cGMP-dependent kinase 2, Na+/H+ exchanger NHE3, and PDZ-adaptor NHERF2 co-assemble in apical membrane microdomains.

AIM: Trafficking, membrane retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII) within detergent-resistant membrane microdomains (DRMs, "lipid rafts") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine. METHODS: Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice, after oral application of the heat-stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X-solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo. RESULTS: In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide-induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII-deficient mice, and strongly reduced in NHERF2-deficient mice. CONCLUSION: NHE3, cGKII, and NHERF2 form a lipid raft-associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft-associated activated cGKII.

Changing the locks on intestinal signaling.

Endogenous peptides and structurally similar bacterial heat-stable enterotoxins (ST) bind guanylate cyclase-C (GC-C), resulting in fluid homeostasis or diarrhea, respectively. In this issue of Cell Host & Microbe, Carey et al., show how bats have evolutionarily maintained homeostatic signaling while avoiding pathogenic effects of ST.

Gut-associated cGMP mediates colitis and dysbiosis in a mouse model of an activating mutation in GUCY2C.

Activating mutations in receptor guanylyl cyclase C (GC-C), the target of gastrointestinal peptide hormones guanylin and uroguanylin, and bacterial heat-stable enterotoxins cause early-onset diarrhea and chronic inflammatory bowel disease (IBD). GC-C regulates ion and fluid secretion in the gut via cGMP production and activation of cGMP-dependent protein kinase II. We characterize a novel mouse model harboring an activating mutation in Gucy2c equivalent to that seen in an affected Norwegian family. Mutant mice demonstrated elevated intestinal cGMP levels and enhanced fecal water and sodium content. Basal and linaclotide-mediated small intestinal transit was higher in mutant mice, and they were more susceptible to DSS-induced colitis. Fecal microbiome and gene expression analyses of colonic tissue revealed dysbiosis, up-regulation of IFN-stimulated genes, and misregulation of genes associated with human IBD and animal models of colitis. This novel mouse model thus provides molecular insights into the multiple roles of intestinal epithelial cell cGMP, which culminate in dysbiosis and the induction of inflammation in the gut.

Diarrheal pathogens trigger rapid evolution of the guanylate cyclase-C signaling axis in bats.

The pathogenesis of infectious diarrheal diseases is largely attributed to enterotoxins that cause dehydration by disrupting intestinal water absorption. We investigated patterns of genetic variation in mammalian guanylate cyclase-C (GC-C), an intestinal receptor targeted by bacterially encoded heat-stable enterotoxins (STa), to determine how host species adapt in response to diarrheal infections. Our phylogenetic and functional analysis of GC-C supports long-standing evolutionary conflict with diarrheal bacteria in primates and bats, with highly variable susceptibility to STa across species. In bats, we further show that GC-C diversification has sparked compensatory mutations in the endogenous uroguanylin ligand, suggesting an unusual scenario of pathogen-driven evolution of an entire signaling axis. Together, these findings suggest that conflicts with diarrheal pathogens have had far-reaching impacts on the evolution of mammalian gut physiology.

The cGMP-Dependent Protein Kinase 2 Contributes to Cone Photoreceptor Degeneration in the Cnga3-Deficient Mouse Model of Achromatopsia.

Mutations in the CNGA3 gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the Cnga3 knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in Cnga3 KO mice. Virus-mediated knockdown or genetic ablation of Prkg2 in Cnga3 KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in Cnga3 KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies.

Expression of PKG2 in ovarian cancer and its effect on epidermal growth factor receptor.

PURPOSE: This research tried to explore the expression level of II cGMP-dependent protein kinase (PKG2) in human ovarian tissue and to clarify the molecular mechanism of EGFR regulation and its clinical significance. METHODS: The expression levels of PKG2 and EGFR in 10 normal ovarian tissues, 14 benign ovarian tumor tissues and 39 epithelial ovarian cancer tissues preserved in the archives of the Affiliated Hospital of Xuzhou Medical University from 2016 to 2018 were detected by real-time fluorescence quantitative (RT-PCR), and the correlation between the expressions of the two genes was analyzed. The expressions of in vitro cultured ovarian cancer cell lines SKOV3, PKG2 and EGFR were detected by RT-PCR and western blot, and the over-expressed PKG2 plasmid and PKG2 small interfering RNA (siRNA) were transfected into the cells, and the protein and phosphorylation of Akt and ERK in EGFR and its downstream signaling pathway were detected by western blot. RESULTS: Compared with normal ovarian tissue, the mRNA and protein expression levels of PKG2 in ovarian cancer tissue and SKOV3 cell line were significantly reduced (p<0.05). However, the mRNA and protein expression levels of EGFR in ovarian cancer tissue and SKOV3 cell line were both high (p<0.05). In addition, after transient transfection of PKG2, the expression changes of PKG2 significantly affected the expression of EGFR, and PKG2 over-expression could significantly inhibit the phosphorylation of Akt and ERK in EGFR and its downstream signaling pathways, thereby affecting cell proliferation. CONCLUSION: PKG2 may play a role in inhibiting EGFR expression in ovarian cancer, but the specific mechanism of its effect on tumor development still needs to be further explored.

Protein synthesis inhibition promotes nitric oxide generation and activation of CGKII-dependent downstream signaling pathways in the retina.

Nitric oxide is an important neuromodulator in the CNS, and its production within neurons is modulated by NMDA receptors and requires a fine-tuned availability of L-arginine. We have previously shown that globally inhibiting protein synthesis mobilizes intracellular L-arginine "pools" in retinal neurons, which concomitantly enhances neuronal nitric oxide synthase-mediated nitric oxide production. Activation of NMDA receptors also induces local inhibition of protein synthesis and L-arginine intracellular accumulation through calcium influx and stimulation of eucariotic elongation factor type 2 kinase. We hypothesized that protein synthesis inhibition might also increase intracellular L-arginine availability to induce nitric oxide-dependent activation of downstream signaling pathways. Here we show that nitric oxide produced by inhibiting protein synthesis (using cycloheximide or anisomycin) is readily coupled to AKT activation in a soluble guanylyl cyclase and cGKII-dependent manner. Knockdown of cGKII prevents cycloheximide or anisomycin-induced AKT activation and its nuclear accumulation. Moreover, in retinas from cGKII knockout mice, cycloheximide was unable to enhance AKT phosphorylation. Indeed, cycloheximide also produces an increase of ERK phosphorylation which is abrogated by a nitric oxide synthase inhibitor. In summary, we show that inhibition of protein synthesis is a previously unanticipated driving force for nitric oxide generation and activation of downstream signaling pathways including AKT and ERK in cultured retinal cells. These results may be important for the regulation of synaptic signaling and neuronal development by NMDA receptors as well as for solving conflicting data observed when using protein synthesis inhibitors for studying neuronal survival during development as well in behavior and memory studies.

cGMP-dependent protein kinase II determines ß-catenin accumulation that is essential for uterine decidualization in mice.

In the early phase of pregnancy, decidualization is an indispensable event after mammal embryo implantation, accompanied by proliferation and differentiation of uterine stromal cells. Type II cGMP-dependent protein kinase (Prkg2) belongs to the family of serine/threonine kinase, which plays multiple roles in cellular signaling pathways to control proliferation and differentiation. However, the regulatory function and molecular mechanism of Prkg2 in decidualization are still unknown. In this study, we show that Prkg2 has a gradually increased expression pattern during peri-implantation and artificial decidualization, and the expression of Prkg2 is induced by estrogen and progesterone in the ovariectomized mouse uteri and primary cultured uterine stromal cells, the process of which is blocked by treating with estrogen receptor (ER) antagonist (ICI-182,780) and progesterone receptor (PR) antagonist (RU-486). Inhibition of Prkg2 activity by HA-100 promotes uterine stromal cell proliferation but compromises decidualization with decreased expression of prolactin family 8, subfamily a, member 2. In addition, the functional regulation of decidualization by Prkg2 is accomplished by its induced phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) at serine-9, which results in accumulation of ß-catenin in the decidual cells. Taken together, our findings demonstrate that estrogen and progesterone upregulate the expression of Prkg2 in uterine stromal cells depending on ER and PR; Prkg2 promotes phosphorylation of GSK-3ß at serine-9 and inactivates it, leading to the accumulation of ß-catenin and promoting the process of decidualization. In addition to revealing the regulatory mechanism of Prkg2 that ensures the success of uterine decidualization, our findings will contribute to the understanding in the maintenance of early pregnancy.

HIV induces synaptic hyperexcitation via cGMP-dependent protein kinase II activation in the FIV infection model.

Over half of individuals infected with human immunodeficiency virus (HIV) suffer from HIV-associated neurocognitive disorders (HANDs), yet the molecular mechanisms leading to neuronal dysfunction are poorly understood. Feline immunodeficiency virus (FIV) naturally infects cats and shares its structure, cell tropism, and pathology with HIV, including wide-ranging neurological deficits. We employ FIV as a model to elucidate the molecular pathways underlying HIV-induced neuronal dysfunction, in particular, synaptic alteration. Among HIV-induced neuron-damaging products, HIV envelope glycoprotein gp120 triggers elevation of intracellular Ca2+ activity in neurons, stimulating various pathways to damage synaptic functions. We quantify neuronal Ca2+ activity using intracellular Ca2+ imaging in cultured hippocampal neurons and confirm that FIV envelope glycoprotein gp95 also elevates neuronal Ca2+ activity. In addition, we reveal that gp95 interacts with the chemokine receptor, CXCR4, and facilitates the release of intracellular Ca2+ by the activation of the endoplasmic reticulum (ER)-associated Ca2+ channels, inositol triphosphate receptors (IP3Rs), and synaptic NMDA receptors (NMDARs), similar to HIV gp120. This suggests that HIV gp120 and FIV gp95 share a core pathological process in neurons. Significantly, gp95's stimulation of NMDARs activates cGMP-dependent protein kinase II (cGKII) through the activation of the neuronal nitric oxide synthase (nNOS)-cGMP pathway, which increases Ca2+ release from the ER and promotes surface expression of AMPA receptors, leading to an increase in synaptic activity. Moreover, we culture feline hippocampal neurons and confirm that gp95-induced neuronal Ca2+ overactivation is mediated by CXCR4 and cGKII. Finally, cGKII activation is also required for HIV gp120-induced Ca2+ hyperactivation. These results thus provide a novel neurobiological mechanism of cGKII-mediated synaptic hyperexcitation in HAND.

PKG II inhibits PDGF-BB triggered biological activities by phosphorylating PDGFRß in gastric cancer cells.

Previous studies revealed that type II cGMP-dependent protein kinase G (PKG II) could inhibit the activation of epidermal growth factor receptor (EGFR) which is a widely investigated RTK. PDGFR belongs to family of receptor tyrosine kinases (RTKs) too. However, the effect of PKG II on PDGFR activation is not clear yet. This study investigated potential regulatory effect of PKG II on activation of PDGFRß and the downstream signaling transductions in gastric cancer. The results from CCK8 assay and Transwell assay indicated that PDGF-BB induced cell proliferation and migration. Activated PKG II reversed the above variations caused by PDGF-BB. Immunoprecipitation and Western blotting results showed that PKG II combined with PDGFRß and phosphorylated this receptor, and thereby inhibited PDGF-BB induced activation of PDGFRß, and MAPK/ERK and PI3K/Akt mediated signal transduction pathways. Based on the prediction by phosphorylation site software, Ser643 and Ser712 were mutated to alanine respectively which prevented phosphorylation at these sites. Mutation at Ser712 abolished the inhibitory function of PKG II on PDGFRß activation but mutation of Ser643 had no such an effect, indicating that Ser712 was PKG II-specific phosphorylating site of PDGFRß. In conclusion, PKG II inhibited PDGFRß activation in gastric cancer via phosphorylating Ser712 of this RTK.

Cyclic Guanosine Monophosphate (cGMP)-Dependent Protein Kinase II Blocks Epidermal Growth Factor (EGF)/Epidermal Growth Factor Receptor (EGFR)-Induced Biological Effects on Osteosarcoma Cells.

BACKGROUND The present work was performed to detect the potential inhibitory effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase II (PKG II) on epidermal growth factor (EGF) receptor-induced biological activity and related signal cascades in osteosarcoma cells. MATERIAL AND METHODS We transfected the osteosarcoma MG-63 cell line with an adenoviral vector encoding PKG II cDNA (Ad-PKGII) and incubated the transfected cells with 250 µM 8-pCPT-cGMP to activate the PKG II. We stimulated the MG-63 cells with100 ng/ml EGF, and then detected their proliferation using a CCK-8 assay. Transwell assay was used to examine MG-63 cell migration; and Western blot analysis was used to detect expression of matrix metalloproteinase 9 (MMP-9) and activation of ERK and Akt. RESULTS Stimulating cells by 100 ng/ml EGF promoted MG-63 cell proliferation and migration, ERK and Akt phosphorylation, and MMP-9 expression. These effects of EGF were inhibited in MG-63 cells infected with Ad-PKGII and incubated with 8-pCPT-cGMP. CONCLUSIONS Our results demonstrate that Ad-PKGII infection significantly inhibited EGF-induced proliferation and migration, as well as the associated-signal cascades; which indicates that PKG II might be a potential anti-cancer factor.

Inhibition of Cgkii Suppresses Seizure Activity and Hippocampal Excitation by Regulating the Postsynaptic Delivery of Glua1.

BACKGROUND/AIMS: The imbalance between excitation and inhibition is a defining feature of epilepsy. GluA1 is an AMPA receptor subunit that can strengthen excitatory synaptic transmission when upregulated in the postsynaptic membrane, which has been implicated in the pathogenesis of epilepsy. cGKII, a cGMP-dependent protein kinase, regulates the GluA1 levels at the plasma membrane. METHODS: To explore the role of cGKII in epilepsy, we investigated the expression of cGKII in patients with temporal lobe epilepsy (TLE) and in a pilocarpine-induced rat model and then performed behavioral, histological, and electrophysiological analyses by applying either a cGKII agonist or inhibitor in the hippocampus of the animal model. RESULTS: cGKII expression was upregulated in the epileptogenic brain tissues of both humans and rats. Pharmacological activation or inhibition of cGKII induced changes in epileptic behaviors in vivo and epileptic discharges in vitro. Further studies indicated that cGKII activation disrupted the balance of excitation and inhibition due to strengthened AMPAR-mediated excitatory synaptic transmission. Moreover, cGKII regulated epileptic seizures by phosphorylating GluA1 at Ser845 to modulate the expression and function of GluA1 in the postsynaptic membrane. CONCLUSION: These results suggest that cGKII plays a key role in seizure activity and could be a potential therapeutic target for epilepsy.

Transepithelial Fluid and Salt Re-Absorption Regulated by cGK2 Signals.

Transepithelial fluid and salt re-absorption in epithelial tissues play an important role in fluid and salt homeostasis. In absorptive epithelium, fluid and salt flux is controlled by machinery mainly composed of epithelial sodium channels (ENaC), cystic fibrosis transmembrane conductance regulator (CFTR), Na⁺/H⁺ exchanger (NHE), aquaporin, and sodium potassium adenosine triphosphatase (Na⁺/K⁺-ATPase). Dysregulation of fluid and salt transport across epithelium contributes to the pathogenesis of many diseases, such as pulmonary edema and cystic fibrosis. Intracellular and extracellular signals, i.e., hormones and protein kinases, regulate fluid and salt turnover and resolution. Increasing evidence demonstrates that transepithelial fluid transport is regulated by cyclic guanosine monophosphate-dependent protein kinase (cGK) signals. cGK2 was originally identified and cloned from intestinal specimens, the presence of which has also been confirmed in the kidney and the lung. cGK2 regulates fluid and salt through ENaC, CFTR and NHE. Deficient cGK2 regulation of transepithelial ion transport was seen in acute lung injury, and cGK2 could be a novel druggable target to restore edematous disorder in epithelial tissues.

PKG II effectively reversed EGF-induced protein expression alterations in human gastric cancer cell lines.

Epidermal growth factor receptor (EGFR) plays an important role in gastric cancer (GC) progression. Our previous data demonstrated that type II cGMP-dependent protein kinase (PKG II) could block the EGF-EGFR axis as well as down-stream signaling pathways, for example, MAPK, PI3 K, and PLC in GC cells. However, the exact mechanisms of PKG II against cancer remain unclear. Therefore, the present work was to address the above question. Human GC cell line AGS was infected with adenoviral construct encoding cDNA of PKG II (Ad-PKG II) to up-regulate PKG II and then treated with 8-pCPT-cGMP. Two-dimensional electrophoresis (2-DE) was used to analyze the changes of protein expression in the cells. The results showed that 17 proteins had more than twofold changes in EGF-treated group compared with control. However, Ad-PKG II could effectively reversed the changes. Furthermore, far upstream element-binding protein 1 (FUBP1) and MarvelD3 were chosen and PKG II activation reversed EGF/EGFR-induced up-regulation of FUBP1 and downregulation of MarvelD3, respectively. MarvelD3 silence effectively abolished the inhibitory effect of PKG II on EGF-triggered migration. These data indicated that the inhibitory effect of PKG II partially was associated with MarvelD3.

Structural Basis of Analog Specificity in PKG I and II.

Cyclic GMP analogs, 8-Br, 8-pCPT, and PET-cGMP, have been widely used for characterizing cellular functions of cGMP-dependent protein kinase (PKG) I and II isotypes. However, interpreting results obtained using these analogs has been difficult due to their low isotype specificity. Additionally, each isotype has two binding sites with different cGMP affinities and analog selectivities, making understanding the molecular basis for isotype specificity of these compounds even more challenging. To determine isotype specificity of cGMP analogs and their structural basis, we generated the full-length regulatory domains of PKG I and II isotypes with each binding site disabled, determined their affinities for these analogs, and obtained cocrystal structures of both isotypes bound with cGMP analogs. Our affinity and activation measurements show that PET-cGMP is most selective for PKG I, whereas 8-pCPT-cGMP is most selective for PKG II. Our structures of cyclic nucleotide binding (CNB) domains reveal that the B site of PKG I is more open and forms a unique π/π interaction through Arg285 at ß4 with the PET moiety, whereas the A site of PKG II has a larger ß5/ß6 pocket that can better accommodate the bulky 8-pCPT moiety. Our structural and functional results explain the selectivity of these analogs for each PKG isotype and provide a starting point for the rational design of isotype selective activators.

Type II cGMP­dependent protein kinase inhibits the migration, invasion and proliferation of several types of human cancer cells.

Previous studies have indicated that type II cyclic guanosine monophosphate (cGMP)­dependent protein kinase (PKG II) could inhibit the proliferation and migration of gastric cancer cells. However, the effects of PKG II on the biological functions of other types of cancer cells remain to be elucidated. Therefore, the aim of the present study was to investigate the effects of PKG II on cancer cells derived from various types of human tissues, including A549 lung, HepG2 hepatic, OS­RC­2 renal, SW480 colon cancer cells and U251 glioma cells. Cancer cells were infected with adenoviral constructs coding PKG II (Ad­PKG II) to up­regulate PKG II expression, and treated with 8­(4­chlorophenylthio) (8­pCPT)­cGMP to activate the kinase. A Cell Counting kit 8 assay was used to detect cell proliferation. Cell migration was measured using a Transwell assay, whereas a terminal deoxynucleotidyl transferase 2'­deoxyuridine, 5'­triphosphate nick­end labeling assay was used to detect cell apoptosis. A pull­down assay was used to investigate the activation of Ras­related C3 botulinum toxin substrate (Rac) 1 and western blotting was used to detect the expression of proteins of interest. The present results demonstrated that EGF (100 ng/ml, 24 h) promoted the proliferation and migration of cancer cells, and it suppressed their apoptosis. In addition, treatment with EGF enhanced the activation of Rac1, and up­regulated the protein expression of proliferating cell nuclear antigen, matrix metalloproteinase (MMP)2, MMP7 and B­cell lymphoma (Bcl)­2, whereas it down­regulated the expression of Bcl­2­associated X protein. Transfection of cancer cells with Ad­PKG II, and PKG II activation with 8­pCPT­cGMP, was identified to counteract the effects triggered by EGF. The present results suggested that PKG II may exert inhibitory effects on the proliferation and migration of various types of cancer cells.

Linaclotide activates guanylate cyclase-C/cGMP/protein kinase-II-dependent trafficking of CFTR in the intestine.

The transmembrane receptor guanylyl cyclase-C (GC-C), expressed on enterocytes along the intestine, is the molecular target of the GC-C agonist peptide linaclotide, an FDA-approved drug for treatment of adult patients with Irritable Bowel Syndrome with Constipation and Chronic Idiopathic Constipation. Polarized human colonic intestinal cells (T84, CaCo-2BBe) rat and human intestinal tissues were employed to examine cellular signaling and cystic fibrosis transmembrane conductance regulator (CFTR)-trafficking pathways activated by linaclotide using confocal microscopy, in vivo surface biotinylation, and protein kinase-II (PKG-II) activity assays. Expression and activity of GC-C/cGMP pathway components were determined by PCR, western blot, and cGMP assays. Fluid secretion as a marker of CFTR cell surface translocation was determined using in vivo rat intestinal loops. Linaclotide treatment (30 min) induced robust fluid secretion and translocation of CFTR from subapical compartments to the cell surface in rat intestinal loops. Similarly, linaclotide treatment (30 min) of T84 and CaCo-2BBe cells increased cell surface CFTR levels. Linaclotide-induced activation of the GC-C/cGMP/PKGII signaling pathway resulted in elevated intracellular cGMP and pVASPser239 phosphorylation. Inhibition or silencing of PKGII significantly attenuated linaclotide-induced CFTR trafficking to the apical membrane. Inhibition of protein kinase-A (PKA) also attenuated linaclotide-induced CFTR cell surface trafficking, implying cGMP-dependent cross-activation of PKA pathway. Together, these findings support linaclotide-induced activation of the GC-C/cGMP/PKG-II/CFTR pathway as the major pathway of linaclotide-mediated intestinal fluid secretion, and that linaclotide-dependent CFTR activation and recruitment/trafficking of CFTR from subapical vesicles to the cell surface is an important step in this process.

Type II cGMP-dependent protein kinase negatively regulates fibroblast growth factor signaling by phosphorylating Raf-1 at serine 43 in rat chondrosarcoma cells.

Although type II cGMP-dependent protein kinase (PKGII) is a major downstream effector of cGMP in chondrocytes and attenuates the FGF receptor 3/ERK signaling pathway, its direct target proteins have not been fully explored. In the present study, we attempted to identify PKGII-targeted proteins, which are associated with the inhibition of FGF-induced MAPK activation. Although FGF2 stimulation induced the phosphorylation of ERK1/2, MEK1/2, and Raf-1 at Ser-338 in rat chondrosarcoma cells, pretreatment with a cell-permeable cGMP analog strongly inhibited their phosphorylation. On the other hand, Ser-43 of Raf-1 was phosphorylated by cGMP in a dose-dependent manner. Therefore, we examined the direct phosphorylation of Raf-1 by PKGII. Wild-type PKGII phosphorylated Raf-1 at Ser-43 in a cGMP-dependent manner, but a PKGII D412A/R415A mutant, which has a low affinity for cGMP, did not. Finally, we found that a phospho-mimic mutant, Raf-1 S43D, suppressed FGF2-induced MAPK pathway. These results suggest that PKGII counters FGF-induced MEK/ERK activation through the phosphorylation of Raf-1 at Ser-43 in chondrocytes.

AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2.

K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function.

Type II cGMP­dependent protein kinase inhibits EGF­induced JAK/STAT signaling in gastric cancer cells.

Previous research has demonstrated that type II cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF)­initiated signal transduction of MAPK­mediated, PI3K/Akt­mediated and PLCγ1­mediated pathways through blocking EGF­induced phosphorylation/activation of EGF receptor (EGFR). As EGF/EGFR signaling also initiated signal transduction of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT)­mediated pathway, the present study was performed to investigate whether PKG II exerts an inhibitory effect this pathway. AGS human gastric cancer cell line was infected with adenoviral constructs encoding the cDNA of PKG II (Ad­PKG II), to increase the expression of PKG II, and treated with 8­pCPT­cGMP to activate the kinase. Western blotting was performed to detect the phosphorylation/activation of EGFR, JAK1, JAK2, STAT1 and STAT3 and the expression of cell cycle­associated proteins, including cyclin D1 and cyclin E. EGF­induced cell cycle changes were detected by flow cytometry. Transcriptional activity was determined by a reporter gene assay. The results demonstrated that EGF treatment increased the phosphorylation of EGFR, JAK1, JAK2, STAT1 and STAT3, increased the expression levels of cyclin D1 and cyclin E, promoted the cells to enter S phase, and stimulated transcriptional activity in the cells. Increased PKG II activity through infecting the cells with Ad­PKG II and activating the kinase with 8­pCPT­cGMP efficiently reversed the changes caused by EGF. The results suggest that PKG II inhibits EGF­induced signal transduction of the JAK/STAT­mediated pathway and further confirms that PKG II may be a cancer inhibitor.