CoPK32 is a novel stress-responsive protein kinase in the mushroom Coprinopsis cinerea

Biochimica et Biophysica Acta 1810 (2011) 620–629

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n

CoPK32 is a novel stress-responsive protein kinase in the mushroom Coprinopsis cinerea Keisuke Kaneko a, Yasunori Sugiyama a, Yusuke Yamada a, Noriyuki Sueyoshi a, Akira Watanabe a, Yasuhiko Asada a, Atsuhiko Ishida b, Isamu Kameshita a,⁎ a b

Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan

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Article history: Received 15 October 2010 Received in revised form 7 March 2011 Accepted 24 March 2011 Available online 1 April 2011 Keywords: Basidiomycetes Coprinopsis cinerea (Coprinus cinereus) Mushroom Osmotic stress Protein kinase Stress response

a b s t r a c t Background: In a previous study, we conducted an expression cloning screen of a cDNA library prepared from Coprinopsis cinerea mycelia using Multi-PK antibodies and detected a wide variety of Ser/Thr protein kinases. One of the isolated clones, CMZ032, was found to encode a putative Ser/Thr protein kinase designated CoPK32. In the present study, we investigated the biochemical properties and physiological significance of CoPK32. Methods: CoPK32 was expressed in Escherichia coli, and its biochemical properties were examined. The effects of high osmotic stresses on the growth of C. cinerea and on the endogenous CoPK32 activity in mycelia were also examined. Results: CoPK32 showed autophosphorylation activity and effectively phosphorylated exogenous protein substrates. CoPK32S, a splice variant that was 18 amino acids shorter than CoPK32, showed much lower protein kinase activity than CoPK32. The catalytic properties of CoPK32 deletion mutants suggested that the C-terminal region of CoPK32 was important for the kinase activity and recognition of substrates. CoPK32 was highly expressed in the actively growing region of the mycelial colony. When mycelia were stimulated by high osmotic stresses, endogenous CoPK32 was markedly activated and the mycelial growth was severely inhibited. The activation of CoPK32 activity by high osmotic stresses was abrogated by SB202190 or SB239063 as well-known inhibitors of p38 mitogen-activated protein kinase. Conclusions: CoPK32 is involved in the stress response pathway in mycelia of C. cinerea in response to environmental stresses. General significance: In C. cinerea, protein kinases such as CoPK32 play important roles in signal transduction pathways involved in stress responses. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Coprinopsis cinerea is one of the model organisms that are commonly used for morphological and molecular biological studies in basidiomycete mushrooms. Basidiomycete mushrooms exhibit unique developmental processes during their life cycles. Dikaryotic mycelia arise from fusion of monokaryons of different mating types, and fruiting bodies are subsequently formed in response to environmental stresses including light exposure, changes in temperature and nutrient depletion [1,2]. Although it has been suggested that Rasmediated cAMP signaling pathways play roles in fruiting body formation in the basidiomycete fungus Schizophyllum commune [3],

Abbreviations: CaMK, Ca2+/calmodulin-dependent protein kinase; MAPK, mitogenactivated protein kinase; MAPKAPK, mitogen-activated protein kinase-activated protein kinase; MBP, myelin basic protein; PKA, cAMP-dependent protein kinase ⁎ Corresponding author. Tel./fax: + 81 87 891 3120. E-mail address: [email protected] (I. Kameshita). 0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2011.03.018

little is known about the detailed molecular mechanisms of the signal transduction pathways for fruiting body formation. It is widely accepted that the development and differentiation of various organisms are governed by protein phosphorylation. In the Hedgehog signaling pathway, which is essential for mammalian embryogenesis, protein kinases such as cAMP-dependent protein kinase (PKA) and casein kinases play important roles in the signal transduction [4]. In social amoebas, differentiation processes including aggregation and multicellular development are known to be regulated by the cAMP signaling pathway, which involves several protein kinases [5]. In the basidiomycete Cryptococcus neoformans, some protein kinases such as Kic1 and Cbk1 are involved in the control of cell polarity and morphogenesis [6]. The formation of asexual spores termed conidia is critical in the life cycle of many fungi including basidiomycetes. It has been suggested that PKA, mitogenactivated protein kinase (MAPK) or Ca2+/calmodulin-dependent protein kinases (CaMKs) are involved in the signaling pathway responsible for conidial germination in some fungi [7]. Prompted by

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these findings, we assumed that protein phosphorylation would also be involved in the development of fruiting bodies from vegetative mycelia of basidiomycetes. However, there are only a few biochemical studies dealing with the biological significance of protein kinases in the life cycle of mushrooms. Since the AmutBmut strain of C. cinerea is a mutant that produces fruiting bodies without prior mating with another strain, it is often used for developmental studies of C. cinerea. In a previous study, we examined the expression profile of Ser/Thr protein kinases in the AmutBmut strain. Using Multi-PK antibodies, which can detect a wide variety of Ser/Thr protein kinases, we found differential expressions of some protein kinases during the developmental processes [8]. These observations suggested that a variety of protein kinases play roles in individual developmental stages of C. cinerea. To further explore the roles of the protein kinases, we isolated 14 cDNA clones from a mycelial cDNA library of a homokaryotic mutant strain by expression cloning using Multi-PK antibodies as probes [9]. Of these 14 cDNAs, two isolated clones (CMZ012 and CMZ032) were found to encode novel protein kinases. CMZ012 encodes CoPK12, which is a novel CaMK in C. cinerea. When the activity of CaMKs including CoPK12 was inhibited, suppression of mycelial growth was observed, suggesting that a Ca2+-mediated signaling pathway is involved in the proliferation of C. cinerea [9]. In the present study, we analyzed the other clone, CMZ032. This clone encodes CoPK32, which shows high homology to fission yeast Srk1 and budding yeast Rck2, which are known to be mitogenactivated protein kinase-activated protein kinase (MAPKAPK) in yeasts. MAPKAPK is a downstream protein kinase of MAPK, suggesting that CoPK32 is a member of the MAPK cascade in C. cinerea. In yeasts, Rck2 and Srk1 are regulated by activated MAPK and mediate gene expression in response to environmental stresses [10,11]. In mushrooms, investigations of MAPK and its downstream targets are extremely scarce, and their physiological roles remain to be clarified. Although a recent study found that the expression level of MAPK increases during development in the basidiomycete Lentinula edodes [12], its physiological function is still unclear. In the present study, we found that CoPK32 is a novel stress-responsive protein kinase in C. cinerea. We also evaluated the gene expression profiles and biochemical properties of CoPK32, together with its functional role in stress-response pathways. 2. Materials and methods 2.1. Materials [γ-32P]ATP (111 TBq/mmol) was purchased from PerkinElmer. ATP, SB202190, SB239063, Cy3-labeled anti-mouse IgG, lysing enzymes from Trichoderma harzianum, myelin basic protein (MBP), α-casein, BSA and histone type IIA were obtained from Sigma-Aldrich. SB202474 was purchased from Merck. Goat anti-mouse IgG + A+M conjugated with horseradish peroxidase and an anti-His6 antibody were obtained from ICN Pharmaceuticals and Roche Diagnostics, respectively. Myosin light chain was prepared as described previously [13]. Multi-PK antibodies, M8C and M1C, were obtained from two hybridoma cell lines that were established as described previously [14]. 2.2. Strains, growth conditions and preparation of crude extracts of C. cinerea

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one mycelial plug was inoculated on MYG agar containing specified concentrations of KCl or mannitol and grown under the conditions described above. Mycelia on the surface of agar were gently scraped and collected with a spatula. Primordia and fruiting bodies were picked up with forceps. Crude extracts of these specimens were prepared as described previously [8]. When the growing mycelia were stimulated using aqueous solutions, the plates were filled with 4 ml of specified concentrations of KCl, NaCl, sucrose, mannitol or H2O2 and incubated at room temperature for 30 min. After removal of the stimulant solutions, the mycelia were immediately harvested. When UV irradiation (8 W) and heat treatment (42 °C) were carried out, growing mycelia were incubated under these conditions for 30 min and then harvested. 2.3. Cloning of CoPK32 and CoPK32S CMZ032, the cDNA encoding CoPK32, was obtained from expression cloning in a previous study [9]. A SMART RACE cDNA amplification kit (Clontech) was used to obtain the full-length coding sequence for CoPK32 [9]. Briefly, the 5'-end of the mycelial cDNA obtained from C. cinerea strain 326 was amplified by PCR with a genespecific primer (5'-GTA CGG TCG CTT TAT TGG GTC TTG GA-3') and a universal primer mix with the 5'-RACE first-strand cDNA as the template. A sense primer (5'-AAT TCA TCC TTT TTC GAT CCC CC-3') and an antisense primer (5'-GAA TGG ACC AGG GGA TAT CAA TAG C-3') were designed from the sequences outside of the open reading frame, respectively. A full-length cDNA was obtained by PCR using these primers and a 5'-RACE ready cDNA library as a template. The PCR product was cloned into a pGEM-T Easy vector, and 12 independent clones were sequenced. These clones included two open reading frames with lengths of 1869 and 1815 bp, which were designated pGEMCoPK32 and pGEMCoPK32S, respectively. 2.4. Plasmid constructions To generate pET-23a(+) vectors (Novagen) encoding CoPK32 and CoPK32S tagged with His6 at their C-terminals, PCR amplification (30 cycles of denaturation for 10 s at 96 °C, annealing for 10 s at 60 °C and extension for 3 min at 72 °C) was carried out with pGEMCoPK32 or pGEMCoPK32S as the template using the following primers: sense primer (5'-GCT AGC ATG CCT CCA ACC GGC GT-3') and antisense primer (5'-GAG CTC GCA AGG GCC GTC ACA GAT GGA-3'). The NheI ¼ (underlined)–SacI (double-underlined) fragment was inserted into the NheI–SacI sites of pET-23a(+), and the resulting products were designated pETCoPK32 and pETCoPK32S, respectively. Two C-terminal deletion mutants of CoPK32, CoPK32(1–487) and CoPK32(1–401) were prepared by an inverse PCR method [16] with a sense primer (5'-GCG AGC TCC GTC GAC AAG CT-3') and antisense primers (5'-GTT AAG CTC GCT AAG GAA GCC CC-3' for CoPK32(1–487) or 5'-GCA CCA AGG GTG TGC GAG G-3' for CoPK32(1–401)). PCR amplifications were carried out as described above except that the extension time was 7 min and pETCoPK32 was used as a template. After gel purification, the amplified products were phosphorylated and self-ligated by T4 DNA polynucleotide kinase and T4 DNA ligase (Nippon Gene). A deletion mutant missing the sequence for residues 447–486 was generated using a PrimeSTAR Mutagenesis Basal Kit (TaKaRa) according to the manufacturer's instructions. 2.5. Expression and purification of recombinant enzymes

The homokaryotic strain 326 (AmutBmut) and standard homokaryotic strain 5302 (A2B2) of C. cinerea [15] were kindly provided by Dr. T. Kamada of Okayama University. Four mycelial plugs were inoculated and grown on MYG agar (1% malt extract, 0.4% yeast extract, 0.4% glucose, 2% agar) in 8.5-cm plastic dishes under a 12h:12-h light/dark cycle at 28 °C. For observation of mycelial growth,

Escherichia coli BL21(DE3) cells transformed with pETCoPK32, pETCoPK32S and other expression plasmids were grown at 18 °C to an A600 of 0.6–0.8, and then isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.1 mM. After shaking for 24 h at 18 °C, the bacteria were harvested by centrifugation and suspended in

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20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 40 containing 1 mM phenylmethylsulfonyl fluoride. Recombinant proteins expressed in the bacteria were purified using a HiTrap Chelating HP column (GE Healthcare Bio-Sciences) as described previously [9]. The purified enzymes were stored in aliquots at −80 °C until use.

focusing were incubated in the standard reaction mixture (10 μl) containing 20 mM MgCl2 and 100 ng of CoPK32 or 25 ng of CoPK32 (1–401). After incubation at 30 °C for 1 h, an equal volume of 2× SDS– PAGE sample buffer was added to the phosphorylation mixture to stop the reaction. The phosphorylated proteins were subjected to SDS–PAGE and visualized by autoradiography.

2.6. Antibody production An antibody against CoPK32 was produced by immunizing BALB/c mice with purified CoPK32. The immunization was carried out essentially as described previously [9]. All of the mice were kept in specific pathogen-free rooms that were controlled for both temperature and light. The experiments were approved by the Kagawa University Institutional Animal Care and Use Committee. 2.7. RT-PCR Total RNA was prepared from 1-g samples of representative developmental stages of C. cinerea strain 326 using 1 ml of TRIzol reagent (Invitrogen) with mortars and pestles. cDNAs were synthesized from 1 μg of the total RNA with iScript Reverse Transcriptase (Bio-Rad) and used as templates. A pair of primers (5'-ATG CCT CCA ACC GGC GTC GT-3' and 5'-TGC ACC TCT TTG AGA ATA TTC GCC-3') for simultaneous amplification of CoPK32 and CoPK32S was used for PCR (35 cycles) with rTaq polymerase (TaKaRa). pETCoPK32 and pETCoPK32S were used as positive controls. The amplified fragments were subjected to 2% agarose gel electrophoresis in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA) and stained with ethidium bromide. An actin gene (GenBank ID: BAA86222) was used as an internal standard for the RT-PCR analyses. 2.8. Protein determination, SDS–PAGE and Western blotting Protein concentrations were determined by the method of Bensadoun and Weinstein using BSA as a standard [17]. SDS–PAGE was carried out essentially according to the method of Laemmli [18] in slab gels consisting of a 7%, 10% or 12% acrylamide separating gel and a 3% stacking gel. Western blotting analysis was carried out as described previously [9]. 2.9. Fractionation of crude extracts using a MicroRotofor Liquid phase isoelectric focusing using a MicroRotofor (Bio-Rad) was carried out essentially as described previously [19]. A crude extract of C. cinerea mycelia (2.5 mg) was precipitated with trichloroacetic acid, and the precipitate was rinsed with acetone and solubilized with 2.5 ml of an IEF buffer (7 M urea, 2 M thiourea, 5 mM dithiothreitol, 4% CHAPS, 2% Pharmalyte pH 3–10). The protein solution was fractionated using the MicroRotofor and electrophoresed for 2.5 h at a constant power of 1 W at room temperature. After the electrophoresis, the protein fractions from each compartment (200 μl) were collected and precipitated with trichloroacetic acid. The precipitates were rinsed with acetone, dissolved in 40 μl of homogenizing buffer and used for protein kinase assays. 2.10. Protein kinase assay Autophosphorylation of CoPK32 and CoPK32S was carried out using a standard reaction mixture (10 μl) consisting of 40 mM HepesNaOH (pH 8.0), 5 mM Mg(CH3COO)2, 0.1 mM EGTA, 2 mM dithiothreitol, 100 μM [γ-32P]ATP and 100 ng of enzymes. The reactions were started by the addition of the enzymes, incubated at 30 °C for 30 min and stopped by the addition of 10 μl of 2× SDS–PAGE sample buffer. Phosphorylation of protein substrates (500 ng) was carried out in the standard reaction mixture (10 μl) containing 100 ng of enzymes at 30 °C for 30 min. Mycelial extracts (4 μl) fractionated by isoelectric

2.11. Detection of endogenous CoPK32 kinase activity Endogenous CoPK32 was immunoprecipitated from a crude extract of C. cinerea mycelia (50 μg) using an anti-CoPK32 antibody and protein G-Sepharose beads (10 μl; GE Healthcare Bio-Sciences). The beads were washed five times with 500 μl of PBS (2.68 mM KCl, 1.47 mM KH2PO4, 137 mM NaCl, 8.1 mM Na2HPO4) containing 0.05% Tween 20 and then once with the standard reaction mixture without ATP. The washed beads were incubated with 1 μg of histone in 20 μl of the standard reaction mixture containing 100 μM [γ-32P]ATP at 30 °C for 1 h. After the incubation, the reaction was stopped by the addition of 5 μl of 5× SDS–PAGE sample buffer. The phosphorylation of histone by endogenous CoPK32 was analyzed by autoradiography. 2.12. Immunocytochemistry of CoPK32 in hyphal cells The localization of CoPK32 was analyzed using a previously described method for hyphal cells [20,21] with minor modifications. C. cinerea mycelia were grown on an MYG agar plate with cover glasses placed near the mycelial plugs. Growing hyphae reaching the center of the cover glasses were fixed in 4% (w/v) formaldehyde containing 5% (w/v) PEG6000, 50 mM NaH2PO4-NaOH (pH 6.5), 5 mM MgCl2 and 5 mM EGTA at room temperature for 20 min. The cover glasses were washed three times with PBS, and then soaked in 0.4% (w/v) lysing enzymes, 50 mM NaH2PO4–NaOH (pH 6.5) and 5 mM MgCl2 at room temperature for 3 min to partly digest the cell walls. Next, the cover glasses were washed three times with PBS and soaked in 1% (v/v) Triton X-100, 5 mM EGTA and 1 mM phenylmethylsulfonyl fluoride at room temperature for 20 min. After three rinses with PBS, the samples were incubated with an anti-CoPK32 antibody diluted 1:200 with PBS containing 1% (w/v) BSA at 4 °C for 16 h, followed by incubation with Cy3-labeled anti-mouse IgG (1:1000) and 10 μg/ml Calcofluor White for 8 h to detect the bound primary antibody and hyphal cell walls, respectively. The stained cells were observed under a confocal laser scanning microscope (Olympus FV-1000D).

3. Results 3.1. Molecular cloning of CoPK32 In a previous study, we isolated 14 cDNA clones of putative protein kinases from a mycelial cDNA library of C. cinerea strain 326 by expression cloning using Multi-PK antibodies for Ser/Thr protein kinases [9]. One of the isolated clones was designated CMZ032, and its full-length cDNA containing the entire coding region was obtained using the RACE PCR method. Two splice variants of CMZ032 were found, designated CoPK32 and CoPK32 short (CoPK32S), that encoded proteins containing 12 subdomains specific to protein kinases. CoPK32 and CoPK32S consisted of 623 and 605 amino acids, respectively, and the only difference between them was that the former had an insertion sequence of 18 amino acids between subdomains II and III while the latter did not (Fig. 1). When BLAST homology searches were carried out on the basis of the CoPK32 sequence, fission yeast Srk1 and budding yeast Rck2 showed 47% and 36% identity to CoPK32, respectively. Since Srk1 and Rck2 are known to be stress-responsive MAPKAPK homologues in yeasts [10,11], CoPK32 appears to be a homologue of MAPKAPK in C. cinerea.

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Fig. 1. Primary structure of CoPK32. (A) Schematic illustrations of CoPK32 (GenBank ID: BAG74357) and CoPK32S (GenBank ID: BAG74358). The catalytic domains are shown by black boxes. (B) The amino acid sequence of CoPK32 was aligned with those of fission yeast Srk1 (GenBank ID: CAA22861) and budding yeast Rck2 (GenBank ID: EEU06778) using CLUSTAL W. Identical amino acids are shaded in black, and gaps inserted into the sequences are indicated by dots. Twelve subdomains specific to protein kinases are shown by underlines and Roman numerals. An 18-amino-acid insertion sequence between subdomains II and III of CoPK32 is indicated by asterisks. The double underlined sequence indicates a putative autoinhibitory domain.

3.2. Heterologous expression of CoPK32 in bacteria Recombinant CoPK32 and CoPK32S were expressed in soluble forms in E. coli cells carrying pETCoPK32 and pETCoPK32S, respectively. These proteins were purified by chromatography using a

HiTrap Chelating HP column. When purified CoPK32 and CoPK32S were subjected to SDS–PAGE followed by Coomassie brilliant blue staining, the protein bands showed slightly different electrophoretic mobilities with molecular masses of around 70 kDa (Fig. 2A). Western blotting analyses of the purified proteins with an anti-His6 antibody or

Fig. 2. Electrophoretic analyses of CoPK32. (A–D) Recombinant CoPK32 (left lane) and CoPK32S (right lane) were subjected to 7% SDS–PAGE and detected by Coomassie brilliant blue (A, 1 μg). Western blotting analyses were carried out with an anti-His6 antibody (B, 100 ng CoPK32), a Multi-PK antibody (C, 10 ng CoPK32) and an anti-CoPK32 antibody (D, 5 ng CoPK32). (E) Autophosphorylation activities of CoPK32 and CoPK32S. Approximately 100 ng of purified enzymes was incubated with 100 μM [γ-32P]ATP in a standard phosphorylation mixture (10 μl). After incubation at 30 °C for 30 min, the reactions were stopped by the addition of 10 μl of 2× SDS–PAGE sample buffer. Phosphorylated proteins were resolved by SDS–PAGE and detected by autoradiography.

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a Multi-PK antibody detected single immunoreactive bands corresponding to the Coomassie brilliant blue-stained bands (Fig. 2B and C). When an antibody specific for CoPK32 was used for the Western blotting analyses, similar immunoreactive bands were detected (Fig. 2D). The autophosphorylation activities of these enzymes were also examined. When the enzymes were incubated under autophosphorylation conditions in the presence of [γ-32P]ATP, a prominent radioactive band of CoPK32, but not of CoPK32S, was detected by autoradiography (Fig. 2E), indicating that CoPK32 has stronger autophosphorylation activity than CoPK32S.

tip cells of mycelia, including branching cells, and was hardly detected in the other regions. When the antibody was preincubated with the recombinant CoPK32 used for immunization, the immunoreactive signals were greatly diminished (Fig. 4C and F), thereby eliminating the possibility that the detected signals were caused by non-specific binding of the antibody. Taken together, these findings suggest that CoPK32 is specifically expressed in actively growing mycelia, especially in the hyphal tip cells.

3.3. Expression of CoPK32 during the life cycle of C. cinerea

As described above, we found that the autophosphorylation activity of CoPK32S was much lower than that of CoPK32 (Fig. 2E). To examine the protein kinase activities of CoPK32 and CoPK32S toward exogenous protein substrates, canonical protein substrates such as MBP and histone were used. CoPK32 effectively phosphorylated these proteins, whereas CoPK32S hardly phosphorylated them (Fig. 5). Although the physiological significance of CoPK32S remains unclear, we mainly focused on the enzymatic properties of CoPK32 in the following experiments. The C-terminal region of MAPKAPK is known to be an autoinhibitory domain and is important for the interaction with an upstream kinase MAPK [10,22]. To examine the role of the C-terminal domain of CoPK32, two mutants of CoPK32 (CoPK32(1–487) and CoPK32(1–401)), in which the C-terminal regions were sequentially deleted, were prepared (Fig. 6A). These mutants were expressed in bacteria and purified by chelate affinity column chromatography. SDS–PAGE analysis of the purified enzymes showed that they had the apparent molecular weights predicted from their primary sequences (Fig. 6B). When the kinase activities of these enzymes were examined using MBP as a substrate, CoPK32(1–401) showed about 5-fold higher activity than full-length CoPK32, while the kinase activities toward histone were only slightly changed (Fig. 6C). These findings suggest that the C-terminal region of CoPK32 is important for substrate recognition. To ascertain whether the C-terminal region plays a role in substrate recognition, the substrate specificities of CoPK32 and

To examine the changes in gene expression of CoPK32 and CoPK32S during the life cycle of C. cinerea, we prepared total RNA from mycelia, primordia and fruiting bodies and carried out RT-PCR analyses. As shown in Fig. 3B, the CoPK32 gene was abundantly expressed in mycelia, while the CoPK32S gene was only modestly expressed (Fig. 3B). No significant expression of CoPK32 and CoPK32S was observed in the later stages. Crude extracts prepared from C. cinerea were analyzed by Western blotting using the anti-CoPK32 antibody. Endogenous CoPK32 was only detected in mycelia and not in the later stages (Fig. 3C), in good agreement with the RT-PCR data (Fig. 3B). Moreover, endogenous CoPK32 was similarly detected in mycelia of wild-type C. cinerea strain 5302, which was used as a standard strain (data not shown). Since CoPK32 was highly expressed in mycelia, the gene expression profile of CoPK32 was investigated in a growing colony with different ages. The growing colony was divided into five zones, and the expression of CoPK32 in each region was analyzed by Western blotting with the anti-CoPK32 antibody. CoPK32 was only detected in the outer growing regions and not in the inner or central resting regions (Fig. 3D and E). Since the Western blotting analyses suggested that CoPK32 was abundantly and specifically expressed in the growing hyphae, the cellular localization of CoPK32 was examined by immunocytochemistry. In both the mutant strain 326 (Fig. 4A and B) and wild-type strain 5302 (Fig. 4D and E), CoPK32 was only detected in the hyphal

3.4. Biochemical properties of CoPK32 and its mutants

Fig. 3. Detection of CoPK32 during the developmental process of a C. cinerea homokaryotic strain. (A) Schematic illustrations of the amplified gene fragment of CoPK32. A pair of primers specific to the cDNAs of CoPK32 and CoPK32S used for the RT-PCR analysis is shown by arrows. The cDNA corresponding to nucleotides 412–465 (54 bp in length) of the CoPK32 insertion sequence is indicated by a gray box. (B) RT-PCR analysis of CoPK32 and CoPK32S mRNAs expressed in C. cinerea. The RT-PCR analysis was carried out using approximately 1 μg of total RNAs from mycelia (My), primordia (Pri) and fruiting bodies (Fru). pETCoPK32 (K32) and pETCoPK32S (K32S) were used as positive controls (upper panel). Expression of the actin gene as an internal standard is also shown (lower panel). The PCR products were stained with ethidium bromide. (C) Approximately 20 μg of crude extracts prepared from mycelia and later representative developmental stages were subjected to 10% SDS–PAGE and analyzed by Western blotting with an anti-CoPK32 antibody. (D) A photograph showing the growing colony divided into five zones (1–5) from the outer region to the central region. (E) Crude extracts (20 μg) prepared from the divided regions 1–5 of mycelia were subjected to 10% SDS–PAGE and analyzed by Western blotting with an anti-CoPK32 antibody.

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Fig. 4. Detection of endogenous CoPK32 in hyphal cells of the homokaryotic mutant strain 326 (A–C) and homokaryotic wild-type strain 5302 (D–F). The hyphal cells were stained with an anti-CoPK32 antibody that had been preincubated with recombinant CoPK32 (C and F) or BSA (B and E), or not preincubated (A and D). Endogenous CoPK32 was visualized by confocal microscopy (Cy3, left panels). The hyphal cell wall was stained with Calcofluor White (CW, middle panels). Merged images are shown in the right panels. Scale bars: 20 μm.

CoPK32(1–401) were compared using C. cinerea mycelial extracts fractionated by isoelectric focusing with a MicroRotofor. After kinase reactions using various fractions of the mycelial extract as substrates, the reaction mixtures were subjected to SDS–PAGE, and the phosphoproteins were detected by autoradiography. As shown in Fig. 7A, full-length CoPK32 preferentially phosphorylated basic proteins with pI values of around 10. In contrast, CoPK32(1–401) phosphorylated a variety of proteins with a wider range of pI values (Fig. 7B).

If the C-terminal region comprising residues 402–487 contains an autoinhibitory domain, the region could have a characteristic secondary structure that interacts with its catalytic core. Therefore, we analyzed the secondary structure consensus sequence of CoPK32 using the NPS@ (Network Protein Sequence Analysis) server, which is available on the website http://npsa-pbil.ibcp.fr. The predicted secondary structure of the C-terminal region comprising residues 447–468 contained a helix-turn-helix motif, which is highly conserved in yeast homologues (Fig. 1B). When the protein kinase

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3.5. Possible involvement of CoPK32 in stress-response pathways in C. cinerea

Fig. 5. Catalytic properties of CoPK32. Approximately 100 ng of CoPK32 (A) or CoPK32S (B) was incubated with 500 ng of protein substrates (MBP, myosin light chain (MLC), histone and α-casein) in a standard reaction mixture (10 μl) containing 100 μM [γ-32P] ATP. After incubation at 30 °C for 30 min, the reactions were stopped by the addition of 10 μl of 2× SDS–PAGE sample buffer. The phosphorylated proteins were resolved by SDS–PAGE and visualized by autoradiography. A control reaction was also carried out in the absence of a protein substrate (−). The radioactive bands corresponding to autophosphorylated CoPK32 and CoPK32S are indicated by asterisks.

activity of a deletion mutant devoid of this region, CoPK32(Δ447– 468), was assessed, the kinase activities toward not only MBP but also histone were increased (Fig. 6C). To further confirm the substrate specificity of CoPK32(Δ447–468), its kinase activity was examined using the fractionated extracts used in the experiments shown in Fig. 7. Similar patterns of autoradiography to those obtained with fulllength CoPK32 were observed (data not shown). These findings indicate that the substrate specificities of the mutant and full-length CoPK32 were the same and that the C-terminal residues 447–468 are important for regulation of the protein kinase activity.

Since CoPK32 was assumed to be a MAPKAPK homologue in C. cinerea, we examined whether several environmental stresses affected the CoPK32 activity. Prominent activation of CoPK32 in mycelia was observed after treatments with 1 M KCl and 1 M H2O2, while no significant activation was observed after ultraviolet irradiation and heat stress (Fig. 8A). It is likely that KCl acted as an osmolyte to stimulate osmotic stress pathways. To confirm this, NaCl, sucrose and mannitol were used as osmolytes to examine the influence of high osmotic stress on the CoPK32 activity in mycelia. All of these solutions markedly activated the endogenous CoPK32 activity (Fig. 8B). Furthermore, the expression level of CoPK32 was greatly increased by KCl stimulation while that of CoPK12, a CaMK homologue in C. cinerea, as a loading control was not changed (Fig. 8C). The effects of high osmotic stresses on the growth of C. cinerea was investigated using MYG agar plates containing KCl or mannitol at concentrations ranging from 0 to 0.75 M. The growth of mycelia on the plates was inhibited by KCl and mannitol in dose-dependent manners (Fig. 9A). When growing mycelia were treated with various concentrations of KCl or mannitol, the CoPK32 activity in the mycelia was markedly activated as the concentrations of the osmolytes increased (Fig. 9B). These findings supported the notion that CoPK32 is a stress-responsive MAPKAPK in C. cinerea. If this is the case, the activation of CoPK32 induced by high concentrations of osmolytes should be abolished by an inhibitor of p38 MAPK, which is known to be involved in stress-responsive signaling via a MAPK pathway [22–24]. To examine this possibility, we examined the effects of two p38 MAPK inhibitors, SB202190 and SB239063, together with the negative control compound SB202474, on the activation of CoPK32 induced by 0.5 M KCl. As shown in Fig. 10B, the increased histone kinase activity of CoPK32 induced by 0.5 M KCl was markedly abrogated by the p38 MAPK inhibitors, but not by the negative control SB202474 (lower panels). Since the histone kinase activity of CoPK32 was not directly inhibited by these compounds (Fig. 10A), the observed effects of the p38 inhibitors should be caused by inhibition of the upstream MAPK of C. cinerea corresponding to mammalian p38 MAPK. Taken together, all of these findings suggest that CoPK32 functions as a stress-responsive MAPKAPK in C. cinerea. 4. Discussion

Fig. 6. Catalytic properties of CoPK32 mutants truncated in the C-terminal region. (A) Schematic illustrations of full-length CoPK32 (CoPK32(FL)) and its C-terminal deletion mutants (CoPK32(1–487), CoPK32(1–401) and CoPK32(Δ447–468)). The catalytic domains are shown by black boxes. (B) Approximately 100 ng of purified kinases was subjected to 10% SDS–PAGE and analyzed by Western blotting with an antiHis6 antibody. (C) Approximately 100 ng of the kinases was incubated with 500 ng of MBP or histone in a standard phosphorylation mixture (10 μl) containing 100 μM [γ-32P] ATP at 30 °C for 30 min. The phosphorylated proteins were analyzed by autoradiography (upper and middle panels). The phosphorylated protein bands were quantified using a BAS1800 Bio-Image Analyzer (lower panel). The CoPK32 activities are presented as the relative ratios of the radioactivity incorporated into MBP (black bars) and histone (gray bars), when the activities mediated by CoPK32(FL) are set as 1 (lower panel). Data are means ± SD values from three independent experiments.

To analyze the functions of protein kinases in basidiomycetes, we previously isolated 14 clones from a mycelial cDNA library of the C. cinerea AmutBmut strain by expression cloning with Multi-PK antibodies [9]. In this study, we examined the expression profile, biochemical properties and biological functions of the novel protein kinase CoPK32, which is encoded by one of the clones, CMZ032. In addition to CoPK32, we also found its splice variant CoPK32S, in which a sequence of 18 amino acids between subdomains II and III of CoPK32 was deleted. Although both CoPK32 and CoPK32S completely share the subdomains specific to protein kinases, the kinase activity of CoPK32S was much lower than that of CoPK32, indicating that the insertion sequence of CoPK32 is critical for its protein kinase activity. The detailed mechanism for how the insertion sequence affects the kinase activity remains to be clarified. Interestingly, BLAST homology searches revealed that homologous proteins to both CoPK32 and its splice variant CoPK32S were found in the basidiomycetes S. commune and C. neoformans. The findings that both CoPK32 homologues and their splice variants are conserved in basidiomycete mushrooms imply some functional significance of the splice variant CoPK32S in basidiomycetes. However, the kinase activity of CoPK32S was much lower than that of CoPK32, suggesting that CoPK32 is more functionally important for the hyphal cells of C. cinerea.

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Fig. 7. Substrate specificity of CoPK32(FL) and its C-terminal deletion mutant CoPK32(1–401). A crude extract (2.5 mg) prepared from C. cinerea mycelia was loaded onto a MicroRotofor and separated into 10 fractions on the basis of the pI values of the endogenous proteins. The fractionated proteins were dissolved in homogenizing buffer (4 μl) and incubated with 100 ng of CoPK32 or 25 ng of CoPK32(1–401) in the presence of 100 μM [γ-32P]ATP at 30 °C for 30 min. Each sample was separated by SDS–PAGE using a 10% gel, and the [32P]phosphate incorporated into the endogenous proteins was detected by autoradiography. As controls, the protein kinases were incubated as described above but without the protein substrates (−). The bands corresponding to autophosphorylated CoPK32(FL) and CoPK32(1–401) are indicated by asterisks.

MAPKAPKs in mammals and yeasts have an autoinhibitory domain in their C-terminal regions [10,22]. Each MAPKAPK is phosphorylated by an upstream MAPK, leading to a conformational change in the autoinhibitory domain of the MAPKAPK to unmask its substratebinding site [25]. In the present study, we found that C. cinerea CoPK32 was activated by deletion of residues 447–468 in the C-terminal region. This finding suggests that CoPK32 also possesses an autoinhibitory domain. The region comprising residues 447–468 showed 50% and 59% sequence identity to Rck2 and Srk1, respectively (Fig. 1B). A computer prediction of the secondary structure of this region showed that it had a helix-turn-helix motif, in good agreement with the fact that mammalian MAPKAPKs have a similar structure at the C-terminal region comprising residues 352–368, which is a portion

Fig. 8. Effects of environmental stresses on CoPK32 activity. (A) Activity of endogenous CoPK32 under various stress conditions. Extracts were prepared from growing mycelia of C. cinerea strain 326 after treatment with 1 M KCl, 1 M H2O2, ultraviolet irradiation (8 W) or high temperature (42 °C) for 30 min. Endogenous CoPK32 coprecipitated with anti-CoPK32 antibody-coupled protein G-Sepharose (10 μl) was resuspended in 20 μl of a standard phosphorylation mixture containing 1 μg of histone and incubated at 30 °C for 1 h. The reactions were stopped by the addition of 5 μl of 5× SDS–PAGE sample buffer, and phosphorylated histone was evaluated by SDS–PAGE followed by autoradiography. (B) Activity of endogenous CoPK32 under high osmotic conditions. Crude extracts were prepared from mycelia treated with 1 M each of KCl, NaCl, sucrose or mannitol (4 ml each) for 30 min, and endogenous CoPK32 activity was detected as described above. (C) Effects of high osmotic stress on expression of CoPK32. Growing mycelia were harvested immediately after soaking in 4 ml of 0.5 M KCl for the indicated times. Proteins in crude extracts of mycelia (20 μg) were resolved by SDS–PAGE, and analyzed by Western blotting with an anti-CoPK32 antibody (upper panel). Western blotting analysis with an anti-CoPK12 antibody was also carried out to ensure equal amount of loading (lower panel).

of the autoinhibitory domain containing a nuclear export signal. This finding also suggests that the corresponding region comprising residues 452–468 of CoPK32 contains a nuclear export signal, similar to the case for mammalian MAPKAPKs [26]. It is interesting to note that the protein kinase activity of CoPK32 toward MBP was markedly activated by deletion of residues 402–487 in the C-terminal region, while the kinase activity toward histone was unchanged (Fig. 6C). These observations led us to investigate the substrate specificity of mutant protein kinases in which a portion of the C-terminal region was deleted. Using full-length and deletion

Fig. 9. Effects of high osmotic stress on mycelial growth. (A) High osmotic stress causes inhibition of mycelial growth. Mycelia of C. cinerea strain 326 were grown in 8.5-cm agar plates containing 0, 0.4 and 0.75 M KCl or mannitol for 5 days. (B) CoPK32 activity in mycelia treated with varying concentrations of osmolytes. Extracts were prepared from growing mycelia of C. cinerea treated with the indicated concentrations of KCl or mannitol. Endogenous CoPK32 was immunoprecipitated with anti-CoPK32 antibodycoupled protein G-Sepharose, and the resin (10 μl) containing the purified CoPK32 was resuspended in 20 μl of a standard phosphorylation mixture containing 1 μg of histone. The kinase reaction was carried out at 30 °C for 1 h. After the reaction, phosphorylated histone was resolved by SDS–PAGE and detected by autoradiography.

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Fig. 10. Effects of p38 MAPK inhibitors on the activation of CoPK32 induced by high osmotic stress. (A) Effects of mammalian p38 MAPK inhibitors SB202190 and SB239063 and the negative control SB202474 on CoPK32 activity in vitro. Recombinant CoPK32 (100 ng) was incubated in a standard phosphorylation mixture (10 μl) with 500 ng of histone as a protein substrate and 100 μM of inhibitors at 30 °C for 30 min. After the reaction, phosphorylated histone was detected by autoradiography. As a control, dimethyl sulfoxide was used instead of the compounds. (B) Effects of MAPK inhibitors on the activity of CoPK32 in mycelia under high osmotic stress conditions. Growing mycelia on MYG agar plates were pretreated with 2 ml of each compound at room temperature for 10 min. Subsequently, 2 ml of H2O or 1 M KCl (final concentration: 0. 5 M) were added to the pretreated mycelia. After incubation at room temperature for 20 min, the solutions were removed and the mycelia were immediately harvested as described above. The obtained crude extracts (50 μg) were incubated with anti-CoPK32 antibodycoupled protein G–Sepharose. Endogenous CoPK32 in crude extract was detected by Western blotting with an anti-CoPK32 antibody (upper panel), and assayed with 1 μg of histone under standard phosphorylation conditions. After the reaction, phosphorylated histone was resolved by SDS–PAGE and detected by autoradiography (lower panels). The graph shows the relative histone kinase activities of the endogenous CoPK32 under the indicated conditions. The CoPK32 activities are represented as relative values, for which the activity under the control conditions is set as 1. Data are means ± SD values from three independent experiments.

mutants of CoPK32, we compared the kinase activities toward endogenous proteins fractionated by MicroRotofor. Although the mutant CoPK32(1–401) phosphorylated substrate proteins with a wide range of pIs, the full-length CoPK32 and CoPK32(Δ447–468) only phosphorylated basic proteins (Fig. 7 and data not shown). Therefore, CoPK32 may prefer to phosphorylate basic proteins in hyphal cells through its C-terminal region. Taken together, these data imply that the C-terminal region of CoPK32 is important for not only autoregulation of the kinase activity but also its substrate recognition. In mammalian cells, hyperosmotic conditions elicit the activation of MAPK signaling. Activated MAPK phosphorylates downstream targets such as MAPKAPK and transcription factors, leading to the induction of apoptosis and inflammation [22]. In fungi, bacteria and plants, some environmental stresses can be transduced into the cells by two-component signal transduction systems [27]. In these systems, a histidine protein kinase undergoes autophosphorylation at a specific His residue, and a response regulator protein catalyzes the transfer of the phosphoryl group from the phosphorylated His residue to one of its own Asp residues to transduce the stress signal. In lower eukaryotes, the two-component systems interface with more conventional signaling systems such as MAPK cascades. [28]. In budding yeasts, osmotic stress activates a MAPK homologue, Hog1, which subsequently phosphorylates Rck2 to induce osmoadaptive responses, thereby allowing the cells to survive in a high osmolarity environment [10]. In fission yeasts, a MAPK homologue, Sty1,

phosphorylates a MAPKAPK, Srk1, to induce the dissociation of the Srk1–Sty1 complex and the activation of Srk1, leading to cell cycle arrest under high osmotic stress. Subsequently, the activated Srk1 is degraded, while the active Sty1 activates the transcription of core environmental stress-response genes [29]. In C. cinerea, we found that CoPK32, which shows homology with Srk1 and Rck2, was expressed in the actively growing region of the colony and was also activated by oxidative and high osmotic stresses (Fig. 8A and B). Furthermore, we found that high osmotic conditions increased the expression of CoPK32 (Fig. 8C) and enhanced its kinase activity (Fig. 9B). These observations are in accordance with previous studies showing that elevation of Srk1 expression is dependent on Sty1 MAPK in S. pombe [11] and that elevation of Rck2 expression is dependent on Hog1 MAPK in Candida albicans [30]. Furthermore, we found that the osmotic stress-dependent activation of CoPK32 activity under high osmotic conditions was very sensitive to p38 MAPK inhibitors (Fig. 10B). Taken together, the present findings strongly suggest that CoPK32 is involved in environmental stress responses in the basidiomycete mushroom C. cinerea as a MAPKAPK homologue. Upon stimulation by environmental stresses such as high osmolarity, CoPK32 is possibly phosphorylated by an upstream p38 MAPK to become activated. Although the detailed mechanism for how CoPK32 induces an osmoadaptive response is currently unclear, CoPK32 may be involved in the regulation of gene expression or of cell cycle progression for an adaptive response in C. cinerea. Comprehensive identification of endogenous CoPK32 substrates in C. cinerea mycelia may provide some clues to the biological functions of CoPK32 in regulating the growth rate of the mycelia in C. cinerea under environmental stress.

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