Transcription factor AP-2 activity is modulated by protein kinase A-mediated phosphorylation.
(9/7238)
We recently reported that APOE promoter activity is stimulated by cAMP, this effect being mediated by factor AP-2 [Garcia et al. (1996) J. Neurosci. 16, 7550-7556]. Here, we study whether cAMP-induced phosphorylation modulates the activity of AP-2. Recombinant AP-2 was phosphorylated in vitro by protein kinase A (PKA) at Ser239. Mutation of Ser239 to Ala abolished in vitro phosphorylation of AP-2 by PKA, but not the DNA binding activity of AP-2. Cotransfection studies showed that PKA stimulated the effect of AP-2 on the APOE promoter, but not that of the S239A mutant. Therefore, cAMP may modulate AP-2 activity by PKA-induced phosphorylation of this factor. (+info)
Regulation of cardiac L-type Ca2+ channel by coexpression of G(alpha s) in Xenopus oocytes.
(10/7238)
Activation of G(alpha s) via beta-adrenergic receptors enhances the activity of cardiac voltage-dependent Ca2+ channels of the L-type, mainly via protein kinase A (PKA)-dependent phosphorylation. Contribution of a PKA-independent effect of G(alpha s) has been proposed but remains controversial. We demonstrate that, in Xenopus oocytes, antisense knockdown of endogenous G(alpha s) reduced, whereas coexpression of G(alpha s) enhanced, currents via expressed cardiac L-type channels, independently of the presence of the auxiliary subunits alpha2/delta or beta2A. Coexpression of G(alpha s) did not increase the amount of alpha1C protein in whole oocytes or in the plasma membrane (measured immunochemically). Activation of coexpressed beta2 adrenergic receptors did not cause a detectable enhancement of channel activity; rather, a small cAMP-dependent decrease was observed. We conclude that coexpression of G(alpha s), but not its acute activation via beta-adrenergic receptors, enhances the activity of the cardiac L-type Ca2+ channel via a PKA-independent effect on the alpha1C subunit. (+info)
cAMP inhibits translation by inducing Ca2+/calmodulin-independent elongation factor 2 kinase activity in IPC-81 cells.
(11/7238)
Treatment of IPC-81 cells led to inhibition of protein synthesis, which was accompanied by an increase in the average size of polysomes and a decreased rate of elongation, indicating that it involved inhibition of peptide chain elongation. This inhibition was also associated with increased phosphorylation of elongation factor eEF2 (which inhibits its activity) and enhanced Ca2+/calmodulin-independent activity of eEF2 kinase. Previous work has shown that phosphorylation of eEF2 kinase by cAMP-dependent protein kinase (cAPK) in vitro induces such activator-independent activity, and the present data show that such a mechanism can occur in intact cells to link physiological levels of cAPK activation with inhibition of protein synthesis. (+info)
L-arginine stimulation of glucose-induced insulin secretion through membrane depolarization and independent of nitric oxide.
(12/7238)
The mechanism of L-arginine stimulation of glucose-induced insulin secretion from mouse pancreatic islets was studied. At 16.7 mmol/l glucose, L-arginine (10 mmol/l) potentiated both phases 1 and 2 of glucose-induced insulin secretion. This potentiation of glucose-induced insulin secretion was mimicked by the membrane depolarizing agents tetraethylammonium (TEA, 20 mmol/l) and K+ (60 mmol/l), which at 16.7 mmol/l glucose obliterated L-arginine (10 mmol/l) modulation of insulin secretion. Thus L-arginine may potentiate glucose-induced insulin secretion by stimulation of membrane depolarization. At 3.3 mmol/l glucose, L-arginine (10 mmol/l) failed to stimulate insulin secretion. In accordance with membrane depolarization by the electrogenic transport of L-arginine, however, L-arginine (10 mmol/l) stimulation of insulin secretion was enabled by the K+ channel inhibitor TEA (20 mmol/l), which potentiates membrane depolarization by L-arginine. Furthermore, L-arginine (10 mmol/l) stimulation of insulin secretion was permitted by forskolin (10 micromol/l) or tetradecanoylphorbol 13-acetate (0.16 micromol/l), which, by activation of protein kinases A and C respectively sensitize the exocytotic machinery to L-arginine-induced Ca2+ influx. Thus glucose may sensitize L-arginine stimulation of insulin secretion by potentiation of membrane depolarization and by activation of protein kinase A or protein kinase C. Finally, L-arginine stimulation of glucose-induced insulin secretion was mimicked by NG-nitro-L-arginine methyl ester (10 mmol/l), which stimulates membrane depolarization but inhibits nitric oxide synthase, suggesting that L-arginine-derived nitric oxide neither inhibits nor stimulates insulin secretion. In conclusion, it is suggested that L-arginine potentiation of glucose-induced insulin secretion occurs independently of nitric oxide, but is mediated by membrane depolarization, which stimulates insulin secretion through protein kinase A- and C-sensitive mechanisms. (+info)
beta3-adrenoceptor control the cystic fibrosis transmembrane conductance regulator through a cAMP/protein kinase A-independent pathway.
(13/7238)
In human cardiac myocytes, we have previously identified a functional beta3-adrenoceptor in which stimulation reduces action potential duration. Surprisingly, in cardiac biopsies obtained from cystic fibrosis patients, beta3-adrenoceptor agonists produced no effects on action potential duration. This result suggests the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) chloride current in the electrophysiological effects of beta3-adrenoceptor stimulation in non-cystic fibrosis tissues. We therefore investigated the control of CFTR activity by human beta3-adrenoceptors in a recombinant system: A549 human cells were intranuclearly injected with plasmids encoding CFTR and beta3-adrenoceptors. CFTR activity was functionally assayed using the 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescent probe and the patch-clamp technique. Injection of CFTR-cDNA alone led to the expression of a functional CFTR protein activated by cAMP or cGMP. Co-expression of CFTR (but not of mutated DeltaF508-CFTR) with high levels of beta3-adrenoceptor produced an increased halide permeability under base-line conditions that was not further sensitive to cAMP or beta3-adrenoceptor stimulation. Patch-clamp experiments confirmed that CFTR channels were permanently activated in cells co-expressing CFTR and a high level of beta3-adrenoceptor. Permanent CFTR activation was not associated with elevated intracellular cAMP or cGMP levels. When the expression level of beta3-adrenoceptor was lowered, CFTR was not activated under base-line conditions but became sensitive to beta3-adrenoceptor stimulation (isoproterenol plus nadolol, SR 58611, or CGP 12177). This later effect was not prevented by protein kinase A inhibitors. Our results provide molecular evidence that CFTR but not mutated DeltaF508-CFTR is regulated by beta3-adrenoceptors expression through a protein kinase A-independent pathway. (+info)
Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.
(14/7238)
Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment. (+info)
The type and the localization of cAMP-dependent protein kinase regulate transmission of cAMP signals to the nucleus in cortical and cerebellar granule cells.
(15/7238)
cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases, typically determined by their specific regulatory subunits. In the brain the major regulatory isoform RIIbeta and the RII-anchor protein, AKAP150 (rat) or 75 (bovine), are differentially expressed. Cortical neurons express RIIbeta and AKAP75; conversely, granule cerebellar cells express predominantly RIalpha and RIIalpha. Cortical neurons accumulate PKA catalytic subunit and phosphorylated cAMP responsive element binding protein very efficiently into nuclei upon cAMP induction, whereas granule cerebellar cells fail to do so. Down-regulation of RIIbeta synthesis by antisense oligonucleotides inhibited cAMP-induced nuclear signaling in cortical neurons. Expression in cerebellar granule cells of RIIbeta and AKAP75 genes by microinjection of specific expression vectors, markedly stimulated cAMP-induced transcription of the lacZ gene driven by a cAMP-responsive element promoter. These data indicate that the composition of PKA in cortical and granule cells underlies the differential ability of these cells to transmit cAMP signals to the nucleus. (+info)
cAMP-dependent protein kinase and phosphoproteins in mammalian mitochondria. An extension of the cAMP-mediated intracellular signal transduction.
(16/7238)
Evidence has been obtained for the occurrence of a cAMP-dependent serine protein kinase associated with the inner membrane/matrix of mammalian mitochondria. The catalytic site of this kinase is localized at the inner side of the inner membrane, where it phosphorylates a number of mitochondrial proteins. One of these has been identified as the AQDQ subunit of complex I. cAMP-dependent phosphorylation of this protein promotes the activity of complex I and mitochondrial respiration. A 5 bp duplication in the nuclear gene encoding this protein has been found in a human patient, which eliminates the phosphorylation site. PKA anchoring proteins have recently been identified in the outer membrane of mammalian mitochondria, which could direct phosphorylation of proteins at contact sites with other cell structures. (+info)