Interaction of cCMP with the cGK,cAK and MAPK Kinases in Murine Tissues

cAMP and cGMP are well established second messengers that are essential for numerous (patho)physiological processes. These purine cyclic nucleotides activate cAK and cGK, respectively. Recently, the existence of cCMP was described, and a possible function for this cyclic nucleotide was investigated. It was postulated that cCMP plays a role as a second messenger. However, the functions regulated by cCMP are mostly unknown. To elucidate probable functions, cCMP-binding and -activated proteins were identified using different methods. We investigated the effect of cCMP on purified cyclic nucleotide-dependent protein kinases and lung and jejunum tissues of wild type (WT), cGKI-knockout (cGKI KO) and cGKII-knockout (cGKII KO) mice. The catalytic activity of protein kinases was measured by a (γ-³²P) ATP kinase assay. Cyclic nucleotide-dependent protein kinases (cAK, cGKI and cGKII) in WT tissue lysates were stimulated by cCMP. In contrast, there was no stimulation of phosphorylation in KO tissue lysates. Competitive binding assays identified cAK, cGKI, and cGKII as cCMP-binding proteins. An interaction between cCMP/MAPK and a protein-protein complex of MAPK/cGK were detected via cCMP affinity chromatography and co-immunoprecipitation, respectively. These complexes were abolished or reduced in jejunum tissues from cGKI KO or cGKII KO mice. In contrast, these complexes were observed in the lung tissues from WT, cGKI KO and cGKII KO mice. Moreover, cCMP was also able to stimulate the phosphorylation of MAPK. These results suggest that MAPK signaling is regulated by cGMP-dependent protein kinases upon activation by cCMP. Based on these results, we propose that additional cCMP-dependent protein kinases that are capable of modulating MAPK signaling could exist. Hence, cCMP could potentially act as a second messenger in the cAK/cGK and MAPK signaling pathways and play an important role in physiological processes of the jejunum and lung.     

IRAG is essential for relaxation of receptor-triggered smooth muscle contraction by cGMP kinase

Signalling by cGMP-dependent protein kinase type I (cGKI) relaxes various smooth muscles modulating thereby vascular tone and gastrointestinal motility. cGKI-dependent relaxation is possibly mediated by phosphorylation of the inositol 1,4,5-trisphosphate receptor I (IP(3)RI)-associated protein (IRAG), which decreases hormone-induced IP(3)-dependent Ca(2+) release. We show now that the targeted deletion of exon 12 of IRAG coding for the N-terminus of the coiled-coil domain disrupted in vivo the IRAG-IP(3)RI interaction and resulted in hypomorphic IRAG(Delta12/Delta12) mice. These mice had a dilated gastrointestinal tract and a disturbed gastrointestinal motility. Carbachol- and phenylephrine-contracted smooth muscle strips from colon and aorta, respectively, of IRAG(Delta12/Delta12) mice were not relaxed by cGMP, while cAMP-mediated relaxation was unperturbed. Norepinephrine-induced increases in [Ca(2+)](i) were not decreased by cGMP in aortic smooth muscle cells from IRAG(Delta12/Delta12) mice. In contrast, cGMP-induced relaxation of potassium-induced smooth muscle contraction was not abolished in IRAG(Delta12/Delta12) mice. We conclude that cGMP-dependent relaxation of hormone receptor-triggered smooth muscle contraction essentially depends on the interaction of cGKI-IRAG with IP(3)RI.     

Molecular determinants of the interaction between the inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG) and cGMP kinase Ibeta

Cyclic GMP-dependent protein kinase I (cGKI) affects the inositol 1,4,5-trisphosphate (InsP(3))-dependent release of intracellular calcium by phosphorylation of IRAG (inositol 1,4,5-trisphophate receptor-associated cGMP kinase substrate). IRAG is present in a macromolecular complex with the InsP(3) receptor type I (InsP(3)RI) and cGKIbeta. The specificity of the interaction between these three proteins was investigated by using the yeast two-hybrid system and by co-precipitation of expressed proteins. The amino-terminal region containing the leucine zipper (amino acids 1-53) of cGKIbeta but not that of cGKIalpha or cGKII interacted with the sequence between amino acids 152 and 184 of IRAG in vitro and in vivo most likely through electrostatic interaction. cGKIbeta did not interact with the InsP(3)RI, but co-precipitated the InsP(3)RI in the presence of IRAG indicating that IRAG bound to the InsP(3)RI and to cGKIbeta. cGKIbeta phosphorylated up to four serines in IRAG. Mutation of these four serines to alanine showed that cGKIbeta-dependent phosphorylation of Ser(696) is necessary to decrease calcium release from InsP(3)-sensitive stores. These results show that cGMP induced reduction of cytosolic calcium concentrations requires cGKIbeta and phosphorylation of Ser(696) of IRAG.     

Regulation of Sensory Neuron Precursor Proliferation by Cyclic GMP-Dependent Protein Kinase

Abstract: Cyclic GMP (cGMP) is a molecular messenger involved in diverse cellular processes. Recently, cGMP-dependent protein kinase (cGK) type II was determined to be a regulator of endochondral ossification and bone growth, identifying a role for cGMP in the regulation of cellular proliferation. Here, we demonstrate the presence of cGK type I (cGKI) in cells of the developing trigeminal ganglia. cGKI occurs in some proliferating precursors as evidenced by double labeling with an antibody to cGKI and 5-bromo-2'-deoxyuridine(BrdU) incorporation. Inhibition of cGKI with KT5823 or Rp -8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate ( Rp -8-pCPT-cGMPS) in chick embryos results in a 30–40% decrease in trigeminal ganglia cell number, and this effect is independent of nitric oxide synthase (NOS). In addition, inhibition of cGKI with Rp -8-pCPT-cGMPS results in a 60% decrease in BrdU incorporation in the trigeminal ganglia of embryonic day 5 chicks. We find that PC12 cells expressing cGKI proliferate more rapidly and incorporate more BrdU than do control cells. The cGKI inhibitor Rp -8-pCPT-cGMPS decreases proliferation and BrdU incorporation in transfected PC12 cells but has no effect on control cells. The PC12 cells do not express NOS, indicating that this effect is also independent of NOS. Thus, cGKI regulates the proliferation of sensory neurons as a result of activation of a NOS-independent pathway, representing a novel pathway by which the number of sensory neurons is regulated.     

Autoinhibition and isoform-specific dominant negative inhibition of the type II cGMP-dependent protein kinase

In the absence of cyclic nucleotides, the cAMP-dependent protein kinase and cGMP-dependent protein kinases (cGKs) suppress phosphotransfer activity at the catalytic cleft by competitive inhibition of substrate binding with a pseudosubstrate sequence within the holoenzyme. The magnitude of inhibition can be diminished by autophosphorylation near this pseudosubstrate sequence. Activation of type I cGK (cGKI) and type II cGK (cGKII) are differentially regulated by their cyclic nucleotide-binding sites. To address the possibility that the distinct activation mechanisms of cGKII and cGKI result from differences in the autophosphorylation of the inhibitory domain, we investigated the effects of autophosphorylation on the kinetics of activation. Unlike the type I cGKs (cGKIalpha and Ibeta), cGKII autophosphorylation did not alter the basal activity, nor the sensitivity of the enzyme to cyclic nucleotide activation. To determine residues responsible for autoinhibition of cGKII, Ala was substituted for basic residues (Lys(122), Arg(118), and Arg(119)) or a hydrophobic residue (Val(125)) within the putative pseudosubstrate domain of cGKII. The integrity of these residues was essential for full cGKII autoinhibition. Furthermore, a cGKII truncation mutant containing this autoinhibitory region demonstrated a nanomolar IC(50) toward a constitutively active form of cGKII. Finally, we present evidence that the dominant negative properties of this truncation mutant are specific to cGKII when compared with cAMP-dependent protein kinase Calpha and cGKIbeta. These findings extend the known differences in the activation mechanisms among cGK isoforms and allow the design of an isoform-specific cGKII inhibitor.     

Direct evidence for cross-activation of cGMP-dependent protein kinase by cAMP in pig coronary arteries.

Elevation of either cAMP or cGMP causes smooth muscle relaxation. Whether these effects are mediated through cAMP-dependent protein kinase (cAK), cGMP-dependent protein kinase (cGK), or both is unknown. Pig coronary arteries were treated with sodium nitroprusside (SNP) or atrial natriuretic factor (ANF), relaxants which elevate cGMP, and with isoproterenol or forskolin, relaxants which elevate cAMP. Incubation of the arteries with 10 microM SNP produced a 3.3-fold increase in cGMP without altering cAMP; the cGK activity ratio (-cGMP/+cGMP) in these extracts was increased by 2.6-fold as determined by a newly developed assay, while the cAK activity ratio (-cAMP/+cAMP) was unchanged. The increase in cGK activity ratio by SNP was concentration-dependent and was nearly maximal at 30 s. Treatment of the tissue with 10 nM ANF also increased the cGK activity ratio (2.3-fold), but not that of cAK. 100 microM isoproterenol caused a 2.9-fold elevation of cAMP with no change in cGMP, but both cAK and cGK activity ratios were increased (2.3- and 1.6-fold, respectively). The increase in the cGK activity ratio could be mimicked by cAMP addition to control tissue extracts at the concentration measured in extracts of the isoproterenol-treated tissue. Forskolin (1 and 10 microM) also increased the cGK activity ratio (1.9- and 4.9-fold). The increases in cGK activity observed in extracts suggest that moderate elevation of either cGMP or cAMP causes intracellular cGK activation, thus producing relaxation of vascular smooth muscle.     

Molecular cloning and predicted full-length amino acid sequence of the type I beta isozyme of cGMP-dependent protein kinase from human placenta. Tissue distribution and developmental changes in rat

In this study we report the isolation and characterization of three overlapping cDNA clones for the type I beta isozyme of cGMP-dependent protein kinase (cGK) from human placenta libraries. The composite sequence was 3740 nucleotides long and contained 58 nucleotides from the 5'-noncoding region, an open reading frame of 2061 bases including the stop codon, and a 3'-noncoding region of 1621 nucleotides. The predicted full-length human type I beta cGK protein contained 686 amino acids including the initiator methionine, and had an estimated molecular mass of 77,803 Da. On comparison to the published amino acid sequence of bovine lung I alpha, human placenta I beta cGK differed by only two amino acids in the carboxyl-terminal region (amino acids 105-686). In contrast, the amino-terminal region of the two proteins was markedly different (only 36% similarity), and human I beta cGK was 16 amino acids longer. In a specific region in the amino-terminus (amino acids 63-75), 12 out of 13 amino acids of the human I beta cGK were identical to the partial amino acid sequence recently published for a new I beta isoform of cGK from bovine aorta. Northern blot analysis demonstrated a human I beta cGK mRNA, 7 kb in size, in human uterus and weakly in placenta. An mRNA of 7 kb was also observed in rat cerebellum, cerebrum, lung, kidney, and adrenal, whereas an mRNA doublet of 7.5 and 6.5 kb were observed in rat heart. Comparison of Northern and Western blot analyses demonstrated that the mRNA and protein for cerebellar cGK increased during the development of rats from 5 to 30 days old, whereas the 6.5 kb mRNA in rat heart declined.     

Defective smooth muscle regulation in cGMP kinase I-deficient mice.

Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP-dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross-activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP-dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI-deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.     

Inhibition of cGMP-dependent protein kinase II by its own splice isoform

cGMP- and cAMP-dependent protein kinases (cGK I, cGK II, and cAK) are important mediators of many signaling pathways that increase cyclic nucleotide concentrations and ultimately phosphorylation of substrates vital to cellular functions. Here we demonstrate a novel mRNA splice isoform of cGK II arising from alternative 5' splicing within exon 11. The novel splice variant encodes a protein (cGK II Delta(441-469)) lacking 29 amino acids of the cGK II Mg-ATP-binding/catalytic domain, including the conserved glycine-rich loop consensus motif Gly-x-Gly-x-x-Gly-x-Val which interacts with ATP in the protein kinase family of enzymes. cGK II Delta(441-469) has no intrinsic enzymatic activity itself, however, it antagonizes cGK II and cGK I, but not cAK. Thus, the activation and cellular functions of cGK II may be determined not only by intracellular cGMP levels but also by alternative splicing which may regulate the balance of expression of cGK II versus its own inhibitor, cGK II Delta(441-469).     

A novel interaction of cGMP-dependent protein kinase I with troponin T

cGMP-dependent protein kinase (cGK) is a major intracellular receptor of cGMP and is implicated in several signal transduction pathways. To identify proteins that participate in the cGMP/cGK signaling pathway, we employed the yeast two-hybrid system with cGK Ialpha as bait. cDNAs encoding slow skeletal troponin T (skTnT) were isolated from both mouse embryo and human skeletal muscle cDNA libraries. The skTnT protein interacted with cGK Ibeta but not with cGK II nor cAMP-dependent protein kinase. The yeast two-hybrid and in vitro binding assays revealed that the N-terminal region of cGK Ialpha, containing the leucine zipper motif, is sufficient for the association with skTnT. In vivo analysis, mutations in cGK Ialpha, which disrupted the leucine zipper motif, were shown to completely abolish the binding to skTnT. Furthermore, cGK I also interacted with cardiac TnT (cTnT) but not with cardiac troponin I (cTnI). Together with the observations that cTnI is a good substrate for cGK I and is effectively phosphorylated in the presence of cTnT in vitro, these findings suggest that TnT functions as an anchoring protein for cGK I and that cGK I may participate in the regulation of muscle contraction through phosphorylation of TnI.     

IRAG and novel PKG targeting in the cardiovascular system

Signaling by nitric oxide (NO) determines several cardiovascular functions including blood pressure regulation, cardiac and smooth muscle hypertrophy, and platelet function. NO stimulates the synthesis of cGMP by soluble guanylyl cyclases and thereby activates cGMP-dependent protein kinases (PKGs), mediating most of the cGMP functions. Hence, an elucidation of the PKG signaling cascade is essential for the understanding of the (patho)physiological aspects of NO. Several PKG signaling pathways were identified, meanwhile regulating the intracellular calcium concentration, mediating calcium desensitization or cytoskeletal rearrangement. During the last decade it emerged that the inositol trisphosphate receptor-associated cGMP-kinase substrate (IRAG), an endoplasmic reticulum-anchored 125-kDa membrane protein, is a main signal transducer of PKG activity in the cardiovascular system. IRAG interacts specifically in a trimeric complex with the PKG1β isoform and the inositol 1,4,5-trisphosphate receptor I and, upon phosphorylation, reduces the intracellular calcium release from the intracellular stores. IRAG motifs for phosphorylation and for targeting to PKG1β and 1,4,5-trisphosphate receptor I were identified by several approaches. The (patho)physiological functions for the regulation of smooth muscle contractility and the inhibition of platelet activation were perceived. In this review, the IRAG recognition, targeting, and function are summarized compared with PKG and several PKG substrates in the cardiovascular system.     

Mechanisms involved in the relaxation of bovine aortic endothelial cells

The importance of endothelial cell contraction in the regulation of vascular biology is being increasingly recognized. Our group has demonstrated that reactive oxygen species, particularly hydrogen peroxide, which are released in pathological conditions such as ischemia-reperfusion, are able to induce contraction in bovine aortic endothelial cells (BAEC). The cGMP-dependent relaxation of contractile cells depends on the ability of the cyclic nucleotide to interfere with intracellular calcium; however, this is not the only mechanism involved. The present experiments were designed to analyse the mechanism by which cGMP induces relaxation in BAEC. Sodium nitroprusside (SNP), an activator of soluble guanylate cyclase, as well as atrial natriuretic (ANP) and C-type natriuretic (CNP) peptides, activators of particulate guanylate cyclase, blunted the hydrogen peroxide-induced contraction of BAEC and myosin light chain phosphorylation. The inhibitory effect was more marked with SNP and CNP than with ANP, and the action of SNP and CNP were partially reversed by blocking soluble and particulate guanylate cyclases, respectively. Dibutyryl cGMP (db-cGMP), a cGMP analogue, mimicked the effect of SNP and CNP. Cyclic GMP-dependent protein kinase (cGK) protein levels and activity were measured. Hydrogen peroxide induced a significant reduction in cGK activity without any change in protein level. This effect was completely reversed by preincubation with db-cGMP. Calyculin A, a myosin light chain phosphatase inhibitor, prevented the cGMP-induced relaxation of BAEC. SNP, CNP and db-cGMP also partially prevented the hydrogen peroxide-induced increase in intracellular calcium levels. Catalase completely blocked this effect. In summary, the present results support a role for those metabolites which activate guanylate cyclases in the relaxation of BAEC, and suggest that the cGMP-induced BAEC relaxation could be due, at least partially, to the stimulation of cGK and/or myosin light chain phosphatase activity, and to calcium blockade.     

Properties of a cGMP-dependent monomeric protein kinase from bovine aorta

A form of cGMP-dependent protein kinase (cGK) that was different from previously described cGK was purified from bovine aorta smooth muscle. The partial amino-terminal sequencing of this enzyme indicated that it was derived by endogenous proteolysis of the type I beta isozyme of cGK. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this form migrated as a smaller protein (Mr = 70,000) than the parent cGK (Mr = 80,000), and since the calculated nondenatured Mr was approximately 89,000 compared to Mr = 170,000 for the dimeric native enzyme, it represented a monomeric form of cGK. The monomer bound approximately 2 mol of [3H]cGMP per mol of monomer, although it had only one rapid component in [3H]cGMP dissociation assays as compared to one rapid and one slow component for the native cGK. The specific catalytic activity of the kinase was similar to that of the native enzyme, suggesting that the catalytic domain was essentially intact. The monomeric cGK incorporated significant 32P when incubated with Mg2+ and [gamma-32P]ATP in the presence of cGMP, although the phosphorylation proceeded at a slower rate than that obtained with native cGK. In contrast to previous reports of monomeric forms of cGK, this monomer was highly cGMP-dependent, although it had a slightly higher Ka (0.8 microM) for cGMP than that of the native enzyme (0.4 microM) and a low Hill coefficient of 1.0 (1.6 for the native enzyme). The cGMP dependence of the monomer did not decrease with dilution, implying that the cGMP dependence was not due to monomer-monomer interactions in the assay. The results indicated that the catalytic domain, cGMP binding domain(s), and inhibitory domain of cGK interact primarily within the same subunit rather than between subunits of the dimer as previously hypothesized for dimeric cGK.     

Nitric oxide sensitivity in pulmonary artery and airway smooth muscle: a possible role for cGMP responsiveness

We aimed to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Porcine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used in concentration-response studies to the NO donor (Z)-1-[N-2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM consistently exhibited greater relaxation at a given DETA-NO concentration (NO responsiveness) than TSM NO responsiveness, with DETA-NO log EC(50) being -6.55 +/- 0.11 and -5.37 +/- 0.13 for PASM and TSM, respectively (P < 0.01). We determined relationships between tissue cGMP concentration ([cGMP](i)) and relaxation using the particulate guanylyl cyclase agonist atrial natriuretic peptide. Atrial natriuretic peptide resulted in nearly complete relaxation, with no detectable increase in [cGMP](i) in PASM and only 20% relaxation (10-fold increase in [cGMP](i)) in TSM, indicating that TSM is less cGMP responsive than PASM. Total cGMP-dependent protein kinase I (cGKI) mRNA expression was greater in PASM than in TSM (2.23 +/- 0.36 vs. 0.93 +/- 0.31 amol mRNA/mug total RNA, respectively; P < 0.01), but total cGKI protein expression was not significantly different (0.56 +/- 0.07 and 0.49 +/- 0.04 ng cGKI/mug protein, respectively). The phosphotransferase assay for the soluble fraction of tissue homogenates demonstrated no difference in the cGMP EC(50) between PASM and TSM. The maximal phosphotransferase activity indexed to the amount of total cGKI in the homogenate differed significantly between PASM and TSM (1.61 +/- 0.15 and 1.04 +/- pmol.min(-1).ng cGKI(-1), respectively; P < 0.05), suggesting that cGKI may be regulated differently in the two tissues. A novel intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is thus greater cGMP responsiveness from increased cGKI-specific activity in PASM.     

Cysteine-rich protein 2, a novel substrate for cGMP kinase I in enteric neurons and intestinal smooth muscle

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.     

Functional characteristics of urinary tract smooth muscles in mice lacking cGMP protein kinase type I

Nitric oxide (NO)-mediated smooth muscle relaxation is mediated by cGMP through activation of cGMP-dependent protein kinase I (cGKI). We studied the importance of cGKI for lower urinary tract function in mice lacking the gene for cGKI (cGKI-/-) and in litter-matched wild-type mice (cGKI+/+) in vitro and in vivo. cGKI deficiency did not result in any changes in bladder gross morphology or weight. Urethral strips from cGKI-/- mice showed an impaired relaxant response to nerve-derived NO. The cGMP analog 8-bromo-cGMP (8-BrcGMP) and the NO-donor SIN-1 relaxed the wild-type urethra (50-60%) but had only marginal effects in the cGKI-deficient urethra. Bladder strips from cGKI-/- mice responded normally to electrical field stimulation and to carbachol but not to 8-BrcGMP. In vivo, the cGKI-deficient mice showed bladder hyperactivity characterized by decreased intercontraction intervals and nonvoiding bladder contractions. Loss of cGKI abolishes NO-cGMP-dependent relaxations of urethral smooth muscle and results in hyperactive voiding. These data suggest that certain voiding disturbances may be associated with impaired NO-cGKI signaling.