Structural insights into the allosteric activation mechanism of cGMP-dependent protein kinase I

Cyclic GMP-dependent protein kinase (PKG) is a key transducer in the NO-cGMP signaling pathway. In this line, PKG has been considered an important drug target for treating hypertensive cardiovascular and pulmonary diseases. However, the investigation of PKG’s allosteric activation mechanism has been hampered by a lack of structural information. One of the fundamental questions on the cGMP-dependent activation of PKG is how the enzyme can distinguish cGMP over cAMP and selectively respond to cGMP. To ensure proper signaling, PKG must have developed unique features to ensure its activation upon the right activation signal. In this thesis, the cGMP-selective activation mechanism of PKG was studied through determining crystal structures of three truncated constructs of the regulatory domain [CNB-A (92-227), CNB-B (271-369), and CNB-A/B (92-351)] of PKG Iβ in the absence or presence of cyclic nucleotides. Herein, two individual CNB domain structures with biochemical data revealed that the C-terminal CNB domain (CNB-B) is responsible for cGMP selectivity, while the N-terminal CNB-domain (CNB-A) has a higher binding affinity for both cGMP and cAMP without showing any selectivity. Based on these crystal structures, mutagenesis studies were performed in which the critical residues for cyclic nucleotide selectivity and activation were identified. Furthermore, we discovered that the conformational changes of the C-terminal helix of the CNB-B that bridges between the regulatory and catalytic domains including the hydrophobic capping interaction are crucial for PKG activation. In addition, to observe the global conformation of the activated R-domain, I solved a co-crystal structure of the CNB-A/B with cGMP. Although a monomeric construct was crystallized, the structure displays a dimer. Strikingly, the CNB-A domain and its bound cGMP provide a key interface for this dimeric interaction. Using small angle X-ray scattering (SAXS), the existence of the cGMP-mediated dimeric interface within the CNB domains was confirmed. Furthermore, measuring cGMP-binding affinities (EC50) of the dimeric interface mutants as well as determining activation constants (Ka) revealed that the interface formation is important for PKG activation. To conclude, this thesis study provides a new mechanistic insight in PKG activation along with a newly found interface that can be targeted for designing PKG-specific activity modulators.

Diacylglycerol-induced protection against injury during ischemia and reperfusion can be achieved without activation of protein kinase C in the rat heart: Comparative studies with ischemic preconditioning

The role of protein kinase C (PKC) activation in ischemic preconditioning remains controversial. Since diacylglycerol is the endogenous activator of PKC and as such might be expected cardioprotective, we have investigated whether: (i) the diacylglycerol analog 1,2-dioctanoyl-sn-glycerol (DOG) can protect against injury during ischemia and reperfusion; (ii) any effect is mediated via PKC activation; and (iii) the outcome is influenced by the time of administration. Isolated rat hearts were perfused with buffer at 37°C and paced at 400 bpm. In Study 1, hearts (n=6/group) were subjected to one of the following: (1) 36 min aerobic perfusion (controls); (2) 20 min aerobic perfusion plus ischemic preconditioning (3 min ischemia/3 min reperfusion+5 min ischemia/5 min reperfusion); (3) aerobic perfusion with buffer containing DOG (10 μM) given as a substitute for ischemic preconditioning; (4) aerobic perfusion with DOG (10 μM) during the last 2 min of aerobic perfusion. All hearts then were subjected to 35 min of global ischemia and 40 min reperfusion. A further group (5) were perfused with DOG (10 μM) for the first 2 min of reperfusion. Ischemic preconditioning improved postischemic recovery of LVDP from 24±3% in controls to 71±2% (P<0.05). Recovery of LVDP also was enhanced by DOG when given just before ischemia (54±4%), however, DOG had no effect on the recovery of LVDP when used as a substitute for ischemic preconditioning (22±5%) or when given during reperfusion (29±6%). In Study 2, the first four groups of study were repeated (n=4–5/group) without imposing the periods of ischemia and reperfusion, instead hearts were taken for the measurement of PKC activity (pmol/min/mg protein±SEM). PKC activity after 36 min in groups (1), (2), (3) and (4) was: 332±102, 299±63, 521±144, and 340±113 and the membrane:cytosolic PKC activity ratio was: 5.6±1.5, 5.3±1.8, 6.6±2.7, and 3.9±2.1 (P=NS in each instance). In conclusion, DOG is cardioprotective but under the conditions of the present study is less cardioprotective than ischemic preconditioning, furthermore the protection does not appear to necessitate PKC activation prior to ischemia.

Growth of melanocytic cells is associated with down-regulation of protein kinase C α,δ and ε isoforms: Possible role of diacylglycerol

Protein kinase C (PKC) down-regulation has been shown to correlate with the growth of murine melanocytic cells in culture (Brooks, G., Wilson, R. E., Dooley, T. P., Goss, M. W., and Hart, I. R. (1991) Cancer Res. 51, 3281-3288). We now show that PKC alpha, delta, epsilon, and zeta isoforms are present at the protein level in quiescent, non-transformed Mel-ab melanocytes, maintained in the absence of phorbol ester. Proliferation of Mel-ab cells, achieved by incubation in the continual presence of phorbol 12,13-dibutyrate, was associated with a down-regulation of the PKC alpha, delta, and epsilon isozymes. Examination of two transformed syngeneic lines (the B16 murine melanoma and the long terminal repeat Ras.2 line), that grew in the absence of exogenous phorbol esters, showed that PKC alpha protein levels were either partially down-regulated or unaffected, the PKC delta and epsilon isoforms were down-regulated completely, and the levels of PKC zeta protein remained unaltered relative to quiescent Mel-ab cells. Basal levels of total diacylglycerol were elevated 5-fold in B16 melanoma cells compared with levels found in quiescent or proliferating Mel-ab melanocytes and appear to arise largely from the breakdown of phosphatidylinositol phospholipids accompanied by a significant rise in phospholipase C activity. Hourly treatments of quiescent Mel-ab melanocytes with the synthetic diacylglycerol analogue, 1,2-dioctanoyl-sn-glycerol, for 24 h, resulted in an induction of DNA synthesis which was associated with a significant down-regulation of PKC levels mediated largely via post-translational rather than transcriptional mechanisms. These results show for the first time that specific isoforms of PKC are down-regulated at the protein level during proliferation of murine melanocytic cells and suggest that the constitutive down-regulation of PKC in transformed melanoma cells may arise as a consequence of elevated endogenous phosphatidylinositol-derived diacylglycerol levels.

The effects of phorbol esters with different activities on protein kinase C

The sapintoxins are a series of naturally occurring fluorescent phorbol esters with a range of selective biological activities (e.g. pro-inflammatory but non-tumour promoting). Their ability to activate protein kinase C (PKC) in vitro has been studied. Both tumour promoting and non-promoting phorbol derivatives activate the enzyme in vitro at low concentrations. 12-deoxyphorbol-13-phenylacetate-20 acetate (DOPPA) acts as a partial agonist in the activation of protein kinase C. Structurally distinct phorbol esters may therefore preferentially activate different forms of protein kinase C. -sapinine, a biologically inactive compound, binds to protein kinase C without stimulating the enzyme and prevents subsequent activation by phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (TPA).

The role of 80K/MARCKS, a specific substrate of protein kinase C, in cell growth and tumour progression

Since its discovery more than a decade ago [Wu et al., 1982; Rozengurt et al., 1983], the 80-87 kDa myristoylated a lanine-rich C-kinase substrate (80K/MARCKS) protein has attracted a great deal of attention from researchers interested in cell growth and tumour progression. However, despite its ubiquitous distribution, a definitive functional role for 80K/MARCKS has not been found. The purpose of this review is to describe the properties, distribution and regulation of 80K/MARCKS and to discuss some of the most recent findings, both from our laboratory and from others, that have suggested a functional role for this protein in modulating cell growth and tumour progression. Furthermore, I will present data from our laboratory that implicates 80K/MARCKS as a novel tumour suppressor in cells of melanocyte origin.

GTP binding and intramolecular regulation by the ROC domain of Death Associated Protein Kinase 1

The ROCO proteins are a family of large, multidomain proteins characterised by the presence of a Ras of complex proteins (ROC) domain followed by a COR, or C-terminal of ROC, domain. It has previously been shown that the ROC domain of the human ROCO protein Leucine Rich Repeat Kinase 2 (LRRK2) controls its kinase activity. Here, the ability of the ROC domain of another human ROCO protein, Death Associated Protein Kinase 1 (DAPK1), to bind GTP and control its kinase activity has been evaluated. In contrast to LRRK2, loss of GTP binding by DAPK1 does not result in loss of kinase activity, instead acting to modulate this activity. These data highlight the ROC domain of DAPK1 as a target for modifiers of this proteins function, and casts light on the role of ROC domains as intramolecular regulators in complex proteins with implications for a broad range of human diseases.

Protease-activated receptor 2 sensitizes TRPV1 by protein kinase Cepsilon- and A-dependent mechanisms in rats and mice

Proteases that are released during inflammation and injury cleave protease-activated receptor 2 (PAR2) on primary afferent neurons to cause neurogenic inflammation and hyperalgesia. PAR2-induced thermal hyperalgesia depends on sensitization of transient receptor potential vanilloid receptor 1 (TRPV1), which is gated by capsaicin, protons and noxious heat. However, the signalling mechanisms by which PAR2 sensitizes TRPV1 are not fully characterized. Using immunofluorescence and confocal microscopy, we observed that PAR2 was colocalized with protein kinase (PK) Cepsilon and PKA in a subset of dorsal root ganglia neurons in rats, and that PAR2 agonists promoted translocation of PKCepsilon and PKA catalytic subunits from the cytosol to the plasma membrane of cultured neurons and HEK 293 cells. Subcellular fractionation and Western blotting confirmed this redistribution of kinases, which is indicative of activation. Although PAR2 couples to phospholipase Cbeta, leading to stimulation of PKC, we also observed that PAR2 agonists increased cAMP generation in neurons and HEK 293 cells, which would activate PKA. PAR2 agonists enhanced capsaicin-stimulated increases in [Ca2+]i and whole-cell currents in HEK 293 cells, indicating TRPV1 sensitization. The combined intraplantar injection of non-algesic doses of PAR2 agonist and capsaicin decreased the latency of paw withdrawal to radiant heat in mice, indicative of thermal hyperalgesia. Antagonists of PKCepsilon and PKA prevented sensitization of TRPV1 Ca2+ signals and currents in HEK 293 cells, and suppressed thermal hyperalgesia in mice. Thus, PAR2 activates PKCepsilon and PKA in sensory neurons, and thereby sensitizes TRPV1 to cause thermal hyperalgesia. These mechanisms may underlie inflammatory pain, where multiple proteases are generated and released.

Endothelin-1 and fibroblast growth factors stimulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes. The potential role of the cascade in the integration of two signaling pathways leading to myocyte hypertrophy.

Maximally effective concentrations of endothelin-1 (ET-1), acidic FGF (aFGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA) activated mitogen-activated protein kinase (MAPK) by 3-4-fold in crude extracts of myocytes cultured from neonatal rat heart ventricles. Maximal activation was achieved after 5 min. Thereafter, MAPK activity stimulated by ET-1 or aFGF declined to control values within 1-2 h, whereas activation by TPA was more sustained. Two peaks of MAPK activity (a 42- and a 44-kDa MAPK) were resolved in cells exposed to ET-1 or aFGF by fast protein liquid chromatography on a Mono Q column. One major and one minor peak of MAPK kinase (MAPKK) was stimulated by ET-1 or aFGF. Cardiac myocytes expressed protein kinase C (PKC)-alpha, -delta, -epsilon and -zeta as shown immunoblotting. Exposure to 1 microM TPA for 24 h down-regulated PKC-alpha, -delta, and -epsilon, but not PKC-zeta. This maneuver wholly abolished the activation of MAPK on re-exposure to TPA but did not affect the response to aFGF. The effect of ET-1 was partially down-regulated. ET-1 stimulated phospho[3H]inositide hydrolysis 18-fold, whereas aFGF stimulated by only 30%. Agonists which initially utilize dissimilar signaling pathways may therefore converge at the level of MAPKK/MAPK and this may be relevant to the hypertrophic response of the heart.

Activation of the mitogen-activated protein kinase cascade by pertussis toxin-sensitive and -insensitive pathways in cultured ventricular cardiomyocytes.

The involvement of pertussis toxin (PTX)-sensitive and -insensitive pathways in the activation of the mitogen-activated protein kinase (MAPK) cascade was examined in ventricular cardiomyocytes cultured from neonatal rats. A number of agonists that activate heterotrimeric G-protein-coupled receptors stimulated MAPK activity after exposure for 5 min. These included foetal calf serum (FCS), endothelin-1 (these two being the most effective of the agonists examined), phenylephrine, endothelin-3, lysophosphatidic acid, carbachol, isoprenaline and angiotensin II. Activation of MAPK and MAPK kinase (MEK) by carbachol returned to control levels within 30-60 min, whereas activation by FCS was more sustained. FPLC on Mono Q showed that carbachol and FCS activated two peaks of MEK and two peaks of MAPK (p42MAPK and p44MAPK). Pretreatment of cells with PTX for 24 h inhibited the activation of MAPK by carbachol, FCS and lysophosphatidic acid, but not that by endothelin-1, phenylephrine or isoprenaline. Involvement of G-proteins in the activation of the cardiac MAPK cascade was demonstrated by the sustained (PTX-insensitive) activation of MAPK (and MEK) after exposure of cells to AlF4-. AlF4- activated PtdIns hydrolysis, as did endothelin-1, endothelin-3, phenylephrine and FCS. In contrast, the effect of lysophosphatidic acid on PtdIns hydrolysis was small and carbachol was without significant effect even after prolonged exposure. We conclude that PTX-sensitive (i.e. Gi/G(o)-linked) and PTX-insensitive (i.e. Gq/Gs-linked) pathways of MAPK activation exist in neonatal ventricular myocytes. FCS may stimulate the MAPK cascade through both pathways.

Expression of protein kinase C isoforms during cardiac ventricular development.

The expression of protein kinase C (PKC) isoforms (PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta) was studied by immunoblotting in whole ventricles of rat hearts during postnatal development (1-26 days) and in the adult. PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta were detected in ventricles of 1-day-old rats, although PKC-alpha and PKC-beta 1 were only barely detectable. All isoforms were rapidly downregulated during development, with abundances relative to total protein declining in the adult to < 25% of 1-day-old values. PKC-beta 1 was not detectable in adult ventricles. The specific activity of PKC was also downregulated. The rat ventricular myocyte becomes amitotic soon after birth but continues to grow, increasing its protein content 40- to 50-fold between the neonate and the 300-g adult. An important question is thus whether the amount of PKC per myocyte is downregulated. With the use of isolated cells, immunoblotting showed that the contents per myocyte of PKC-alpha and PKC-epsilon increased approximately 10-fold between the neonatal and adult stages. In rat ventricles, the rank of association with the particulate fraction was PKC-delta > PKC-epsilon > PKC-zeta. Association of these isoforms with the particulate fraction was less in the adult than in the neonate. In primary cultures of ventricular myocytes prepared from neonatal rat hearts, 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited translocation of PKC-alpha, PKC-delta, and PKC-epsilon from the soluble to the particulate fraction in < 1 min, after which time no further translocation was observed. Prolonged exposure (16 h) of myocytes to 1 microM TPA caused essentially complete downregulation of these isoforms, although downregulation of PKC-epsilon was slower than for PKC-delta. In contrast, PKC-zeta was neither translocated nor downregulated by 1 microM TPA. Immunoblotting of human ventricular samples also revealed downregulation of PKC relative to total protein during fetal/postnatal development.