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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Urea (200-400 milliosmolar) activates transcription, translation of, and trans-activation by the immediate-early gene transcription factor Egr-1 in a renal epithelial cell-specific fashion. The effect at the transcriptional level has been attributed to multiple serum response elements and their adjacent Ets motifs located within the Egr-1 promoter. Elk-1, a principal ternary complex factor and Ets domain-containing protein, is a substrate of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. In the renal medullary mIMCD3 cell line, urea (200-400 milliosmolar) activated both ERK1 and ERK2 as determined by in-gel kinase assay and immune-complex kinase assay of epitope-tagged] ERK1 and ERK2. Importantly, urea did not affect abundance of either ERK. Urea-inducible Egr-1 transcription was a consequence of ERK activation because the ERK-specific inhibitor, PD98059, abrogated transcription from the murine Egr-1 promoter in a luciferase reported gene assay. In addition, activators of protein kinase A, including forskolin and 8-Br-cAMP, which are known to inhibit ERK-mediated events, also inhibited urea-inducible Egr-1 transcription. Furthermore, urea-inducible activation of the physiological ERK substrate and transcription factor, Elk-1, was demonstrated through transient cotransfection of a chimeric Elk-1/GAL4 expression plasmid and a GAL4-driven luciferase reporter plasmid. Taken together, these data indicate that, in mIMCD3 cells, urea activates ERKs and the ERK substrate, Elk-1, and that ERK inhibition abrogates urea-inducible Egr-1 transcription. These data are consistent with a model of urea-inducible renal medullary gene expression wherein sequential activation of ERKs and Elk-1 results in increased transcription of Egr-1 through serum response element/Ets motifs.
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PMID:Urea-inducible Egr-1 transcription in renal inner medullary collecting duct (mIMCD3) cells is mediated by extracellular signal-regulated kinase activation. 885 40

We examined the behaviour in vivo of native, specifically phosphorylated, and multimeric vitronectin to determine the effects of these modifications on its turnover, distribution and molecular behaviour. In normal rabbits, the plasma half-life (T1/2) of antigenically detected vitronectin was 8.00 +/- 1.26 h (mean +/- s.d.), with a fractional catabolic rate (FCR) of 18.77 +/- 1.57%/h and extravascular/intravascular ratio (EV/IV) of 1.00 (0.48-1.60, median and range). For vitronectin selectively phosphorylated by protein kinase A, T1/2 was 8.87 +/- 0.48 h, with a significantly smaller FCR of 10.85 +/- 0.71%/h (P < 0.005) and an EV/IV of 0.28 (0.15-0.36) (P < 0.05 compared with antigenically detected vitronectin). In vitro, phosphorylation had no effect on the affinity of vitronectin for heparin-Sepharose, while complement activation with cobra venom factor (CVF) led to a two-fold enrichment of 32P-vitronectin within the SC5b-9 complex. In vivo CVF caused a rapid decrease in the circulating levels of 32P-vitronectin and was accompanied by the prompt appearance of a high mol. wt species consistent with SC5b-9. Despite specific incorporation of 32P-vitronectin into SC5b-9, both forms of the molecule had similar inhibitory effects on C9-mediated haemolysis of EAC1-7 cells. Urea-activated vitronectin was rapidly cleared from circulation with less than 15% remaining after 1 h while protein-bound label accumulated in the spleen, lung and liver. These results demonstrate that vitronectin is a rapidly metabolized protein whose in vivo behaviour is markedly altered when phosphorylated or activated to form multimers and SC5b-9.
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PMID:The behaviour of human vitronectin in vivo: effects of complement activation, conformation and phosphorylation. 891 93

Two forms of phospholipase D (PLD) have been found to be present in nuclei isolated from rat hepatocytes by measuring phosphatidylbutanol produced from exogenous radiolabeled phosphatidylcholine in the presence of butanol. In nuclear lysates from either rat liver or ascites hepatoma AH 7974 cells, the PLD activity was markedly stimulated by a recombinant ADP-ribosylation factor (rARF) in the presence of the guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and phosphatidylinositol 4, 5-bisphosphate. ATP and phorbol-12-myristate 13-acetate had no synergistic effect on this PLD activity. On the other hand, the nuclear PLD was stimulated by unsaturated fatty acids, especially by oleic acid. The ARF-dependent nuclear PLD activity was increased in the S-phase of the regenerating rat liver after partial hepatectomy and also was much higher in AH 7974 cells than in the resting rat liver. In contrast, the levels of the oleate-dependent PLD activity remained constant throughout the cell cycle in liver regeneration. The intranuclear levels of the stimulating proteins of the nuclear PLD activity, e.g. ARF, RhoA, and protein kinase Cdelta increased in the S-phase of the regenerating liver. These results suggested that the nuclear ARF-dependent PLD activity may be associated with cell proliferation.
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PMID:Nuclear ADP-ribosylation factor (ARF)- and oleate-dependent phospholipase D (PLD) in rat liver cells. Increases of ARF-dependent PLD activity in regenerating liver cells. 903 May 90

Inner medullary collecting duct (IMCD) cells adapt to a hypertonic environment by synthesizing transporters that allow for accumulation of organic osmolytes. To examine for activation of additional mitogen-activated protein (MAP) kinases, extracts of IMCD-3 cells subjected to a hypertonic medium (600 mosmol/kgH2O) for 15 min were fractionated by Mono Q fast-performance liquid chromatography and assayed with the epidermal growth factor receptor [EGFR-(662-681)] peptide as substrate. Three peaks of activity were identified. Western blotting revealed that these peaks coincided with Jun NH2-terminal kinase (JNK), extracellular signal-regulated protein kinases, ERK1 and ERK2, and p38 MAP kinase. To assess the functional significance of ERK2 activation in IMCD-3 cells, the effect of PD-098059, an inhibitor of the upstream regulatory protein kinase MAP/ERK kinase (MEK) was assessed. PD-098059 inhibited ERK activation by hypertonicity. Yet, the stimulation of inositol uptake, a marker of adaptation, after 16 h was unaltered. Direct measurements of JNK activity [phosphorylation of GST-cJun-(1-79)] revealed a marked (20- to 40-fold) increase in activity as medium osmolality was increased from 300 to 900 mosmol/kgH2O with either NaCl or mannitol. Urea induced a more modest increase in activity. The response is prompt and detected as early as 2 min after exposure, reaching a maximum activation at 10-15 min. Downregulation of cellular protein kinase C (PKC) by chronic exposure to phorbol esters only minimally attenuated the JNK response to hyperosmolality, indicating a lack of involvement of PKC. We conclude that, in IMCD-3 cells, inhibition of ERK activation by hyperosmolality does not prevent osmoregulatory increase in inositol transport. This is not consistent with a role for ERKs in the response. The roles for JNK and p38 have not been ruled out, and these pathways may represent the initiating event in the subsequent transcription of organic osmolyte transporter genes and adaptation to extracellular hypertonicity.
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PMID:Multiple mitogen-activated protein kinases are regulated by hyperosmolality in mouse IMCD cells. 908 72

Phosphorylation of buffalo sperm chromatin proteins under optimum conditions (8 mM Mg2+, pH 8.0, and at 30 degrees C) using [gamma-32P]ATP and endogenous protein kinase activity was linear for 15 min incubation time and up to 330 micrograms protein. The 32P transferred from [gamma-32P]ATP was located in protein as a phosphoester bond. Fractionation with 1.2 M NaCl-4 M urea-0.2 M 2-mercaptoethanol-1 mM PMSF followed by acid treatment solubilized 87% of the total chromatin proteins termed "sperm histones." The remaining 21% nonhistone protein was tightly bound to DNA. Follow-up of the label showed 91% of the 32P in sperm histone and 9% with DNA-associated proteins. Histone kinase activity was solubilized with 0.35 M NaCl, which extracted 70% of the initial enzyme activity associated with chromatin. Of the different histones tested as substrates, histone kinase phosphorylated only histone H3 and, therefore, is highly specific for arginine rich histone. It also phosphorylates the acidic protein, casein. Cyclic AMP at concentrations up to 50 microM had no effect on the phosphorylation of histone H3. Phosphoamino acid analysis using histone H3 as the substrate showed serine to be the acceptor amino acid for phosphoester link.
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PMID:Histone kinase activity of buffalo sperm chromatin. 914 Jun 15

cAMP is important for the initiation of mammalian sperm motility. Previously we established that a type II cAMP-dependent protein kinase is tightly associated with the fibrous sheath of rat sperm. This unique cytoskeletal structure surrounds the 9+2 axonemal network in the principal piece of the flagellum. Association of the kinase to the fibrous sheath is mediated via its regulatory subunit, RII. An RII-binding overlay procedure was used to document that RII could specifically associate with fibrous sheath polypeptides of 120 and 80 kDa. In this study, we report the cloning of a rat testis-specific, developmentally regulated, RII-binding protein (TAKAP-80). A 1.2-kb cDNA clone, isolated by screening a rat testis expression library with 32P-labeled RII, hybridized to a 1.8-kb mRNA transcript present exclusively in testis. This transcript appeared at detectable levels at 30 days after birth. Over the next 10 days the mRNA levels increased greatly. This time interval corresponds to the initiation of spermiogenesis. The complete nucleotide sequence of TAKAP-80 cDNA was obtained by polymerase chain reaction and contained a continuous open reading frame of 502 amino acids. The deduced amino acid sequence showed a clear demarcation of charged and hydrophobic amino acid residues. Amino acids 1-147 of the protein contained 45% charged residues, with lysine and arginine predominating. Similarly, amino acids 268-502 also contained a high percentage of charged amino acids (35%). In contrast, amino acids 148-267 were mostly hydrophobic and contained clusters of a repeating PXXP motif where X was predominantly valine and alanine or sometimes proline. The 1.2-kb cDNA clone was inserted into the pRSET vector and expressed as a His6 tag fusion protein in Escherichia coli. The recombinant protein was soluble and bound RIIalpha, RIIbeta and type IIalpha holoenzyme by the RII-binding overlay procedure. Deletion analysis revealed that the high-affinity interaction site for RII was contained within amino acids 258-378 of TAKAP-80. Antibodies prepared against the fusion protein recognized an 80-kDa protein present in the urea-insoluble particulate fraction of rat testis and in purified fibrous sheath preparations isolated from rat epididymal sperm. Levels of the 80-kDa immunoreactive protein were significantly higher in mature (60 days old) compared with immature (30 days old) rat testis, correlating with the mRNA levels.
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PMID:Cloning and characterization of a testis-specific, developmentally regulated A-kinase-anchoring protein (TAKAP-80) present on the fibrous sheath of rat sperm. 920 34

Specific serine and threonine residues of recombinant human beta-casein produced in Escherichia coli were shown to be phosphorylated in vivo when human casein kinase II was coexpressed in the same plasmid. All of the phosphorylated forms found in the native protein were also detected in the recombinant protein. The phosphorylation of recombinant human beta-casein was confirmed by immunoblots, fast protein liquid chromatography, urea-polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis, and liquid chromatography-mass spectrometry. The results indicate that the substrate specificity of casein kinase II in vivo was unaffected in its recombinant form. This is the first demonstration of in vivo phosphorylation of specific residues of a multiphosphorylated protein produced in E. coli with a single plasmid.
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PMID:Expression and characterization of phosphorylated recombinant human beta-casein in Escherichia coli. 922 16

The human phospholipase D1 (hPLD1) has recently been cloned. Although recent data have implicated PLD in receptor-stimulated secretion, the regulation of the activity of PLD enzymes remains to be clarified. Purified hPLD1 is activated by several cytosolic cofactors among which are protein kinase Calpha, ARF, and RhoA. In human granulocytes, a strong correlation between tyrosine phosphorylation of proteins and PLD activity has been established. In this study, the presence of hPLD1 in HL-60 granulocytes and its phosphorylation on tyrosine residues have been studied. We generated antipeptide antibodies (Abs) specific for hPLD1 but not PLD2 as shown by Western blotting (WB) of recombinant PLD1 and PLD2. These Abs identified the presence of hPLD1 in HL-60 cells with the bulk of it being detected in the membranes and only a minor fraction in the cytosol. The hPLD1 Abs detected a major band at 120 kDa (PLD1a) and a minor band at 115 kDa (PLD1b). The specificity of the Abs was confirmed using PLD antisera neutralized with the immunizing peptides. The two forms of hPLD1 were consistently detected by immunoprecipitation under nondenaturing and denaturing conditions following a WB analysis with hPLD1 Abs. Following exposure of HL-60 cells to peroxides of vanadate (V4+-OOH), an inhibitor of tyrosine phosphatases, hPLD1 was immunoprecipitated under nondenaturing conditions from HL-60 cell lysates and assayed for tyrosine phosphorylation by WB. hPLD1 comigrated with a 120-kDa tyrosine phosphorylated protein by gel electrophoresis. Other tyrosine-phosphorylated peptides of 160, 140, 135, 90, and 75-80 kDa were also detected in hPLD1 immune complexes. hPLD1 and the associated tyrosine-phosphorylated proteins were not immunoprecipitated by neutralized hPLD1 Abs. Using denaturing conditions, the PLD immunoprecipitates were sequentially immunoblotted with anti-PLD1 and anti-phosphotyrosine Abs. PLD1a and PLD1b were detected, and the major PLD1a protein was superimposable with a major tyrosine-phosphorylated protein detected at 120 kDa. Conversely, PLD1a and PLD1b were recovered, at least in part, in the anti-phosphotyrosine immunoprecipitates. These results provide evidence that two PLD1 forms are expressed in human granulocytes. Furthermore, in response to stimulation by V4+-OOH, PLD1 was tyrosine-phosphorylated and associated with several, presently undefined, tyrosine-phosphorylated proteins.
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PMID:Human phospholipase D1 can be tyrosine-phosphorylated in HL-60 granulocytes. 925 84

We have previously demonstrated that the phospholipase C-coupled m3-muscarinic receptor is phosphorylated in an agonist-sensitive manner by a protein kinase of approximately 40 kDa purified from porcine cerebellum (Tobin, A. B., Keys, B., and Nahorski, S. R. (1996) J. Biol Chem. 271, 3907-3916). This kinase, called muscarinic receptor kinase (MRK), is distinct from second messenger-regulated protein kinases and from beta-adrenergic receptor kinase and other members of the G-protein-coupled receptor kinase family. In the present study we propose that MRK is casein kinase 1alpha (CK1alpha) based on the following evidence: 1) the amino acid sequence from two proteolytic peptide fragments derived from purified MRK corresponded exactly to sequences within CK1alpha. 2) Casein kinase activity co-eluted with MRK activity from the final two chromatography steps in the purification of porcine brain MRK. 3) Recombinant CK1alpha expressed in Sf9 cells is able to phosphorylate both casein and the bacterial fusion protein, Ex-m3, that contains a portion of the third intracellular loop of the m3-muscarinic receptor downstream of glutathione S-transferase. 4) Partially purified CK1alpha increased the level of muscarinic receptor phosphorylation in an agonist-sensitive manner when reconstituted with membranes from Chinese hamster ovary-m3 cells expressing the human recombinant m3-muscarinic receptor. 5) Partially-purified CK1alpha phosphorylated rhodopsin, contained in urea-treated bovine rod outer segment membranes, and the extent of phosphorylation was increased in the presence of light. These data demonstrate that the kinase previously called MRK is CK1alpha, and that CK1alpha offers an alternative protein kinase pathway from that of the G-protein-coupled receptor kinase family for the stimulus-dependent phosphorylation of the m3-muscarinic receptor, rhodopsin, and possibly other G-protein-coupled receptors.
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PMID:Stimulus-dependent phosphorylation of G-protein-coupled receptors by casein kinase 1alpha. 925 10

The RIalpha subunit of cAMP-dependent protein kinase is maintained as an asymmetric dimer by a dimerization motif at the N terminus. Based on resistance to proteolysis and expression as a discrete domain in Escherichia coli, this motif is defined as residues 12-61. This motif is chemically, kinetically, and thermally stable. The two endogenous interchain disulfide bonds between Cys16 and Cys37 in RIalpha are extremely resistant to reduction even in 8 M urea, indicating that they are well shielded from the reducing environment of the cell. The disulfide bonds were present in recombinant RIalpha as well as when the dimerization domain alone was expressed in E. coli, emphasizing the unusual stability of this motif and the disulfide bonds. Although 100 mM dithiothreitol was sufficient to reduce the disulfide bonds, it did not abolish dimerization. In addition, a stable dimer also still formed when Cys37 was replaced with His, confirming unambiguously the original antiparallel alignment of the disulfide bonds. Thus, both in vitro and in vivo, disulfide bonds are not required for dimerization. Circular dichroism of the dimerization domain indicated a high content of a thermostable alpha-helix. Based on the CD data, trypsin resistance of the fragment, location of the disulfide bonds, and amphipathic helix predictions, potential models are discussed. A new alignment of the dimerization domains of RI, RII, and cGMP-dependent protein kinase elucidates fundamental similarities as well as significant differences among these three domains.
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PMID:A stable alpha-helical domain at the N terminus of the RIalpha subunits of cAMP-dependent protein kinase is a novel dimerization/docking motif. 935 2


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