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)

A protein kinase was isolated from spinach thylakoid membranes by solubilization with octyl glucoside and cholate. The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, and sucrose density centrifugation, followed by affinity chromatography on either Affi-Gel blue (yielding denatured enzyme) or on histone cross-linked to Sepharose (yielding active enzyme). Electrophoresis on denaturing polyacrylamide gels, followed by staining with silver, revealed the kinase as a single band corresponding to an apparent molecular mass of 64 kDa. The active enzyme underwent autophosphorylation and could be detected by autoradiography following incubation with [gamma-32P]ATP and Mg2+ ion. The specific phosphotransferase activity of purified kinase was approximately 30 nmol of phosphate min-1 (mg protein)-1 with lysine-rich histone (III-S or V-S) as substrate; casein was phosphorylated at approximately 30% of this rate. The physiological substrate for the kinase is presumed to be light-harvesting chlorophyll a/b protein complex. In solubilized form, this was phosphorylated at approximately 10% of the rate observed with histone III-S as substrate, or 10-100 times slower than the estimated rate of phosphorylation of the light-harvesting complex in situ. Possible reasons for this shortfall are considered. The kinase is proposed as the principal effector of thylakoid protein phosphorylation and associated State transition phenomena.
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PMID:Purification and characterization of a membrane-bound protein kinase from spinach thylakoids. 373 55

The claim of Racker and co-workers (Lin, Z. F., Lucero, H. A., and Racker, E. (1982) J. Biol. Chem. 257, 12153-12156 and Lucero, H. A., Lin, Z. F., and Racker, E. (1982) J. Biol. Chem. 257, 12157-12160) that two protein kinases, designated CPK1 (25 kDa) and CPK2 (38 kDa), are present in spinach thylakoid membranes was investigated in light of results from this laboratory (Coughlan, S. J., and Hind, G. (1986) J. Biol. Chem. 261, 11378-11385) showing that 75-80% of the measurable protein kinase activity of isolated thylakoids is attributable to a protein kinase of 64 kDa apparent molecular mass. Extraction of thylakoid membranes with octyl glucoside/cholate according to the procedure of Lin et al. (Lin, Z. F., Lucero, H. A., and Racker, E. (1982) J. Biol. Chem. 257, 12153-12156) released proteins assignable to CPK1 and CPK2 on the basis of photoaffinity labeling with 8-azido-[32P]ATP. The 64-kDa protein kinase was present in this extract and accounted for greater than 80% of the total phosphotransferase activity toward lysine-rich histone as substrate; it was not labeled by the photoaffinity reagent. The three presumptive kinases were purified by ammonium sulfate precipitation, sucrose density gradient centrifugation, hydroxylapatite chromatography, and affinity chromatography. CPK1 was specifically eluted from Cibacron blue-Sepharose by 10 mM ATP; it electrophoresed on denaturing polyacrylamide gels as a single band with apparent molecular mass of 25 kDa. Its specific activity toward lysine-rich histone as substrate was approximately 250 pmol of phosphate transferred (mg protein)-1 min-1. The 64-kDa protein kinase was eluted from the affinity column by 1% (w/v) lithium dodecyl sulfate or from a histone IIIs-Sepharose affinity column by 0.25 M NaCl. Its specific activity towards lysine-rich histone was 100-200 times greater than that of CPK1. CPK2 eluted from the Cibacron blue affinity column in 10 mM NADP+; it had an apparent molecular mass of 38 kDa, possessed NADPH-dependent diaphorase activity (specific activity: 225 nmol of ferricyanide reduced (mg protein)-1 min-1), and cross-reacted with immunoglobulin raised against purified ferredoxin:NADP+ oxidoreductase, with which it was thus identified. Kinase activity was not detectable in CPK2 or in reductase isolated by conventional procedures.
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PMID:Protein kinases of the thylakoid membrane. 377 22

A glucose-dependent phosphorylation of a 68kDa islet-cell protein was observed in islet-cell homogenates. In the presence of [gamma-32P]ATP the protein was phosphorylated only in the presence of alpha-D-glucose; other sugars were ineffective. Activation of the phosphorylation was half-maximal at 0.34 mM-glucose, 7 microM-ATP and 0.3 mM-Mg2+. Although the addition of glucose 6-phosphate in this design did not stimulate phosphorylation of the islet-cell protein, addition of glucose 6-phosphate to the radioactively labelled 68kDa protein rapidly removed (chased) the 32P label. The addition of presynthesized glucose 6-[32P]phosphate phosphorylated the 68kDa band in the islet-cell homogenate and also phosphorylated purified skeletal-muscle phosphoglucomutase. Phosphoglucomutase labelled thus by 32P was indistinguishable from the islet-cell phosphoprotein on electrophoretic gels. The 32P incorporated into both the islet-cell protein and the purified skeletal-muscle phosphoglucomutase was chased similarly by hexose phosphates. The purified phosphoglucomutase could also be phosphorylated by cyclic AMP-dependent protein kinase or by a mannoheptulose-insensitive process by the islet-cell cytosol. The phosphoenzyme formed thus was also dephosphorylated by D-glucose 6-phosphate and alpha-D-glucose 1-phosphate, suggesting that this may be a mechanism for generation of glucose 1,6-bisphosphate.
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PMID:Glucose-stimulated protein phosphorylation in the pancreatic islet. 623 88

The effect of 8-bromo cyclic adenosine 3':5'-monophosphate (8-Br-cAMP) on sugar and amino acid transport was investigated in wild-type Chinese hamster ovary (CHO) cells and in mutants selected for resistance to cAMP inhibition of cell growth. In wild type cells, both 3-O-methyl-D-glucose and alpha-aminoisobutyric acid transport were decreased in cells treated for 24 h with 8-Br-cAMP; kinetic analysis indicated that a decrease in Vmax, without a significant change in Km, accounted for the lower transport capacity of 8-Br-cAMP treated cells. Among the different transport systems contributing to amino acid entry, "alanine" preferring transport system (system A) appeared to be specifically affected. The sensitivity of transport processes to 8-Br-cAMP was tested in three cAMP-resistant cell lines. When tested for their capacity to phosphorylate histones in crude extracts, one strain had apparently normal amounts of protein kinase activity, one strain had a decreased enzyme sensitivity to cAMP, and one strain had little or no enzyme activity. In all three mutants, no effect of 8-Br-cAMP on 3-O-methyl glucose and alpha-aminoisobutyric acid transport could be observed, regardless of the level of cAMP-dependent protein kinase activity. These data do not indicate whether the effect of cAMP on nutrient transport in CHO cells is the cause or consequence of growth inhibition. However, they support the conclusion that, in CHO cells, the presence of a normally functioning cAMP-dependent protein kinase appears to be necessary but may not be sufficient to observe the effects of cAMP on nutrient transport as well as cell shape and cell growth.
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PMID:Mechanism of cyclic AMP effect on nutrient transport in Chinese hamster ovary cells. A genetic approach. 625 Oct 43

It is known that the administration of parathyroid hormone to dogs results in phosphaturia and decreased phosphate transport in brush-border vesicles isolated from the kidneys of those dogs. Parathyroid hormone has been shown to activate adenylate cyclase at the basal-lateral membrane of the renal proximal tubular cell. It has been postulated that parathyroid hormone-induced phosphaturia is effected through phosphorylation of brush-border protein by membrane-bound cAMP-dependent protein kinase. An experimental system was designed such that phosphorylation of brush-border vesicles and Na+-stimulated solute transport could be studied in the same preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane vesicles revealed cAMP-dependent phosphorylation of 2 protein bands (Mr = 96,000 and 62,000), which was enhanced by exposure of the inside of the membrane vesicles to ATP and cAMP. Cyclic AMP-dependent phosphorylation of brush-border vesicles was accompanied by inhibition of Na+-stimulated Pi but not D-glucose transport or 22Na+ uptake. When renal brush-border vesicles from parathyroidectomized and normal dogs were phosphorylated in vitro in the presence and absence of cAMP, both the cAMP-dependent phosphorylation and inhibition of Na+-stimulated Pi transport were greater in vesicles isolated from kidneys of parathyroidectomized dogs relative to control animals. We conclude that the cAMP-dependent phosphorylation of brush-border membrane-vesicle proteins is associated with specific inhibition of Na+-stimulated Pi transport. The phosphaturic action of parathyroid hormone (PTH) could be mediated through the cAMP-dependent phosphorylation of specific brush-border membrane proteins.
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PMID:Cyclic AMP-dependent protein phosphorylation in canine renal brush-border membrane vesicles is associated with decreased phosphate transport. 627 74

Alloxan was found to inhibit a Ca2+- and calmodulin-dependent protein kinase recently identified in pancreatic islets. This effect of alloxan may be specifically related to the inhibitory action of alloxan on insulin secretion from islets since: 1) in islet-cell subcellular fractions, alloxan at micromolar concentrations irreversibly inhibits the Ca2+- and calmodulin-dependent protein kinase activity; 2) pretreatment of intact islets with alloxan at concentrations that inhibit insulin secretion similarly inhibits the protein kinase activity; and 3) alloxan inhibition of both insulin secretion and protein kinase activity in intact islets can be prevented by D-glucose. This inhibition by alloxan appears to be a direct effect on the enzyme since alloxan treatment of either the islet homogenate or the microsomal fraction enriched in protein kinase activity inhibited the kinase activity with similar concentration dependence. These results suggest that alloxan-induced inhibition of a Ca2+- and calmodulin-dependent protein kinase may represent a critical inhibitory site which mediates alloxan-induced inhibition of insulin secretion.
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PMID:Alloxan inhibition of a Ca2+- and calmodulin-dependent protein kinase activity in pancreatic islets. 634 20

Human lung cancer transplanted into athymic mice contains predominantly an acidic variant (designated B1) of lysosomal arylsulfatase B. B1 enzyme was suggested to be phosphorylated and sialylated (Gasa, S., Makita, A., Kameya, T., Kodama, T., Koide, T., Tsumuraya, M., and Komai, T. (1981) Eur. J. Biochem. 116, 497-503). In order to determine the localization of phosphate in B1 enzyme, we labeled in vivo the transplanted tumor with [32P]H3PO4 or [3H]glucosamine and purified B1 enzyme by immunoprecipitation. Bio-Gel chromatography of the labeled B1 enzyme treated with endoglycosidase H demonstrated that both the excluded and included materials were labeled with 32P and 3H. From acid hydrolysate of the excluded materials, phosphorylated serine and threonine were detected. Protein phosphorylation of arylsulfatase was confirmed by in vitro labeling experiments with [gamma-32P]ATP. By incubation of the tumor homogenate with ATP followed by isolation of the enzymes, B1 enzyme had a significant amount of radioactivity, whereas the B enzyme had little; by exogenous protein kinase, partially purified B enzyme was phosphorylated 35 times more than B1 enzyme. Acid hydrolysate of the included materials in the Bio-Gel column demonstrated mannose 6-phosphate and an unknown phosphorylated compound which migrates more than Man-6-P on electrophoresis and chromatography.
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PMID:Phosphorylation on protein and carbohydrate moieties of a lysosomal arylsulfatase B variant in human lung cancer transplanted into athymic mice. 640 42

We used the specific tyrosine kinase inhibitor genistein to define the involvement of tyrosine phosphorylation in the regulation of chloride transport in the rectal gland of the dogfish shark, a model for chloride secretion via a cystic fibrosis transmembrane conductance regulator (CFTR)-like channel. In the perfused gland, genistein (100 microM) promptly increased chloride secretion from basal values of 159 +/- 36 to 966 +/- 49 mueq.h-1.g-1 (P < 0.0001). Bumentanide fully reversed genistein-induced secretion. In primary culture monolayers of rectal gland tubular cells, genistein, but not the inactive 7-glucoside form, genistin, increased short-circuit current in a dose-dependent manner, from basal values of 2.7 +/- 4.3 to 104 +/- 10 microA/cm2 (P < 0.0001). Apically applied genistein induced significantly greater chloride secretion than basolateral addition. Genistein did not increase the adenosine 3',5'-cyclic monophosphate (cAMP) content of either perfused glands or cultured monolayers. Using an anti-phosphotyrosine antibody, we observed phosphorylation of multiple proteins. Four peptides, with molecular masses of 250, 210, 55, and 53 kDa, responded to genistein treatment with a decrease in tyrosine phosphorylation. These data demonstrate the following: 1) genistein induces bumetanide-sensitive chloride secretion in both perfused rectal glands and cultured tubular cells; 2) these effects are not accompanied by an elevation of tissue cAMP, indicating that genistein-induced secretion is not mediated by the cAMP-protein kinase A pathway; and 3) genistein-sensitive peptides are present in the rectal gland cell and are candidates for involvement in the regulation of chloride secretion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tyrosine phosphorylation is a novel pathway for regulation of chloride secretion in shark rectal gland. 748 46

Genes involved in the phospholipid synthesis of Saccharomyces cerevisiae, such as PEM1, PEM2, PSS, and INO1, are coordinately repressed by myo-inositol and choline. In order to investigate this regulation, we transformed wild-type yeast with a PEM1 promoter-lacZ fusion and isolated two mutants, named ric1 and ric2 (regulation by myo-inositol and choline), exhibiting decreased PEM1 expression. The lowered PEM1 expression in the mutants was monitored in colonies in terms of their failure fully to develop blue color on 5-bromo-4-chloro-3-indolyl-beta-galactopyranoside-containing agar. ric1 mutant was auxotrophic for myo-inositol, indicating that INO1 expression was also affected, whereas ric2 mutant required myo-inositol only in the presence of choline. The RIC1 gene was isolated by complementation of the Ino- phenotype of ric1 mutant and its identity was confirmed by genetic cross between the original ric1 mutant and a gene disruptant. The RIC1 gene was sequenced and found to be identical with the previously identified gene, SNF2/SWI2/GAM1/TYE3, which is known to encode a general transcription factor required for the expression of various genes including INO1. Analysis using various lacZ fusion constructs containing promoters for genes in phospholipid synthesis revealed that the expression of myo-inositol-choline-regulated genes, PEM1, PEM2, PSS, CKI, and INO1, was markedly decreased in the snf2/swi2/gam1/tye3/ric1 background, but the expression of a constitutive gene, PIS, was not. We conclude that SNF2/SWI2/GAM1/TYE3/RIC1 is a positive regulatory gene required for the expression of not only INO1 gene, but also of myo-inositol-choline-regulated genes in general.
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PMID:The SNF2/SWI2/GAM1/TYE3/RIC1 gene is involved in the coordinate regulation of phospholipid synthesis in Saccharomyces cerevisiae. 760 26

The extracellular acidification rate of the human bone marrow cell line, TF-1, increases rapidly in response to a bolus of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF). Extracellular acidification rates were measured using a silicon microphysiometer. This instrument contains micro-flow chambers equipped with potentiometric sensors to monitor pH. The cells are immobilized in a fibrin clot sandwiched between two porous polycarbonate membranes. The membranes are part of a disposable plastic "cell capsule" that fits into the microphysiometer flow chamber. The GM-CSF activated acidification burst is dose dependent and can be neutralized by pretreating the cytokine with anti-GM-CSF antibody. The acidification burst can be resolved kinetically into at least two components. A rapid component of the burst is due to activation of the sodium/proton antiporter as evidenced by its elimination in sodium-free medium and in the presence of amiloride. A slower component of the GM-CSF response is a consequence of increased glycolytic metabolism as demonstrated by its dependence on D-glucose as a medium nutrient. Okadaic acid (a phospho-serine/threonine phosphatase inhibitor), phorbol 12-myristate 13-acetate (PMA, a protein kinase C (PKC) activator), and ionomycin (a calcium ionophore) all produce metabolic bursts in TF-1 cells similar to the GM-CSF response. Pretreatment of TF-1 cells with PMA for 18 h resulted in loss of the GM-CSF acidification response. Although this treatment is reported to destroy protein kinase activity, we demonstrate here that it also down-regulates expression of high-affinity GM-CSF receptors on the surface of TF-1 cells. In addition, GM-CSF driven TF-1 cell proliferation was decreased after the 18 h PMA treatment. Short-term treatment with PMA (1-2 h) again resulted in loss of the GM-CSF acidification response, but without a decrease in expression of high-affinity GM-CSF receptors. Evidence for involvement of PKC in GM-CSF signal transduction was obtained using calphostin C, a specific inhibitor of PKC, which inhibited the GM-CSF metabolic burst at a subtoxic concentration. Genistein and herbimycin A, tyrosine kinase inhibitors, both inhibited the GM-CSF response of TF-1 cells, but only at levels high enough to also inhibit stimulation by PMA. These results indicate that GM-CSF activated extracellular acidification of TF-1 cells is caused by increases in sodium/proton antiporter activity and glycolysis, through protein kinase signalling pathways which can be both activated and down-regulated by PMA.
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PMID:GM-CSF triggers a rapid, glucose dependent extracellular acidification by TF-1 cells: evidence for sodium/proton antiporter and PKC mediated activation of acid production. 767 63


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