Kss1 is concentrated in the nucleus and that its distribution is not altered detectably during signaling.     

MAP kinase and cAMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea

Many fungal pathogens invade plants using specialized infection structures called appressoria that differentiate from the tips of fungal hyphae contacting the plant surface. We demonstrate a role for a MAP kinase that is essential for appressorium formation and infectious growth in Magnaporthe grisea, the fungal pathogen responsible for rice blast disease. The PMK1 gene of M. grisea is homologous to the Saccharomyces cerevisiae MAP kinases FUS3/KSS1, and a GST-Pmk1 fusion protein has kinase activity in vitro. pmk1 mutants of M. grisea fail to form appressoria and fail to grow invasively in rice plants. pmk1 mutants are still responsive to cAMP for early stages of appressorium formation, which suggests Pmk1 acts downstream of a cAMP signal for infection structure formation. PMK1 is nonessential for vegetative growth and sexual and asexual reproduction in culture. Surprisingly, when expressed behind the GAL1 promoter in yeast, PMK1 can rescue the mating defect in a fus3 kss1 double mutant. These results demonstrate that PMK1 is part of a highly conserved MAP kinase signal transduction pathway that acts cooperatively with a cAMP signaling pathway for fungal pathogenesis.     

Dynamics and organization of MAP kinase signal pathways

In the budding yeast, Saccharomyces cerevisiae, four separate but structurally related mitogen-activated protein kinase (MAPK) activation pathways are known. The best understood of these regulates mating. Pheromone binding to receptor informs cells of the proximity of a mating partner and induces differentiation to a mating competent state. The MAPK activation cascade mediating this signal is made up of Ste11 (a MEK kinase [MEKK]), Ste7 (a MAPK/ERK kinase [MEK]), and the redundant MAPK-related Fus3 and Kss1 enzymes. Another MAPK activation pathway is important for cell integrity and regulates cell wall construction. This cascade consists of Bck1 (a MEKK), the redundant Mkk1 and Mkk2 enzymes (MEKs), and Mpk1 (a MAPK). We exploited these two pathways to learn about the coordination and signal transmission fidelity of MAPK activation cascades. Two lines of evidence suggest that the activities of the mating and cell integrity pathways are coordinated during mating differentiation. First, cells deficient in Mpk1 are susceptible to lysis when they make a mating projection in response to pheromone. Second, Mpk1 activation during pheromone induction coincides with projection formation. The mechanism underlying this coordination is still unknown to us. Our working model is that projection formation generates a mobile second messenger for activation of the cell integrity pathway. Analysis of a STE7 mutation gave us some unanticipated but important insights into parameters important for fidelity of signal transmission. The Ste7 variant has a serine to proline substitution at position 368. Ste7-P368 has higher basal activity than the wild-type enzyme but still requires Ste11 for its function. Additionally, the proline substitution enables the variant to transmit the signal from mammalian Raf expressed in yeast. This novel activity suggests that Ste7-P368 is inherently more permissive than Ste7 in its interactions with MEKKs. Yet, Ste7-P368 cross function in the cell integrity pathway occurs only when it is highly overproduced or when Ste5 is missing. This behavior suggests that Ste5, which has been proposed to be a tether for the kinases in the mating pathway, contributes to Ste7 specificity and fidelity of signal transmission.     

Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics. International Federation of Clinical Chemistry Scientific Division Committee on Molecular Biology Techniques

Random mutations were generated in the G-protein-coupled receptor (Ste2p) for the tridecapeptide pheromone (alpha-factor) of Saccharomyces cerevisiae. These mutants were screened for variants that responded to antagonists. Because multiple mutations were detected in each mutant receptor recovered from the screen, site-directed mutagenesis was used to create single-site mutant receptors. Three receptors containing mutations F55V, S219P, and S259P were analyzed for their biological responses to various alpha-factor analogs and for their ligand binding profiles. Cells expressing each of the mutant receptors responded to alpha-factor as well as or better than wild-type cells in a growth arrest assay. In contrast, the binding of alpha-factor to the F55V and S219P mutant receptors was at least 10-fold reduced in comparison to wild-type receptor indicating a complex non-linear correlation between binding affinity and biological activity. Cells expressing mutant receptors responded to some normally inactive analogs in biological assays, despite the fact that these analogs had a low affinity for Ste2p. The analysis of these mutant receptors confirms previous findings that the first and sixth transmembrane regions of Ste2p are important for ligand interaction, ligand specificity, and/or receptor activation to initiate the signal transduction pathway. Changes in binding affinity of pheromone analogs to wild-type and mutant receptors indicate that residue 55 of Ste2p is involved with both ligand binding and signal transduction.     

Role of cyclooxygenase-2 for fluid secretion by the inflamed gallbladder mucosa

Heterotrimeric GTP-binding protein (G-protein)-coupled receptors are able to induce a variety of responses including cell proliferation, differentiation, and activation of several intracellular kinase cascades. Prominent among these kinases are the activation of mitogen-activated protein (MAP) kinase, including the extracellular signal-regulated kinases (ERKs), ERK1 and ERK2 (p44mapk and p42mapk, respectively); stress-activated protein kinases (SAPKs/JNKs); and p38 kinase. These receptors signal through G-proteins. Recent data have shown that the activation of mitogen-activated protein/ERK kinase induced by G-protein-coupled receptors is mediated by both Galpha and Gbetagamma subunits involving a common signaling pathway with receptor-tyrosine-kinases. Gbetagamma-mediated mitogen-activated protein kinase activation is mediated by activation of phosphoinositide 3-kinase, followed by a tyrosine phosphorylation event, and proceeds in a sequence of events that involve functional association among the adaptor proteins Shc, Grb2, and Sos. SAPKs/JNKs and p38 are able to be activated by Gbetagamma proteins in a pathway involving Rho family proteins including RhoA, Rac1, and Cdc42.     

The Ca2+-dependent binding of calmodulin to an N-terminal motif of the heterotrimeric G protein beta subunit

Ca2+ ion concentration changes are critical events in signal transduction. The Ca2+-dependent interactions of calmodulin (CaM) with its target proteins play an essential role in a variety of cellular functions. In this study, we investigated the interactions of G protein betagamma subunits with CaM. We found that CaM binds to known betagamma subunits and these interactions are Ca2+-dependent. The CaM-binding domain in Gbetagamma subunits is identified as Gbeta residues 40-63. Peptides derived from the Gbeta protein not only produce a Ca2+-dependent gel mobility shifting of CaM but also inhibit the CaM-mediated activation of CaM kinase II. Specific amino acid residues critical for the binding of Gbetagamma to CaM were also identified. We then investigated the potential function of these interactions and showed that binding of CaM to Gbetagamma inhibits the pertussis toxin-catalyzed ADP-ribosylation of Galphao subunits, presumably by inhibiting heterotrimer formation. Furthermore, we demonstrated that interaction with CaM has little effect on the activation of phospholipase C-beta2 by Gbetagamma subunits, supporting the notion that different domains of Gbetagamma are responsible for the interactions of different effectors. These findings shed light on the molecular basis for the interactions of Gbetagamma with Ca2+-CaM and point to the potential physiological significance of these interactions in cellular functions.