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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNA polymerase (RNAP) II is a multisubunit enzyme composed of several different subunits. Phosphorylation of the C-terminal domain (CTD) of the largest subunit is tightly regulated. In quiescent or in exponentially growing cells, both the unphosphorylated (IIa) and the multiphosphorylated (IIo) subunits of RNAP II are found in equivalent amounts as the result of the equilibrated antagonist action of protein kinases and phosphatases. In Drosophila and mammalian cells, heat shock markedly modifies the phosphorylation of the RNAP II CTD. Mild heat shocks result in dephosphorylation of the RNAP II CTD. This dephosphorylation is blocked in the presence of actinomycin D, as the CTD dephosphorylation observed in the presence of protein kinase inhibitors. Thus, heat shock might inactivate CTD kinases which are operative at normal growth temperatures, as some protein kinase inhibitors do. In contrast, severe heat shocks are found to increase the amount of phosphorylated subunit independently of the transcriptional activity of the cells. Mild and severe heat shocks activate protein kinases, which then phosphorylate, in vitro and in vivo, the CTD fused to beta-galactosidase. Most of the heat-shock-activated CTD kinases present in cytosolic lysates co-purify with the activated mitogen-activated protein (MAP) kinases, p42mapk and p44mapk. The weak CTD kinase activation occurring upon mild heat shock might be insufficient to compensate for the heat inactivation of the already existing CTD kinases. However, under severe stress, the MAP kinases are strongly heat activated and might prevail over the phosphatases. A survey of different cells and different heat-shock conditions shows that the RNAP II CTD hyperphosphorylation rates follow the extent of MAP kinase activation. These observations lead to the proposal that the RNAP II CTD might be an in vivo target for the activated p42mapk and p44mapk MAP kinases.
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PMID:Phosphorylation state of the RNA polymerase II C-terminal domain (CTD) in heat-shocked cells. Possible involvement of the stress-activated mitogen-activated protein (MAP) kinases. 758 77

The largest subunit of RNA polymerase (RNAP) II contains at it C-terminus an unusual domain comprising tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This C-terminal domain (CTD) can undergo phosphorylation at multiple sites giving rise to a form of the enzyme designated RNAP IIO. The unphosphorylated form is designated RNAP IIA. The largest subunits of RNAPs IIO and IIA are designated IIo and IIa, respectively. In quiescent NIH 3T3 fibroblasts, subunits IIo and IIa are present in comparable amounts. Upon serum stimulation, the amount of subunit IIo increases markedly and remains elevated for several hours. The increase of subunit IIo also occurs in transcription-inhibited cells and, therefore, is not a consequence of serum-activated transcription. This observation suggests that serum stimulation activates a CTD kinase and/or inhibits a CTD phosphatase. This hypothesis is supported by the finding that serum stimulates phosphorylation of a beta-galactosidase-CTD fusion protein expressed in these cells. Furthermore, an enhanced CTD kinase activity was discovered in lysates from serum-stimulated fibroblasts and was found to copurify with MAP kinases on a Mono Q column and to bind to anti-MAP kinase antibodies. The idea that MAP kinases phosphorylate the CTD in vivo is supported by the observation that subunit IIa, but not subunit IIb which lacks the CTD, is phosphorylated at multiple sites by purified MAP kinase. Consequently, the MAP kinases are a new class of CTD kinases which appear to be involved in the phosphorylation of RNAP II following serum stimulation. This phosphorylation may contribute to the transcriptional activation of serum-stimulated genes.
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PMID:Enhanced phosphorylation of the C-terminal domain of RNA polymerase II upon serum stimulation of quiescent cells: possible involvement of MAP kinases. 795 47

In Xenopus, normal mesoderm formation depends on signaling through the fibroblast growth factor (FGF) tyrosine kinase receptor. An important signaling pathway from receptor tyrosine kinases involves Ras/Raf/MAP kinase. However, the downstream pathway that occurs in the nucleus to finally trigger gene expression for mesoderm formation remains unknown. We report here that a high level of activator protein-1 (AP-1)-dependent transcriptional activity is detected during the early development of Xenopus embryos. Injection of a dominant negative mutant jun (DNM-jun or TAM67) RNA into the two-cell stage embryos inhibited endogenous AP-1 activity and blocked normal embryonic development with severe posterior truncation in tadpoles. The inhibition of AP-1 activity and the phenotypic change induced by TAM67 was rescued by co-injection of wild-type c-jun RNA, but not by the control beta-galactosidase RNA. The FGF-stimulated mesoderm induction was markedly inhibited in animal cap explants from the embryos injected with TAM67. Activin induction of mesoderm, on the other hand, was normal in the embryos injected with TAM67 RNA. These findings suggest that AP-1 mediates FGF, but not activin, receptor signaling during mesoderm induction and the AP-1/Jun is a key signaling molecule in the development of posterior structure.
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PMID:AP-1/jun is required for early Xenopus development and mediates mesoderm induction by fibroblast growth factor but not by activin. 862 31

Raf-1 is a serine/threonine specific kinase that integrates signaling by a large number of mitogens to elicit a transcriptional response in the nucleus. Activated Raf-1 phosphorylates and activates MAPK/ERK kinase Mek), thus initiating the Mek--> MAP kinase cascade, which ultimately results in the phosphorylation and activation of transcription factors by MAP kinase. Here we have characterized the mechanism by which monoclonal antibody URP26K, which binds to an epitope in the Raf-1 kinase domain, inhibits intracellular signal transduction. This antibody preferentially immunoprecipitated the underphosphorylated, non-activated form of Raf-1 from quiescent cells. Baculovirus-expressed Raf-1 immunoprecipitated with URP26K was largely refractory to phosphorylation and activation mediated by protein kinase C (PKC)alpha or the tyrosine kinase Lck. In addition, URP26K reduced the binding of Raf-1 to its substrate Mek in vitro, but did not disturb the association of Raf-1 with Ras. Microinjection of URP26K into Rat-1 cells blocked DNA synthesis initiated by serum, insulin and various purified growth factors, but it did not block DNA synthesis initiated by v-ras. Microinjected URP26K also impaired the expression of stably transfected beta-galactosidase reporter genes regulated by minimal promoter elements. These results demonstrate, (i) that the URP26K monoclonal antibody inhibits Raf-1 by preventing activating Raf-1 phosphorylation and/or association with its substrate Mek, (ii) that inhibition of Raf-1 by URP26K does not interfere with Ras-induced DNA synthesis. In contrast to dominant negative Raf-1 mutants, which also block Ras signaling by binding to the Ras effector domain, antibody mediated Raf-1 inhibition thus reveals a branchpoint of mitogenic signaling at the level of Ras.
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PMID:Inhibition of Raf-1 signaling by a monoclonal antibody, which interferes with Raf-1 activation and with Mek substrate binding. 880 5

Phosphatidylinositol 3-kinase (PI3K) activation is necessary for many insulin-induced metabolic and mitogenic responses. However, it is unclear whether PI3K activation is sufficient for any of these effects. To address this question we increased PI3K activity in differentiated 3T3-L1 adipocytes by adenovirus-mediated expression of both the inter-SH2 region of the regulatory p85 subunit of PI3K (iSH2) and the catalytic p110 alpha subunit (p110). Coexpression resulted in PI3K activity that exceeded insulin-stimulated activity by two- to fivefold in cytosol, total membranes, and the low density microsome (LDM) fraction, the site of greatest insulin stimulation. While insulin increased glucose transport 15-fold, coexpression of iSH2-p110 increased transport (5.2-) +/- 0.7-fold with a parallel increase in GLUT4 translocation to the plasma membrane. Constitutive activation of PI3K had no effect on maximally insulin-stimulated glucose transport. Neither basal nor insulin-stimulated activity of glycogen synthase or mitogen-activated protein kinase was altered by iSH2-p110 coexpression. DNA synthesis was increased twofold by insulin in control 3T3-L1 adipocytes transduced with beta-galactosidase-encoding recombinant adenovirus, while iSH2-p110 coexpression increased DNA synthesis fivefold. These data indicate that (i) increased PI3K activity is sufficient to activate some but not all metabolic responses to insulin, (ii) activation of PI3K to levels exceeding the effect of insulin in adipocyte LDM results in only a partial stimulation of glucose transport, and (iii) increased PI3K activity in the absence of growth factor or oncoprotein stimulation is a potent stimulus of DNA synthesis.
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PMID:Differential effects of constitutively active phosphatidylinositol 3-kinase on glucose transport, glycogen synthase activity, and DNA synthesis in 3T3-L1 adipocytes. 897 99

Abnormal migration and proliferation of arterial smooth muscle cells may be a central event in inflammatory proliferative arterial diseases such as atherosclerosis and restenosis after angioplasty. The proto-oncogene c-H-ras is considered to be a key transducer in various growth-signaling events. We constructed an adenoviral vector (AdexCAHRasY57) expressing a potent dominant-negative mutated form of c-H-ras in which tyrosine replaces aspartic acid at residue 57. Infection of smooth muscle cells with AdexCAHRasY57 produced a large quantity of H-ras-p21, completely inhibited serum-stimulated activation of mitogen-activated protein kinase, and abolished the DNA synthesis in response to serum mitogens. However, a surge of intracellular Ca2+ concentration in response to platelet-derived growth factor was not affected, suggesting that some cellular functions were preserved. When we applied AdexCAHRasY57 into balloon-injured rat carotid arteries from inside the lumen, neointimal formation was significantly reduced (neointima/media ratio: 0.28) compared with that (1.50) in arteries treated with either injury alone or injury and infection with a control adenovirus, AdexCALacZ, expressing bacterial beta-galactosidase. Our results suggest that adenovirus-mediated arterial transfer of dominant-negative H-ras may be a practical form of effective molecular intervention for proliferative arterial diseases.
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PMID:Adenovirus-mediated transfer of a dominant-negative H-ras suppresses neointimal formation in balloon-injured arteries in vivo. 915 53

The hepatic stellate cell (HSC), following a fibrogenic stimulus, is transformed from a quiescent to an activated cell. Cytokines induce NFkappaB activity in activated but not in quiescent HSCs with subsequent expression of NFkappaB-responsive genes, such as intercellular adhesion molecule (ICAM)-1 and interleukin (IL)-6. We investigated the effect of proteasome inhibitors and an IkappaB super-repressor on the cytokine mediated activation of NFkappaB, ICAM-1, and IL-6 in activated HSCs. Culture-activated HSCs were stimulated with IL-1beta or tumor necrosis factor alpha (TNFalpha) in the presence or absence of proteasome inhibitors, ALLN or MG-132, or after infection with an adenovirus expressing the IkappaB super-repressor (Ad5IkappaB) or beta-galactosidase (Ad5LacZ) as a control. NFkappaB activity was evaluated by immunofluorescence and by electrophoretic mobility shift assay. The steady state level of cytoplasmic IkappaB protein was measured by Western Blot. ICAM-1 and IL-6 expression was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbant assay. Proteasome inhibitors, which block the degradation of IkappaB, and the Ad5IkappaB, which provides an exogenous nondegradable IkappaB, block the stimulation of NFkappaB activity by TNFalpha and IL-1beta in activated HSCs. These reagents block the subsequent nuclear translocation of p65 NFkappaB and induction of ICAM-1 and IL-6 by cytokines. The specificities of the proteasome inhibitors and the IkappaB super-repressor are demonstrated by their failure to block c-Jun N-terminal kinase induction by cytokines. Cytokine-induced stimulation of NFkappaB, ICAM-1, and IL-6 is blocked by proteasome inhibitors and Ad5IkappaB in activated HSCs. Inhibition of IkappaBalpha degradation is a potential target for anti-inflammatory therapy in the liver and might influence the activation process of HSCs following fibrotic stimuli.
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PMID:Inhibition of NFkappaB in activated rat hepatic stellate cells by proteasome inhibitors and an IkappaB super-repressor. 958 6

Prostate cancer is the most commonly diagnosed neoplasm in men. LNCaP cells continue to possess many of the molecular characteristics of in situ prostate cancer. These cells lack ras mutations, and mitogen-activated protein kinase (MAPK) is not extensively phosphorylated in these cells. To determine the effects of ras/raf/MAPK pathway activation in these cells, we transfected LNCaP cells with an activatable form of c-raf-1(deltaRaf-1:ER). Activation of deltaRaf-1:ER, with resultant MAPK activation, reduced plating efficiency and soft agarose cloning efficiency 30-fold in LNCaP cells. Cell cycle distribution showed an accumulation of cells in G1 and was associated with the induction of CDK inhibitor p21WAF1/CIP1 at the protein and mRNA levels. p21WAF1/CIP1 mRNA stability was increased after deltaRaf-1:ER activation. In addition, activated deltaRaf-1:ER induced the senescence associated-beta-galactosidase in LNCaP cells. These data demonstrate that raf activation can activate growth inhibitory pathways leading to growth suppression in prostate carcinoma cells and also suggest that raf/MEK/MAPK pathway activation, rather than inhibition, may be a therapeutic target for some human prostate cancer cells.
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PMID:Raf-1-induced cell cycle arrest in LNCaP human prostate cancer cells. 1002 6

In response to extracellular stimuli, mitogen-activated protein kinase (MAPK, also known as ERK) translocates from the cytoplasm to the nucleus. MAP kinase kinase (MAPKK, also know as MEK), which possesses a nuclear export signal (NES), acts as a cytoplasmic anchor of MAPK. Here we show evidence that tyrosine (Tyr190 in Xenopus MPK1/ERK2) phosphorylation of MAPK by MAPKK is necessary and sufficient for the dissociation of the MAPKK-MAPK complex, and that the dissociation of the complex is required for the nuclear translocation of MAPK. We then show that nuclear entry of MAPK through a nuclear pore occurs via two distinct mechanisms. Nuclear import of wild-type MAPK (mol. wt 42 kDa) was induced by activation of the MAPK pathway even in the presence of wheat germ agglutinin or dominant-negative Ran, whereas nuclear import of beta-galactosidase (beta-gal)-fused MAPK (mol. wt 160 kDa), which occurred in response to stimuli, was completely blocked by these inhibitors. Moreover, while a dimerization-deficient mutant of MAPK was able to translocate to the nucleus upon stimulation, this mutant MAPK, when fused to beta-gal, became unable to enter the nucleus. These results suggest that monomeric and dimeric forms of MAPK enter the nucleus by passive diffusion and active transport mechanisms, respectively.
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PMID:Two co-existing mechanisms for nuclear import of MAP kinase: passive diffusion of a monomer and active transport of a dimer. 1050 67

We have previously shown that CO(2) stimulation of the renal Na-HCO(3) cotransporter (NBC) activity is abrogated by general inhibitors of protein tyrosine kinases. The more selective inhibitor herbimycin also blocked this effect at concentrations known to preferentially inhibit Src family kinases (SFKs). We therefore examined a role for SFKs in CO(2)-stimulated NBC activity. To this end, we engineered OK cells to express the COOH-terminal Src kinase (Csk), a negative regulator of SFKs. CO(2) stimulated NBC activity normally in beta-galactosidase-expressing and untransfected control cells. In contrast, Csk-expressing cells had normal baseline NBC activity that was not stimulated by CO(2). CO(2) stimulation increased both total SFK activity and specific tyrosine phosphorylation of Src. The specific MEK1/2 inhibitor PD-98059 completely inhibited the CO(2) stimulation of NBC activity as well as the accompanying phosphorylation and activation of ERK1/2. Our data suggest the involvement of both SFKs, probably Src, and the "classic" MAPK pathway in mediating CO(2)-stimulated NBC activity in renal epithelial cells.
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PMID:Regulation of the renal Na-HCO(3) cotransporter. XI. Signal transduction underlying CO(2) stimulation. 1051 83


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