Identification of mycobacterial GarA as a substrate of protein kinase G from M. tuberculosis using a KESTREL-based proteome wide approach.

Signal transduction in bacteria is generally mediated via two-component systems. These systems depend on the transfer of a phosphate molecule from a donor to an acceptor by histidine kinases, thereby activating the acceptor to allow downstream signaling/activation. Several bacterial genomes, including the genome of M. tuberculosis, were shown to encode eukaryotic-like kinases. To better understand the function of these kinases and the regulatory networks within which they operate, identification of downstream targets is essential. We here present a straightforward approach for the identification of bacterial Ser/Thr-kinase substrates. This approach is based on the KESTREL (Kinase Tracking and Substrate Elucidation) procedure combined with reversed-phase chromatography and two-dimensional gel electrophoresis. Using this method, GarA was identified as one potential substrate for the mycobacterial Ser/Thr-protein kinase G (PknG). These results show that the modified KESTREL approach can be successfully employed for the identification of substrates for bacterial Ser/Thr-kinases.

Purification and functional analysis of protein kinase G-1α using a bacterial expression system.

3',5' Cyclic guanosine monophosphate (cGMP)-dependent protein kinase G-1α (PKG-1α) is an enzyme that is a target of several anti-hypertensive and erectile dysfunction drugs. Binding of cGMP to PKG-1α produces a conformational change that leads to enzyme activation. Activated PKG-1α performs important roles both in blood vessel vasodilation and in maintaining the smooth muscle cell in a differentiated contractile state. Recombinant PKG-1α has been expressed and purified using Sf9-insect cells. However, attempts at purifying full length protein in a soluble and active form in prokaryotes have thus far been unsuccessful. These attempts have been hampered by the lack of proper eukaryotic protein folding machinery in bacteria. In this study, we report the successful expression and purification of PKG-1α using a genetically engineered Escherichia coli strain, Rosetta-gami 2(DE3), transduced with full-length human PKG-1α cDNA containing a C-terminal histidine tag. PKG-1α was purified to homogeneity using sequential nickel affinity chromatography, gel filtration and ion exchange MonoQ columns. Protein identity was confirmed by immunoblot analysis. N-terminal sequencing using Edman degradation demonstrated that the purified protein was full length. Analysis of enzyme kinetics, using a nonlinear regression curve, identified that, at constant cGMP levels (10µM) and varying ATP concentrations, PKG-1α had a maximal velocity (V(max)) of 5.02±0.25pmol/min/µg and a Michaelis-Menten constant (K(m)) of 11.78±2.68µM ATP. Recent studies have suggested that endothelial function can be attenuated by oxidative and/or nitrosative stress but the role of PKG-1α under these conditions is unclear. We found that PKG-1α enzyme activity was attenuated by exposure to the NO donor, spermine NONOate, hydrogen peroxide, and peroxynitrite but not by superoxide, suggesting that the attenuation of PKG-1α activity may be an under-appreciated mechanism underlying the development of endothelial dysfunction in a number of cardiovascular diseases.

cGMP-binding prepares PKG for substrate binding by disclosing the C-terminal domain.

Type I cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) is involved in the nitric oxide/cGMP signaling pathway. PKG has been identified in many different species, ranging from unicelölular organisms to mammals. The enzyme serves as one of the major receptor proteins for intracellular cGMP and controls a variety of cellular responses, ranging from smooth-muscle relaxation to neuronal synaptic plasticity. In the absence of a crystal structure, the three-dimensional structure of the homodimeric 152-kDa kinase PKG is unknown; however, there is evidence that the kinase adopts a distinct cGMP-dependent active conformation when compared to the inactive conformation. We performed mass-spectrometry-based hydrogen/deuterium exchange experiments to obtain detailed information on the structural changes in PKG I alpha induced by cGMP activation. Site-specific exchange measurements confirmed that the autoinhibitory domain and the hinge region become more solvent exposed, whereas the cGMP-binding domains become more protected in holo-PKG (dimeric PKG saturated with four cGMP molecules bound). More surprisingly, our data revealed a specific disclosure of the substrate-binding region of holo-PKG, shedding new light into the kinase-activation process of PKG.

Intraflagellar transport particles participate directly in cilium-generated signaling in Chlamydomonas.

Primary cilia are widely used for signal transduction during development and in homeostasis and are assembled and maintained by intraflagellar transport (IFT). Here, we have dissected the role of IFT in signaling within the flagella (structural and functional counterparts of cilia) of the biflagellated green alga Chlamydomonas. Using a conditional IFT mutant enables us to deplete the IFT machinery from intact, existing flagella. We identify a cGMP-dependent protein kinase (CrPKG) within flagella as the substrate of a protein tyrosine kinase activated by flagellar adhesion during fertilization. We demonstrate that flagellar adhesion stimulates association of CrPKG with a new flagellar compartment. Moreover, formation of the compartment requires IFT, and IFT particles themselves are part of the compartment. Our results lead to a model in which the IFT machinery is required not only for assembling cilia and flagella but also for organizing a signaling pathway within the organelles during cilium-generated signaling.

Biochemical evidence for a cGMP-regulated protein kinase in Pharbitis nil.

It is known that the level of cGMP is modulated in response to a number of stimuli in plant cells but intracellular events distal to cGMP metabolism are not clear. Cyclic GMP-dependent protein kinase (Pk-G) is a major effector of cGMP action in animals and yeasts. We wanted to determine whether such kinase is present in plant cells. A soluble protein kinase was isolated from seedlings of Pharbitis nil and purified following purification methods including anion-exchange and affinity-chromatography. The enzyme consists of a single polypeptide of M(r) 70 kDa as determined by SDS-PAGE. From conventional modulators only cyclic GMP, when applied in low concentration, was able to accelerate the enzyme activity in the presence of histones. The enzyme autophosphorylated on serine and threonine residues and phosphorylated some substrates only on serine residues. Mixture of histones and histones H2B, H3 were the best phosphate acceptors. The process of autophosphorylation was accelerated by a low concentration of cGMP and reduced by high concentration of this second messenger. Antibodies raised against catalytic domain of animals Pk-G I alpha and beta cross-reacted with protein kinase from Pharbitis nil tissue. These data, taken together, demonstrate the presence of functional enzyme, which activity is regulated by cGMP and allow to classify this protein kinase as a member of the second messenger regulated group of enzymes.

Molecular characterization of a membrane-bound cGMP dependent protein kinase from the silk moth Bombyx mori.

The cGMP signalling pathway has been suggested to be involved in the signal transduction of various physiological functions in insects; olfaction, antidiuresis and eclosion. However, the cGMP signalling mechanism has remained elusive. We isolated two cDNAs of the cGMP dependent protein kinase, designated BmPKG-Ialpha and BmPKG-Ibeta. The deduced amino acid sequences indicate that both BmPKG-Ialpha and BmPKG-Ibeta appear to consist of an amino terminal region, a cGMP binding domain and a protein kinase domain. Transcripts of BmPKG-Ialpha and BmPKG-Ibeta were detected in various tissues: flight muscles, antennae, midgut, legs, head, thoracic ganglia and Malphighian tubules. Recombinant BmPKG-Ialpha bound to lipid membranes, while BmPKG-Ialpha with a deleted amino terminal region failed to bind to lipid membranes.

Regulation of body size and behavioral state of C. elegans by sensory perception and the EGL-4 cGMP-dependent protein kinase.

The growth and behavior of higher organisms depend on the accurate perception and integration of sensory stimuli by the nervous system. We show that defects in sensory perception in C. elegans result in abnormalities in the growth of the animal and in the expression of alternative behavioral states. Our analysis suggests that sensory neurons modulate neural or neuroendocrine functions, regulating both bodily growth and behavioral state. We identify genes likely to be required for these functions downstream of sensory inputs. Here, we characterize one of these genes as egl-4, which we show encodes a cGMP-dependent protein kinase. We demonstrate that this cGMP-dependent kinase functions in neurons of C. elegans to regulate multiple developmental and behavioral processes including the orchestrated growth of the animal and the expression of particular behavioral states.

Purification and molecular characterization of cGMP-dependent protein kinase from Apicomplexan parasites. A novel chemotherapeutic target.

The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (Compound 1) inhibits the growth of Eimeria spp. both in vitro and in vivo. The molecular target of Compound 1 was identified as cGMP-dependent protein kinase (PKG) using a tritiated analogue to purify a approximately 120-kDa protein from lysates of Eimeria tenella. This represents the first example of a protozoal PKG. Cloning of PKG from several Apicomplexan parasites has identified a parasite signature sequence of nearly 300 amino acids that is not found in mammalian or Drosophila PKG and which contains an additional, third cGMP-binding site. Nucleotide cofactor regulation of parasite PKG is remarkably different from mammalian enzymes. The activity of both native and recombinant E. tenella PKG is stimulated 1000-fold by cGMP, with significant cooperativity. Two isoforms of the parasite enzyme are expressed from a single copy gene. NH(2)-terminal sequence of the soluble isoform of PKG is consistent with alternative translation initiation within the open reading frame of the enzyme. A larger, membrane-associated isoform corresponds to the deduced full-length protein sequence. Compound 1 is a potent inhibitor of both soluble and membrane-associated isoforms of native PKG, as well as recombinant enzyme, with an IC(50) of <1 nm.

Guanosine 3':5'-cyclic monophosphate-dependent particulate protein kinase activity from yeast (Saccharomyces cerevisiae).

Continuing our studies on cGMP in growing yeast we detected a particulate cGMP dependent protein kinase (Pk-G), which was solubilized by detergents and NaCl. It achieves maximum activity at 25 degrees C and pH = 6.8, high concentrations of substrate proteins or cGMP produce saturation. Casein and histones are appropriate substrates, phosphatase-pretreated histone H-2a provokes outstandingly high activity. Pk-G differs from cAMP-dependent protein kinase (Pk-A) with respect to pH optimum, temperature tolerance above 50 degrees C, and stability. Partial purification is achieved by chromatography with DEAE-cellulose, Sepharose, and cGMP-substituted Sepharose. The latter step also markedly removes Pk-A. At least three proteins with Pk-G-activity and high cGMP-affinity are separated by polyacrylamide-gel-electrophoresis. Their apparent molecular masses, as deduced from comigrating marker proteins, differ considerably from those of other Pk-G's, but also of Pk-A's.

Regulation of ciliary beat frequency by both PKA and PKG in bovine airway epithelial cells.

Ciliary beating is required for the maintenance of lung mucociliary transport. We investigated the role of cyclic nucleotide-dependent protein kinases in stimulating ciliary beat frequency (CBF) in bovine bronchial epithelial cells (BBECs). cAMP-dependent protein kinase (PKA) activity and cGMP-dependent protein kinase (PKG) activity were distinguished after DEAE-Sephacel chromatography of BBEC extracts. cAMP levels and PKA activity are increased in BBECs stimulated with 0.01-1 mM isoproterenol, with a corresponding increase in CBF. cGMP levels and PKG activity are increased in BBECs stimulated with 0.1-10 microM sodium nitroprusside, with a corresponding increase in CBF. Direct protein kinase-activating analogs of cAMP and cGMP (dibutyryl cAMP and 8-bromo-cGMP, respectively) also activate their specific kinases and stimulate CBF. Preincubation of BBECs with inhibitors of PKA or PKG [KT-5720 or Rp-8-(p-chlorophenylthio)-guanosine 3',5'-cyclic monophosphothioate] results in the inhibition of specific kinase activity as well as in the inhibition of CBF. These studies suggest that the activation of either PKA or PKG can lead to the stimulation of CBF in bovine airway epithelium.

Ligand-induced conformational changes in cyclic nucleotide phosphodiesterases and cyclic nucleotide-dependent protein kinases.

Three methods have been used to assess the conformational effects associated with ligand binding to two unrelated cyclic nucleotide receptor proteins: the cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE or PDE5A) and the cGMP-dependent protein kinase (PKG). The methods should be applicable to other proteins and to other types of modification such as phosphorylation. The procedures use either ion-exchange chromatography, size-exclusion chromatography, or native gel electrophoresis of these proteins in the absence and presence of regulatory ligands. Measurements from these respective approaches allow documentation of changes in the quaternary structure, surface electronegativity, and relative compactness (Stokes radius) of the protein molecule. The combined data allow the changes in protein conformation to be quantitated in terms of alterations in the axial ratio or length/width dimension of the molecule. The methods can be applied to partially purified proteins and to proteins that are available in limited quantities. Conformational changes due to stable modifications of proteins can be potentially examined in crude extracts of intact cells. Each of the methods can be tailored to optimize resolution of a particular protein under a variety of conditions. Activity measurements, Coomassie brilliant blue or silver staining of gels, radioautography, or Western blot analysis can be used for detection of the protein.

Endogenous type II cGMP-dependent protein kinase exists as a dimer in membranes and can Be functionally distinguished from the type I isoforms.

In mammalian tissues two types of cGMP-dependent protein kinase (cGK) have been identified. In contrast to the dimeric cGK I, cGK II purified from pig intestine was shown previously to behave as a monomer. However, recombinant rat cGK II was found to have hydrodynamic parameters indicative of a homodimer. Chemical cross-linking studies showed that pig cGK II in intestinal membranes has a dimeric structure as well. However, after purification, cGK II was found to be partly proteolyzed into C-terminal monomeric fragments. Phosphorylation studies in rat intestinal brush borders revealed that the potency of cGMP analogs to stimulate or inhibit native cGK II in vitro (i.e. 8-(4-chlorophenylthio)-cGMP > cGMP > beta-phenyl-1,N2-etheno-8-bromo-cGMP > beta-phenyl-1,N2-etheno-cGMP and Rp-8-(4-chlorophenylthio)-cGMPs > Rp-beta-phenyl-1, N2-etheno-8-bromo-cGMPs, respectively) correlated well with their potency to stimulate or inhibit cGK II-mediated Cl- secretion across intestinal epithelium but differed strikingly from their potency to affect cGK I activity. These data show that the N terminus of cGK II is involved in dimerization and that endogenous cGK II displays a distinct activation/inhibition profile with respect to cGMP analogs, which permits a pharmacological dissection between cGK II- and cGK I-mediated physiological processes.

Cyclic GMP-dependent protein kinase activation in the absence of negative inotropic effects in the rat ventricle.

1. It has been suggested that activation of cyclic GMP-dependent protein kinase (PKG) is a necessary step in the chain of events leading to the production of negative inotropy by muscarinic receptor agonists in mammalian ventricles, and that some cyclic GMP-elevating agents, such as sodium nitroprusside (SNP), fail to exert a negative inotropic effect because they elevate cyclic GMP levels in a pool that does not activate the kinase. This hypothesis was tested in the present study by monitoring the effects of carbachol, SNP and atrial natriuretic peptide (ANP) on contractility, cyclic GMP content and PKG activity in rat intact ventricular preparations and freshly isolated ventricular cardiomyocytes. 2. The presence of PKG in both the intact vehicle and in isolated ventricular cardiomyocytes was confirmed by MonoQ anion exchange chromatography and Western blotting. The elution profile indicated that the conditions of the PKG assay were selective for measuring PKG activity. 3. Carbachol induced a marked negative inotropic effect in intact, perfused hearts and ventricular strips in the presence of isoproterenol. The negative inotropic effect of carbachol was not associated with significant changes in cyclic GMP content or PKG activity in intact ventricular tissue, or in PKG activity in isolated cardiomyocytes. 4. SNP and ANP significantly increased cyclic GMP levels and activated PKG in intact ventricular preparations. Both drugs also activated PKG in isolated cardiomyocytes. However, neither drug had any negative inotropic effect in isoprenaline-stimulated perfused hearts and ANP did not change the contractility of isoprenaline-stimulated isolated cardiomyocytes. 5. The results of this study demonstrate that the negative inotropic effects of muscarinic receptor agonists can occur in the absence of significant activation of PKG. Conversely, marked increases in ventricular cyclic GMP content and PKG activity caused by SNP or ANP were not accompanied by a negative inotropic effect. 6. These results suggest that increases in cyclic GMP levels and activation of PKG do not play important roles in the regulation of rat ventricular contractility by muscarinic receptor agonists.

Biochemical properties and cellular localization of the Drosophila DG1 cGMP-dependent protein kinase.

The protein encoded by the Drosophila cGMP-dependent protein kinase gene, DG1, was expressed in Sf9 cells. cGMP (10 microM) stimulated histone H2B phosphorylation by the DG1 protein kinase 20-fold. Maximal activity was observed at 40-50 mM Mg2+. The concentrations of cGMP, cAMP, cIMP, 8-bromo-cGMP, and 8-bromo-cAMP that gave 50% activation were 0.19 +/- 0.06, 11.7 +/- 2.8, 5.3 +/- 1.5, 0.04 +/- 0. 01, and 0.62 +/- 0.06 microM, respectively. cGMP activation was cooperative with a Hill coefficient (nH) of 1.28 +/- 0.10, whereas activation by cAMP was not cooperative. DG1 kinase expressed in Sf9 cells was found to be a dimer with an amino-terminal dimerization domain. It also autophosphorylated in a reaction stimulated by cGMP and cAMP. Immunoadsorbed DG1 protein from fly extracts was also capable of autophosphorylation, and this assay was used to quantitate the DG1 kinase in extracts from heads and bodies of adults and whole embryos. Activity was highest in heads of either sex and male bodies, intermediate in female bodies, and lowest in embryos. These results were in accord with DG1 mRNA abundance. Tissue distribution of the DG1 kinase was investigated by immunohistochemistry. In embryos, specific immunoreactivity was observed in large cells scattered along the anterior-posterior axis at stage 13. Prominent staining of adult heads was restricted to the proximal level of the lamina cortex.

Fast and slow cyclic nucleotide-dissociation sites in cAMP-dependent protein kinase are transposed in type Ibeta cGMP-dependent protein kinase.

Both cyclic GMP-dependent protein kinase (cGK) and cyclic AMP-dependent protein kinase (cAK) contain two distinct cyclic nucleotide-binding sites referred to as fast and slow sites based on cyclic nucleotide dissociation behavior. In cAK, the fast site lies amino-terminal to the slow site, and sequence homologies between cAK and cGK have suggested similar positioning for the sites in cGK. Recombinant human type Ibeta cGK (wild type (WT) cGK) was overexpressed, and the properties of purified WT cGK and native type Ibeta cGK were similar. cGK was mutated singly at Thr-193 (T193A, T193V, and T193S) and Thr-317 (T317A, T317V, and T317S), which have been predicted to provide cGMP specificity in the cGMP-binding sites of cGK; a double mutant (T193A/T317A) was produced also. Compared with WT cGK, half-maximal activation (Ka) of mutant cGKs by cGMP was increased 2- (T317A), 27- (T193A), or 63-fold (T193A/T317A), but the Ka for cAMP of these mutants was essentially unchanged. The T193A and T193V mutants had a large increase in the rate of the slow component of [3H]cGMP dissociation, but in the T317A and T317V mutants, there was no change in the slow component. The T193S and T317S mutants had only minor effects on [3H]cGMP dissociation, thus establishing the importance of the hydroxyl group of Thr-193 and -317 for cGMP binding to cGK. Thus, in type Ibeta cGK, the slow cGMP-binding site is identified as the amino-terminal site in contrast to the order assigned to the fast and slow cAMP-binding sites of cAK.

Differential regulation of distinct types of gap junction channels by similar phosphorylating conditions.

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.

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 I alpha 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 ATP gamma S (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 cGKI alpha, is a potential activator of chloride transport in CFTR-expressing cell types.

Expression, purification, and characterization of the cGMP-dependent protein kinases I beta and II using the baculovirus system.

Detailed studies of differences in distinct cGMP kinase isoforms are highly dependent on expression of large amounts of these enzyme isoforms that are not easily purified by conventional methods. Here cGMP-dependent protein kinases, the type I beta soluble form from human placenta, and the type II membrane-associated form from rat intestine, were each expressed in a baculovirus/Sf9 cell system and purified in milligram amounts by affinity chromatography. The expressed recombinant proteins displayed characteristics like those of their native counterparts. cGK I beta was expressed as a 76 kDa protein predominantly found in the cytosol fraction, whereas cGK II was expressed as an 86 kDa protein predominantly associated with the membrane fraction. The apparent Ka and Vmax of cGMP for activation of cGK I beta were 0.5 microM and 3.4 mumol/min/mg, and for cGK II were 0.04 microM and 1.8 mumol/min/mg.