Suzanne M. Lohmann

Distinct and specific functions of cGMP-dependent protein kinases.

cGMP-dependent protein kinases I and II conduct signals from widespread signaling systems. Whereas the type I kinase mediates numerous effects of natriuretic peptides and nitric oxide in cardiovascular cells, the type II kinase transduces signals from the Escherichia coli heat-stable enterotoxin, STa, and from the endogenous intestinal peptide, guanylin, stimulating Cl- conductance of the cystic fibrosis transmembrane conductance regulator (CFTR). Although the two kinases may be interchangeable for several functions, CFTR regulation specifically requires the type II kinase.

Physiology and Pathophysiology of Vascular Signaling Controlled by Cyclic Guanosine 3′,5′-Cyclic Monophosphate–Dependent Protein Kinase

Despite impressive medical advances1 that have led to diminished cardiovascular death rates in some countries over the past 20 years, cardiovascular disease remains the leading cause of death in developed countries such as the United States. This promises to worsen as a result of aging populations; the incipient obesity and type II diabetes epidemic; sedentary lifestyle; and continued abuse of tobacco, alcohol, and other substances. Cardiovascular disease is clearly multifactorial, and the approach to its prevention necessarily likewise. Candidates for prevention include cyclic guanosine 3′,5′-cyclic monophosphate (cGMP)–dependent signaling networks initiated by natriuretic peptides (NPs) and nitric oxide (NO), which demonstrate characteristics deemed worthy of diagnostic and therapeutic exploitation. cGMP signaling contributes to the function and interaction of several vascular cell types, and its dysfunction could be involved in major destructive processes such as atherosclerosis, hypertension, diabetic complications, (re)stenosis, and tissue infarction, as well as the undermining of clinical therapy like in the case of nitrate tolerance. This review takes a focused look at key elements of the cGMP signaling cascade in vascular tissue, particularly recent advances in our knowledge of cGMP-dependent protein kinase (cGK, also known as PKG) function. Finally, we discuss the potential of clinical monitoring of cGK activity for assessing the functional status of cGMP signaling and for guiding the design of therapeutic strategies to improve vascular function. One of the 2 major synthetic pathways for cGMP generation from guanosine 5′-triphosphate (GTP) is directed by NPs (Figure 1), consisting of atrial (ANP), B-type (BNP), and C-type (CNP) natriuretic peptides, which act via the membrane receptor guanylate cyclases GC-A (highest affinity for ANP, BNP) and GC-B (highest affinity for CNP). ANP and BNP, released from the heart by mechanical stretch in response to increased atrial pressure/volume, and CNP, released from the endothelium, can all cause smooth muscle (SM) …

Inhibition of calcineurin-NFAT hypertrophy signaling by cGMP-dependent protein kinase type I in cardiac myocytes.

Recent investigation has focused on identifying signaling pathways that inhibit cardiac hypertrophy, a major risk factor for cardiovascular morbidity and mortality. In this context, nitric oxide (NO), signaling via cGMP and cGMP-dependent protein kinase type I (PKG I), has been recognized as a negative regulator of cardiac myocyte (CM) hypertrophy. However, the underlying mechanisms are poorly understood. Here, we show that PKG I inhibits CM hypertrophy by targeting the calcineurin-NFAT signaling pathway. Calcineurin, a Ca2+-dependent phosphatase, promotes hypertrophy in part by activating NFAT transcription factors which induce expression of hypertrophic genes, including brain natriuretic peptide (BNP). Activation of PKG I by NO/cGMP in CM suppressed NFAT transcriptional activity, BNP induction, and cell enlargement in response to α1-adrenoreceptor stimulation but not in response to adenoviral expression of a Ca2+-independent, constitutively active calcineurin mutant, thus demonstrating NO-cGMP-PKG I inhibition of calcineurin-NFAT signaling upstream of calcineurin. PKG I suppressed single L-type Ca2+-channel open probability, [Ca2+]i transient amplitude, and, most importantly, L-type Ca2+-channel current-induced NFAT activation, indicating that PKG I targets Ca2+-dependent steps upstream of calcineurin. Adenoviral expression of PKG I enhanced NO/cGMP inhibitory effects upstream of calcineurin, confirming that PKG I mediates NO/cGMP inhibition of calcineurin-NFAT signaling. In CM overexpressing PKG I, NO/cGMP also suppressed BNP induction and cell enlargement but not NFAT activation elicited by constitutively active calcineurin, which is consistent with additional, NFAT-independent inhibitory effect(s) of PKG I downstream of calcineurin. Inhibition of calcineurin-NFAT signaling by PKG I provides a framework for understanding how NO inhibits cardiac myocyte hypertrophy.

Cyclic GMP-Dependent Protein Kinases and the Cardiovascular System Insights From Genetically Modified Mice

Signaling cascades initiated by nitric oxide (NO) and natriuretic peptides (NPs) play an important role in the maintenance of cardiovascular homeostasis. It is currently accepted that many effects of these endogenous signaling molecules are mediated via stimulation of guanylyl cyclases and intracellular production of the second messenger cGMP. Indeed, cGMP-elevating drugs like glyceryl trinitrate have been used for more than 100 years to treat cardiovascular diseases. However, the molecular mechanisms of NO/NP signaling downstream of cGMP are not completely understood. Recent in vitro and in vivo evidence identifies cGMP-dependent protein kinases (cGKs) as major mediators of cGMP signaling in the cardiovascular system. In particular, the analysis of conventional and conditional knockout mice indicates that cGKs are critically involved in regulating vascular remodeling and thrombosis. Thus, cGKs may represent novel drug targets for the treatment of human cardiovascular disorders.

Presynaptic and Postsynaptic Roles of NO, cGK, and RhoA in Long-Lasting Potentiation and Aggregation of Synaptic Proteins

Recent results suggest that long-lasting potentiation at hippocampal synapses involves the rapid formation of clusters or puncta of presynaptic as well as postsynaptic proteins, both of which are blocked by antagonists of NMDA receptors and an inhibitor of actin polymerization. We have investigated whether the increase in puncta involves retrograde signaling through the NO-cGMP-cGK pathway and also examined the possible roles of two classes of molecules that regulate the actin cytoskeleton: Ena/VASP proteins and Rho GTPases. Our results suggest that NO, cGMP, cGK, actin, and Rho GTPases including RhoA play important roles in the potentiation and act directly in both the presynaptic and postsynaptic neurons, where they contribute to the increase in puncta of synaptic proteins. cGK phosphorylates synaptic VASP during the potentiation, whereas Rho GTPases act both in parallel and upstream of cGMP, in part by maintaining the synaptic localization of soluble guanylyl cyclase.

Molecular cloning and predicted full‐length amino acid sequence of the type Iβ isozyme of cGMP‐dependent protein kinase from human placenta

In this study we report the isolation and characterization of three overlapping cDNA clones for the type Iβ 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 Iβ 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 Iα, human placenta Iβ 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 Iβ 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 Iβ cGK were identical to the partial amino acid sequence recently published for a new Iβ isoform of cGK from bovine aorta. Northern blot analysis demonstrated a human Iβ 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.

Functional analysis of cGMP-dependent protein kinases I and II as mediators of NO/cGMP effects

Abstract NO and cGMP have emerged as important signal transduction mediators of the effects of certain hormones, inter-/intracellular signals, toxins and drugs. However, a major challenge is to define relevant criteria for determining which of the many NO and/or cGMP effects are dependent on cGMP-dependent protein kinases (cGKs). Important criteria include that: (1) the cell types/tissues investigated contain at least one form of cGK which is activated by the cGMP-elevating agent in the intact cell system; (2) specific activators/inhibitors of cGKs mimic/block the effects of cGMP-elevating agents in the intact cell system; and (3) the cGMP effect is absent or blunted in cGK-deficient systems, or can be reconstituted by the introduction of active cGKs.Previously, analysis of cGK activity in intact cells has been very difficult. However, the analysis of vasodilator-stimulated phosphoprotein (VASP) phosphorylation by polyclonal antibodies and newly developed monoclonal antibodies, each of which specifically recognize different phosphorylation sites, allows the quantitative measurement of cGK activity in intact cells. With the use of these methods, the properties of certain cGK mutants, cGK activators (cGMP, 8-Br-cGMP, 8-pCPT-cGMP) as well as various “specific cGK inhibitors” (KT 5823, Rp-8Br-PET-cGMPS, Rp-8-pCPT-cGMPS, H8 and H89) were investigated. Although these “specific cGK inhibitors” have been widely used to establish or rule out functional roles of cGKs, very few studies have actually addressed the efficiency/specificity of such compounds in intact cells. Our results demonstrate that these inhibitors are useful tools only when used in combination with other experimental approaches and biochemical evidence.

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 Iβ mutant (cGK Iβ-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 Iβ-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.

Isotype-specific activation of cystic fibrosis transmembrane conductance regulator-chloride channels by cGMP-dependent protein kinase II

Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type Iα cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl− channel in excised, inside-out membrane patches from NIH-3T3 fibroblasts and from a rat intestinal cell line (IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the presence of cGMP and ATP, cGKII was found to mimic the effect of the catalytic subunit of cAMP-dependent protein kinase (cAK) on opening CFTR-Cl− channels, albeit with different kinetics (2-3-min lag time, reduced rate of activation). By contrast, cGKI or a monomeric cGKI catalytic fragment was incapable of opening CFTR-Cl− channels and also failed to potentiate cGKII activation of the channels. The cAK activation but not the cGKII activation was blocked by a cAK inhibitor peptide. The slow activation by cGKII could not be ascribed to counteracting protein phosphatases, since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A, nor ATPγS (adenosine 5′-O-(thiotriphosphate)), producing stable thiophosphorylation, was able to enhance the activation kinetics. Channels preactivated by cGKII closed instantaneously upon removal of ATP and kinase but reopened in the presence of ATP alone. Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino acids 645-835) could be phosphorylated to a similar extent with only minor kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very subtle differences in site-specificity between the cGK isoforms. These results indicate that cGKII, in contrast to cGKIα, is a potential activator of chloride transport in CFTR-expressing cell types.

Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator.

Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life-threatening secretory diarrhea. The intracellular effector of cGMP actions has not been clarified. Recently we cloned the cDNA for a rat intestinal type II cGMP dependent protein kinase (cGK II) which is highly enriched in intestinal mucosa. Here we show that cGK II mRNA and protein are restricted to the intestinal segments from the duodenum to the proximal colon, with the highest amounts of cGK II protein in duodenum and jejunum. cGK II mRNA and protein decreased along the villus to crypt axis in the small intestine, whereas substantial amounts of both were found in the crypts of cecum. In intestinal epithelia, cGK II was specifically localized in the apical membrane, a major site of ion transport regulation. In contrast to cGK II, cGK I was localized in smooth muscle cells of the villus lamina propria. Short circuit current (ISC), a measure of Cl- secretion, was increased to a similar extent by STa and by 8-Br-cGMP, a selective activator of cGK, except in distal colon and in monolayers of T84 human colon carcinoma cells in which cGK II was not detected. In human and mouse intestine, the cyclic nucleotide-regulated Cl- conductance can be exclusively accounted for by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Viewed collectively, the data suggest that cGK II is the mediator of STa and cGMP effects on Cl- transport in intestinal-epithelia.