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)

cAMP-dependent protein kinase, derived from either calf lens or bovine heart, promotes the phosphorylation of three lens plasma membrane proteins of molecular mass 28 kDa, 26 kDa and 18 kDa. Correlation of the maximal level of phosphorylation of these components with the Coomassie blue staining intensity of fractionated lens membranes suggests that the phosphorylation of the 28 kDa and 18 kDa components may be approximately stoichiometric. The protein kinase substrates could be dephosphorylated by a cardiac sarcoplasmic-reticulum-bound protein phosphatase activity. The 26 k Da component comigrated with MP26, the major lens membrane component that has been localized to the lens fiber cell junction. Treatment of phosphorylated lens membranes with chymotrypsin did not suggest that any of the three major phosphorylated components was derived from the partial proteolysis of a larger phosphoprotein. After electrophoretic separation of phosphorylated proteins, treatment with N-chlorosuccinimide confirmed that there was little similarity in the structure of the three phosphoproteins. Chymotrypsin did, however, reveal a cryptic phosphorylation site in a 22 kDa fragment that appeared to be derived from MP26. Treatment of phosphorylated membranes with reducing agents resulted in the disappearance of the 28 kDa phosphorylated component and the appearance of a new phosphorylated component of 18 kDa; neither MP26 nor the original 18 kDa component was affected by such treatment. It is not clear whether the original 18 kDa phosphoprotein, present in unreduced samples, is the same as that generated with reducing agents from the 28 kDa phosphorylated lens membrane component.
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PMID:Characterization of the bovine lens plasma membrane substrates for cAMP-dependent protein kinase. 299 Sep 30

Activities of glycogen synthase (total) and branching enzyme in slow (soleus) muscle are higher than those in fast (vastus lateralis) muscle, while those of phosphorylase kinase (total), phosphorylase (total) and debranching enzyme are reversed. The active form ratio of glycogen synthase is higher in fast muscle, while those of phosphorylase kinase and phosphorylase are higher in slow muscle. Activities of cAMP-dependent protein kinase and protein phosphatase in slow muscle are higher than those in fast muscle. These results suggest that glycogen metabolizing enzymes in slow muscle, distinct from those in fast muscle, are regulated more strongly by cAMP-dependent protein kinase rather than by protein phosphatase.
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PMID:Comparison of enzyme activities on glycogen metabolism in rabbit slow and fast muscles. 299 76

We report potent inhibition of the Mg(II).ATP-dependent protein phosphatase, Fc.M, by the regulatory subunit dimer of type II cAMP-dependent protein kinase, RII2. The protein kinase catalytic subunit has no effect on phosphatase activity and is unable to substitute for kinase FA in the kinase FA- and Mg(II).ATP-mediated phosphatase activation reaction. Phosphatase inhibition was investigated as a function of RII2 concentration. The results suggest that RII2 both inhibits the active phosphatase and inhibits phosphatase activation. The inhibition is shown to be noncompetitive with respect to substrate (phosphorylase a). The potential physiological significance of this inhibition is discussed in terms of phosphorylation/dephosphorylation cascade systems involving this kinase and phosphatase.
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PMID:Inhibition of the Mg(II).ATP-dependent phosphoprotein phosphatase by the regulatory subunit of cAMP-dependent protein kinase. 299 70

The results of the present study permit the explanation of one of the mechanisms of the interconnection between the regulatory systems of cAMP and 2-5A. cAMP-dependent regulation of 2'-PDE was found to involve phosphorylation of the specific protein inhibitor. Originally, a similar way of regulation of the enzyme activity was discovered for protein phosphatase I. This enzyme has a specific protein inhibitor type 1, which is phosphorylated by cAMP-dependent protein kinase and is activated by phosphorylation (18). It is interesting that the molecular weights of 2'-PDE protein inhibitor and of the inhibitor type 1 of protein phosphatase I are essentially the same. There is also a certain similarity between the above described mechanism and phosphorylation of the regulatory subunit of cAMP-dependent protein kinase type 2. The regulatory subunit can also act as a protein inhibitor of the enzyme and change its properties as a result of phosphorylation (19). The results obtained permit as well a more detailed explanation for cAMP-dependent inhibition of cell proliferation. Evidently, cAMP elevation causes activation of cAMP-dependent phosphorylation which, in turn, leads to the induction of 2-5A synthetase and inhibition of 2'-PDE. As a result of variations in the activities of these enzymes, the level of 2-5A rises. The latter brings about the changes characteristic of the resting state. They involve activation of RNase L and the succeeding acceleration of RNA hydrolysis, inhibition of protein synthesis and cell proliferation. The resting state is characterized by a rapid turnover of macromolecules due to their intensive degradation (20). The above described scheme suggested that the rapid turnover of RNA during inhibition of cell proliferation can be partially accounted for by activation of 2-5A-dependent RNase L. Thus, it can be thought that at least one of the mechanisms of the antiproliferative effect of cAMP-dependent phosphorylation of proteins involves cAMP-dependent elevation of intracellular 2-5A. Evidently, a number of properties of the resting cells are determined by the elevated content of 2-5A. Finally, it should be noted that the interconnection between the systems of cAMP and 2-5A is a multiple process. We have earlier demonstrated (12) that 2-5A activates cAMP phosphodiesterase in NIH 3T3 cell homogenates. These data suggest that the mutual regulation of cAMP and 2-5A levels involves the negative feedback mechanism (Fig. 8).
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PMID:Regulation of 2-5 A phosphodiesterase activity by cAMP-dependent phosphorylation: mechanism and biological role. 300 Jan 46

In intact goldfish xanthophores, the phosphorylation of a pigment organelle (carotenoid droplet) protein, p57, appears to play an important role in adrenocorticotropin (ACTH)- or cAMP-induced pigment organelle dispersion while the dephosphorylation of this protein upon withdrawal of ACTH or cAMP is implicated in pigment aggregation. In this paper, we report the cAMP-dependent phosphorylation of this protein in cell-free extracts of xanthophores as determined by the incorporation of 32P from [gamma-32P]ATP. As is the case in intact cells, p57 is the predominant protein phosphorylated in the presence of cAMP. The cAMP-dependent protein kinase which phosphorylates p57 is not bound to the isolated organelles but is found in the soluble portion of the cell extracts. Hence, the phosphorylation of p57 requires the carotenoid droplets bearing the substrate, soluble extract containing the kinase, cAMP (half-maximal activation at 0.5 microM), and Mg2+ (optimal at 5 mM or higher). The presence of protein phosphatase(s) in these extracts was shown indirectly by the stimulation of phosphorylation by fluoride. The phosphorylation of p57 does not appear to require a cell-specific kinase as soluble extracts of goldfish dermal nonpigment cells also phosphorylate p57 associated with isolated carotenoid droplets. Furthermore, using a constant amount of carotenoid droplets, a linear relationship was demonstrated between the rate of p57 phosphorylation and the amount of extract present in the assays. These results suggest that p57 is phosphorylated directly by a cAMP-dependent protein kinase and that the activity of this enzyme is important in regulating the intracellular movement of the pigment organelles of the xanthophore.
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PMID:Regulation of pigment organelle translocation. II. Participation of a cAMP-dependent protein kinase. 300 26

Calmodulin-dependent protein phosphatase purified from bovine cardiac muscle catalyzed the rapid dephosphorylation of Ser-95 of bovine cardiac cAMP-dependent protein kinase regulatory subunit (RII). The kinetic constants determined for the reaction (Km = 20 microM; Vmax = 2 mumol min-1 mg-1) are comparable to those determined for other good substrates of this phosphatase. Because little is known about the determinants of substrate specificity for the calmodulin-dependent phosphatase, various phosphopeptides were used to investigate the structural features important for substrate recognition. Limited proteolysis of phospho-RII with trypsin and chymotrypsin yielded fragments (residues 93-400 and 91-400, respectively) that were poor substrates, whereas digestion with Staphylococcal aureus V8 protease produced three phosphopeptides that were all dephosphorylated as rapidly as intact RII. The sequence of the shortest phosphopeptide produced by S. aureus V8 protease was determined by sequence analysis to be Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Cys-Ala-Glu, corresponding to residues 81-99 of RII. Synthetic phosphopeptides corresponding to residues 81-99, 85-99, 90-99, and 91-99 were prepared to determine the minimum sequence necessary for substrate recognition. Only the 19-residue peptide (81-99) was dephosphorylated with kinetics comparable to RII (Km = 26 microM, Vmax = 1.7 mumol min-1 mg-1). Structural analysis of this peptide indicates that an amphipathic beta-sheet structure may be an important structural determinant for some substrates of the calmodulin-dependent phosphatase.
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PMID:Dephosphorylation of cAMP-dependent protein kinase regulatory subunit (type II) by calmodulin-dependent protein phosphatase. Determinants of substrate specificity. 301 43

We have examined protein phosphatase activities that are present during the cellular differentiation of Dictyostelium. Utilizing differential centrifugation, ion exchange, gel filtration, and concanavalin A affinity chromatography we found a number of distinct protein phosphatase activities. Three peaks of soluble Kemptide phosphatase activity and a very broad and heterogeneous soluble histone phosphatase activity were resolved by anion exchange chromatography. Histone phosphatase was associated with the particulate fraction, while Kemptide phosphatase was not. The protein phosphatase activities were able to dephosphorylate sites that had been phosphorylated by the cyclic AMP-dependent protein kinase. Therefore it is possible that their function in vivo may be to oppose the action of the cAMP-dependent protein kinase. In addition several paranitrophenyl phosphate phosphatase activities are shown to be largely separable from the protein phosphatases. An apparent heat-stable inhibitor of histone phosphatase is shown to be artifactual in that instead of interacting with the enzyme it acts by complexing with histone.
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PMID:Chromatographic resolution of soluble and particulate protein phosphatases from Dictyostelium discoideum. 301 27

Immunoaffinity purified pp60v-src was found to activate the MgATP-dependent protein phosphatase in the presence of MgATP. Although preliminary evidence suggested that phosphorylation of the inhibitor-2 subunit on tyrosine residues was responsible for the activation, preincubation of the pp60v-src preparation at 41 degrees C resulted in a rapid loss of its protein kinase activities towards both casein and inhibitor-2 while its ability to activate the protein phosphatase complex was relatively insensitive to this treatment. This result demonstrated that pp60v-src was not responsible for activation of the MgATP-dependent protein phosphatase. A protein kinase activity which phosphorylated glycogen synthase on serine residues was detected in the pp60v-src preparation. The protein kinase was active in the presence of inhibitors of phosphorylase kinase, glycogen synthase kinase 5/casein kinase II, and cAMP-dependent protein kinase. It is, therefore, likely that activation of the MgATP-dependent protein phosphatase resulted from the presence of a glycogen synthase kinase 3 like activity in the pp60v-src preparation. Our results illustrate the importance of applying multiple criteria to link the phosphorylation of a protein with an observed change in its activity.
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PMID:Apparent activation of the MgATP-dependent protein phosphatase by pp60v-src. Identification of an activity like that of glycogen synthase kinase 3 in immunoaffinity purified pp60v-src preparations. 301 36

High-affinity antibodies against calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase and protein phosphatase (calcineurin) were purified and characterized. Rabbit anti-phosphodiesterase antibody did not react with other phosphodiesterases or with the regulatory subunits of cAMP-dependent protein kinase. Affinity-purified goat anti-calcineurin antibody recognized both the 61-kDa catalytic subunit and the 18-kDa Ca2+-binding subunit of the phosphatase. Neither antibody reacted with CaM, several CaM-binding proteins (calmodulin-dependent protein kinase, myosin light chain kinase, fodrin), or other cytosolic proteins from brain. The antibodies were used to compare the cellular localization of these two CaM-dependent enzymes in rat brain. Both calcineurin and phosphodiesterase were found predominantly in nerve cells; however, phosphodiesterase was restricted to very specific neuronal populations. Phosphodiesterase was prominent in the somatic cytoplasm and dendrites of regional output neurons--e.g., cerebellar Purkinje cells and hippocampal and cortical pyramidal cells. The extensive and uniform staining in the dendrites was consistent with postsynaptic localization and suggested an important function for this enzyme in neurons that integrate multiple convergent inputs. Calcineurin was present in virtually all classes of neurons, with immunoreactivity confined primarily to cell bodies. Both diffuse cytoplasmic staining and characteristic punctate staining of cell bodies were observed; the latter suggested compartmentalization of calcineurin at or near the plasma membrane. The results of this study demonstrate that calcineurin and phosphodiesterase are differentially localized in the central nervous system. Thus, the expression and compartmentalization of CaM-binding proteins may be highly regulated and specific for particular differentiated nerve cell types.
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PMID:Differential localization of calmodulin-dependent enzymes in rat brain: evidence for selective expression of cyclic nucleotide phosphodiesterase in specific neurons. 302 62

C-protein purified from chicken cardiac myofibrils was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase to nearly 3 mol [32P]phosphate/mol C protein. Digestion of 32P-labeled C-protein with trypsin revealed that the radioactivity was nearly equally distributed in three tryptic peptides which were separated by reversed-phase HPLC. Fragmentation of 32P-labeled C-protein with CNBr showed that the isotope was incorporated at different ratios in three CNBr fragments which were separated on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphorylation was present in both serine and threonine residues. Incubation of 32P-labeled C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of the [32P]phosphate. The major site(s) dephosphorylated by either one of the phosphatases was a phosphothreonine residue(s) apparently located on the same tryptic peptide and on the same CNBr fragment. CNBr fragmentation also revealed a minor phosphorylation site which was dephosphorylated by either of the phosphatases. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A at high concentrations could completely dephosphorylate C-protein. These results demonstrate that C-protein phosphorylated with cAMP-dependent protein kinase can be dephosphorylated by protein phosphatases 1 and 2A. It is suggested that the enzyme responsible for dephosphorylation of C-protein in vivo is phosphatase 2A.
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PMID:Dephosphorylation of cardiac myofibril C-protein by protein phosphatase 1 and protein phosphatase 2A. 303 83


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