Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nicotinic acetylcholine receptor (nAChR) is phosphorylated to a high stoichiometry on tyrosine residues both in vitro and in vivo. Moreover, tyrosine phosphorylation has been shown to regulate the functional properties of the receptor. We report here the purification and characterization of a protein tyrosine phosphatase that dephosphorylates tyrosine-phosphorylated nAChR from Torpedo electroplax, a tissue highly enriched in the nAChR. The 32P-labeled tyrosine phosphorylated nAChR was used as a substrate to monitor the enzyme activity during purification. The protein tyrosine phosphatase activity was purified using three consecutive cation-exchange columns (phosphocellulose, S Sepharose Fast Flow, Bio-Rex 70), followed by two affinity matrices (p-aminobenzylphosphonic acid-agarose and thiophosphotyrosyl nAChR-Sepharose 4B). The enzyme activity was purified to homogeneity, with an overall purification of 25,000-fold and a yield of 20%. The purified enzyme had an apparent molecular mass of 43 kDa on sodium dodecyl sulfate-polyacrylamide gels and migrated as a monomer during Superose 12 chromatography. It had a neutral pH optimum and a specific activity of 18 mumol/mg of protein/min, with a Km of 4.7 microM for tyrosine-phosphorylated nAChR. The phosphatase was specific for tyrosine phosphorylated nAChR; it showed no activity towards the nAChR phosphorylated on serine residues by cAMP-dependent protein kinase. The enzyme also dephosphorylated 32P-labeled poly(Glu-Tyr) (4:1). However, it did not dephosphorylate p-nitrophenylphosphate. The tyrosine phosphatase was inhibited by ammonium molybdate (IC50 of 2 microM), sodium vanadate (IC50 of 150 microM) and the divalent cations Mg2+, Mn2+, and Ca2+ at millimolar concentrations, but not by 100 microM ZnCl or 10 mM NaF. Poly-(Glu, Tyr) (4:1) and heparin inhibited the enzyme activity at micromolar concentrations. These unique properties of the purified enzyme suggest that it may be a novel protein tyrosine phosphatase that specifically dephosphorylates the nAChR.
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PMID:Purification and characterization of a protein tyrosine phosphatase which dephosphorylates the nicotinic acetylcholine receptor. 165 33

Nuclear histone kinase activity, specifically histone H1 phosphotransferase activity, was shown to increase in synchronous Chinese hamster cells from the G1/S boundary to late G2/early M phase. Chromatin extracts purified by DEAE-Sephacel chromatography showed a cAMP-independent kinase activity that demonstrated cell cycle dependence and high specificity for histone H1 as the phosphate acceptor in the presence of [gamma-32P] ATP. This activity was purified approximately 40-fold. Using as substrates calf thymus histone H1 subfractions resolved by Bio-Rex 70 ion exchange chromatography, phosphorylation by the nuclear histone H1 kinase indicated that 32P incorporation into H1-2 was at least twice that for H1-1 and H1-3 subfractions. Both amino- and carboxy-terminal fragments generated by N-bromosuccinimide cleavage were phosphorylated. Phosphoamino acid analysis showed phosphothreonine to be approximately twice as abundant as phosphoserine. Histone H1 kinase activity was not activated by cyclic nucleotides, nor inhibited by cAMP-dependent protein kinase inhibitors or regulatory subunits. There was no effect on activity by Ca2+ alone or in the presence of calmodulin or diacylglycerol. Kinase activity was inhibited by nonhydrolyzable analogs of ATP such as adenyl-5'-yl imidodiphosphate, by 5'-p-fluorosulfonylbenzoyladenosine which binds to the ATP binding site of the enzyme, and by quercetin. Column fractions enriched in histone H1 kinase were labeled with 5'-p-fluorosulfonylbenzoyl[8-14C]adenosine, and peptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band, Mr 67,000, was specifically labeled and may represent the H1 kinase catalytic subunit.
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PMID:Histone H1 kinase in exponential and synchronous populations of Chinese hamster fibroblasts. 300 70

Topoisomerase I (Topo I) is involved in many cellular functions that involve unwinding of supercoiled DNA, such as transcription and replication. Topo I is also the target of autoimmune antibodies in progressive systemic sclerosis (scleroderma), and abnormal regulation of Topo I may influence the excessive production of collagen found in scleroderma. Topo I is phosphorylated in vivo at serine residues and, in vitro, the activity of Topo I is increased by phosphorylation by casein kinase type II (CKII) and protein kinase C (PKC). In this study, a protein kinase activity from rat liver nuclei is shown to copurify with Topo I during Bio-Rex 70 cation exchange chromatography. The kinase can phosphorylate Topo I at serine residues, resulting in a threefold increase in topoisomerase activity. A relatively tight association between this kinase and Topo I is demonstrated by the ability to coprecipitate the kinase with scleroderma autoimmune anti-Topo I antibodies. The kinase activity is similar to CKII since it is Ca2+ and cyclic nucleotide independent, it can utilize either ATP or GTP as phosphate donor, and it can phosphorylate casein and phosvitin, but not histones. However, unlike typical CKII, the Topo I-associated kinase could utilize Mn2+ almost as well as Mg2+, it is not stimulated by polyamines, and it does not appear to undergo autophosphorylation. In conclusion, we demonstrate that rat liver Topo I is relatively tightly associated with a CKII-like protein kinase that can phosphorylate and activate Topo I. These findings provide corroborative evidence that CKII, or a CKII-like protein kinase, is a physiologic regulator of Topo I.
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PMID:A casein kinase type II (CKII)-like nuclear protein kinase associates with, phosphorylates, and activates topoisomerase I. 826 Jan 98

The 2'-5' oligoadenylate synthetases and the protein kinase PKR are both interferon-induced, double-stranded RNA-dependent proteins that play important roles in the antiviral effects of the interferons and in cellular growth control. Both enzymes are activated by natural or synthetic dsRNAs and by single-stranded RNAs that possess extensive secondary structure. This report describes the effects of the small Epstein-Barr virus-encoded RNA EBER-1 on the regulation of 2-5(A) synthetase activity. We demonstrate that EBER-1 RNA binds to and activates the human 40-kDa 2-5(A) synthetase in a dose-dependent manner. The efficiency of EBER-1 as an activator of 2-5(A) synthetase is approximately 25% of that of the synthetic double-stranded RNA poly(I)/poly(C), and poly(I)/poly(C) further stimulates enzyme activity even in the presence of a high concentration of EBER-1. Conversely, EBER-1 neither stimulates nor inhibits 2-5(A) synthetase that has been activated by a high concentration of poly(I)/poly(C). Competitive binding assays suggest that the relative affinity of the enzyme for poly(I)/poly(C) is considerably higher than that for EBER-1. Our data indicate that EBER-1, like VAI RNA of adenovirus, TAR RNA of HIV-1, and Rex-RE RNA of HTLV-1, is able to activate the 2-5(A) synthetases. The significance of why several viruses may activate the 2-5(A) synthetase/RNase L-mediated RNA degradation pathway is discussed.
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PMID:Activation of the interferon-inducible (2'-5') oligoadenylate synthetase by the Epstein-Barr virus RNA, EBER-1. 1032 41

Human and mouse embryonic stem cells (HESCs and MESCs, respectively) self-renew indefinitely while maintaining the ability to generate all three germ-layer derivatives. Despite the importance of ESCs in developmental biology and their potential impact on tissue replacement therapy, the molecular mechanism underlying ESC self-renewal is poorly understood. Here we show that activation of the canonical Wnt pathway is sufficient to maintain self-renewal of both HESCs and MESCs. Although Stat-3 signaling is involved in MESC self-renewal, stimulation of this pathway does not support self-renewal of HESCs. Instead we find that Wnt pathway activation by 6-bromoindirubin-3'-oxime (BIO), a specific pharmacological inhibitor of glycogen synthase kinase-3 (GSK-3), maintains the undifferentiated phenotype in both types of ESCs and sustains expression of the pluripotent state-specific transcription factors Oct-3/4, Rex-1 and Nanog. Wnt signaling is endogenously activated in undifferentiated MESCs and is downregulated upon differentiation. In addition, BIO-mediated Wnt activation is functionally reversible, as withdrawal of the compound leads to normal multidifferentiation programs in both HESCs and MESCs. These results suggest that the use of GSK-3-specific inhibitors such as BIO may have practical applications in regenerative medicine.
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PMID:Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. 1470 26

PtdIns(3, 4, 5)P(3)-dependent Rac exchanger (P-Rex) 1 is a guanine nucleotide exchange factor (GEF) for the small GTPase Rac. P-Rex1 is activated by G protein betagamma subunits (Gbetagamma), and the Gbetagamma-induced activation is inhibited by cAMP-dependent protein kinase A (PKA). However, the details of regulatory mechanism of P-Rex1 remain to be clarified. In the present study, we investigated the mechanism of activation and inhibition of P-Rex1 using various truncated and alanine-substituted mutants and found that the domain-domain interaction of P-Rex1 is important for Gbetagamma-induced activation and PKA-induced inhibition. Immunoprecipitation analysis showed that the second Disheveled/EGL-10/Pleckstrin (DEP) and first PSD-95/Dlg/ZO-1 (PDZ) domains of P-Rex1 associate with the inositol polyphosphate-4-phosphatase (IP4P) domain. Carboxyl-terminal truncation on the IP4P domain or mutations in the protein-binding pocket of the first PDZ domain abolished the association. Analysis of in vitro guanine nucleotide exchange assay, PAK1/2 phosphorylation, and Rac-specific actin reorganization revealed that Gbetagamma could activate a complex of the P-Rex1 mutant lacking the IP4P domain and the isolated IP4P domain as well as full-length P-Rex1. Moreover, PKA phosphorylation prevented the domain-domain interaction and Gbetagamma-binding. These results provide a new insight into the regulation of other Rho-family GEFs and cell responses induced by the heterotrimeric G protein.
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PMID:Domain-domain interaction of P-Rex1 is essential for the activation and inhibition by G protein betagamma subunits and PKA. 1851 84

In the present study, the potential of selenium to enhance stem cell behavior through improvement of human adipose tissue-derived stromal cells (ATSCs) and the associated molecular mechanism was evaluated. Selenium-induced improvement in stem cell behavior of human ATSCs caused expression of several genes, indicating downregulated mature cell marker proteins coupled with increased cell growth and telomerase activities after the overexpression of Rex1, Nanog, OCT4, SOX2, KLF4, and c-Myc. Also, selenium-treated ATSCs significantly downregulated p53 and p21 tumor suppressor gene products. Selenium induced active growth and growth enhanced by the activation of signal proteins in ATSCs via the inhibition of reactive oxygen species-mediated phospho-stress-activated protein kinase/c-Jun N-terminal protein kinase activation. The selenium-induced activation of extracellular regulated kinases 1/2 and Akt in ATSCs resulted in a subsequent induction of the expression of stemness transcription factors, particularly Rex1, Nanog, and Oct4, along with definitive demethylation on regulatory regions of Rex-1, Nanog, and Oct4. The results of our small interfering RNA knockdown experiment showed that Rex1 plays a major role in the proliferation of selenium-induced ATSCs. Selenium-treated ATSCs also exhibited more profound differentiation into mesodermal and neural lineages. We performed a direct comparison of gene expression profiles in control ATSCs and selenium-treated ATSCs and delineated specific members of important growth factor, signaling, cell adhesion, and transcription factor families. The observations of improved life span and multipotency of selenium-treated ATSCs clearly indicate that selenium-treated ATSCs represent an extraordinarily useful candidate cell source for tissue regeneration. Disclosure of potential conflicts of interest is found at the end of this article.
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PMID:IFATS collection: Selenium induces improvement of stem cell behaviors in human adipose-tissue stromal cells via SAPK/JNK and stemness acting signals. 2473 3

This study was undertaken to establish rat embryonic stem (ES) cells from parthenogenetically developing blastocysts. Ten blastocysts were prepared by treatment of ovulated rat oocytes with ionomycin and cycloheximide, and three alkaline phosphatase-positive ES cell lines were established using the N2B27 medium supplemented with mitogen activated protein kinase kinase inhibitor PD0325901, glycogen synthase kinase 3 inhibitor CHIR99021, rat leukemia inhibitory factor, and forskolin. Expression of stem cell marker genes (Oct-4, rNanog, Fgf-4, and Rex-1) was confirmed in all three ES cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR). Combined bisulfite restriction analysis showed that the differentially methylated region locus of five imprinted genes (H19, Meg3IG, Igf2r, Peg5, and Peg10) in these ES cells remained to be demethylated or was hypomethylated, which was similar to that in control ES cells established from normal blastocysts. Characteristics of the parthenogenetic blastocyst-derived ES cells were successfully transmitted to the next generation through a chimeric rat for one of the three ES cell lines. This is the first report on germline-competent (genuine) ES cells derived from parthenogenetically developing rat blastocysts.
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PMID:Derivation of embryonic stem cell lines from parthenogenetically developing rat blastocysts. 2401 May 70