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)

Cystic fibrosis (CF) is a common lethal genetic disease that manifests itself in airway and other epithelial cells as defective chloride ion absorption and secretion, resulting at least in part from a defect in a cyclic AMP-regulated, outwardly-rectifying Cl- channel in the apical surface. The gene responsible for CF has been identified and predicted to encode a membrane protein termed the CF transmembrane conductance regulator (CFTR). Identification of a cryptic bacterial promoter within the CFTR coding sequence led us to construct a complementary DNA in a low-copy-number plasmid, thereby avoiding the deleterious effects of CFTR expression on Escherischia coli. We have used this cDNA to express CFTR in vitro and in vivo. Here we demonstrate that CFTR is a membrane-associated glycoprotein that can be phosporylated in vitro by cAMP-dependent protein kinase. Polyclonal and monoclonal antibodies directed against distinct domains of the protein immunoprecipitated recombinant CFTR as well as the endogenous CFTR in nonrecombinant T84 cells. Partial proteolysis fingerprinting showed that the recombinant and non-recombinant proteins are indistinguishable. These data, which establish several characteristics of the protein responsible for CF, will now enable CFTR function to be studied and will provide a basis for diagnosis and therapy.
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PMID:Expression and characterization of the cystic fibrosis transmembrane conductance regulator. 169 61

Rabbit skeletal muscle membranes contain a protein which inhibits the cAMP-dependent protein kinase. The activity of the partially purified membrane protein is characterized by an IC-50 of 10 to 30 nM with respect to the inhibition of the activity of the catalytic subunit of the cAMP-dependent protein kinase and is sensitive to treatment with heat, acid, alkali and trypsin. The active fractions contain proteins ranging from 40 to 120 kDa, analysed by SDS-gel electrophoresis.
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PMID:Characterization and partial purification of a membrane protein from rabbit skeletal muscle which inhibits the cAMP-dependent protein kinase. 173 83

The yeast VPS15 gene encodes a novel protein kinase homolog that is required for the sorting of soluble hydrolases to the yeast vacuole. In this study, we extend our previous mutational analysis of the VPS15 gene and show that alterations of specific Gps15p residues, that are highly conserved among all protein kinase molecules, result in the biological inactivation of Vps15p. Furthermore, we demonstrate here that short C-terminal deletions of Vps15p result in a temperature-conditional defect in vacuolar protein sorting. Immediately following the temperature shift, soluble vacuolar hydrolases, such as carboxypeptidase Y and proteinase A, accumulate as Golgi-modified precursors within a saturable intracellular compartment distinct from the vacuole. This vacuolar protein sorting block is efficiently reversed when mutant cells are shifted back to the permissive temperature; the accumulated precursors are rapidly processed to their mature forms indicating that they have been delivered to the vacuole. This rapid and efficient reversal suggests that the accumulated vacuolar protein precursors were present within a normal transport intermediate in the vacuolar protein sorting pathway. In addition, this protein delivery block shows specificity for soluble vacuolar enzymes as the membrane protein, alkaline phosphatase, is efficiently delivered to the vacuole at the non-permissive temperature. Interestingly, the C-terminal Vps15p truncations are not phosphorylated in vivo suggesting that the phosphorylation of Vps15p may be critical for its biological activity at elevated temperatures. The rapid onset and high degree of specificity of the vacuolar protein delivery block in these mutants suggests that the primary role of Vps15p is to regulate the sorting of soluble hydrolases to the yeast vacuolar compartment.
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PMID:A genetic and structural analysis of the yeast Vps15 protein kinase: evidence for a direct role of Vps15p in vacuolar protein delivery. 175 16

Effects of endotoxin administration on the ATP-dependent Ca2+ uptake by canine cardiac sarcoplasmic reticulum (SR) were investigated. Results obtained 4 h after endotoxin administration show that ATP-dependent Ca2+ uptake by cardiac SR was decreased by 27-43% (p less than 0.05). Kinetic analysis indicates that the Vmax values for Ca2+ and for ATP were significantly decreased while the S0.5 and the Hill coefficient values were not affected during endotoxin shock. Magnesium (1-5 mM) stimulated while vanadate (25-250 microM) inhibited the ATP-dependent Ca2+ uptake, but the Mg(2+)-stimulated and the vanadate-inhibited activities remained significantly lower in the endotoxin-treated animals. Phosphorylation of SR by the exogenously added catalytic subunit of the cAMP-dependent protein kinase or by the addition of calmodulin stimulated the ATP-dependent Ca2+ uptake activities both in the control and endotoxin-injected dogs. However, the phosphorylation-stimulated activities remained significantly lower in the endotoxin-injected dogs. Dephosphorylation of SR decreased the ATP-dependent Ca2+ uptake, but the half-time required for the maximal dephosphorylation was reduced by 31% (p less than 0.05) 4 h post-endotoxin. These data indicate that endotoxin administration impairs the ATP-dependent Ca2+ uptake in canine cardiac SR and the endotoxin-induced impairment in the SR calcium transport is associated with a mechanism involving a defective phosphorylation and an accelerated dephosphorylation of SR membrane protein. Since ATP-dependent Ca2+ uptake by cardiac SR plays an important role in the regulation of the homeostatic levels of the contractile calcium, our findings may provide a biochemical explanation for myocardial dysfunction that occurs during endotoxin shock.
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PMID:Impaired calcium uptake by cardiac sarcoplasmic reticulum and its underlying mechanism in endotoxin shock. 177 Sep 48

Conductive transport of chloride ion is important in controlling ion and fluid secretion by exocrine tissues. The current study was directed at identifying proteins in the intestinal brush-border membrane that may be involved with conductive chloride transport. Reaction of total brush-border membrane protein with phenyl-isothiocyanate inhibited conductive chloride transport into brush-border membrane vesicles. The conductive transport process was protected from this inhibition by including the ligands Cl- and alpha-phenylcinnamate in the reaction mixture. Brush-border membrane protein protected by this procedure and labeled with fluorescein had an apparent molecular mass in the region of 130 and 23 kDa on separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation of brush-border membrane protein with [gamma-32P] ATP and endogenous protein kinase under conditions causing activation of chloride conductance in membrane vesicles caused the transfer of 32P to several proteins, including ones in the same molecular size range (130 and 23 kDa) as those identified by the fluorescein labeling procedure. Conductive chloride transport in porcine intestinal brush-border vesicles may occur via proteins identified by this differential labeling procedure.
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PMID:Candidate proteins for conductive chloride transport in porcine ileal brush-border membrane. 191 21

Estradiol receptors are discovered in nuclei, cytoplasm and plasmatic membranes of the target cells. Estradiol activates the transmembrane polyphosphoinositide system: it stimulates the protein kinase C translocation from cytosol to cell membranes, the membrane protein phosphorylation being elevated. Transmembrane adenylate cyclase system is also activated. The cAMP system stimulation by estradiol is mediated by protein kinase C, phosphorylating a protein of adenylate cyclase complex. Estradiol causes protein kinases A translocation into the cell nuclei and enhances the protein kinase NII activity. The role of protein phosphorylation, activated by steroid hormones, in the transcription and protein synthesis regulation, is discussed.
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PMID:[The participation of transmembrane messenger systems in the action of steroid hormones on target cells]. 196 59

The anthraquinone dye reactive blue 2 was found to be a potent inhibitor of a protein kinase isolated and purified from thylakoids. This enzyme was also inhibited in situ, with corresponding inhibition of ATP-dependent quenching of the chlorophyll fluorescence. The mode of inhibition was noncompetitive, with a Ki of 8 microM for the membrane-bound kinase, and 6 microM for the purified kinase. The inhibitor did not modify the substrate preference of the endogenous kinase and could be removed from the membrane by washing. Unlike reactive blue 2, the enzyme did not partition into detergent micelles and is therefore presumably not a hydrophobic, intrinsic membrane protein.
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PMID:Reactive blue 2 is a potent inhibitor of a thylakoid protein kinase. 202 69

In Torpedo marmorata electroplaque, an extrinsic membrane protein of apparent mass 43,000 daltons colocalizes with the cytoplasmic face of the nicotinic acetylcholine receptor (AChR) in approximately 1:1 stoichiometry. We show that this 43K protein can be phosphorylated in vitro by endogenous protein kinases present in AChR-rich membranes. The extent of 43K protein phosphorylation exceeds that of the subunits of the AChR, well-established substrates for enzymatic phosphorylation. We demonstrate that significant 43K phosphoprotein exists in vivo. The kinetics of phosphate incorporation mediated by endogenous kinases differed significantly from those of the AChR subunits, suggesting that different phosphorylation cascades are involved. Use of specific inhibitors of a variety of protein kinases indicated that endogenous cAMP-dependent protein kinase catalyzes phosphorylation of the 43K protein in vitro. All of the phosphate incorporated into 43K protein was accounted for by phosphoserine (0.65 mol/mol of 43K protein). Potential structural and functional consequences of 43K protein phosphorylation are discussed.
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PMID:Serine-specific phosphorylation of nicotinic receptor associated 43K protein. 203 28

Sphingosine inhibition of [3H] [N3-Me-His] TRH (MeTRH) binding, previously shown to be independent of its effects on protein kinase-C, has been further characterized in GH3 cell membranes and in a partially purified, digitonin-solubilized receptor preparation. In membranes, as in intact cells, sphingosine inhibited [3H]MeTRH binding by decreasing receptor affinity, but, in contrast to its effect in intact cells, did not affect the number of available binding sites. The inhibition of binding was linear up to 75 microM sphingosine (in the presence of 100 microM BSA at 0.1 mg membrane protein/ml), yielding an apparent Ki of 51 microM. Since GTP decreases the affinity for MeTRH binding in GH3 cell membranes, we studied interactions between GTP and sphingosine. While the effects of low concentrations of GTP gamma S and sphingosine were additive, sphingosine inhibition of MeTRH binding surpassed and was not affected by the addition of maximally inhibitory concentrations of GTP gamma S. Also, sphingosine (75 microM) did not affect the ability of a maximally effective dose of TRH to stimulate the low Km GTPase (vehicle, +35 +/- 5%; sphingosine, +32 +/- 10%); there was a 25% decrease in total GTPase activity in the presence of sphingosine. MeTRH binding to digitonin-solubilized receptors, which had properties similar to those described previously by others, including no effect of GTP on binding, was inhibited by sphingosine. In solubilized receptors, as in membranes, sphingosine caused a decrease in apparent affinity without changes in the number of binding sites. These data suggest that sphingosine interacts directly with the TRH receptor [or an associated factor(s) in the receptor complex] to decrease affinity by a mechanism that does not involve uncoupling of G-proteins.
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PMID:Sphingosine interacts directly with the receptor complex to inhibit thyrotropin-releasing hormone binding. 210 32

The main intrinsic membrane protein of the lens fiber cell, MIP, has been previously shown to be phosphorylated in preparations of lens fragments. Phosphorylation occurred on serine residues near the cytoplasmic C-terminus of the molecule. Since MIP is thought to function as a channel protein in lens plasma membranes, possibly as a cell-to-cell channel protein, phosphorylation could regulate the assembly or gating of these channels. We sought to identify the specific serines which are phosphorylated in order to help identify the kinases involved in regulating MIP function. To this end we purified a peptide fragment from native membranes that had not been subjected to any exogenous kinases or kinase activators. Any phosphorylation detected in these fragments must be due to cellular phosphorylation and thus is termed in vivo phosphorylation. Purified membranes were also phosphorylated with cAMP-dependent protein kinase to determine the mobility of phosphorylated and unphosphorylated MIP-derived peptides on different HPLC columns and to determine possible cAMP-dependent protein kinase phosphorylation sites. Lens membranes, which contain 50-60% of the protein as MIP, were digested with lysylendopeptidase C. Peptides were released from the C-terminal region of MIP and a major product of 21-22 kDa remained membrane-associated. Separation of the lysylendopeptidase-C-released peptides on C8 reversed-phase HPLC demonstrated that one of these fragments, corresponding to residues 239-259 in MIP, was partially phosphorylated. The phosphorylated and nonphosphorylated forms of this peptide were separated on QAE HPLC. In vivo phosphorylation sites were found at residues 243 and 245 through phosphoserine modification via ethanethiol and sequence analysis. Phosphorylation was never detected on serine 240. The phosphorylation level of serine 243 could be increased by incubation of membranes with cAMP-dependent protein kinase under standard assay conditions. Other kinases that phosphorylate serines found near acidic amino acids must be responsible for the in vivo phosphorylation demonstrated at serine 245.
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PMID:Amino acid sequence of in vivo phosphorylation sites in the main intrinsic protein (MIP) of lens membranes. 217 1


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