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G L Johnson

Publications and source records attributed to G L Johnson.

At least 91 records · Page 5Linked to original sources
PubMed1996

Molecular cloning of mitogen-activated protein/ERK kinase kinases (MEKK) 2 and 3. Regulation of sequential phosphorylation pathways involving mitogen-activated protein kinase and c-Jun kinase.

Mitogen-activated protein/ERK kinase kinases (MEKKs) phosphorylate and activate protein kinases which in turn phosphorylate and activate the p42/44 mitogen-activated protein kinase (MAPK), c-Jun/stress-activated protein kinases (JNKs), and p38/Hog1 kinase. We have isolated the cDNAs for two novel mammalian MEKKs (MEKK 2 and 3). MEKK 2 and 3 encode proteins of 69.7 and 71 kDa, respectively. The kinase domains encoded in the COOH-terminal moiety are 94% conserved; the NH2-terminal moieties are approximately 65% homologous, suggesting this region may encode sequences conferring differential regulation of the two kinases. Expression of MEKK 2 or 3 in HEK293 cells results in activation of p42/44MAPK and JNK but not of p38/Hog1 kinase. Immunoprecipitated MEKK 2 phosphorylated the MAP kinase kinases, MEK 1, and JNK kinase. Titration of MEKK 2 and 3 expression in transfection assays indicated that MEKK 2 preferentially activated JNK while MEKK 3 preferentially activated p42/44MAPK. These findings define a family of MEKK proteins capable of regulating sequential protein kinase pathways involving MAPK members.

Amino Acid Sequence↗
PubMed1996

Signal transduction pathways regulated by mitogen-activated/extracellular response kinase kinase kinase induce cell death.

Mitogen-activated/extracellular response kinase kinase (MEK) kinase (MEKK) is a serine-threonine kinase that regulates sequential protein phosphorylation pathways, leading to the activation of mitogen-activated protein kinases (MAPK), including members of the Jun kinase (JNK)/stress-activated protein kinase (SAPK) family. In Swiss 3T3 and REF52 fibroblasts, activated MEKK induces cell death involving cytoplasmic shrinkage, nuclear condensation, and DNA fragmentation characteristic of apoptosis. Expression of activated MEKK enhanced the apoptotic response to ultraviolet irradiation, indicating that MEKK-regulated pathways sensitize cells to apoptotic stimuli. Inducible expression of activated MEKK stimulated the transactivation of c-Myc and Elk-1. Activated Raf, the serine-threonine protein kinase that activates the ERK members of the MAPK family, stimulated Elk-1 transactivation but not c-Myc; expression of activated Raf does not induce any of the cellular changes associated with MEKK-mediated cell death. Thus, MEKK selectively regulates signal transduction pathways that contribute to the apoptotic response.

3T3 Cells↗
PubMed1996

Interleukin-8 regulation of the Ras/Raf/mitogen-activated protein kinase pathway in human neutrophils.

Interleukin-8 (IL-8), the prototypic member of the CXC subfamily of chemokines, induces in neutrophils chemotaxis, the respiratory burst, granule release, and increased cell adhesion. The IL-8 receptor is a seven-transmembrane spanning receptor coupled to specific heterotrimeric G proteins including Gi and G16. IL-8 stimulation of its receptor on neutrophils activates Ras GTP loading and the mitogen-activated protein kinase (MAPK) pathway including Raf-1 and B-Raf. The properties of IL-8 stimulation of the MAPK pathway differ from those observed for chemoattractants such as C5a. Even though Ras GTP loading is similar for IL-8 and C5a, the maximal activation of Raf-1 and B-Raf is approximately 2-fold and 3-7-fold, respectively, less for IL-8 than that observed for C5a. Raf-1 activation is rapid but transient, returning to near basal levels by 10 min. B-Raf activation is slower in onset and does not return to basal levels for nearly 30 min. IL-8 activation of MAPK follows a time course suggesting an involvement of both Raf-1 and B-Raf. Surprisingly, wortmannin, at low concentrations, inhibits Raf-1, B-Raf, and MAPK activation in response to IL-8 and C5a demonstrating a role for phosphatidylinositol 3-kinase in the activation of Raf kinases in G protein-coupled receptor systems in human neutrophils. Furthermore, wortmannin inhibits IL-8 stimulated granule release and neutrophil adherence. These findings demonstrate the control of Raf kinases, the MAPK pathway and specific neutrophil functions by phosphatidylinositol 3-kinase enzymes.

Androstadienes↗
PubMed1996

Intramuscular administration of ceftiofur sodium versus intramammary infusion of penicillin/novobiocin for treatment of Streptococcus agalactiae mastitis in dairy cows.

OBJECTIVE: To determine the efficacy of intramuscular administration of ceftiofur sodium as treatment for intramammary infections attributable to Streptococcus agalactiae, compared with that for a standard treatment of intramammary infusion of penicillin/novobiocin. DESIGN: Prospective, randomized, controlled trial. ANIMALS: 72 lactating Holstein cows with intramammary infections caused by S agalactiae from 5 commercial dairies in Michigan. PROCEDURE: In 36 of 72 infected cows, ceftiofur was administered (2.2 mg/kg of body weight, IM, q 24 h) for 5 days; 150 mg of novobiocin and 100,000 U of procaine penicillin G was infused daily into each mammary gland of the other 36 cows for 2 days. Milk samples were collected aseptically at approximately 4 and 8 weeks after initial treatment. If cows were determined to be infected at 4 weeks after initial treatment, the treatment was repeated. RESULTS: The cure rate at 4 weeks (91.7%) and at 8 weeks (96.8%) after initial treatment for the penicillin/novobiocin-treated cows was significantly (P < 0.0001) higher, compared with that of the ceftiofur-treated cows (2.8 and 9.1%, respectively). Somatic cell counts were significantly (P < 0.0001) lower in the penicillin/novobiocin-treated group after treatment. CLINICAL IMPLICATIONS: Intramuscular administration of ceftiofur is not efficacious as a treatment to eliminate intramammary infections caused by S agalactiae and should not be used to reduce the prevalence of this organism in dairy herds.

Animals↗
PubMed1996

Discordant signal transduction and growth inhibition of small cell lung carcinomas induced by expression of GTPase-deficient G alpha 16.

Small cell lung carcinoma (SCLC) accounts for 20-25% of primary lung cancers and is rapidly growing, widely metastatic, and rarely curable. Autocrine stimulation of multiple G protein-coupled neuropeptide receptor systems contributes to the transformed growth of SCLC. The ability of neuropeptide receptors to stimulate phospholipase C and mobilize intracellular Ca2+ indicates that Gq family members of heterotrimeric G proteins are a convergence point mediating autocrine signaling by multiple neuropeptides in SCLC. Expression of a GTPase-deficient, constitutive active form of an alpha q family member, alpha 16Q212L, in SCLC markedly inhibited growth of the cells in soft agar and tumor formation in nude mice. SCLC lines expressing alpha 16Q212L exhibited 2-4-fold elevated basal phospholipase C activity, but neuropeptide and hormone-regulated intracellular Ca2+ mobilization was nearly abolished. The data suggest that Ca2+ mobilization is an obligatory signal in neuropeptide-stimulated growth of SCLC. In addition, the proline-directed c-Jun NH2-terminal kinases/stress-activated protein kinases, which are members of the mitogen-activated protein kinase family, were stimulated approximately 2-fold in parental SCLC in response to exogenous neuropeptides and muscarinic agonists and were constitutively activated to the same degree in alpha 16Q212L-expressing SCLC. Thus, alpha 16Q212L expression induced desensitizaton of neuropeptide-stimulated Ca2+ signaling and persistent activation of the c-Jun NH2-terminal kinase/stress-activated protein kinase pathway. We propose that the induction of discordant signaling by selective perturbation of receptor-regulated effector systems leads to the inhibition of SCLC cell growth.

Amino Acid Sequence↗
PubMed1996

Bombesin-like peptide receptors in human bronchial epithelial cells.

Northern blot and RNAse protection assays previously failed to detect bombesin-like peptide (BLP) receptors in normal human lung tissue, but by RT/PCR cultured human bronchial epithelial (HBE) cells expressed all three BLP receptor subtypes, predominantly neuromedin B (NMB) receptor. By RT/PCR, we found expression of all three BLP receptor subtypes by human lung tissue and confirmed NMB receptor expression in six out of six HBE samples. However, transformed HBE BEAS B2B cells expressed only gastrin-releasing peptide (GRP) receptors; saturable, high-affinity (Kd = 3.5 nM) specific [125I]GRP binding confirmed functional GRP receptor, with M(r) = 75 kDa and immunologic cross-reactivity with GRP receptor from human small-cell lung carcinoma (SCLC) NCI-H345 cells. Altered regulation of BLP receptors may accompany transformation of normal lung cells to cancer.

3T3 Cells↗
PubMed1996

Late echocardiographic findings following childhood chemotherapy with normal serial cardiac monitoring.

PURPOSE: Late development of myocardial dysfunction years following successful treatment of childhood malignancy with anthracyclines is well documented. There have been few studies of late cardiac performance in children in whom serial monitoring during treatment suggested normal cardiac performance, and those studies that do exist rely on the results of extensive evaluation. It was our purpose to determine whether findings consistent with known late cardiac changes could be discovered in these patients by echocardiographic monitoring similar to that routinely performed during treatment. PATIENTS AND METHODS: A total 28 consecutive asymptomatic patients who had completed anthracycline therapy at least 3 years previously, had been free of malignant disease since the completion of therapy, and who had had normal serial echocardiographic studies during and at completion of treatment were restudied by echocardiography. Of these 28, 12 had undergone mediastinal radiation as part of their acute treatment. RESULTS: Four patients (14%) of the study group were found to have abnormally low values for left ventricular shortening and ejection fractions. All four had also received mediastinal radiation. The remaining 24 patients, while having values for shortening fraction within the normal range, had, as a group, experienced a significant decrease in echocardiographic left ventricular shortening since completion of treatment. In these patients, left ventricular wall thickness had not increased commensurate with growth in body size and left ventricular cavity dimension. CONCLUSIONS: The known incidence of late asymptomatic cardiac dysfunction is confirmed despite the presence of persistently normal echocardiographic monitoring studies during and at completion of anthracycline treatment. Additionally, as a population, these patients show impaired myocardial growth over time, placing them at risk for future myocardial failure. Normal echocardiographic monitoring studies during antineoplastic treatment in children may not necessarily predict that patients will be free of the development of late cardiac dysfunction. Routine serial echocardiographic monitoring can, however, be helpful in the long-term management of these patients.

Adolescent↗
PubMed1996

Constitutive activation of a phosphoinositidase C-linked G protein in murine fibroblasts decreases agonist-stimulated Ca2+ mobilization.

We compared Ca2+ signaling and inositol polyphosphate metabolism in NIH-3T3 cells stably transfected with cDNA encoding either the wild-type G protein G16 alpha subunit or a GTPase-deficient alpha 16 subunit (Q212L-alpha 16). Constitutive activation of phosphoinositidase C (PIC) in cells expressing Q212L-alpha 16 was demonstrated by 1) an increased basal level of [3H]inositol polyphosphates, 2) an enhanced rate of [3H]inositol polyphosphate accumulation in cells treated with 10 mM LiCl, and 3) an increased rate of incorporation of [3H]inositol into cell lipids. Q212L-alpha 16 cells had a diminished cell growth rate. Basal intracellular Ca2+ concentration was equivalent in Fura-2 acetoxymethyl ester-loaded Q212L-alpha 16 cells compared with controls; however, calcium release in Q212L-alpha 16 cells exposed to ionomycin, ATP (a G protein-linked agonist), or platelet-derived growth factor (a tyrosine kinase-linked agonist) was decreased. Permeabilized, 45Ca-loaded Q212L-alpha 16 cells released less 45Ca at each concentration of inositol-1,4,5-trisphosphate than did control cells. Accordingly, the total amount of inositol trisphosphate (IP3) receptor protein was decreased in Q212L-alpha 16 cells relative to controls. These data demonstrate that Q212L-alpha 16 cells maintain physiological levels of cytoplasmic calcium and partially loaded Ca2+ stores in the face of constitutively active PIC. This is accomplished, at least in part, by down-regulation of IP3 receptor number. Thus, diminution in cell growth rate in Q212L-alpha 16 cells seems to be attributable to a combination of at least two effects: a direct effect of PIC activation leading to partial depletion of Ca2+ stores and an indirect, adaptive response resulting in a decreased IP3 receptor number.

3T3 Cells↗
PubMed1996

CT evaluation of the acute abdomen: bowel pathology spectrum of disease.

CT has become the primary imaging modality for the evaluation of the patient with clinical symptoms of an acute abdomen and a confusing clinical picture. Because these patients may have a range of various pathologies, CT has been used successfully to define the presence of disease and localize it to a specific organ or organ system. In this article, we review the various processes that resulted in acute abdomen focusing on the small bowel and colon. Specific entities discussed include appendicitis, diverticulitis, Crohn disease, and ulcerative colitis. Other less common processes, including pseudomembranous colitis, intussusception, and bowel ischemia are also discussed. The specific role of CT scanning and specific CT signs are discussed and addressed. The value of CT in relationship to other modalities and clinical evaluation is discussed and key statistics provided.

Abdomen, Acute↗
PubMed1995

Selective activation of c-Jun kinase mitogen-activated protein kinase by CD40 on human B cells.

The B cell surface antigen receptor, surface IgM (sIgM), is involved in B cell activation and proliferation. CD40 is involved in regulating IgE production and B cell survival. Cross-linking of B cell sIgM activates the Ras/Raf/p42erk2 pathway. In contrast, ligation of CD40 by antibody or soluble gp39 (CD40 ligand) leads to activation of the c-Jun kinase (JNK)/stress-activated protein kinase pathway. JNK/stress-activated protein kinase activation correlated with the stimulation of MEK kinase activity. CD40 does not activate the p42erk2 pathway, and sIgM fails to regulate the JNK/stress-activated protein kinase pathway in B cells. Thus, two important cell surface receptors involved in controlling specific B cell response differentially regulate sequential protein kinase pathways involving different members of the mitogen-activated protein kinase family. Anti-CD40 also rescued B cell apoptosis induced by anti-IgM. CD40 ligation did not affect the sIgM stimulation of p42erk2 activity. Conversely, sIgM ligation did not influence CD40 stimulation of JNK/stress-activated protein kinase. These results suggest that independent, parallel protein kinase response pathways are involved in the integration of sIgM and CD40 control of B cell phenotype and function.

Antibodies↗
PubMed1995

G alpha 12 and G alpha 13 stimulate Rho-dependent stress fiber formation and focal adhesion assembly.

Rho, a member of the Ras superfamily of GTP-binding proteins, regulates actin polymerization resulting in the formation of stress fibers and the assembly of focal adhesions. In Swiss 3T3 cells, heterotrimeric G protein-coupled receptors for lysophosphatidic acid and gastrin releasing peptide stimulate Rho-dependent stress fiber and focal adhesion formation. The specific heterotrimeric G protein subunits mediating Rho-dependent stress fiber and focal adhesion formation have not been defined previously. We have expressed GTPase-deficient, constitutively activated G protein alpha subunits and mixtures of beta and gamma subunits in Swiss 3T3 cells. Measurement of actin polymerization and focal adhesion formation indicated that GTPase-deficient alpha 12 and alpha 13, but not the activated forms of alpha 12 or alpha q stimulated stress fiber and focal adhesion assembly. Combinations of beta and gamma subunits were unable to stimulate stress fiber or focal adhesion formation. G alpha 12- and alpha 13-mediated stress fiber and focal adhesion assembly was inhibited by botulinum C3 exoenzyme, which ADP-ribosylates and inactivates Rho, indicating that alpha 12 and alpha 13, but not other G protein alpha subunits or beta gamma complexes, regulate Rho-dependent responses. The results define the integration of G12 and G13 with the regulation of the actin cytoskeleton.

3T3 Cells↗
PubMed1995

Mitogen-activated protein kinase activation requires two signal inputs from the human anaphylatoxin C5a receptor.

The anaphylatoxin C5a receptor activates the Ras/Raf/mitogen-activated protein (MAP) kinase pathway in human neutrophils. The signal pathways involved in Ras/Raf/MAP kinase activation in response to C5a and other chemoattractant receptors is poorly understood. Stimulation of the C5a receptor expressed in HEK293 cells results in modest MAP kinase activation, which is inhibited by pertussis toxin-catalyzed ADP-ribosylation of G(i). Coexpression of the C5a receptor and the G16 alpha subunit (alpha 16) results in the G16-mediated activation of phospholipase C beta and a robust MAP kinase activation. Pertussis toxin treatment of C5a receptor/alpha 16-cotransfected cells inhibits C5a stimulation of MAP kinase activity approximately 60% relative to the control response. Similarly, the protein kinase C inhibitor, GF109203X inhibits activation of MAP kinase activation in C5a receptor/alpha 16-cotransfected cells by 60%; the protein kinase C inhibitor does not affect the modest C5a receptor response in the absence of alpha 16 expression. These results demonstrate that two independent signals are required for the maximal activation of MAP kinase by G protein-coupled receptors.

Antigens, CD↗
PubMed1995

Activation of Jun kinase/stress-activated protein kinase by GTPase-deficient mutants of G alpha 12 and G alpha 13.

Signal transduction pathways regulated by G12 and G13 heterotrimeric G proteins are largely unknown. Expression of activated, GTPase-deficient mutants of alpha 12 and alpha 13 alter physiological responses such as Na+/H+ exchanger activity, but the effector pathways controlling these responses have not been defined. We have found that the expression of GTPase-deficient mutants of alpha 12 (alpha 12Q229L) or alpha 13 (alpha 13Q226L) leads to robust activation of the Jun kinase/stress-activated protein kinase (JNK/SAPK) pathway. Inducible alpha 12Q229L and alpha 13Q226L expression vectors stably transfected in NIH 3T3 cells demonstrated JNK/SAPK activation but not extracellular response/mitogen-activated protein kinase activation. Transient transfection of alpha 12Q229L and alpha 13Q226L also activated the JNK/SAPK pathway in COS-1 cells. Expression of the GTPase-deficient mutant of alpha q (alpha qQ209L) but not alpha i (alpha iQ205L) or alpha s (alpha sQ227L) was also able to activate the JNK/SAPK pathway. Functional Ras signaling was required for alpha 12Q229L and alpha 13Q226L activation of the JNK/SAPK pathway; expression of competitive inhibitory N17Ras inhibited JNK/SAPK activation in response to both alpha 12Q229L and alpha 13Q226L. The results describe for the first time a Ras-dependent signal transduction pathway involving JNK/SAPK regulated by alpha 12 and alpha 13.

3T3 Cells↗
PubMed1995

Differential calcium dependence in the activation of c-Jun kinase and mitogen-activated protein kinase by muscarinic acetylcholine receptors in rat 1a cells.

Carbachol stimulation of the muscarinic acetylcholine m1 receptor (m1R), stably expressed in Rat 1a fibroblasts, resulted in a calcium-dependent activation of c-Jun kinase (JNK). Stimulation of the muscarinic acetylcholine m2 receptor (m2R), stably expressed in Rat 1a fibroblasts, resulted in a G1-mediated activation of JNK that was weak relative to that observed with the m1R. Chelation of calcium inhibited the m2R-mediated activation of JNK but not the robust m2R stimulation of mitogen-activated protein kinase (MAPK) activity. These findings demonstrate a role for the second messenger, calcium, in the differential regulation of the activity of JNK and MAPK in Rat 1a cells.

Animals↗
PubMed1995

Regulation of recombinant MEK1 and MEK2b expressed in Escherichia coli.

Mitogen-activated protein kinase (MAPK) activation is an important signal involved in regulating cellular proliferation and/or differentiation. The immediate upstream kinase MAPK kinase, referred to as MEK, activates MAPK by phosphorylation on specific tyrosine and threonine residues. To date, two MEK's have been cloned and characterized, MEK1 and MEK2. Here we report the cloning of MEK2b from mouse pituitary cDNA. Rat and mouse MEK2 amino acid sequences vary by only three amino acids; the three changes are conserved in the MEK1 sequence. Analysis of recombinant MEK2b and MEK1 demonstrated similar activation by upstream kinases and phosphotransferase activity toward MAPK, while they differed in autophosphorylation and the ability to serve as a substrate for MAPK. The findings demonstrate significant differences in potential regulatory mechanisms of MEK1 and MEK2/2b but not in their activation by upstream regulators.

Amino Acid Sequence↗
PubMed1995

Direct interaction between Ras and the kinase domain of mitogen-activated protein kinase kinase kinase (MEKK1).

Mitogen-activated protein kinase kinase kinase (MEKK1) is a serine-threonine kinase that regulates sequential protein kinase pathways involving stress-activated protein kinases and mitogen-activated protein kinases. MEKK1 is activated in response to growth factor stimulation of cells and by expression of activated Ras. We demonstrate that the kinase domain of MEKK1 (MEKKCOOH) binds to GST-RasV12 in a GTP-dependent manner. Purified bacterially expressed MEKKCOOH binds to GST-RasV12(GTP gamma S) (GTP gamma S is guanosine 5'-3-O-(thio)triphosphate), demonstrating a direct interaction of the two proteins. A Ras effector domain peptide blocks the binding of MEKKCOOH to GST-RasV12(GTP gamma S). MEKKCOOH complexed with GST-RasV12(GTP gamma S) is capable of phosphorylating MEK1. These findings indicate that MEKK1 directly binds Ras.GTP. Thus, Ras interacts with protein kinases of both the Raf and MEKK families.

Animals↗
PubMed1995

Differential modulation of bombesin-stimulated phospholipase C beta and mitogen-activated protein kinase activity by [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P.

Mitogenic stimulation of Swiss 3T3 fibroblasts with bombesin results in receptor-mediated activation of a complex array of effectors, including phospholipase C beta and mitogen-activated protein (MAP) kinase. Incubation of Swiss 3T3 fibroblasts with the 11-amino acid [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide inhibited bombesin-stimulated cell proliferation and phospholipase C beta activation even at high bombesin concentrations. The peptide did not inhibit the activation of phospholipase C beta by a GTPase-deficient form of the Gq-like protein, G16, indicating that the peptide does not inhibit phospholipase C beta and is acting at a point upstream of the activated form of the G protein alpha subunit. The peptide inhibited MAP kinase activation at low bombesin concentrations, but unlike phospholipase C beta, this inhibition could be overcome with 30 nM bombesin. In control Swiss 3T3 cells, bombesin did not measurably activate Ras or Raf-1 above basal levels. Following incubation of the cells with the [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide, 50 nM bombesin activated Raf-1 4-6-fold over basal levels. Platelet-derived growth factor-stimulated activities of PLC, Ras, Raf-1, and MAP kinase were unaltered after incubation of Swiss 3T3 cells with the [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide, as was platelet-derived growth factor-stimulated growth of the Swiss 3T3 cells. Thus, the peptide behaves as an antagonist that differentially inhibited phospholipase C beta and MAP kinase signal transduction pathways. The growth arrest observed with the peptide indicates that the bombesin-stimulated activation of MAP kinase is not sufficient to support mitogenesis in Swiss 3T3 cells.

3T3 Cells↗