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)

1H, 13C, and 15N NMR resonances of the SH2 domain of Grb2/Ash in both the free form and the form complexed with a phosphotyrosine-containing peptide derived from the EGF receptor were assigned by analysis of multi-dimensional, double- and triple-resonance NMR experiments. From the chemical shift changes of individual residues upon peptide binding, the binding site for the peptide was mapped on the structure of Grb2/Ash SH2. The peptide was not recognized by the groove formed by the BG and EF loops, suggesting that the EGFR peptide does not bind to Grb2/Ash SH2 in an extended conformation. This was supported by analysis of the binding affinity of mutants where residues on the BG and EF loops were changed to alanine. The present results are consistent with the recently reported structures of Grb2/Ash SH2 complexed with BCR-Abl and Shc-derived phosphotyrosine containing peptides, where the peptide forms a turn conformation. This shows that the specific conformation of the phosphotyrosine-containing sequence is required for the SH2 binding responsible for downstream signaling.
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PMID:Solution structure of the SH2 domain of Grb2/Ash complexed with EGF receptor-derived phosphotyrosine-containing peptide. 1034 19

Two serine residues in the beta-adrenergic receptor (beta-AR) have been proposed to form hydrogen bonds with the catechol moiety of the ligand and contribute to the activation of the receptor. These conserved serine residues in the dopamine (DA) and norepinephrine transporters (DAT and NET, respectively) have also been shown to affect substrate transport in the rat DAT. In the present work, hydrogen bonding interactions between the corresponding serine residues in the human NET (hNET), 354 and 357, and the hydroxyl groups on the substrate were systematically evaluated by examining the transport and binding properties of DA and several single hydroxyl analogues of DA at wild-type and serine-to-alanine-substituted transporters. A comparison of [3H]nisoxetine binding at the serine 354 mutant, in which K(D) increased 70-fold from the wild-type value, with the binding of DA, m-tyramine (m-TYR), and p-tyramine (p-TYR) at mutant 354, where the increase in Ki was less dramatic, revealed that serine 354 is more influential in inhibitor than substrate binding. The binding of m-TYR and p-TYR at the serine 354 and serine 357 mutants did not show a direct interaction between one serine and one substrate catechol hydroxyl group. DA, m-TYR, and p-TYR binding affinity did not deviate from the wild-type value at the serine 357 and double mutant transporters. At these two transporters, however, the Km of DA uptake increased, suggesting that the roles of serine 357 and serine 354 in substrate transport are different from their roles in binding. The K'm for induced efflux of DA decreased at the serine 357 mutant compared with the wild-type, whereas the K'm at the serine 354 mutant was the same as that of the wild-type. Further investigation of the role of substrate hydroxyls in the transport process revealed no difference between the transport of m-TYR or p-TYR, as measured indirectly through their induced efflux of DA, at any of the mutants. Although these serines are influential in inhibitor and substrate binding to the transporter and substrate uptake and efflux, they do not appear to be involved in a direct hydrogen bond interaction with substrate, suggesting that the pattern of distinct hydrogen bonding interactions at the beta-AR does not exist at the hNET.
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PMID:Effects of serine mutations in transmembrane domain 7 of the human norepinephrine transporter on substrate binding and transport. 1042 62

Several mutations were identified in the kinase domain of the RET proto-oncogene in patients with multiple endocrine neoplasia (MEN) 2B, familial medullary thyroid carcinoma (FMTC) or sporadic medullary thyroid carcinoma. We introduced seven mutations (glutamic acid 768-->aspartic acid (E768D), valine 804-->leucine (V804L), alanine 883-->phenylalanine (A883F), serine 891-->alanine (S891A), methionine 918-->threonine (M918T), alanine 919-->proline (A919P) and E768D/A919P) into the short and long isoforms of RET cDNA and transfected the mutant cDNAs into NIH3T3 cells. The transforming activity of the long isoform of Ret with each mutation was much higher that that of its short isoform. Based on the levels of the transforming activity, these mutant RET genes were classified into two groups; a group with high transforming activity (A883F, M918T and E768D/A919P) and a group with low transforming activity (E768D, V804L, S891A and A919P) (designated high group and low group). Interestingly, the level of transforming activity correlated with clinical phenotypes; high group Ret with the A883F or M918T mutation and low group Ret with the E768D, V804L or S891A mutation were associated with the development of MEN 2B and FMTC, respectively. In addition, we found that substitution of phenylalanine for tyrosine 905 present in the kinase domain abolished both transforming and autophosphorylation activities of low group Ret whereas it did not affect the activities of high group Ret.
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PMID:Biological and biochemical properties of Ret with kinase domain mutations identified in multiple endocrine neoplasia type 2B and familial medullary thyroid carcinoma. 1044 57

We investigated the role of the I-E-N-K-L (amino acids 1057-1061) sequence amino-terminal to Tyr1062 in Ret for binding of the Shc phosphotyrosine-binding (PTB) domain. Substitution of Ser for Ile1057 (I1057S), Ala for Asn1059 (N1059A), or Pro for Leu1061 (L1061P) in this sequence significantly decreased the transforming activity of Ret with the multiple endocrine neoplasm type 2A (MEN2A) mutation as well as the binding affinity of Shc to it in vivo and in vitro, indicating that these three amino acids play a role in Shc binding. In addition, as the RET protooncogene is translated as three isoforms of 1114 amino acids (Ret 51), 1106 amino acids (Ret 43), and 1072 amino acids (Ret 9) that differ from one another in the sequence carboxyl-terminal to Tyr1062, we examined whether these sequence differences influence the binding affinity of Shc to Ret. As a result, we found that the transforming activity of Ret 43 isoform with the MEN2A mutation and the binding affinity of Shc to it were very low, although the consensus sequence for the binding of the Shc PTB domain is conserved in the Ret 43 isoform. This finding suggested that the sequence carboxyl-terminal to Tyr1062 in Ret could also influence the binding affinity to Shc.
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PMID:The role of amino acids surrounding tyrosine 1062 in ret in specific binding of the shc phosphotyrosine-binding domain. 1046 68

Affinity chromatography, employing the extracellular domain of the Sea receptor, was used to enrich Sea-binding proteins from chicken serum. One isolated protein bound both a Sea-immunoglobulin fusion protein and an antisera raised against murine macrophage stimulating protein. Amino-terminal sequencing of the dual-reactive protein yielded sequences which were identical to the predicted alpha and beta subunits of chicken macrophage stimulating protein. The partially purified chicken macrophage stimulating protein caused autophosphorylation of the Sea receptor. Previous work showed that recombinant expression of fully activatible human or mouse macrophage stimulating protein required a specific Cys to Ala substitution (Wahl, R. C., Costigan, V. J., Batac, J. P., Chen, K., Cam, L., Courchesne, P. L., Patterson, S. D. Zhang, K., and Pacifici, R. E. (1997) J. Biol. Chem. 272, 1-4). Therefore, we expressed both the wild type and the specific Cys to Ala form of chicken macrophage stimulating protein as recombinant proteins. After proteolytic activation, only conditioned media from COS cells transfected with the C665A chicken macrophage stimulating protein, but not from wild type chicken macrophage-stimulating protein, or control vector, was detected by the Sea-immunoglobulin fusion protein in Western blotting experiments. Conditioned media containing the C665A chicken macrophage-stimulating protein readily caused Sea phosphorylation, while conditioned media containing the wild type chicken macrophage-stimulating protein was only effective at inducing receptor phosphorylation at high concentrations. In addition to receptor phosphorylation, the C665A chicken macrophage-stimulating protein induced phosphorylation of Shc, Erk1, and Erk 2. We conclude that macrophage-stimulating protein is a ligand of the Sea receptor protein-tyrosine kinase.
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PMID:Chicken macrophage stimulating protein is a ligand of the receptor protein-tyrosine kinase Sea. 1047 93

The extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) comprises four subdomains (I-IV) and mediates binding of several different polypeptide ligands, including EGF, transforming growth factor-alpha, and heparin-binding EGF. Previous studies have predominantly implicated subdomain III in ligand binding. To investigate a possible role for sequences in subdomain IV, we constructed several mutant EGFRs in which clusters of charged or aromatic amino acids were replaced with alanine. Analysis of stably transfected Chinese hamster ovary cells expressing mutant EGFRs confirmed that they were present on the cell surface at levels approaching that of the wild-type receptor. Although tyrosine phosphorylation of most mutants was markedly induced by EGF, a cluster mutation (mt25) containing four alanine substitutions in the span of residues 521-527 failed to respond. EGF-induced tyrosine phosphorylation of an alternative mutant (DeltaEN) with amino acids 518-589 deleted was also greatly diminished. Larger doses of EGF or heparin-binding EGF induced only weak tyrosine phosphorylation of mt25, whereas the response to transforming growth factor-alpha was undetectable. These results suggest that mt25 might be defective with respect to either ligand binding or receptor dimerization. Quantitative analyses showed that binding of (125)I-EGF to mt25 and DeltaEN was reduced to near background levels, whereas binding of EGF to other cluster mutants was reduced 60-70% compared with wild-type levels. Among the mutants, only mt25 and DeltaEN failed to form homodimers or to transphosphorylate HER2/Neu in response to EGF treatment. Collectively, our results are the first to provide direct evidence that discrete subdomain IV residues are required for normal binding of EGF family ligands. Significantly, they were obtained with the full-length receptor in vivo, rather than a soluble truncated receptor, which has been frequently used for structure/function studies of the EGFR extracellular region.
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PMID:Mutagenesis reveals a role for epidermal growth factor receptor extracellular subdomain IV in ligand binding. 1049 95

Saccharomyces cerevisiae Cet1p is the prototype of a family of metal-dependent RNA 5'-triphosphatases/NTPases encoded by fungi and DNA viruses; the family is defined by conserved sequence motifs A, B, and C. We tested the effects of 12 alanine substitutions and 16 conservative modifications at 18 positions of the motifs. Eight residues were identified as important for triphosphatase activity. These were Glu-305, Glu-307, and Phe-310 in motif A (IELEMKF); Arg-454 and Lys-456 in motif B (RTK); Glu-492, Glu-494, and Glu-496 in motif C (EVELE). Four acidic residues, Glu-305, Glu-307, Glu-494, and Glu-496, may comprise the metal-binding site(s), insofar as their replacement by glutamine inactivated Cet1p. E492Q retained triphosphatase activity. Basic residues Arg-454 and Lys-456 in motif B are implicated in binding to the 5'-triphosphate. Changing Arg-454 to alanine or glutamine resulted in a 30-fold increase in the K(m) for ATP, whereas substitution with lysine increased K(m) 6-fold. Changing Lys-456 to alanine or glutamine increased K(m) an order of magnitude; ATP binding was restored when arginine was introduced. Alanine in lieu of Phe-310 inactivated Cet1p, whereas Tyr or Leu restored function. Alanine mutations at aliphatic residues Leu-306, Val-493, and Leu-495 resulted in thermal instability in vivo and in vitro. A second S. cerevisiae RNA triphosphatase/NTPase (named Cth1p) containing motifs A, B, and C was identified and characterized. Cth1p activity was abolished by E87A and E89A mutations in motif A. Cth1p is nonessential for yeast growth and, by itself, cannot fulfill the essential role played by Cet1p in vivo. Yet, fusion of Cth1p in cis to the guanylyltransferase domain of mammalian capping enzyme allowed Cth1p to complement growth of cet1Delta yeast cells. This finding illustrates that mammalian guanylyltransferase can be used as a vehicle to deliver enzymes to nascent pre-mRNAs in vivo, most likely through its binding to the phosphorylated CTD of RNA polymerase II.
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PMID:Mutational analyses of yeast RNA triphosphatases highlight a common mechanism of metal-dependent NTP hydrolysis and a means of targeting enzymes to pre-mRNAs in vivo by fusion to the guanylyltransferase component of the capping apparatus. 1050 29

We report a novel case of multiple endocrine neoplasia type 2A (MEN 2A) associated with two mutations of the protooncogene RET. One affects codon 634 and causes a cysteine to arginine substitution; the second at codon 640 causes an alanine to glycine substitution in the transmembrane region. The two mutations were present on the same RET allele and were detected in germline and tumor DNA. Both mutations were de novo, i.e. they were not found in the DNA of the parents or relatives. Immunohistochemical and RT-PCR analysis showed that the pheochromocytoma expressed calcitonin as well as both RET alleles. A cell line established from the tumor and propagated in culture sustained the expression of RET and calcitonin, as did the original pheochromocytoma. Because the patient presented with medullary thyroid carcinoma and pheochromocytoma without parathyroid gland involvement, we speculate that this clinical picture could be correlated with the two RET mutations and to the unusual calcitonin production. This is the first report of a MEN 2A case due to two mutations of the RET gene and associated with a calcitonin-producing pheochromocytoma.
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PMID:A novel case of multiple endocrine neoplasia type 2A associated with two de novo mutations of the RET protooncogene. 1052 89

The interaction of tumor necrosis factor-alpha (TNFalpha) with its receptor sets in motion downstream signaling events including the activation of members of the mitogen-activated protein kinase (MAPK) family. In this study, we show that p42(mapk/erk2) phosphorylates sequences present within the cytoplasmic domain of CD120a (p55). By using a GST-CD120a-(207-425) fusion protein as substrate, phosphorylation was induced following stimulation of mouse macrophages with TNFalpha, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and zymosan particles and was blocked by immunodepletion of p42(mapk/erk2) and by specific inhibition of p42(mapk/erk2) activation with PD098059. Transfection of COS-7 cells with CD120a (p55), wild-type p42(mapk/erk2), and constitutively active MEK-1 followed by metabolic labeling with [(32)P]orthophosphate indicated that p42(mapk/erk2) phosphorylated the cytoplasmic domain of CD120a (p55) in intact cells. As a consequence of phosphorylation, CD120a (p55) expression at the plasma membrane and Golgi apparatus was lost and the receptor accumulated in intracellular tubular structures associated with the endoplasmic reticulum. Mutation of the four Ser and Thr ERK consensus phosphorylation sites to Ala residues inhibited the ability of the receptor to redistribute to intracellular tubules in a p42(mapk/erk2)-dependent fashion; whereas mutation of the phosphorylation sites to Asp and Glu residues mimicked the effect of receptor phosphorylation. These findings thus indicate that the phosphorylation of CD120a (p55) alters the subcellular localization of the receptor and may thereby result in changes in its signaling properties.
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PMID:Phosphorylation of tumor necrosis factor receptor CD120a (p55) by p42(mapk/erk2) induces changes in its subcellular localization. 1055 65

Staphylococcal enterotoxin H (SEH) has been described as a superantigen by sequence homology with the SEA subfamily and briefly characterized for its in vivo activity. In this study, we demonstrate that SEH is a potent T cell mitogen and inducer of T cell cytotoxicity that possesses unique MHC class II-binding properties. The apparent affinity of SEH for MHC class II molecules is the highest affinity ever measured for a staphylococcal enterotoxin (Bmax1/2 approximately 0.5 nM for MHC class II expressed on Raji cells). An excess of SEA or SEAF47A, which has reduced binding to the MHC class II alpha-chain, is able to compete for binding of SEH to MHC class II, indicating an overlap in the binding sites at the MHC class II beta-chain. The binding of SEH to MHC class II is like SEA, SED, and SEE dependent on the presence of zinc ions. However, SEH, in contrast to SEA, binds to the alanine-substituted DR1 molecule, betaH81A, believed to have impaired zinc-bridging capacity. Furthermore, alanine substitution of residues D167, D203, and D208 in SEH decreases the affinity for MHC class II as well as its in vitro potency. Together, this indicates an MHC class II binding site on SEH with a different topology as compared with SEA. These unique binding properties will be beneficial for SEH to overcome MHC class II isotype variability and polymorphism as well as to allow an effective presentation on APCs also at low MHC class II surface expression.
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PMID:Staphylococcal enterotoxin H displays unique MHC class II-binding properties. 1058 65


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