Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases.
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PMID:FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors. 1062 55

To evaluate the influence of individual susceptibility factors on the level of polyaromatic (PAH) hydrocarbon DNA adducts and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutants in peripheral lymphocytes, 70 coke-oven workers exposed to PAH were genotyped for four metabolic enzyme polymorphisms of potential importance in PAH metabolism. The examined genetic polymorphisms concerned glutathione S-transferases M1 (GSTM1; gene deletion; 96 workers), T1 (GSTT1; gene deletion), P1 (GSTP1; Ile-->Val substitution at codon 104 or Ile-->Val at codon 104 and Val-->Ala at codon 113), and microsomal epoxide hydrolase (EPHX; Tyr-->His substitution at codon 113 and His-->Arg at codon 139). The workers were classified in a high- and low-exposure group on the basis of urinary concentration of 1-pyrenol. The GSTM1 null genotype increased the number of DNA adducts in smoking coke-oven workers with high PAH exposure. DNA adducts were affected by PAH-exposure in non-smokers and in GSTM1 null smokers and by smoking in GSTM1 null individuals. In a multiple linear regression analysis, the interaction of the GSTM1 genotype was statistically significant (p = 0.04) with smoking (yes/no) and of borderline significance (p = 0.06) with PAH-exposure (high/low). As smoking also increased urinary 1-pyrenol, the genotype modification seemed to concern DNA adducts due to smoking rather than occupational exposure. GSTT1 positive individuals showed an elevated level of DNA adducts in comparison with GSTT1 null subjects (p = 0.04), and EPHX genotypes associated with slow hydroxylation reaction yielded a higher (p = 0.05) HPRT mutant frequency than fast EPHX genotypes; these findings were, however, based on small numbers of subjects and need to be clarified in further studies. In conclusion, our findings indicate that homozygous deletion of GSTM1 results in an increased sensitivity to genotoxic PAHs in tobacco smoke, which is seen as an increase in aromatic DNA adducts in blood mononuclear cells.
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PMID:Influence of GSTM1, GSTT1, GSTP1, and EPHX gene polymorphisms on DNA adduct level and HPRT mutant frequency in coke-oven workers. 1063 83

The role of signal transducer and activator of transcription (STAT) signaling pathways in the interleukin-6 (IL-6)-induced morphological differentiation of PC12-E2 cells was assessed using wild type and dominant negative mutants of Stat1 and Stat3, containing Tyr --> Phe (YF), Ser --> Ala (SA), and the double mutations (DM), respectively. FS3-YF or FS3-DM markedly inhibited the IL-6-induced response, but overexpression of FS3-SA caused only a modest inhibition. Expression of all Stat3 mutants had no effect on NGF-induced neurite outgrowth. Overexpression of wild type Stat1 protein inhibited IL-6 activated DNA binding complexes containing Stat3 homodimers, which may explain the partial negative effect of Stat1 on IL-6-induced neurite outgrowth. Specificity of these STAT constructs was confirmed using luciferase reporter gene assays, which showed that IL-6-activated transcription was blocked by expression of FS3-YF and FS3-DM and that FS1 enhanced the interferon gamma-activated transcription. Thus, in PC12-E2 cells, Stat3 homodimers are preferentially activated by IL-6, indicating a role for Stat3 in the regulation of cellular differentiation. Furthermore, IL-6 induced robust neurite outgrowth in PC12-E2 cells expressing dominant negative forms of RAS or SHC or in cells pretreated with the mitogen-activated protein kinase mitogen-activated protein kinase kinase inhibitor, PD98059. Thus, activation of the Stat3 signaling pathway, but not RAS/ERK dependent pathways, is essential for differentiation of PC12-E2 cells by IL-6.
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PMID:Activation of the Stat3 signaling pathway is required for differentiation by interleukin-6 in PC12-E2 cells. 1063 20

Elk-1, a member of the TCF family of Ets domain proteins, contains a C-terminal transcriptional activation domain with multiple copies of the MAPK core consensus sequence S/T-P. This region is phosphorylated by MAP kinases in vitro and in vivo, but the extent and kinetics of phosphorylation at the different sites have not been investigated in detail. We prepared antisera against the phosphorylated forms of residues T353, T363, T368, S383, S389 and T417. The antisera specifically recognize the phosphorylated Elk-1 C terminus and are specific for their cognate sites, as assessed by peptide competition and mutagenesis experiments. Analysis of cells stably expressing Elk-1 in vivo shows that following serum or TPA stimulation, residues T353, T363, T368, S383, S389 and T417 become phosphorylated with similar kinetics. Mutation of any one site does not prevent phosphorylation of the others. Mutation to alanine of S383, F378 or W379, which virtually abolishes transcriptional activation by Elk-1, does not affect phosphorylation of any sites tested. Analysis of Elk-1 using two-dimensional gel electrophoresis shows that following ERK activation Elk-1 receives at least six phosphates in addition to those present prior to stimulation. We propose that the Elk-1 C-terminal regulatory domain becomes stoichiometrically phosphorylated following growth factor stimulation.
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PMID:ERK activation induces phosphorylation of Elk-1 at multiple S/T-P motifs to high stoichiometry. 1063 5

Transmembrane domains of receptor tyrosine kinases are increasingly seen as key modulatory elements in signaling pathways. The present work addresses problems surrounding expression, isolation, secondary structure recovery, and assembly into membranes, of the relatively large quantities of transmembrane peptides needed to investigate these pathways by NMR spectroscopy. We demonstrate significant correspondence between SDS-PAGE behavior of such peptides and their (2)H NMR spectra in lipid bilayer membranes. A 50-residue peptide, Neu(exp), containing the transmembrane portion of the receptor tyrosine kinase, Neu, was designed for expression in Escherichia coli. The sequence also contained 11-12 amino acids from each side of the transmembrane domain. The common problem of low expressivity of transmembrane peptides was encountered-likely associated with membrane toxicity of the desired gene product. This difficulty was overcome by expressing the peptide as a TrpE fusion protein in a pATH vector to target expression products to inclusion bodies, and subsequently removing the TrpE portion by cyanogen bromide cleavage. Inclusion bodies offered the additional benefits of reduced proteolytic degradation and simplified purification. The presence of a hexa-His tag allowed excellent recovery of the final peptide, while permitting use of denaturing solvents and avoiding the need for HPLC with its attendant adsorption losses. Isolated expressed peptides were found to be pure, but existed as high oligomers rich in beta-structure as evidenced by CD spectroscopy and SDS-PAGE behavior. Dissolution in certain acidic organic solvents led to material with increased alpha-helix content, which behaved in detergent as mixtures of predominantly monomers and dimers-a situation often considered to exist in cell membranes. For purposes of NMR spectroscopy, peptide alanine residues were deuterated in high yield during expression. The same acidic organic solvents used to dissolve and dissociate expressed transmembrane peptides proved invaluable for their assembly into lipid bilayers. Analogous transmembrane peptides from the human receptor tyrosine kinase, ErbB-2, demonstrated related phenomena.
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PMID:Expression and membrane assembly of a transmembrane region from Neu. 1067 38

Multiple endocrine neoplasia (MEN) type 2B mutations have been reported at methionine 918 or alanine 883 in the tyrosine kinase domain of the RET proto-oncogene. Recently, a new combination of two germline missense mutations at valine 804 and tyrosine 806 was identified in a patient with MEN 2B-like clinical phenotypes including medullary thyroid carcinoma, mucosal neuroma, and marfanoid habitus. In this case, valine 804 and tyrosine 806 were replaced with methionine and cysteine, respectively. In the present study, biological activities of RET with these new mutations were compared with those with known MEN 2A or MEN 2B mutations. The transforming activity of RET with the V804M/Y806C mutation was about 8- to 13-fold higher than that of RET with a single V804M or Y806C mutation. Like RET with the M918T or A883F MEN 2B mutation, the transforming activity of RET with the V804M/Y806C mutation was not affected by substitution of phenylalanine for tyrosine 905 that abolished the activity of RET with the MEN 2A mutation. On the other hand, substitution of phenylalanine for tyrosines 864 and 952 drastically diminished the activity of RET with the V804M/Y806C, M918T or A883F mutation, suggesting that these three mutant proteins have similar biological properties.
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PMID:A two-hit model for development of multiple endocrine neoplasia type 2B by RET mutations. 1067 86

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms arising in the gastrointestinal tract. GISTs express the KIT receptor tyrosine kinase, and many cases have activating mutations in the KIT juxtamembrane region. We now report an analysis of KIT cDNA and genomic sequences in eight GISTs that lack juxtamembrane region mutations. Six cases contained heterozygous exon 9 mutations in which six nucleotides, encoding Ala-Tyr, were duplicated. The other two cases contained homozygous exon 13 missense mutations, resulting in substitution of Glu for Lys(642), that were associated with constitutive KIT tyrosine phosphorylation. Sequence analysis of DNAs from nonneoplastic companion tissues revealed that both the exon 9 and exon 13 mutations were somatic. These are the first descriptions, in any tumor, of mutations in KIT exons encoding the C-terminal end of the extracellular domain and the first part of the split kinase domain. These findings indicate that KIT may be activated by mutations in at least three domains-extracellular, juxtamembrane, and kinase-in GISTs.
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PMID:KIT extracellular and kinase domain mutations in gastrointestinal stromal tumors. 1070 94

HePTP is a tyrosine specific protein phosphatase that is strongly expressed in activated T-cells. It was recently demonstrated that in transfected T-cells HePTP impairs TCR-mediated activation of the MAP-kinase family members ERK2 and p38 and it was suggested that both ERK and p38 MAP-kinases are substrates of HePTP. The HePTP gene has been mapped to human chromosome 1q32.1. Abnormalities in this region are frequently found in various hematopoietic malignancies. HePTP is highly expressed in acute myeloid leukemia and its expression in fibroblasts resulted in transformation. To address a possible involvement of HePTP in hematopoietic malignancies we sought to identify HePTP substrate(s) in leukemic cells. Using substrate trapping mutants we have identified the MAP-kinase ERK2 as a specific target of HePTP in the myelogenous leukemia cell line K562. Tyrosine phosphorylated ERK2, but not ERK1, p38, or JNK1, efficiently bound to catalytically inactive HePTP mutants in which the active site cysteine (HePTP-C/S) or the conserved aspartic acid residue (HePTP-D/A) had been exchanged for serine and alanine, respectively. Moreover, the interaction of ERK2 with HePTP trapping mutants was dependent on ERK2 tyrosine phosphorylation, indicating that HePTP is specifically targeted to activated ERK2. Using a deletion mutant of HePTP (HePTP-dLD), in which 14 amino acid residues within the N-terminus are missing, we show that regions outside the catalytic domain are also required for the interaction. Furthermore, overexpression of HePTP in K562 cells and fibroblasts interfered with PMA or growth factor induced MAP-kinase activation and HePTP efficiently dephosphorylated active ERK2 on the tyrosine residue in the activation loop in vitro. Together, these data identify ERK2 as a specific and direct target of HePTP and are consistent with a model in which HePTP negatively regulates ERK2 activity as part of a feedback mechanism. Oncogene (2000) 19, 858 - 869.
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PMID:The MAP-kinase ERK2 is a specific substrate of the protein tyrosine phosphatase HePTP. 1070 94

The thesis of this work was to compare the aminopeptidases activity in the amniotic fluid obtained during the physiological labor and during the labor of pregnant with EPH-gestosis, in presence of the beta-naphtlamidic L-amino-acids (alanine, leucine, phenylalanine, tyrosine, histidine, cysteine) chromogenic substrates. It was assumed the 3-7 times increase in the aminopeptidases activity counted to the proteins from the labored with EPH-gestosis comparing to the labored in physiological labor. Among the used substrates the highest activity in both groups of labored women was measured in presence of substrates with exact amino-acids in this order: L-Ala > L-Leu > L-Phe > L-Tyr > L-His > L-Cys.
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PMID:[Aminopeptidases activity in the amniotic fluid in women in labor with EPH-gestosis]. 1076 94

Certain point mutations within the hydrophobic transmembrane domains of class I receptor tyrosine kinases have been associated with oncogenic transformation in vitro and in vivo [Gullick, J., and Srinivasan, R. (1998) Breast Cancer Res. Treat. 52, 43-53]. An important example is the replacement of a single (hydrophobic) valine by (charged) glutamate in the rat protein, Neu, and in the homologous human protein, ErbB-2. It has been suggested that the oncogenic nature of this Val-->Glu substitution may derive from alteration of the transmembrane domain's ability to take part in direct side-to-side associations. In the present work, we examined the basis of this phenomenon by studying transmembrane portions of ErbB-2 in fluid bilayer membranes. An expression system was designed to produce such peptides from the wild-type ErbB-2, and from an identical region of the transforming mutant in which Val(659) is replaced by Glu. All peptides were 50-mers, containing the appropriate transmembrane domain plus contiguous stretches of amino acids from the cytoplasmic and extracellular domains. Deuterium heteronuclear probes were incorporated into alanine side chains (thus, each alanine -CH(3) side chain became -CD(3)). Given the presence of natural alanine residues at positions 648 and 657 within ErbB-2, this approach afforded heteronuclear probes within the motif Ser(656)AlaValValGlu(660), thought to be important for homodimer formation, and nine residues upstream of this site. Further peptides were produced, by site-directed mutagenesis, to confirm spectral assignments and to provide an additional probe location at position 670 (11 residues downstream of the motif region). On SDS-polyacrylamide gels, the transmembrane peptides migrated as predominant monomers in equilibrium with smaller populations of homodimers/-oligomers. CD spectra of both wild-type and transforming mutant peptides were consistent with the transmembrane portions being basically alpha-helical. (2)H NMR spectra of each transmembrane peptide were obtained in fluid phospholipid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) from 35 to 65 degrees C. Results were consistent with the concept that the glutamic acid residue characterizing the mutant is uncharged at neutral pH. Narrowed spectral components from species rotating rapidly and symmetrically within the membrane appeared to represent monomeric peptide. Mutation of Val(659) to Glu within the hydrophobic domain induced changes in side chain angulation of at least 6-8 degrees at Ala(657) (i.e., within the five amino acid motif thought to be involved in homodimer formation), and downstream of this site to residue 670. There was little evidence of effect at the upstream site (Ala(648)) at the membrane surface. This result argues that the transforming mutation is associated with significant intramolecular rearrangement of the monomeric transmembrane helix-extending over some four helix turns-which could influence its lateral associations. In addition, temperature effects on spectral quadrupole splittings suggested that there is greater peptide backbone flexibility for the wild-type transmembrane region.
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PMID:Val(659)-->Glu mutation within the transmembrane domain of ErbB-2: effects measured by (2)H NMR in fluid phospholipid bilayers. 1082 74


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