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 functional significance of the extracellular amino-terminal region and of three highly conserved aromatic residues present in the fifth (TM-V) and sixth (TM-VI) transmembrane domains of the rat cholecystokinin (CCK)B receptor, transfected in Cos-7 cells, was investigated by site-directed mutagenesis. The amino-terminal region of the CCKB receptor seemed to be weakly involved in CCK binding in that the affinities of CCK8 and selective agonists and antagonists were not modified by truncation of this region. Substitution of Phe347 in TM-VI with alanine produced a mutant receptor that displays the same affinity and selectivity as the wild-type receptor for agonists, but a slightly increased affinity for the selective CCKB antagonist L-365,260. However, the addition of saturating CCK8 concentrations to cells expressing this mutant did not result in the production of inositol phosphates, demonstrating the critical role of Phe347 in CCKB receptor to G protein coupling. Substitution of Phe227 with alanine was without effect on the affinities of CCKB ligands and on phosphoinositide turnover but modified the affinity of the CCKA antagonist L-364,718. Residue Trp351 located within the CCKB receptor TM-VI is involved in the binding of CCK8 and CCK4 and of the CCK4-based antagonist PD-134,308, as illustrated by the decreased affinities of these ligands in W351A mutant. The lower affinity for CCK8 observed with this mutated CCKB receptor accounts for the higher EC50 value for phosphotidylinositol hydrolysis. This study suggests that at least part of the binding site for the agonist is located inside the transmembrane domain of the CCKB receptor, partially overlapping that of antagonists, and gives new insights into the regions involved in the transduction process.
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PMID:Binding sites and transduction process of the cholecystokininB receptor: involvement of highly conserved aromatic residues of the transmembrane domains evidenced by site-directed mutagenesis. 958 14

Germline mutations in the RET proto-oncogene have been shown to be the underlying cause of multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Some cases of sporadic medullary thyroid carcinoma (sporadic MTC) are reported to have specific codon 918, 883 and 768 mutations of the RET gene in tumor tissues. We examined RET gene mutations in 40 Japanese cases who had previously undergone surgery for sporadic MTC. DNA extracted from formalin-fixed tumor tissues and corresponding normal thyroid tissues or peripheral blood leukocytes was analyzed for mutations of exon 10, 11, 13, 14 and 16 of the RET gene by DNA sequencing and by mutation-specific restriction enzyme analysis. Germline RET point mutations were found in six of 40 cases (15%), cysteine residues at codon 618 in two, codon 634 in three and valine residue at codon 804 in one, and were newly identified as heritable MTC. Of the remaining 34 sporadic MTC cases, four (12%) had tumor-specific RET point mutations. Two were found in exon 16; one case showed an ATG to ACG (Met to Thr) mutation at codon 918, and the other showed two point mutations, ATG to ACG (Met to Thr) at codon 918 and GCA to GTA (Ala to Val) at codon 919 with loss of the wild-type allele, suggesting that both alleles at the RET locus were altered. The other two were found in exon 13; one case showed a CCG to TCG (Pro to Ser) mutation at codon 766 and the other showed a silent mutation, GTC to GTT (Val) at codon 778 with loss of the wild-type allele. There was no association of sporadic mutations with recurrence or prognosis in patients with sporadic MTC. The low rate of somatic RET mutation at codon 918 in our sporadic MTC suggests that as yet unknown factors may be involved. Genetic alterations in both alleles may have an important role in small fraction of sporadic MTCs.
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PMID:Novel point mutations and allele loss at the RET locus in sporadic medullary thyroid carcinomas. 961 47

A fully functional cysteine-free derivative of the intrinsically stable anti-HER2 scFv fragment hu4D5-8 was generated by replacing the disulfide forming cysteine residues in VH and VL with the amino acid combination valine-alanine in both domains. The antigen binding properties, determined by ELISA and BIAcore measurements, were not affected by removal of the disulfide bonds. The thermodynamic stability of the disulfide-containing scFv of 8.1 kcal/mol is decreased upon complete reduction of both disulfides to 2.7 kcal/mol, while that of the valine-alanine variant is somewhat higher (about 3.8 kcal/ mol). Our results suggest that, in principle, a disulfide-free fully functional derivative of any scFv can be obtained, as long as the corresponding disulfide-containing scFv has a high enough thermodynamic stability.
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PMID:An intrinsically stable antibody scFv fragment can tolerate the loss of both disulfide bonds and fold correctly. 963 57

UV irradiation leads to severe damage, such as cutaneous inflammation, immunosuppression, and cancer, but it also results in a gene induction protective response termed the UV response. The signal triggering the UV response was thought to originate from DNA damage; recent findings, however, have shown that it is initiated at or near the cell membrane and transmitted via cytoplasmic kinase cascades to induce gene transcription. Urokinase-type plasminogen activator (uPA) was the first protein shown to be UV inducible in xeroderma pigmentosum DNA repair-deficient human cells. However, the underlying molecular mechanisms responsible for the induction were not elucidated. We have found that the endogenous murine uPA gene product is transcriptionally upregulated by UV in NIH 3T3 fibroblast and F9 teratocarcinoma cells. This induction required an activator protein 1 (AP1) enhancer element located at -2.4 kb, since deletion of this site abrogated the induction. We analyzed the contribution of the three different types of UV-inducible mitogen-activated protein (MAP) kinases (ERK, JNK/SAPK, and p38) to the activation of the murine uPA promoter by UV. MEKK1, a specific JNK activator, induced transcription from the uPA promoter in the absence of UV treatment, whereas coexpression of catalytically inactive MEKK1(K432M) and of cytoplasmic JNK inhibitor JIP-1 inhibited UV-induced uPA transcriptional activity. In contrast, neither dominant negative MKK6 (or SB203580) nor PD98059, which specifically inhibit p38 and ERK MAP kinase pathways, respectively, could abrogate the UV-induced effect. Moreover, our results indicated that wild-type N-terminal c-Jun, but not mutated c-Jun (Ala-63/73), was able to mediate UV-induced uPA transcriptional activity. Taken together, we show for the first time that kinases of the JNK family can activate the uPA promoter. This activation links external UV stimulation and AP1-dependent uPA transcription, providing a transcription-coupled signal transduction pathway for the induction of the murine uPA gene by UV.
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PMID:UV irradiation induces the murine urokinase-type plasminogen activator gene via the c-Jun N-terminal kinase signaling pathway: requirement of an AP1 enhancer element. 967 63

A new point mutation, TCG(Ser)-->GCG(Ala) in codon 891, exon 15 of the RET protooncogene was revealed in two patients from a pedigree with familial medullary thyroid carcinoma (FMTC), but not in healthy persons. A linkage analysis with two well-known and two new intragene polymorphisms showed that informative polymorphic markers, the phenotypic expression of the disease, and the mutation are cosegregated in the studied pedigree. Two new polymorphisms, G/A at position-24 of intron 14 and C/T in codon 836 of exon 14, were found in the RET protooncogene. The frequencies of allele 1 of the polymorphic site in codon 836 were the same (0.96) in the Russian and German populations. This was also characteristic of two polymorphisms revealed earlier, namely, the sites in codons 691 (0.80 and 0.81, respectively) and 904 (0.21 and 0.22). However, the frequency of allele 1 of the polymorphisms in intron 14 differed significantly (0.87 and 0.77, respectively).
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PMID:[A new inherited RET proto-oncogene mutation associated with familial medullary thyroid carcinoma and polymorphisms in adjacent regions]. 977 62

Platelet-derived growth factor beta receptor (PDGFbetaR) is a transmembrane receptor tyrosine kinase involved in a variety of cellular functions. We have generated a constitutively activated murine PDGFbetaR containing a valine to alanine substitution at residue 536, located in the cytoplasmic juxtamembrane domain. When this mutant receptor (PR-V536A) was expressed in Ba/F3 cells, it allowed the cells to survive and proliferate in the absence of IL-3 or PDGF, and tyrosine phosphorylation of PR-V536A was increased markedly compared with that of the wild-type PDGFbetaR in the absence of ligand and similar to that observed in ligand-activated PDGFbetaR. PR-V536A displayed increased tyrosine kinase activity in vitro toward an exogenous substrate, and the tyrosine kinase activity of the receptor was required for the constitutive activation of the mutant. This valine to alanine substitution also activated a PDGFbetaR mutant unable to bind PDGF. Alanine substitutions at positions homologous to V536 of the murine PDGFbetaR also activated other members of the PDGF receptor subfamily. The amino acid sequence of this region revealed a strong similarity to WW domains present in other signal transduction proteins. Furthermore, GST fusion proteins containing the juxtamembrane region of the PDGFR specifically associated with peptides containing the WW domain consensus recognition sequence PPXY. The results suggest that the cytoplasmic juxtamembrane domain plays a role in the regulation of receptor activity and function, perhaps by participating in protein-protein interactions.
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PMID:A single amino acid substitution in a WW-like domain of diverse members of the PDGF receptor subfamily of tyrosine kinases causes constitutive receptor activation. 984 97

The extracellular receptor stimulated kinase ERK2 (p42(MAPK))-phosphorylated human cAMP-specific phosphodiesterase PDE4D3 at Ser579 and profoundly reduced ( approximately 75%) its activity. These effects could be reversed by the action of protein phosphatase PP1. The inhibitory state of PDE4D3, engendered by ERK2 phosphorylation, was mimicked by the Ser579-->Asp mutant form of PDE4D3. In COS1 cells transfected to express PDE4D3, challenge with epidermal growth factor (EGF) caused the phosphorylation and inhibition of PDE4D3. This effect was blocked by the MEK inhibitor PD98059 and was not apparent using the Ser579-->Ala mutant form of PDE4D3. Challenge of HEK293 and F442A cells with EGF led to the PD98059-ablatable inhibition of endogenous PDE4D3 and PDE4D5 activities. EGF challenge of COS1 cells transfected to express PDE4D3 increased cAMP levels through a process ablated by PD98059. The activity of the Ser579-->Asp mutant form of PDE4D3 was increased by PKA phosphorylation. The transient form of the EGF-induced inhibition of PDE4D3 is thus suggested to be due to feedback regulation by PKA causing the ablation of the ERK2-induced inhibition of PDE4D3. We identify a novel means of cross-talk between the cAMP and ERK signalling pathways whereby cell stimuli that lead to ERK2 activation may modulate cAMP signalling.
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PMID:The MAP kinase ERK2 inhibits the cyclic AMP-specific phosphodiesterase HSPDE4D3 by phosphorylating it at Ser579. 1002 32

Mutations at aspartic acid 631 in Ret were reported in sporadic pheochromocytoma and medullary thyroid carcinoma. We replaced this aspartic acid with four other amino acids including tyrosine, glycine, asparagine, and alanine and investigated the transforming activity of these mutant cDNAs. Among them, RET cDNA with a mutation of aspartic acid to tyrosine (D631Y) that was reported in sporadic pheochromocytoma showed high transforming activity. The D631Y mutation activated Ret by inducing its disulfide-linked dimerization in the transfectant as observed for multiple endocrine neoplasia (MEN) 2A mutations at cysteine 609, 611, 618, 620, 630, or 634. Further mutation analysis suggested that cysteine 630 or 634 could be involved in the disulfide-linked Ret dimerization induced by the D631Y mutation.
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PMID:Mechanism of Ret activation by a mutation at aspartic acid 631 identified in sporadic pheochromocytoma. 1004 54

Serine phosphorylation of signal transducers and activators of transcription (STAT) 1 and 3 modulates their DNA-binding capacity and/or transcriptional activity. Earlier we suggested that STAT5a functional capacity could be influenced by the mitogen-activated protein kinase (MAPK) pathway. In the present study, we have analyzed the interactions between STAT5a and the MAPKs, extracellular signal-regulated kinases ERK1 and ERK2. GH treatment of Chinese hamster ovary cells stably transfected with the GH receptor (CHOA cells) led to rapid and transient activation of both STAT5a and ERK1 and ERK2. Pretreatment of cells with colchicine, which inhibits tubulin polymerization, did not inhibit STAT5a translocation to the nucleus and ERK1/2 activation. In vitro precipitation with a glutathione-S-transferase-fusion protein containing the C-terminal transactivation domain of STAT5a showed GH-regulated association of ERK1/2 with the fusion protein, while this was not seen when serine 780 in STAT5a was changed to alanine. In vitro phosphorylation of the glutathione-S-transferase-fusion proteins using active ERK only worked when the fusion protein contained wild-type STAT5a sequence. The same experiment, performed with full-length wild-type STAT5a and the corresponding S780A mutant, showed that serine 780 is the only substrate in full-length STAT5a for active ERK. In coimmunoprecipitation experiments, larger amounts of STAT5a-ERK1/2 complexes were detected in cytosol from untreated CHOA cells than in cytosol from GH-treated cells, suggesting the presence of preformed STAT5a-ERK1/2 complexes in unstimulated cells. Transfection experiments with COS cells showed that kinase-inactive ERK1 decreased GH stimulation of STAT5-regulated reporter gene expression. These observations show, for the first time, direct physical interaction between ERK and STAT5a and also clearly identify serine 780 as a target for ERK. Furthermore, it is also established that serine phosphorylation of STAT5a transactivation domain, via the MAPK pathway, is a means of modifying GH-induced transcriptional activation.
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PMID:Extracellular signal-regulated kinase (ERK) interacts with signal transducer and activator of transcription (STAT) 5a. 1019 62

KIT receptor kinase activity is repressed, prior to stem cell factor binding, by unknown structural constraints. Using site-directed mutagenesis, we examined the role of KIT intracellular juxtamembrane residues Met-552 through Ile-563 in controlling receptor autophosphorylation. Alanine substitution for Tyr-553, Trp-557, Val-559, or Val-560, all sitting along the hydrophobic side of an amphipathic alpha-helix (Tyr-553-Ile-563) predicted by the Chou-Fasman algorithm, resulted in substantially increased spontaneous receptor phosphorylation, revealing inhibitory roles for these residues. Alanine substitution for other residues, most of which are on the hydrophilic side of the helix, caused no or slightly increased basal receptor phosphorylation. Converting Tyr-553 or Trp-557 to phenylalanine generated slight or no elevation, respectively, in basal KIT phosphorylation, indicating that the phenyl ring of Tyr-553 and the hydrophobicity of Trp-557 are critical for the inhibition. Although alanine substitution for Lys-558 had no effect on receptor phosphorylation, its substitution with proline produced high spontaneous receptor phosphorylation, suggesting that the predicted alpha-helical conformation is involved in the inhibition. A synthetic peptide comprising Tyr-553 through Ile-563 showed circular dichroism spectra characteristic of alpha-helix, supporting the structural prediction. Thus, the KIT intracellular juxtamembrane region contains important residues which, in a putative alpha-helical conformation, exert inhibitory control on the kinase activity of ligand-unoccupied receptor.
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PMID:Inhibition of spontaneous receptor phosphorylation by residues in a putative alpha-helix in the KIT intracellular juxtamembrane region. 1022 3


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