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)

Purified fructose-1,6-bisphosphatase from Saccharomyces cerevisiae was phosphorylated in vitro by purified yeast cAMP-dependent protein kinase. Maximal phosphorylation was accompanied by an inactivation of the enzyme by about 60%. In vitro phosphorylation caused changes in the kinetic properties of fructose-1,6-bisphosphatase: 1) the ratio R(Mg2+/Mn2+) of the enzyme activities measured at 10 mM Mg2+ and 2 mM Mn2+, respectively, decreased from 2.6 to 1.2; 2) the ratio R(pH 7/9) of the activities measured at pH 7.0 and pH 9.0, respectively, decreased from 0.62 to 0.38, indicating a shift of the pH optimum to the alkaline range. However, the affinity of the enzyme for its inhibitors fructose-2,6-bisphosphate (Fru-2,6-P2) and AMP, expressed as the concentration required for 50% inhibition, was not changed. The maximum amount of phosphate incorporated into fructose-1,6-bisphosphatase was 0.6-0.75 mol/mol of the 40-kDa subunit. Serine was identified as the phosphate-labeled amino acid. The initial rate of in vitro phosphorylation of fructose-1,6-bisphosphatase, obtained with a maximally cAMP-activated protein kinase, increased when Fru-2,6-P2 and AMP, both potent inhibitors of the enzyme, were added. As Fru-2,6-P2 and AMP did not affect the phosphorylation of histone by cAMP-dependent protein kinase, the inhibitors must bind to fructose-1,6-bisphosphatase in such a way that the enzyme becomes a better substrate for phosphorylation. Nevertheless, Fru-2,6-P2 and AMP did not increase the maximum amount of phosphate incorporated into fructose-1,6-bisphosphatase beyond that observed in the presence of cAMP alone.
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PMID:Phosphorylation and inactivation of yeast fructose-1,6-bisphosphatase by cyclic AMP-dependent protein kinase from yeast. 299 82

Membrane-permeant cAMP derivatives (dibutyryl- and 8-bromo-cAMP) increase gap-junctional conductance within minutes when applied to voltage-clamped pairs of rat hepatocytes. Glucagon also increases junctional conductances, but the response has a more rapid onset and is more rapidly reversible. The glucagon effect can be prevented by intracellular injection of the protein inhibitor of the cAMP-dependent protein kinase (Walsh inhibitor), indicating that the catalytic subunit of cAMP-dependent protein kinase is directly involved. The 27-kDa major gap junction polypeptide is phosphorylated when liver cells dissociated into small groups are incubated with 32P. Addition of 8-bromo-cAMP to cells increases the incorporation of 32P into the 27-kDa junctional protein. Serine is the amino acid residue that is phosphorylated. When isolated liver gap junctions are incubated in the presence of catalytic subunit of the cAMP-dependent protein kinase, the 27-kDa gap junction polypeptide is phosphorylated with low stoichiometry on serine. The rapid increases in gap junctional conductance caused by agents that elevate cAMP and phosphorylation of the gap junction protein by cAMP-dependent protein kinase suggest that cAMP-dependent phosphorylation of the gap junction channel modulates the conductance of liver gap junctions.
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PMID:cAMP increases junctional conductance and stimulates phosphorylation of the 27-kDa principal gap junction polypeptide. 301 Mar 11

A polycation-dependent protein kinase was found to be associated with purified phytochrome preparations from etiolated Avena seedlings. This kinase and three mammalian protein kinases, the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and a Ca2+-activated phospholipid-dependent protein kinase, were used to probe light-induced conformational changes in 124-kilodalton Avena phytochrome in vitro. The red absorbing form of phytochrome (Pr) was found to be a substrate for all four protein kinases. Although the far-red absorbing form of phytochrome (Pfr) was as good a substrate as Pr with the cAMP-dependent protein kinase, the Pfr form was poorly phosphorylated by the other three protein kinases. Serine is the major amino acid residue phosphorylated on phytochrome regardless of the form of phytochrome used as substrate. Peptide mapping revealed that the sites of phosphorylation catalyzed by the cAMP-dependent protein kinase differ for Pr and Pfr forms of phytochrome. For the Pr form, the preferred site(s) of phosphorylation was near the amino terminus of the 124-kilodalton subunit. Upon photo-conversion to Pfr, this site can no longer be phosphorylated easily and a new phosphorylation site in the COOH-terminal nonchromophore domain of the molecule becomes accessible to the cAMP-dependent protein kinase. These studies of the phosphorylation of phytochrome provide a new means to study the effect of light absorption by phytochrome on the molecular conformation of the protein. The potential physiological implications of differential phosphorylation of Pr and Pfr await elucidation.
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PMID:Phosphorylation of Avena phytochrome in vitro as a probe of light-induced conformational changes. 374 79

Two intrinsic membrane proteins of calf lens fiber cells can be phosphorylated by a soluble bovine lens cAMP-dependent protein kinase and rabbit muscle cAMP-dependent protein kinase. After electrophoresis of the phosphorylated membranes, 32P comigrates with the lens main intrinsic protein at 26-27 kDa and with a minor band of protein that migrates at 19-20 kDa. 32P is also found with proteins that, based on the molecular sizes, are likely multimers of the 19-kDa and 26-kDa proteins. Upon boiling in NaDodSO4, all the radioactivity is found at the top of the gel, suggesting that both phosphoproteins are intrinsic membrane proteins. Serine is the only phospho amino acid detected in both proteins regardless of the source of protein kinase. The phosphorylation sites of both proteins are lost upon cleavage with trypsin and chymotrypsin. The smaller phosphoprotein is likely not a crystallin, because antibodies directed against alpha-, beta-, or gamma-crystallins do not cross-react with the 19-kDa protein. The 19-kDa 32P-labeled protein does not migrate coincident with calf alpha-crystallin.
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PMID:Phosphorylation of lens fiber cell membrane proteins. 388 45

The principal (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive (L-type) calcium channels is present in full-length (212 kDa) and COOH-terminal truncated (190 kDa) forms, which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. Immunoprecipitation of the calcium channel from rabbit muscle myotubes in primary cell culture followed by phosphorylation with cA-PK, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed comparable phosphorylation of three COOH-terminal phosphopeptides found in the purified full-length alpha 1 subunit. Stimulation of muscle myotubes with a permeant cAMP analogue, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate, prior to immunoprecipitation of alpha 1 results in a 60-80% reduction of cA-PK catalyzed "back" phosphorylation of each of these sites in vitro in calcium channels purified from the cells, indicating that these sites are phosphorylated in vivo in response to increased intracellular cAMP. Serine 687, the most rapidly phosphorylated site in the truncated 190-kDa alpha 1 subunit, was observed as a minor phosphopeptide whose level of phosphorylation was not significantly affected by stimulation of endogenous cA-PK in the myotubes. The COOH-terminal sites, designated tryptic phosphopeptides 4, 5, and 6, were identified as serine 1757 (phosphopeptides 4 and 6) and 1854 (phosphopeptide 5) by a combination of protease cleavage, phosphorylation of synthetic peptides and fusion proteins, specific immunoprecipitation, and phosphopeptide mapping. Phosphorylation of serines 1757 and 1854 in the COOH-terminal region of the 212-kDa alpha 1 subunit in intact skeletal muscle cells may play a pivotal role in the regulation of calcium channel function by cA-PK.
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PMID:Sites of selective cAMP-dependent phosphorylation of the L-type calcium channel alpha 1 subunit from intact rabbit skeletal muscle myotubes. 760 7

In the yeast Saccharomyces cerevisiae, genetic studies suggest that the RIM1 gene encodes a positive regulator of meiosis. rim1 mutations cause reduced expression of IME1, which is required for expression of many meiotic genes, and thus lead to a partial defect in meiosis and spore formation. We report the sequence of RIM1 and functional analysis of its coding region. The RIM1 gene product (RIM1) contains three regions similar to C2H2 zinc fingers. Serine substitutions for cysteine in each of the putative zinc fingers abolish RIM1 function. The carboxyl-terminus of RIM1 is enriched in acidic amino acids and is required for full RIM1 activity. RIM1 also contains two putative cAMP-dependent protein kinase (cAPK) phosphorylation sites. At one site, substitution of alanine for serine does not affect RIM1 activity; at the other site, this substitution impairs activity. This analysis of RIM1 suggests that the protein may function as a transcriptional activator. We have used the cloned RIM1 gene to create a complete rim1 deletion. This null allele, like previously isolated rim1 mutations, causes a partial meiotic defect. In addition to RIM1, maximum IME1 expression requires the MCK1 and IME4 gene products. Defects associated with rim1, mck1, and ime4 mutations in expression of a meiotic reporter gene (ime2-lacZ) and in sporulation are additive. These findings suggest that RIM1 acts independently of MCK1 and IME4 to stimulate IME1 expression.
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PMID:Molecular characterization of the yeast meiotic regulatory gene RIM1. 836 97

Cyclic nucleotide phosphodiesterase (PDE) is an important regulator of the cellular concentrations of the second messengers cyclic AMP (cAMP) and cGMP. Insulin activates the 3B isoform of PDE in adipocytes in a phosphoinositide 3-kinase-dependent manner; however, downstream effectors that mediate signaling to PDE3B remain unknown. Insulin-induced phosphorylation and activation of endogenous or recombinant PDE3B in 3T3-L1 adipocytes have now been shown to be inhibited by a dominant-negative mutant of the serine-threonine kinase Akt, suggesting that Akt is necessary for insulin-induced phosphorylation and activation of PDE3B. Serine-273 of mouse PDE3B is located within a motif (RXRXXS) that is preferentially phosphorylated by Akt. A mutant PDE3B in which serine-273 was replaced by alanine was not phosphorylated either in response to insulin in intact cells or by purified Akt in vitro. In contrast, PDE3B mutants in which alanine was substituted for either serine-296 or serine-421, each of which lies within a sequence (RRXS) preferentially phosphorylated by cAMP-dependent protein kinase, were phosphorylated by Akt in vitro or in response to insulin in intact cells. Moreover, the serine-273 mutant of PDE3B was not activated by insulin when expressed in adipocytes. These results suggest that PDE3B is a physiological substrate of Akt and that Akt-mediated phosphorylation of PDE3B on serine-273 is important for insulin-induced activation of PDE3B.
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PMID:Insulin-induced phosphorylation and activation of cyclic nucleotide phosphodiesterase 3B by the serine-threonine kinase Akt. 1045 75

During activation of adrenocortical cells by adrenocorticotrophic hormone (ACTH), tyrosine dephosphorylation of paxillin is stimulated and this correlates with protrusion of filopodial structures and a decreased number of focal adhesions. These effects are inhibited by Na(3)VO(4), a phosphotyrosine phosphatase inhibitor [Vilgrain, Chinn, Gaillard, Chambaz and Feige (1998) Biochem. J. 332, 533-540]. However, the tyrosine phosphatases involved in these processes remain to be identified. In this study, we provide evidence that the Src homology domain (SH)2-containing phosphotyrosine phosphatase (SHP)2, but not SHP1, is expressed in adrenocortical cells and is phosphorylated upon ACTH challenge. ACTH (10(-8) M) treatment of (32)P-labelled adrenocortical cells resulted in an increase in phosphorylated SHP2. By probing SHP2-containing immunoprecipitates with an antibody to phosphoserine we found that SHP2 was phosphorylated on serine in ACTH-treated cells in a dose- and time-dependent manner. Furthermore, using an in vitro kinase assay, we showed that SHP2 was a target for cAMP-dependent protein kinase (PKA). Serine was identified as the only target amino acid phosphorylated in SHP2. Phosphorylation of SHP2 by PKA resulted in a dramatic stimulation of phosphatase activity measured either with insulin receptor substrate-1 or with the synthetic peptide [(32)P]poly(Glu/Tyr) as substrate. In an in-gel assay of SHP2-containing immunoprecipitates, phosphorylated in vitro by PKA or isolated from adrenocortical cells treated with 10 nM ACTH, a pronounced activation of SHP2 activity was shown. These observations clearly support the idea that a PKA-mediated signal transduction pathway contributes to SHP2 regulation in adrenocortical cells and point to SHP2 as a possible mediator of the effects of ACTH.
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PMID:Adrenocorticotrophic hormone stimulates phosphotyrosine phosphatase SHP2 in bovine adrenocortical cells: phosphorylation and activation by cAMP-dependent protein kinase. 1108 42

Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo, and could be phosphorylated in vitro on serine residues with a significant increase of enzyme activity, but no phosphorylation sites were identified. In this work the identification of the specific phosphorylation sites of rat GNMT is reported. Three different preparations of rat GNMT were analyzed: (1) purified from liver by standard methods of protein purification, (2) prepared from isolated hepatocytes and from liver tissue by immunoprecipitation, and (3) recombinant protein expressed in Escherichia coli. We measured the molecular weights of protein isoforms using electrospray mass spectrometry and used liquid chromatography-tandem mass spectrometry (LC-MS/MS) of peptides resulting from tryptic and chymotryptic digests. We also performed chemical analysis of phosphoamino acids and protein sequencing. In all samples, the phosphorylated serine residues 71, 182, and 241 were found. In GNMT prepared from liver tissue and hepatocytes an S9 additional residue was found to be phosphorylated. In hepatocytes and in recombinant GNMT S139 was detected. Serine 9 was also identified as a target for cAMP-dependent protein kinase in vitro. The positions of these phosphorylated residues in the tertiary structure of GNMT indicate their possible effect on enzyme conformation and activity.
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PMID:Identification of phosphorylation sites in glycine N-methyltransferase from rat liver. 1652 97

Adipocyte fatty acid-binding protein (AFABP/aP2) forms a physical complex with the hormone-sensitive lipase (HSL) and AFABP/aP2-null mice exhibit reduced basal and hormone-stimulated lipolysis. To identify the determinants affecting the interaction fluorescence resonance energy transfer (FRET) imaging was used in conjunction with a mutagenesis strategy to evaluate the roles AFABP/aP2 fatty acid binding and HSL phosphorylation have in complex formation as well as determine the HSL binding site on AFABP/aP2. The nonfatty acid binding mutant of AFABP/aP2 (R126Q) failed to form a FRET-competent complex with HSL either under basal or forskolin-stimulated conditions, indicating that lipid binding is required for association. Once bound to HSL and on the surface of the lipid droplet, YFP-AFABP/aP2 (but not YFP-HSL) exhibited energy transfer between the fusion protein and BODIPY-C12-labeled triacylglycerol. Serine to alanine mutations at the two PKA phosphorylation sites of HSL (659 and 660), or at the AMPK phosphorylation sites (565), blocked FRET between HSL and AFABP/aP2. Substitution of isoleucine for lysine at position 21 of AFABP/aP2 (K21I), but not 31 (K31I), resulted in a non-HSL-binding protein indicating that residues on helix alphaI of AFABP/aP2 define a component of the HSL binding site. These results indicate that the ligand-bound form of AFABP/aP2.interacts with the activated, phosphorylated HSL and that the association is likely to be regulatory; either delivering FA to inhibit HSL (facilitating feedback inhibition) or affecting multicomponent complex formation on the droplet surface.
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PMID:Interaction of the adipocyte fatty acid-binding protein with the hormone-sensitive lipase: regulation by fatty acids and phosphorylation. 1778 68


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