Nitric oxide regulation of gene transcription via soluble guanylate cyclase and type I cGMP-dependent protein kinase.
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Nitric oxide (NO) regulates the expression of multiple genes but in most cases its precise mechanism of action is unclear. We used baby hamster kidney (BHK) cells, which have very low soluble guanylate cyclase and cGMP-dependent protein kinase (G-kinase) activity, and CS-54 arterial smooth muscle cells, which express these two enzymes, to study NO regulation of the human fos promoter. The NO-releasing agent Deta-NONOate (ethanamine-2,2'-(hydroxynitrosohydrazone)bis-) had no effect on a chloramphenicol acetyltransferase (CAT) reporter gene under control of the fos promoter in BHK cells transfected with an empty vector or in cells transfected with a G-kinase Ibeta expression vector. In BHK cells transfected with expression vectors for guanylate cyclase, Deta-NONOate markedly increased the intracellular cGMP concentration and caused a small (2-fold) increase in CAT activity; the increased CAT activity appeared to be from cGMP activation of cAMP-dependent protein kinase. In BHK cells co-transfected with guanylate cyclase and G-kinase expression vectors, CAT activity was increased 5-fold in the absence of Deta-NONOate and 7-fold in the presence of Deta-NONOate. Stimulation of CAT activity in the absence of Deta-NONOate appeared to be largely from endogenous NO since we found that: (i) BHK cells produced high amounts of NO; (ii) CAT activity was partially inhibited by a NO synthase inhibitor; and (iii) the inhibition by the NO synthase inhibitor was reversed by exogenous NO. In CS-54 cells, we found that NO increased fos promoter activity and that the increase was prevented by a guanylate cyclase inhibitor. In summary, we found that NO activates the fos promoter by a guanylate cyclase- and G-kinase-dependent mechanism. (+info)
Regulation of myosin phosphatase by a specific interaction with cGMP- dependent protein kinase Ialpha.
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Contraction and relaxation of smooth muscle are regulated by myosin light-chain kinase and myosin phosphatase through phosphorylation and dephosphorylation of myosin light chains. Cyclic guanosine monophosphate (cGMP)-dependent protein kinase Ialpha (cGKIalpha) mediates physiologic relaxation of vascular smooth muscle in response to nitric oxide and cGMP. It is shown here that cGKIalpha is targeted to the smooth muscle cell contractile apparatus by a leucine zipper interaction with the myosin-binding subunit (MBS) of myosin phosphatase. Uncoupling of the cGKIalpha-MBS interaction prevents cGMP-dependent dephosphorylation of myosin light chain, demonstrating that this interaction is essential to the regulation of vascular smooth muscle cell tone. (+info)
Nitric oxide and cGMP regulate gene expression in neuronal and glial cells by activating type II cGMP-dependent protein kinase.
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Nitric oxide (NO) and cGMP have been implicated in many neuronal functions, including regulation of gene expression, but little is known about the downstream targets of NO/cGMP in the nervous system. We found that type II cGMP-dependent protein kinase (G-kinase), which is widely expressed in the brain, mediated NO- and cGMP-induced activation of the fos promoter in cells of neuronal and glial origin; the enzyme was ineffective in regulating gene expression in fibroblast-like cells. The effect of G-kinase II on gene expression did not require calcium uptake but was synergistically enhanced by calcium. G-kinase II was membrane associated and did not translocate to the nucleus; however, a soluble G-kinase II mutant translocated to the nucleus and regulated gene expression in fibroblast-like cells. Soluble G-kinase I also regulates fos promoter activity, but membrane targeting of G-kinase I prevented the enzyme from translocating to the nucleus and regulating transcription in multiple cell types, including glioma cells; this suggests that cell type-specific factor(s) that mediate the transcriptional effects of extranuclear G-kinase II are not regulated by G-kinase I. Our results suggest that G-kinase I and II control gene expression by different mechanisms and that NO effects on neuronal plasticity may involve G-kinase II regulation of gene expression.-Gudi, T., Hong, G. K.-P., Vaandrager, A. B., Lohmann, S. M., Pilz, R. B. Nitric oxide and cGMP regulate gene expression in neuronal and glial cells by activating type II cGMP-dependent protein kinase. (+info)
Identification of a conserved residue responsible for the autoinhibition of cGMP-dependent protein kinase Ialpha and beta.
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We isolated a constitutively active form of cGMP-dependent protein kinase Ialpha (cGK Ialpha) by PCR-driven random mutagenesis. The replacement of Ile-63 by Thr in the autoinhibitory domain results in the enhancement of autophosphorylation and the basal kinase activity in the absence of cGMP. The hydrophobicity at position 63 is essential for the inactive state of cGK Ialpha, and Ile-78 of cGK Ibeta is also required for the autoinhibitory property. Furthermore, cGK Ialpha (Ile-63-Thr) is constitutively active in vivo. These findings suggest that a conserved residue in the autoinhibitory domain was involved in the autoinhibition of both cGK Is. (+info)
Binding and phosphorylation of a novel male germ cell-specific cGMP-dependent protein kinase-anchoring protein by cGMP-dependent protein kinase Ialpha.
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cGMP-dependent protein kinase (cGK) is a major cellular receptor of cGMP and plays important roles in cGMP-dependent signal transduction pathways. To isolate the components of the cGMP/cGK signaling pathway such as substrates and regulatory proteins of cGK, we employed the yeast two-hybrid system using cGK-Ialpha as a bait and isolated a novel male germ cell-specific 42-kDa protein, GKAP42 (42-kDa cGMP-dependent protein kinase anchoring protein). Although the N-terminal region (amino acids 1-66) of cGK-Ialpha is sufficient for the association with GKAP42, GKAP42 could not interact with cGK-Ibeta, cGK-II, or cAMP-dependent protein kinase. GKAP42 mRNA is specifically expressed in testis, where it is restricted to the spermatocytes and early round spermatids. Endogenous cGK-I is co-immunoprecipitated with anti-GKAP42 antibody from mouse testis tissue, suggesting that cGK-I physiologically interacts with GKAP42. Immunocytochemical observations revealed that GKAP42 is localized to the Golgi complex and that cGK-Ialpha is co-localized to the Golgi complex when coexpressed with GKAP42. Although both cGK-Ialpha and -Ibeta, but not cAMP-dependent protein kinase, phosphorylated GKAP42 in vitro, GKAP42 was a good substrate only for cGK-Ialpha in intact cells, suggesting that the association with kinase protein is required for the phosphorylation in vivo. Finally, we demonstrated that the kinase-deficient mutant of cGK-Ialpha stably associates with GKAP42 and that binding of cGMP to cGK-Ialpha facilitates their release from GKAP42. These findings suggest that GKAP42 functions as an anchoring protein for cGK-Ialpha and that cGK-Ialpha may participate in germ cell development through phosphorylation of Golgi-associated proteins such as GKAP42. (+info)
Cysteine-rich protein 2, a novel substrate for cGMP kinase I in enteric neurons and intestinal smooth muscle.
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Nitric oxide/cGMP/cGMP kinase I (cGKI) signaling causes relaxation of intestinal smooth muscle. In the gastrointestinal tract substrates of cGKI have not been identified yet. In the present study a protein interacting with cGKIbeta has been isolated from a rat intestinal cDNA library using the yeast two-hybrid system. The protein was identified as cysteine-rich protein 2 (CRP2), recently cloned from rat brain (Okano, I., Yamamoto, T., Kaji, A., Kimura, T., Mizuno, K., and Nakamura, T. (1993) FEBS Lett. 333, 51-55). Recombinant CRP2 is specifically phosphorylated by cGKs but not by cAMP kinase in vitro. Co-transfection of CRP2 and cGKIbeta into COS cells confirmed the phosphorylation of CRP2 in vivo. Cyclic GMP kinase I phosphorylated CRP2 at Ser-104, because the mutation to Ala completely prevented the in vivo phosphorylation. Immunohistochemical analysis using confocal laser scan microscopy showed a co-localization of CRP2 and cGKI in the inner part of the circular muscle layer, in the muscularis mucosae, and in specific neurons of the myenteric and submucosal plexus. The co-localization together with the specific phosphorylation of CRP2 by cGKI in vitro and in vivo suggests that CRP2 is a novel substrate of cGKI in neurons and smooth muscle of the small intestine. (+info)
Activation of cGMP-dependent protein kinase Ibeta inhibits interleukin 2 release and proliferation of T cell receptor-stimulated human peripheral T cells.
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Several major functions of type I cGMP-dependent protein kinase (cGK I) have been established in smooth muscle cells, platelets, endothelial cells, and cardiac myocytes. Here we demonstrate that cGK Ibeta is endogenously expressed in freshly purified human peripheral blood T lymphocytes and inhibits their proliferation and interleukin 2 release. Incubation of human T cells with the NO donor, sodium nitroprusside, or the membrane-permeant cGMP analogs PET-cGMP and 8-pCPT-cGMP, activated cGK I and produced (i) a distinct pattern of phosphorylation of vasodilator-stimulated phosphoprotein, (ii) stimulation of the mitogen-activated protein kinases ERK1/2 and p38 kinase, and, upon anti-CD3 stimulation, (iii) inhibition of interleukin 2 release and (iv) inhibition of cell proliferation. cGK I was lost during in vitro culturing of primary T cells and was not detectable in transformed T cell lines. The proliferation of these cGK I-deficient cells was not inhibited by even high cGMP concentrations indicating that cGK I, but not cGMP-regulated phosphodiesterases or channels, cAMP-dependent protein kinase, or other potential cGMP mediators, was responsible for inhibition of T cell proliferation. Consistent with this, overexpression of cGK Ibeta, but not an inactive cGK Ibeta mutant, restored cGMP-dependent inhibition of cell proliferation of Jurkat cells. Thus, the NO/cGMP/cGK signaling system is a negative regulator of T cell activation and proliferation and of potential significance for counteracting inflammatory or lymphoproliferative processes. (+info)
Gene transfer of cGMP-dependent protein kinase I enhances the antihypertrophic effects of nitric oxide in cardiomyocytes.
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NO acting through soluble guanylyl cyclase and cGMP formation is a negative regulator of cardiomyocyte hypertrophy. Downstream targets mediating the inhibitory effects of NO/cGMP on cardiomyocyte hypertrophy have not been elucidated. In addition to its antihypertrophic effects, NO promotes apoptosis in cardiomyocytes, presumably through cGMP-independent pathways. We investigated the role of cGMP-dependent protein kinase (PKG) in the antihypertrophic and proapoptotic effects of NO. Incubation of neonatal rat cardiomyocytes with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) (250 micromol/L) or the PKG-selective cGMP analog 8-pCPT-cGMP (500 micromol/L) activated endogenous PKG type I, as shown by the site-specific phosphorylation of vasodilator-stimulated phosphoprotein, a well-characterized PKG substrate. SNAP (250 micromol/L) and 8-pCPT-cGMP (500 micromol/L) modestly attenuated the hypertrophic response to alpha(1)-adrenergic stimulation with phenylephrine. Although a high concentration of SNAP (1000 micromol/L) promoted apoptosis in cardiomyocytes, as evidenced by the formation of histone-associated DNA fragments, antihypertrophic concentrations of SNAP (250 micromol/L) and 8-pCPT-cGMP (500 micromol/L) did not promote cell death. Because chronic activation downregulated endogenous PKG I, we explored whether gene transfer of PKG I would enhance the sensitivity of cardiomyocytes to the antihypertrophic effects of NO/cGMP. Indeed, after adenoviral overexpression of PKG Ibeta, SNAP (250 micromol/L) and 8-pCPT-cGMP (500 micromol/L) completely suppressed the hypertrophic response to alpha(1)-adrenergic stimulation. As observed in noninfected cells, SNAP (250 micromol/L) and 8-pCPT-cGMP (500 micromol/L) did not promote apoptosis in cardiomyocytes overexpressing PKG Ibeta. Moreover, overexpression of PKG Ibeta did not enhance the proapoptotic effects of 1000 micromol/L SNAP, implying PKG-independent effects of NO on apoptosis. Endogenous PKG I mediates antihypertrophic but not proapoptotic effects of NO in a cell culture model of cardiomyocyte hypertrophy. Adenoviral gene transfer of PKG I selectively enhances the antihypertrophic effects of NO without increasing the susceptibility to apoptosis. (+info)