Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has been suggested that the signal transduction pathway initiated by apoA-I activates key proteins involved in cellular lipid efflux. We investigated apoA-I-mediated cAMP signaling in cultured human fibroblasts induced with (22R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Treatment of stimulated fibroblasts with apoA-I for short periods of time (<or=45 min) increased ATP binding cassette A1 (ABCA1) phosphorylation in a concentration-dependent manner. Concomitantly, apoA-I increased the intracellular level of cAMP in a concentration- and time-dependent manner. The maximal cAMP level was reached within 10 min at 10 microg/ml apoA-I representing a 1-fold increase. The ability of apoA-I to mediate cAMP production was only observed in stimulated fibroblasts. Furthermore, overexpression of ABCA1 in Chinese hamster ovary cells resulted in a 1.5-fold increase in apoA-I-mediated cAMP accumulation as compared with untransfected cells. In contrast, forskolin increased cAMP production significantly in unstimulated fibroblasts as well as in untransfected Chinese hamster ovary cells. Pharmacological inhibition of protein kinase A (H89) completely blocked apoA-I-mediated ABCA1 phosphorylation. Naturally occurring mutations of ABCA1 associated with Tangier disease (C1477R, 2203X, and 2145X) severely reduced apoA-I-mediated cAMP production, ABCA1 phosphorylation, (125)I-apoA-I binding, and lipid efflux, without affecting forskolin-mediated cAMP elevation. In contrast, the protein kinase A catalytic subunit was able to phosphorylate ABCA1 similarly from mutant and normal cell lines in vitro. Together, our results indicate that apoA-I activates ABCA1 phosphorylation through the cAMP/protein kinase A-dependent pathway, apoA-I-mediated cAMP production required high level expression of functional ABCA1, and Tangier disease mutants have defective apoA-I-mediated cAMP signaling. These findings suggest that apoA-I may activate cAMP signaling through G protein-coupled ABCA1 transporter.
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PMID:Apolipoprotein A-I activates cellular cAMP signaling through the ABCA1 transporter. 1470 24

The fungal pathogen Candida albicans switches from a yeast-like to a filamentous mode of growth in response to a variety of environmental conditions. We examined the morphogenetic behavior of C. albicans yeast cells lacking the BCY1 gene, which encodes the regulatory subunit of protein kinase A. We cloned the BCY1 gene and generated a bcy1 tpk2 double mutant strain because a homozygous bcy1 mutant in a wild-type genetic background could not be obtained. In the bcy1 tpk2 mutant, protein kinase A activity (due to the presence of the TPK1 gene) was cyclic AMP independent, indicating that the cells harbored an unregulated phosphotransferase activity. This mutant has constitutive protein kinase A activity and displayed a defective germinative phenotype in N-acetylglucosamine and in serum-containing medium. The subcellular localization of a Tpk1-green fluorescent protein (GFP) fusion protein was examined in wild-type, tpk2 null, and bcy1 tpk2 double mutant strains. The fusion protein was observed to be predominantly nuclear in wild-type and tpk2 strains. This was not the case in the bcy1 tpk2 double mutant, where it appeared dispersed throughout the cell. Coimmunoprecipitation of Bcy1p with the Tpk1-GFP fusion protein demonstrated the interaction of these proteins inside the cell. These results suggest that one of the roles of Bcy1p is to tether the protein kinase A catalytic subunit to the nucleus.
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PMID:Candida albicans lacking the gene encoding the regulatory subunit of protein kinase A displays a defect in hyphal formation and an altered localization of the catalytic subunit. 1487 49

The NF-kappaB p50/p50 homodimer is mainly associated with transcriptional repression. Previously, we demonstrated that phosphorylation of NF-kappaB p50 Ser(337) is critical for DNA binding. Here, we report that p50 Ser(337) is constitutively phosphorylated by the protein kinase A catalytic subunit (PKAc) in three different cell types, which may account for the constant binding of p50/p50 to DNA in unstimulated cells. This was demonstrated first by showing that treatment of cells with PKAc-specific inhibitors blocked p50/p50 DNA binding. Second, phosphorylation of p50 by PKAc was prevented by substitution of Ser(337) to alanine. Third, both p50 and PKAc proteins as well as kinase activity that phosphorylates p50 were found to co-fractionate following gel filtration chromatography. Finally, PKAc and p50 were shown to be able to reciprocally co-immunoprecipitate one another, and their physical association was blocked by a PKA catalytic site inhibitory peptide. This indicates that phosphorylation of p50 Ser(337) involves direct contact with the PKAc catalytic center. In contrast to the dramatic elevation of nuclear p50/p65 heterodimers induced by tumor necrosis factor alpha, DNA binding of p50/p50 homodimers was not greatly altered. Taken together, these findings reveal for the first time that there is a direct interaction between PKAc and p50 that accounts for constitutive phosphorylation of p50 Ser(337) and the existence of DNA bound p50/p50 in the nuclei of most resting cells. This mechanism of DNA binding by p50/p50 following phosphorylation of Ser(337) by PKAc may represent an important means for maintaining stable negative regulation of NF-kappaB gene expression in the absence of extracellular stimulation.
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PMID:DNA binding of repressor nuclear factor-kappaB p50/p50 depends on phosphorylation of Ser337 by the protein kinase A catalytic subunit. 1564 94

Filamentous fungal genomes contain two distantly related cyclic AMP-dependent protein kinase A catalytic subunits (PKAs), but only one PKA is found to play a principal role. In Aspergillus nidulans, PkaA is the primary PKA that positively functions in vegetative growth and spore germination but negatively controls asexual sporulation and production of the mycotoxin sterigmatocystin. In this report, we present the identification and characterization of pkaB, encoding the secondary PKA in A. nidulans. Although deletion of pkaB alone does not cause any apparent phenotypic changes, the absence of both pkaB and pkaA is lethal, indicating that PkaB and PkaA are essential for viability of A. nidulans. Overexpression of pkaB enhances hyphal proliferation and rescues the growth defects caused by DeltapkaA, indicating that PkaB plays a role in vegetative growth signaling. However, unlike DeltapkaA, deletion of pkaB does not suppress the fluffy-autolytic phenotype resulting from DeltaflbA. While upregulation of pkaB rescues the defects of spore germination resulting from DeltapkaA in the presence of glucose, overexpression of pkaB delays spore germination. Furthermore, upregulation of pkaB completely abolishes spore germination on medium lacking a carbon source. In addition, upregulation of pkaB enhances the level of submerged sporulation caused by DeltapkaA and reduces hyphal tolerance to oxidative stress. In conclusion, PkaB is the secondary PKA that has a synthetic lethal interaction with PkaA, and it plays an overlapping role in vegetative growth and spore germination in the presence of glucose but an opposite role in regulating asexual sporulation, germination in the absence of a carbon source, and oxidative stress responses in A. nidulans.
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PMID:The pkaB gene encoding the secondary protein kinase A catalytic subunit has a synthetic lethal interaction with pkaA and plays overlapping and opposite roles in Aspergillus nidulans. 1608 51

The enzyme 5-lipoxygenase initiates the synthesis of leukotrienes from arachidonic acid. Protein kinase A phosphorylates 5-lipoxygenase on Ser(523), and this reduces its activity. We report here that phosphorylation of Ser(523) also shifts the subcellular distribution of 5-lipoxygenase from the nucleus to the cytoplasm. Phosphorylation and redistribution of 5-lipoxygenase could be produced by overexpression of the protein kinase A catalytic subunit alpha, by pharmacological activators of protein kinase A, and by prostaglandin E(2). Mimicking phosphorylation by replacing Ser(523) with glutamic acid caused cytoplasmic localization; replacement of Ser(523) with alanine prevented phosphorylation and redistribution in response to protein kinase A activation. Because Ser(523) is positioned within the nuclear localization sequence-518 of 5-lipoxygenase, the ability of protein kinase A to phosphorylate and alter the localization of green fluorescent protein fused to the nuclear localization sequence-518 peptide was also tested. Site-directed replacement of Ser(523) with glutamic acid within the peptide impaired nuclear accumulation; overexpression of the protein kinase A catalytic subunit alpha and pharmacological activation of protein kinase caused phosphorylation of the fusion protein at Ser(523), and the phosphorylated protein was found chiefly in the cytoplasm. Taken together, these results indicate that phosphorylation of Ser(523) inhibits the nuclear import function of a nuclear localization sequence, resulting in the accumulation of 5-lipoxygenase enzyme in the cytoplasm. As cytoplasmic localization can be associated with reduced leukotriene synthetic capacity, phosphorylation of Ser(523) serves to inhibit leukotriene production by both impairing catalytic activity and by placing the enzyme in a site that is unfavorable for action.
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PMID:Phosphorylation by protein kinase a inhibits nuclear import of 5-lipoxygenase. 1623 Mar 55

Previous studies in transgenic mice and with isolated ryanodine receptors (RyR) have indicated that Ca2+-calmodulin-dependent protein kinase II (CaMKII) can phosphorylate RyR and activate local diastolic sarcoplasmic reticulum (SR) Ca2+ release events (Ca2+ sparks) and RyR channel opening. Here we use relatively controlled physiological conditions in saponin-permeabilized wild type (WT) and phospholamban knockout (PLB-KO) mouse ventricular myocytes to test whether exogenous preactivated CaMKII or endogenous CaMKII can enhance resting Ca2+ sparks. PLB-KO mice were used to preclude ancillary effects of CaMKII mediated by phospholamban phosphorylation. In both WT and PLB-KO myocytes, Ca2+ spark frequency was increased by both preactivated exogenous CaMKII and endogenous CaMKII. This effect was abolished by CaMKII inhibitor peptides. In contrast, protein kinase A catalytic subunit also enhanced Ca2+ spark frequency in WT, but had no effect in PLB-KO. Both endogenous and exogenous CaMKII increased SR Ca2+ content in WT (presumably via PLB phosphorylation), but not in PLB-KO. Exogenous calmodulin decreased Ca2+ spark frequency in both WT and PLB-KO (K0.5 approximately 100 nmol/L). Endogenous CaMKII (at 500 nmol/L [Ca2+]) phosphorylated RyR as completely in <4 minutes as the maximum achieved by preactivated exogenous CaMKII. After CaMKII activation Ca2+ sparks were longer in duration, and more frequent propagating SR Ca2+ release events were observed. We conclude that CaMKII-dependent phosphorylation of RyR by endogenous associated CaMKII (but not PKA-dependent phosphorylation) increases resting SR Ca2+ release or leak. Moreover, this may explain the enhanced SR diastolic Ca2+ leak and certain triggered arrhythmias seen in heart failure.
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PMID:Ca2+/Calmodulin-dependent protein kinase II phosphorylation of ryanodine receptor does affect calcium sparks in mouse ventricular myocytes. 1691 97

We have previously shown that protein kinase A of the medically important zygomycete Mucor rouxii participates in fungal morphology through cytoskeletal organization. As a first step towards finding the link between protein kinase A and cytoskeletal organization we here demonstrate the cloning of the Rho1 gene and the characterization of its protein product. The RHO1 protein primary sequence shows 70-85% identity with fungal RHO1 or mammalian RhoA. Two protein kinase A phosphorylation sequences in adequate context are predicted, Ser73 and Ser135. The peptide IRRNSQKFV, containing Ser135 proved to be a good substrate for M. rouxii protein kinase A catalytic subunit. The over-expressed Rho1 fully complements a Saccharomyces cerevisiae null mutant. The endogenous protein was identified by western blot against a developed antibody and by ADP-ribosylation. Localization in germlings was visualized by immunofluorescence; the protein was localized in patches in the mother cell surface and excluded from the germ tube. Measurement of Rho1 expression during germination indicates that Rho1, at both the mRNA and protein levels, correlates with differentiation and not with growth. Rho1 has been shown to be the regulatory protein of the beta-1,3-glucan synthase complex in fungi in which beta-1,3-glucans are major components of the cell wall. Even though glucans have not been detected in zygomycetes, caspofungin, an echinochandin known to be an inhibitor of beta-1,3-glucan synthase complex, is shown here to have a negative effect on growth and to produce an alteration on morphology when added to M. rouxii growth culture medium. This result has an important impact on the possible participation of beta-1,3-glucans on the regulation of morphology of zygomycetes.
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PMID:Mucor rouxii Rho1 protein; characterization and possible role in polarized growth. 1708 Feb 89

The molecular mechanisms for the suppression of corticotropin-releasing hormone (CRH) gene expression by glucocorticoid remain to be clarified albeit the well-known physiological role of the glucocorticoid-induced negative feedback regulation of the gene. In this study, we examined the effect of glucocorticoid on CRH gene transcription using the human BE(2)C neuronal cell line, which expresses the CRH gene and produces CRH peptide intrinsically. Dexamethasone, a specific ligand for the glucocorticoid receptor (GR), potently suppressed human CRH 5'-promoter activity. The effect was GR-dependent, and was completely antagonized by antiglucocorticoid RU38486. Treatment with neither sodium butyrate nor trichostatin A abolished the suppression, thus making the possible involvement of histone deacetylase (HDACs) unlikely. The suppression was not influenced by the deletion or mutation of the proposed negative glucocorticoid-response element (nGRE) but was completely eliminated by that of cAMP-response element. Finally, overexpression of protein kinase A catalytic subunit antagonized the glucocorticoid suppression, whereas overexpression of GR enhanced it. Taken together, our data suggest that: (1) glucocorticoid exerts its negative effect on CRH gene transcription in a GR-dependent manner, but the GR-mediated inhibition appears to be independent of the nGRE; (2) HDACs do not play a significant role in the glucocorticoid repression; (3) some of the inhibitory events may take place through transrepression of protein kinase A by GR.
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PMID:Molecular mechanisms for corticotropin-releasing hormone gene repression by glucocorticoid in BE(2)C neuronal cell line. 1716 83

Rodent Oatp2 is a hepatic uptake transporter for such compounds as cardiac glycosides. In the present study, we found that fasting resulted in a 2-fold induction of Oatp2 expression in liver of mice. Because the cAMP-protein kinase A (PKA) signaling pathway is activated during fasting, the role of this pathway in Oatp2 induction during fasting was examined. In Hepa-1c1c7 cells, adenylyl cyclase activator forskolin as well as two cellular membrane-permeable cAMP analogs, dibutyryl cAMP and 8-bromo-cAMP, induced Oatp2 mRNA expression in a time- and dose-dependent manner. These three chemicals induced reporter gene activity in cells transfected with a luciferase reporter gene construct containing a 7.6-kilobase (kb) 5'-flanking region of mouse Oatp2. Transient transfection of cells with 5'-deletion constructs derived from the 7.6-kb Oatp2 promoter reporter gene construct, as well as 7.6-kb constructs in which a consensus cAMP response element (CRE) half-site CGTCA (-1808/-1804 bp) was mutated or deleted, confirms that this CRE site was required for the induction of luciferase activity by forskolin. Luciferase activity driven by the Oatp2 promoter containing this CRE site was induced in cells cotransfected with a plasmid encoding the protein kinase A catalytic subunit. Cotransfection of cells with a plasmid encoding the dominant-negative CRE binding protein (CREB) completely abolished the inducibility of the reporter gene activity by forskolin. In conclusion, induction of Oatp2 expression in liver of fasted mice may be caused by activation of the cAMP-dependent signaling pathway, with the CRE site (-1808/-1804) and CREB being the cis- and trans-acting factors mediating the induction, respectively.
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PMID:Activation of cAMP-dependent signaling pathway induces mouse organic anion transporting polypeptide 2 expression. 1724 98

Cardiac ventricular myocytes extrude a sizeable amount of their total Mg(2+) content upon stimulation by beta-adrenergic agonists. This extrusion occurs within a few minutes from the application of the agonist, suggesting the operation of rapid and abundantly represented Mg(2+) transport mechanisms in the cardiac sarcolemma. The present study was aimed at characterizing the operation of these transport mechanisms under well defined conditions. Male Sprague-Dawley rats were used to purify a biochemical standardized preparation of sealed rat cardiac sarcolemmal vesicles. This experimental model has the advantage that trans-sarcolemmal cation transport can be studied under specific extra- and intra-vesicular ionic conditions, in the absence of intracellular organelles, and buffering or signaling components. Magnesium ion (Mg(2+)) transport was assessed by atomic absorbance spectrophotometry. The results reported here indicate that: (1) sarcolemma vesicles retained trapped intravesicular Mg(2+) in the absence of extravesicular counter-ions; (2) the addition of Na(+) or Ca(2+) induced a rapid and concentration-dependent Mg(2+) extrusion from the vesicles; (3) co-addition of maximal concentrations of Na(+) and Ca(2+) resulted in an additive Mg(2+) extrusion; (4) Mg(2+ )extrusion was blocked by addition of amiloride or imipramine; (5) pre-treatment of sarcolemma vesicles with alkaline phosphatase at the time of preparation completely abolished Na(+)- but not Ca(2+)-induced Mg(2+) extrusion; (6) Na(+)-dependent Mg(2+) transport could be restored by stimulating vesicles loaded with protein kinase A catalytic subunit and ATP with membrane-permeant cyclic-AMP analog; (7) extra-vesicular Mg(2+) could be accumulated in exchange for intravesicular Na(+) via a mechanism inhibited by amiloride or alkaline phosphatase treatment; (8) Mg(2+) accumulation could be restored via cAMP/protein kinase A protocol. Overall, these data provide compelling evidence for the operation of distinct Na(+)- and Ca(2+)-dependent Mg(2+) extrusion mechanisms in sarcolemma vesicles. The Na(+)-dependent mechanism appears to be specifically activated via protein kinase A/cAMP-dependent phosphorylation process, and can operate in either direction based upon the cation concentration gradient across the sarcolemma. The Ca(2+)-dependent mechanism, instead, only mediates Mg(2+) extrusion in a cAMP-independent manner.
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PMID:Functional characterization of two distinct Mg(2+) extrusion mechanisms in cardiac sarcolemmal vesicles. 1741 22


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