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:2.7.11.24 (mitogen-activated protein kinase)
95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When PC12D cells, a subline of PC12 cells, were cultured with nerve growth factor (NGF), outgrowth of neurites was promoted even when RNA synthesis was blocked. This property of PC12D cells may enable us to resolve the mechanism of the outgrowth of neurites that is induced in a transcription-independent manner. The outgrowth of neurites from PC12D cells was also stimulated in response to fibroblast growth factor (FGF) and was slightly stimulated in response to epidermal growth factor (EGF). The brief exposure of intact PC12D cells not only to NGF but also to FGF or to EGF stimulated a protein kinase activity in extracts of such cells that catalyzed phosphorylation of microtubule-associated protein 1 (MAP-1) and MAP-2 in vitro. Similar dose-response relationships for the effects of NGF and of FGF on the activation of the kinase and on the outgrowth of neurites were observed. The effects of combinations of NGF and GFG or EGF were not additive in terms of either the outgrowth of neurites or the increase in the kinase activity. Treatment of cells with phorbol 12-myristate 13-acetate (PMA) also stimulated the kinase activity that phosphorylated MAPs in vitro. However, the level of the enzymatic activity that resulted from the combined treatment of cells with PMA and NGF was additive, as is the case with dibutyryl cyclic AMP and NGF. These findings suggest that NGF, FGF, and EGF may stimulate the activity of the same MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Activation of microtubule-associated protein kinase in PC12D cells in response to both fibroblast growth factor and epidermal growth factor and concomitant stimulation of the outgrowth of neurites. 131 Jul 25

Nerve growth factor-stimulated mitogen-activated protein kinase (pp42/44MAP) kinase was characterized by sequential column chromatography on DEAE-Sephacel, phenyl-Sepharose CL4B, and S-200. The kinase displayed an apparent molecular mass of 42 kDa and reacted with an antiphosphotyrosine antibody. Peptide mapping of myelin basic protein revealed the presence of one phosphopeptide that was phosphorylated on Thr-97. pp42/44MAP kinase activity was dependent on Mg2+ and inhibited by K252a both in vitro and in vivo. Nerve growth factor-stimulated kinase activation was diminished by down-regulation of protein kinase C with 200 nM 12-phorbol 13-myristate acetate or with staurosporine (1 nM), a protein kinase C inhibitor. Genistein, a protein tyrosine kinase inhibitor, blocked nerve growth factor-mediated neurite extension as well as diminished activation of pp42/44MAP kinase. Our data demonstrate that activation of this kinase system by nerve growth factor displays a requirement for both protein kinase C as well as protein tyrosine kinase. In addition, other agents that are capable of promoting neurite outgrowth in PC12 cells, such as fibroblast growth factor or dibutyryl cyclic AMP, do so independently of activating this kinase system.
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PMID:pp42/44MAP kinase is a component of the neurogenic pathway utilized by nerve growth factor in PC12 cells. 132 67

When the supernatant fractions from extracts of control and nerve growth factor (NGF)- or dibutyryl cyclic AMP-treated PC12D cells were applied to DEAE-Sepharose columns and proteins were eluted with a gradient of NaCl, three separate peaks of kinase activity that phosphorylated microtubule-associated proteins (MAPs) were recovered. Enhancement of the kinase activity in peak 1 was noted in the case of dibutyryl cyclic AMP-treated cells. In contrast, the kinase activity in the third peak was markedly elevated, in terms of the ability to phosphorylate MAP1 and MAP2, in the case of the extract from NGF-treated cells. This activity was designated previously as NGF-dependent MAP kinase. The apparent molecular mass of the active kinase was 45-50 kDa. The apparent Km value was 35 microM for ATP with either MAP1 or MAP2 as substrate. When the kinase activity in the fractions from the DEAE-Sepharose column was assayed in the presence of Mn2+ instead of Mg2+, another NGF-stimulated kinase activity was detected in the fractions eluted by a lower concentration of NaCl than that which eluted the Mg(2+)-activated kinase. Other growth factors, namely, epidermal growth factor and basic fibroblast growth factor, also stimulated the activity of NGF-dependent MAP kinase. Possible involvement of the kinase in the outgrowth of neurites has been suggested. The NGF-induced activation of NGF-dependent MAP kinase was blocked by the presence of K-252a. In contrast, the activation of NGF-dependent MAP kinase by basic fibroblast growth factor and by epidermal growth factor was not blocked, but actually stimulated by K-252a, a result that correlates well with the analogous actions of the drug on the outgrowth of neurites that is induced by these growth factors. The latter observation strengthens the possibility of a close relationship between the outgrowth of neurites and the activation of NGF-dependent MAP kinase.
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PMID:Chromatographic resolution and characterization of a nerve growth factor-dependent kinase that phosphorylates microtubule-associated proteins 1 and 2 in PC12 cells. 132 17

Casein kinase II (CKII) is one of several protein kinases that become activated before germinal-vesicle breakdown in maturing sea-star oocytes. Echinoderm CKII was purified over 11,000-fold with a recovery of approximately 10% by sequential fractionation of the oocyte cytosol on tyrosine-agarose, heparin-agarose, casein-agarose and MonoQ. The purified enzyme contained 45, 38 and 28 kDa polypeptides, which corresponded to its alpha, alpha' and beta subunits respectively. The beta-subunit was autophosphorylated on one major tryptic peptide on serine residues, whereas the alpha'-subunit incorporated phosphate into at least two tryptic peptides primarily on threonine residues. Western-blotting analysis of sea-star oocyte extracts with two different anti-peptide antibodies that recognized conserved regions of the alpha-subunit indicated that the protein levels of the alpha- and alpha'-subunits of CKII were unchanged during oocyte maturation. The purified CKII was partly inactivated (by 25%) by preincubation with protein-serine/threonine phosphatase 2A, but protein-tyrosine phosphatases had no effect. The beta-subunit of CKII was phosphorylated on a serine residue(s) up to 0.54 mol of P/mol of beta-subunit by purified protein kinase C, and this correlated with a 1.5-fold enhancement of its phosphotransferase activity with phosvitin as a substrate. CKII was not a substrate for the maturation-activated myelin basic protein kinase p44mpk from sea-star oocytes, nor for cyclic-AMP-dependent protein kinase. These studies point to possible regulation of CKII by protein phosphorylation.
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PMID:Purification and characterization of echinoderm casein kinase II. Regulation by protein kinase C. 159 Jul 72

Early in an infection the bloodstream forms of the African trypanosome Trypanosoma brucei brucei are long, slender and rapidly dividing. Later, non-dividing, short, stumpy forms may be found. In this report we described biochemical differences between the two parasite populations in the phosphorylation of their proteins in vitro. Compared with the slender populations, the non-dividing stumpy forms of the parasites exhibit decreased phosphorylation of an 80 kDa protein and enhanced phosphorylation of 37 kDa and 42 kDa proteins (pp37 and pp42). These changes occurred regardless of whether the stumpy trypanosomes were generated naturally during the course of the infection or induced by difluoromethylornithine treatment. The phosphorylation of pp37 and pp42 occurs on serine and threonine residues and is totally dependent upon the presence of Mn2+ or Mg2+. However, excess Mn2+ or Mg2+ inhibits phosphorylation. Maximal phosphorylation of pp42 occurs with 1 mm-Mn2+ or 10 mm-Mg2+, whereas that of pp37 occurs with 50 mM-Mn2+ or greater than 100 mm-Mg2+. The phosphorylation of pp37 is greatly enhanced by KCl, whereas that of pp42 is only slightly increased by this salt. Ca2+, calmodulin, phospholipids and cyclic AMP have no discernible effect upon the phosphorylation of pp42 or pp37 in vitro, whereas heparin, suramin, polylysine, polyarginine and polyamines all inhibit phosphorylation. Thus the enzymes that phosphorylate pp42 and pp37 have properties similar to, but distinct from, those of mammalian casein kinase II. Since the casein-kinase-like activity is higher in the slender than in the stumpy forms, the enhanced phosphorylation of pp42 and pp37 in the non-dividing parasites is probably a result of the enhanced synthesis of these acidic proteins.
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PMID:Phosphorylation differences among proteins of bloodstream developmental stages of Trypanosoma brucei brucei. 201 86

We have established a subline of PC12 cells (PC12D) that extend neurites very quickly in response not only to nerve growth factor (NGF) but also to cyclic AMP (cAMP) in the same way as primed PC12 cells (NGF-pretreated cells). When phosphorylation of brain microtubule proteins by extracts of these cells was monitored, two distinct kinase activities were found to be increased [from three-to eightfold in terms of phosphorylation of microtubule-associated protein (MAP) 2] by a brief exposure of cells to NGF or to dibutyryl cAMP (dbcAMP). The effect of the combined stimulation with both NGF and dbcAMP was additive in terms of the phosphorylation of MAP2. The apparent molecular mass of the kinase activated by dbcAMP was 40 kDa, and this kinase appears to be cAMP-dependent protein kinase. The molecular mass of the kinase activated by NGF was 50 kDa. The latter was activated to a measurable extent after 5 min of exposure of cells to NGF: it required Mg2+ for activity but not Mn2+ or Ca2+. This kinase appears to be distinct from previously reported kinases in PC12 cells, and it has been designated as NGF-dependent MAP kinase, although its physiological substrates are not known at present. An inhibitor of protein kinases, K-252a, selectively inhibited the outgrowth of neurites from PC12D cells in response to NGF but not to dbcAMP. When this inhibitor was added to the incubation medium of cells exposed simultaneously to NGF or dbcAMP, the increase in activity of the NGF-dependent MAP kinase was selectively abolished.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A nerve growth factor-dependent protein kinase that phosphorylates microtubule-associated proteins in vitro: possible involvement of its activity in the outgrowth of neurites from PC12 cells. 216 66

Microtubule protein prepared by cycles of assembly-disassembly contains a cyclic AMP-dependent protein kinase that phosphorylates the high-molecular-weight microtubule-associated protein MAP-2. The polyamine spermine at 2mM affected the phosphorylation of MAP-2 in a manner that depended on the cyclic AMP concentration. At cyclic AMP concentrations below 10(-6) M, spermine increased the rate of phosphorylation, while at cyclic AMP concentrations above 10(-6) M, spermine decreased the rate of phosphorylation. Spermine also decreased the final extent of cyclic AMP-dependent phosphorylation but did not affect the protein substrate specificity of the microtubule-associated protein kinase. MAP-2 was the principal substrate both in the presence and in the absence of spermine. Because of these results, we propose that microtubule protein phosphorylation may be regulated in vivo by spermine as well as by cyclic AMP levels.
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PMID:Polyamine regulation of the microtubule-associated protein kinase. 299 32

A consensus cyclic AMP response element (CRE) in the murine prostaglandin synthase-2 (PGS2) promoter is essential for pgs2 gene expression induced by pp60v-src, the v-src oncogene product. In this study, we investigate (i) the transcription factors active at the PGS2 "CRE site" in response to v-src activation and (ii) the signal transduction pathways by which pp60v-src activates these transcription factors. Transient transfection assays with pgs2 promoter/luciferase reporter chimeric genes suggest that c-Jun mediates v-src-induced pgs2 gene expression. Antibody supershift experiments demonstrate that c-Jun can participate in a complex with the pgs2 promoter CRE site. Moreover, in vitro immuno-complex assays demonstrate that pp60v-src expression strongly activates c-Jun N-terminal kinase (JNK1) enzyme activity. Serines 63 and 73, the sites of c-Jun phosphorylation by JNK, are essential for v-src-induced, pgs2 promoter-mediated luciferase expression. Cotransfection studies with plasmids expressing wild-type JNK, dominant-negative JNK, and dominant-negative MEKK-1 confirm that activation of the Ras/MEKK-1/JNK/c-Jun pathway is required for v-src-induced pgs2 gene expression. Overexpression of either wild-type ERK-1 or ERK-2 proteins also potentiate v-src-mediated luciferase expression driven by the pgs2 promoter, and expression of dominant-negative mutants of ERK-1, ERK-2, or Raf-1 attenuate this response. Thus, in response to v-src expression, a Ras/MEKK-1/JNK signal transduction pathway activating c-Jun and a Ras/Raf-1/ERK pathway converge to mediate pgs2 gene expression via the CRE site in the pgs2 promoter.
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PMID:v-src induces prostaglandin synthase 2 gene expression by activation of the c-Jun N-terminal kinase and the c-Jun transcription factor. 749 26

Growth factor stimulation of the mitogen-activated protein (MAP) kinase pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve growth factor-induced MAP kinase activation nor differentiation in PC12 pheochromocytoma cells. Instead, in PC12 cells cAMP activates MAP kinase. Since one of the major differences between the Ras/Raf/MAP kinase cascades of these cell types is the expression of B-Raf in PC12 cells, we compared the effects of cAMP on Raf-1 and B-Raf. In PC12 cells maintained in serum-containing medium, B-Raf was refractory to inhibition by cAMP, whereas Raf-1 was effectively inhibited. In contrast, both B-Raf and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The effect of cAMP is thus dependent upon growth conditions, with B-Raf being resistant to cAMP inhibition in the presence of serum. These results were extended by studies of Rat-1 fibroblasts into which B-Raf had been introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effectively inhibited. In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of MAP kinase and mitogenesis. These results indicate that Raf-1 and B-Raf are differentially sensitive to inhibition by cAMP and that B-Raf expression can contribute to cell type-specific differences in the regulation of the MAP kinase pathway. In contrast to the situation in PC12 cells, cAMP by itself did not stimulate MAP kinase in B-Raf-expressing Rat-1 cells. The activation of MAP kinase by cAMP in PC12 cells was inhibited by the expression of a dominant negative Ras mutant, indicating that cAMP acts on a target upstream of Ras. Thus, it appears that a signaling component upstream of Ras is also require for cAMP stimulation of MAP kinase in PC12 cells.
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PMID:Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells. 756 4

Activation of mitogen-activated protein kinase (MAP kinase) plays an important role in the cellular effects of nerve growth factor (NGF). Although the precise pathway by which NGF activates MAP kinase is not clear, several enzymes have been identified that may form a linear phosphorylation cascade, in which MAP kinase is activated by MAP kinase kinase (MEK). A key enzyme that links the ras-GTP complex to MEK is widely believed to be the raf kinase. However, immunoprecipitation experiments in PC-12 cells revealed that raf is not the major NGF-dependent MEK kinase [Zheng, Ohmichi, Saltiel and Guan (1994) Biochemistry 33, 5595-5599]. We have identified a protein kinase from PC-12 cells that catalyses both the phosphorylation and activation of MEK. This activity is stimulated 3-fold in cells treated with NGF. The partial purification on FPLC and characterization of this MEK kinase indicate that it is distinct from raf, MEK, MAP kinase and other previously described NGF-stimulated protein kinases. The activity of this enzyme is unaffected by direct addition to the assay of heparin, staurosporine, K252A and the heat-stable cyclic AMP-dependent kinase peptide inhibitor, but is slightly inhibited by NaF and calcium ions. Comparison of its behaviour on gel permeation and sucrose-density gradients indicates a molecular mass in the region of 50,000 Da. Moreover, isoelectric focusing of the enzyme revealed a pI of approx. 7.3. The kinase activity is specific for ATP as substrate with a Km of 11 microM, and requires Mg2+ as a cofactor. Analysis of the activation of this enzyme in PC-12 cells transfected with a dominant inhibitory mutant of p21ras suggests that this MEK kinase resides downstream of ras in the MAP kinase activation pathway. Moreover, site-directed mutation of the residues on MEK that are phosphorylated by raf does not completely abrogate phosphorylation by the MEK kinase, suggesting that this enzyme may share some phosphorylation sites with raf, but also phosphorylates MEK on other sites.
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PMID:Nerve growth factor stimulates a novel protein kinase in PC-12 cells that phosphorylates and activates mitogen-activated protein kinase kinase (MEK). 773 91


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