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Biomedical subjects

M J Weber

Publications and source records attributed to M J Weber.

At least 55 records · Page 3Linked to original sources
PubMed1995

SCH 51344 inhibits ras transformation by a novel mechanism.

A pyrazolo-quinoline compound, 6-methoxy-4-[2-[(2-hydroxyethoxyl)-ethyl]amino]-3-methyl-1M-pyrazo lo [3,4-b]quinoline (SCH 51344), was identified based on its ability to derepress human smooth muscle alpha-actin promoter activity in ras-transformed cells. In this study, we show that SCH 51344 reverts several key aspects of ras transformation, such as morphological changes, actin filament organization, and anchorage-independent growth, and also inhibits Val-12 Ras-induced maturation of Xenopus oocytes. SCH 51344 is also a potent inhibitor of the anchorage-independent growth of human tumor lines known to contain multiple genetic alterations in addition to activated ras genes. We have sought to determine whether SCH 51344 disrupts the signaling pathway that activates mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK) in normal and ras-transformed fibroblast cells. NIH 3T3 cells transformed by different oncogenes, which have products that participate at different steps of the Ras signaling pathway, were tested in a soft-agar colony formation assay to determine which step of the pathway is inhibited by SCH 51344. Our results indicate that SCH 51344 inhibits the ability of v-abl, v-mos, H-ras, v-raf, and mutant active MAP kinase kinase-transformed NIH 3T3 cells to grow in soft agar. Only v-fos-transformed cells were found to be resistant to the treatment of SCH 51344. SCH 51344 treatment had very little effect, if any, on the activation of MAP kinase kinase, MAP kinase, and p90RSK activity in response to growth factor stimulation. Treatment of ras-transformed cells with SCH 51344 led to stimulation of serum response factor DNA binding activity and activation of serum response element-dependent gene transcription, accounting for its ability to activate alpha-actin promoter activity in ras-transformed cells. Our results indicate that SCH 51344 inhibits ras transformation by a novel mechanism and acts at a point either downstream or parallel to extracellular signal-regulated kinase-dependent Ras signaling pathway.

3T3 Cells↗
PubMed1995

Requirement for MAP kinase (ERK2) activity in interferon alpha- and interferon beta-stimulated gene expression through STAT proteins.

Activation of early response genes by interferons (IFNs) requires tyrosine phosphorylation of STAT (signal transducers and activators of transcription) proteins. It was found that the serine-threonine kinase mitogen-activated protein kinase (MAPK) [specifically, the 42-kilodalton MAPK or extracellular signal-regulated kinase 2 (ERK2)] interacted with the alpha subunit of IFN-alpha/beta receptor in vitro and in vivo. Treatment of cells with IFN-beta induced tyrosine phosphorylation and activation of MAPK and caused MAPK and Stat1 alpha to coimmunoprecipitate. Furthermore, expression of dominant negative MAPK inhibited IFN-beta-induced transcription. Therefore, MAPK appears to regulate IFN-alpha and IFN-beta activation of early response genes by modifying the Jak-STAT signaling cascade.

Binding Sites↗
PubMed1995

Reversal of Raf-1 activation by purified and membrane-associated protein phosphatases.

The Raf-1 protein kinase participates in transduction of mitogenic signals, but its mechanisms of activation are incompletely understood. Treatment of human Raf-1 purified from insect Sf9 cells co-expressing c-H-Ras and Src(Y527F) (in which phenylalanine replaces tyrosine at residue 527) with either serine-threonine or tyrosine phosphatases resulted in enzymatic inactivation of Raf-1. Inactivation of purified Raf-1 was blocked by addition of either the 14-3-3 zeta protein or heat shock protein 90. Loading of plasma membranes from transformed cells with guanosine triphosphate (GTP) resulted in inactivation of endogenous or exogenous Raf-1; inactivation was blocked by inclusion of protein phosphatase inhibitors. These results suggest the existence of protein phosphatases in the cell membrane that are regulated by GTP and are responsible for Raf-1 inactivation.

14-3-3 Proteins↗
PubMed1995

An incomplete program of cellular tyrosine phosphorylations induced by kinase-defective epidermal growth factor receptors.

Although signaling by the epidermal growth factor (EGF) receptor is thought to be dependent on receptor tyrosine kinase activity, it is clear that mitogen-activated protein (MAP) kinase can be activated by receptors lacking kinase activity. Since analysis of the signaling pathways used by kinase-defective receptors could reveal otherwise masked capabilities, we examined in detail the tyrosine phosphorylations and enzymes of the MAP kinase pathway induced by kinase-defective EGF receptors. Following EGF stimulation of B82L cells expressing a kinase-defective EGF receptor mutant (K721M), we found that ERK2 and ERK1 MAP kinases, as well as MEK1 and MEK2 were all activated, and SHC became prominently tyrosine-phosphorylated. By contrast, kinase-defective receptors failed to induce detectable phosphorylations of GAP (GTPase-activating protein), p62, JAK1, or p91STAT1, all of which were robustly phosphorylated by wild-type receptors. These data demonstrate that kinase-defective receptors induce several protein tyrosine phosphorylations, but that these represent only a subset of those seen with wild-type receptors. This suggests that kinase-defective receptors activate a heterologous tyrosine kinase with a specificity different from the EGF receptor. We found that kinase-defective receptors induced ErbB2/c-Neu enzymatic activation and ErbB2/c-Neu binding to SHC at a level even greater than that induced by wild-type receptors. Thus, heterodimerization with and activation of endogenous ErbB2/c-Neu is a possible mechanism by which kinase-defective receptors stimulate the MAP kinase pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Biochemical analysis of MEK activation in NIH3T3 fibroblasts. Identification of B-Raf and other activators.

Numerous potential activators of MEK have been identified, including c-Raf-1, B-Raf, c-Mos, and a family of MEK kinases. However, little information gives insight into the activators actually utilized in vivo. To address this, we have used column chromatography and a coupled MEK activation assay to identify in NIH3T3 cells, two major MEK activators, and a third insulin-specific activator. The first MEK activator has an apparent M(r) of 40,000-50,000, was immunologically distinct from A-Raf, B-Raf, c-Raf-1, c-MEKK, c-Mos, MEK1, and MEK2, and was rapidly activated by serum, platelet-derived growth factor (PDGF), insulin, thrombin, and phorbol ester. The second MEK activator was identified as B-Raf. Activation of 93-95 kDa B-Raf was observed in column fractions and B-Raf immunoprecipitates from cytosolic and particulate fractions after stimulation with serum or PDGF, but not insulin. c-Raf-1 from cytosol did not exhibit MEK activator activity; however, c-Raf-1 immunoprecipitates from the particulate fraction revealed MEK activator activity that was enhanced after stimulation with PDGF or phorbol ester, but not serum or insulin. Both c-Mos and c-MEKK were present in NIH3T3 fibroblasts but did not show MEK activator activity. These data provide direct evidence that 93-95-kDa B-Raf isozymes and an unidentified 40-50-kDa MEK activator are major agonist-specific MEK activators in NIH3T3 fibroblasts.

3T3 Cells↗
PubMed1995

Human choline acetyltransferase gene: localization of alternative first exons.

Two overlapping cosmids containing the 5' end of human choline acetyltransferase (ChAT) gene have been cloned. Using heterologous probes, we localized two alternative first exons homologous to rodent ChAT exons R and M (Misawa et al.: J Biol Chem 267:20392-20399, 1992). The sequence of rodent exon N was not conserved in the human gene. Northern blot analysis of mRNA purified from the human neuroepithelioma cell lines LA-N2 and MC-I-XC revealed that both exons R and M were transcribed in mRNA species of 6.0 and 2.5 kb. Only the 6-kb species was detected with both R- and M-specific probes in the neuroepithelioma cell line CHP126. Reverse transcription-polymerase chain reaction (RT-PCR) analysis suggested that the major mRNA species in MC-I-XC and CHP126 cells contained the proximal part of exon M spliced to exon 1, which contains the alternative ACG initiation codon. RT-PCR also allowed the characterization of a mRNA species containing exon R spliced to exon 1, but no species containing both exon R and the distal part of exon M could be detected. RT-PCR was also used to evidence an alternative exon (tentatively numbered exon 8) in the coding sequence.

Alternative Splicing↗
PubMed1995

A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

Amino Acid Sequence↗
PubMed1994

Partial purification of a mitogen-activated protein kinase kinase activator from bovine brain. Identification as B-Raf or a B-Raf-associated activity.

A classical biochemical approach was taken to identify mitogen-activated protein kinase kinase (MEK) activators in bovine brain. Fractionation revealed the presence of one major MEK-stimulating activity that was distinct from c-Raf-1 and MEK kinase. Similar results were obtained using bovine adrenal chromaffin cells, and in both cases, immunoblotting and immunoprecipitation experiments demonstrated co-purification of MEK activator with B-Raf. Partially purified MEK activator stimulated phosphorylation of MEK1 on residues tentatively identified as serine 218 and serine 222. Little or no MEK activator was associated with c-Raf-1 in bovine brain or chromaffin cells, although this protein was expressed, suggesting that B-Raf might be the major MEK activator in cells of neural origin.

Adrenal Glands↗
PubMed1994

c phosphorylation and activation of the IGF-I receptor in src-transformed cells.

Using a panel of src mutants partially defective for malignant transformation, our laboratory has previously identified the insulin-like growth factor (IGF-I) receptor as a protein whose tyrosine phosphorylation correlates with transformation by src in embryonic chick cells (Kozma et al., 1990; Kozma and Weber, 1990). It has not been clear, however, whether src-induced phosphorylation altered the enzymatic or signaling properties of the IGF-I receptor and thus whether the IGF-I receptor could be a functionally significant target for pp60v-src. To examine the effect of src expression on the activity of the IGF-I receptor, the human IGF-I receptor was expressed in Rat-1 fibroblasts co-expressing the temperature-sensitive v-src mutant, tsLA29. The IGF-I receptor exhibited an elevated level of tyrosine phosphorylation in src transformed cells even in the absence of IGF-I treatment. Increased receptor phosphorylation occurred rapidly when cells expressing a temperature-conditional src mutant were shifted from the restrictive to the permissive temperature. Src-induced phosphorylation of the receptor was correlated with an increase in the in vitro tyrosine kinase activity of the receptor, both toward itself and exogenous substrates. The src-induced increase in receptor activity was shown to be dependent on tyrosine phosphorylation, as treatment with a tyrosine-specific phosphatase lowered receptor activity. A kinase-defective mutant of the IGF-I receptor also became constitutively phosphorylated in src-transformed cells, ruling out a possible autocrine mechanism for this phosphorylation. Collectively these data indicate that pp60v-src induces ligand-independent phosphorylation and activation of the IGF-I receptor by an intracellular mechanism, consistent with the possibility that receptor phosphorylation could contribute to the genesis of the transformed phenotype.

Animals↗
PubMed1994

Differential tyrosine phosphorylation of JAK1, JAK2, and STAT1 by growth hormone and interferon-gamma in IM-9 cells.

Both the growth hormone (GH) and interferon gamma (IFN gamma) receptors are members of the cytokine receptor family that activate tyrosine phosphorylation despite the lack of a tyrosine kinase domain. Recently, the Janus kinase (JAK) family of tyrosine kinases have been shown to play an integral role in intracellular signaling by the cytokine receptors. We demonstrate that, in the human IM-9 lymphocyte, both JAK1 and JAK2 are tyrosine-phosphorylated in response to IFN gamma, whereas only JAK2 is tyrosine-phosphorylated in response to GH. Furthermore, dimerization of the GH receptor appears to be necessary for GH stimulated tyrosine phosphorylation of JAK2. We provide two lines of evidence that the JAK2 kinases can be regulated independently by GH and IFN gamma in IM-9 cells: 1) desensitization of JAK2 to GH stimulation does not affect the IFN gamma stimulated tyrosine phosphorylation of JAK2; and 2) JAK2 tyrosine phosphorylation by GH and IFN gamma is additive to that seen with either hormone alone. Furthermore, we demonstrate that although IFN gamma activates the tyrosine phosphorylation of the p91 signal transducer and activator of transcription (STAT1) in IM-9 cells, GH does not. GH does activate the tyrosine phosphorylation of a 93-kDa protein that appears to be distinct from STAT1.

Cells, Cultured↗
PubMed1994

Performance and limits of the mixed-phase assay for chloramphenicol acetyltransferase at low [3H]acetyl-CoA concentration.

We have explored the possibility of increasing the sensitivity of the mixed-phase assay for chloramphenicol acetyltransferase (CAT) by using a low concentration (3.75 microM) of isotopically undiluted [3H]acetyl-CoA (200 mCi/mmol). Using extracts of PC12 cells transiently transfected with a plasmid CMV-CAT, we found that the assay was linear with time for about 8 h, unless 25% of the substrate was exhausted. Under the conditions of the assay, the tritiated substrate was relatively stable, as 75% was still available for the reaction after a 20-h incubation at 37 degrees C under the toluene phase in the absence of cell extract. CAT activity could be reliably measured with 4-8 ng protein of cell extract, corresponding to 50-100 transfected cells. We determined the range of linearity of the initial rate with the volume of cell extract and showed that, above a certain value, the rate becomes limited by the diffusion of 3H-acetylated chloramphenicol in the organic phase. The sensitivity of the new assay compared favorably with that of the previously described CAT assays and approached that of the luciferase assay.

Acetyl Coenzyme A↗
PubMed1994

Membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of Ras.

A pathway by which calcium influx through voltage-sensitive calcium channels leads to mitogen-activated protein kinase (MAPK) activation has been characterized. In PC12 cells, membrane depolarization leading to calcium influx through L-type calcium channels activates the dual specificity MAPK kinase MEK1, which phosphorylates and activates MAPK. Calcium influx leads within 30 s to activation of the small guanine nucleotide-binding protein Ras. Moreover, activation of MAPK in response to calcium influx is inhibited by the dominant negative mutant RasAsn17, indicating that Ras activity is required for calcium signaling to MAPK. Ras is also activated by release of calcium from intracellular stores and by membrane depolarization of primary cortical neurons. The pleiotropic regulatory potential of both Ras and the MAPK pathway suggests that they may be central mediators of calcium signaling in the nervous system.

Amino Acid Sequence↗
PubMed1994

Multiple promoters of human choline acetyltransferase and aromatic L-amino acid decarboxylase genes.

The promoter regions of human choline acetyltransferase (ChAT) and aromatic L-amino acid decarboxylase (AADC) genes have been analyzed by transient transfection assays. AADC gene is transcribed from two alternative noncoding first exons, 1N and 1NN, expressed in pheochomocytoma and hepatoma cells, respectively. 5' flanking sequences of exon 1 N (from 9000 to 147 bp) display promoter activity in SK-N-BE neuroblastoma cells, but not in MC-I-XC cholinergic neuroepithelioma cells, and in AADC-rich non-neuronal cells. On the contrary, 5' flanking sequences of exon 1 NN (from 1117 to 119 bp) display high promoter activity in human hepatoma cells HepG2, but not in SK-N-BE cells, suggesting high degrees of specificity of promoters N and NN for AADC-expressing neuronal and non-neuronal cells, respectively. Preliminary evidence suggests that leukemia inhibitory factor suppresses the activity of the neuronal promoter in cultured sympathetic neurons. Two alternative first exons, R and M, have been localized in human ChAT gene, and the corresponding promoters characterized in cholinergic PC12 and NG-108-15 cells, and in non-cholinergic neuro2A cells. Several positively or negatively acting cis elements have been localized in the two promoters, as well as a cAMP-inducible, enhancer-like element in the second intron. Among the various cell lines studied, there was no correlation between promoter activities and the expression of the endogenous ChAT gene, suggesting that the fine-tuning of ChAT gene expression is controlled by silencer elements which remain to be localized.

Animals↗
PubMed1994

RAS and RAF-1 form a signalling complex with MEK-1 but not MEK-2.

Recent studies have demonstrated the existence of a physical complex containing p21ras (RAS), p74raf-1 (RAF-1), and MEK-1. Although it is clear that formation of this complex depends on the activation state of RAS, it is not known whether this complex is regulated by the activation state of the cell and whether MEK-2 is also present in the complex. To analyze the regulation and specificity of this complex, we utilized immobilized RAS to probe lysates of cultured NIH 3T3 fibroblasts and analyzed the proteins complexing with RAS following serum starvation or stimulation. Complex formation among RAS, RAF-1, and MEK-1 was dependent only on RAS:GMP-PNP and not on cell stimulation. Incubations of lysates with immobilized RAS depleted all RAF-1 from the lysate but bound only a small fraction of cytosolic MEK-1, and further MEK-1 could bind immobilized RAS only if exogenous RAF-1 was added to the lysate. This indicates that binding of MEK-1 to RAS depends on the presence of RAF-1 or an equivalent protein. In contrast to MEK-1, MEK-2 was not detected in the RAS signalling complex. A proline-rich region of MEK-1 containing a phosphorylation site appears to be essential for signalling complex formation. Consistent with the preferential binding of MEK-1 to RAS:RAF-1, the basal activity of MEK-1 in v-ras-transformed cells was found to be elevated sixfold, whereas MEK-2 was elevated only twofold, suggesting that the RAS signalling pathway favors MEK-1 activation.

3T3 Cells↗
PubMed1994

Growth hormone and erythropoietin differentially activate DNA-binding proteins by tyrosine phosphorylation.

Binding of growth hormone (GH) and erythropoietin (EPO) to their respective receptors results in receptor clustering and activation of tyrosine kinases that initiate a cascade of events resulting not only in the rapid tyrosine phosphorylation of several proteins but also in the induction of early-response genes. In this report, we show that GH and EPO induce the tyrosine phosphorylation of cellular proteins with molecular masses of 93 kDa and of 91 and 84 kDa, respectively, and that these proteins form DNA-binding complexes which recognize an enhancer that has features in common with several rapidly induced genes such as c-fos. Assembly of the protein complexes required tyrosine phosphorylation, which occurred within minutes after addition of ligand. The activated complexes translocated from the cytoplasm to the nucleus. The protein activated by GH is antigenically similar to p91, a protein common to several transcription complexes that are activated by interferons and other cytokines. In contrast, the proteins activated by EPO are distinct from p91. These findings establish the outlines for a cytokine-induced intracellular signaling pathway, which begins with ligand-induced receptor clustering that activates one or more tyrosine kinases. These data are the first to demonstrate that GH- and EPO-activated tyrosine-phosphorylated proteins can specifically recognize a well-defined enhancer and therefore provide a mechanism for rapidly transducing signals from the membrane to the nucleus.

Base Sequence↗
PubMed1993

Dual phosphorylation and autophosphorylation in mitogen-activated protein (MAP) kinase activation.

p42mapk [mitogen activated protein (MAP) kinase; extracellular signal-regulated protein kinase (ERK)] is a serine/threonine-specific protein kinase that is activated by dual tyrosine and threonine phosphorylation in response to diverse agonists. Both the tyrosine and threonine phosphorylations are necessary for full enzymic activity. A MAP kinase activator recently purified and cloned has been shown to be a protein kinase (MAP kinase kinase) that is able to induce the dual phosphorylation of MAP kinase on both the regulatory tyrosine and threonine sites in vitro. In the present paper we have utilized MAP kinase mutants altered in the sites of regulatory phosphorylation to show, both in vivo and in vitro, that phosphorylation of the tyrosine and the threonine can occur independently of one another, with no required order of phosphorylation. We also utilized kinase-defective variants of MAP kinase with mutations in either the ATP-binding loop or the catalytic loop, and obtained data suggesting that the activity or structure of the catalytic loop of MAP kinase plays an important role in its own dual phosphorylation.

Amino Acid Sequence↗
PubMed1993

Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate.

Mitogen-activated protein (MAP) kinases p42mapk and p44mapk are activated in cells stimulated with epidermal growth factor (EGF) and other agents. A principal pathway for MAP kinase (MAPK) activation by EGF consists of sequential activations of the guanine nucleotide exchange factor Sos, the guanosine triphosphate binding protein Ras, and the protein kinases Raf-1, MAPK kinase (MKK), and MAPK. Because adenosine 3',5'-monophosphate (cAMP) does not activate MAPK and has some opposing physiologic effects, the effect of increasing intracellular concentrations of cAMP with forskolin and 3-isobutyl-1-methylxanthine on the EGF-stimulated MAPK pathway was studied. Increased concentrations of cAMP blocked activation of Raf-1, MKK, and MAPK in Rat1hER fibroblasts, accompanied by a threefold increase in Raf-1 phosphorylation on serine 43 in the regulatory domain. Phosphorylation of Raf-1 in vitro and in vivo reduces the apparent affinity with which it binds to Ras and may contribute to the blockade by cAMP.

1-Methyl-3-isobutylxanthine↗