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)

G protein beta and gamma subunits (Gbeta and Ggamma) form a complex that is involved in various signaling pathways. We reported that the C-terminal 10 amino acids of Gbeta are required for association with Ggamma (Yamauchi, J., Kaziro, Y., and Itoh, H. (1995) Biochem. Biophys. Res. Commun., 214, 694-700). To evaluate further the significance of the C-terminal region of Gbeta in the formation of a Gbetagamma complex and its signal transduction, we constructed several C-terminal mutants and expressed them in human embryonal kidney 293 cells. The mutant lacking the C-terminal 2 amino acids (DeltaC2) failed to associate with Ggamma, whereas deletion of the C-terminal amino acid (DeltaC1), replacement of Trp at -2 position by Ala (W339A), and addition of six histidines ((His)6) at the C terminus did not affect the association with Ggamma. We also studied the effect of these mutations on the activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). Co-expression of the DeltaC2 or (His)6 mutant with Ggamma did not activate MAPK/ERK at all, whereas the DeltaC1 or W339A mutant showed the MAPK/ERK activation. The JNK/SAPK activity was stimulated by the W339A, DeltaC2, or (His)6 mutant, but not by the DeltaC1 mutant. These results suggest that the C-terminal region of Gbeta participates differentially in the signaling for MAPK/ERK and JNK/SAPK activations in mammalian cells.
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PMID:C-terminal mutation of G protein beta subunit affects differentially extracellular signal-regulated kinase and c-Jun N-terminal kinase pathways in human embryonal kidney 293 cells. 906 14

MEK kinase 1 (MEKK1) shares sequence identity with the yeast kinases Ste11 and Byr2, and is capable of phosphorylation and activation of both mitogen-activated protein/extracellular signal-related protein kinase (MAP/ERK) kinase (MEK) and stress-activated protein kinase (SAPK)/ERK kinase (SEK) in vitro. In vivo, however, MEKK1 predominantly activates the SEK/SAPK kinase cascade. Mechanisms of activation of MEKK1 are unclear. We have identified a major site of autophosphorylation (Thr-575) within the 'activation loop' of MEKK1 between the kinase subdomains VII and VIII. Phosphatase treatment of a constitutively active MEKK1 decreased kinase activity by 59%. Dephosphorylated T575 was rapidly re-(auto)phosphorylated by MEKK1. Mutation of T575 to alanine decreased MEKK1 transphosphorylation activity with a SEK substrate to approx. 30% of wild-type. Mutation of a second threonine residue (Thr-587) to alanine eliminated the phosphorylation of MEK or SEK substrate but not autophosphorylation. MEKK1 autophosphorylation is an intramolecular reaction because active MEKK1 cannot transphosphorylate a kinase-inactive MEKK1. Inactive MEKK1 was not phosphorylated on Thr-575 within cells, suggesting that the phosphorylation of Thr-575 in vivo results from autophosphorylation rather than phosphorylation by an upstream kinase. Autoactivation of MEKK1 via autophosphorylation of Thr-575 might be an immediate response to initial kinase activation through non-phosphorylation mechanisms.
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PMID:Regulation of the activity of MEK kinase 1 (MEKK1) by autophosphorylation within the kinase activation domain. 907 60

The staphylococcal enterotoxins, SEA and SEE, bind one zinc atom per molecule of protein. The presence of this metal atom enhances the binding of the toxins to MHC class II molecules, presumably through an interaction with histidine 81 of the beta chain. L cell transfectants expressing HLA-DR1 and HLA-DR7 molecules, with mutations in either the alpha1 or beta1 domains, were tested for their ability to bind SEA and present it to T cells. Cells expressing DR1 molecules with alanine at positions 77, 78, 80, 83, 84 and 85, or serine at position 79 could all bind SEA and present it to either polyclonal or monoclonal T cells. Most point mutations within the alpha-helical portion of the DR7 beta chain had no effect on binding and presentation. However, substitution of histidine 81 with alanine, glutamate, or aspartate, abrogated SEA binding as well as T cell stimulation by the superantigen. This effect was also observed when the non-polymorphic aspartate, at position 76 was changed to alanine. Mutation of the asparagine at position 82 had an intermediate effect. Point mutations of the DR alpha chain had little effect on binding of SEA as determined by a flow cytometric assay. However, mutation of lysine at position 39 of the alpha chain and, to a lesser extent methionine at position 36, markedly decreased the ability of SEA to stimulate toxin-responsive mouse T cell hybridomas. Finally, the monoclonal antibody, L243 binds to the alpha chain of HLA-DR, and was able to block T cell activation by SEA without blocking SEA binding. These data support the model whereby HLA-DR has two binding sites for SEA. A low affinity site, present on the alpha chain, is required for T cell stimulation by the superantigen, but is insufficient to mediate toxin binding. High affinity binding of HLA-DR to SEA occurs solely through residues on the beta chain, including both histidine 81 and aspartate 76.
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PMID:Functional activity of staphylococcal enterotoxin A requires interactions with both the alpha and beta chains of HLA-DR. 912 63

We have investigated a phosphatase-sensitive sequential epitope of the nucleoprotein (N), one of the phosphoproteins of rabies virus, which is recognized by the monoclonal antibody (MAb) #5-2-26. The epitope was shared in common by all of the rabies virus strains we tested, including the HEP, ERA, CVS and Japanese strains (Nishigahara and Komatsukawa). Thin layer chromatography of the acid hydrolyzates of 32P-labeled N protein showed that the protein contained phosphoserine and phosphothreonine at a molar ratio of about 4 to 1, while no phosphotyrosine was detected. Immunoprecipitation studies with several deletion mutants of the N protein showed that the epitope is located in a region spanning from amino acid 344 to 415. If the phosphatase-sensitive epitope is located at or near the phosphoamino acid, the location of the latter could be narrowed further to a region from amino acid 354 to 389 by comparing the amino-acid sequences among the viral strains. To examine this assumption, point mutation was introduced by amino-acid substitution with alanine at either of five potential phosphorylation sites (i.e., positions 354, 375, 377, 386 and 389) in the 354-389 region. Among those, only one substitution, at position 389, greatly affected the antigenicity. Substitution of serine-389 by threonine also reduced the antigenicity. These results strongly suggest that serine-389 is a phosphorylation site and essential for constructing or stabilizing the antigenic structure for MAb 5-2-26.
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PMID:Identification of a phosphatase-sensitive epitope of rabies virus nucleoprotein which is recognized by a monoclonal antibody 5-2-26. 913 Feb 35

The serum response element (SRE), which is pivotal for transcriptional up-regulation of the c-fos protooncogene, is constitutively occupied by a protein complex comprising the serum response factor and a ternary complex factor (TCF). Phosphorylation of the TCFs Elk-1 and Sap-1a by the ERK and JNK subclasses of MAP kinases triggers c-fos transcription. We demonstrate here that Elk-1 is barely activated by a third subclass of MAP kinases (p38), most likely because the critical residues Ser383 and Ser389 are poorly phosphorylated by p38 MAP kinase. In contrast, the TCF Sap-1a is efficiently phosphorylated by p38 MAP kinase in vitro and in vivo on the homologous residues Ser381 and Ser387. Mutation of these sites to alanine severely reduces c-fos SRE-dependent transcription mediated by Sap-1a and p38 MAP kinase. Thus, Sap-1a may be an important target for mitogens, stress and apoptotic signals to elicit a nuclear response. However, signaling from p38 MAP kinase to Sap-1a or from Sap-1a to the basal transcription machinery does not occur in all cell types nor at promoters other than the c-fos SRE, which may ensure the specificity of signaling.
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PMID:Convergence of MAP kinase pathways on the ternary complex factor Sap-1a. 913 Jul 7

The ternary complex factor (TCF) subfamily of ETS-domain transcription factors form ternary complexes with the serum response factor (SRF) and the c-fos SRE. Extracellular signals are relayed via MAP kinase signal transduction pathways through the TCF component of the ternary complex. Protein-protein interactions between TCFs and SRF play an essential role in formation of this ternary complex. A 30 amino acid sequence encompassing the TCF B-box is sufficient to mediate interactions with SRF. In this study we have identified amino acids which are critical for this interaction and derived a molecular model of the SRF binding interface. Alanine scanning of the Elk-1 B-box reveals five predominantly hydrophobic residues which are essential for binding to SRF and for ternary complex formation in vitro and in vivo. These amino acids are predicted to lie on one face of an alpha-helix. Peptides encompassing the B-box retain biological activity and have helix-forming propensity. alpha-Helix and ternary complex formation is disrupted by the introduction of helix-breaking proline residues. Our results are consistent with a model in which the Elk-1 B-box forms an inducible alpha-helix which presents a hydrophobic face for interaction with SRF. We discuss the wider applicability of our results to similar short protein-protein interaction motifs found in other transcription factors.
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PMID:Molecular characterization of the B-box protein-protein interaction motif of the ETS-domain transcription factor Elk-1. 917 56

RB 101 (N-((R,S)-2-benzyl-3[(S)(2-amino-4-methylthio)butyldithio]-1-oxopr opyl)-L-phenylalanine benzyl ester) is a full inhibitor of the enkephalin-catabolizing enzymes, which induces strong naloxone-reversible antinociceptive responses after i.v. or i.p. administration, but is only slightly active after oral administration. Chemical modifications were introduced on this compound, resulting in molecules such as RB 120 (N-((S)-2-benzyl-3[(S)(2-amino-4-methylthio)butyldithio]-1-oxoprop yl)-L-alanine benzyl ester), which was selected for a complete study, after oral administration, in various assays commonly used to select analgesics: mouse hot plate test, rat tail-flick test, electrical stimulation of the tail in rats, paw pressure test on inflamed paws in rats, acetic acid-induced writhing test and the formalin test in mice. RB 120 induced potent dose-dependent antinociceptive responses in all these tests after oral administration. The differences in antinociceptive effects induced by RB 120 in the various assays is probably related to the amount of enkephalins released and to the efficiency of peptidase inactivation in particular brain regions implicated in the control of a given nociceptive input. The goal of discovering orally active analgesics endowed with a potency similar to that of morphine but devoid of its major side-effects, seems now to have been reached with mixed neutral endopeptidase/aminopeptidase N (NEP/APN) inhibitors, although these compounds have yet to be evaluated in clinical trials.
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PMID:Pain-suppressive effects on various nociceptive stimuli (thermal, chemical, electrical and inflammatory) of the first orally active enkephalin-metabolizing enzyme inhibitor RB 120. 946 29

The role of glutamate-219 in the a-subunit of the Escherichia coli F0F1-ATPase was examined using site-directed mutagenesis. The replacement of Glu-219 by lysine, alanine or glycine resulted in a partially functional F0F1-ATPase. Combining any of these mutations with the substitution of glutamate for Gln-252 did not result in any increase in function. These findings rule out a proposal that glutamate at position 252 can functionally replace glutamate at position 219 [S.B. Vik, B.J. Antonio, J. Biol. Chem. 269 (1994) 30364-30369]. All the single and double mutants grew better at 25 degrees C than at 37 degrees C, suggesting a role for Glu-219 in maintaining the structure of the F0.
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PMID:Glutamate residues at positions 219 and 252 in the a-subunit of the Escherichia coli ATP synthase are not functionally equivalent. 951 21

Transmission of zucchini yellow mosaic virus (ZYMV) by aphids was examined by introducing mutations within the highly conserved proline-threonine-lysine (PTK) motif of the helper component proteinase (HC-Pro) using a cDNA full-length clone. Replacement of proline by alanine (ATK) in the PTK motif abolished transmission almost completely both from plants and from membranes. Substitution of the basic lysine by glutamic acid (PTE) did not reduce the rate of transmission compared with the wild-type. Replacement of threonine by valine (PVK) or serine (PSK) resulted in a rate of transmission that was lower than that of the wild-type. The rate was lower for PSK than for PVK. Western blot comparison did not permit attribution of HC-Pro functionality in transmission to its level in the host. The HC-Pro of strains that effected transmission (with the wild-type PTK motif, and with the mutated PTE and PVK motifs) could also bind in vitro to virions of ZYMV. HC-Pro with a PSK motif, which was less effective in assisting transmission, could bind only weakly to virions, while HC-Pro of the almost non-transmissible strains (with PAK and ATK motifs) did not bind at all. Interestingly, positive binding was recorded for transmission-defective ZYMV-Ct, which has a PTK motif but has glutamic acid instead of lysine in the lysine-leucine-serine-cysteine (KLSC) motif. These findings support the 'bridge hypothesis', and confirm the binding of the HC-Pro to the virion. The possible role of the PTK and KLSC motifs in binding to the virus and to the mouthparts of the aphid is discussed.
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PMID:Mutations in the HC-Pro gene of zucchini yellow mosaic potyvirus: effects on aphid transmission and binding to purified virions. 956 86

Neutral endopeptidase-24.11 (NEP; neprilysin; EC 3.4.24.11) and endothelin-converting enzyme (ECE) are related zinc metallopeptidases involved in the processing of biologically active peptides. Only ECE, however, exists as a disulphide-linked homodimer. The covalent linkage in rat ECE is between Cys412 in each subunit, which is equivalent to Glu403 in rabbit NEP. Here we report that directed mutagenesis of Glu403 to cysteine in rabbit NEP creates a disulphide-linked homodimer, as revealed by transient transfection in COS-1 cells and SDS/PAGE of a membrane fraction. Under reducing conditions, both the mutant (E403C) and the wild-type NEP migrate as a polypeptide of 92 kDa. However, under non-reducing conditions, the Mr of the wild type remains unchanged, whereas that of the mutant is doubled. Co-transfection of wild-type ECE and E403C NEP cDNA did not result in the production of a NEP-ECE heterodimer. Comparison of the kinetic constants for wild-type and E403C mutant NEP with either [D-Ala2,Leu5]enkephalin or 3-carb oxypropanoyl-alanyl-alanyl- leucine-4-nitroanilide(Suc-Ala-Ala-Leu-NH-Np) as substrate show a decrease of approx. 50% in Vmax/Km for the mutant form. The IC50 value for inhibition of the mutant by phosphoramidon or thiorphan is increased 3-fold and 5-fold respectively. Although NEP and ECE exhibit only about 40% identity and differ substantially in substrate specificity and some other characteristics, these data indicate that they have considerable similarity in three-dimensional structure, allowing dimer formation in the mutant NEP with the disulphide link probably occurring in a hydrophilic surface loop.
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PMID:Mutagenesis of Glu403 to Cys in rabbit neutral endopeptidase-24.11 (neprilysin) creates a disulphide-linked homodimer: analogy with endothelin-converting enzyme. 958 75


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