cGMP-dependent Protein Kinase Type Research Articles

α1G-dependent T-type Ca2+ current antagonizes cardiac hypertrophy through a NOS3-dependent mechanism in mice

In noncontractile cells, increases in intracellular Ca2+ concentration serve as a second messenger to signal proliferation, differentiation, metabolism, motility, and cell death. Many of these Ca2+-dependent regulatory processes operate in cardiomyocytes, although it remains unclear how Ca2+ serves as a second messenger given the high Ca2+ concentrations that control contraction. T-type Ca2+ channels are reexpressed in adult ventricular myocytes during pathologic hypertrophy, although their physiologic function remains unknown. Here we generated cardiac-specific transgenic mice with inducible expression of alpha1G, which generates Cav3.1 current, to investigate whether this type of Ca2+ influx mechanism regulates the cardiac hypertrophic response. Unexpectedly, alpha1G transgenic mice showed no cardiac pathology despite large increases in Ca2+ influx, and they were even partially resistant to pressure overload-, isoproterenol-, and exercise-induced cardiac hypertrophy. Conversely, alpha1G-/- mice displayed enhanced hypertrophic responses following pressure overload or isoproterenol infusion. Enhanced hypertrophy and disease in alpha1G-/- mice was rescued with the alpha1G transgene, demonstrating a myocyte-autonomous requirement of alpha1G for protection. Mechanistically, alpha1G interacted with NOS3, which augmented cGMP-dependent protein kinase type I activity in alpha1G transgenic hearts after pressure overload. Further, the anti-hypertrophic effect of alpha1G overexpression was abrogated by a NOS3 inhibitor and by crossing the mice onto the Nos3-/- background. Thus, cardiac alpha1G reexpression and its associated pool of T-type Ca2+ antagonize cardiac hypertrophy through a NOS3-dependent signaling mechanism.

Response to Letter Regarding Article, “Ventricular Phosphodiesterase-5 Expression Is Increased in Patients With Advanced Heart Failure and Contributes to Adverse Ventricular Remodeling After Myocardial Infarction in Mice”

We appreciate the comments by Cingolani et al on a possible involvement of Na+/H+ exchanger (NHE-1) in signaling mechanisms downstream of cGMP that can mediate cardioprotection after myocardial infarction. These authors previously reported that NHE-1 expression and activity are increased after myocardial infarction in rats (as a result of myocardial acidosis) and that sustained …

CGMP-dependent protein kinase I, the circadian clock, sleep and learning

The second messenger cGMP controls cardiovascular and gastrointestinal homeostasis in mammals. However, its physiological relevance in the nervous system is poorly understood.1 Now, we have reported that the cGMP-dependent protein kinase type I (PRKG1) is implicated in the regulation of the timing and quality of sleep and wakefulness.2 Prkg1 mutant mice showed altered distribution of sleep and wakefulness as well as reduction in rapid-eye-movement sleep (REMS) duration and in non-REMS consolidation. Furthermore, the ability to sustain waking episodes was compromised. These observations were also reflected in wheel-running and drinking activity. A decrease in electroencephalogram power in the delta frequency range (1–4 Hz) under baseline conditions was observed, which was normalized after sleep deprivation. Together with the finding that circadian clock amplitude is reduced in Prkg1 mutants these results indicate a decrease of the wake-promoting output of the circadian system affecting sleep. Because quality of sleep might affect learning we tested Prkg1 mutants in several learning tasks and find normal spatial learning but impaired object recognition memory in these animals. Our findings indicate that Prkg1 impinges on circadian rhythms, sleep and distinct aspects of learning.

MiR‐21 plays a Role in Smooth Muscle Cell Proliferation induced by Hypoxia

Chronic hypoxia is an important contributing factor to the development of pulmonary hypertension. Hypoxia triggers pulmonary vascular smooth muscle cell (PVSMC) remodeling from a differentiated state to a proliferative, dedifferentiated state. cGMP‐dependent protein kinase type 1 (Prkg1) is involved in the regulation of vasomotor tone and remodelling. MicroRNAs play important roles in various biologic processes in cells, where they regulate apoptosis, proliferation, differentiation, development & metabolism. However, the role of microRNAs in hypoxia‐mediated Prkg1 down‐regulation in smooth muscle cells and SMC proliferation is unknown. The objective of the study was to characterize miRNA regulation in PVSMC response to hypoxia, affecting cell proliferation. This is important in understanding the molecular mechanisms triggered by cell exposure to low oxygen tension in Persistent Pulmonary Hypertension (PPH) in neonates. We used human PVSMCs as an in vitro model system and studied the effects of hypoxic (3% oxygen) exposure. Cells were processed for mRNA, protein and microRNA studies. We found reduced mRNA and protein expression of SMC markers such as α‐Smooth muscle actin and calponin as well as Prkg1. Quantitative PCR analyses showed that 24h hypoxia treatment induced miR‐21 upregulation. Our studies suggest that miR‐21 plays a role in hypoxia‐induced smooth muscle cell proliferation. This work was supported by R01 HL075187‐05A1

CGMP-Dependent Protein Kinase Type I Is Implicated in the Regulation of the Timing and Quality of Sleep and Wakefulness

Many effects of nitric oxide (NO) are mediated by the activation of guanylyl cyclases and subsequent production of the second messenger cyclic guanosine-3′,5′-monophosphate (cGMP). cGMP activates cGMP-dependent protein kinases (PRKGs), which can therefore be considered downstream effectors of NO signaling. Since NO is thought to be involved in the regulation of both sleep and circadian rhythms, we analyzed these two processes in mice deficient for cGMP-dependent protein kinase type I (PRKG1) in the brain. Prkg1 mutant mice showed a strikingly altered distribution of sleep and wakefulness over the 24 hours of a day as well as reductions in rapid-eye-movement sleep (REMS) duration and in non-REM sleep (NREMS) consolidation, and their ability to sustain waking episodes was compromised. Furthermore, they displayed a drastic decrease in electroencephalogram (EEG) power in the delta frequency range (1–4 Hz) under baseline conditions, which could be normalized after sleep deprivation. In line with the re-distribution of sleep and wakefulness, the analysis of wheel-running and drinking activity revealed more rest bouts during the activity phase and a higher percentage of daytime activity in mutant animals. No changes were observed in internal period length and phase-shifting properties of the circadian clock while chi-squared periodogram amplitude was significantly reduced, hinting at a less robust oscillator. These results indicate that PRKG1 might be involved in the stabilization and output strength of the circadian oscillator in mice. Moreover, PRKG1 deficiency results in an aberrant pattern, and consequently a reduced quality, of sleep and wakefulness, possibly due to a decreased wake-promoting output of the circadian system impinging upon sleep.

The Commonly Used cGMP-dependent Protein Kinase Type I (cGKI) Inhibitor Rp-8-Br-PET-cGMPS Can Activate cGKI in Vitro and in Intact Cells

Small-molecule modulators of cGMP signaling are of interest to basic and clinical research. The cGMP-dependent protein kinase type I (cGKI) is presumably a major mediator of cGMP effects, and the cGMP analogue Rp-8-Br-PET-cGMPS (Rp-PET) (chemical name: beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer) is currently considered one of the most permeable, selective, and potent cGKI inhibitors available for intact cell studies. Here, we have evaluated the properties of Rp-PET using cGKI-expressing and cGKI-deficient primary vascular smooth muscle cells (VSMCs), purified cGKI isozymes, and an engineered cGMP sensor protein. cGKI activity in intact VSMCs was monitored by cGMP/cGKI-stimulated cell growth and phosphorylation of vasodilator-stimulated phosphoprotein. Unexpectedly, Rp-PET (100 microm) did not efficiently antagonize activation of cGKI by the agonist 8-Br-cGMP (100 microm) in intact VSMCs. Moreover, in the absence of 8-Br-cGMP, Rp-PET (100 microm) stimulated cell growth in a cGKIalpha-dependent manner. Kinase assays with purified cGKI isozymes confirmed the previously reported inhibition of the cGMP-stimulated enzyme by Rp-PET in vitro. However, in the absence of the agonist cGMP, Rp-PET partially activated the cGKIalpha isoform. Experiments with a fluorescence resonance energy transfer-based construct harboring the cGMP binding sites of cGKI suggested that binding of Rp-PET induces a conformational change similar to the agonist cGMP. Together, these findings indicate that Rp-PET is a partial cGKIalpha agonist that under certain conditions stimulates rather than inhibits cGKI activity in vitro and in intact cells. Data obtained with Rp-PET as cGKI inhibitor should be interpreted with caution and not be used as sole evidence to dissect the role of cGKI in signaling processes.

Cyclic Guanosine Monophosphate-dependent Protein Kinase I Promotes Adhesion of Primary Vascular Smooth Muscle Cells

The cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase type I (cGKI) pathway regulates many cellular functions. The current study shows that 8-Br-cGMP stimulates the number of attached primary but not that of subcultured murine vascular smooth muscle cells (VSMCs). These effects of 8-Br-cGMP require the presence of cGKI. In agreement with previous studies, cGKI inhibited the number of cells in repeatedly passaged murine VSMCs. Activation of the cGMP/cGKI pathway in freshly isolated primary VSMCs slightly decreased apoptosis and strongly increased cell adhesion. The stimulation of cell adhesion by cGKI involves an inhibition of the RhoA/Rho kinase pathway and increased exposure of beta(1) and beta(3) integrins on the cell surface. Together, these results identify a novel proadhesive function of cGMP/cGKI signaling in primary VSMCs and suggest that the opposing effects of this pathway on VSMC number depend on the phenotypic context of the cells.

Anemia and splenomegaly in cGKI-deficient mice

To explore the functional significance of cGMP-dependent protein kinase type I (cGKI) in the regulation of erythrocyte survival, gene-targeted mice lacking cGKI were compared with their control littermates. By the age of 10 weeks, cGKI-deficient mice exhibited pronounced anemia and splenomegaly. Compared with control mice, the cGKI mutants had significantly lower red blood cell count, packed cell volume, and hemoglobin concentration. Anemia was associated with a higher reticulocyte number and an increase of plasma erythropoietin concentration. The spleens of cGKI mutant mice were massively enlarged and contained a higher fraction of Ter119(+) erythroid cells, whereas the relative proportion of leukocyte subpopulations was not changed. The Ter119(+) cGKI-deficient splenocytes showed a marked increase in annexin V binding, pointing to phosphatidylserine (PS) exposure at the outer membrane leaflet, a hallmark of suicidal erythrocyte death or eryptosis. Compared with control erythrocytes, cGKI-deficient erythrocytes exhibited in vitro a higher cytosolic Ca(2+) concentration, a known trigger of eryptosis, and showed increased PS exposure, which was paralleled by a faster clearance in vivo. Together, these results identify a role of cGKI as mediator of erythrocyte survival and extend the emerging concept that cGMP/cGKI signaling has an antiapoptotic/prosurvival function in a number of cell types in vivo.

Role of Smooth Muscle cGMP/cGKI Signaling in Murine Vascular Restenosis

Nitric oxide (NO) is of crucial importance for smooth muscle cell (SMC) function and exerts numerous, and sometimes opposing, effects on vascular restenosis. Although cGMP-dependent protein kinase type I (cGKI) is a principal effector of NO, the molecular pathway of vascular NO signaling in restenosis is unclear. The purpose of this study was to examine the functional role of the smooth muscle cGMP/cGKI signaling cascade in restenosis of vessels. Tissue-specific mouse mutants were generated in which the cGKI protein was ablated in SMCs. We investigated whether the absence of cGKI in SMCs would affect vascular remodeling after carotid ligation or removal of the endothelium. No differences were detected between the tissue-specific cGKI mutants and control mice at different time points after vascular injury on a normolipidemic or apoE-deficient background. In line with these results, chronic drug treatment of injured control mice with the phosphodiesterase-5 inhibitor sildenafil elevated cGMP levels but had no influence on the ligation-induced remodeling. The genetic and pharmacological manipulation of the cGMP/cGKI signaling indicates that this pathway is not involved in the protective effects of NO, suggesting that NO affects vascular remodeling during restenosis via alternative mechanisms.

Activation of HIV-1 expression and replication by cGMP dependent protein kinase type 1-β (PKG1β)

The effect of cGMP (cyclic GMP) dependent protein kinase 1-β (PKG1-β) and cGMP analogues on transcriptional activity and replication of human immunodeficiency virus type 1 (HIV-1) was investigated. Transfection of PKG1β expression plasmid increased expression from an HIV-1 LTR-reporter as well as from an infectious HIV-1 molecular clone, pNL4-3. Treatment of HIV-1 AD8-infected monocyte derived macrophages (MDMs) with cGMP agonists and cGMP antagonists caused respectively increased and decreased virus replication. These findings provide evidence that cGMP and PKG serve to regulate HIV-1 infection in human cells.

Activation of the cGMP/cGKI signalling pathway in primary murine VSMCs accelerates growth

The endogenous signaling molecule nitric oxide (NO) exerts many of its actions via the cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase type I (cGKI) pathway. Recently, the analysis of an in vivo mouse model for atherosclerosis suggested that activation of cGKI in vascular smooth muscle cells (VSMCs) promotes the phenotypic modulation of medial VSMCs and, thus, vascular lesion formation. In contrast, many in vitro studies demonstrate an anti-proliferative role for cGKI. In the current study, primary and subcultured wild-type and cGKI-deficient VSMCs in response to the membrane permeable cGMP analogue 8-Br-cGMP were compared. In agreement with the common view an anti-proliferative effect of cGKI was found in repeatedly passaged VSMCs (P5–11). In contrast, the analysis of primary VSMCs revealed that activation of cGKI in primary VSMCs strongly promotes growth. Analysing proliferation, apoptosis, cytoskeletal dynamics and various signaling pathways indicated that an increase in cell adhesion is a major mechanism for cGKI-mediated growth in primary VSMCs. This pro-adhesive effect of cGKI might be mediated via an inhibition of Rho kinase (ROCK) and enhanced integrin signaling. These processes might contribute to inhibit anoikis, the programmed cell death induced by the loss of cell/matrix interactions. from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany. 15–17 June 2007

Analysis of cGKI inhibition by Rp-cGMP analogs

Although it is well established that cGMP has important (patho-)physiological functions, the downstream mediators of cGMP signalling are not well understood. The cGMP-dependent protein kinase type I (cGKI) is an attractive candidate receptor for cGMP. A common approach to distinguish between cGKI-dependent and cGKI-independent cGMP effects is the use of pharmacological inhibitors. However, it is increasingly recognized that some of these drugs, such as KT5823, may be less specific than previously thought or may not at all inhibit cGKI activity in intact cells. The Rp-cGMP analogs are reported to inhibit cGKI by binding to the cGMP-binding sites. In this study, we have analysed the effects of two popular RpcGMP analogs, Rp-8-Br-PET-cGMPS and Rp-8-pCPTcGMPS, on basal and cGMP/cGKI-stimulated growth of vascular smooth muscle cells. Surprisingly, neither compound showed a clear inhibition of cell growth and cGKI activity in intact cells as monitored by growth assays and phosphorylation of vasodilator-stimulated phosphoprotein (VASP), respectively. In vitro kinase assays with purified cGKI demonstrated that both Rp-cGMP analogs are partial agonists for cGKI rather than antagonists (see Figure 1). Thus, it appears difficult to interpret data obtained from experiments with Rp-8-Br-PET-cGMPS and Rp-8pCPT-cGMPS. Both compounds should be used with caution as inhibitors. from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany. 15–17 June 2007

Vascular remodelling in the absence and presence of cGKI

The signaling molecule nitric oxide (NO) is of crucial importance for smooth muscle cell (SMC) function and exerts numerous, sometimes opposing effects on vascular proliferative diseases, such as atherosclerosis and restenosis. Under physiological conditions many effects of NO are mediated via the cyclic guanosine-3',5'-monophosphate (cGMP)/cGMP-dependent protein kinase type I (cGKI) pathway. In the cardiovascular system protective effects of NO signaling have been generally attributed to cGMP/cGKI. In contrast, novel genetic evidence from the analysis of atherosclerosis-prone mice indicated that endogenous cGKI accelerated plaque growth and, thus, was pro-atherogenic. However, in restenosis after intraarterial interventions, such as balloon angioplasty or stent placement the mediator(s) of NO signals, in particular the role of endogenous SMC cGKI is unclear. Towards a better understanding of cGKI and its function in restenosis the Cre-loxP site-specific recombination system was used to generate conditional cardiac and SMC cGKI knockout mice. To test whether the lack of cGKI in SMCs affects the injury-induced vascular remodelling conditional mutants and control mice were analysed by morphometry and immunohistochemistry at different time points after injury, in a normolipidemic situation as well as on an apolipoprotein E-deficient genetic background. In addition, by continuous administration of sildenafil the cGMP/cGKI pathway was chronically activated in wild type mice subjected to the injury.

Analysis of mice with genetic modifications of PKG I

The signaling molecule nitric oxide (NO) exerts both beneficial and deleterious effects on the vasculature; however, the molecular signaling pathways are incompletely understood. In smooth muscle cells (SMCs), the second messenger cyclic guanosine monophosphate (cGMP) mediates many effects of NO via the activation of cGMPdependent protein kinase type I (cGKI). The cGKI gene, prkg1, encodes two isoforms, cGKIα and cGKIβ, both of which are expressed in SMCs. The isoforms differ only in their individual amino-terminal ends, which mediate the interaction with partner proteins and target the kinases to different subcellular compartments. Conventional knockouts carrying a cGKI null mutation (cGKI-/-) show multiple phenotypes. These animals are difficult to analyse, since most die before adulthood. Recently, two mouse lines were generated that express either the cGKIα or cGKIβ isoform exclusively in SMCs of cGKI-/mice. These mouse lines allow an isoform specific of individual smooth muscle functions in adult healthy animals. 8Br-cGMP treatment of aortic SMCs from rescued mice reduced norepinephrineand depolarization-induced Ca2+-increases to wild type levels. In addition, the 8-BrcGMP-induced relaxation of hormone contracted aorta and jejunum was intact in the rescued animals. Finally, telemetric blood pressure recordings in awake, freely moving mice revealed no differences between rescued and control (wild type) mice. Taken together, these results suggest that both isoforms can rescue a major part of the cGMP/cGKI signaling in SMCs and either isoform can compensate for the other in the modulation of vascular tone. Irrespective of the reconstituting isozyme, 50% of the rescued cGKIα and cGKIβ mice die within 52 weeks. Abnormalities found in the mouse lines that possibly cause premature death will be discussed. from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany. 15–17 June 2007

Novel GKAPs for cGMP-dependent protein kinase type II: identification and functional significance

Many protein kinases are maintained in specific subcellular microenvironments by association to anchoring proteins. Proper functioning of the cGMP-dependent protein kinase II (cGK II) involves membrane targeting through myristoylation. To determine whether additional binding to anchoring proteins is also important for its targeting, in analogy with G-kinase anchoring proteins (GKAPs) for cGK I, and to identify potential new components of the cGMP/cGK signalling pathway we performed a yeast twohybrid screen of novel cGK II binding partners. By screening a brain cDNA library using cGK II as a bait the intermediate filament protein GFAP (glial fibrillary acidic protein) was obtained as a positive clone. The interaction was confirmed by overlayand pull-down assays and by immunoprecipitation. Interestingly, the highly homologous intermediate filament protein vimentin has previously been identified as a high affinity binding partner for cGK Iα. We determined which domains are required for the interaction and found, surprisingly, that the least conserved N-terminal domain of the cGK's (aa 1–108 of cGKII and 1–94 of cGK Iα) interacts with the most strongly conserved core domain of the intermediate filaments (aa 237–386 of GFAP). The physiological function of the interaction is under current investigation.

Analysis of smooth muscle function in IRAG knockout mice

Signaling by NO/cGMP relaxes various smooth muscles, modulating thereby vascular tone and gastrointestinal motility. An important signaling pathway of cGMPdependent protein kinase type I (cGKI)-dependent relaxation is mediated by IRAG (IP3 Receptor Associated cGKI substrate) which is highly expressed in smooth muscle tissues. In order to elucidate the physiological role of IRAG we generated IRAG knockout mice by targeted deletion of exon 3.

Differential regulation of the nitric oxide–cGMP pathway exacerbates postischaemic heart injury in stroke‐prone hypertensive rats

Using a working perfused heart model, we investigated the hypothesis that alterations in the NO-cGMP pathway may exacerbate postischaemic mechanical dysfunction in the hypertrophied heart. Ischaemia for 25 min followed by reperfusion for 30 min produced marked cardiac mechanical dysfunction in both stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY). Exogenous treatment with S-nitroso-N-acetyl-dl-penicillamine (SNAP), a NO donor, had beneficial effects on the cardiac dysfunction induced by ischaemia-reperfusion (I/R) in the WKY heart, but the cardioprotective effect of SNAP was eliminated by guanylyl cyclase inhibitor. Cardiac cGMP levels were increased by SNAP or ischaemia in WKY. In contrast, in SHRSP hearts, SNAP could not alleviate the cardiac dysfunction caused by I/R. Pre-ischaemia, the cardiac cGMP level was significantly higher in SHRSP than in WKY; however, no significant difference was found after SNAP and ischaemia. The myocardial Ca(2+)-dependent NO synthase (NOS) activity increased at the end of ischaemia in WKY. Conversely, the Ca(2+)-independent NOS activity and protein levels were upregulated by I/R in the SHRSP myocardium. In the SHRSP hearts, non-selective NOS and selective Ca(2+)-independent NOS inhibitors or antioxidant treatment alleviated cardiac dysfunction caused by I/R. Moreover, mRNA expression and Western blotting analysis of cGMP-dependent protein kinase type I showed more deterioration of SHRSP hearts compared with WKY. These results suggest that: (1) the NO-dependent cardioprotective effect is depressed; and (2) overproduction of NO derived from Ca(2+)-independent NOS contributes to postischaemic heart injury in the hypertrophied heart of hypertensive status.

CGMP-dependent Protein Kinase Type I Inhibits TAB1-p38 Mitogen-activated Protein Kinase Apoptosis Signaling in Cardiac Myocytes

Cardiac myocyte apoptosis during ischemia and reperfusion (I/R) is tightly controlled by a complex network of stress-responsive signaling pathways. One pro-apoptotic pathway involves the interaction of the scaffold protein TAB1 with p38 mitogen-activated protein kinase (p38 MAPK) leading to the autophosphorylation and activation of p38 MAPK. Conversely, NO and its second messenger cGMP protect cardiac myocytes from apoptosis during I/R. We provide evidence that the cGMP target cGMP-dependent protein kinase type I (PKG I) interferes with TAB1-p38 MAPK signaling to protect cardiac myocytes from I/R injury. In isolated neonatal cardiac myocytes, activation of PKG I inhibited the interaction of TAB1 with p38 MAPK, p38 MAPK phosphorylation, and apoptosis induced by simulated I/R. During I/R in vivo, mice with a cardiac myocyte-restricted deletion of PKG I displayed a more pronounced interaction of TAB1 with p38 MAPK and a stronger phosphorylation of p38 MAPK in the myocardial area at risk during reperfusion and more apoptotic cardiac myocytes in the infarct border zone as compared with wild-type littermates. Notably, adenoviral expression of a constitutively active PKG I mutant truncated at the N terminus(PKGI-DeltaN1-92) did not inhibit p38 MAPK phosphorylation and apoptosis induced by simulated I/R in vitro, indicating that the N terminus of PKG I is required. As shown by co-immunoprecipitation experiments in HEK293 cells, cGMP-activated PKG I, but not constitutively active PKG I-DeltaN1-92 or PKG I mutants carrying point mutations in the N-terminal leucine-isoleucine zipper, interacted with p38 MAPK, and prevented the binding of TAB1 to p38 MAPK. Together, our data identify a novel interaction between the cGMP target PKG I and the TAB1-p38 MAPK signaling pathway that serves as a defense mechanism against myocardial I/R injury.

ICln159 Folds into a Pleckstrin Homology Domain-like Structure: INTERACTION WITH KINASES AND THE SPLICING FACTOR LSm4

ICln is a multifunctional protein involved in regulatory mechanisms as different as membrane ion transport and RNA splicing. The protein is water-soluble, and during regulatory volume decrease after cell swelling, it is able to migrate from the cytosol to the cell membrane. Purified, water-soluble ICln is able to insert into lipid bilayers to form ion channels. Here, we show that ICln₁₅₉, a truncated ICln mutant, which is also able to form ion channels in lipid bilayers, belongs to the pleckstrin homology (PH) domain superfold family of proteins. The ICln PH domain shows unusual properties as it lacks the electrostatic surface polarization seen in classical PH domains. However, similar to many classical PH domain-containing proteins, ICln interacts with protein kinase C, and in addition, interacts with cAMP-dependent protein kinase and cGMP-dependent protein kinase type II but not cGMP-dependent protein kinase type Iβ. A major phosphorylation site for all three kinases is Ser-45 within the ICln PH domain. Furthermore, ICln₁₅₉ interacts with LSm4, a protein involved in splicing and mRNA degradation, suggesting that the ICln₁₅₉ PH domain may serve as a protein-protein interaction platform.

Genetic dissection of signaling via cGMP-dependent protein kinases

Increasing evidence suggests that the second messenger cGMP plays a central role in health and disease, but the downstream mechanisms of cGMP-dependent signaling are not well understood. The cGMP-dependent protein kinase type I (cGKI) is an attractive candidate mediator of cGMP signaling, but the pharmacological analysis of cGKI function is limited by the lack of highly specific cGKI activators and inhibitors. To study the (patho)physiological roles of cGKI in vivo, conventional and conditional cGKI knockout mouse models have been established. Whereas cGKI null mutants have a decreased life span (50% of these mice die before 5 to 6 weeks of age), tissue-specific mutants lacking cGKI selectively in the cerebellum, hippocampus, cardiomyocytes, or smooth muscle cells are fully viable allowing one to perform long-term experiments and to study the cell type-specific role of cGKI in adult animals. The analysis of cGKI-deficient mouse models showed that this protein kinase contributes to many (but not all) effects of cGMP in the cardiovascular, gastrointestinal, and nervous system. For instance, cGKI is involved in the acute regulation of smooth muscle tone and platelet aggregation, but is most likely not essential for basal blood pressure control. Of particular interest, cGKI signaling contributes to long-lasting changes in neuronal plasticity, ranging from axonal pathfinding during embryogenesis to the adaptation of synaptic activity during learning and nociception.