Regulation of squid visual phospholipase C by activated G-protein alpha.

Phospholipase C (PLC) is the key enzyme in the phototransduction cascade of invertebrate rhabdomeric photoreceptors. In addition to 130 kDa PLC, a 95 kDa protein recognized by antibody against the catalytic site of PLC was found in the squid retina. The PLC-like 95 kDa protein (95 kDa PLC) was produced from 130 kDa PLC by an intrinsic protease in the presence of calcium. The 130 kDa PLC was stimulated by the active form of Gq-class G-protein alpha (Gq alpha), but the 95 kDa PLC was not, although their PLC activity was similar. A 35 kDa fragment, the counterpart of 95 kDa PLC, was not recognized by antibodies against catalytic site or N-terminal site of the 130 kDa PLC, indicating that the cleavage site is on the C-terminal side beyond the catalytic site. In the presence of a large excess of the 35 kDa fragment, 95 kDa PLC was stimulated by Gq alpha to a similar extent as intact 130 kDa PLC. These results indicate that the C-terminal polypeptide of PLC is necessary for regulation of its enzyme activity by Gq alpha. The uncoupling of PLC from Gq alpha, caused by limited proteolysis, is therefore a candidate regulatory mechanism of the phototransduction cascade in rhabdomeric photoreceptors.     

Stimulation of phospholipase C by guanine-nucleotide-binding protein beta gamma subunits.

We have previously shown that soluble fractions obtained from human HL-60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5'-[3-O-thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743-748]. To investigate whether this stimulation was due to a soluble alpha subunit of a heterotrimeric guanine-nucleotide-binding protein or a soluble low-molecular-mass GTP-binding protein, we have examined the effect of purified guanine-nucleotide-binding protein beta gamma dimers on the phospholipase-C-mediated formation of inositol phosphates by HL-60 cytosol. We found that beta gamma subunits, purified from bovine retinal transducin (beta gamma t), markedly stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by beta gamma t was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin alpha subunit and was additive to activation of phospholipase C by guanosine 5'-[3-O-thio]triphosphate. Beta gamma t also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane-bound, detergent-solubilized phospholipase C from HL-60 cells. Stimulation of soluble HL-60 phospholipase C was not restricted to beta gamma t, but was also observed with highly purified beta gamma subunits from bovine brain. Fractionation of HL-60 cytosol by anion-exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in HL-60 granulocytes. Only one of these forms was sensitive to stimulation by beta gamma t, demonstrating that stimulation of phospholipase C by beta gamma subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein beta gamma subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine-nucleotide-binding proteins.     

Stimulation of cellular signaling and G protein subunit dissociation by G protein betagamma subunit-binding peptides

We previously developed peptides that bind to G protein betagamma subunits and selectively block interactions between betagamma subunits and a subset of effectors in vitro (Scott, J. K., Huang, S. F., Gangadhar, B. P., Samoriski, G. M., Clapp, P., Gross, R. A., Taussig, R., and Smrcka, A. V. (2001) EMBO J. 20, 767-776). Here, we created cell-permeating versions of some of these peptides by N-terminal modification with either myristate or the cell permeation sequence from human immunodeficiency virus TAT protein. The myristoylated betagamma-binding peptide (mSIRK) applied to primary rat arterial smooth muscle cells caused rapid activation of extracellular signal-regulated kinase 1/2 in the absence of an agonist. This activation did not occur if the peptide lacked a myristate at the N terminus, if the peptide had a single point mutation to eliminate betagamma subunit binding, or if the cells stably expressed the C terminus of betaARK1. A human immunodeficiency virus TAT-modified peptide (TAT-SIRK) and a myristoylated version of a second peptide (mSCAR) that binds to the same site on betagamma subunits as mSIRK, also caused extracellular signal-regulated kinase activation. mSIRK also stimulated Jun N-terminal kinase phosphorylation, p38 mitogen-activated protein kinase phosphorylation, and phospholipase C activity and caused Ca2+ release from internal stores. When tested with purified G protein subunits in vitro, SIRK promoted alpha subunit dissociation from betagamma subunits without stimulating nucleotide exchange. These data suggest a novel mechanism by which selective betagamma-binding peptides can release G protein betagamma subunits from heterotrimers to stimulate G protein pathways in cells.     

Conformational activation of radixin by G13 protein alpha subunit

G(13) protein, one of the heterotrimeric guanine nucleotide-binding proteins (G proteins), regulates diverse and complex cellular responses by transducing signals from the cell surface presumably involving more than one pathway. Yeast two-hybrid screening of a mouse brain cDNA library identified radixin, a member of the ERM family of three closely related proteins (ezrin, radixin, and moesin), as a protein that interacted with Galpha(13). Interaction between radixin and Galpha(13) was confirmed by in vitro binding assay and by co-immunoprecipitation technique. Activated Galpha(13) induced conformational activation of radixin, as determined by binding of radixin to polymerized F-actin and by immunofluorescence in intact cells. Finally, two dominant negative mutants of radixin inhibited Galpha(13)-induced focus formation of Rat-1 fibroblasts but did not affect Ras-induced focus formation. Our results identifying a new signaling pathway for Galpha(13) indicate that ERM proteins can be activated by and serve as effectors of heterotrimeric G proteins.     

Cell cycle-dependent phosphorylation and ubiquitination of a G protein alpha subunit

A diverse array of external stimuli, including most hormones and neurotransmitters, bind to cell surface receptors that activate G proteins. Mating pheromones in yeast Saccharomyces cerevisiae activate G protein-coupled receptors and initiate events leading to cell cycle arrest in G(1) phase. Here, we show that the Gα subunit (Gpa1) is phosphorylated and ubiquitinated in response to changes in the cell cycle. We systematically screened 109 gene deletion strains representing the non-essential yeast kinome and identified a single kinase gene, ELM1, as necessary and sufficient for Gpa1 phosphorylation. Elm1 is expressed in a cell cycle-dependent manner, primarily at S and G(2)/M. Accordingly, phosphorylation of Gpa1 in G(2)/M phase leads to polyubiquitination in G(1) phase. These findings demonstrate that Gpa1 is dynamically regulated. More broadly, they reveal how G proteins can simultaneously regulate, and become regulated by, progression through the cell cycle.     

Regulation of phospholipase D2 activity by protein kinase C alpha

It has been well documented that protein kinase C (PKC) plays an important role in regulation of phospholipase D (PLD) activity. Although PKC regulation of PLD1 activity has been studied extensively, the role of PKC in PLD2 regulation remains to be established. In the present study it was demonstrated that phorbol 12-myristate 13-acetate (PMA) induced PLD2 activation in COS-7 cells. PLD2 was also phosphorylated on both serine and threonine residues after PMA treatment. PKC inhibitors Ro-31-8220 and bisindolylmaleimide I inhibited both PMA-induced PLD2 phosphorylation and activation. However, G? 6976, a PKC inhibitor relatively specific for conventional PKC isoforms, almost completely abolished PLD2 phosphorylation by PMA but only slightly inhibited PLD2 activation. Furthermore, time course studies showed that phosphorylation of PLD2 lagged behind its activation by PMA. Concentration curves for PMA action on PLD2 phosphorylation and activation also showed that PLD2 was activated by PMA at concentrations at which PMA didn't induce phosphorylation. A kinase-deficient mutant of PKCalpha stimulated PLD2 activity to an even higher level than wild type PKCalpha. Co-expression of wild type PKCalpha, but not PKCdelta, greatly enhanced both basal and PMA-induced PLD2 phosphorylation. A PKCdelta-specific inhibitor, rottlerin, failed to inhibit PMA-induced PLD2 phosphorylation and activation. Co-immunoprecipitation studies indicated an association between PLD2 and PKCalpha under basal conditions that was further enhanced by PMA. Time course studies of the effects of PKCalpha on PLD2 showed that as the phosphorylation of PLD2 increased, its activity declined. In summary, the data demonstrated that PLD2 is activated and phosphorylated by PMA and PKCalpha in COS-7 cells. However, the phosphorylation is not required for PKCalpha to activate PLD2. It is suggested that interaction rather than phosphorylation underscores the activation of PLD2 by PKC in vivo and that phosphorylation may contribute to the inactivation of the enzyme.     

Neuronal expression of a newly identified Drosophila melanogaster G protein alpha 0 subunit.

Guanine nucleotide-binding proteins (G proteins) mediate signals between activated cell-surface receptors and cellular effectors. A bovine G-protein alpha-subunit cDNA has been used to isolate similar sequences from Drosophila genomic and cDNA libraries. One class, which we call DG alpha 0, hybridized to position 47A on the second chromosome of Drosophila. The nucleotide sequence of the protein coding region of one cDNA has been determined, revealing an alpha subunit that is 81% identical with rat alpha 0. The cDNA hybridizes strongly to a 3.8 kb mRNA and weakly with a 5.3 kb message. Antibodies raised against a trp-E-DG alpha 0 fusion protein recognized a 39,000 Da protein in Drosophila extracts. In situ hybridization to adult Drosophila sections combined with immunohistochemical studies revealed expression throughout the optic lobes and central brain and in the thoracic and abdominal ganglia. DG alpha 0 message and protein were also detected in the antennae, oocytes, and ovarian nurse cells. The neuronal expression of this gene is similar to mammalian alpha 0, which is most abundantly expressed in the brain.     

Differential regulation of G protein alpha subunit trafficking by mono- and polyubiquitination

Previously we used mass spectrometry to show that the yeast G protein alpha subunit Gpa1 is ubiquitinated at Lys-165, located within a subdomain not present in other G alpha proteins (Marotti, L. A., Jr., Newitt, R., Wang, Y., Aebersold, R., and Dohlman, H. G. (2002) Biochemistry 41, 5067-5074). Here we describe the functional role of Gpa1 ubiquitination. We find that Gpa1 expression is elevated in mutants deficient in either proteasomal or vacuolar protease function. Vacuolar protease pep4 mutants accumulate monoubiquitinated Gpa1, and much of the protein is localized within the vacuolar compartment. In contrast, proteasome-defective rpt6/cim3 mutants accumulate polyubiquitinated Gpa1, and in this case the protein exhibits cytoplasmic localization. Cells that lack Ubp12 ubiquitin-processing protease activity accumulate both mono- and polyubiquitinated forms of Gpa1. In this case, Gpa1 accumulates in both the cytoplasm and vacuole. Finally, a Gpa1 mutant that lacks the ubiquitinated subdomain remains unmodified and is predominantly localized at the plasma membrane. These data reveal a strong relationship between the extent of ubiquitination and trafficking of the G protein alpha subunit to its site of degradation.     

Signal transduction by Tga3, a novel G protein alpha subunit of Trichoderma atroviride

Trichoderma species are used commercially as biocontrol agents against a number of phytopathogenic fungi due to their mycoparasitic characterisitics. The mycoparasitic response is induced when Trichoderma specifically recognizes the presence of the host fungus and transduces the host-derived signals to their respective regulatory targets. We made deletion mutants of the tga3 gene of Trichoderma atroviride, which encodes a novel G protein alpha subunit that belongs to subgroup III of fungal Galpha proteins. Deltatga3 mutants had changes in vegetative growth, conidiation, and conidial germination and reduced intracellular cyclic AMP levels. These mutants were avirulent in direct confrontation assays with Rhizoctonia solani or Botrytis cinerea, and mycoparasitism-related infection structures were not formed. When induced with colloidal chitin or N-acetylglucosamine in liquid culture, the mutants had reduced extracellular chitinase activity even though the chitinase-encoding genes ech42 and nag1 were transcribed at a significantly higher rate than they were in the wild type. Addition of exogenous cyclic AMP did not suppress the altered phenotype or restore mycoparasitic overgrowth, although it did restore the ability to produce the infection structures. Thus, T. atroviride Tga3 has a general role in vegetative growth and can alter mycoparasitism-related characteristics, such as infection structure formation and chitinase gene expression.     

Regulation of phospholipase C by G proteins

Specific phospholipase C enzymes can hydrolyse phosphatidylinositol 4,5-bisphosphate into two products: inositol 1,4,5-trisphosphate, which regulates the release of intracellular calcium stores, and diacylglycerol, which can stimulate protein kinase C. A new group of G proteins, the G_q subfamily, have recently been shown to mediate the regulation of this activity by a variety of hormones. How do different members of this family modulate unique phospholipase C isozymes? What is the mechanism of this regulation? How might the G_q subfamily act to modulate other important second messenger pathways? The tools to answer these questions are being rapidly developed.     

Immunolocalization of the G protein alpha subunit encoded by the GPA1 gene in Arabidopsis.

Heterotrimeric GTP binding proteins (G proteins) are important signal transducers in lower eukaryotes and in animal cells. In plants, the occurrence of GTP binding proteins has been reported, but their biological function remains unclear. Two genes coding for G protein alpha subunits have been cloned: GPA1 in Arabidopsis and TGA1 in tomato. To gain some insights into the function of GPA1, we describe an extensive immunolocalization of GPalpha1, the gene product of GPA1, during Arabidopsis development. Our results show that the GPalpha1 is present through all stages of development and in all organs examined, with the exception of mature seeds. It is expressed in roots, floral stem, rosette leaves, cauline leaves, flowers, and seed pods. Interestingly, the level of GPalpha1 protein is higher in immature organs than in mature organs. GPalpha1 is present at a high level in the root meristem and elongation zone, in the shoot and floral meristems, and in the leaf primordium and floral organ (sepal, petal, stamen, and gynoecium) primordia. During flower development, dividing microspores, but not mature pollen, show high levels of GPalpha1. During pollination, GPalpha1 is present in the growing pollen tubes. The protein is also present in nectaries and developing ovules and, after fertilization, in developing embryos. In mature tissue, GPalpha1 is preferentially found in the vascular system but is also present in other cell types. The complexity of the GPalpha1 localization pattern suggests that GPalpha1 might be involved in different signaling pathways depending on the developmental stage.     

Dual regulation of phospholipase D1 by protein kinase C alpha in vivo

The regulation of phospholipase D1 (PLD1), which has been shown to be activated by protein kinase C (PKC) alpha, was investigated in the human melanoma cell lines. In G361 cell line, which lacks PKCalpha, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced PLD activation was potentiated by introducing PKCalpha by the adenovirus vector. The kinase-negative PKCalpha elevated TPA-induced PLD activity less significantly than the wild type. A PKC specific inhibitor GF109203X lowered PLD activation in the cells expressing PKCalpha, but did not prevent PLD potentiation induced by the kinase-negative PKCalpha. Expression of PKCbetaII and the kinase-negative PKCbetaII enhanced TPA-stimulated PLD activity moderately in MeWo cell line, in which PKCbetaII is absent. Furthermore, the TPA treatment increased the association of PKCalpha, PKCbetaII, and their kinase-negative mutants with PLD1 in melanoma cells. These results indicate that PLD1 is dually regulated through phosphorylation as well as through the protein-protein interaction by PKCalpha, and probably by PKCbetaII, in vivo.     

Isolation and sequence analysis of TGA1 cDNAs encoding a tomato G protein alpha subunit.

We have isolated cDNAs for a gene coding for a G protein alpha subunit from tomato (Lycopersicon esculentum, cv. VF36). This gene, named TGA1, was isolated using a cDNA of the Arabidopsis thaliana G protein alpha subunit-encoding gene, GPA1, as a DNA probe. The sequences of four cDNA clones indicate that the deduced amino acid (aa) sequence of the gene product (TG alpha 1) has 384 aa (44906 Da). The predicted TG alpha 1 protein exhibits similarity to all known G protein alpha subunits. The aa are 84.6% identical and 93% similar (identical and conservative changes) to A. thaliana GP alpha 1, and 34% identical and 59% similar to mammalian transducins. Furthermore, it has all of the consensus regions for a GTP-binding protein. Finally, hybridizations of tomato genomic DNA indicate that TGA1 is a single-copy gene.     

One-step affinity purification of the G protein betagamma subunits from bovine brain using a histidine-tagged G protein alpha subunit

An efficient one-step affinity purification of bovine brain G protein betagamma subunits (betagamma's) is described. The betagamma's, in a detergent extract of brain membranes, are first dissociated from the alpha subunits (alpha's), reassociated with decahistidine-tagged alphail produced in bacteria, and then adsorbed onto Ni2+-nitrilotriacetic acid-agarose via the histidine tag. This mild adsorption retained the high activity of the ligand alpha's, in contrast to the commonly used chemical crosslinking methods. A wash step with a buffer containing chaotropic ions (SCN-) completely removed contaminating proteins that were refractory to washes with high concentrations of detergents, after which the highly purified betagamma's were eluted with a buffer containing Al3+, Mg2+, and F- ions. The obtained betagamma's were found to be fully functional, as assessed by their ability to support pertussis toxin-catalyzed ADP-ribosylation of alphail. Since the combination of the mild adsorption via the histidine tag and the wash with chaotropic ions can be easily applied to purifying betagamma's from various animal tissues, this new chromatographic method is expected to facilitate the purification of other membrane proteins that bind to Galpha and/or Galphabetagamma.     

Activation of protein kinase D by signaling through Rho and the alpha subunit of the heterotrimeric G protein G13

Protein kinase D (PKD/PKCmu) immunoprecipitated from COS-7 cells transiently transfected with either a constitutively active mutant of Rho (RhoQ63L) or the Rho-specific guanine nucleotide exchange factor pOnco-Lbc (Lbc) exhibited a marked increase in basal activity. Addition of aluminum fluoride to cells co-transfected with PKD and wild type Galpha(13) also induced PKD activation. Co-transfection of Clostridium botulinum C3 toxin blocked activation of PKD by RhoQ63L, Lbc, or aluminum fluoride-stimulated Galpha(13). Treatment with the protein kinase C inhibitors GF I or Ro 31-8220 prevented the increase in PKD activity induced by RhoQ63L, Lbc, or aluminum fluoride-stimulated Galpha(13). PKD activation in response to Galpha(13) signaling was also completely prevented by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Co-expression of C. botulinum C3 toxin and a COOH-terminal fragment of Galpha(q) that acts in a dominant-negative fashion blocked PKD activation in response to agonist stimulation of bombesin receptor. Expression of the COOH-terminal region of Galpha(13) also attenuated PKD activation in response to bombesin receptor stimulation. Our results show that Galpha(13) contributes to PKD activation through a Rho- and protein kinase C-dependent signaling pathway and indicate that PKD activation is mediated by both Galpha(q) and Galpha(13) in response to bombesin receptor stimulation.     

Activation of protein kinase D by signaling through the alpha subunit of the heterotrimeric G protein G(q)

Protein kinase D (PKD/PKCmu) immunoprecipitated from COS-7 cells transiently transfected with a constitutively active alpha subunit of G(q) (Galpha(q)Q209L) exhibited a marked increase in basal activity, which was not further enhanced by treatment of the cells with phorbol 12,13-dibutyrate. In contrast, transient transfection of COS-7 cells with activated Galpha(12)Q229L or Galpha(13)Q226L neither promoted PKD activation nor interfered with the increase of PKD activity induced by phorbol 12,13-dibutyrate. The addition of aluminum fluoride to cells co-transfected with PKD and wild type Galpha(q) induced a marked increase in PKD activity, which was comparable with that induced by expression of Galpha(q)Q209L. Treatment with the protein kinase C inhibitor GF I or Ro 31-8220 prevented the increase in PKD activity induced by aluminum fluoride. Expression of a COOH-terminal fragment of Galpha(q) that acts in a dominant negative fashion attenuated PKD activation in response to agonist stimulation of bombesin receptor. PKD activation in response to either Galpha(q) or bombesin was completely prevented by mutation of Ser(744) and Ser(748) to Ala in the kinase activation loop of PKD. Our results show that Galpha(q) activation is sufficient to stimulate sustained PKD activation via protein kinase C and indicate that the endogenous Galpha(q) mediates PKD activation in response to acute bombesin receptor stimulation.     

Dominant-negative inhibition of pheromone receptor signaling by a single point mutation in the G protein alpha subunit

In yeast, two different constitutive mutants of the G protein alpha subunit have been reported. Gpa1(Q323L) cannot hydrolyze GTP and permanently activates the pheromone response pathway. Gpa1(N388D) was also proposed to lack GTPase activity, yet it has an inhibitory effect on pheromone responsiveness. We have characterized this inhibitory mutant (designated Galpha(ND)) and found that it binds GTP, interacts with G protein betagamma subunits, and exhibits full GTPase activity in vitro. Although pheromone leads to dissociation of the receptor from wild-type G protein, the same treatment promotes stable association of the receptor with Galpha(ND). We conclude that agonist binding to the receptor promotes the formation of a nondissociable complex with Galpha(ND), and in this manner prevents activation of the endogenous wild-type G protein. Dominant-negative mutants may be useful in matching specific receptors and their cognate G proteins and in determining mechanisms of G protein signaling specificity.     

Immunological identification of the alpha subunit of G13, a novel guanine nucleotide binding protein.

An antiserum (13CB) was generated against a synthetic peptide, HDNLKQLMLQ, which is predicted to represent the C-terminal decapeptide of the alpha subunit of the novel G-protein, G13. Competitive ELISA indicated that the antiserum reacted with this peptide but that it showed minimal ability to recognize peptides which represent the equivalent regions of the pertussis toxin-insensitive G-proteins, Gq + G11, G12, G15 + G16, GL1 (also called G14) as Gz, and well as other G-proteins. Immunoblots of human platelet membranes with antiserum 13CB identified a single 43-kDa polypeptide, and while this immunoreactivity was abolished by the presence of the cognate peptide it was not modified by the presence of peptides corresponding to the equivalent region of other G-proteins. Immunoreactivity corresponding to G13 alpha was detected in a range of cell types with human platelets having the highest levels of this polypeptide.     

Activation of the phosphatidylinositol 3-kinase Vps34 by a G protein alpha subunit at the endosome

In the yeast Saccharomyces cerevisiae, the G protein beta gamma subunits are essential for pheromone signaling. The Galpha subunit Gpa1 can also promote signaling, but the effectors in this pathway are not well characterized. To identify candidate Gpa1 effectors, we expressed the constitutively active Gpa1(Q323L) mutant in each of nearly 5000 gene-deletion strains and measured mating-specific responses. Our analysis reveals a requirement for both the catalytic (Vps34) and regulatory (Vps15) subunits of the sole phosphatidylinositol 3-kinase in yeast. We demonstrate that Gpa1 is present at endosomes, where it interacts directly with both Vps34 and Vps15 and stimulates increased production of phosphatidylinositol 3-phosphate. Notably, Vps15 binds to GDP-bound Gpa1 and is predicted to have a seven-WD repeat structure similar to that of known G protein beta subunits. These findings reveal two new components of the pheromone signaling pathway. More remarkably, these proteins appear to comprise a preformed effector-G beta subunit assembly and function at the endosome rather than at the plasma membrane.