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)

A putative protein kinase gene (PfPK2) has been isolated from the human parasite Plasmodium falciparum by using a mixed oligonucleotide pool which corresponds to a highly conserved region of serine/threonine protein kinases. The complete nucleotide sequence of 5 kb suggests the existence of a second transcriptional unit besides that of the PfPK2 gene, separated by a highly (A+T)-rich region and transcribed in a different orientation. No intron sequence exists in PfPK2. The predicted amino acid sequence of PfPK2 contains features characteristic of eukaryotic serine/threonine protein kinases. Within its putative catalytic domain it shares 33%, 30%, and 28% amino acid identities with rat calcium-calmodulin-dependent protein kinase, human protein kinase C, and bovine cAMP-dependent protein kinase, respectively. Outside the catalytic domain, however, PfPK2 has no homology with regulatory domains of other protein kinases, indicating PfPK2 might be modulated by signals different from those of higher eukaryotes or might be associated with other regulatory subunits. Using a specific antiserum raised in rabbits against a recombinant fragment of the protein expressed in Escherichia coli, PfPK2 was found to be expressed in a stage-specific fashion and mainly localized in the parasitic membrane.
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PMID:Molecular cloning, stage-specific expression and cellular distribution of a putative protein kinase from Plasmodium falciparum. 137 3

We have investigated the role of protracted phosphatase inhibition and the consecutive protracted protein phosphorylation on neuronal viability. We found that in primary cultures of cerebellar granule neurons, the protracted (24-h) inhibition of the serine/threonine protein phosphatases 1 and 2A (EC 3.1.3.16) by treatment of the cultures with okadaic acid (OKA; 5-20 nM) caused neurotoxicity that could be inhibited by the protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) or by the previous down-regulation of the neuronal protein kinase C (PKC; ATP:protein phosphotransferase; EC 2.7.1.37). PKC was down-regulated by exposure of the cultures for 24 h to 100 nM phorbol 12-myristate 13-acetate (TPA). The effect of the drugs used in the viability studies on the pattern of protein phosphorylation was measured by quantitative autoradiography. In particular, the 50- and 80-kDa protein bands showed dramatic changes in the degree of phosphorylation: increase by OKA and brief TPA treatment; decrease by H7 or 24 h of TPA treatment; and inhibition of the OKA-induced increase by H7 or 24 h of TPA treatment. The results suggest that the protracted phosphorylation, in particular that mediated by PKC, may lead to neuronal death and are in line with our previous suggestion that prolonged PKC translocation is operative in glutamate neurotoxicity.
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PMID:Pathological phosphorylation causes neuronal death: effect of okadaic acid in primary culture of cerebellar granule cells. 140 5

The catalytic subunit of cAMP-dependent protein kinase contains two stable phosphorylation sites, Thr-197 and Ser-338 (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). Thr-197 is very resistant to dephosphorylation and thus cannot typically be autophosphorylated in vitro once the stable subunit is formed. Ser-338 is slowly dephosphorylated and can be rephosphorylated autocatalytically. In addition to these two stable phosphorylation sites, a new site of autophosphorylation, Ser-10, was identified. Phosphorylation at Ser-10 does not have a major effect on activity, and phosphates from Ser-10 or Ser-338 are not transferred to physiological substrates such as the type II regulatory subunit. Autophosphorylation at Ser-10 is associated with one of the two major isoelectric variants of the catalytic subunit. The form having the more acidic pI can be autophosphorylated at Ser-10 while the more basic form of the catalytic subunit cannot. Phosphorylation at Ser-10 does not account for the two isoenzyme forms. Since the reason for two isoelectric variants of the catalytic subunit is still unknown, it is not possible to provide a structural basis for the difference in accessibility of Ser-10 to phosphorylation. Either Ser-10 is not accessible in the more basic form of the catalytic subunit or some other type of post- or cotranslational modification causes Ser-10 to be a poor substrate. Whether the myristoyl group at the amino-terminal Gly is important for Ser-10 autophosphorylation remains to be established. The isoenzyme forms of the catalytic subunit do not correspond to the gene products coded for by the C alpha and C beta genes.
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PMID:Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase. 146 17

Using homologous probes for the cloning of related genes within the family of guanine nucleotide-binding protein-coupled receptors, we have cloned the gene for the rhesus macaque D1 dopamine receptor. By using the rat D1 receptor coding sequence as a probe under high stringency conditions, the rhesus D1 receptor gene was isolated from a lambda EMBL3 rhesus genomic DNA library. The rhesus D1 dopamine receptor gene is intronless and encodes a 446-amino acid protein that contains two consensus sites for asparagine-linked glycosylation (Asn-5 and Asn-176) and two consensus sites for cAMP-dependent protein kinase phosphorylation (Thr-136 and Thr-268). The primary amino acid sequence of the rhesus D1 dopamine receptor shows an extremely high degree of similarity (99.6%) to the human D1 receptor. Genomic DNA analyses conducted with high and reduced stringency hybridizations indicate that the rhesus macaque D1 receptor is a member of a large multigene family. Like the human D1 receptor mRNA, the rhesus D1 receptor mRNA is approximately 4 kilobases in size and is localized predominantly in the caudate, with lesser amounts in the hippocampus and cortex. The rhesus D1 receptor coding region was inserted into the cytomegalovirus promoter-driven expression vector pcDNA-1, and the recombinant (pcDNA-D1) was cotransfected with the selectable marker pRSVneo, conferring G418 resistance, into D1 receptor-deficient C6 glioma cells. Analyses of the selected transfectant demonstrate the expression of a high affinity, functional D1 dopamine receptor. The D1 receptor radioligand [3H]SCH 23390 bound transfectant membranes with an affinity (Kd), of 0.3 nM; the D2-selective ligand spiperone, the dopamine receptor ligand clozapine, and the serotonin receptor antagonist ketanserin bound with considerably lower affinities (102, 80, and 95 nM, respectively). Both dopamine and the D1-selective agonist SKF 38393 inhibited the binding of [3H]SCH 23390 to transfectant cell membranes; the binding of these agonists was sensitive to GTP. Dopamine potently stimulated the accumulation of cAMP in transfected C6 cells, whereas SKF 38393 was a partial agonist in these cells. Also, the density of recombinant D1 receptors on the transfectant cells was decreased 40% upon treatment with 10 microM dopamine, indicating that occupation of recombinant D1 receptors by agonists alters surface expression of the receptors.
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PMID:Molecular cloning and expression of the rhesus macaque D1 dopamine receptor gene. 153 68

The intracellular events which are involved in controlling the G1 to S phase transition during the eucaryotic cell cycle are important to define in order to understand the mechanisms by which mitogenic and growth arrest-inducing agents control cell growth. Because a change in protein kinase activity is associated with the initial response of cells to mitogenic stimulants and growth factors, we used a kinase renaturation assay to identify specific protein kinases which are modulated as human T cells make the G1 to S phase transition after mitogenic stimulation with lectin. We identified four protein serine/threonine kinases of 180, 97, 85, and 38 kilodaltons which are increased in activity as these cells enter S phase. A-55 kDa serine/threonine kinase (PK55) was shown to have maximal activity during G0 and its activity was reduced by 95% upon movement into S phase. PK55 is inducible in human T cells by removal of interleukin 2 and low serum incubation which arrests cells in G1 phase, indicating that it is closely associated with G1 phase growth arrest. Furthermore, a similar PK55 activity was induced upon growth arrest in HL-60 cells treated with dimethyl sulfoxide and in Daudi cells treated with interferon alpha. Because the cAMP-dependent protein kinase (PK-A) family has been shown to be antiproliferative to lectin stimulated T cells, we were interested in determining whether PK55 was in fact an isozyme of PK-A. Comparative analysis using a specific peptide inhibitor of PK-A activity revealed that PK55 is catalytically distinct from PK-A. This data suggest that increases in PK55 may be associated with the growth-arrested state and further that PK55 is distinct from PK-A.
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PMID:Specific protein kinases modulated during T cell mitogenesis. Activity of a 55-kDa serine kinase is associated with growth arrest in human T cells. 153 85

Mutations were identified in the catalytic subunit (C) of the cAMP-dependent protein kinase (EC 2.7.1.37) that block inactivation by regulatory subunit (R) without compromising catalytic activity. Randomly mutagenized mouse C expression vectors were screened functionally for clones that stimulated gene induction in the presence of excess R. Point mutations in the C coding sequence were identified that result in a His----Gln substitution at amino acid 87 (His87Gln) and a Trp----Arg change at amino acid 196 (Trp196Arg). In contrast to wild-type C, both mutants retained partial activity in the presence of excess R isoform RI alpha, although only Trp196Arg retained partial activity in the presence of excess R isoform RII alpha. A C expression vector that included both mutations was fully active in promoting gene induction and was virtually unaffected by an 80-fold excess of either RI alpha or RII alpha. These results demonstrate that mutations at His-87 and Trp-196 alter R interactions with C at a site that is not involved in substrate recognition or enzymatic activity. In contrast to these randomly generated mutations, a site-specific alteration of the autophosphorylated Thr-197 to an Ala resulted in an 80% loss of biological activity and partial resistance to R inhibition. The location and proximity of His-87 and Trp-196 in the crystal structure of C suggest a surface domain that may interact with a region of R that is outside of the substrate/pseudosubstrate site.
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PMID:Mutations in the catalytic subunit of cAMP-dependent protein kinase result in unregulated biological activity. 158 9

Essential to signal transduction are mechanisms of "cross-talk" to coordinate different pathways. This study shows that stimulation of serine/threonine protein kinases activates protein-tyrosine phosphatase (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). More than 95% of intracellular PTPase was in the particulate fraction of various cell lines and was extracted with detergent as a 150-kDa complex that contained a 55-kDa catalytic subunit. The complex was activated by protease digestion, which changed its substrate specificity coincident with reduction in size. The complex was dissociated by treatment of the membrane fraction with 3 M LiBr. Treatment of intact cells with isoproterenol, forskolin, or cAMP analogues to stimulate cAMP-dependent protein kinase (PKA) or with phorbol ester or dioctanoylglycerol to stimulate Ca2+/phospholipid-dependent protein kinase (PKC) produced activation of membrane PTPase complex without a change in its size. Inhibition of protein-serine/threonine phosphatases with okadaic acid or fluoride also resulted in activation of the membrane PTPase. These results support a model for regulation of PTPase by phosphorylation and dephosphorylation of serine/threonine residues in a regulatory component complexed with the 55-kDa PTPase catalytic subunit. This mechanism may be important in regulating sensitivity to extracellular signals transduced via tyrosine phosphorylation and in the synchronization of events during the cell cycle.
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PMID:Activation of membrane protein-tyrosine phosphatase involving cAMP- and Ca2+/phospholipid-dependent protein kinases. 165 Apr 78

The regulation of the plasma membrane Ca2+ pump by hormones via phosphorylation in intact cells has not been clearly established. We now present evidence that the Ca2+ pump is phosphorylated on both serine and threonine residues in unstimulated and stimulated cultured rat aortic endothelial cells. Among the stimuli tested, the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was most potent and increased the level of phosphorylation threefold, while the cAMP-dependent protein kinase activator 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) stimulated the phosphorylation 1.6-fold. Two-dimensional tryptic phosphopeptide maps of the Ca2+ pump from unstimulated and CPT-cAMP-stimulated cells have identical patterns (five phosphopeptides) while PMA-stimulated cells have three additional phosphopeptides. Isoproterenol-, ATP-, angiotensin II-, and bradykinin-stimulated cells also have increased levels of Ca2+ pump phosphorylation. Stimuli-induced phosphorylation of the Ca2+ pump was rapid (5-10 min) and was concomitant with stimulated calcium efflux from the same cells. This is the first direct evidence that the plasma membrane Ca2+ pump in intact cells is regulated by various hormones or agonists via cAMP-dependent protein kinase or protein kinase C phosphorylation.
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PMID:Hormone-induced phosphorylation of the plasma membrane calcium pump in cultured aortic endothelial cells. 165 40

Previous studies identified proline-directed protein kinase (PDPK) as a growth factor-sensitive serine/threonine protein kinase that is active in the cytosol of proliferative cells and tissues during interphase. In this communication, we report that the regulatory subunit (RII) of bovine cardiac muscle cAMP-dependent protein kinase (PKA) is a putative substrate for the multifunctional PDPK. Purified RII is readily phosphorylated by PDPK in vitro in a time-dependent, enzyme-dependent manner to a stoichiometry approaching 0.7 mol phosphate/mol RII subunit protein. The major RII phosphorylation site is identified as a threonine residue located within a large hydrophobic tryptic peptide that is predicted to contain the cAMP binding domains. In contrast to the reported effects of RII autophosphorylation, kinetic analysis of RII function following phosphorylation by PDPK indicates that the inhibitory potency of RII toward the catalytic subunit of PKA in a reassociation assay is increased in proportion to the degree of phosphorylation. Further studies indicate that the cAMP-dependent activation of the RII2C2 holoenzyme is inhibited by PDPK phosphorylation. Taken together, the results of these studies indicate that phosphorylation of RII by PDPK attenuates the activity of PKA. This antagonistic interaction suggests a biochemical mechanism by which a growth factor-activated signaling system may function to modulate cAMP-dependent cellular responses.
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PMID:Phosphorylation of RII subunit and attenuation of cAMP-dependent protein kinase activity by proline-directed protein kinase. 165 46

Cyclic AMP regulates a variety of cellular responses through activation of the catalytic subunit of cAMP-dependent protein kinase. The cDNAs for two protein isoforms of the catalytic subunit, C alpha and C beta, were placed into expression vectors, and their ability to stimulate cAMP-dependent transcription of the human enkephalin promoter was examined in transiently transfected CV-1 cells. Expression vectors for C alpha and C beta that were directed by the human cytomegalovirus promoter produced up to 350- and 200-fold increases in chloramphenicol acetyltransferase activity, respectively, when cotransfected with the ENKAT-12 reporter plasmid. Transcriptional activation was shown to be dependent upon functional kinase activity by point mutations in catalytic subunit vectors which eliminated activation. Transcriptional activation by C alpha and C beta was eliminated when the cAMP response elements (CREs) were deleted from the native enkephalin promoter, but activation was recovered when this region was replaced with an oligonucleotide containing two copies of the somatostatin CRE consensus TGACGTCA. C alpha expression vectors were found to produce 2-fold greater transcriptional activation than C beta expression vectors. These results were most likely due to the cellular kinase activity produced by the catalytic subunit expression vectors and did not appear to be dependent on CRE motif or substrate specificity. In vitro mutagenesis indicates that neither C alpha nor C beta requires N-terminal myristylation for transcriptional activation, but threonine-197 is critical to subunit function.
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PMID:Regulation of the human enkephalin promoter by two isoforms of the catalytic subunit of cyclic adenosine 3',5'-monophosphate-dependent protein kinase. 165 33


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