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)

Epidermal growth factor (EGF) stimulates adenylyl cyclase in the heart via activation of the stimulatory GTP-binding protein Gs. Therefore, employing peptides corresponding to regions in the cytosolic domain of the EGF receptor, we have investigated the ability of sequences within the EGF receptor to activate Gs. A 13-aa peptide (EGFR-13) corresponding to the juxtamembrane region in the cytosolic domain of the EGF receptor stimulated GTP binding and GTPase activity of Gs. This peptide did not stimulate GTP binding to Gi but increased the GTPase activity of this protein. Additionally, phosphorylation of the protein kinase C site (threonine residue) within EGFR-13 decreased the ability of the peptide to stimulate Gs and increase GTPase activity of Gi. Further, in functional assays of Gs employing S49 cyc- cell membranes, EGFR-13 increased the ability of Gs to stimulate adenylyl cyclase; phospho-EGFR-13 and a 14-aa peptide corresponding to a sequence in the cytosolic domain of the EGF receptor did not alter the functional activity of Gs. Hence, the juxtamembrane region of the EGF receptor can activate Gs and, by stimulating GTPase activity of Gi, inactivates this latter G protein. Phosphorylation of the threonine residue within this region attenuates the activity of the peptide as a modulator of G-protein function.
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PMID:A region in the cytosolic domain of the epidermal growth factor receptor antithetically regulates the stimulatory and inhibitory guanine nucleotide-binding regulatory proteins of adenylyl cyclase. 789 52

In Drosophila and Caenorhabditis, signal transduction pathways initiated by the activation of receptor-protein tyrosine kinases can mediate developmental fate decisions. In order to examine whether similar mechanisms are employed during mammalian embryogenesis, we undertook a search for novel protein kinases expressed during heart development in the mouse. The primitive mouse heart is formed between 7.75 and 8.5 days post coitum (dpc) and consists of myocardial and endocardial cells. A reverse transcriptase polymerase chain reaction-based approach was used to amplify protein kinase specific products from cDNAs obtained from 8.5 dpc heart tissue. Twenty independent PCR products corresponding to either protein serine/threonine or tyrosine kinases were identified. In this report, we describe the characterization of two of the genes corresponding to the novel PCR products (designated Hek2 and msk). Hek2 encodes the mouse ortholog of human HEK2, a recently identified member of the eph receptor-protein tyrosine kinase gene family. Prior to and at the time of heart formation (7.5-8.0 dpc), Hek2 is expressed in the cranial (rostral) region of the embryo from which a subpopulation of cells will give rise to the rudimentary heart. Between 8.0 and 9.5 dpc, Hek2 mRNA expression is observed in myocardial cells, head mesenchyme and paraxial mesoderm. Hek2 transcripts are not detected in endocardial cells. After 9.5 dpc, Hek2 expression is downregulated. msk (for myocardial SNF1-like kinase) encodes a putative protein serine/threonine kinase most similar to the yeast gene SNF1. msk mRNA expression is restricted to myocardial cells and their progenitors in the 7.75-8.5 dpc developing heart. Subsequently, msk mRNA expression is rapidly downregulated. The patterns of Hek2 and msk expression suggest that these protein kinases may function during development of the primitive heart.
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PMID:Identification of novel protein kinases expressed in the myocardium of the developing mouse heart. 789 99

Multiple endocrine neoplasia type 2B (MEN 2B) is a human cancer syndrome characterized by medullary thyroid carcinoma (MTC), pheochromocytomas, mucosal neuromas, ganglioneuromas of the intestinal tract, and skeletal and ophthalmic abnormalities. It appears both as an inherited disorder and as de novo disease. Sequence analysis of germ-line DNA from MEN 2B patients revealed the existence of the same point mutation in the RET protooncogene in 34 unrelated individuals. This sequence difference was not observed in 93 unaffected individuals, including the normal parents of 14 de novo MEN 2B patients. The mutation (ATG-->ACG) results in the replacement of methionine with threonine within the catalytic core region of the tyrosine kinase domain. We propose that this amino acid replacement effects substrate interactions and results in dominant oncogenic activity by the RET protein. Missense mutations in the extracellular ligand-binding domain of the RET protooncogene previously have been associated with two other disorders [MEN 2A and familial MTC (FMTC)] in which MTC is observed. MEN 2B represents the third form of heritable MTC known to be an allele of RET. Alterations in two different functional domains of the putative receptor protein tyrosine kinase are implicated in development of MTC.
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PMID:Single missense mutation in the tyrosine kinase catalytic domain of the RET protooncogene is associated with multiple endocrine neoplasia type 2B. 790 17

The activity of cyclin-dependent kinases (cdks) depends on the phosphorylation of a residue corresponding to threonine 161 in human p34cdc2. One enzyme responsible for phosphorylating this critical residue has recently been purified from Xenopus and starfish. It was termed CAK (for cdk-activating kinase), and it was shown to contain p40MO15 as its catalytic subunit. In view of the cardinal role of cdks in cell cycle control, it is important to learn if and how CAK activity is regulated during the somatic cell cycle. Here, we report a molecular characterization of a human p40MO15 homologue and its associated CAK activity. We have cloned and sequenced a cDNA coding for human p40MO15, and raised specific polyclonal and monoclonal antibodies against the corresponding protein expressed in Escherichia coli. These tools were then used to demonstrate that p40MO15 protein expression and CAK activity are constant throughout the somatic cell cycle. Gel filtration suggests that active CAK is a multiprotein complex, and immunoprecipitation experiments identify two polypeptides of 34 and 32 kD as likely complex partners of p40MO15. The association of the three proteins is near stoichiometric and invariant throughout the cell cycle. Immunocytochemistry and biochemical enucleation experiments both demonstrate that p40MO15 is nuclear at all stages of the cell cycle (except for mitosis, when the protein redistributes throughout the cell), although the p34cdc2/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis. The absence of obvious changes in CAK activity in exponentially growing cells constitutes a surprise. It suggests that the phosphorylation state of threonine 161 in p34cdc2 (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin substrates, and by phosphatase(s).
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PMID:Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase). 792 89

Exposure of non-excitatory cells to the tyrosine kinase (PTK) inhibitors, genistein, herbimycin A, and tyrphostin, induced at least two families of K+ currents. The first, a TEA-insensitive slow-inactivating K+ current, is induced within 3 min following treatment with 140 mM genistein or 100 nM herbimycin A. The second current, a TEA-sensitive delayed rectifier, is induced within 30 min following treatment with 50 mM genistein or 10 nM herbimycin A. Currents with similar biophysical and pharmacological characteristics are induced in these cells following exposure to ionizing radiation. The radiation-induced currents are inhibited by pretreatment with the free radical scavenger, N-Acetyl L-Cysteine, or by pretreatment with the protein kinase C inhibitor, staurosporine; those induced by PTK inhibitors are not. The latter, therefore, do not appear to be mediated through free radicals or require serine/threonine phosphorylation for activation. Once the channels are activated by the PTK inhibitors, phosphorylation of the channel at serine/threonine residues results in slower inactivation of the induced current. We propose that protein tyrosine phosphorylation of the K+ channel protein itself or of a factor that interacts with it maintains the K+ channels of non-excitatory cells in a closed state. Following exposure to ionizing radiation, free radical-induced activation of serine/threonine kinase(s) results in phosphorylation of the channel and/or inactivation of a tyrosine kinase that in turn leads to activation of the K+ channels.
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PMID:Modulation of potassium channels by protein tyrosine kinase inhibitors. 792 99

Transitions of the cell cycle are controlled by cyclin-dependent protein kinases (cdks) whose phosphorylation on the Thr residue included in the conserved sequence YTHEVV dramatically increases the activity. A kinase responsible for this specific phosphorylation, called CAK for cdk-activating kinase, has been recently purified from starfish and Xenopus oocytes and shown to contain the MO15 gene product as a catalytic subunit. In the present paper, we have cloned the human homolog of Xenopus p40MO15 by probing a HeLa cell cDNA library with degenerate oligonucleotides deduced from Xenopus and starfish MO15 sequences. Human and Xenopus MO15 displayed a strong homology showing 86% identity with regard to amino acid sequences. Northern blot analysis of RNA extracts from a series of human tissues as well as from cultured rodent fibroblasts revealed a unique 1.4 kb MO15 mRNA. No variation in the amount of MO15 transcript or protein was found along the entire course of the fibroblast cell cycle. Fluorescence in situ hybridization on human lymphocyte metaphases showed two distinct chromosomal locations of human MO15 gene at 5q12-q13 and 2q22-q24. By using gene tagging and mammalian cell transfection, we demonstrate that the KRKR motif located at the carboxy terminal end of MO15 is required for nuclear targeting of the protein. Mutation of KRKR to NGER retains MO15 in the cytoplasmic compartment, whilst the wild-type protein is detected exclusively in the nucleus. Interestingly, we demonstrate that the nuclear targeting of MO15 is necessary to confer the protein its CAK activity. In contrast to the wild-type, the NLS-mutated MO15 expressed in Xenopus oocytes is unable to generate CAK as long as the nuclear envelope is not broken. The nuclear localization of both the MO15 gene product and CAK activity may imply that cdks activation primarily occurs in the cell nucleus.
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PMID:Cloning, expression and subcellular localization of the human homolog of p40MO15 catalytic subunit of cdk-activating kinase. 793 35

MEK, a dual specificity threonine/tyrosine kinase, has been postulated to be a convergent point for signaling from receptor protein tyrosine kinases (RTKs) and G-protein-coupled receptors. In contrast to yeast and mammalian cells where several MEKs have been isolated, only one Drosophila MEK (D-Mek) has been characterized to date. Previous studies have shown that D-Mek acts in the Torso RTK signaling pathway. To demonstrate that D-Mek also operates downstream of other RTKs, we generated a temperature-sensitive allele of D-mek (D-mekts) by site-directed mutagenesis based on the amino acid change of a yeast cdc2ts mutation. Using D-mekts, we show that in addition to its role in Torso signaling, D-Mek operates in the Sevenless and in the Drosophila epidermal growth factor RTK pathways. Because loss-of-function mutations in D-mek and the upstream receptors give rise to similar phenotypes, it suggests that D-mek is the only MEK activated by Drosophila RTKs. In addition, we demonstrate that different RTK pathways respond differently to alteration in D-Mek activity.
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PMID:A temperature-sensitive MEK mutation demonstrates the conservation of the signaling pathways activated by receptor tyrosine kinases. 795 87

The 13 amino acid EGFR-sequence AENAEYLRVAPQS-NH2 containing the in vivo autophosphorylated Tyr 1171, was synthesized by Fmoc continuous-flow SPPS with and without N-terminal Boc protection. In addition to the native sequence, peptides in which tyrosine was exchanged by serine and threonine were prepared. Global phosphorylation of the unprotected hydroxyl amino acids on the resin with di-tert-butyl-N,N-diethylphosphoramidite and 1H-tetrazole followed by in situ oxidation of the resulting phosphites with tert-butyl hydroperoxide or with dibenzoyl tetrasulfide resulted in the tyrosine-, serine- and threonine-phosphorylated and -thiophosphorylated sequences, respectively. The quality of the products after phosphorylation with N-terminal protection was better than without. Whereas the serine- and threonine-thiophosphate group was stable, tyrosine-thiophosphate turned out to be hydrolytically labile under acidic conditions. The rate of hydrolysis was determined with the tyrosine-thiophosphorylated model dipeptide Ac-Tyr-Gly-OH between pH 0.1 and 8. Hydrolysis was fastest at pH 3, with a half-time of 12.5 h at room temperature. The tyrosine-thiophosphate group was completely stable at pH 8.
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PMID:Solid-phase syntheses of phosphorylated and thiophosphorylated peptides related to an EGFR sequence. 807 Sep 68

Ras proteins exert their mitogenic and oncogenic effects through activation of downstream protein kinases. An important question is how Ras-generated signals reach the nucleus to activate downstream target genes. AP-1, a heterodimeric complex of Jun and Fos proteins, which activates mitogen-inducible genes, is a major nuclear target of Ras. Ras can stimulate AP-1 activity by inducing c-fos transcription, a process which is probably mediated by the ERK1 and -2 mitogen-activated protein (MAP) kinases, which phosphorylate the transcription factor Elk-1/TCF. Besides inducing transcription from fos and jun genes, mitogens and Ras proteins enhance AP-1 activity through phosphorylation of c-Jun. Phosphorylation of the c-Jun activation domain leads to c-jun induction through an autoregulatory loop. Ras- and ultra-violet-responsive protein kinases that phosphorylate c-Jun on serine residues at positions 63 and 73 and stimulate its transcriptional activity have been identified. These proline-directed kinases, termed JNKs, are novel MAP kinases. It is not clear, however, whether c-Jun is the only recipient and JNK the only transducer of the Ras signal to AP-1 proteins. A short sequence surrounding the major JNK phosphorylation site of c-Jun is conserved in c-Fos and is part of its activation domain, suggesting that c-Fos may be similarly regulated. Here we show that Ras does indeed augment the transcriptional activity of c-Fos through phosphorylation at Thr 232, the homologue of Ser 73 of c-Jun. However, this is mediated by a novel Ras- and mitogen-responsive proline-directed protein kinase that is different from JNKs and ERKs. Therefore, at least three types of proline-directed kinases transmit Ras- and mitogen-generated signals to the transcriptional machinery.
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PMID:c-Fos transcriptional activity stimulated by H-Ras-activated protein kinase distinct from JNK and ERK. 807 47

The eukaryotic cell cycle is regulated by the sequential activation of cyclin-dependent kinases (CDKs). CDK activation is dependent on cyclin binding and phosphorylation of a conserved threonine (T161 in Cdc2) mediated by the CDK-activating kinase CAK. A CDK-related kinase, MO15 (ref. 10), has been identified as the catalytic subunit of CAK (refs 11-13). Here we use a yeast two-hybrid screen to show that a new human cyclin (cyclin H) is a MO15-associated protein. Cyclin H is a major MO15 partner in vivo and enhances the kinase activity of MO15 towards Cdk2/cyclin A. These findings demonstrate that a cyclin/kinase complex can function as a regulator of other cyclin/kinase complexes, and suggest that cyclin/kinase cascades may exist.
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PMID:A cyclin associated with the CDK-activating kinase MO15. 807 87


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