cGMP-dependent Protein Kinase Type Research Articles

Functional reconstitution of vascular smooth muscle cells with cGMP-dependent protein kinase I isoforms.

The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation. Smooth muscle expresses two isoforms of cGKI, cGKIalpha and cGKIbeta, but the specific role of each isoform in vascular smooth muscle cells (VSMCs) is poorly understood. We have used a genetic deletion/rescue strategy to analyze the functional significance of cGKI isoforms in the regulation of the cytosolic Ca(2+) concentration by NO/cGMP in VSMCs. Cultured mouse aortic VSMCs endogenously expressed both cGKIalpha and cGKIbeta. The NO donor diethylamine NONOate (DEA-NO) and the membrane-permeable cGMP analogue 8-bromo-cGMP inhibited noradrenaline-induced Ca(2+) transients in wild-type VSMCs but not in VSMCs genetically deficient for both cGKIalpha and cGKIbeta. The defective Ca(2+) regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKIalpha and enhanced green fluorescent protein (EGFP) but not by a cGKIbeta-EGFP construct. Fluorescence imaging indicated that the cGKIalpha-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKIbeta-EGFP protein was more homogeneously distributed in the cytoplasm. These results suggest that one component of NO/cGMP-induced smooth muscle relaxation is the activation of the cGKIalpha isoform, which decreases the noradrenaline-stimulated cytosolic Ca(2+) level.

Active Rho Kinase (ROK-α) Associates with Insulin Receptor Substrate-1 and Inhibits Insulin Signaling in Vascular Smooth Muscle Cells

Recent studies from our laboratory have shown that insulin stimulates myosin-bound phosphatase (MBP) in vascular smooth muscle cells (VSMCs) by decreasing site-specific phosphorylation of the myosin-bound subunit (MBS) of MBP via nitric oxide/cGMP-mediated Rho/Rho kinase inactivation. Here we tested potential interactions between Rho kinase and insulin signaling pathways. In control VSMCs, insulin inactivates ROK-alpha, the major Rho kinase isoform in VSMCs, and inhibits thrombin-induced increase in ROK-alpha association with the insulin receptor substrate-1 (IRS-1). Hypertension (in spontaneous hypertensive rats) or expression of an active RhoA(V14) up-regulates Rho kinase activity and increases ROK-alpha/IRS-1 association resulting in IRS-1 serine phosphorylation that leads to inhibition of both insulin-induced IRS-1 tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3-kinase) activation. In contrast, expression of dominant negative RhoA or cGMP-dependent protein kinase type I alpha inactivates Rho kinase, abolishes ROK-alpha/IRS-1 association, and potentiates insulin-induced tyrosine phosphorylation and PI3-kinase activation leading to decreased MBS(T695) phosphorylation and decreased MBP inhibition. Collectively, these results suggest a novel function for ROK-alpha in insulin signal transduction at the level of IRS-1 and potential cross-talk between cGMP-dependent protein kinase type I alpha, Rho/Rho kinase signaling, and insulin signaling at the level of IRS-1/PI3-kinase.

Structure and function of the heat-stable enterotoxin receptor/guanylyl cyclase C.

Guanylyl cyclase C (GC-C) was found to function as the principal receptor for heat-stable enterotoxins (STa), major causative factors in E. coli-induced secretory diarrhea. GC-C is enriched in intestinal epithelium, but was also detected in other epithelial tissues. The enzyme belongs to the family of receptor guanylyl cyclases, and consists of an extracellular receptor domain, a single transmembrane domain, a kinase homology domain, and a catalytic domain. GC-C is modified by N-linked glycosylation and, at least in the small intestine, by proteolysis, resulting in a STa receptor that is coupled non-covalently to the intracellular domain. So far two endogenous ligands of mammalian GC-C have been identified i.e. the small cysteine-rich peptides guanylin and uroguanylin. The guanylins are released in an auto- or paracrine fashion into the intestinal lumen but may also function as endocrine hormones in gut-kidney communication and as regulators of ion transport in extra-intestinal epithelia. They are thought to activate GC-C by inducing a conformational change in the extracellular portion of the homotrimeric GC-C complex, which allows two of the three intracellular catalytic domains to dimerize and form two active catalytic clefts. In the intestine, activation of GC-C results in a dual action: stimulation of Cl and HCO3 secretion, through the opening of apical CFTR Cl channels; and inhibition of Na absorption, through blockade of an apical Na/H exchanger. The principal effector of the GC-C effect on ion transport is cGMP dependent protein kinase type II, which together with GC-C and the ion transporters, may form a supramolecular complex at the apical border of epithelial cells.

Localization of cGMP-dependent protein kinase type II in rat brain

In brain, signaling pathways initiated by atrial natriuretic peptide, or transmitters which stimulate nitric oxide synthesis, increase cGMP as their second messenger. One important class of target molecules for cGMP is cGMP-dependent protein kinases, and in the present study, biochemical and immunocytochemical analyses demonstrate the widespread distribution of type II cGMP-dependent protein kinase in rat brain, from the cerebral cortex to the brainstem and cerebellum. Also, colocalization of cGMP-dependent protein kinase type II with its activator, cGMP, was found in several brain regions examined after in vitro stimulation of brain slices with sodium nitroprusside. In western blots, cGMP-dependent protein kinase type II was observed in all brain regions examined, although cerebellar cortex and pituitary contained comparatively less of the kinase. Immunocytochemistry revealed cGMP-dependent protein kinase type II in certain neurons, and occasionally in putative oligodendrocytes and astrocytes, however, its most striking and predominant localization was in neuropil. Electron microscopy examination of neuropil in the medial habenula showed localization of the kinase in both axon terminals and dendrites. As a membrane-associated protein, cGMP-dependent protein kinase type II often appeared to be transported to cell processes to a greater extent than being retained in the cell body. Thus, immunocytochemical labeling of cGMP-dependent protein kinase type II often did not coincide with the localization of kinase mRNA previously observed by others using in situ hybridization. We conclude that in contrast to cGMP-dependent protein kinase type I, which has a very restricted localization to cerebellar Purkinje cells and a few other sites, cGMP-dependent protein kinase type II is a very ubiquitous brain protein kinase and thus a more likely candidate for relaying myriad cGMP effects in brain requiring protein phosphorylation.

Regulation of Human Endothelial Cell Focal Adhesion Sites and Migration by cGMP-dependent Protein Kinase I

cGMP-dependent protein kinase type I (cGK I), a major constituent of the atrial natriuretic peptide (ANP)/nitric oxide/cGMP signal transduction pathway, phosphorylates the vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins involved in regulation of the actin cytoskeleton. Here we demonstrate that stimulation of human umbilical vein endothelial cells (HUVECs) by both ANP and 8-(4-chlorophenylthio)guanosine 3':5'-monophosphate (8-pCPT-cGMP) activates transfected cGK I and causes detachment of VASP and its known binding partner (zyxin) from focal adhesions in >60% of cells after 30 min. The ANP effects, but not the 8-pCPT-cGMP effects, reversed after 3 h of treatment. In contrast, a catalytically inactive cGK Ibeta mutant (cGK Ibeta-K405A) was incapable of mediating these effects. VASP mutated (Ser/Thr to Ala) at all three of its established phosphorylation sites (vesicular stomatitis virus-tagged VASP-AAA mutant) was not phosphorylated by cGK I and was resistant to detaching from HUVEC focal adhesions in response to 8-pCPT-cGMP. Furthermore, activation of cGK I, but not of mutant cGK Ibeta-K405A, caused a 1.5-2-fold inhibition of HUVEC migration, a dynamic process highly dependent on focal adhesion formation and disassembly. These results indicate that cGK I phosphorylation of VASP results in loss of VASP and zyxin from focal adhesions, a response that could contribute to cGK alteration of cytoskeleton-regulated processes such as cell migration.

Serine 19 of Human 6-Pyruvoyltetrahydropterin Synthase Is Phosphorylated by cGMP Protein Kinase II

6-Pyruvoyltetrahydropterin synthase (PTPS) participates in tetrahydrobiopterin cofactor biosynthesis. We previously identified in a PTPS-deficient patient an inactive PTPS allele with an Arg(16) to Cys codon mutation. Arg(16) is located in the protein surface exposed phosphorylation motif Arg(16)-Arg-Ile-Ser, with Ser(19) as the putative phosphorylation site for serine-threonine protein kinases. Purification of recombinant PTPS-S19A from bacterial cells resulted in an active enzyme (k(cat)/K(m) = 6.4 x 10(3) M(-1) s(-1)), which was similar to wild-type PTPS (k(cat)/K(m) = 4.1 x 10(3) M(-1) s(-1)). In assays with purified enzymes, wild-type but not PTPS-S19A was a specific substrate for the cGMP-dependent protein kinase (cGK) type I and II. Upon expression in COS-1 cells, PTPS-S19A was stable but not phosphorylated and had a reduced activity of approximately 33% in comparison to wild-type PTPS. Extracts from several human cell lines, including brain, contained a kinase that bound to and phosphorylated immobilized wild-type, but not mutant PTPS. Addition of cGMP stimulated phosphotransferase activity 2-fold. Extracts from transfected COS-1 cells overexpressing cGKII stimulated Ser(19) phosphorylation more than 100-fold, but only 4-fold from cGKI overexpressing cells. Moreover, fibroblast extracts from mice lacking cGKII exhibited significantly reduced phosphorylation of PTPS. These results suggest that Ser(19) of human PTPS may be a substrate for cGKII phosphorylation also in vivo, a modification that is essential for normal activity.

Downregulation of soluble guanylyl cyclase in young and aging spontaneously hypertensive rats.

Endothelial dysfunction, as observed in hypertension and atherosclerosis, is associated with a reduction in the bioavailability of endothelium-derived nitric oxide (NO). We tested the hypothesis that alterations in the soluble guanylyl cyclase (sGC) pathway may also contribute to the pathogenesis of hypertension. Therefore, we investigated the expression and activity of sGC in young (6 weeks) and aging (17 months) spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto rats (WKY). Endothelium-independent relaxation of aortic rings in response to the sGC activator YC-1 was attenuated in SHR, and expression of both alpha(1) and beta(1) subunits of heterodimeric sGC and the basal contents of cGMP were reduced specifically in SHR aorta. Moreover, mRNA expression of the cGMP receptor and effector protein cGMP-dependent protein kinase type Ialpha (cGKIalpha) was also reduced. Interestingly, downregulation of both sGC and cGKIalpha expression was observed in young, ie, normotensive SHR, whereas impairment of the endothelium-independent relaxation was found only in aging SHR. Accordingly, similar cGMP levels were reached in response to YC-1 in young SHR and young WKY, suggesting a compensatory increased sensitivity or effectiveness of the sGC pathway in young SHR. In aging SHR, however, increased sensitivity to YC-1 no longer compensated for the impairment of endothelium-independent relaxation, suggesting that other mechanisms were involved. In fact, endothelium-independent relaxations were partially restored by superoxide dismutase, suggesting a pathophysiological role of superoxide production, particularly at later disease stages. Thus, tissue-specific downregulation of components of the sGC/cGMP pathway is an early event in the pathogenesis of hypertension.

Atrial natriuretic peptide-stimulated Ca2+ reabsorption in rabbit kidney requires membrane-targeted, cGMP-dependent protein kinase type II.

Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca2+ reabsorption via a novel mechanism specifically involving type II cGMP-dependent protein kinase (cGK II). ANP and the NO donor, sodium nitroprusside (SNP), markedly increased Ca2+ uptake in freshly immunodissected rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). Although readily increasing cGMP, ANP and SNP did not affect Ca2+ and Na+ reabsorption in primary cultures of these segments. Immunoblot analysis demonstrated that cGK II, and not cGK I, was present in freshly isolated CNT and CCD but underwent a complete down-regulation during the primary cell culture. However, upon adenoviral reexpression of cGK II in primary cultures, ANP, SNP, and 8-Br-cGMP readily increased Ca2+ reabsorption. In contrast, no cGMP-dependent effect on electrogenic Na+ transport was observed. The membrane localization of cGK II proved to be crucial for its action, because a nonmyristoylated cGK II mutant that was shown to be localized in the cytosol failed to mediate ANP-stimulated Ca2+ transport. The Ca2+-regulatory function of cGK II appeared isotype-specific because no cGMP-mediated increase in Ca2+ transport was observed after expression of the cytosolic cGK Ibeta or a membrane-bound cGK II/Ibeta chimer. These results demonstrate that ANP- and NO-stimulated Ca2+ reabsorption requires membrane-targeted cGK II.

Phosphorylation of vasodilator-stimulated phosphoprotein: a consequence of nitric oxide- and cGMP-mediated signal transduction in brain capillary endothelial cells and astrocytes

There is contradictory information on the relevance of nitric oxide (NO) and cGMP for the function of brain capillary endothelial cells (BCEC) forming the blood–brain barrier (BBB). Therefore, NO/cGMP-mediated signal transduction was investigated in cell cultures of BCEC and of astrocytes (AC) inducing BBB properties in BCEC. Constitutive, Ca 2+-activated isoforms of NO synthase (NOS) were found in BCEC (endothelial NOS: eNOS) and in AC (neuronal NOS: nNOS), leading to increased NO release after incubation with the Ca 2+-ionophore A23187. Both cell types expressed inducible NOS (iNOS) after incubation with cytokines. Soluble guanylate cyclase (sGC) was detected in both cell types. NO-dependent cGMP formation were observed in BCEC and, less pronounced, in AC. Furthermore, both cell types formed cGMP independently of NO via stimulation of particulate guanylate cyclase (pGC). cGMP-dependent protein kinase (PKG) type Iβ, but not type II, was expressed in BCEC and AC. In BCEC, vasodilator-stimulated phosphoprotein (VASP) was detected, an established substrate of PKG and associated with microfilaments and cell–cell contacts. Phosphorylation of VASP was intensified by increased intracellular cGMP concentrations. The results indicate that BCEC and, to a smaller degree, AC can form NO and cGMP in response to different stimuli. In BCEC, NO/cGMP-dependent phosphorylation of VASP is demonstrated, thus providing a possibility of influencing cell–cell contacts.

Regulation of sensory neuron precursor proliferation by cyclic GMP-dependent protein kinase.

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.

Expression of cGMP-dependent protein kinase I and its substrate VASP in neointimal cells of the injured rat carotid artery.

Neointimal fibroproliferative lesions after balloon angioplasty remain a major clinical problem, frequently leading to restenosis of initially successfully dilated coronary arteries. The cGMP-dependent protein kinase type I (cGMP-PK I) and its substrate vasodilator-stimulated phosphoprotein (VASP), molecular targets of the atrial natriuretic factor (ANF) and nitric oxide (NO) signalling pathways, are likely to be involved in various aspects of vascular wall regulation and restenosis formation. To investigate the occurrence of cGMP-PK I and VASP in neointimal cells in situ, we performed immunohistochemistry and immunoblotting experiments on denuded rat carotid arteries. Although the soluble cGMP-PK I showed a homogeneous distribution throughout the neointima, VASP apparently was more concentrated in smooth muscle cells (SMCs) lining the artery lumen, possibly reflecting enhanced growth factor stimulation of luminal SMCs. The membrane-associated cGMP-PK type II could not be detected in both the non-injured vessel wall and the restenotic tissue. The presence of both cGMP-PK I and VASP, major regulators of the actin cytoskeleton and cell motility, in neointimal tissue suggest that this emerging signal transduction pathway could be a target for the regulation and control of restenosis.

CGMP Stimulation of Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channels Co-expressed with cGMP-dependent Protein Kinase Type II but Not Type Iβ

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was analyzed after expression of cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I- efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Ibeta or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e. g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.

Guanosine 3',5'-cyclic monophosphate-dependent protein kinase II mediates heat-stable enterotoxin-provoked chloride secretion in rat intestine

BACKGROUND & AIMS: Escherichia coli heat-stable enterotoxins (STa) provoke electrogenic Cl- secretion in the intestine through a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent signal transduction pathway. The cGMP receptor involved in the activation of the Cl- channel is not known with certainty but may comprise either adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (cAK) or cGMP-dependent protein kinase (cGK) type II. The aim of this study was to discriminate between these possibilities using specific kinase inhibitors.METHODS: Intestinal electrogenic Cl- secretion was determined by measuring short-circuit current (Isc) in a Ussing chamber.RESULTS: The general protein kinase inhibitors staurosporine and H-8 inhibited rat cGK II activity in vitro with 50% inhibitory concentration values of 4 nmol/L and 3 mumol/L, respectively, which are lower than those reported for cAK. Both staurosporine and H-8, when added to rat proximal colon at concentrations that did not affect the Isc response to 8-bromo-cAMPS, inhibited the STa- and 8-bromo-cGMP- provoked Isc response for more than 80%. Furthermore, the relative specific cGK inhibitor Rp isomer of 8-(chlorophenylthio)-cGMP, but not the cAK inhibitor RP isomer of (Rp) 8-bromo-cAMPS, inhibited the Isc response to submaximal levels of STa in rat proximal colon.CONCLUSIONS: These data provide further evidence for an important role of cGK II in STa-mediated Cl- secretion in native rat intestinal epithelium.(Gastroenterology 1997 Feb;112(2):437-43)

Expression of the human cGMP-dependent protein kinase II gene is lost upon introduction of SV40 T antigen or immortalization in human cells

We have cloned a human cGMP-dependent protein kinase type II cDNA to examine its gene expression in terms of cellular senescence and/or immortalization. The genetic locus was mapped to band 4q21 by FISH. Northern blot analysis revealed that expression of the type II gene was markedly decreased or lost in mortal or immortal human fibroblasts producing SV40 T antigen. Also in various immortalized cell lines tested, the gene was not expressed. In normal diploid fibroblasts, the gene was constitutively expressed during cell-cycle and population doubling levels (PDLs).