Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.12 (PKG)
 2,515 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Small GTPase Rho and cGMP/cGMP-dependent protein kinase (cGK) pathways exert opposing effects in specific systems such as vascular contraction and growth. However, the direct interaction between these pathways has remained elusive. We demonstrate that cGK phosphorylates RhoA in vitro at Ser188, the same residue phosphorylated by cAMP-dependent protein kinase. In HeLa cells transfected with constitutively active cGK (C-cGK), stress fiber formation induced by lysophosphatidic acid or V14RhoA was blocked. By contrast, C-cGK failed to inhibit stress fiber formation in cells transfected with mutant RhoA with substitution of Ser188 to Ala. C-cGK did not affect actin reorganization induced by Rac1 or Rho-associated kinase, one of the effectors for RhoA. Furthermore, C-cGK expression inhibited the membrane translocation of RhoA. Collectively, our findings suggest that cGK phosphorylates RhoA at Ser188 and inactivates RhoA signaling. The physiological relevance of the direct interaction between RhoA and cGK awaits further investigation.
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PMID:cGMP-dependent protein kinase phosphorylates and inactivates RhoA. 1116 91

The erectile response of the penis depends on a balance between vasoconstrictor agents which cause cavernosal smooth muscle to contract limiting blood inflow, and vasodilators which relax cavernosal smooth muscle leading to increased blood inflow and erection. This review emphasizes the role of vasoconstrictors in the penis and shows that both endothelin-1 (ET-1) and the alpha-adrenergic agonist, methoxamine (METHOX) exert strong vasoconstrictor actions in the cavernosal circulation. We recently reported the vasoconstrictor actions of exogenous ET-1 and METHOX to be mediated by the RhoA/Rho-kinase pathway in the cavernosal circulation. While it is widely held that the nitric oxide-cyclic GMP-protein kinase G (NO-cGMP-PKG) pathway mediates vasorelaxation and penile erection, the interaction between this pathway and the vasoconstrictor process remains to be fully elucidated. Our studies also have shown that, during erection, the vasoconstrictor action of METHOX and ET-1 are inhibited and that NO is likely responsible for this inhibition. We hypothesize that the NO-cGMP-PKG pathway controls erection by acting in two distinct ways-by lowering intracellular levels of calcium leading to vasorelaxation and by inhibiting Rho-kinase mediated vasoconstriction.
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PMID:Vasoconstrictors in erectile physiology. 1178 44

NO induces vasodilation through cGMP-dependent protein kinase--dependent and --independent mechanisms. A recent study demonstrated that recombinant cGMP-dependent protein kinase can phosphorylate the small G protein, RhoA, thus inhibiting its activity. Additionally, sodium nitroprusside was found to reverse the phenylephrine-induced translocation of RhoA, which is further indicative of the inhibition of RhoA activity. RhoA is known to be involved in the Ca(2+) sensitization of vascular smooth muscle through the actions of one of its downstream effectors, Rho-kinase. This study examined whether NO endogenously induces the relaxation of intact rat aorta via the inhibition of the Rho-kinase--mediated Ca(2+)-sensitizing pathway. Endogenous Rho-kinase inhibitor activity was inhibited by the selective compound Y-27632. Treatment of endothelium-intact rat aorta with Y-27632 (1 micromol/L) resulted in an attenuation of maximal force generated in response to phenylephrine. In endothelium-denuded rings, however, 1 micromol/L Y-27632 was ineffective at inhibiting the phenylephrine-induced contraction. Additionally, 1 micromol/L Y-27632 was significantly less effective at inhibiting the phenylephrine-induced contraction of endothelium-intact rings in the presence of inhibitors of NO synthase or guanylate cyclase (N(omega)-nitro-L-arginine and 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, respectively). Interestingly, sodium nitroprusside restored the ability of 1 micromol/L Y-27632 to attenuate phenylephrine-induced contraction. Rho-kinase inhibition was also found to increase the sensitivity of the endothelium-denuded aorta to sodium nitroprusside. These data demonstrate that NO inhibits Rho-kinase activity in the intact rat aorta, supporting the hypothesis that endogenous NO-mediated vasodilation occurs through the inhibition of Rho-kinase constrictor activity in the intact rat aorta.
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PMID:Nitric oxide induces dilation of rat aorta via inhibition of rho-kinase signaling. 1188 86

RhoA, in its active GTP-bound form, stimulates transcription through activation of the serum-response factor (SRF). We found that cGMP inhibited serum-induced Rho.GTP loading and transcriptional activation of SRF-dependent reporter genes in smooth muscle and glial cells in a cGMP-dependent protein kinase (G-kinase)-dependent fashion. Serum stimulation of the SRF target gene vinculin was also blocked by cGMP/G-kinase. G-kinase activation inhibited SRF-dependent transcription induced by upstream RhoA activators including Galpha(13) and p115RhoGEF, with Galpha(13)-induced Rho.GTP loading inhibited by G-kinase. G-kinase had no effect on the high activation levels of RhoA(63L) or the double mutant RhoA(63L,188A) but inhibited transcriptional activation by these two RhoA mutants to a similar extent, suggesting an effect downstream of RhoA and independent of RhoA Ser(188) phosphorylation. Constitutively active forms of the Rho effectors Rho kinase (ROK), PKN, and PRK-2 induced SRF-dependent transcription in a cell type-specific fashion with ROK being the most efficient; G-kinase inhibited transcription induced by all three effectors without affecting ROK catalytic activity. G-kinase had no effect on RhoA(63L)-induced morphological changes in glial cells, suggesting distinct transcriptional and cytoskeletal effectors of RhoA. We conclude that G-kinase inhibits SRF-dependent transcription by interfering with RhoA signaling; G-kinase acts both upstream of RhoA, inhibiting serum- or Galpha(13)-induced Rho activation, and downstream of RhoA, inhibiting steps distal to the Rho targets ROK, PKN, and PRK-2.
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PMID:cGMP-dependent protein kinase inhibits serum-response element-dependent transcription by inhibiting rho activation and functions. 1211 92

The small G protein RhoA is a convergence point for multiple signals that regulate smooth muscle cell functions. NO plays a major role in the structure and function of the normal adult vessel wall, mainly through modulation of gene transcription. This study was thus performed to analyze in vitro and in vivo the effect of NO signaling on RhoA expression in arterial smooth muscle cells. In rat or human artery smooth muscle cells, sodium nitroprusside or 8-(2-chlorophenylthio)-cGMP induced a rise in RhoA mRNA and protein expression, which was inhibited by the cGMP-dependent protein kinase (PKG) inhibitor (R(p))-8-bromo-beta-phenyl-1,N(2)-ethenoguanosine 3':5'-phosphorothioate. The NO/PKG stimulation of RhoA expression involved both an increase in RhoA protein stability and stimulation of rhoA gene transcription. Cloning and functional analysis of the human rhoA promoter revealed that the effect of NO/PKG involved phosphorylation of ATF-1 and subsequent binding to the cAMP-response element. Chronic inhibition of NO synthesis in N(omega)-nitro-l-arginine-treated rats induced a strong decrease in RhoA mRNA and protein expression in aorta and pulmonary artery associated with inhibition of RhoA-mediated Ca(2+) sensitization. These effects were prevented by oral administration of the cGMP phosphodiesterase inhibitor sildenafil. These results show that NO/PKG signaling positively controls RhoA expression and suggest that the basal release of NO is necessary to maintain RhoA expression and RhoA-dependent functions in vascular smooth muscle cells.
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PMID:RhoA expression is controlled by nitric oxide through cGMP-dependent protein kinase activation. 1252 25

The role of RhoA in myosin light-chain (MLC)(20) dephosphorylation and smooth muscle relaxation by PKA and PKG was examined in freshly dispersed and cultured smooth muscle cells expressing wild-type RhoA, constitutively active Rho(V14), and phosphorylation site-deficient Rho(A188). Activators of PKA (5,6-dichloro-1-beta-ribofuranosyl benzimidazole 3',5'-cyclic monophosphothionate, Sp-isomer; cBIMPS) or PKG [8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP), sodium nitroprusside (SNP)] or both PKA and PKG (VIP) induced phosphorylation of constitutively active Rho(V14) and agonist (ACh)- or GTPgammaS-stimulated wild-type RhoA but not Rho(A188). Phosphorylation was accompanied by translocation of membrane-bound wild-type RhoA and Rho(V14) to the cytosol and complete inhibition of ACh-stimulated Rho kinase and phospholipase D activities, RhoA/Rho kinase association, MLC(20) phosphorylation, and sustained muscle contraction. Each of these events was blocked depending on the agent used, by the PKG inhibitor KT5823 or the PKA inhibitor myristoylated PKI. Inhibitors were used at a concentration (1 microM) previously shown by direct measurement of kinase activity to selectively inhibit the corresponding kinase. In muscle cells overexpressing the active phosphorylation site-deficient mutant Rho(A188), MLC(20) phosphorylation was partly inhibited by SNP, VIP, cBIMPS, and 8-pCPT-cGMP, suggesting the existence of an independent inhibitory mechanism downstream of RhoA. Results demonstrate that dephosphorylation of MLC(20) and smooth muscle relaxation are preferentially mediated by PKG- and PKA-dependent phosphorylation and inactivation of RhoA.
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PMID:Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially mediated by phosphorylation of RhoA. 1273 49

Regulation of vascular smooth muscle cell contractile state is critical for the maintenance of blood vessel tone. Abnormal vascular smooth muscle cell contractility plays an important role in the pathogenesis of hypertension, blood vessel spasm, and atherosclerosis. Myosin phosphatase, the key enzyme controlling myosin light chain dephosphorylation, regulates smooth muscle cell contraction. Vasoconstrictor and vasodilator pathways inhibit and activate myosin phosphatase, respectively. G-protein-coupled receptor agonists can inhibit myosin phosphatase and cause smooth muscle cell contraction by activating RhoA/Rho kinase, whereas NO/cGMP can activate myosin phosphatase and cause smooth muscle cell relaxation by activation of cGMP-dependent protein kinase. We have used yeast two-hybrid screening to identify a 116-kDa human protein that interacts with both myosin phosphatase and RhoA. This myosin phosphatase-RhoA interacting protein, or M-RIP, is highly homologous to murine p116RIP3, is expressed in vascular smooth muscle, and is localized to actin myofilaments. M-RIP binds directly to the myosin binding subunit of myosin phosphatase in vivo in vascular smooth muscle cells by an interaction between coiled-coil and leucine zipper domains in the two proteins. An adjacent domain of M-RIP directly binds RhoA in a nucleotide-independent manner. M-RIP copurifies with RhoA and Rho kinase, colocalizes on actin stress fibers with RhoA and MBS, and is associated with Rho kinase activity in vascular smooth muscle cells. M-RIP can assemble a complex containing both RhoA and MBS, suggesting that M-RIP may play a role in myosin phosphatase regulation by RhoA.
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PMID:Myosin phosphatase-Rho interacting protein. A new member of the myosin phosphatase complex that directly binds RhoA. 1450 64

Cyclic GMP, produced in response to nitric oxide and natriuretic peptides, is a key regulator of vascular smooth muscle cell contractility, growth, and differentiation, and is implicated in opposing the pathophysiology of hypertension, cardiac hypertrophy, atherosclerosis, and vascular injury/restenosis. cGMP regulates gene expression both positively and negatively at transcriptional as well as at posttranscriptional levels. cGMP-regulated transcription factors include the cAMP-response element binding protein CREB, the serum response factor SRF, and the nuclear factor of activated T cells NF/AT. cGMP can regulate CREB directly, through phosphorylation by cGMP-dependent protein kinase, or indirectly, through activation of mitogen-activated protein kinase pathways; regulation of SRF and NF/AT by cGMP is indirect, through modulation of RhoA and calcineurin signaling, respectively. Downregulation of the RNA-binding protein HuR by cGMP leads to destabilization of guanylate cyclase mRNA, but this posttranscriptional mechanism may affect many more cGMP-regulated genes. In this review, we discuss the role of cGMP-regulated gene expression in (patho)physiological processes most relevant to the cardiovascular system, such as regulation of vascular tone, cardiac hypertrophy, phenotypic modulation of vascular smooth muscle cells, and regulation of cell proliferation and apoptosis.
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PMID:Regulation of gene expression by cyclic GMP. 1464 34

Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca(2+) sensitization/desensitization in smooth muscle. Ca(2+) sensitization induced by activation of G protein-coupled receptors acting through RhoA involves phosphorylation of Thr-696 (of the human isoform) of the myosin targeting subunit (MYPT1) of SMPP-1M inhibiting activity. In contrast, agonists that elevate intracellular cGMP and cAMP promote Ca(2+) desensitization in smooth muscle through apparent activation of SMPP-1M. We show that cGMP-dependent protein kinase (PKG)/cAMP-dependent protein kinase (PKA) efficiently phosphorylates MYPT1 in vitro at Ser-692, Ser-695, and Ser-852 (numbering for human isoform). Although phosphorylation of MYPT1 by PKA/PKG has no direct effect on SMPP-1M activity, a primary site of phosphorylation is Ser-695, which is immediately adjacent to the inactivating Thr-696. In vitro, phosphorylation of Ser-695 by PKA/PKG appeared to prevent phosphorylation of Thr-696 by MYPT1K. In ileum smooth muscle, Ser-695 showed a 3-fold increase in phosphorylation in response to 8-bromo-cGMP. Addition of constitutively active recombinant MYPT1K to permeabilized smooth muscles caused phosphorylation of Thr-696 and Ca(2+) sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism of Ca(2+) desensitization in smooth muscle that involves mutual exclusion of phosphorylation, whereby phosphorylation of Ser-695 prevents phosphorylation of Thr-696 and therefore inhibition of SMPP-1M.
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PMID:Smooth muscle phosphatase is regulated in vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of Serine 695 in response to cyclic nucleotides. 1519 81

The contractile tone of the vascular smooth muscle plays an important role on the regulation of the blood pressure as well as the local perfusion of the important organs such as the heart and brain. The importance of the Ca(2+) sensitivity in the regulation of the vascular tone has been established by the development of the simultaneous measurements of intracellular Ca(2+) concentration ( [Ca(2+)](i) ) and tension as well as that of the receptor coupled permeabilized preparation in the late 1980s. Recently, the mechanisms underlying the regulation of Ca(2+) sensitivity have been revealed. The increase in the Ca(2+) sensitivity involves the myosin phosphatase (MLCP) inhibition mediated by rhoA-rho kinase system and PKC-CPI system. The decrease in the Ca(2+) sensitivity involves the PKA-mediated inhibition of myosin light chain kinase, the PKG-mediated activation of MLCP, and PKA- or PKG-mediated inactivation of rhoA. In this article, the regulation of the Ca(2+) sensitivity of the contractile apparatus of the vascular smooth muscle will be briefly reviewed.
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PMID:[Regulation of vascular tone by the modulation of Ca2+ sensitivity]. 1577 34


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