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)

Polyclonal (RP) and monoclonal (Ab) antibodies were raised against synthetic peptides (or fusion proteins) corresponding to amino acid sequences unique to human and mouse retinoic acid receptor-beta (RAR beta) isoforms. Antibodies directed against the A2 region [Ab6 beta 2(A2), Ab7 beta 2(A2), and RP beta 2(A2)], the D2 region [RP beta(D2)], or the F region [Ab8 beta(F)2, RP beta(F)1, and RP beta(F)2] were selected. The monoclonal and polyclonal antibodies directed against the D2 and F regions specifically immunoprecipitated and recognized by Western blotting all human and mouse RAR beta isoforms (mRAR beta 1, -beta 2, -beta 3, and -beta 4), produced in COS-1 cells transfected with expression vectors containing the corresponding RAR beta cDNA. Furthermore, in gel retardation assays, the monoclonal antibodies supershifted RAR beta protein-RA response element oligonucleotide complexes. Antibodies directed against the A2 region were specific for the RAR beta 2 isoform. The above antibodies allowed us to detect the presence of mRAR beta 2 proteins in mouse embryos and to show that their presence in embryonal carcinoma cells (F9 and P19 cell lines) is dependent upon RA treatment. The antibodies were also used to demonstrate that RAR beta proteins produced by transfection in COS-1 cells are phosphorylated. RAR beta 2 phosphorylation was not affected by RA treatment, whereas the phosphorylation of RAR beta 1 and RAR beta 3 isoforms was greatly enhanced by RA. We also show that, in contrast to RAR alpha 1 and RAR gamma 1, RAR beta 2 proteins contain phosphotyrosine residues and are only weakly phosphorylated in vitro by cAMP-dependent protein kinase. These results support our previous proposal that the various receptors have distinct functions in the RA-signaling pathway.
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PMID:Retinoic acid receptor-beta: immunodetection and phosphorylation on tyrosine residues. 128 41

G-proteins couple hormonal activation of receptors to the regulation of specific enzymes and ion channels. Gs and Gi are G-proteins which regulate the stimulation and inhibition, respectively, of adenylyl cyclase. We have constructed two chimeric cDNAs in which different lengths of the alpha subunit of Gs (alpha s) have been replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). One chimera, referred to as alpha i(54)/s' replaces the NH2-terminal 61 amino acids of alpha s with the first 54 residues of alpha i. Within this sequence there are 7 residues unique to alpha s, and 16 of the remaining 54 amino acids are nonhomologous between alpha i and alpha s. The second chimera, referred to as alpha i/s(Bam), replaces the first 234 amino acids of alpha s with the corresponding 212 residues of alpha i. Transient expression of alpha i(54)/s in COS-1 cells resulted in an 18- to 20-fold increase in cyclic AMP (cAMP) levels, whereas expression of either alpha i/s(Bam) or the wild-type alpha s polypeptide resulted in only a 5- to 6-fold increase in cellular cAMP levels. COS-1 cells transfected with alpha i showed a small decrease in cAMP levels. Stable expression of the chimeric alpha i(54)/s polypeptide in Chinese hamster ovary (CHO) cells constitutively increased both cAMP synthesis and cAMP-dependent protein kinase activity. CHO clones expressing transfected alpha i/s(Bam) or the wild-type alpha s and alpha i cDNAs exhibited cAMP levels and cAMP-dependent protein kinase activities similar to those in control CHO cells. Therefore, the alpha i(54)/s chimera behaves as a constitutively active alpha s polypeptide, whereas the alpha i/s(Bam) polypeptide is regulated similarly to wild-type alpha s. Expression in cyc-S49 cells, which lack expression of wild-type alpha s, confirmed that the alpha i(54)/s polypeptide is a highly active alpha s molecule whose robust activity is independent of any change in intrinsic GTPase activity. The difference in phenotypes observed upon expression of alpha i(54)/s or alpha i/s(Bam) indicates that the NH2-terminal moieties of alpha s and alpha i function as attenuators of the effector enzyme activator domain which is within the COOH-terminal half of the alpha subunit. Mutation at the NH2 terminus of alpha s relieves the attenuator control of the Gs protein and results in a dominant active G-protein mutant.
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PMID:Mutation of the Gs protein alpha subunit NH2 terminus relieves an attenuator function, resulting in constitutive adenylyl cyclase stimulation. 169 62

Highly degenerate oligonucleotide primers designed from regions conserved between protein-serine kinases have been used specifically to amplify human epithelial (HeLa) cDNA by the polymerase chain reaction (PCR). Of several novel cDNA fragments encoding putative kinases thus isolated, one was further characterised. Screening of human fibroblast and bovine brain cDNA libraries with the PCR fragment yielded several clones with an open reading frame of 479 amino acids containing all of the conserved sequence motifs of protein-serine kinases. The predicted protein was most similar to the protein kinase C (PKC)/cAMP-dependent protein kinase (PKA) families and its gene has thus been termed pkb. Expression of the pkb gene is general but highest in brain, heart and lung. Translation of pkb RNA in vitro generated a 57-kDa protein (PKB) recognised by antisera raised to a bacterially expressed PKB/TrpE fusion protein. Transfection of COS cells with the kinase cDNA resulted in the synthesis of a 60-kDa protein which was partially purified by Mono Q anion-exchange chromatography. Column fractions containing PKB-immunoreactive protein exhibited elevated histone H1 kinase activity compared with similar fractions from control cells, demonstrating the enzymatic activity of this protein kinase.
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PMID:Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families. 153 86

Human cDNA clones for a heat-stable protein kinase inhibitor (PKI) protein of the cAMP-dependent protein kinase (PKA) were isolated using a mouse PKI cDNA fragment. Two human cDNA clones of 1.7 and 2.0 kb were sequenced and shown to encode the entire open reading frame of 228 nucleotides. Together these clones comprised 2147 nucleotides of the mRNA. The deduced amino acid sequence of the human clones showed 100% identity to the rabbit skeletal muscle PKI protein and 97% identity to the mouse brain PKI. The mouse and human PKI cDNAs shared nucleotide homology in their 3' untranslated regions as well as in the 32 nucleotides immediately 5' of the translation initiation site. Northern blot analysis of human skeletal muscle RNA with a human cRNA probe detected a major mRNA of approximately 4.0 kb. Transient overexpression in COS cells verified that a heat-stable inhibitor of protein kinase was produced by he human PKI cDNA, and protein extracts from the transfected COS cells inhibited both the C alpha and C beta isoforms of the PKA catalytic subunit with equal efficacy. Functional expression of the human PKI protein was further studied by assaying the ability of PKI expression vectors to inhibit PKA catalytic subunit stimulation of transcription from the human enkephalin promoter. In these studies, elimination of a conserved alternative translation start site in the 5' untranslated region of PKI was shown to potentiate the inhibitory activity of the PKI expression vector.
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PMID:Inhibition of protein kinase-A by overexpression of the cloned human protein kinase inhibitor. 177 Sep 51

Phosphorylation of purified Na+,K(+)-ATPase by cAMP-dependent protein kinase (protein kinase A) decreases the activity of this enzyme. We have now shown, using several experimental approaches, that a highly conserved seryl residue on the catalytic (alpha) subunit of Na+,K(+)-ATPase, corresponding to Ser943 of the rat alpha 1 isoform, is the phosphorylation site for protein kinase A. cDNAs corresponding to wild-type Na+,K(+)-ATPase and Na+,K(+)-ATPase in which Ser943 was mutated to Ala were transfected into COS cells. Treatment of the transfected cells with forskolin plus 3-isobutyl-1-methylxanthine resulted in a decrease in the activity of the wild-type enzyme but not in that of the mutated enzyme. The results suggest that, in intact cells, the activity of the Na+,K(+)-ATPase is regulated in part by signal transduction pathways that use protein kinase A-dependent phosphorylation of the Na+,K(+)-ATPase alpha subunit.
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PMID:Identification of the phosphorylation site for cAMP-dependent protein kinase on Na+,K(+)-ATPase and effects of site-directed mutagenesis. 751 Jul 9

Treatment of acute promyelocytic leukemia (APL) blasts with cyclic adenosine monophosphate (cAMP) analogs, in combination with all-trans retinoic acid (ATRA), results in the upregulation of the expression of leukocyte alkaline phosphatase (LAP), a marker for the differentiation of the granulocyte. The synergistic interaction between the cyclic nucleotide analogs and the retinoid is not unique to APL cells, as it is observed also in the peripheral granulocytes of chronic myelogenous leukemia (CML) patients. The molecular mechanisms underlying LAP induction were studied in NB4, an immortalized APL cell line. Induction of LAP enzymatic activity is dependent on the time of exposure and on the concentrations of dibutyryl-cAMP or 8-bromo-cAMP and ATRA, two factors that influence the kinetics of appearance of detectable levels of the enzyme. Augmentation of LAP levels by ATRA and cAMP is the result of both transcriptional and early posttranscriptional events and requires de novo protein synthesis. LAP induction correlates with augmentation in the levels of the type I catalytic subunit of cAMP-dependent protein kinase transcript and with granulocytic differentiation. The transcriptional component of the process leading to increased LAP gene expression was reproduced in its main features by transient transfection experiments performed in COS-7 cells using the normal retinoic acid receptor type alpha (RAR-alpha) or the APL-specific aberrant form (PML-RAR) and the upstream promoter of the liver/bone/kidney (L/B/K)-type alkaline phosphatase gene. The promoter is upregulated by treatment with ATRA, and this upregulation is further increased by cAMP analogs.
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PMID:All-trans retinoic acid and cyclic adenosine monophosphate cooperate in the expression of leukocyte alkaline phosphatase in acute promyelocytic leukemia cells. 778 Jan 46

Although the protein kinase inhibitors (PKIs) are known to be potent and specific inhibitors of the catalytic (C) subunit of cAMP-dependent protein kinase, little is known about their physiological roles. Glutamate 203 of the C alpha isoform (C alpha E203) has been implicated in the binding of the arginine 15 residue of the skeletal isoform of PKI (PKI alpha R15) (Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N., Taylor, S.S., and Sowadski, J. M. (1991) Science 253, 414-420). To investigate the role of C alpha E203 in the binding of PKI and in vivo C-PKI interactions, in vitro mutagenesis was used to change the C alpha E203 codon of the murine C alpha cDNA to alanine and glutamine codons. Initially, the C alpha E203 mutant proteins were expressed and purified from Escherichia coli. C alpha E203 is not essential for catalysis as all of the C subunit mutants were enzymatically active. The mutation of Glu203 did increase the apparent Km for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) severalfold but did not affect the apparent Km for ATP. The Vmax(app) was not affected by the mutation of C alpha E203. The mutation of C alpha E203 compromised the ability of PKI alpha (5-24), PKI alpha, and PKI beta to inhibit phosphotransferase activity. PKI alpha was altered using in vitro mutagenesis to probe the role of Arg15 in interacting with C alpha E203. The PKI alpha R15A mutant was reduced in its inhibition of C alpha. Preliminary studies of the expression of these C alpha mutants in COS cells gave similar results. These results suggest that the C alpha E203 mutants may be useful in assessing the role of PKI in vivo.
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PMID:Glutamic acid 203 of the cAMP-dependent protein kinase catalytic subunit participates in the inhibition by two isoforms of the protein kinase inhibitor. 790 1

We report that the human 1,25-dihydroxyvitamin D3 receptor is an efficient substrate for cAMP-dependent protein kinase, in vitro. This phosphorylation reaction is rapid and neither dependent upon nor significantly affected by the presence of the 1,25-dihydroxyvitamin D3 ligand. Preliminary mapping experiments utilizing C-terminal truncation mutants reveal that the primary site(s) of phosphorylation, in vitro, is localized between amino acids 133 and 201. Cotransfection of the catalytic subunit of murine cAMP-dependent protein kinase and the human 1,25-dihydroxyvitamin D3 receptor into monkey kidney (COS-7) cells not only results in a dramatic kinase-dependent increase in receptor phosphorylation but also elicits an attenuation in 1,25-dihydroxyvitamin D3-dependent transcriptional activation of a reporter gene. These observations suggest a potential role for cAMP-dependent protein kinase in the modulation of 1,25-dihydroxyvitamin D3 receptor-mediated gene regulation.
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PMID:Phosphorylation of the human 1,25-dihydroxyvitamin D3 receptor by cAMP-dependent protein kinase, in vitro, and in transfected COS-7 cells. 838 50

Two isoforms of the catalytic subunit of cAMP-dependent protein kinase, Calpha and Cbeta1, are known to be widely expressed in mammals. Although much is known about the structure and function of Calpha, few studies have addressed the possibility of a distinct role for the Cbeta proteins. The present study is a detailed comparison of the biochemical properties of these two isoforms, which were initially expressed in Escherichia coli and purified to homogeneity. Cbeta1 demonstrated higher Km values for some peptide substrates than did Calpha, but Cbeta1 was insensitive to substrate inhibition, a phenomenon that was observed with Calpha at substrate concentrations above 100 microM. Calpha and Cbeta1 displayed distinct IC50 values for the alpha and beta isoforms of the protein kinase inhibitor, protein kinase inhibitorpeptide, and the type IIalpha regulatory subunit (RIIalpha). Of particular interest, purified type II holoenzyme containing Cbeta1 exhibited a 5-fold lower Ka value for cAMP (13 nM) than did type II holoenzyme containing Calpha (63 nM). This latter result was extended to in vivo conditions by employing a transcriptional activation assay. In these experiments, luciferase reporter activity in COS-1 cells expressing RIIalpha2Cbeta12 holoenzyme was half-maximal at 12-fold lower concentrations of 8-(4-chlorophenylthio)-cAMP and 5-fold lower concentrations of forskolin than in COS-1 cells expressing RIIalpha2Calpha2 holoenzyme. These results provide evidence that type II holoenzyme formed with Cbeta1 is preferentially activated by cAMP in vivo and suggest that activation of the holoenzyme is determined in part by interactions between the regulatory and catalytic subunits that have not been described previously.
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PMID:The major catalytic subunit isoforms of cAMP-dependent protein kinase have distinct biochemical properties in vitro and in vivo. 866 89

Activation of the TAL1 (or SCL) gene, initially identified through its involvement by a recurrent chromosomal translocation, is the most frequent gain-of-function mutation recognized in T-cell acute lymphoblastic leukemia. The translational products of this gene contain the basic domain helix-loop-helix motif characteristic of a family of transcription factors that bind to a consensus nucleotide sequence termed the E-box. Previous work established that the TAL1 proteins are phosphorylated exclusively on serine and identified Ser122 as a substrate for the mitogen-activated protein kinase ERK-1. We provide evidence that an additional serine residue, Ser172, located in a conserved region proximal to the DNA binding domain and sharing homology with a similarly positioned sequence in the HLH oncoprotein LYL1, can be phosphorylated in vitro and in vivo by the catalytic subunit of cAMP-dependent protein kinase. Phosphorylation was found to alter TAL1 DNA binding activity in a target-dependent manner that was influenced by both the specific CANNTG E-box core motif and its flanking sequences. In contrast, the ability of TAL1 to interact with the E2A gene product E12 and its subcellular localization in transfected COS cells were unaffected by Ser172 phosphorylation. These results suggest this serine residue has a regulatory function and indicate a mechanism by which phosphorylation could affect DNA binding site discrimination.
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PMID:Target-dependent effect of phosphorylation on the DNA binding activity of the TAL1/SCL oncoprotein. 911 Oct 58


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