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)

Protein phosphorylation is considered an early cellular mechanism of signal transduction by surface immunoglobulins (sIg) and other receptors of B cells. Using intact human peripheral blood B cells of young subjects labeled with orthophosphate, increased phosphorylation levels of serine/threonine and tyrosine substrates were demonstrated on indicator phosphoproteins corresponding to the CD20 isoforms and microtubule-associated protein 2 kinase after cross-linking sIg and costimulation with phorbol diesters. By contrast, stimulated B cells from certain elderly subjects displayed substantial alterations in the phosphorylation patterns of serine/threonine or tyrosine indicator phosphoproteins. Also, age-related impairments in sIg stimulated mobilization of cytosolic protein kinase C (PKC) enzymatic activity and in cytosolic calcium [Ca2+]i responses of B cells were observed with the altered phosphorylation reactions. Comparison of the substrate phosphorylation profiles to the proliferative responses of stimulated B cells from individual elderly subjects suggested a model of signal transduction in which differing stimuli have different dependencies on phosphorylation reactions. Diminished proliferative responses after sIg ligation coincided with decreased phosphorylations of either tyrosine or serine/threonine indicator substrates. However, the decreased proliferative responses of B cells from elderly subjects with substantial reductions of tyrosine phosphorylation after sIg ligation were enhanced by the direct stimulation of serine/threonine kinase activity with phorbol diesters or CD40 ligation. Experiments with kinase inhibitors evaluated the relative dependency of different B cell stimuli on tyrosine and serine/threonine phosphorylation reactions. The proliferative responses of normal B cells to sIg ligation were quite sensitive to the tyrosine kinase inhibitor genistein whereas those observed following costimulations with phorbol diesters or CD40 ligation were more resistant. However, treatment of B cells with H7, an inhibitor of PKC activity, led to a more uniform reduction of B-cell responses after different stimuli. Results from RNase protection assays of c-myc expression also suggested that different B-cell stimuli might utilize distinct intracellular signaling pathways. Both the type of stimuli and mode of sIg ligation were important in determining the stimulated levels of c-myc mRNA expression. Thus, the current findings suggest that age-related defects are present in human B cell signaling pathways as reflected by tyrosine and serine/threonine phosphorylation reactions. Also, these age-related defects can coexist with altered mobilization of PKC enzymatic activity and with alterations in [Ca2+]i and proliferative responses.
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PMID:Signal transduction in human B cells during aging: alterations in stimulus-induced phosphorylations of tyrosine and serine/threonine substrates and in cytosolic calcium responsiveness. 180 9

Basic fibroblast growth factor (bFGF) plays an important role in development of the central nervous system and is neurotropic for a variety of neurons. In this study, we investigated whether bFGF is neurotropic for GT1 GnRH neuronal cell lines and if these cells express functional FGF receptors (FGFRs). The GT1 cell lines generated by genetically targeted tumorigenesis display highly differentiated properties of GnRH neurons. Addition of 2 and 10 ng/ml bFGF increased neurite outgrowth of GT1-7 cells and resulted in a significant increase of GT1 cell survival in serum-free medium. However, bFGF had no effect on [3H]thymidine incorporation at 24 or 48 h. RNase protection assays using riboprobes specific for murine FGFRs 1-3 showed that GT1 cells express FGFRs 1 and 3 but not 2. Occupancy of FGFRs with 10 ng/ml bFGF stimulated the sustained tyrosine phosphorylation of both the 42- and 44-kilodalton mitogen-activated protein kinases (MAPKs) for up to 6 h as shown by Western blot analysis. In addition, phosphorylation of the MAPKs was associated with enzyme activation as shown by an in-gel MAPK assay. GT1-1 and GT1-7 cells also express messenger RNA for bFGF, although the level of bioactive bFGF synthesized by GT1 cells appears suboptimal because GT1 cells can further respond to exogenously added bFGF. Thus, we have demonstrated that bFGF is a neurotropic factor in GT1 GnRh neuronal cell lines, raising the possibility that bFGF may play a role in the neurobiology of GnRH neurons.
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PMID:Basic fibroblast growth factor is a neurotropic factor in GT1 gonadotropin-releasing hormone neuronal cell lines. 764 90

The cholecystokinin-B and gastrin receptor is encoded by a single gene composed of five exons and spanning over 10 kilobases on human chromosome 11p 15.5-->15.4. Exon 4 has two possible alternative splicing donor sites that seem to be conserved in other species such as the canine, rat, Mastomys, and mouse. They could generate two receptor isoforms (short- and long-form), which differ in their putative third cytoplasmic domain of the serpentine G-protein-coupled receptors. In the present study, we examined whether an alternative splicing is operated in a tissue-specific manner and whether two receptor isoforms have functional differences. RNase-protection assay and S1 nuclease mapping demonstrated the preferential expression of the short-form in the human brain as well as the digestive organs, stomach and pancreas. The two putative isoforms of the cholecystokinin-B/gastrin receptor expressed in mouse fibroblasts showed the same characteristics in their ligand-bindings, the major signal transduction such as phosphoinositides production, cytoplasmic Ca2+ increase, tyrosine phosphorylation of focal adhesion kinase, activation of mitogen-activated protein kinase, and the induction of early-responsive genes such as c-fos, c-myc, and c-jun. Moreover, the ligand-dependent trophic effect was seen in both receptor isoforms. Taken together with the absence of tissue-specific expression of two receptor isoforms, these results suggest a species-specific dominant splice donor site in exon 4 of the human receptor gene.
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PMID:Functional characterization of two cholecystokinin-B/gastrin receptor isoforms: a preferential splice donor site in the human receptor gene. 784 14

An antisense oligodeoxynucleotide (ODN) approach was used to investigate whether mitogen-activated protein kinase (MAPK) is necessary for the hypertrophic response in cardiac myocytes. A phosphorothioate-protected 17-mer directed against the initiation of translation sites of the p42 and p44 MAPK isoform mRNAs was introduced into cultured cardiac myocytes by liposomal transfection. At an antisense ODN concentration of 0.2 mumol/L, p42 MAPK protein was reduced by 82% (immunoblot) after 48 hours, and p42 and p44 MAPK activities were reduced by 44% and 60%, respectively. The same concentration of anti-MAPK ODN inhibited development of the morphological features of hypertrophy (sarcomerogenesis, increased cell size) in myocytes exposed to phenylephrine. Phenylephrine-induced activation of the atrial natriuretic factor (ANF) promoter (measured by the activity of a transfected ANF promoter/luciferase reporter gene) and induction of ANF mRNA (measured by RNase protection assay) were also attenuated. We conclude that MAPK is important for the development of the hypertrophic phenotype in this model of hypertrophy.
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PMID:Depletion of mitogen-activated protein kinase using an antisense oligodeoxynucleotide approach downregulates the phenylephrine-induced hypertrophic response in rat cardiac myocytes. 863 45

The expression of the angiogenic growth factors, vascular endothelial cell growth factor (VEGF) and placenta growth factor (PIGF) was demonstrated in isolated human term cytotrophoblast and in vitro differentiated syncytiotrophoblast. RNase protection assays demonstrated VEGF expression in both cytotrophoblast and syncytiotrophoblast while prominent PIGF expression was detected in both types of trophoblast by Northern blot analyses. VEGF expression increased approximately eightfold in trophoblast cultured under hypoxic conditions (1 per cent O2) yet PIGF expression decreased 73 +/- 5.5 per cent in the same trophoblast. These results suggest distinct regulatory mechanisms govern expression of VEGF and PIGF in trophoblast. Characterization of the VEGF/PIGF receptors, KDR and flt-1, revealed the presence of flt-1 mRNA in isolated cytotrophoblast and in vitro differentiated syncytiotrophoblast. KDR was not detected in the isolated trophoblast. Exogenous rhVEGF induced c-Jun N-terminal kinase (JNK) activity in the normal trophoblast indicating that the flt-1 receptors on trophoblast are functional. Trophoblast-derived VEGF/PIGF could act in a paracrine fashion to promote uterine angiogenesis and vascular permeability within the placental bed. In addition, presence of function flt-1 on normal trophoblast suggests that VEGF/PIGF functions in an autocrine manner to perform an as yet undefined role in trophoblast invasion, differentiation, and/or metabolic activity during placentation.
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PMID:Vascular endothelial growth factor, placenta growth factor and their receptors in isolated human trophoblast. 936 1

The family of Tyr/Thr protein phosphatases, called dual-specificity phosphatases, have been implicated in the feedback regulation of the MAP kinase cascade by dephosphorylating the MAP kinases. Using low stringent cDNA screening we have isolated a chicken homologue of the CL100 phosphatase also called MAP kinase phosphatase 1 (MKP-1). The chicken MKP-1 has 84% and 85.5% identity to the rat and human amino acid sequence, respectively. Using RNase protection assay and in situ hybridization we have found that MKP-1 mRNA is expressed at low levels in most tissues during development. In embryonic dorsal root and sympathetic ganglia MKP-1 mRNA expression increases with age. The expression in large cells in dorsal root ganglia suggests that it is neurons which express MKP-1 mRNA. We also show that MKP-1 mRNA is induced in dissociated embryonic sympathetic neurons after nerve growth factor stimulation. In addition, our results show that MKP-1 mRNA is induced after NGF stimulation of fibroblasts expressing the NGF receptor TrkA, suggesting that MKP-1 is upregulated after activation of the TrkA receptor. These data show that the MKP-1 gene is regulated in a tissue and temporal specific fashion with strong expression in the developing peripheral ganglia, and suggest that the activation of MKP-1 mRNA expression by NGF is a ubiquitously induced response to TrkA activation, independent of the cellular origin or type on which the TrkA receptor is active.
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PMID:MAP kinase phosphatase-1 mRNA is expressed in embryonic sympathetic neurons and is upregulated after NGF stimulation. 960 44

Fatty acid synthase (FAS) is a critical enzyme in de novo lipogenesis. It catalyzes the seven steps in the conversion of malonyl-CoA and acetyl-CoA to palmitate. We have shown that the rate of FAS transcription is induced dramatically when fasted animals are refed with a high carbohydrate, fat-free diet or when streptozotocin-diabetic mice are given insulin. The FAS promoter was up-regulated by insulin through the proximal insulin response sequence containing an E-box motif at the -65-base pair position. Binding of upstream stimulatory factors to the -65 E-box is functionally required for insulin regulation of the FAS promoter. In the present study, we characterized signaling pathways in the insulin stimulation of FAS transcription using specific inhibitors for various signaling molecules and transfecting engineered phosphatidylinositol (PI) 3-kinase subunits and protein kinase B (PKB)/Akt. PD98059 and rapamycin, which inhibit MAP kinase and P70 S6 kinase, respectively, had little effect on the insulin-stimulated FAS promoter activity in 3T3-L1 adipocytes. On the other hand, wortmannin and LY294002, which specifically inactivate PI 3-kinase, strongly inhibited the insulin-stimulated FAS promoter activity. As shown in RNase protection assays, LY294002 also inhibited insulin stimulation of the endogenous FAS mRNA levels in 3T3-L1 adipocytes. Cotransfection of expression vectors for the constitutively active P110 subunit of PI 3-kinase resulted in an elevated FAS promoter activity in the absence of insulin and a loss of further insulin stimulation. Transfecting a dominant negative P85 subunit of PI 3-kinase decreased FAS promoter activity and blocked insulin stimulation. Furthermore, cotransfected wild-type PKB/Akt increased FAS promoter activity in the absence of insulin and a loss of insulin responsiveness of the FAS promoter. On the other hand, kinase-dead PKB/Akt acted in a dominant negative manner to decrease the FAS promoter activity and abolished its insulin responsiveness. These results demonstrate that insulin stimulation of fatty acid synthase promoter is mediated by the PI 3-kinase pathway and that PKB/Akt is involved as a downstream effector.
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PMID:Insulin stimulation of the fatty acid synthase promoter is mediated by the phosphatidylinositol 3-kinase pathway. Involvement of protein kinase B/Akt. 973 10

Microinjection of Onconase or RNase A into NIH/3T3 cells was used to study the intracellular actions of these two proteins. Onconase preferentially killed actively growing cells in both microinjection and cell culture experiments. Moreover, agents that increased the number of cells in S phase such as serum or microinjected signal transduction mediators (Ras, protein kinase C, and mitogen-activated protein kinase) enhanced Onconase cytotoxicity. Conversely, agents that decreased these proliferative pathways (dibutyryl cAMP and protein kinase A) correspondingly diminished Onconase cytotoxicity in microinjection experiments. These results were also mimicked in cell culture experiments since log-phase v-ras-transformed NIH/3T3 cells were more sensitive to Onconase (IC50 of 7 microg/ml) than parental NIH/3T3 fibroblasts (IC50 of 40 microg/ml). Based on those data we postulated that Onconase-mediated cell death in NIH/3T3 cells was related to events occurring at two or more points in the cell cycle preferentially associated with late G1/S and S phases. In contrast, quiescent NIH/3T3 cells were more sensitive to microinjected RNase A than log phase cells and positive mediators of proliferative signal transduction did not enhance RNase A-mediated cytotoxicity. Taken together, these results demonstrate that these two RNases use different pathways and/or mechanisms to elicit cytotoxic responses in NIH/3T3 cells. Predictions formulated from these studies can be tested for relevance to RNase actions in different target tumor cells.
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PMID:Cell cycle-related differences in susceptibility of NIH/3T3 cells to ribonucleases. 1004 64

To understand how the TNF receptor-associated factor 1 (TRAF1) is transcriptionally regulated, in vitro DNA binding assays, promoter-reporter gene assays, and RNase protection assays were performed with the human TRAF1 gene. Binding of NF-kappaB to three of five putative binding sites within the human TRAF1 promoter was found in electrophoretic mobility shift assay studies, and analysis of TRAF1 gene promoter luciferase constructs confirmed the functional importance of these elements. Moreover, triggering of TNF-R1, CD40, and the interleukin-1 receptor resulted in transcription of the TRAF1 gene, whereas receptors that are not activators or only poor activators of NF-kappaB in HeLa cells failed to show a significant TRAF1 induction. Because it has been shown that members of the TRAF family are involved in activation of NF-kappaB and the c-Jun N-terminal kinase (JNK) by the interleukin-1 receptor and members of the TNF receptor superfamily, a role of TRAF1 in receptor cross-talk and/or feedback regulation of activated receptor signaling complexes can be suggested. In fact, we found that TNF-induced activation of JNK is prolonged in transfectants overexpressing TRAF1, whereas overexpression of a deletion mutant of TRAF1 in which the N-terminal part had been replaced by the green fluorescent protein interfered with TNF-induced activation of NF-kappaB and JNK.
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PMID:The human tumor necrosis factor (TNF) receptor-associated factor 1 gene (TRAF1) is up-regulated by cytokines of the TNF ligand family and modulates TNF-induced activation of NF-kappaB and c-Jun N-terminal kinase. 1038 49

Angiotensin II and hypertension increase vascular oxidant stress. We examined how these might affect expression of the extracellular superoxide dismutase (ecSOD), a major form of vascular SOD. In mice, angiotensin II infusion (1.1 mg/kg for 7 days) increased systolic blood pressure from 107+/-3 to 152+/-9 mm Hg and caused a 3-fold increase in ecSOD, but there was no change in the cytosolic Cu/Zn SOD protein, as determined by Western blot analysis. This was associated with a similar increase in ecSOD mRNA as assessed by RNase protection assay and was prevented by losartan. Induction of ecSOD by angiotensin II was not due to hypertension alone, because hypertension caused by norepinephrine (5.6 mg. kg-1. d-1) had no effect on ecSOD. Similarly, exposure of mouse aortas to angiotensin II (100 nmol/L) in organoid culture increased ecSOD by approximately 2-fold. In the organoid culture, angiotensin II-induced upregulation of ecSOD was prevented by losartan (10 micromol/L) and PD985059 (30 micromol/L), a specific inhibitor of p42/44 MAP kinase kinase. Angiotensin II activates the NADH/NADPH oxidase; however, diphenyleneiodonium chloride (10 micromol/L), an inhibitor of this oxidase, did not prevent p42/44 MAP kinase phosphorylation or ecSOD induction by angiotensin II. Finally, in human aortic smooth muscle cells, angiotensin II moderately increased transcriptional rate (as assessed by nuclear run-on analysis) but markedly increased ecSOD mRNA stability. Thus, angiotensin II increases ecSOD expression independent of hypertension, and this increase involves both an increase in ecSOD transcription and stabilization of ecSOD mRNA. This effect of angiotensin II on ecSOD expression may modulate the oxidative state of the vessel wall in pathological processes in which the renin-angiotensin system is activated.
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PMID:Modulation of extracellular superoxide dismutase expression by angiotensin II and hypertension. 1040 Sep 7


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