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:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate), a phosphoprotein substrate for cAMP-dependent protein kinase, is unevenly distributed in adult rat brain. Using immunoblotting and phosphorylation in vitro followed by immunoprecipitation, ARPP-21 was found to be enriched in caudate-putamen, substantia nigra, nucleus accumbens and olfactory tubercle. Intermediate levels were found in cerebral cortex and hippocampus. ARPP-21 was very low in most other brain areas and was not detected in any of the peripheral tissues studied. Following unilateral lesion of the caudate-putamen with quinolinic acid, a marked decrease in the levels of ARPP-21 was observed in both the lesioned caudate-putamen (-75%) and the ipsilateral substantia nigra (-70%) compared with the unlesioned side. This result demonstrates the enrichment of ARPP-21 in striatonigral neurons. In slices of caudate-putamen, substantia nigra or cerebral cortex incubated in vitro, the phosphorylation of ARPP-21 was enhanced by 8-Br-cAMP, a stable analog of cAMP. In striatal slices, forskolin, a compound which stimulates adenylate cyclase directly, enhanced the phosphorylation of ARPP-21 with an EC50 of 0.5 microM. In conclusion, ARPP-21 is a neuron-specific phosphoprotein enriched in specific brain areas which are known to receive a rich dopaminergic innervation and to contain high levels of D1 dopamine receptors. The phosphorylation of ARPP-21 is likely to mediate some of the intracellular effects of neurotransmitters which stimulate adenylate cyclase in these regions, in particular dopamine and vasoactive intestinal peptide.
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PMID:ARPP-21, a cAMP-regulated phosphoprotein enriched in dopamine-innervated brain regions: tissue distribution and regulation of phosphorylation in rat brain. 196 23

The lipid fraction ("fat cake") of rat epididymal adipocytes contains a prominent phosphoprotein (62 kDaapp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) that is multiply phosphorylated by cAMP-dependent protein kinase in vivo, at which point it migrates as a 65/67-kDaapp doublet by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is by far the most heavily radiolabeled protein in the cell. Western blot analysis of various tissues with immunopurified antibodies purified from antisera raised against the 62-kDa species suggests that the protein is specific for adipocytes. This protein, which we term perilipin, is found in differentiated cultured 3T3-L1 adipocytes, but not in their precursor 3T3-L1 fibroblasts. Immunocytochemical studies with specific antiserum shows that the perilipin is closely associated with the periphery of lipid storage droplets in cultured adipocytes. Given its adipocyte specificity, acute regulation by hormones, and subcellular location, we speculate that perilipin plays a role in the specialized lipid storage function of adipocytes.
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PMID:Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets. 204 Jun 38

Caldesmon is a calmodulin- and actin-binding protein present in both smooth and non-muscle tissue. The present study demonstrates that platelet caldesmon is a substrate for cAMP-dependent protein kinase (protein kinase A). Purified platelet caldesmon has an apparent molecular mass of 82 kDa on sodium dodecyl sulfate-polyacrylamide gels and can be phosphorylated in vitro by the catalytic subunit of protein kinase A to a level of 2 mol of phosphate/mol of caldesmon. Phosphorylation of caldesmon by protein kinase A results in a shift in the apparent molecular mass of the protein to 86 kDa. When caldesmon was immunoprecipitated from intact platelets treated with prostacyclin (PGI2) the same shift in apparent molecular mass of caldesmon was observed. Comparison of two-dimensional tryptic phosphopeptide maps of caldesmon phosphorylated in vitro by protein kinase A with caldesmon immunoprecipitated from intact platelets verified that protein kinase A was responsible for the observed increase in caldesmon phosphorylation in PGI2-treated platelets. The present study demonstrates that although caldesmon is basally phosphorylated in the intact platelet, activation of protein kinase A by PGI2 results in the significant incorporation of phosphate into two new sites. In addition, the effects of phorbol ester, collagen, and thrombin on caldesmon phosphorylation were also examined. Although phorbol ester treatment results in a significant increase in caldesmon phosphorylation apparently by protein kinase C, treatment of intact platelets with thrombin or collagen does not result in an increase in caldesmon phosphorylation.
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PMID:Caldesmon phosphorylation in intact human platelets by cAMP-dependent protein kinase and protein kinase C. 205 Jun 83

Chromatography of wild-type yeast extracts on DEAE-cellulose columns resolves two populations of glycogen synthase I (glucose-6-P-independent) and D (glucose-6-P-dependent) (Huang, K. P., Cabib, E. (1974) J. Biol. Chem. 249, 3851-3857). Extracts from a glycogen-deficient mutant strain, 22R1 (glc7), yielded only the D form of glycogen synthase. Glycogen synthase D purified from either wild-type yeast or from this glycogen-deficient mutant displayed two polypeptides with molecular masses of 76 and 83 kDa on sodium dodecyl sulfate-gel electrophoresis in a protein ratio of about 4:1. Phosphate analysis showed that glycogen synthase D from either strain of yeast contained approximately 3 phosphates/subunit. The 76- and 83-kDa bands of the mutant strain copurified through a variety of procedures including nondenaturing gel electrophoresis. These two polypeptides showed immunological cross-reactivity and similar peptide maps indicating that they are structurally related. The relative amounts of these two forms remained constant during purification and storage of the enzyme and after treatment with cAMP-dependent protein kinase or with protein phosphatases. The two polypeptides were phosphorylated to similar extent in vitro by the catalytic subunit of mammalian cyclic AMP-dependent protein kinase. Phosphorylation of the enzyme in the presence of labeled ATP followed by tryptic digestion and reversed phase high performance liquid chromatography yielded two labeled peptides from each of the 76- and 83-kDa subunits. Treatment of wild-type yeast with Li+ increased the glycogen synthase activity, measured in the absence of glucose-6-P, by approximately 2-fold, whereas similar treatment of the glc7 mutant had no effect. The results of this study indicate that the GLC7 gene is involved in a pathway that regulates the phosphorylation state of glycogen synthase.
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PMID:Purification and characterization of glycogen synthase from a glycogen-deficient strain of Saccharomyces cerevisiae. 211 10

Dehydroepiandrosterone (DHEA) treatment is effective in preventing or delaying the onset of various genetic and induced disorders of mice and rats. Associated with the beneficial therapeutic effects exerted by action of this steroid is the development of hepatomegaly. To determine whether the changes associated with hepatomegaly also involve alterations in activities of tissue enzymes, we evaluated the effects of DHEA (0.45% in food, w/w) on hepatic protein kinases, phosphatases, and lipogenic enzymes in mice of various strains. The rates of fatty acid and cholesterol syntheses also were evaluated. DHEA administration resulted in profound changes in the sodium dodecylsulfate-polyacrylamide gel electrophoresis patterns of endogenous radiophosphorylated proteins obtained by incubation of liver homogenates with (gamma-32P]ATP. These changes were dependent upon the medium used for homogenization. Thus, when homogenates of liver tissue of DHEA-treated mice were prepared in Tris buffer containing sucrose (0.25 M) there was a marked decrease in phosphorylation of the proteins of relative molecular weight approximately 116,000 (Mr approximately 116,000), approximately 82,000, approximately 80,000, approximately 58,000, approximately 56,000, approximately 48,000, approximately 34,000, and approximately 31,000 compared with controls. With liver homogenates of DHEA-treated mice prepared in Tris buffer alone, there was a marked increase in phosphorylation of the proteins of Mr approximately 70,000, approximately 49,000, approximately 34,000, approximately 31,000, and 28,000 compared with controls. Moreover, the specific activity of kinases for endogenous protein acceptors in liver of control mice was higher than that in liver of DHEA-treated animals. The specific activities of casein kinase, cAMP-dependent protein kinase, and cGMP-dependent protein kinase remained unchanged with DHEA treatment, but the specific activity of histone kinase was increased approximately 30%. Long-term administration of DHEA also was associated with increases in the specific activities of liver AMPase and GTPase (approximately two times), but not of other nucleotidases, alkaline phosphatase, acid phosphatase, glucose-6-phosphatase, or phosphotyrosine phosphatase. The activity of hepatic NADP-linked malic enzyme was increased significantly (two to three times) by DHEA treatment of female mice of three different strains, but was unchanged in male C57BL/6 mice. The specific activities of hepatic glucose-6-phosphate dehydrogenase, NADP-linked isocitrate dehydrogenase, and ATP-citrate lyase were not affected significantly by DHEA treatment of mice. The rate of hepatic lipogenesis, determined by incorporation of tritium from 3H2O into fatty acids, was decreased approximately 70% in DHEA-treated mice, while the rate of cholesterol synthesis was increased approximately 44% compared with controls.
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PMID:Dehydroepiandrosterone feeding and protein phosphorylation, phosphatases, and lipogenic enzymes in mouse liver. 215 82

The activation of cyclic adenosine 3'5'-monophosphate (cAMP)-dependent protein kinases has been implicated as an integral mechanism in stimulus-secretion coupling in the anterior pituitary. Therefore, we have investigated phosphorylation of endogenous protein substrates both in the presence and absence of cAMP in cell-free extracts of the rodent anterior pituitary. Specific phosphoprotein substrates in the rat anterior pituitary, which are phosphorylated by a cAMP-dependent protein kinase in vitro, were identified. Cyclic AMP potentiated the phosphorylation of proteins with apparent molecular weights of 85,000, 77,000, 63,000, 53,000, 39,000, and 33,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins with apparent molecular weights of 124,000, 93,000, 48,000, and 43,000 were phosphorylated only in the presence of cAMP and not in the basal condition. The results highlight endogenous protein substrates that may potentially be involved in cAMP-dependent stimulus-secretion coupling in the anterior pituitary.
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PMID:Cyclic AMP-dependent protein phosphorylation in the rat anterior pituitary. 216 Jul 42

Relaxin is a member of the insulin family of polypeptide hormones and is known to exert its biological effects on various parts of the mammalian reproductive system. Biologically active human relaxin has been chemically synthesized based on the nucleotide sequence obtained from an ovarian cDNA clone. In the present study synthetic human relaxin was radiolabled by phosphorylation with cAMP-dependent protein kinase and [gamma-32P]ATP to a specific activity of 5000 Ci/mmol. The phosphorylated relaxin was purified on cation exchange high performance liquid chromatography and was shown to co-migrate with relaxin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mass spectrometry revealed a single phosphorylated site on the B chain of relaxin. The 32P-relaxin was able to bind to a goat anti-relaxin antibody, and this binding could be displaced by unlabeled relaxin in a concentration-dependent manner. A comparison of the concentration responses of cellular cAMP production stimulated by relaxin and phosphorylated relaxin in a primary human uterine cell line showed that phosphorylation did not affect the in vitro biological efficacy of relaxin. This made it suitable for in situ autoradiographic localization of relaxin binding sites in rat uterine, cervical, and brain tissue sections. Displacement of the binding of 100 pM 32P-relaxin by 100, 10, and 3 nM unlabeled relaxin, but not by 100 nM insulin, insulin-like growth factor-I, and an insulin-like growth factor-I analog, demonstrated the high affinity and specificity of such binding. We conclude that 32P-labeled human relaxin is biologically and immunologically active and that this novel probe binds reversibly and with high affinity to classical (e.g. uterus) and unpredicted (e.g. brain) tissues.
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PMID:Preparation of biologically active 32P-labeled human relaxin. Displaceable binding to rat uterus, cervix, and brain. 216 Sep 76

This study characterized the various adenosine 3',5'-cyclic monophosphate (cAMP)-binding proteins in epithelial cells cultured from human sweat glands to identify potential pathways of cAMP action in this tissue. The cAMP-binding proteins were identified by specific labeling with the photoprobe 8-azido-[32P]-cAMP and visualized by autoradiography after electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Three cAMP-binding proteins, with molecular masses of 48, 52, and 54 kDa, were identified in subcellular fractions of the cultured sweat glands. On the basis of their relative electrophoretic mobilities and reactivity with specific antisera, the 48-kDa protein was identified as the regulatory subunit of the type 1 cAMP-dependent protein isozyme, and the 54-kDa protein was identified as the regulatory subunit of the type 2 cAMP-dependent protein kinase isozyme. The 52-kDa cAMP-binding protein appeared to be a distinct isoform of the regulatory subunit, possibly related to the subclass associated with neural tissue. As determined from the relative distributions of the photolabel, the 48-, 52-, and 54-kDa binding proteins were present in the cell cytosol at a ratio of 1:2:1. The 48- and 52-kDa proteins bound the photoprobe with apparent dissociation constants (Kd) of 20-40 nM, whereas the 54-kDa protein bound the photoprobe with much lower affinity (Kd 230 nM). Only the 48- and 52-kDa proteins were associated with cell membranes. They were present in the membrane fractions in approximately equal amounts and bound the photoprobe with apparent Kd values of greater than 100 nM.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:cAMP-binding proteins in epithelial cells cultured from human sweat glands. 216 95

Immunoprecipitation, radiophosphorylation and SDS-PAGE autoradiography enable the characterization of sodium channel polypeptides in the central nervous system of insects belonging to four phylogenetically distinct orders: grasshoppers, cockroaches, flies and moth larvae. It has been shown that the insect sodium channels: (1) Are recognized by the previously described (Gordon et al. (1988) Biochemistry 27, 7032-7038) site directed antibodies corresponding to a highly conserved segment linking the homologous domains III and IV in the vertebrate sodium channel alpha subunits. (2) Serve as substrates for phosphorylation by cAMP-dependent protein kinase. (3) Are devoid of disulfide linkage to smaller subunits unlike sodium channels in vertebrate brain. (4) Are glycoproteins as shown in the grasshopper by the decrease of apparent molecular weight following endoglycosidase F treatment and specific binding to the lectins concanavalin A and wheat germ agglutinin. (5) Reveal a diversity with regard to their (a) apparent molecular masses which range from 240 to 280 kDa and (b) V8 proteinase digestion phosphopeptides indicating either differences in the positioning of the enzymatic cleavage and/or phosphorylation sites. These results provide the first evidence for structural diversity of sodium channel subtypes among various insect orders and are compared to their mammalian counterparts.
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PMID:Sodium channel polypeptides in central nervous systems of various insects identified with site directed antibodies. 216 10

In isolated, 32Pi-loaded, rat adipocytes, we have examined phosphorylation of the major cAMP-dependent protein kinase (A-kinase) substrate, a protein that appears to be associated with the lipid storage droplet and migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 65-67-kDa doublet. In control cells, a strong phosphorylation signal is detected as the (+/- cAMP) A-kinase activity ratio ranges from approximately 0.1 to approximately 0.3-0.4 with increasing isoproterenol concentrations. By contrast, insulin-treated cells exhibiting A-kinase activity ratios over the range of 0.1-0.25 contain less 32P in the 65-67-kDa protein than control cells exhibiting identical A-kinase activity ratios. At higher activity ratios (greater than 0.3), this reduction in phosphorylation of the 65-67-kDa protein by insulin disappears. It is concluded that insulin stimulates a phosphatase activity that acts on the 65-67-kDa protein. Insulin actions aside, these studies reveal two interesting phenomena. 1) Whereas elevated, steady-state A-kinase activities are established rapidly (1-2 min) upon isoproterenol stimulation, phosphorylation of the 65-67-kDa substrate proceeds through a burst, followed by a decline to a steady-state level by 10-12 min. An "adaptation" mechanism, providing for a constant response to a constant stimulus, may underlie this lack of parallelism between the time course of phosphorylation and A-kinase activity. 2) Removal of [32Pi] orthophosphate immediately before isoproterenol stimulation leads to a rapid (t approximately 10 min) loss in labeling of the 65-67-kDa protein, whereas the phosphorylation state of other phosphoproteins are not changed. These data suggest that elevation of A-kinase activity leads to a rapid exchange of external Pi with an ATP pool that is used by A-kinase.
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PMID:Control of endogenous phosphorylation of the major cAMP-dependent protein kinase substrate in adipocytes by insulin and beta-adrenergic stimulation. 217 32


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