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Studies on the role of protein kinase A in humoral immune response of Galleria mellonella larvae
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Journal of Insect Physiology 52 (2006) 744–753 www.elsevier.com/locate/jinsphys
Studies on the role of protein kinase A in humoral immune response of Galleria mellonella larvae Ma"gorzata Cytryn´ska, Agnieszka Zdybicka-Barabas, Teresa Jakubowicz Department of Invertebrate Immunology, Institute of Biology, Maria Curie-Sk!odowska University, Akademicka 19, 20-033 Lublin, Poland Received 21 February 2006; received in revised form 4 April 2006; accepted 4 April 2006
Abstract Protein kinase A (PKA) activity was detected in the fat body of Galleria mellonella larvae by a non-radioactive method using a specific peptide substrate—kemptide. The enzyme activity was stimulated by cAMP and its analogues: BzcMP, 8-Chl-cAMP and 8-Br-cAMP in concentrations of 1–4 mM. Cyclic GMP was not effective in PKA activation. A two-fold increase in PKA activity was detected in the fat body of G. mellonella LPS-challenged larvae. Selective, membrane-permeable PKA inhibitors, H89 and Rp-8-Br-cAMPS, inhibited protein kinase A activity in the fat body of G. mellonella larvae in vitro and in vivo. The inhibition of PKA activity in vivo was correlated with a considerable lowering of haemolymph antibacterial activity and a decrease in lysozyme content in the fat body of immune challenged larvae. The use of phospho-motif antibodies recognising PKA phosphorylation consensus site allowed identification of four potential PKA phosphorylation substrates of 79, 45, 40 and 36 kDa in G. mellonella fat body. r 2006 Elsevier Ltd. All rights reserved. Keywords: Galleria mellonella; Protein kinase A; Lysozyme; Antibacterial activity; H89; Rp-8-Br-cAMPS
1. Introduction Insect immunity relies on humoral and cellular innate defence mechanisms. The cellular response results in phagocytosis, nodulation or encapsulation of non-self bodies. The humoral response generates antimicrobial proteins and peptides synthesised in the fat body and haemocytes and subsequently secreted into the haemolymph. Molecular mechanisms leading to the induction of antimicrobial peptide synthesis are not fully elucidated. In Drosophila melanogaster two independent immune signalling pathways, Toll/Dif and imd/Relish, were described, both of which activate the Rel family of transcription factors similar to the human NF-kB factors (Imler and Hoffmann, 2000; Khush et al., 2001; Hetru et al., 2003; Hultmark, 2003; Leclerc and Reichhart, 2004; Iwanaga and Lee, 2005). The Rel family of Drosophila transcription factors comprises Dorsal, Dif and Relish. Most members Corresponding author. Tel.: +48 81 537 50 89; fax: +48 81 537 50 50.
E-mail addresses: [email protected] (M. Cytryn´ska), [email protected] (T. Jakubowicz). 0022-1910/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2006.04.002
of Rel family share 300 amino acid N-terminal homology region containing a potential protein kinase A (PKA) phosphorylation site (Ghosh and Karin, 2002). It was shown that the transcriptional activity of certain vertebrate Rel family members was regulated by PKA (Zhong et al., 1998; Hou et al., 2003). Norris and Manley (1992) have found that nuclear transport and activation of Drosophila morphogen Dorsal resulted from phosphorylation of Dorsal by PKA. Moreover, phosphorylation of Dorsal at Ser312 by PKA enhanced nuclear import through facilitating an interaction with importin (Briggs et al., 1998). There are data indicating that PKA may play a regulatory role in humoral as well as cellular immune response in invertebrates. Cecropin B gene expression was triggered by the membrane-permeable cAMP analogue, dibutyryl-cAMP, and inhibited by a selective PKA inhibitor, H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide), in isolated Bombyx mori haemocytes (Shimabukuro et al., 1996; Taniai et al., 1996). PKA was implicated to be involved in the activation of the marine mussel Mytilus galloprovincialis haemocytes by bacterial lipopolysaccharide (LPS) and interleukin-2
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(Cao et al., 2004). A cyclic AMP signalling pathway was engaged in modulation of phagocytosis in oyster Crassostrea gigas haemocytes by noradrenaline (Lacoste et al., 2001). The use of PKA inhibitor, H89, also demonstrated that PKA is involved in regulation of adhesion properties of greater wax moth Galleria mellonella haemocytes. Inhibition of PKA increased the adhesion of granulocytes, but not of plasmatocytes, to glass slides (Zakarian et al., 2003). It was demonstrated that active PKA limited the G. mellonella haemocyte response in vitro and in vivo. The use of Rp-8-Br-cAMPS (Rp-isomer of 8-Bromoadenosine 30 ,50 -cyclic monophosphorothioate) for PKA inhibition increased the number of haemocytes with adherent bacteria and enhanced phagocytosis of bacteria in vitro. It also reduced haemocyte counts and induced nodulation in vivo (Brooks and Dunphy, 2005). Compounds elevating cellular cAMP concentration such as adenylate cyclase activators and cAMP-phosphodiesterases inhibitors impaired removal of Bacillus subtilis cells by G. mellonella haemocytes in vivo (Marin et al., 2005). It was concluded that a high level of cAMP impaired non-self response in G. mellonella larvae and suggested that PKA could be a mediator of the cellular immune response. In this paper, we report the results of studies on PKA activity in the fat body of the greater wax moth G. mellonella larvae. We investigated a possible role of PKA in the humoral immune response of this insect. Two membrane-permeable PKA inhibitors with different mechanisms of action were used, H89 and Rp-8-Br-cAMPS. H89 inhibits PKA activity in a competitive fashion with ATP (Chijiwa et al., 1990) while Rp-8-Br-cAMPS interacts with cAMP-binding sites in R subunits and prevents PKA holoenzyme dissociation (Gjertsen et al., 1995). We studied the correlation between PKA activity and an antimicrobial activity level in the haemolymph and the lysozyme content in the fat body of immune challenged G. mellonella larvae. Furthermore, we used specific antibodies directed against the consensus phosphorylation site of PKA for identification of potential PKA phosphorylation substrates in G. mellonella fat body. 2. Materials and methods
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apyrogenic water, respectively. In some experiments, fifteen minutes after inhibitor administration, the larvae were injected with 2.5 mg of Escherichia coli LPS (Serotype 026:B6, Sigma) in 3 ml of apyrogenic water. After the treatment, the larvae were kept at 30 1C in the dark on sterile Petri plates and the haemolymph was collected after the time indicated in the text. Prior to haemolymph collection, the insects were chilled for 15 min at 4 1C and surface disinfected with 70% ethanol. Haemolymph samples were obtained by puncturing the larval abdomen with a sterile needle. Out-flowing haemolymph was immediately transferred into sterile and chilled Eppendorf tubes containing a few crystals of phenylthiourea (PTU) to prevent melanisation. The haemocyte-free haemolymph was obtained by centrifugation at 200g for 5 min to pellet haemocytes without disruption and the supernatant was subsequently centrifuged at 20,000g for 15 min at 4 1C to pellet cell debris. The obtained haemocyte-free haemolymph was used immediately for testing antibacterial activity. 2.2. Antibacterial activity assay For antibacterial activity tests the LPS defective, streptomycin and ampicillin resistant mutant of E. coli K12, strain D31 was used (Boman et al., 1974). The antibacterial activity in the haemolymph was detected using solid agar plates containing viable E. coli cells as described (Hoffmann et al., 1981). To improve the sensitivity of the method, hen egg white lysozyme (EWL) at a concentration of 2.0 mg ml1 of the medium was added (Chalk and Suliaman, 1998; Cytryn´ska et al., 2001). Each well on the Petri dish was filled with 4 ml of haemolymph four times diluted with sterile water. The agar plates were then incubated at 37 1C for 24 h. The diameters of E. coli D31 growth inhibition zones were measured and the level of antimicrobial activity was calculated using the algorithm described by Hultmark et al. (1982). For evaluation of antibacterial activity synthetic cecropin B (Sigma) was used as a standard. 2.3. Isolation of fat bodies and preparation of cell-free extracts
2.1. Insect culture and haemolymph collection Larvae of the greater wax moth G. mellonella (Lepidoptera: Pyralidae) were reared on a natural diet—honeybee nest debris at 30 1C in the dark. Last instar larvae (250–300 mg in weight) were used throughout the study. For PKA activity inhibition in vivo the larvae were injected with 1.5 nmol of H89 (Sigma) in 3 ml of 30% dimethyl sulphoxide (DMSO) solution or 1.5 nmol of Rp8-Br-cAMPS (Sigma) in 3 ml of apyrogenic water (the approximate concentration of inhibitors in larval haemolymph—20 mM). Control animals were injected with the same volume of 30% DMSO solution (an average final concentration of DMSO in larval haemolymph—1.3%) or
For fat body isolation the larvae were anaesthesized by submerging in ice-cold apyrogenic water and then surface disinfected with 70% ethanol. The fat bodies were dissected on Petri plates under sterile ice-cold physiological saline (172 mM KCl, 68 mM NaCl, 5 mM NaHCO3, pH 6.1, osmolarity 420 mOsm) (Vilcinskas and Matha, 1997). After dissection the fat body was transferred into sterile, chilled Eppendorf tube containing 1 ml of physiological saline. Then the solution was removed and the fat body was frozen in liquid nitrogen. Cell-free extracts of fat bodies were prepared in ice-cold PKA buffer (50 mM Tris-HCl pH 7.4, 10 mM b-glycerophosphate, 2.5 mM sodium pyrophosphate, 0.5 mM
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ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 6 mM b-mercaptoethanol). Frozen fat bodies were thawed on ice and homogenised using a pellet pestle motor (Sigma-Aldrich). The obtained extracts were centrifuged at 20,000g for 15 min at 4 1C and supernatants were collected. For PKA activity assay the supernatants were used immediately. For immunoblotting an appropriate volume of Laemmli sample buffer (Laemmli, 1970) was added and the samples were stored at 20 1C. 2.4. PKA activity assay PKA activity was measured by a non-radioactive method using PepTags Assay (Promega) mainly in accordance with manufacturer’s instructions. In this assay, PKA phosphorylation substrate peptide-kemptide in modified, specially coloured version was used (named PepTags A1 Peptide). The reaction mixture in the volume of 25 ml contained: 20 mM Tris–HCl pH 7.4, 10 mM MgCl2, 1 mM ATP, 1 mM cAMP, 2 mg of PepTags A1 Peptide and 20 mg of fat body total proteins. For serine phosphatases inhibition, sodium pyrophosphate and b-glycerophosphate, each at the final concentration of 2.5 mM, were added to the reaction mixture. In the cases indicated in the text, cAMP was replaced with cAMP analogues or cGMP. For PKA activity inhibition in vitro, H89 and Rp-8-BrcAMPS were used in the concentrations indicated in the text. As for H89, which was dissolved in 30% DMSO, a control mixture contained an appropriate concentration of DMSO and concerning H89 mode of action, the ATP concentration was diminished to 0.35 mM. Each reaction was performed at room temperature for 30 min and then stopped by heating the incubation mixture at 95 1C for 10 min. PepTags A1 Peptide fluorescence was visualised after 20 min of horizontal electrophoresis in 0.8% agarose by using the UV transilluminator. During electrophoresis the unmodified substrate migrated towards the negative electrode while the PKA phosphorylated towards the positive one. For identification of PKA phosphorylation substrates in fat body cell-free extracts in vitro, incubation was performed without kemptide in the reaction mixture (25 ml) containing: 20 mM Tris–HCl pH 7.4, 10 mM MgCl2, 0.1 mM ATP, 4 mM cAMP, 5 mM b-mercaptoethanol and 40 mg of fat body total proteins. After 30 min of incubation at room temperature the reaction was stopped by addition of 12.5 ml Laemmli sample buffer. The samples were then analysed by immunoblotting as described below. 2.5. Immunoblotting Samples of fat body extracts (40 mg or 80 mg of total protein) were subjected to 13.8% SDS/PAGE and electroblotted onto Immobilon membranes (Millipore) for 90 min at 350 mA. For lysozyme identification, the membranes were probed with polyclonal antibodies (1:1000) to
G. mellonella lysozyme, a generous gift of Prof. I. H. Lee, Department of Life Science, Hoseo University, South Korea. As second antibodies, alkaline phosphatase-conjugated goat anti-rabbit IgGs (1:30,000) were used and immunoreactive bands were visualised by incubation with p-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3indolyl phosphate. Phosphorylated PKA substrates were detected with polyclonal Phospho-(Ser/Thr) PKA Substrate Antibody (1:1000) (Cell Signaling Technology, Beverly, MA). These antibodies recognised phosphorylated threonine and serine residues in PKA consensus phosphorylation motif RXXT and RRXS where R is arginine, X any amino acid, T threonine and S serine. As second antibodies, horseradish peroxidase-conjugated goat anti-rabbit IgGs (1:2000) were used and immunoreactive bands were visualised using chemiluminescent reagent and peroxide according to the manufacturer protocol (New England Biolabs. Inc., Beverly, MA). 2.6. Other methods Polyacrylamide gel electrophoresis of proteins was performed by 13.8% glycine SDS/PAGE according to Laemmli (1970). The protein concentration was estimated by Bradford method using bovine serum albumin (BSA) as a standard (Bradford, 1976). The densitometric analysis of bands was performed using Quantity One computer imaging system (BioRad, Hercules, CA). All data are presented as means7standard deviation (SD) for at least three experiments. 3. Results 3.1. PKA activity in larval fat body after treatment with PKA inhibitors It is well documented that insect fat body is the main tissue in which humoral immune response components are synthesised and for this reason PKA activity in G. mellonella fat body was investigated. The enzyme activity in vitro was measured in fat body cell-free extracts using PKA specific peptide substrate-kemptide in the presence of cAMP, cAMP analogues or cGMP. The results presented in Fig. 1 clearly showed that the enzyme was activated by 1 mM concentrations of cAMP, 8-Br-cAMP (8-Bromoadenosine-30 ,50 -cyclic monophosphate), 8-ChlcAMP (8-Chloroadenosine-30 ,50 -cyclic monophosphate) and BzcMP (ribofuranosylbenzimidazole 30 :50 -cyclic monophosphate). The level of kemptide phosphorylation in the presence of cAMP and its analogues used was comparable. On the contrary, in the presence of cGMP the kemptide phosphorylation level was much lower. Even at a concentration of cGMP as high as 10 mM, the kemptide phosphorylation level reached only 24.4% of that obtained
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Fig. 1. Protein kinase A activity in fat body of Galleria mellonella larvae. PKA activity was measured using kemptide as a phosphorylation substrate in the presence of indicated concentrations of cAMP, cGMP, BzcMP, 8-Chl-cAMP and 8-Br-cAMP as described in the Materials and Methods section. Agarose gel (photo A) was taken immediately after phosphorylated and non-phosphorylated kemptide resolution and the fragment of the gel containing phosphorylated kemptide (marked as pkemptide) is demonstrated. The diagram (B) presents kemptide phosphorylation level as revealed by densitometric analysis of the bands containing phosphorylated kemptide. Kemptide phosphorylation level measured without cyclic nucleotide addition was subtracted. Bars ¼ 7SD of four independent experiments.
in the presence of 4 mM cAMP (Fig. 1). This indicated that cGMP was not as effective as cAMP in G. mellonella fat body PKA activation. It could also suggest that protein kinase G, which is activated by cGMP, was not involved in kemptide modification. The studied enzyme activity was considerably reduced in vitro by two selective PKA inhibitors, H89 (Fig. 2A) and Rp-8-Br-cAMPS (Fig. 2B), confirming that PKA was responsible for kemptide modification. Its activity was inhibited in the presence of 0.1 and 1 mM H89 by 32.4% and 57%, respectively, in comparison to PKA activity measured in the presence of DMSO (Fig. 2A). DMSO alone decreased kemptide phosphorylating activity by 30.2%. In the presence of Rp-8-Br-cAMPS in the concentrations of 0.1 and 1 mM, the fat body PKA activity was inhibited by 65.5% and 80%, respectively (Fig. 2B). In the following experiments, we investigated the level of PKA activity in fat bodies of larvae treated with H89 and Rp-8-Br-cAMPS for different periods of time (Fig. 3). It was found that both inhibitors used in vivo reduced PKA activity 15 min after treatment. The enzyme activity returned to the control level 30 min after inhibitor
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Fig. 2. Inhibition of Galleria mellonella fat body PKA activity in vitro by H89 (A) and Rp-8-Br-cAMPS (B). PKA activity was measured in fat body cell-free extracts in the presence of 1 mM concentration of cAMP as described in Materials and Methods. The diagrams present the densitometric analysis of the bands containing phosphorylated kemptide. The insets—photos of agarose gel fragments containing phosphorylated kemptide bands. Bars ¼ 7SD of three independent experiments.
administration. H89 injection caused an enzyme activity reduction by 54.2% in comparison to the activity measured in DMSO-treated larvae (Fig. 3A). It should be mentioned here that, similarly to the in vitro experiments, DMSO injection caused reduction in kemptide phosphorylation level by 33.3% in comparison to untreated larvae. Rp-8-BrcAMPS was also effective in PKA inhibition in vivo and its administration decreased the fat body enzyme activity by 44.2% (Fig. 3B). 3.2. PKA activity in fat body of immune-challenged G. mellonella larvae The above observations prompted us to study PKA activity in the fat bodies of immune-challenged G. mellonella larvae. We found a considerable increase in PKA activity level after LPS injection (Fig. 4A, B). The highest, about two-fold, increase in PKA activity level was detected in fat bodies isolated 15–30 min after the immune challenge. When the larvae were injected with a selective PKA inhibitor, Rp-8-Br-cAMPS, before LPS treatment, a considerable reduction in enzyme activity was detected 15 min after the challenge (63.6% of inhibition). A strong
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Fig. 3. The in vivo effect of H89 (A) and Rp-8-Br-cAMPS (B) on PKA activity in Galleria mellonella larvae fat body. The larvae (five individuals per each group) were injected with H89 or DMSO solution (control) (A) and Rp-8-Br-cAMPS or water (control) (B) as described in Materials and Methods. Fat bodies were isolated at the indicated time points (one cell-free extract was prepared from fat bodies collected at the individual time point) and PKA activity was measured. The densitometric analysis of the bands containing phosphorylated kemptide was performed and the results are presented as the kemptide phosphorylation level. The first hatched bar represents PKA activity in fat body of non-treated larvae. Bars ¼ 7SD of three experiments. The insets present agarose gel fragments containing phosphorylated kemptide bands.
reduction of PKA activity (46.9–50%) was also detected 6–24 h after the challenge (Fig. 4A, B). When the cell-free extracts of fat bodies isolated from LPS-injected larvae were tested by immunoblotting with specific antibodies recognising PKA phosphorylation consensus site, a transient increase in phosphorylation level of at least four proteins of 79, 45, 40 and 36 kDa was noticed, additionally confirming the in vivo activation of PKA at the immune challenge conditions (Fig. 4C). The increase in phosphorylation level of 45 and 79 kDa proteins was detected within the first hour, whereas the 36 and 40 kDa proteins appeared 3–8 h after challenge. Studies are in progress to identify these proteins and to determine their function in G. mellonella fat body cells. We also observed cAMP stimulated phosphorylation of the 36 kDa protein in vitro in cell-free extract of non-immune fat body. 3.3. Lysozyme content in fat body of G. mellonella larvae treated with PKA inhibitors It is well documented that lysozyme is a very important factor of insect innate immunity (Hultmark, 1996). This protein is synthesised in insect fat body and haemocytes and released into the haemolymph. When G. mellonella larvae were immune challenged with LPS, lysozyme was
Fig. 4. PKA activity in fat body of immune-challenged Galleria mellonella larvae. The larvae (five individuals per each group) were injected with water, LPS or PKA inhibitor and LPS as described in Materials and Methods. Fat bodies were isolated at the indicated time points (one cellfree extract was prepared from fat bodies collected at the individual time point) and PKA activity was measured. Diagram (A) presents the densitometric analysis of the bands containing phosphorylated kemptide and photo (B) shows fragments of the agarose gels containing phosphorylated kemptide bands. The first hatched bar represents PKA activity in the fat body of non-treated larvae. Bars ¼ 7SD of three experiments. (C) Detection of potential PKA substrates in the fat body of LPS challenged larvae (in vivo) and in cell-free extracts of non-immune fat body incubated with (+) and without () cAMP addition (in vitro). The immunoblotting analysis of cell-free extracts of fat body (40 mg of total protein) was performed as described in Materials and Methods. The arrows mark four protein bands of transient increase in phosphorylation level.
detected in the fat body starting from 5–6 h after challenge (data not shown). In this paper, we tested the lysozyme content in fat body of immune-challenged G. mellonella larvae pre-treated with PKA inhibitors. To follow the lysozyme protein level, the immunoblotting technique with anti-G. mellonella lysozyme antibodies was used. It was found that the lysozyme content in the larval fat body gradually increased starting from 4 h, reaching the highest-level 24 h after DMSO–LPS treatment (Fig. 5A). On the contrary, in the fat body of larvae pre-treated with H89 before LPS injection, the
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Fig. 5. Immunodetection of lysozyme in Galleria mellonella immunechallenged larvae pre-treated with H89 (A) and Rp-8-Br-cAMPS (B) in vivo. The larvae (five individuals per each group) were injected with PKA inhibitors and LPS as described in Materials and Methods. Fat bodies were isolated at the indicated time points (one cell-free extract was prepared from fat bodies collected at the individual time point) and prepared for immunoblotting. Samples containing 80 mg of total fat body protein were resolved by SDS/PAGE and transferred onto Immobilon membranes. The membranes were probed with anti G. mellonella lysozyme antibodies. The lysozyme band is marked by an arrow. The diagrams present the densitometric analysis of the bands containing lysozyme. Bars ¼ 7SD of three experiments.
lysozyme appeared later (5 h after challenge) and the protein level detected was much lower in comparison to DMSO–LPS-injected larvae. The densitometric analysis revealed that in the fat body of H89–LPS-treated larvae obtained 5, 6, 8 and 24 h after challenge, the lysozyme content reached 15.4%, 36.5%, 71.8% and 46.4%, respectively, of the content measured for the fat bodies of DMSO–LPS-treated larvae (Fig. 5A). Similar results were obtained when Rp-8-Br-cAMPS was used for PKA activity inhibition in vivo (Fig. 5B). The lysozyme was detected in the fat body of water pre-treated immune larvae 6–24 h after the challenge, whereas in the fat body of inhibitor-injected larvae only traces of the protein were detected and the lysozyme content reached only 24.5–42.5% of that measured in immune fat body. The presented results suggested that the changes in lysozyme content detected after PKA inhibitors injection, could be caused, at least in part, by fat body PKA activity inhibition. 3.4. Antibacterial activity in haemolymph of G. mellonella larvae treated with PKA inhibitors As shown above, the immune challenge of G. mellonella larvae led to an increase in fat body PKA activity, and injection of PKA inhibitor before the challenge caused a strong reduction in enzyme activity. The insect fat body is the main site of antimicrobial protein and peptide synthesis and release into the haemolymph. We tested whether the observed changes in fat body PKA activity were correlated with the level of antibacterial activity in the haemolymph. The activity inhibiting growth of E. coli D31 was measured
Fig. 6. Antibacterial activity in the haemolymph of Galleria mellonella immune-challenged larvae pre-treated with H89 (A) and Rp-8-Br-cAMPS (B) in vivo. The larvae (ten individuals per each group) were injected with PKA inhibitors and LPS as described in Materials and Methods. The haemolymph was collected from 10 individuals together at the indicated time points (20 ml/larva) and used for antimicrobial activity assay as described. Bars ¼ 7SD of five independent experiments.
in the haemolymph of immune-challenged larvae pretreated with H89 or Rp-8-Br-cAMPS before LPS administration (Fig. 6). It was found that the antimicrobial activity level in the haemolymph of animals pre-treated with PKA inhibitors was much lower in comparison to the haemolymph of immune larvae. This effect was especially evident during the first 8 h after the challenge. Anti-E. coli activity level in the haemolymph of H89–LPS-treated larvae constituted 47.6–62.5% of the level measured for DMSO–LPS-treated ones (Fig. 6A). In the haemolymph of Rp-8-Br-cAMPS–LPS-treated larvae the antimicrobial activity level constituted 68.2–74.2% of that measured for H2O–LPS-treated animals (Fig. 6B). The obtained results indicated that the lower level of haemolymph antimicrobial activity was correlated with PKA activity inhibition by H89 and Rp-8-Br-cAMPS in vivo. In the case of H89–LPSinjected larvae, 24 h after the challenge, the antimicrobial activity level was comparable to that detected in the DMSO–LPS-treated ones, while in the haemolymph of Rp8-Br-cAMPS–LPS-treated larvae a strong increase in antibacterial activity measured 24 h after immune-challenge was detected.
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4. Discussion Reversible protein phosphorylation regulates a large number of physiological processes in living organisms. Involvement of protein kinases in regulation of the immune response is well documented. In mammalian systems, mitogen-activated protein (MAP) kinases are engaged in the initiation phase of innate immunity and activation of adaptive immunity. MAP kinases participate in signalling pathways leading to the production of inflammatory cytokines, e.g. tumour necrosis factor a (TNF a), interleukin-1 and -12 (Dong et al., 2002). Similarly to the mammalian system, MAP kinases JNK and p38 were activated in response to pathogen recognition in Drosophila (Sluss et al., 1996; Han et al., 1998). Recently, the role of JNK MAP kinase in regulation of the G. mellonella humoral immune response was documented by Wojda et al. (2004). Different protein kinases, e.g. IkB kinase (IKK), Akt kinase, PI-3 kinase, CKII and PKA can be involved in NF-kB signalling pathways in mammalian and insect innate immunity (reviewed in Karin, 1999; Silverman and Maniatis, 2001). It was suggested that protein kinase C and tyrosine protein kinases participate in regulation of the immune response mechanisms (Johansson and So¨derha¨ll, 1993; Weinstein et al., 1993; Charalambidis et al., 1995; Lanz-Mendoza et al., 1996). Literature data indicate PKA participation in regulation of the immune response of vertebrates and invertebrates (Fujihara et al., 1993; Muroi and Suzuki, 1993; Shimabukuro et al., 1996; Chen et al., 1999; Regenhard et al., 2001; Cao et al., 2004; reviewed in Torgersen et al., 2002). The involvement of PKA in the modulation of G. mellonella haemocytes adhesion properties in vitro (Zakarian et al., 2003) and non-self cellular response in vivo (Brooks and Dunphy, 2005; Marin et al., 2005) was shown, suggesting its role in the cellular immune response regulation. In the present study, PKA involvement in the regulation of the humoral immune response of G. mellonella was investigated. Our studies provide evidence for PKA activity in G. mellonella fat body, the tissue in which humoral immune response components are synthesised. The enzyme was activated in vitro in the presence of cAMP and certain cAMP analogues such as 8-Br-cAMP and 8-Chl-cAMP, which are membrane-permeable cAMP analogues widely used in signal transduction research. 8-Chl-cAMP was also extensively tested as an anticancer agent (Schwede et al., 2000). Additionally, G. mellonella fat body PKA was activated by BzcMP indicating that, similarly to mammalian cAMP-dependent protein kinase and yeast S. cerevisiae PKA, the studied enzyme does not require the 6-amino group and the 1 and 3 nitrogenes in the adenine ring to be activated (Yagura et al., 1980; Øgreid et al., 1985; Cytryn´ska et al., 1999). G. mellonella fat body PKA was hardly activated in vitro by cGMP. It was consistent with the studies showing that in higher Eucaryota two different protein kinases, PKA and protein kinase G, respond to cAMP and cGMP, respectively (Weber et al., 1989;
Hofmann et al., 1992). Tsuzuki and Newburgh (1974) suggested that in the central nervous system of G. mellonella two different protein kinases responded to cAMP and cGMP. They also suggested the presence of cIMP-dependent protein kinase in this tissue of G. mellonella. It is worth mentioning here that PKA activated by both cyclic nucleotides, cAMP and cGMP, was reported for several arthropod species: Melanoplus sanquinipes (Vardanis, 1983), Locusta migratoria (Beenakkers et al., 1978), Periplaneta americana (Takahashi and Hanaoka, 1977), Amblyoma americanum (Mane et al., 1985), Orconectes limosus (Christ, 1985). In some unicellular Eucaryota like yeast Saccharomyces cerevisiae and Pichia pastoris, PKA can be also almost equally activated by cAMP and cGMP (Cytryn´ska et al., 1999; Frajnt et al., 2003). The PKA in fat body of G. mellonella larvae was inhibited in vitro by two selective PKA inhibitors, H89 and Rp-8-Br-cAMPS. At a 1 mM concentration of H89 enzyme activity was inhibited by 57%. It is known that H89 inhibits PKA activity in a competitive fashion with ATP with the inhibition constant of 0.048 mM when purified cAMP-dependent protein kinase catalytic subunit from bovine heart and histone IIb as a substrate are used (Chijiwa et al., 1990). In our in vitro experiments, 0.05 mM concentration of H89 inhibited fat body PKA activity by 25%. It is strong inhibition if we consider that the ATP concentration used in our experiments (0.35 mM) was much higher than used in Chijiwa et al. (1990) experiments (5–50 mM). The second selective PKA inhibitor, Rp-8-BrcAMPS, decreased the kemptide phosphorylation level by 65.5% and 80%, respectively, for the concentration of 0.1 and 1 mM. This was consistent with the studies presented by Gjertsen et al. (1995) for purified cAMP-dependent protein kinase I from rabbit muscle. Both inhibitors were widely used for selective PKA inhibition in vitro and in vivo in a large number of studies (Chijiwa et al., 1990; Shimabukuro et al., 1996; Lacoste et al., 2001; Regenhard et al., 2001; Malagoli et al., 2002; Zakarian et al., 2003; Brooks and Dunphy, 2005; Marin et al., 2005). We showed that both compounds were also effective in G. mellonella fat body PKA inhibition in vivo. The enzyme activity inhibition was detected within the first 15 min after inhibitor administration, suggesting a fast response of fat body cells. The activity level was reduced by 54 and 44% in the case of H89 and Rp-8-Br-cAMPS treatment, respectively. The inhibitory effect of Rp-8-Br-cAMPS was much stronger when activity of PKA was tested in the fat body of immune-challenged larvae. PKA activity inhibition was sustained for prolonged periods of time and correlated with changes in the level of humoral immune response components of G. mellonella larvae. The inhibition of fat body PKA activity by H89 and Rp8-Br-cAMPS in vivo was correlated with a decrease in fat body lysozyme content. The observed effect could be caused by a partial inhibition of lysozyme synthesis in the fat body and/or with an increased rate of protein secretion.
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Whether PKA is involved in the regulation of these processes in the insect fat body requires further studies. In promoters of insect immune genes, including lysozyme genes, a conserved consensus sequence, similar to kB site was found (Hultmark, 1996; Engstro¨m, 1998) indicating that Rel transcription factors are involved in the control of these genes expression. As PKA regulates at least certain Rel factors activity, one can speculate that the enzyme could also be involved in the regulation of lysozyme gene expression in G. mellonella fat body. The role of PKA in the regulation of LPS-activated lysozyme gene expression was documented by Regenhard et al. (2001) for chicken lysozyme gene in myelomonocytic cells. The inhibition of PKA activity in G. mellonella fat body in vivo by H89 or Rp-8-Br-cAMPS, prior to immune challenge, coincided with the lowering of antibacterial activity in the haemolymph. The low level of bacterial growth inhibiting activity was sustained until 8 h after the challenge and returned to the control level after 24 h. We thus hypothesise that PKA is involved in modulation of the humoral immune response in G. mellonella larvae. The involvement of PKA in the regulation of cecropin B gene expression was shown in silkworm B. mori by Shimabukuro et al. (1996) and Taniai et al. (1996). We demonstrated that after immune challenge PKA activity in G. mellonella fat body considerably increased, suggesting a role for PKA in regulation of the immune response of this insect. To identify potential PKA phosphorylation substrates in the fat body of G. mellonella larvae we used methodology based on phospho-motif antibodies (Grønborg et al., 2002; Berwick and Tavare´, 2004). Antibodies specific for PKA phosphorylation consensus site with serine or threonine in phosphorylated state recognised several proteins in the fat body of G. mellonella larvae. Some of these proteins were phosphorylated to a similar level in both immune and nonimmune larvae. In addition, at least four fat body proteins of 79, 45, 40 and 36 kDa were transiently phosphorylated in vivo in LPS-challenged larvae. This observation provided another argument for the participation of PKA in regulation of the immune response of G. mellonella larvae. It is worth mentioning here that, during immune response, changes in protein phosphorylation caused by other protein kinases were reported. Wojda et al. (2004) showed changes in the phosphorylation level of fat body JNK MAP kinase observed after LPS immune stimulation of G. mellonella larvae. It was reported that LPS-stimulated exocytosis of non-self recognition protein from Ceratitis capitata haemocytes was dependent on protein tyrosine phosphorylation (Charalambidis et al., 1995) and changes in tyrosine phosphorylation of two haemocyte proteins of 19 and 22kDa were detected (Zervas et al., 1998). Changes in protein phosphorylation induced in human macrophages after LPS exposure were also described by Weinstein et al. (1993). The results presented in this paper demonstrated the PKA activity in the fat body of G. mellonella larvae. The
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inhibition of PKA activity in vivo by two selective PKA inhibitors, H89 and Rp-8-Br-cAMPS, was correlated with a lower level of haemolymph antimicrobial activity and a considerable decrease in lysozyme content in larval fat body. Moreover, phosphorylation changes of at least four potential PKA substrates in the fat body of immune challenged larvae were detected. The presented results clearly indicate the participation of PKA in humoral immune response regulation of greater wax moth G. mellonella larvae. Acknowledgements This work was financially supported by the grant from the State Committee for Scientific Research (KBN, Poland) No. 2 P04C 080 29. References Beenakkers, A.M.T., Van der Horst, D.J., Van Marewijk, W.J.A., 1978. Regulation of release and metabolic function of the adipokinetic hormone in insects. In: Gaillard, P.J., Boer, H.H. (Eds.), Comparative Endocrinology. Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 445–448. Berwick, D.C., Tavare´, J.M., 2004. Identifying protein kinase substrates: hunting for the organ-grinder’s monkeys. Trends in Biochemical Sciences 29, 227–232. Boman, H.G., Nilsson-Faye, I., Paul, K., Rasmuson Jr., T., 1974. Insect immunity. 1. Characteristics of an inducible cell-free antibacterial reaction in hemolymph of Samia cynthia pupae. Infection and Immunity 10, 136–145. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Briggs, L.J., Stein, D., Goltz, J., Corrigan, V.C., Efthymiadis, A., Hu¨bner, S., Jans, D.A., 1998. The cAMP-dependent protein kinase site (Ser312) enhances Dorsal nuclear import through facilitating nuclear localization sequence/importin interaction. The Journal of Biological Chemistry 273, 22745–22752. Brooks, C.L., Dunphy, G.B., 2005. Protein kinase A affects Galleria mellonella (Insecta: Lepidoptera) larval haemocyte non-self responses. Immunology and Cell Biology 83, 150–159. Cao, A., Martinez, J.I.R., Barcia, R., 2004. Implication of PKA and PKC in the activation of the haemocytes of Mytilus galloprovincialis Lmk. by LPS and IL-2. Molecular Immunology 41, 45–52. Chalk, R., Suliaman, W.Y., 1998. Antimicrobial peptides from small insects: methods for insect culture and for the detection, visualization, isolation and sequencing of active hemolymph peptides. In: Wiesner, A., Dunphy, G.B., Marmaras, V.J., Morishima, I., Sugumaran, M., Yamakawa, M. (Eds.), Techniques in Insect Immunology. SOS Publications, USA, pp. 109–124. Charalambidis, N.D., Zervas, C.G., Lambropoulou, M., Katsoris, P.G., Marmaras, V.J., 1995. Lipopolysaccharide-stimulated exocytosis of nonself recognition protein from insect hemocytes depends on protein tyrosine phosphorylation. European Journal of Cell Biology 67, 32–41. Chen, C.C., Chiu, K.T., Sun, Y.T., Chen, W.C., 1999. Role of the cyclic AMP-protein kinase A pathway in lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macrophages. Involvement of cyclooxygenase-2. The Journal of Biological Chemistry 274, 31559–31564. Chijiwa, T., Mishima, A., Hagiwara, M., Sano, M., Hayashi, K., Inoue, T., Naito, K., Toshioka, T., Hidaka, H., 1990. Inhibition of forskolininduced neurite outgrowth and protein phosphorylation by a newly
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Journal of Insect Physiology 52 (2006) 744–753 www.elsevier.com/locate/jinsphys
Studies on the role of protein kinase A in humoral immune response of Galleria mellonella larvae Ma"gorzata Cytryn´ska, Agnieszka Zdybicka-Barabas, Teresa Jakubowicz Department of Invertebrate Immunology, Institute of Biology, Maria Curie-Sk!odowska University, Akademicka 19, 20-033 Lublin, Poland Received 21 February 2006; received in revised form 4 April 2006; accepted 4 April 2006
Abstract Protein kinase A (PKA) activity was detected in the fat body of Galleria mellonella larvae by a non-radioactive method using a specific peptide substrate—kemptide. The enzyme activity was stimulated by cAMP and its analogues: BzcMP, 8-Chl-cAMP and 8-Br-cAMP in concentrations of 1–4 mM. Cyclic GMP was not effective in PKA activation. A two-fold increase in PKA activity was detected in the fat body of G. mellonella LPS-challenged larvae. Selective, membrane-permeable PKA inhibitors, H89 and Rp-8-Br-cAMPS, inhibited protein kinase A activity in the fat body of G. mellonella larvae in vitro and in vivo. The inhibition of PKA activity in vivo was correlated with a considerable lowering of haemolymph antibacterial activity and a decrease in lysozyme content in the fat body of immune challenged larvae. The use of phospho-motif antibodies recognising PKA phosphorylation consensus site allowed identification of four potential PKA phosphorylation substrates of 79, 45, 40 and 36 kDa in G. mellonella fat body. r 2006 Elsevier Ltd. All rights reserved. Keywords: Galleria mellonella; Protein kinase A; Lysozyme; Antibacterial activity; H89; Rp-8-Br-cAMPS
1. Introduction Insect immunity relies on humoral and cellular innate defence mechanisms. The cellular response results in phagocytosis, nodulation or encapsulation of non-self bodies. The humoral response generates antimicrobial proteins and peptides synthesised in the fat body and haemocytes and subsequently secreted into the haemolymph. Molecular mechanisms leading to the induction of antimicrobial peptide synthesis are not fully elucidated. In Drosophila melanogaster two independent immune signalling pathways, Toll/Dif and imd/Relish, were described, both of which activate the Rel family of transcription factors similar to the human NF-kB factors (Imler and Hoffmann, 2000; Khush et al., 2001; Hetru et al., 2003; Hultmark, 2003; Leclerc and Reichhart, 2004; Iwanaga and Lee, 2005). The Rel family of Drosophila transcription factors comprises Dorsal, Dif and Relish. Most members Corresponding author. Tel.: +48 81 537 50 89; fax: +48 81 537 50 50.
E-mail addresses: [email protected] (M. Cytryn´ska), [email protected] (T. Jakubowicz). 0022-1910/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2006.04.002
of Rel family share 300 amino acid N-terminal homology region containing a potential protein kinase A (PKA) phosphorylation site (Ghosh and Karin, 2002). It was shown that the transcriptional activity of certain vertebrate Rel family members was regulated by PKA (Zhong et al., 1998; Hou et al., 2003). Norris and Manley (1992) have found that nuclear transport and activation of Drosophila morphogen Dorsal resulted from phosphorylation of Dorsal by PKA. Moreover, phosphorylation of Dorsal at Ser312 by PKA enhanced nuclear import through facilitating an interaction with importin (Briggs et al., 1998). There are data indicating that PKA may play a regulatory role in humoral as well as cellular immune response in invertebrates. Cecropin B gene expression was triggered by the membrane-permeable cAMP analogue, dibutyryl-cAMP, and inhibited by a selective PKA inhibitor, H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide), in isolated Bombyx mori haemocytes (Shimabukuro et al., 1996; Taniai et al., 1996). PKA was implicated to be involved in the activation of the marine mussel Mytilus galloprovincialis haemocytes by bacterial lipopolysaccharide (LPS) and interleukin-2
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(Cao et al., 2004). A cyclic AMP signalling pathway was engaged in modulation of phagocytosis in oyster Crassostrea gigas haemocytes by noradrenaline (Lacoste et al., 2001). The use of PKA inhibitor, H89, also demonstrated that PKA is involved in regulation of adhesion properties of greater wax moth Galleria mellonella haemocytes. Inhibition of PKA increased the adhesion of granulocytes, but not of plasmatocytes, to glass slides (Zakarian et al., 2003). It was demonstrated that active PKA limited the G. mellonella haemocyte response in vitro and in vivo. The use of Rp-8-Br-cAMPS (Rp-isomer of 8-Bromoadenosine 30 ,50 -cyclic monophosphorothioate) for PKA inhibition increased the number of haemocytes with adherent bacteria and enhanced phagocytosis of bacteria in vitro. It also reduced haemocyte counts and induced nodulation in vivo (Brooks and Dunphy, 2005). Compounds elevating cellular cAMP concentration such as adenylate cyclase activators and cAMP-phosphodiesterases inhibitors impaired removal of Bacillus subtilis cells by G. mellonella haemocytes in vivo (Marin et al., 2005). It was concluded that a high level of cAMP impaired non-self response in G. mellonella larvae and suggested that PKA could be a mediator of the cellular immune response. In this paper, we report the results of studies on PKA activity in the fat body of the greater wax moth G. mellonella larvae. We investigated a possible role of PKA in the humoral immune response of this insect. Two membrane-permeable PKA inhibitors with different mechanisms of action were used, H89 and Rp-8-Br-cAMPS. H89 inhibits PKA activity in a competitive fashion with ATP (Chijiwa et al., 1990) while Rp-8-Br-cAMPS interacts with cAMP-binding sites in R subunits and prevents PKA holoenzyme dissociation (Gjertsen et al., 1995). We studied the correlation between PKA activity and an antimicrobial activity level in the haemolymph and the lysozyme content in the fat body of immune challenged G. mellonella larvae. Furthermore, we used specific antibodies directed against the consensus phosphorylation site of PKA for identification of potential PKA phosphorylation substrates in G. mellonella fat body. 2. Materials and methods
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apyrogenic water, respectively. In some experiments, fifteen minutes after inhibitor administration, the larvae were injected with 2.5 mg of Escherichia coli LPS (Serotype 026:B6, Sigma) in 3 ml of apyrogenic water. After the treatment, the larvae were kept at 30 1C in the dark on sterile Petri plates and the haemolymph was collected after the time indicated in the text. Prior to haemolymph collection, the insects were chilled for 15 min at 4 1C and surface disinfected with 70% ethanol. Haemolymph samples were obtained by puncturing the larval abdomen with a sterile needle. Out-flowing haemolymph was immediately transferred into sterile and chilled Eppendorf tubes containing a few crystals of phenylthiourea (PTU) to prevent melanisation. The haemocyte-free haemolymph was obtained by centrifugation at 200g for 5 min to pellet haemocytes without disruption and the supernatant was subsequently centrifuged at 20,000g for 15 min at 4 1C to pellet cell debris. The obtained haemocyte-free haemolymph was used immediately for testing antibacterial activity. 2.2. Antibacterial activity assay For antibacterial activity tests the LPS defective, streptomycin and ampicillin resistant mutant of E. coli K12, strain D31 was used (Boman et al., 1974). The antibacterial activity in the haemolymph was detected using solid agar plates containing viable E. coli cells as described (Hoffmann et al., 1981). To improve the sensitivity of the method, hen egg white lysozyme (EWL) at a concentration of 2.0 mg ml1 of the medium was added (Chalk and Suliaman, 1998; Cytryn´ska et al., 2001). Each well on the Petri dish was filled with 4 ml of haemolymph four times diluted with sterile water. The agar plates were then incubated at 37 1C for 24 h. The diameters of E. coli D31 growth inhibition zones were measured and the level of antimicrobial activity was calculated using the algorithm described by Hultmark et al. (1982). For evaluation of antibacterial activity synthetic cecropin B (Sigma) was used as a standard. 2.3. Isolation of fat bodies and preparation of cell-free extracts
2.1. Insect culture and haemolymph collection Larvae of the greater wax moth G. mellonella (Lepidoptera: Pyralidae) were reared on a natural diet—honeybee nest debris at 30 1C in the dark. Last instar larvae (250–300 mg in weight) were used throughout the study. For PKA activity inhibition in vivo the larvae were injected with 1.5 nmol of H89 (Sigma) in 3 ml of 30% dimethyl sulphoxide (DMSO) solution or 1.5 nmol of Rp8-Br-cAMPS (Sigma) in 3 ml of apyrogenic water (the approximate concentration of inhibitors in larval haemolymph—20 mM). Control animals were injected with the same volume of 30% DMSO solution (an average final concentration of DMSO in larval haemolymph—1.3%) or
For fat body isolation the larvae were anaesthesized by submerging in ice-cold apyrogenic water and then surface disinfected with 70% ethanol. The fat bodies were dissected on Petri plates under sterile ice-cold physiological saline (172 mM KCl, 68 mM NaCl, 5 mM NaHCO3, pH 6.1, osmolarity 420 mOsm) (Vilcinskas and Matha, 1997). After dissection the fat body was transferred into sterile, chilled Eppendorf tube containing 1 ml of physiological saline. Then the solution was removed and the fat body was frozen in liquid nitrogen. Cell-free extracts of fat bodies were prepared in ice-cold PKA buffer (50 mM Tris-HCl pH 7.4, 10 mM b-glycerophosphate, 2.5 mM sodium pyrophosphate, 0.5 mM
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ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 6 mM b-mercaptoethanol). Frozen fat bodies were thawed on ice and homogenised using a pellet pestle motor (Sigma-Aldrich). The obtained extracts were centrifuged at 20,000g for 15 min at 4 1C and supernatants were collected. For PKA activity assay the supernatants were used immediately. For immunoblotting an appropriate volume of Laemmli sample buffer (Laemmli, 1970) was added and the samples were stored at 20 1C. 2.4. PKA activity assay PKA activity was measured by a non-radioactive method using PepTags Assay (Promega) mainly in accordance with manufacturer’s instructions. In this assay, PKA phosphorylation substrate peptide-kemptide in modified, specially coloured version was used (named PepTags A1 Peptide). The reaction mixture in the volume of 25 ml contained: 20 mM Tris–HCl pH 7.4, 10 mM MgCl2, 1 mM ATP, 1 mM cAMP, 2 mg of PepTags A1 Peptide and 20 mg of fat body total proteins. For serine phosphatases inhibition, sodium pyrophosphate and b-glycerophosphate, each at the final concentration of 2.5 mM, were added to the reaction mixture. In the cases indicated in the text, cAMP was replaced with cAMP analogues or cGMP. For PKA activity inhibition in vitro, H89 and Rp-8-BrcAMPS were used in the concentrations indicated in the text. As for H89, which was dissolved in 30% DMSO, a control mixture contained an appropriate concentration of DMSO and concerning H89 mode of action, the ATP concentration was diminished to 0.35 mM. Each reaction was performed at room temperature for 30 min and then stopped by heating the incubation mixture at 95 1C for 10 min. PepTags A1 Peptide fluorescence was visualised after 20 min of horizontal electrophoresis in 0.8% agarose by using the UV transilluminator. During electrophoresis the unmodified substrate migrated towards the negative electrode while the PKA phosphorylated towards the positive one. For identification of PKA phosphorylation substrates in fat body cell-free extracts in vitro, incubation was performed without kemptide in the reaction mixture (25 ml) containing: 20 mM Tris–HCl pH 7.4, 10 mM MgCl2, 0.1 mM ATP, 4 mM cAMP, 5 mM b-mercaptoethanol and 40 mg of fat body total proteins. After 30 min of incubation at room temperature the reaction was stopped by addition of 12.5 ml Laemmli sample buffer. The samples were then analysed by immunoblotting as described below. 2.5. Immunoblotting Samples of fat body extracts (40 mg or 80 mg of total protein) were subjected to 13.8% SDS/PAGE and electroblotted onto Immobilon membranes (Millipore) for 90 min at 350 mA. For lysozyme identification, the membranes were probed with polyclonal antibodies (1:1000) to
G. mellonella lysozyme, a generous gift of Prof. I. H. Lee, Department of Life Science, Hoseo University, South Korea. As second antibodies, alkaline phosphatase-conjugated goat anti-rabbit IgGs (1:30,000) were used and immunoreactive bands were visualised by incubation with p-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3indolyl phosphate. Phosphorylated PKA substrates were detected with polyclonal Phospho-(Ser/Thr) PKA Substrate Antibody (1:1000) (Cell Signaling Technology, Beverly, MA). These antibodies recognised phosphorylated threonine and serine residues in PKA consensus phosphorylation motif RXXT and RRXS where R is arginine, X any amino acid, T threonine and S serine. As second antibodies, horseradish peroxidase-conjugated goat anti-rabbit IgGs (1:2000) were used and immunoreactive bands were visualised using chemiluminescent reagent and peroxide according to the manufacturer protocol (New England Biolabs. Inc., Beverly, MA). 2.6. Other methods Polyacrylamide gel electrophoresis of proteins was performed by 13.8% glycine SDS/PAGE according to Laemmli (1970). The protein concentration was estimated by Bradford method using bovine serum albumin (BSA) as a standard (Bradford, 1976). The densitometric analysis of bands was performed using Quantity One computer imaging system (BioRad, Hercules, CA). All data are presented as means7standard deviation (SD) for at least three experiments. 3. Results 3.1. PKA activity in larval fat body after treatment with PKA inhibitors It is well documented that insect fat body is the main tissue in which humoral immune response components are synthesised and for this reason PKA activity in G. mellonella fat body was investigated. The enzyme activity in vitro was measured in fat body cell-free extracts using PKA specific peptide substrate-kemptide in the presence of cAMP, cAMP analogues or cGMP. The results presented in Fig. 1 clearly showed that the enzyme was activated by 1 mM concentrations of cAMP, 8-Br-cAMP (8-Bromoadenosine-30 ,50 -cyclic monophosphate), 8-ChlcAMP (8-Chloroadenosine-30 ,50 -cyclic monophosphate) and BzcMP (ribofuranosylbenzimidazole 30 :50 -cyclic monophosphate). The level of kemptide phosphorylation in the presence of cAMP and its analogues used was comparable. On the contrary, in the presence of cGMP the kemptide phosphorylation level was much lower. Even at a concentration of cGMP as high as 10 mM, the kemptide phosphorylation level reached only 24.4% of that obtained
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Fig. 1. Protein kinase A activity in fat body of Galleria mellonella larvae. PKA activity was measured using kemptide as a phosphorylation substrate in the presence of indicated concentrations of cAMP, cGMP, BzcMP, 8-Chl-cAMP and 8-Br-cAMP as described in the Materials and Methods section. Agarose gel (photo A) was taken immediately after phosphorylated and non-phosphorylated kemptide resolution and the fragment of the gel containing phosphorylated kemptide (marked as pkemptide) is demonstrated. The diagram (B) presents kemptide phosphorylation level as revealed by densitometric analysis of the bands containing phosphorylated kemptide. Kemptide phosphorylation level measured without cyclic nucleotide addition was subtracted. Bars ¼ 7SD of four independent experiments.
in the presence of 4 mM cAMP (Fig. 1). This indicated that cGMP was not as effective as cAMP in G. mellonella fat body PKA activation. It could also suggest that protein kinase G, which is activated by cGMP, was not involved in kemptide modification. The studied enzyme activity was considerably reduced in vitro by two selective PKA inhibitors, H89 (Fig. 2A) and Rp-8-Br-cAMPS (Fig. 2B), confirming that PKA was responsible for kemptide modification. Its activity was inhibited in the presence of 0.1 and 1 mM H89 by 32.4% and 57%, respectively, in comparison to PKA activity measured in the presence of DMSO (Fig. 2A). DMSO alone decreased kemptide phosphorylating activity by 30.2%. In the presence of Rp-8-Br-cAMPS in the concentrations of 0.1 and 1 mM, the fat body PKA activity was inhibited by 65.5% and 80%, respectively (Fig. 2B). In the following experiments, we investigated the level of PKA activity in fat bodies of larvae treated with H89 and Rp-8-Br-cAMPS for different periods of time (Fig. 3). It was found that both inhibitors used in vivo reduced PKA activity 15 min after treatment. The enzyme activity returned to the control level 30 min after inhibitor
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Fig. 2. Inhibition of Galleria mellonella fat body PKA activity in vitro by H89 (A) and Rp-8-Br-cAMPS (B). PKA activity was measured in fat body cell-free extracts in the presence of 1 mM concentration of cAMP as described in Materials and Methods. The diagrams present the densitometric analysis of the bands containing phosphorylated kemptide. The insets—photos of agarose gel fragments containing phosphorylated kemptide bands. Bars ¼ 7SD of three independent experiments.
administration. H89 injection caused an enzyme activity reduction by 54.2% in comparison to the activity measured in DMSO-treated larvae (Fig. 3A). It should be mentioned here that, similarly to the in vitro experiments, DMSO injection caused reduction in kemptide phosphorylation level by 33.3% in comparison to untreated larvae. Rp-8-BrcAMPS was also effective in PKA inhibition in vivo and its administration decreased the fat body enzyme activity by 44.2% (Fig. 3B). 3.2. PKA activity in fat body of immune-challenged G. mellonella larvae The above observations prompted us to study PKA activity in the fat bodies of immune-challenged G. mellonella larvae. We found a considerable increase in PKA activity level after LPS injection (Fig. 4A, B). The highest, about two-fold, increase in PKA activity level was detected in fat bodies isolated 15–30 min after the immune challenge. When the larvae were injected with a selective PKA inhibitor, Rp-8-Br-cAMPS, before LPS treatment, a considerable reduction in enzyme activity was detected 15 min after the challenge (63.6% of inhibition). A strong
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Fig. 3. The in vivo effect of H89 (A) and Rp-8-Br-cAMPS (B) on PKA activity in Galleria mellonella larvae fat body. The larvae (five individuals per each group) were injected with H89 or DMSO solution (control) (A) and Rp-8-Br-cAMPS or water (control) (B) as described in Materials and Methods. Fat bodies were isolated at the indicated time points (one cell-free extract was prepared from fat bodies collected at the individual time point) and PKA activity was measured. The densitometric analysis of the bands containing phosphorylated kemptide was performed and the results are presented as the kemptide phosphorylation level. The first hatched bar represents PKA activity in fat body of non-treated larvae. Bars ¼ 7SD of three experiments. The insets present agarose gel fragments containing phosphorylated kemptide bands.
reduction of PKA activity (46.9–50%) was also detected 6–24 h after the challenge (Fig. 4A, B). When the cell-free extracts of fat bodies isolated from LPS-injected larvae were tested by immunoblotting with specific antibodies recognising PKA phosphorylation consensus site, a transient increase in phosphorylation level of at least four proteins of 79, 45, 40 and 36 kDa was noticed, additionally confirming the in vivo activation of PKA at the immune challenge conditions (Fig. 4C). The increase in phosphorylation level of 45 and 79 kDa proteins was detected within the first hour, whereas the 36 and 40 kDa proteins appeared 3–8 h after challenge. Studies are in progress to identify these proteins and to determine their function in G. mellonella fat body cells. We also observed cAMP stimulated phosphorylation of the 36 kDa protein in vitro in cell-free extract of non-immune fat body. 3.3. Lysozyme content in fat body of G. mellonella larvae treated with PKA inhibitors It is well documented that lysozyme is a very important factor of insect innate immunity (Hultmark, 1996). This protein is synthesised in insect fat body and haemocytes and released into the haemolymph. When G. mellonella larvae were immune challenged with LPS, lysozyme was
Fig. 4. PKA activity in fat body of immune-challenged Galleria mellonella larvae. The larvae (five individuals per each group) were injected with water, LPS or PKA inhibitor and LPS as described in Materials and Methods. Fat bodies were isolated at the indicated time points (one cellfree extract was prepared from fat bodies collected at the individual time point) and PKA activity was measured. Diagram (A) presents the densitometric analysis of the bands containing phosphorylated kemptide and photo (B) shows fragments of the agarose gels containing phosphorylated kemptide bands. The first hatched bar represents PKA activity in the fat body of non-treated larvae. Bars ¼ 7SD of three experiments. (C) Detection of potential PKA substrates in the fat body of LPS challenged larvae (in vivo) and in cell-free extracts of non-immune fat body incubated with (+) and without () cAMP addition (in vitro). The immunoblotting analysis of cell-free extracts of fat body (40 mg of total protein) was performed as described in Materials and Methods. The arrows mark four protein bands of transient increase in phosphorylation level.
detected in the fat body starting from 5–6 h after challenge (data not shown). In this paper, we tested the lysozyme content in fat body of immune-challenged G. mellonella larvae pre-treated with PKA inhibitors. To follow the lysozyme protein level, the immunoblotting technique with anti-G. mellonella lysozyme antibodies was used. It was found that the lysozyme content in the larval fat body gradually increased starting from 4 h, reaching the highest-level 24 h after DMSO–LPS treatment (Fig. 5A). On the contrary, in the fat body of larvae pre-treated with H89 before LPS injection, the
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Fig. 5. Immunodetection of lysozyme in Galleria mellonella immunechallenged larvae pre-treated with H89 (A) and Rp-8-Br-cAMPS (B) in vivo. The larvae (five individuals per each group) were injected with PKA inhibitors and LPS as described in Materials and Methods. Fat bodies were isolated at the indicated time points (one cell-free extract was prepared from fat bodies collected at the individual time point) and prepared for immunoblotting. Samples containing 80 mg of total fat body protein were resolved by SDS/PAGE and transferred onto Immobilon membranes. The membranes were probed with anti G. mellonella lysozyme antibodies. The lysozyme band is marked by an arrow. The diagrams present the densitometric analysis of the bands containing lysozyme. Bars ¼ 7SD of three experiments.
lysozyme appeared later (5 h after challenge) and the protein level detected was much lower in comparison to DMSO–LPS-injected larvae. The densitometric analysis revealed that in the fat body of H89–LPS-treated larvae obtained 5, 6, 8 and 24 h after challenge, the lysozyme content reached 15.4%, 36.5%, 71.8% and 46.4%, respectively, of the content measured for the fat bodies of DMSO–LPS-treated larvae (Fig. 5A). Similar results were obtained when Rp-8-Br-cAMPS was used for PKA activity inhibition in vivo (Fig. 5B). The lysozyme was detected in the fat body of water pre-treated immune larvae 6–24 h after the challenge, whereas in the fat body of inhibitor-injected larvae only traces of the protein were detected and the lysozyme content reached only 24.5–42.5% of that measured in immune fat body. The presented results suggested that the changes in lysozyme content detected after PKA inhibitors injection, could be caused, at least in part, by fat body PKA activity inhibition. 3.4. Antibacterial activity in haemolymph of G. mellonella larvae treated with PKA inhibitors As shown above, the immune challenge of G. mellonella larvae led to an increase in fat body PKA activity, and injection of PKA inhibitor before the challenge caused a strong reduction in enzyme activity. The insect fat body is the main site of antimicrobial protein and peptide synthesis and release into the haemolymph. We tested whether the observed changes in fat body PKA activity were correlated with the level of antibacterial activity in the haemolymph. The activity inhibiting growth of E. coli D31 was measured
Fig. 6. Antibacterial activity in the haemolymph of Galleria mellonella immune-challenged larvae pre-treated with H89 (A) and Rp-8-Br-cAMPS (B) in vivo. The larvae (ten individuals per each group) were injected with PKA inhibitors and LPS as described in Materials and Methods. The haemolymph was collected from 10 individuals together at the indicated time points (20 ml/larva) and used for antimicrobial activity assay as described. Bars ¼ 7SD of five independent experiments.
in the haemolymph of immune-challenged larvae pretreated with H89 or Rp-8-Br-cAMPS before LPS administration (Fig. 6). It was found that the antimicrobial activity level in the haemolymph of animals pre-treated with PKA inhibitors was much lower in comparison to the haemolymph of immune larvae. This effect was especially evident during the first 8 h after the challenge. Anti-E. coli activity level in the haemolymph of H89–LPS-treated larvae constituted 47.6–62.5% of the level measured for DMSO–LPS-treated ones (Fig. 6A). In the haemolymph of Rp-8-Br-cAMPS–LPS-treated larvae the antimicrobial activity level constituted 68.2–74.2% of that measured for H2O–LPS-treated animals (Fig. 6B). The obtained results indicated that the lower level of haemolymph antimicrobial activity was correlated with PKA activity inhibition by H89 and Rp-8-Br-cAMPS in vivo. In the case of H89–LPSinjected larvae, 24 h after the challenge, the antimicrobial activity level was comparable to that detected in the DMSO–LPS-treated ones, while in the haemolymph of Rp8-Br-cAMPS–LPS-treated larvae a strong increase in antibacterial activity measured 24 h after immune-challenge was detected.
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4. Discussion Reversible protein phosphorylation regulates a large number of physiological processes in living organisms. Involvement of protein kinases in regulation of the immune response is well documented. In mammalian systems, mitogen-activated protein (MAP) kinases are engaged in the initiation phase of innate immunity and activation of adaptive immunity. MAP kinases participate in signalling pathways leading to the production of inflammatory cytokines, e.g. tumour necrosis factor a (TNF a), interleukin-1 and -12 (Dong et al., 2002). Similarly to the mammalian system, MAP kinases JNK and p38 were activated in response to pathogen recognition in Drosophila (Sluss et al., 1996; Han et al., 1998). Recently, the role of JNK MAP kinase in regulation of the G. mellonella humoral immune response was documented by Wojda et al. (2004). Different protein kinases, e.g. IkB kinase (IKK), Akt kinase, PI-3 kinase, CKII and PKA can be involved in NF-kB signalling pathways in mammalian and insect innate immunity (reviewed in Karin, 1999; Silverman and Maniatis, 2001). It was suggested that protein kinase C and tyrosine protein kinases participate in regulation of the immune response mechanisms (Johansson and So¨derha¨ll, 1993; Weinstein et al., 1993; Charalambidis et al., 1995; Lanz-Mendoza et al., 1996). Literature data indicate PKA participation in regulation of the immune response of vertebrates and invertebrates (Fujihara et al., 1993; Muroi and Suzuki, 1993; Shimabukuro et al., 1996; Chen et al., 1999; Regenhard et al., 2001; Cao et al., 2004; reviewed in Torgersen et al., 2002). The involvement of PKA in the modulation of G. mellonella haemocytes adhesion properties in vitro (Zakarian et al., 2003) and non-self cellular response in vivo (Brooks and Dunphy, 2005; Marin et al., 2005) was shown, suggesting its role in the cellular immune response regulation. In the present study, PKA involvement in the regulation of the humoral immune response of G. mellonella was investigated. Our studies provide evidence for PKA activity in G. mellonella fat body, the tissue in which humoral immune response components are synthesised. The enzyme was activated in vitro in the presence of cAMP and certain cAMP analogues such as 8-Br-cAMP and 8-Chl-cAMP, which are membrane-permeable cAMP analogues widely used in signal transduction research. 8-Chl-cAMP was also extensively tested as an anticancer agent (Schwede et al., 2000). Additionally, G. mellonella fat body PKA was activated by BzcMP indicating that, similarly to mammalian cAMP-dependent protein kinase and yeast S. cerevisiae PKA, the studied enzyme does not require the 6-amino group and the 1 and 3 nitrogenes in the adenine ring to be activated (Yagura et al., 1980; Øgreid et al., 1985; Cytryn´ska et al., 1999). G. mellonella fat body PKA was hardly activated in vitro by cGMP. It was consistent with the studies showing that in higher Eucaryota two different protein kinases, PKA and protein kinase G, respond to cAMP and cGMP, respectively (Weber et al., 1989;
Hofmann et al., 1992). Tsuzuki and Newburgh (1974) suggested that in the central nervous system of G. mellonella two different protein kinases responded to cAMP and cGMP. They also suggested the presence of cIMP-dependent protein kinase in this tissue of G. mellonella. It is worth mentioning here that PKA activated by both cyclic nucleotides, cAMP and cGMP, was reported for several arthropod species: Melanoplus sanquinipes (Vardanis, 1983), Locusta migratoria (Beenakkers et al., 1978), Periplaneta americana (Takahashi and Hanaoka, 1977), Amblyoma americanum (Mane et al., 1985), Orconectes limosus (Christ, 1985). In some unicellular Eucaryota like yeast Saccharomyces cerevisiae and Pichia pastoris, PKA can be also almost equally activated by cAMP and cGMP (Cytryn´ska et al., 1999; Frajnt et al., 2003). The PKA in fat body of G. mellonella larvae was inhibited in vitro by two selective PKA inhibitors, H89 and Rp-8-Br-cAMPS. At a 1 mM concentration of H89 enzyme activity was inhibited by 57%. It is known that H89 inhibits PKA activity in a competitive fashion with ATP with the inhibition constant of 0.048 mM when purified cAMP-dependent protein kinase catalytic subunit from bovine heart and histone IIb as a substrate are used (Chijiwa et al., 1990). In our in vitro experiments, 0.05 mM concentration of H89 inhibited fat body PKA activity by 25%. It is strong inhibition if we consider that the ATP concentration used in our experiments (0.35 mM) was much higher than used in Chijiwa et al. (1990) experiments (5–50 mM). The second selective PKA inhibitor, Rp-8-BrcAMPS, decreased the kemptide phosphorylation level by 65.5% and 80%, respectively, for the concentration of 0.1 and 1 mM. This was consistent with the studies presented by Gjertsen et al. (1995) for purified cAMP-dependent protein kinase I from rabbit muscle. Both inhibitors were widely used for selective PKA inhibition in vitro and in vivo in a large number of studies (Chijiwa et al., 1990; Shimabukuro et al., 1996; Lacoste et al., 2001; Regenhard et al., 2001; Malagoli et al., 2002; Zakarian et al., 2003; Brooks and Dunphy, 2005; Marin et al., 2005). We showed that both compounds were also effective in G. mellonella fat body PKA inhibition in vivo. The enzyme activity inhibition was detected within the first 15 min after inhibitor administration, suggesting a fast response of fat body cells. The activity level was reduced by 54 and 44% in the case of H89 and Rp-8-Br-cAMPS treatment, respectively. The inhibitory effect of Rp-8-Br-cAMPS was much stronger when activity of PKA was tested in the fat body of immune-challenged larvae. PKA activity inhibition was sustained for prolonged periods of time and correlated with changes in the level of humoral immune response components of G. mellonella larvae. The inhibition of fat body PKA activity by H89 and Rp8-Br-cAMPS in vivo was correlated with a decrease in fat body lysozyme content. The observed effect could be caused by a partial inhibition of lysozyme synthesis in the fat body and/or with an increased rate of protein secretion.
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Whether PKA is involved in the regulation of these processes in the insect fat body requires further studies. In promoters of insect immune genes, including lysozyme genes, a conserved consensus sequence, similar to kB site was found (Hultmark, 1996; Engstro¨m, 1998) indicating that Rel transcription factors are involved in the control of these genes expression. As PKA regulates at least certain Rel factors activity, one can speculate that the enzyme could also be involved in the regulation of lysozyme gene expression in G. mellonella fat body. The role of PKA in the regulation of LPS-activated lysozyme gene expression was documented by Regenhard et al. (2001) for chicken lysozyme gene in myelomonocytic cells. The inhibition of PKA activity in G. mellonella fat body in vivo by H89 or Rp-8-Br-cAMPS, prior to immune challenge, coincided with the lowering of antibacterial activity in the haemolymph. The low level of bacterial growth inhibiting activity was sustained until 8 h after the challenge and returned to the control level after 24 h. We thus hypothesise that PKA is involved in modulation of the humoral immune response in G. mellonella larvae. The involvement of PKA in the regulation of cecropin B gene expression was shown in silkworm B. mori by Shimabukuro et al. (1996) and Taniai et al. (1996). We demonstrated that after immune challenge PKA activity in G. mellonella fat body considerably increased, suggesting a role for PKA in regulation of the immune response of this insect. To identify potential PKA phosphorylation substrates in the fat body of G. mellonella larvae we used methodology based on phospho-motif antibodies (Grønborg et al., 2002; Berwick and Tavare´, 2004). Antibodies specific for PKA phosphorylation consensus site with serine or threonine in phosphorylated state recognised several proteins in the fat body of G. mellonella larvae. Some of these proteins were phosphorylated to a similar level in both immune and nonimmune larvae. In addition, at least four fat body proteins of 79, 45, 40 and 36 kDa were transiently phosphorylated in vivo in LPS-challenged larvae. This observation provided another argument for the participation of PKA in regulation of the immune response of G. mellonella larvae. It is worth mentioning here that, during immune response, changes in protein phosphorylation caused by other protein kinases were reported. Wojda et al. (2004) showed changes in the phosphorylation level of fat body JNK MAP kinase observed after LPS immune stimulation of G. mellonella larvae. It was reported that LPS-stimulated exocytosis of non-self recognition protein from Ceratitis capitata haemocytes was dependent on protein tyrosine phosphorylation (Charalambidis et al., 1995) and changes in tyrosine phosphorylation of two haemocyte proteins of 19 and 22kDa were detected (Zervas et al., 1998). Changes in protein phosphorylation induced in human macrophages after LPS exposure were also described by Weinstein et al. (1993). The results presented in this paper demonstrated the PKA activity in the fat body of G. mellonella larvae. The
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inhibition of PKA activity in vivo by two selective PKA inhibitors, H89 and Rp-8-Br-cAMPS, was correlated with a lower level of haemolymph antimicrobial activity and a considerable decrease in lysozyme content in larval fat body. Moreover, phosphorylation changes of at least four potential PKA substrates in the fat body of immune challenged larvae were detected. The presented results clearly indicate the participation of PKA in humoral immune response regulation of greater wax moth G. mellonella larvae. Acknowledgements This work was financially supported by the grant from the State Committee for Scientific Research (KBN, Poland) No. 2 P04C 080 29. References Beenakkers, A.M.T., Van der Horst, D.J., Van Marewijk, W.J.A., 1978. Regulation of release and metabolic function of the adipokinetic hormone in insects. In: Gaillard, P.J., Boer, H.H. (Eds.), Comparative Endocrinology. Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 445–448. Berwick, D.C., Tavare´, J.M., 2004. Identifying protein kinase substrates: hunting for the organ-grinder’s monkeys. Trends in Biochemical Sciences 29, 227–232. Boman, H.G., Nilsson-Faye, I., Paul, K., Rasmuson Jr., T., 1974. Insect immunity. 1. Characteristics of an inducible cell-free antibacterial reaction in hemolymph of Samia cynthia pupae. Infection and Immunity 10, 136–145. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Briggs, L.J., Stein, D., Goltz, J., Corrigan, V.C., Efthymiadis, A., Hu¨bner, S., Jans, D.A., 1998. The cAMP-dependent protein kinase site (Ser312) enhances Dorsal nuclear import through facilitating nuclear localization sequence/importin interaction. The Journal of Biological Chemistry 273, 22745–22752. Brooks, C.L., Dunphy, G.B., 2005. Protein kinase A affects Galleria mellonella (Insecta: Lepidoptera) larval haemocyte non-self responses. Immunology and Cell Biology 83, 150–159. Cao, A., Martinez, J.I.R., Barcia, R., 2004. Implication of PKA and PKC in the activation of the haemocytes of Mytilus galloprovincialis Lmk. by LPS and IL-2. Molecular Immunology 41, 45–52. Chalk, R., Suliaman, W.Y., 1998. Antimicrobial peptides from small insects: methods for insect culture and for the detection, visualization, isolation and sequencing of active hemolymph peptides. In: Wiesner, A., Dunphy, G.B., Marmaras, V.J., Morishima, I., Sugumaran, M., Yamakawa, M. (Eds.), Techniques in Insect Immunology. SOS Publications, USA, pp. 109–124. Charalambidis, N.D., Zervas, C.G., Lambropoulou, M., Katsoris, P.G., Marmaras, V.J., 1995. Lipopolysaccharide-stimulated exocytosis of nonself recognition protein from insect hemocytes depends on protein tyrosine phosphorylation. European Journal of Cell Biology 67, 32–41. Chen, C.C., Chiu, K.T., Sun, Y.T., Chen, W.C., 1999. Role of the cyclic AMP-protein kinase A pathway in lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macrophages. Involvement of cyclooxygenase-2. The Journal of Biological Chemistry 274, 31559–31564. Chijiwa, T., Mishima, A., Hagiwara, M., Sano, M., Hayashi, K., Inoue, T., Naito, K., Toshioka, T., Hidaka, H., 1990. Inhibition of forskolininduced neurite outgrowth and protein phosphorylation by a newly
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