Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A protein phosphorylated efficiently in vitro by MAP kinase-activated protein kinase-2 (MAPKAP-K2) was purified from skeletal muscle extracts and identified as the calcium/calmodulin-dependent myosin light chain kinase (MLCK). The phosphorylation site was mapped to Ser(161), a residue shown previously to be autophosphorylated by MLCK. The residue equivalent to Ser(161) became phosphorylated in vivo when rat hindlimbs were stimulated electrically. However, phosphorylation was triggered within seconds, whereas activation of MAPKAP-K2 required several minutes. Moreover, contraction-induced Ser(161) phosphorylation was similar in wild-type or MAPKAP-K2-/- mice. These results indicate that contraction-induced phosphorylation is probably catalyzed by MLCK and not MAPKAP-K2. Ser(161) phosphorylation induced the binding of MLCK to 14-3-3 proteins, but did not detectably affect the kinetic properties of MLCK. The sequence surrounding Ser(161) is unusual in that residue 158 is histidine. Previously, an arginine located three residues N-terminal to the site of phosphorylation was thought to be critical for the specificity of MAPKAP-K2.
 ...
PMID:Identification of a phosphorylation site on skeletal muscle myosin light chain kinase that becomes phosphorylated during muscle contraction. 1179 75

The protein kinase Raf is an important signaling protein. Raf activation is initiated by an interaction with GTP-bound Ras, and Raf functions in signal transmission by phosphorylating and activating a mitogen-activated protein (MAP) kinase kinase named MEK. We identified 13 mutations in the Caenorhabditis elegans lin-45 raf gene by screening for hermaphrodites with abnormal vulval formation or germline function. Weak, intermediate, and strong loss-of-function or null mutations were isolated. The phenotype caused by the most severe mutations demonstrates that lin-45 is essential for larval viability, fertility, and the induction of vulval cell fates. The lin-45(null) phenotype is similar to the mek-2(null) and mpk-1(null) phenotypes, indicating that LIN-45, MEK-2, and MPK-1 ERK MAP kinase function in a predominantly linear signaling pathway. The lin-45 alleles include three missense mutations that affect the Ras-binding domain, three missense mutations that affect the protein kinase domain, two missense mutations that affect the C-terminal 14-3-3 binding domain, three nonsense mutations, and one small deletion. The analysis of the missense mutations indicates that Ras binding, 14-3-3-binding, and protein kinase activity are necessary for full Raf function and suggests that a 14-3-3 protein positively regulates Raf-mediated signaling during C. elegans development.
 ...
PMID:Caenorhabditis elegans lin-45 raf is essential for larval viability, fertility and the induction of vulval cell fates. 1186 55

The metastasis-suppressive activity of Nm23-H1 was previously correlated with its in vitro histidine protein kinase activity, but physiological substrates have not been identified. We hypothesized that proteins that interact with histidine kinases throughout evolution may represent partners for Nm23-H1 and focused on the interaction of Arabidopsis "two-component" histidine kinase ERS with CTR1. A mammalian homolog of CTR1 was previously reported to be c-Raf; we now report that CTR1 also exhibits homology to the kinase suppressor of Ras (KSR), a scaffold protein for the mitogen-activated protein kinase (MAPK) cascade. Nm23-H1 co-immunoprecipitated KSR from lysates of transiently transfected 293T cells and at endogenous protein expression levels in MDA-MB-435 breast carcinoma cells. Autophosphorylated recombinant Nm23-H1 phosphorylated KSR in vitro. Phosphoamino acid analysis identified serine as the major target, and two peaks of Nm23-H1 phosphorylation were identified upon high performance liquid chromatography analysis of KSR tryptic peptides. Using site-directed mutagenesis, we found that Nm23-H1 phosphorylated KSR serine 392, a 14-3-3-binding site, as well as serine 434 when serine 392 was mutated. Phosphorylated MAPK but not total MAPK levels were reduced in an nm23-H1 transfectant of MDA-MB-435 cells. The data identify a complex in vitro histidine-to-serine protein kinase pathway, which may contribute to signal transduction and metastasis.
 ...
PMID:Nm23-H1 metastasis suppressor phosphorylation of kinase suppressor of Ras via a histidine protein kinase pathway. 1210 13

The enhancement of RNA-mediated motor neuron degeneration in transgenic mice by mutating a major mRNA instability determinant in a light neurofilament (NF-L) transgene implicates cognate RNA binding factors in the pathogenesis of motor neuron degeneration. p190RhoGEF is a neuron-enriched guanine exchange factor (GEF) that binds to the NF-L-destabilizing element, to c-Jun N-terminal kinase-interactive protein-1 (JIP-1), and to 14-3-3 and may link neurofilament expression to pathways affecting neuronal homeostasis. This study was undertaken to identify additional RNA species that bind p190RhoGEF and could affect interactions of the exchange factor with NF-L transcripts. The C-terminal domain of p190RhoGEF, containing the RNA-binding site, was expressed as a glutathione S-transferase fusion protein and was used as an affinity probe to isolate interactive RNAs in rat brain extracts. As expected, NF-L mRNA was identified as an RNA specie eluted from the affinity column. In addition, BC1 RNA was also found enriched in the bound RNA fraction. BC1 is a 152-nucleotide RNA that is highly expressed but untranslated in differentiated neurons. We show that BC1 and NF-L mRNA bind to a similar site in the C-terminal domain of p190RhoGEF, and their bindings to p190RhoGEF are readily cross-competed. Moreover, we identify a novel binding site in BC1 to account for its interaction with p190RhoGEF. The findings suggest a novel role of BC1 in differentiated neurons involving RNA-protein interactions of p190RhoGEF.
 ...
PMID:Binding of p190RhoGEF to a destabilizing element on the light neurofilament mRNA is competed by BC1 RNA. 1221 42

Unstimulated PC12 pheochromocytoma cells contain many proteins that bound to 14-3-3s in competition with a 14-3-3-binding peptide. Additional proteins, including one of 39 kDa (p39), became capable of binding to 14-3-3s in phosphatidylinositol 3-kinase-dependent responses to epidermal growth factor or nerve growth factor in vivo. The growth factor regulation was unaffected by inhibitors of the mitogen- or stress-activated protein kinase pathways, or by glucose starvation, but was blocked by amino acid starvation and only partially blocked by rapamycin. p39 in extracts of unstimulated, nutrient-fed cells, but not nutrient-starved cells, was able to bind to 14-3-3s after phosphorylation by protein kinase B (PKB) in vitro. Nutrient starvation did not affect the growth factor-stimulated activation of PKB in vivo. Either cycloheximide (CHX) or the cysteine protease inhibitor, MG132, restored the responsiveness of p39 to growth factors in nutrient-starved cells. In contrast, MG132 could not replace amino acids in supporting the growth factor-stimulated phosphorylation of two downstream targets of mTOR (mammalian target of rapamycin), namely eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and p70 S6 kinase. CHX permitted complete growth factor-stimulated phosphorylation of both 4E-BP1 and p70 S6 kinase in nutrient- starved cells; however, unlike p39, phosphorylation of these proteins was blocked by rapamycin. These findings implicate PKB (or an enzyme with similar specificity) in the growth factor-triggered phosphorylation of p39. In addition, amino acid starvation induces a CHX- and MG132-sensitive pathway that targets p39 and appears to be distinct from the mechanism of regulation of 4E-BP1 and p70 S6 kinase.
 ...
PMID:Regulation of the 14-3-3-binding protein p39 by growth factors and nutrients in rat PC12 pheochromocytoma cells. 1221 78

In Schizosaccharomyces pombe, rad24 and rad25 have been identified to be homologous to mammalian 14-3-3 genes and found to be involved in many cellular events, including checkpoint and meiosis. In the present study, we present evidences that Rad24 and Rad25 act as negative regulators of Byr2 (mitogen-activated protein kinase [MAPK] kinase kinase). Overexpression of rad24 or rad25 reduced mating and sporulation in homothallic wild-type cells. In contrast, the mating and sporulation efficiency of rad24- or rad25-null cells was higher than that of wild-type cells. Deletion of rad24 or rad25 increased sporulation efficiency in ras1-null diploid cells but not in byr2-, ste4-, byr1-, and spk1-null cells. Rad24 and Rad25 had no effect on the activity of constitutively active Byr1(S214DT218D). Rad24 and Rad25 bound to both the N-terminal and the C-terminal domains of Byr2 when these bacterially expressed proteins were examined. The formation of complexes in vivo between Byr2 and either Rad24 or Rad25 was also confirmed by immunocoprecipitation. Furthermore, we showed negative regulation of Byr2 by Rad25, by monitoring the mRNA level of mam2, which is regulated by both the Ras1/MAPK pathway and ste11, in various combinations of mutants. In addition, the cellular localization of Byr2 in living cells was observed by using fusion to green fluorescent protein. Byr2 was mainly localized in the cytoplasm during vegetative growth and then concentrated at the plasma membrane in response to nitrogen starvation. Deletion of rad24 or rad25 fastened the timing of Byr2 translocation. Our results are consistent with the hypothesis that one of the roles of 14-3-3 is to keep Byr2 in the cytoplasm and to affect the timing of Byr2 translocation in response to sexual developmental signal.
 ...
PMID:The 14-3-3 proteins Rad24 and Rad25 negatively regulate Byr2 by affecting its localization in Schizosaccharomyces pombe. 1224 89

Much effort has focused on the identification of MAPK cascades that are activated by the MEKK family of protein kinases. However, direct phosphorylation and regulation of the MEKK proteins has not been shown. To address this question, we have expressed recombinant (His)6FLAG.MEKK3 in Sf9 insect cells and tethered the purified protein to Ni-Sepharose so that we could precipitate interacting proteins and then identify such proteins by liquid chromatography and mass spectrometry (LC-MS). We identified 14-3-3 proteins as interacting with MEKK3, which suggested that (His)6FLAG.MEKK3 was phosphorylated on serine since 14-3-3 proteins are known to associate with phosphorylated proteins. We identified two phosphorylated amino acids at Ser166 and Ser337 of tryptic peptides derived from (His)6FLAG.MEKK3 by using LC-MS. Antibodies were developed that recognize the specific phosphorylated amino acid and with these antibodies, we demonstrate that various stimuli (tumor necrosis factor, arsenite, forskolin, and serum) promote phosphorylation of Ser166 and Ser337. However, neither of these phosphorylated amino acids is required for association with 14-3-3 protein or regulation of MEKK3-dependent ERK and JNK activity. Nonetheless, these results suggest that MEKK3 is a convergence point of multiple upstream signaling pathways.
 ...
PMID:Phosphorylation of the stress-activated protein kinase, MEKK3, at serine 166. 1239 20

Mineral nutrient deficiencies constitute major limitations for plant growth on agricultural soils around the world. To identify genes that possibly play roles in plant mineral nutrition, we recently generated a high-density array consisting of 1,280 genes from tomato (Lycopersicon esculentum) roots for expression profiling in nitrogen (N) nutrition. In the current study, we used the same array to search for genes induced by phosphorus (P), potassium (K(+)), and iron (Fe) deficiencies. RNA gel-blot analysis was conducted to study the time-dependent kinetics for expression of these genes in response to withholding P, K, or Fe. Genes previously not associated with P, K, and Fe nutrition were identified, such as transcription factor, mitogen-activated protein (MAP) kinase, MAP kinase kinase, and 14-3-3 proteins. Many of these genes were induced within 1 h after withholding the specific nutrient from roots of intact plants; thus, RNA gel-blot analysis was repeated for specific genes (transcription factor and MAP kinase) in roots of decapitated plants to investigate the tissue-specific location of the signal triggering gene induction. Both genes were induced just as rapidly in decapitated plants, suggesting that the rapid response to the absence of P, K, or Fe in the root-bathing medium is triggered either by a root-localized signal or because of root sensing of the mineral environment surrounding the roots. We also show that expression of Pi, K, and Fe transporter genes were up-regulated by all three treatments, suggesting coordination and coregulation of the uptake of these three essential mineral nutrients.
 ...
PMID:Rapid induction of regulatory and transporter genes in response to phosphorus, potassium, and iron deficiencies in tomato roots. Evidence for cross talk and root/rhizosphere-mediated signals. 1242 1

It is widely thought that the biological outcomes of Raf-1 activation are solely attributable to the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. However, an increasing number of reports suggest that some Raf-1 functions are independent of this pathway. In this report we show that mutation of the amino-terminal 14-3-3 binding site of Raf-1 uncouples its ability to activate the MEK/ERK pathway from the induction of cell transformation and differentiation. In NIH 3T3 fibroblasts and COS-1 cells, mutation of serine 259 resulted in Raf-1 proteins which activated the MEK/ERK pathway as efficiently as v-Raf. However, in contrast to v-Raf, RafS259 mutants failed to transform. They induced morphological alterations and slightly accelerated proliferation in NIH 3T3 fibroblasts but were not tumorigenic in mice and behaved like wild-type Raf-1 in transformation assays measuring loss of contact inhibition or anchorage-independent growth. Curiously, the RafS259 mutants inhibited focus induction by an activated MEK allele, suggesting that they can hyperactivate negative-feedback pathways. In primary cultures of postmitotic chicken neuroretina cells, RafS259A was able to sustain proliferation to a level comparable to that sustained by the membrane-targeted transforming Raf-1 protein, RafCAAX. In contrast, RafS259A was only a poor inducer of neurite formation in PC12 cells in comparison to RafCAAX. Thus, RafS259 mutants genetically separate MEK/ERK activation from the ability of Raf-1 to induce transformation and differentiation. The results further suggest that RafS259 mutants inhibit signaling pathways required to promote these biological processes.
 ...
PMID:A Raf-1 mutant that dissociates MEK/extracellular signal-regulated kinase activation from malignant transformation and differentiation but not proliferation. 1261 72

The 14-3-3 protein family comprises critical regulatory molecules involved in signaling during cell division, proliferation, and apoptosis. Despite extensive study, the functions of the 14-3-3 proteins in brain remain unclear. 14-3-3gamma, a subtype of the 14-3-3 family of proteins, was thought to be brain- and neuron-specific. Using RNA arbitrarily primed PCR, we identified an upregulated cDNA fragment of the 14-3-3gamma gene in primary cultures of astrocytes. Using Northern blot analysis, we confirmed this fragment was brain-specific. In cultures of astrocytes, 14-3-3gamma genes and proteins were differentially expressed at different ages and the proteins were distributed only in the cytoplasm. These results indicated that 14-3-3gamma was not neuron-specific but also expressed in astrocytes. The function of this protein in brain is unclear. Northern and Western blot analyses demonstrated that 14-3-3gamma mRNA and protein were upregulated in cultured astrocytes in an anaerobic chamber-induced ischemia model. The induction of 14-3-3gamma proteins was neither suppressed by an MAP kinase inhibitor (U0126) nor a PI-3 kinase inhibitor (LY294002). These data indicated that induction of 14-3-3gamma might not involve PI-3 and MAP kinase-dependent pathways. Using coimmunoprecipitation, we demonstrated that endogenous 14-3-3gamma bound to c-Raf-1 and p-Raf 259. As Raf is one of the critical serine/threonine kinases controlling cell growth, differentiation, and death, the binding of 14-3-3gamma to Raf indicates the critical role of this protein in ischemia-induced apoptosis and the changes in signal transduction in astrocytes in culture.
 ...
PMID:14-3-3gamma is upregulated by in vitro ischemia and binds to protein kinase Raf in primary cultures of astrocytes. 1273 Sep 52


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>