Is phosphorylation of the alpha1 subunit at Ser-16 involved in the control of Na,K-ATPase activity by phorbol ester-activated protein kinase C?

The alpha1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufo alpha1 subunits. In cells incubated at 37 degrees C, phorbol 12, 13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally ( approximately 20-30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A(2), and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18 degrees C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing alpha1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase alpha1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.     

Mutation of the Gs protein alpha subunit NH2 terminus relieves an attenuator function, resulting in constitutive adenylyl cyclase stimulation.

G-proteins couple hormonal activation of receptors to the regulation of specific enzymes and ion channels. Gs and Gi are G-proteins which regulate the stimulation and inhibition, respectively, of adenylyl cyclase. We have constructed two chimeric cDNAs in which different lengths of the alpha subunit of Gs (alpha s) have been replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). One chimera, referred to as alpha i(54)/s' replaces the NH2-terminal 61 amino acids of alpha s with the first 54 residues of alpha i. Within this sequence there are 7 residues unique to alpha s, and 16 of the remaining 54 amino acids are nonhomologous between alpha i and alpha s. The second chimera, referred to as alpha i/s(Bam), replaces the first 234 amino acids of alpha s with the corresponding 212 residues of alpha i. Transient expression of alpha i(54)/s in COS-1 cells resulted in an 18- to 20-fold increase in cyclic AMP (cAMP) levels, whereas expression of either alpha i/s(Bam) or the wild-type alpha s polypeptide resulted in only a 5- to 6-fold increase in cellular cAMP levels. COS-1 cells transfected with alpha i showed a small decrease in cAMP levels. Stable expression of the chimeric alpha i(54)/s polypeptide in Chinese hamster ovary (CHO) cells constitutively increased both cAMP synthesis and cAMP-dependent protein kinase activity. CHO clones expressing transfected alpha i/s(Bam) or the wild-type alpha s and alpha i cDNAs exhibited cAMP levels and cAMP-dependent protein kinase activities similar to those in control CHO cells. Therefore, the alpha i(54)/s chimera behaves as a constitutively active alpha s polypeptide, whereas the alpha i/s(Bam) polypeptide is regulated similarly to wild-type alpha s. Expression in cyc-S49 cells, which lack expression of wild-type alpha s, confirmed that the alpha i(54)/s polypeptide is a highly active alpha s molecule whose robust activity is independent of any change in intrinsic GTPase activity. The difference in phenotypes observed upon expression of alpha i(54)/s or alpha i/s(Bam) indicates that the NH2-terminal moieties of alpha s and alpha i function as attenuators of the effector enzyme activator domain which is within the COOH-terminal half of the alpha subunit. Mutation at the NH2 terminus of alpha s relieves the attenuator control of the Gs protein and results in a dominant active G-protein mutant.     

Cellular distribution of constitutively active mutant parathyroid hormone (PTH)/PTH-related protein receptors and regulation of cyclic adenosine 3',5'-monophosphate signaling by beta-arrestin2

PTH promotes endocytosis of human PTH receptor 1 (PTH1Rc) by activating protein kinase C and recruiting beta-arrestin2. We examined the role of beta-arrestin2 in regulating the cellular distribution and cAMP signaling of two constitutively active PTH1Rc mutants, H223R and T410P. Overexpression of a beta-arrestin2-green fluorescent protein (GFP) conjugate in COS-7 cells inhibited constitutive cAMP accumulation by H223R and T410P in a dose-dependent manner, as well as the response to PTH of both mutant and wild-type PTH1Rcs. The cellular distribution of PTH1Rc-GFP conjugates, fluorescent ligands, and ssarrestin2-GFP was analyzed by fluorescence microscopy in HEK-293T cells. In cells expressing either receptor mutant, a ligand-independent mobilization of beta-arrestin2 to the cell membrane was observed. In the absence of ligand, H223R and wild-type PTH1Rcs were mainly localized on the cell membrane, whereas intracellular trafficking of T410P was also observed. While agonists promoted beta-arrestin2-mediated endocytosis of bot PTH1Rc mutants, antagonists were rapidly internalized only with T410P. The protein kinases inhibitor, staurosporine, significantly decreased internalization of ligand-PTH1Rc mutant complexes, although the recruitment of beta-arrestin2 to the cell membrane was unaffected. Moreover, in cells expressing a truncated wild-type PTH1Rc lacking the C-terminal cytoplasmic domain, agonists stimulated translocation of beta-arrestin2 to the cell membrane followed by ligand-receptor complex internalization without associated beta-arrestin2. In conclusion, cAMP signaling by constitutively active mutant and wild-type PTH1Rcs is inhibited by a receptor interaction with beta-arrestin2 on the cell membrane, possibly leading to uncoupling from G(s)alpha. This phenomenon is independent from protein kinases activity and the receptor C-terminal cytoplasmic domain. In addition, there are differences in the cellular localization and internalization features of constitutively active PTH1Rc mutants H223R and T410P.     

A dominant negative G alpha s mutant is rescued by secondary mutation of the alpha chain amino terminus.

The Gs protein alpha subunit, alpha s, stimulates the activity of adenylyl cyclase. The sequence 223Asp-Val-Gly-Gly-Gln227 in the alpha s polypeptide is predicted to interact with the gamma-phosphate of GTP and mediate the conformational change involved in alpha s activation. Mutation of the alpha s polypeptide within this region at Gly225----Thr had two demonstrative phenotypic effects when expressed in COS-1 cells: the mutant alpha s chain was ineffective in activating adenylyl cyclase and inhibited in a concentration-dependent manner the beta-adrenergic receptor stimulation of cAMP synthesis. Thus, the Gly225----Thr mutation alters the ability of GTP to activate the alpha s chain and when overexpressed the mutant polypeptide exerts a dominant negative phenotype. Mutation at the amino terminus which creates a constitutively active alpha s rescued the inhibited state of the Gly225----Thr mutant when both mutations were encoded in the same polypeptide. This finding defines the amino terminus as a functional regulatory domain controlling the properties of the GTP/GDP binding site of G protein alpha subunit polypeptide chains.     

Recombinant Gq alpha. Mutational activation and coupling to receptors and phospholipase C.

Gq mediates hormonal stimulation of phosphoinositide-specific phospholipase C (PI-PLC). We mutated the alpha subunit of Gq (alpha q) to replace arginine 183 with cysteine. Mutations that substitute cysteine for the corresponding arginine residues of alpha s and alpha i2 constitutively activate their respective effector pathways, creating the gsp and gip2 oncogenes. Transient expression of alpha q-R183C in COS-7 and HEK-293 cells constitutively activates PI-PLC, but wild type (WT) alpha q does not. This suggests that the mutated arginines in alpha s, alpha i2, and alpha q share a common function in regulating the active state of these proteins and that the alpha q gene may serve as a target for oncogenic mutations in human tumors. In an attempt to develop an assay for receptor stimulation of recombinant alpha q, we co-expressed receptors with alpha q-WT. We found that the alpha 2-adrenoceptor stimulates PI-PLC activation in HEK-293 cells in a fashion that depends completely on co-expression of alpha q-WT. These findings create an experimental model, similar to that provided for alpha s by S49 cyc- cells, that should make it possible to analyze receptor and effector coupling by mutant alpha q against a null background.     

Study of the constitutively active form of the alpha subunit of rice heterotrimeric G proteins

We used site-directed mutagenesis to engineer two constitutively active forms of the alpha subunit of a rice heterotrimeric G protein. The recombinant proteins produced from these novel cDNAs had GTP-binding activity but no GTPase activity. A chimeric gene for a constitutively active form of the alpha subunit was introduced into the rice mutant d1, which is defective for the alpha-subunit gene. All the transformants essentially showed a wild-type phenotype compared with normal cultivars, although seed sizes were substantially increased and internode lengths also showed some increase.     

Decreased catalytic subunit mRNA levels and altered catalytic subunit mRNA structure in a cAMP-resistant Chinese hamster ovary cell line.

The mechanisms responsible for decreased levels of cAMP-dependent protein kinase activity in a mutant Chinese hamster ovary cell line have been examined. The cAMP-resistant Chinese hamster ovary 10260 cell line was found to possess only 20% of the cAMP-dependent protein kinase activity found in wild-type cells. The presence of decreased concentrations of the catalytic subunit in these cells was confirmed through binding studies using a radiolabeled, heat-stable inhibitor of the kinase. Cloned Chinese hamster ovary catalytic subunit cDNAs were isolated, characterized, and used as hybridization probes to examine the relative concentrations of catalytic subunit mRNAs in the wild-type and 10260 cell lines. A 40-50% decrease in the concentration of the mRNA for the C alpha isozyme of the catalytic subunit was observed in 10260 cells, as compared with wild-type. This decrease in catalytic subunit mRNA concentration probably accounts for a portion of the decreased kinase activity in the mutant cells. Further analysis of C alpha mRNA by polymerase chain reaction confirmed the decreased expression of C alpha mRNA in 10260 cells and further demonstrated the presence of two different species of C alpha mRNA in the 10260 cells. One species of C alpha cDNAs was indistinguishable from the wild-type cDNA, but the other species was shorter. Nucleotide sequence analysis of the amplified cDNAs led to the identification of a 191-base pair deletion in the shorter cDNA. Gene transfer studies using wild-type and 10260 C alpha cDNAs demonstrated that the longer cDNA from the 10260 cells produced wild-type activity, but the shorter cDNA was inactive. These studies suggest that at least two alterations in gene expression are responsible for decreased cAMP-dependent protein kinase activity in the 10260 cell line. One alteration results in an approximately 2-fold decrease in the concentrations of C alpha mRNA in the cells. The other change produces two species of C alpha mRNA; one of the C alpha mRNAs does not encode an active kinase.     

Phorbol ester-induced redistribution of the ASGP receptor is independent of receptor phosphorylation.

Like virtually all endocytic receptors, the human asialoglycoprotein (ASGP) receptor is phosphorylated by protein kinase C at serine residues within the cytoplasmic domains of its two subunits H1 and H2. Activation of protein kinase C by phorbol esters results in hyperphosphorylation and in a concomitant net redistribution of receptors to intracellular compartments (down-regulation) in HepG2 cells. To test whether there is a causal relationship between receptor hyperphosphorylation and redistribution, we examined the effect of phorbol ester treatment on the ASGP receptor composed of either wild-type subunits or of mutant subunits lacking any cytoplasmic serine residues in transfected NIH3T3 fibroblast and COS-7 cells. Although the wild-type subunits were hyperphosphorylated in fibroblast cells, the distribution of neither the wild-type nor the mutant receptors was affected. In contrast, phorbol ester treatment of transfected COS-7 cells induced down-regulation of both wild-type and mutant receptors. These findings indicate that redistribution of the receptor is independent of its cytoplasmic serines and is not caused by receptor phosphorylation.     

Enhanced casein kinase II activity in COS-1 cells upon overexpression of either its catalytic or noncatalytic subunit.

Casein kinase II consists of catalytic (alpha) and regulatory (beta) subunits complexed into a heterotetrameric alpha 2 beta 2 structure. Full-length cDNAs encoding the alpha and beta subunits of human casein kinase II were subcloned into an expression vector containing the cytomegalovirus promotor, yielding the expression constructs pCMV-alpha and pCMV-beta. Northern analyses of total cellular RNA prepared from COS-1 fibroblasts 65 h after transfection with pCMV-alpha or pCMV-beta or with both expression constructs showed marked specific increases in corresponding alpha and beta subunit RNAs. Immunoblot analysis utilizing anti-casein kinase II antiserum of cytosolic extracts prepared from COS-1 cells co-transfected with pCMV-alpha and pCMV-beta showed 2- and 4-fold increases in immunoreactive alpha and beta subunit protein, respectively, relative to vector-transfected cells. These same cytosolic fractions exhibited an average 5-fold increase in casein kinase II catalytic activity. COS-1 cells transfected with pCMV-alpha alone exhibited a 3-fold increase in immunoreactive alpha subunit protein and a nearly 2-fold increase in cytosolic casein kinase II catalytic activity. Transfection with the cDNA coding for the noncatalytic beta subunit alone also caused a near doubling of cytosolic casein kinase II catalytic activity. No increase in immunoreactive alpha subunit protein was observed in pCMV-beta-transfected cells, and no increase in immunoreactive beta subunit protein was observed in pCMV-alpha-transfected cells. These results indicate that a portion of the endogenous cellular casein kinase II protein is not fully active and that raising the concentration of the alpha or beta subunit stimulates this latent activity.     

A PP2A active site mutant impedes growth and causes misregulation of native catalytic subunit expression

Activity of protein phosphatase 2A (PP2A) is tightly regulated and performs a diverse repertoire of cellular functions. Previously we isolated a dominant-negative active site mutant of the PP2A catalytic (C) subunit using a yeast complementation assay. We have established stable fibroblastic cell lines expressing epitope-tagged versions of the wild-type and H118N mutant C subunits and have used these cells to investigate mechanisms that regulate PP2A activity. Cells expressing the mutant C subunit exhibit a decreased growth rate and a prolonged G1 cell cycle phase. The mutant protein is enzymatically inactive, but extracts made from cells expressing the H118N C subunit show normal levels of total PP2A activity in vitro. The H118N mutant shows reduced binding to the regulatory A subunit, but binds normally to the alpha4 protein, a non-canonical regulator of PP2A. Expression of the H118N mutant interferes with the normal control of C subunit abundance, causing accumulation of the endogenous wild-type protein as well as the mutant transgene product. Our results indicate that the H118N mutant isoform retards C subunit turnover and suggest that PP2A C subunit turnover may be important for normal cell cycle progression.     

The PP2A-associated protein alpha4 plays a critical role in the regulation of cell spreading and migration

Compared with kinases, the role of protein phosphatases in regulating biological functions is less well understood. Here we show that alpha4, a non-catalytic subunit of the protein phosphatase 2A, plays a major role in the control of cell spreading, migration, and cytoskeletal architecture. Fibroblasts lacking alpha4 were impaired in their ability to spread and migrate compared with wild-type cells, whereas enforced expression of alpha4 promoted cell spreading and migration. These effects were not restricted to fibroblasts. Using a T cell-specific alpha4 transgenic mouse model, increased alpha4 expression was found to increase lymphocyte motility and chemotaxis. Elevated alpha4 expression results in an increase in the GTP-bound state of Rac1, and GTP-bound Rac1 was dramatically reduced in alpha4-deficient cells. A constitutively active mutant of Rac1 rescued the defects of cell spreading and migration caused by alpha4 deletion, while inhibition of Rac1 blocked the ability of alpha4 to promote cell migration. Together, these data define a novel role for the protein phosphatase 2A regulatory subunit alpha4 in the regulation of cell spreading and migration.     

The S49 Kin- cell line transcribes and translates a functional mRNA coding for the catalytic subunit of cAMP-dependent protein kinase

The S49 mouse lymphoma mutant cell line Kin- is resistant to the cytotoxic effects of elevated cAMP levels, has no detectable cAMP-dependent protein kinase activity, and has depressed levels of cAMP-binding regulatory subunits. We demonstrate that although the Kin- cell line lacks detectable catalytic subunit protein, these cells express wild-type levels of mRNA for both C alpha and C beta catalytic subunit isoforms. Translation of C alpha mRNA appears to be normal in the Kin- cell, based on the observation that C alpha mRNA associates with large polyribosomes in both wild-type and Kin- cells. We cloned the C alpha cDNA from Kin- cells and show that its transient expression in another cell type leads to activation of a cAMP-sensitive luciferase reporter gene, suggesting that functional C alpha protein is made. In addition to having catalytic activity, the C alpha subunit from Kin- cells is inhibited in the presence of mouse RI alpha regulatory subunit, indicating that formation of the holoenzyme complex is normal. We suggest that the mutation responsible for the Kin- phenotype is in a cellular component that directly or indirectly causes Kin- catalytic subunit protein to be degraded rapidly.     

A kinase-negative mutant of S49 mouse lymphoma cells is defective in posttranslational maturation of catalytic subunit of cyclic AMP-dependent protein kinase.

Kinase-negative mutants of S49 mouse lymphoma cells, which lack detectable catalytic (C) subunit of cyclic AMP-dependent protein kinase, nevertheless contain cytoplasmic mRNAs for the two major forms of C subunit, C alpha and C beta. Investigation of the metabolism of C subunits in wild-type and mutant cells was undertaken to identify the step(s) at which C subunit expression was defective in kinase-negative cells. [35S]methionine-labeled C subunits from cytosolic fractions of wild-type S49 cells or C subunit-overexpressing cell lines were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after purification by either affinity chromatography using a peptide inhibitor of C subunit as the ligand or immunoadsorption with an anti-C subunit antiserum. Immunoadsorption revealed electrophoretic forms of C alpha and C beta subunits that migrated faster than those detected in affinity-purified samples; this unexpected heterogeneity suggested that functional activation of C subunit may require posttranslational modification. Immunoadsorption of cytosolic fractions from wild-type cells labeled for various times with [35S]methionine revealed an additional posttranslational maturation step. The bulk of immunoadsorbable C subunit label in cells pulse-labeled for 5 min or less was in an insoluble fraction from which it could be solubilized with a detergent-containing buffer; solubilization of the newly synthesized material proceeded over an incubation period of about 10 min. The primary defect in kinase-negative cells appeared to be in this solubilization step, since about equal C subunit radioactivity was found in detergent extracts of wild-type and kinase-negative cells but very little was found in mutant cytosols. I speculate that an accessory factor required for proper folding of newly synthesized C subunit in defective in the kinase-negative cells.     

Analysis of the dominance of mutations in cAMP-binding sites of murine type I cAMP-dependent protein kinase in activation of kinase from heterozygous mutant lymphoma cells.

Structural lesions in cAMP-binding sites of regulatory (R) subunit of cAMP-dependent protein kinase caused identical increases in apparent constants for cyclic nucleotide-dependent kinase activation in preparations from cells that were hemizygous or heterozygous for mutant R1 subunit expression. No wild-type kinase activation was observed in extracts from heterozygous mutant cells. This "dominance" was investigated by characterizing expression of wild-type and mutant R1 subunits and properties of protein kinase from S49 mouse lymphoma cell mutants heterozygous for expression of wild-type R1 subunits and R1 subunits with a lesion (Glu200) that inactivates cAMP-binding site A. By both studies of cAMP dissociation and two-dimensional gel analysis, wild-type R subunits comprised about 35% of total R1 subunits in heterozygous mutants. Synthesis of wild-type and mutant R1 subunits was equivalent, but wild-type subunits were degraded preferentially. Hydroxylapatite chromatography revealed a novel R1 subunit-containing species from heterozygous mutant preparations whose elution behavior suggested a trimeric kinase consisting of an R1 subunit dimer and one catalytic (C) subunit. Wild-type R1 subunit was found only in dimer and "trimer" peaks; the tetrameric kinase peak contained only mutant R1 subunit. It is concluded that C subunit binds preferentially to mutant R1 subunit in heterozygous cells forming either tetrameric kinase with mutant R1 subunit homodimers or trimeric kinase with R1 subunit heterodimers. This preferential binding results both in suppression of wild-type kinase activation and differential stabilization of mutant R1 subunits.     

Propionyl coenzyme A carboxylase is required for development of Myxococcus xanthus.

A dcm-1 mutant, obtained by transposon mutagenesis of Myxococcus xanthus, could aggregate and form mounds but was unable to sporulate under nutrient starvation. A sequence analysis of the site of insertion of the transposon showed that the insertion lies within the 3' end of a 1,572-bp open reading frame (ORF) designated the M. xanthus pccB ORF. The wild-type form of the M. xanthus pccB gene, obtained from a lambdaEMBL library of M. xanthus, shows extensive similarity to a beta subunit of propionyl coenzyme A (CoA) carboxylase, an alpha subunit of methylmalonyl-CoA decarboxylase, and a 12S subunit of transcarboxylase. In enzyme assays, extracts of the dcm-1 mutant were deficient in propionyl-CoA carboxylase activity. This enzyme catalyzes the ATP-dependent carboxylation of propionyl-CoA to yield methylmalonyl-CoA. The methylmalonyl-CoA rescued the dcm-1 mutant fruiting body and spore development. During development, the dcm-1 mutant cells also had reduced levels of long-chain fatty acids (C16 to C18) compared to wild-type cells.     

Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides.

We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.     

Serine 19 of human 6-pyruvoyltetrahydropterin synthase is phosphorylated by cGMP protein kinase II.

6-Pyruvoyltetrahydropterin synthase (PTPS) participates in tetrahydrobiopterin cofactor biosynthesis. We previously identified in a PTPS-deficient patient an inactive PTPS allele with an Arg(16) to Cys codon mutation. Arg(16) is located in the protein surface exposed phosphorylation motif Arg(16)-Arg-Ile-Ser, with Ser(19) as the putative phosphorylation site for serine-threonine protein kinases. Purification of recombinant PTPS-S19A from bacterial cells resulted in an active enzyme (k(cat)/K(m) = 6.4 x 10(3) M(-1) s(-1)), which was similar to wild-type PTPS (k(cat)/K(m) = 4.1 x 10(3) M(-1) s(-1)). In assays with purified enzymes, wild-type but not PTPS-S19A was a specific substrate for the cGMP-dependent protein kinase (cGK) type I and II. Upon expression in COS-1 cells, PTPS-S19A was stable but not phosphorylated and had a reduced activity of approximately 33% in comparison to wild-type PTPS. Extracts from several human cell lines, including brain, contained a kinase that bound to and phosphorylated immobilized wild-type, but not mutant PTPS. Addition of cGMP stimulated phosphotransferase activity 2-fold. Extracts from transfected COS-1 cells overexpressing cGKII stimulated Ser(19) phosphorylation more than 100-fold, but only 4-fold from cGKI overexpressing cells. Moreover, fibroblast extracts from mice lacking cGKII exhibited significantly reduced phosphorylation of PTPS. These results suggest that Ser(19) of human PTPS may be a substrate for cGKII phosphorylation also in vivo, a modification that is essential for normal activity.     

Activated G alpha q inhibits p110 alpha phosphatidylinositol 3-kinase and Akt

Some Gq-coupled receptors have been shown to antagonize growth factor activation of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. We used a constitutively active Galphaq(Q209L) mutant to explore the effects of Galphaq activation on signaling through the PI3K/Akt pathway. Transient expression of Galphaq(Q209L) in Rat-1 fibroblasts inhibited Akt activation induced by platelet-derived growth factor or insulin treatment. Expression of Galphaq(Q209L) also attenuated Akt activation promoted by coexpression of constitutively active PI3K in human embryonic kidney 293 cells. Galphaq(Q209L) had no effect on the activity of an Akt mutant in which the two regulatory phosphorylation sites were changed to acidic amino acids. Inducible expression of Galphaq(Q209L) in a stably transfected 293 cell line caused a decrease in PI3K activity in p110alpha (but not p110beta) immunoprecipitates. Receptor activation of Galphaq also selectively inhibited PI3K activity in p110alpha immunoprecipitates. Active Galphaq still inhibited PI3K/Akt in cells pretreated with the phospholipase C inhibitor U73122. Finally, Galphaq(Q209L) co-immunoprecipitated with the p110alpha-p85alpha PI3K heterodimer from lysates of COS-7 cells expressing these proteins, and incubation of immunoprecipitated Galphaq(Q209L) with purified recombinant p110alpha-p85alpha in vitro led to a decrease in PI3K activity. These results suggest that agonist binding to Gq-coupled receptors blocks Akt activation via the release of active Galphaq subunits that inhibit PI3K. The inhibitory mechanism seems to be independent of phospholipase C activation and might involve an inhibitory interaction between Galphaq and p110alpha PI3K.     

Induction of erythroid differentiation by altered Galpha16 activity as detected by a reporter gene assay in MB-02 cells

Heterotrimeric G proteins may assume modulatory roles in cellular proliferation and differentiation. The G protein alpha-subunit Galpha16, which is specifically expressed in hematopoietic cells, is highly regulated during differentiation of normal and leukemic cells. In human erythroleukemia cells, suppression of Galpha16 inhibited cellular growth rates. A reporter gene system was established to assess the role of Galpha16 on erythroid differentiation of MB-02 erythroleukemia cells. It is based on transient transfection with a plasmid that expresses green fluorescent protein under the control of the beta-globin promoter. Expression of Galpha16 led to a significant increase in green fluorescent protein-positive cells, as did transfection with a Galpha16 antisense plasmid (154 and 156% of controls, respectively). The GTPase-deficient, constitutively active mutant of Galpha16, Galpha16R186C, further stimulated differentiation to 195% of control values. Because the effect of Galpha16 is triggered most efficiently by the GTP-bound protein, an indirect action through interference of overexpressed Galpha16 with G protein betagamma-subunits can be excluded. The corresponding mutant of Galphaq (GalphaqR182C), the phylogenetically closest family member of Galpha16, had no effect. The data define a specific role for Galpha16-dependent signal transduction in cellular differentiation: deviations from optimal levels of Galpha16 functional activity lead to reduced growth rates and promote differentiation in hematopoietic cells.     

Genetic identification of residues involved in association of alpha and beta G-protein subunits.

The GPA1, STE4, and STE18 genes of Saccharomyces cerevisiae encode the alpha, beta, and gamma subunits, respectively, of a G protein involved in the mating response pathway. We have found that mutations G124D, W136G, W136R, and delta L138 and double mutations W136R L138F and W136G S151C of the Ste4 protein cause constitutive activation of the signaling pathway. The W136R L138F and W136G S151C mutant Ste4 proteins were tested in the two-hybrid protein association assay and found to be defective in association with the Gpa1 protein. A mutation at position E307 of the Gpa1 protein both suppresses the constitutive signaling phenotype of some mutant Ste4 proteins and allows the mutant alpha subunit to physically associate with a specific mutant G beta subunit. The mutation in the Gpa1 protein is adjacent to the hinge, or switch, region that is required for the conformational change which triggers subunit dissociation, but the mutation does not affect the interaction of the alpha subunit with the wild-type beta subunit. Yeast cells constructed to contain only the mutant alpha and beta subunits mate and respond to pheromones, although they exhibit partial induction of the pheromone response pathway. Because the ability of the modified G alpha subunit to suppress the Ste4 mutations is allele specific, it is likely that the residues defined by this analysis play a direct role in G-protein subunit association.