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G L Johnson

Publications and source records attributed to G L Johnson.

At least 145 records · Page 8Linked to original sources
PubMed1993

Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation.

Cyclic AMP (cAMP)-dependent protein kinase A (PKA) stimulates the transcription of many eucaryotic genes by catalyzing the phosphorylation of the cAMP-regulatory element binding protein (CREB). Conversely, the attenuation or inhibition of cAMP-stimulated gene transcription would require the dephosphorylation of CREB by a nuclear protein phosphatase. In HepG2 cells treated with the protein serine/threonine (Ser/Thr) phosphatase inhibitor okadaic acid, dibutyryl-cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) promoter was enhanced over the level of PEPCK gene transcription observed in cells treated with dibutyryl-cAMP alone. This process was mediated, at least in part, by a region of the PEPCK promoter that binds CREB. Likewise, okadaic acid prevents the dephosphorylation of PKA-phosphorylated CREB in rat liver nuclear extracts and enhances the ability of PKA to stimulate transcription from the PEPCK promoter in cell-free reactions. The ability of okadaic acid to enhance PKA-stimulated transcription in vitro was entirely dependent on the presence of CREB in the reactions. The phospho-CREB (P-CREB) phosphatase activity present in nuclear extracts coelutes with protein Ser/Thr phosphatase type 2A (PP2A) on Mono Q, amino-hexyl Sepharose, and heparin agarose columns and was chromatographically resolved from nuclear protein Ser/Thr-phosphatase type 1 (PP1). Furthermore, P-CREB phosphatase activity in nuclear extracts was unaffected by the heat-stable protein inhibitor-2, which is a potent and selective inhibitor of PP1. Nuclear PP2A dephosphorylated P-CREB 30-fold more efficiently than did nuclear PP1. Finally, when PKA-phosphorylated CREB was treated with immunopurified PP2A and PP1, the PP2A-treated CREB did not stimulate transcription from the PEPCK promoter in vitro, whereas the PP1-treated CREB retained the ability to stimulate transcription. Nuclear PP2A appears to be the primary phosphatase that dephosphorylates PKA-phosphorylated CREB.

Amino Acid Sequence↗
PubMed1993

Mapping of sites on the Src family protein tyrosine kinases p55blk, p59fyn, and p56lyn which interact with the effector molecules phospholipase C-gamma 2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase.

Engagement of the B-cell antigen receptor complex induces immediate activation of receptor-associated Src family tyrosine kinases including p55blk, p59fyn, p53/56lyn, and perhaps p56lck, and this response is accompanied by tyrosine phosphorylation of distinct cellular substrates. These kinases act directly or indirectly to phosphorylate and/or activate effector proteins including p42 (microtubule-associated protein kinase) (MAPK), phospholipases C-gamma 1 (PLC gamma 1) and C-gamma 2 (PLC gamma 2), phosphatidylinositol 3-kinase (PI 3-K), and p21ras-GTPase-activating protein (GAP). Although coimmunoprecipitation results indicate that the Src family protein tyrosine kinases interact physically with some of these effector molecules, the molecular basis of this interaction has not been established. Here, we show that three distinct sites mediate the interaction of these kinases with effectors. The amino-terminal 27 residues of the unique domain of p56lyn mediate association with PLC gamma 2, MAPK, and GAP. Binding to PI 3-K is mediated through the Src homology 3 (SH3) domains of the Src family kinases. Relatively small proportions of cellular PI 3-K, PLC gamma 2, MAPK, and GAP, presumably those which are tyrosine phosphorylated, bind to the SH2 domains of these kinases. Comparative analysis of binding activities of Blk, Lyn, and Fyn shows that these kinases differ in their abilities to associate with MAPK and PI 3-K, suggesting that they may preferentially bind and subsequently phosphorylate distinct sets of downstream effector molecules in vivo. Fast protein liquid chromatography Mono Q column-fractionated MAPK maintains the ability to bind bacterially expressed Lyn, suggesting that the two kinases may interact directly.

Animals↗
PubMed1993

A molecular map of G protein alpha chains in microdissected rat nephron segments.

Membrane-associated guanine nucleotide binding proteins regulate many receptor-mediated signals. Heterogeneity of biochemical and functional properties in nephron segments could be due to differences in G protein expression. To ascertain whether such heterogeneity of G proteins is present in various nephron segments, this study examines the distribution and relative abundance of G protein alpha chains in microdissected medullary thick ascending limb, cortical collecting tubules, outer medullary collecting tubules, proximal inner medullary tubules, and distal inner medullary tubules. Reverse transcription and polymerase chain reactions were employed using oligonucleotides encoding highly conserved regions of all known alpha chains. The cDNA was sequenced for alpha chain identification. The alpha i2 versus alpha s distribution was different in the outer medullary collecting tubules, when compared with the medullary thick ascending limb (P < 0.001) or the cortical collecting tubule, the proximal inner medullary tubules, and the distal inner medullary tubules (P < 0.05). These latter four segments did not significantly differ from each other. A similar analysis was applied to the frequently used line of kidney cells, LLC-PK1, whose exact cellular origin remains unclear. Interestingly, we detected both alpha i2 and alpha i3, while only alpha i2 was detected in the rat distal nephron. No alpha o or alpha z reverse transcription PCR products were detected. In contrast alpha 11 and alpha 14 members of the more recently described alpha q family were detected in the outer medullary collecting tubules and the proximal inner medullary tubules, respectively. We conclude that the majority of nephron segments have a relatively constant distribution of G protein alpha chains.

Animals↗
PubMed1993

G protein amino-terminal alpha i2/alpha s chimeras reveal amino acids important in regulating alpha s activity.

Gs and Gi2 are heterotrimeric G proteins that stimulate and inhibit, respectively, the activity of a common effector, adenylyl cyclase. The Gs and Gi2 alpha subunit polypeptides, alpha s and alpha i2, are 65% homologous in primary sequence. A series of alpha i2/alpha s chimeras and alpha s point mutations were used to map sequences in the alpha s polypeptide that regulate alpha s activity. An amino-terminal region controlling the activation of alpha s was determined to reside within residues Lys-25 to Glu-101. Amino-terminal alpha i2/alpha s chimeras that disrupt this region in alpha s result in an activated alpha s. In contrast, replacement of this entire alpha s sequence with the analogous alpha i2 sequence produces a chimera whose activity is similar to that of the wild-type alpha s polypeptide. The regulation of alpha s activation by the amino-terminal sequence is independent of the intrinsic GTPase function. Inhibition of alpha s GTPase function by the mutation Gln-227 to leucine is additive with the amino-terminal chimera mutations. These mutations appear to independently alter the two rate-limiting steps in activation of the G protein alpha subunit, i.e., GTP hydrolysis and GDP dissociation, allowing subsequent GTP binding. Within this region of alpha s, Arg-42 is just amino-terminal to the G-1 sequence comprising part of the GDP/GTP binding pocket. The G-1 sequence interacts with the alpha- and beta-phosphoryl groups of GDP and GTP. Mutation of alpha s Arg-42 to lysine has modest effects on alpha s activation, but when placed in the background of the glutamine to leucine mutation the alpha sR42K+Q227L mutant polypeptide stimulates cAMP synthesis significantly more than observed with alpha sQ227L expression. Specific mutations in the amino terminus, therefore, have the ability to enhance alpha s activation by influencing the rate of adenylyl cyclase activation, which is independent of GTPase activity.

Adenylyl Cyclases↗
PubMed1992

Regulation of the differentiation of teratocarcinoma cells into primitive endoderm by G alpha i2.

The amount of the heterotrimeric G protein subunit G alpha i2 decreases after the induction of F9 teratocarcinoma cells to become primitive endoderm in the presence of retinoic acid (RA). The reduction of the G alpha i2 protein in F9 cells by antisense RNA expression was associated with (i) loss of receptor-mediated inhibition of adenylyl cyclase; (ii) decreased cell doubling time; (iii) induction of a primitive, endoderm-like phenotype in the absence of RA; and (iv) production of the differentiation marker tissue-type plasminogen activator. Expression of a constitutively active, mutant G alpha i2 blocked RA-induced differentiation. These data suggest the involvement of G alpha i2 in the control of stem cell differentiation and provide insight into the involvement of G proteins in growth regulation.

Adenylyl Cyclases↗
PubMed1992

Myristoylation of the G alpha i2 polypeptide, a G protein alpha subunit, is required for its signaling and transformation functions.

GTPase-inhibiting mutations of the alpha subunit (alpha i2) of the G protein, Gi2, result in constitutive activation of alpha i2 signal transduction functions. GTPase-inhibited alpha i2 mutant polypeptides, referred to as gip2 oncoproteins, have glutamine-205 mutated to leucine (alpha i2Q205L). Expression of the alpha i2Q205L polypeptide inhibits adenylyl cyclase stimulation, constitutively activates p42 mitogen-activated protein kinase, and transforms Rat 1a fibroblasts. The alpha i2 polypeptides are N-terminal-myristoylated, but the function of myristoylation is unclear in alpha i2 signal transduction. We have tested the requirement for myristoylation on the ability of the alpha i2Q205L mutant polypeptide to constitutively regulate signal pathways and cell transformation. When expressed in Rat 1a cells, the nonmyristoylated alpha i2Q205L polypeptide is membrane associated but is unable to regulate adenylyl cyclase or p42 mitogen-activated protein kinase and does not induce cellular transformation. We conclude that myristoylation is absolutely necessary for alpha i2Q205L signal transduction and regulation of effector enzymes in the cell.

Adenylate Cyclase Toxin↗
PubMed1992

NH2-terminal modification of the phosphatase 2A catalytic subunit allows functional expression in mammalian cells.

Functional expression of recombinant wild-type phosphatase 2A catalytic subunit has been unsuccessful in the past. A nine-amino-acid peptide sequence (YP-YDVPDYA) derived from the influenza hemagglutinin protein was used to modify the NH2 and/or COOH terminus of the phosphatase 2A catalytic subunit. Addition of the nine-amino-acid sequence at the NH2 terminus allowed recombinant phosphatase 2A expression as a predominantly cytosolic phosphatase 2A enzyme. The 12CA5 monoclonal antibody that recognizes the nine-amino-acid hemagglutinin peptide sequence was used to immunoprecipitate the epitope-tagged phosphatase 2A catalytic subunit. Assay of the immunoprecipitated epitope-tagged phosphatase 2A demonstrated an okadaic acid-sensitive dephosphorylation of [32P] histone H1 and [32P]myelin basic protein similar to that measured with the wild-type enzyme. Functional phosphatase activity could be demonstrated for the NH2-terminal modified phosphatase 2A catalytic subunit following transient expression in COS cells or stable expression in Rat1a cells. In contrast, the COOH-terminal-modified phosphatase 2A catalytic subunit was very poorly expressed. The NH2-, COOH-modified subunit, having the nine-amino-acid hemagglutinin peptide sequence encoded at both termini of the polypeptide, was also expressed as a functional phosphatase 2A enzyme. Thus, NH2-terminal modification of the phosphatase 2A catalytic subunit results in a functional plasmid-expressed enzyme. The unique nine-amino-acid epitope-tag sequence also provides a method to easily resolve the recombinant phosphatase 2A from the endogenous wild-type gene product and related phosphatases expressed in cells.

Amino Acid Sequence↗
PubMed1992

Mitogen-activated protein kinase activation resulting from selective oncogene expression in NIH 3T3 and rat 1a cells.

Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that are rapidly activated in response to a variety of growth factors in many cell types. MAPKs are activated by phosphorylation of both tyrosine and threonine residues. They are proposed to be key integrators of growth factor receptor transduction systems involving conversion of tyrosine kinase signals to serine/threonine kinase activation. We have studied the influence of specific oncogenes on the regulation of MAPK activity in NIH 3T3 and Rat 1a fibroblasts. In NIH 3T3 cells, ras or raf oncogene expression, but not gip2 oncogene expression, induces a significant constitutive MAPK activation. In contrast, in Rat 1a cells, gip2, but not ras or raf oncogene expression, induces a strong constitutive MAPK activation. The findings indicate that, in a cell type-selective manner, different oncoproteins are capable of causing the constitutive activation of MAPK. However, the magnitude of oncogene-induced MAPK activation is not directly correlated with cellular transformation in either cell type. It appears that expression of only a subset of transforming oncogenes in a specific cell type is able to alter the regulation of the MAPK activation pathway. Thus, the network of cytoplasmic serine/threonine kinases will be differentially regulated when the same oncogene is expressed in different cell types.

3T3 Cells↗
PubMed1992

MAP kinase is constitutively activated in gip2 and src transformed rat 1a fibroblasts.

Rat 1a fibroblasts transformed by the Gi2 oncogene, gip2, exhibit a constitutively elevated mitogen-activated protein (MAP) kinase activity that correlates with enhanced tyrosine phosphorylation of the p42 MAP kinase polypeptide. The MAP kinase activity in gip2 transformed cells is 50-60% of the pertussis toxin-sensitive, thrombin-stimulated activity observed in wild-type Rat 1a cells. A similar activation of MAP kinase is observed in src but not ras or raf transformed Rat 1a cells, indicating that the persistent MAP kinase activity results from the action of the specific oncoprotein and is not the consequence of cellular transformation. The enhanced transactivation function of c-Jun characteristic of the transformed phenotype, measured using a collagenase promoter-CAT reporter gene, is observed in gip2, src, ras, and raf transformed Rat 1a cells. The regulatory networks controlled by the four transforming oncogenes therefore alter the activity of specific transcription factors, but only gip2 and src constitutively activate MAP kinase. The findings demonstrate that the catalytic activity of growth factor-regulated cytoplasmic kinases are selectively and stably activated as a consequence of specific oncogene expression.

Animals↗
PubMed1992

Immunoblots with rhodopsin antisera suggest that a purified mu opioid binding protein has structural characteristics of a G-protein-coupled receptor.

A mu opioid binding protein (OBP), previously purified to homogeneity from bovine striatal membranes, was examined by immunoblotting with six antisera against bovine rhodopsin. An antibody against the carboxyl-terminal tail of rhodopsin and one against membrane-associated rhodopsin gave strong signals at the appropriate molecular mass (65 kDa). An antibody directed against the first cytoplasmic loop of rhodopsin was weakly reactive. Three other antibodies did not recognize OBP. This pattern of crossreactivity was identical to that previously seen with beta-adrenergic receptors. The existence of domains in the OBP, which are antigenically similar to those in two other guanine nucleotide regulatory protein-coupled receptors, supports the hypothesis that mu opioid receptors have the structure characteristic of this receptor family.

Animals↗
PubMed1992

The beta-PDGF receptor induces neuronal differentiation of PC12 cells.

Expression of the mouse beta-PDGF receptor by gene transfer confers PDGF-dependent and reversible neuronal differentiation of PC12 pheochromocytoma cells similar to that observed in response to NGF and basic FGF. A common property of the PDGF, NGF, and basic FGF-induced differentiation response is the requirement for constant exposure of cells to the growth factor. To test the hypothesis that a persistent level of growth factor receptor signaling is required for the maintenance of the neuronal phenotype, we examined the regulation of the serine/threonine-specific MAP kinases after either short- (10 min) or long-term (24 h) stimulation with growth factors. Mono Q FPLC resolved two peaks of growth factor-stimulated MAP kinase activity that coeluted with tyrosine phosphorylated 41- and 43-kDa polypeptides. MAP kinase activity was markedly stimulated (approximately 30-fold) within 5 min of exposure to several growth factors (PDGF, NGF, basic FGF, EGF, and IGF-I), but was persistently maintained at 10-fold above basal activity after 24 h only by the growth factors that also induce PC12 cell differentiation (PDGF, NGF, and basic FGF). Thus the beta-PDGF receptor is in a subset of tyrosine kinase-encoded growth factor receptors that are capable of maintaining continuous signals required for differentiation of PC12 cells. These signals include the constitutive activation of cytoplasmic serine/threonine protein kinases.

Animals↗
PubMed1992

Analysis of the fibroblast transformation potential of GTPase-deficient gip2 oncogenes.

Expression of GTPase-deficient Gi2 alpha subunit (alpha i2) mutant polypeptides and overexpression of the wild-type alpha i2 polypeptide in Rat 1a, Swiss 3T3, and NIH 3T3 fibroblasts altered normal growth regulation and induced a loss of contact inhibition. In Rat 1a cells (but not in NIH 3T3 or Swiss 3T3 cells), expression of the GTPase-deficient alpha i2 mutant polypeptides allowed colony formation in soft agar, which correlated with a loss in anchorage dependence and a decreased serum requirement. The altered growth regulatory properties of Rat 1a cells induced by expression of alpha i2 mutant polypeptides was not significantly inhibited by cotransfection with a dominant negative Ha-ras mutant polypeptide (Asn-17rasH), indicating that the activated Gi2 membrane signal transduction protein is uniquely capable of altering the regulation of Rat 1a cell growth by a predominantly c-ras-independent mechanism. The results show that GTPase-deficient alpha i2 mutant polypeptides have the properties of an oncogene that can induce the phenotypic characteristics of transformation in Rat 1a cells but that only a subset of these changes is observed with NIH 3T3 and Swiss 3T3 cells.

3T3 Cells↗
PubMed1992

Activating transcription factor-2 DNA-binding activity is stimulated by phosphorylation catalyzed by p42 and p54 microtubule-associated protein kinases.

Recent studies have detailed the ability of activating transcription factor-2 (ATF-2) to mediate adenoviral E1a stimulation of gene expression; however, an endogenous regulator for the transcriptional activity of this protein has not been described. To characterize the regulation of ATF-2 activity, we have expressed full-length and truncated peptides corresponding to various regions of the ATF-2 protein in bacteria and the baculovirus insect cell system. Bacterially expressed truncated (350-505) but not full-length ATF-2, was able to bind a consensus cAMP response element-containing oligonucleotide, suggesting the N-terminal moiety may serve as a negative regulator of DNA-binding activity. In contrast, the full-length ATF-2 protein expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus was fully competent to bind DNA. Protein phosphatase 2A reversed the DNA-binding activity by dephosphorylating the ATF-2 polypeptide. Microtubule-associated protein kinase catalyzed the phosphorylation and stimulated the DNA-binding activity of bacterially expressed full-length ATF-2. Phosphopeptide mapping of phosphorylated ATF-2 proteins identified a single peptide in the N-terminal moiety of ATF-2 phosphorylated by p42 or p54 microtubule-associated protein kinase. Therefore, we propose that phosphorylation of this regulatory site is sufficient to induce an allosteric structural change in the ATF-2 protein, which allows dimerization and subsequent DNA binding.

Amino Acid Sequence↗
PubMed1991

GTPase-deficient G alpha i2 oncogene gip2 inhibits adenylylcyclase and attenuates receptor-stimulated phospholipase A2 activity.

The GTPase activity of a G protein alpha subunit functions as a timer to control the lifetime of the activated conformation of the protein. Expression of the GTPase-deficient Gi2 alpha subunit oncogene, gip2 (alpha i2Q205L), in Chinese hamster ovary cells inhibited the stimulation of adenylylcyclase and altered the calcium regulation of the Gi2-phospholipase A2 (PLA2) effector complex. The phenotypic consequence of the activated alpha i2 mutant on hormonal stimulation of PLA2 varied depending on the cytoplasmic calcium transient elicited by different Gi2-linked receptors. The stimulation of PLA2 by thrombin, which mobilized calcium only from internal stores, was markedly attenuated in gip2-expressing cells. In contrast, the attenuation of the PLA2 response to ATP, a purinergic agonist which mobilizes calcium from both extracellular space and internal stores, was significantly less than that observed for thrombin. Ionomycin, a calcium ionophore, stimulated PLA2 activity in clones which expressed gip2 to a level similar to that observed in wild-type Chinese hamster ovary cells. Thus, the dominant GTPase-deficient gip2 polypeptide will constitutively inhibit adenylylcyclase but differentially modulate enzymes regulated by calcium and coupled to Gi2.

Adenosine Triphosphate↗
PubMed1991

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.

Adenylyl Cyclases↗
PubMed1991

Genetic and structural analysis of G protein alpha subunit regulatory domains.

Genetic and structural analysis of the alpha chain polypeptides of heterotrimeric G proteins defines functional domains for GTP/GDP binding, GTPase activity, effector activation, receptor contact and beta gamma subunit complex regulation. The conservation in sequence comprising the GDP/GTP binding and GTPase domains among G protein alpha subunits readily allows common mutations to be made for the design of mutant polypeptides that function as constitutive active or dominant negative alpha chains when expressed in different cell types. Organization of the effector activation, receptor and beta gamma contact domains is similar in the primary sequence of the different alpha subunit polypeptides relative to the GTP/GDP binding domain sequences. Mutation within common motifs of the different G protein alpha chain polypeptides have similar functional consequences. Thus, what has been learned with the Gs and Gi proteins and the regulation of adenylyl cyclase can be directly applied to the analysis of newly identified G proteins and their coupling to receptors and regulation of putative effector enzymes.

Adenosine Diphosphate Ribose↗
PubMed1991

The NH2-terminal alpha subunit attenuator domain confers regulation of G protein activation by beta gamma complexes.

Gs and Gi, respectively, activate and inhibit the enzyme adenylyl cyclase. Regulation of adenylyl cyclase by the heterotrimeric Gs and Gi proteins requires the dissociation of GDP and binding of GTP to the alpha s or alpha i subunit. The beta gamma subunit complex of Gs and Gi functions, in part, to inhibit GDP dissociation and alpha subunit activation by GTP. Multiple beta and gamma polypeptides are expressed in different cell types, but the functional significance for this heterogeneity is unclear. The beta gamma complex from retinal rod outer segments (beta gamma t) has been shown to discriminate between alpha i and alpha s subunits (Helman et al: Eur J Biochem 169:431-439, 1987). beta gamma t efficiently interacts with alpha i-like G protein subunits, but poorly recognizes the alpha s subunit. beta gamma t was, therefore, used to define regions of the alpha i subunit polypeptide that conferred selective regulation compared to the alpha s polypeptide. A series of alpha subunit chimeras having NH2-terminal alpha i and COOH-terminal alpha s sequences were characterized for their regulation by beta gamma t, measured by the kinetics of GTP gamma S activation of adenylyl cyclase. A 122 amino acid NH2-terminal region of the alpha i polypeptide encoded within an alpha i/alpha s chimera was sufficient for beta gamma t to discriminate the chimera from alpha s. A shorter 54 amino acid alpha i sequence substituted for the corresponding NH2-terminal region of alpha s was insufficient to support the alpha i-like interaction with beta gamma t. The findings are consistent with our previous observation (Osawa et al: Cell 63:697-706, 1990) that a region in the NH2-terminal moiety functions as an attenuator domain controlling GDP dissociation and GTP activation of the alpha subunit polypeptide and that the attenuator domain is involved in functional recognition and regulation by beta gamma complexes.

Adenylyl Cyclases↗