The mitogen-activated protein kinase p38 pathway is conserved in metazoans: Cloning and activation of p38 of the SAPK2 subfamily from the sponge Suberites domuncula

Biology o! the Cell 92 (2000) 95-104 © 2000 Editions scientifiques et medicales Elsevier SAS. All rights reserved

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Original article

The mitogen-activated protein kinase p38 pathway is conserved in metazoans: Cloning and activation of p38 of the SAPK2 subfamily from the sponge Suberites domuncula* Markus B6hm a, Heinz C. Schr6der a, Isabel M. MLillera, Werner E.G. MiJllera**, Vera Gamulin b alnstitut ffJr Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universit~t Mainz, Duesbergweg 6, 55099 Mainz, Germany blnstitute Rudjer Boskovic, Department of Molecular Genetics, HR-10000 Zagreb, Croatia Received 31 August 1999; accepted 31 January 2000

Our recent data suggest that during auto- and allograft recognition in sponges (Porifera), cytokines are differentially expressed. Since the mitogen-activated protein kinase (MAPK) signal transduction modulates the synthesis and release of cytokines, we intended to identify one key molecule of this pathway. Therefore, a cDNA from the marine sponge Suberites domuncula encoding the MAPK was isolated and analyzed. Its encoded protein is 366 amino acids long (calculated M r 42 209), has a TGY dual phosphorylation motif in protein kinase subdomain VIII and displays highest overall similarity to the m a m m a lian p38 stress activated protein kinase (SAPK2), one subfamily of MAPKs. The sponge protein was therefore termed p38_SD. The overall homology (identity and similarity) between p38_SD and h u m a n p380t (CSBP2) kinase is 82%. One feature of the sponge kinase is the absence of threonine at position 106. In h u m a n p38R MAPK this residue is involved in the interaction with the specific pyridinyl-imidazole inhibitor; T]0 6 is replaced in p38_SD by methionine. Inhibition studies with the respective inhibitor SB 203580 showed that it had no effect on the phosphorylation of the p38 substrate myelin basic protein. A stress responsive kinase Krs_SD similar to m a m m a l i a n Ste20 kinases, upstream regulators of p38, had already previously been found in S. domuncula. The S. domuncula p38 MAPK is phosphorylated after treatment of the animal in hypertonic medium. In contrast, exposure of cells to hydrogen peroxide, heat shock and ultraviolet light does not cause any phosphorylation of p38. It is concluded that sponges, the oldest and most simple multicellular animals, utilize the conserved p38 MAPK signaling pathway, k n o w n to be involved in stress and i m m u n e (inflammatory) responses in higher animals. © 2000 Editions scientifiques et m6dicales Elsevier SAS

evolution / mitogen-activated protein kinase / stress-activated protein kinase / Suberites domuncula/ signal transduction/ Porifera

*The sequence reported here is deposited in the EMBL/GenBank database (Accession no. Y18861) **Correspondence and reprints Abbreviations: aa, amino acid(s); ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; nt, nucleotide(s); SAPK, stress-activated protein kinase; SRF, serum-response factor; TCF, ternary complex factor.

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1. INTRODUCTION The mitogen-activated protein kinases (MAPKs) are proline-directed serine/threonine kinases (ClarkLewis et al., 1991) that are stimulated by a large variety of signals, i.e., mitogens, growth factors, cytokines, ultraviolet- and ionizing radiation, osmotic stress, heat shock, oxidative stress and others. Most MAPKs have small amino- and carboxy termini and display almost the entire eukaryotic protein kinase domain spanning about 300 aa. MAPKs are activated through threonine/ tyrosine phosphorylation at the dual phosphorylation site TxY by dual specificity protein kinases known as MAPK kinases (MAPKK), which in turn are activated by serine phosphorylation by a third class of protein kinases, known as MAPKK kinases (Hunter, 1995). These phosphorylation cascades represent the core units for the transcytoplasmic signal transduction from the cell surface to the nucleus. The MAPK pathway is a conserved eukaryotic signaling module that converts receptor signals into a variety of outputs. There are independently regulated MAPK pathways in vertebrates known as extracellular signal-regulated kinases (ERK), stress-activated protein kinases (SAPK1/JNK (Jun kinases)) and SAPK2 (p38) protein kinase cascades (Kyriakis and Avruch, 1996; Kfiltz, 1998). ERKs are activated by mitogens and growth factors via a Ras dependent pathway (Boulton et al., 1991). In contrast, stress activated MAPKs, called SAPKs (JNK, p38), are stimulated by pro-inflammatory cytokines (tumor necrosis factor-c~, interleukin-1) and cellular stresses (UV irradiation, heat and osmotic shock, etc.) (Kyriakis and Avruch, 1996). Many MAPK substrates are inducible transcription factors and products of immediate early genes, but MAPKs also phosphorylate different cytoplasmic proteins and other protein kinases, including their own kinases (Nishida and Gotoh, 1993; Hill and Triesman, 1995). An extensive phylogenetic and functional classification of almost 100 known MAPKs was recently performed by Ktiltz (1998). ERKs are found in animals, as well as in fungi and plants, and form one phylogenetic subgroup with at least five subfamilies, while the SAPKs subgroup, not present in plants, consists of three subfamilies: SAPK1 (JNK), SAPK2 (p38) and yeast SAPK (YSAPK). The members of the SAPK2 (p38) subfamily are close relatives of YSAPKs, i.e., yeast HOG1 kinase, involved in host protection from hyperosmotic stress (Brewster et al., 1993). SAPKs are thought to have evolved most probably later in evolution, after the animal/fungal lineage split off from the plant ancestor (Kfiltz, 1998). The amino acid separating the two phosphorylation sites in the dual phosphorylation motif TxY located in the protein kinase subdomain VIII, is highly conserved within each subfamily (Ktiltz, 1998). The dual phospho-

p38 of the SAPK2 subfamily from the sponge S. domuncula

Biology of the Cell 92 (2000) 95-104 rylation site in SAPK1 (JNK) members reads TPY, while the SAPK2 (p38) and the YSAPK (HOG1) members possess the TGY motif. SAPKs account for most of the MAPKs expressed in vertebrates (Kfiltz, 1998) and several paralogous SAPK isoforms exist in mammals. However, only few SAPK members are known from invertebrates; SAPK1 (JNK) (Sluss et al., 1996; Riesgo-Escovar et al., 1996), SAPK2 (p38) (Han et al., 1998a, 1998b) from Drosophila melanogaster, and three Caenorhabditis elegans SAPKs, which were found in the data bases (www.ncbi.nlm.nih.gov [Blast]; www.The Sanger Centre Web Server.ac.uk [wormpep data base]). Previously we described that sponges have genes encoding cytokines, e.g., the allograft inflammatory factor 1 and the glutathione peroxidase, which are expressed as response to grafting (Mfiller et al., 1999b). Since some pro-inflammatory cytokines, e.g., interleukin 1, induce in humans transactivation of Elk-1 a n d / o r Sap-la (transcription factors) through their phosphorylation by SAPK2 (p38) and JNK (reviewed in Wasylyk et al., 1998), it was suggested to search also in sponge for a key molecule involved in the cytokine receptor signaling. Here we report that the marine sponge Suberites domuncula, a member of the oldest and simplest phylum of multicellular animals, the Porifera (reviewed in Mfiller, 1995, 1997), possesses a MAPK with the dual phosphorylation motif TGY, which is highly homologous to the mammalian p38c~ kinases of the SAPK2 subfamily; this enzyme family is unique to metazoans and has not been found in non-metazoans. In addition, we demonstrate that this kinase is activated in response to hyperosmotic shock, but not to other stresses, e.g., hydrogen peroxide, temperature shock or ultraviolet light.

2. MATERIALS AND METHODS 2.1. Materials Restriction endonucleases, digoxygenin labeling and detection kit and other enzymes for recombinant DNA techniques were obtained from Boehringer Mannheim (Mannheim, Germany). A )vZAP Express kit for cDNA synthesis and library preparation was purchased from Stratagene (La Jolla, USA) and MaxPlax Packaging extract from Epicentre Technologies (Madison, USA); myelin basic protein (rabbit brain) and the specific inhibitor for p38 kinase SB 203580 was from Sigma (Deisenhofen; Germany) and [y-32p]ATP from Amersham (Amersham, UK). Two antibodies were used: first, the p38 MAP kinase phospho-specific antibody (which recognizes the phosphorylated Thr180/Tyr182 residues), and second, the p38 MAP kinase antibody

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Biology of the Cell 92 (2000) 95-104 (which recognizes the total p38 kinase); both were obtained from N e w England Biolabs (Beverly, USA). Life specimens of S. domuncula (Olivi) (Porifera, Demospongiae, Tetractinomorpha, Hadromerida, Suberitidae) were collected from the Adriatic Sea near Rovinj (Croatia) and then kept in aquaria in Mainz (Germany) at a temperature of 17°C.

2.2. Preparation of cDNA library and isolation of p38 cDNA cDNA library of S. domuncula was prepared in KZAP Express vector between the EcoRI and XhoI sites and packaged in vitro using MaxPlax Packaging Extract. The resulting n u m b e r of independent clones was 1.7 x 107 (Kruse et al., 1997). The complete sponge cDNA, encoding the p38 MAPK, termed SDp38, was isolated from the S. domuncula cDNA library (Kruse et al., 1997) by polymerase chain reaction (PCR). The degenerate reverse primer, directed against the conserved aa segments found in the sequences from the related SAPK2 and YSAK sequences within subdomain VIII (aa179 and aa186 (MTGYVATR) with respect to the h u m a n p38 MAPK p38/CSBP2 (accession n u m b e r L35264), 5'-T/ CCTIGTIGCIACA/GTAICCIGTCAT-3' (where I = inosine), in conjunction with the 5'-end vector-specific primer was used. The PCR reaction was carried out at an initial denaturation at 95°C for 3 min, then 32 amplification cycles at 95°C, for 30 s, 56.3°C for 45 s, 74°C fof 1.5 min, and a final extension step at 74°C for 10 min. The reaction mixture was as described earlier (Wiens et al., 1998). The fragment of = 600 bp was used to isolate the cDNA from the library (Ausubel et al., 1995). The longest insert obtained had a size of 1324 nt (excluding the poly(A) tail). The clone was termed SDp38 and sequenced using an automatic D N A sequenator (LiCor 4200).

2.3. Sequence comparisons Sequences were stored and analyzed using P C / GENE 14.0 p r o g r a m s from IntelliGenetics (Mountain View, USA). H o m o l o g y searches and sequences retrieval were done via Internet server BLAST (NCBI, NIH, Bethesda, USA). Multiple alignment was perf o r m e d with CLUSTAL W ver. 1.7 p r o g r a m (Thompson et al., 1994) and its graphic presentation by the p r o g r a m GeneDoc (Nicholas and Nicholas, 1996). The phylogenetic tree was constructed from an amino acid alignment b y neighbor-joining m e t h o d (Saitou and Nei, 1987) a p p l y i n g the PHYLIP package version 3.5c p r o g r a m (Felsenstein, 1993) and the degree of s u p p o r t for internal branches was further assessed by bootstrap analysis.

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2.4. Exposure of tissue samples from S. domuncula to osmotic shock Tissue samples of 40 g each were exposed to seawater adjusted to 0.25 to 2 M with respect to NaC1. After the indicated time intervals samples were taken and immediately frozen in liquid nitrogen. Then, the tissue samples were homogenized in ice-cold lysis-buffer (1 x Tris-buffered saline (TBS), p H 7.5, 1 m M EDTA, 1% Nonidet-P40, 10 m M NaF, i m M phenylmethylsulfonyl fluoride, 10 ~tg/mL aprotinin, and i mM sodium orthovanadate); after standing on ice for 30 min, the suspension was centrifuged (15000 g for 15 min at 4°C). The supernatant was collected and subjected to Western blot analysis.

2.5. Exposure of cells from S. domuncula to other stressors Cells from S. domuncula were obtained as described (Mtiller et al., 1999a). A suspension of 2 x 106 cells/mL in 6 mL of seawater was transferred into 60 m m petri dishes. The cultures were kept at 17°C.

2.5.1. Exposure to hydrogen peroxide Cells were exposed to 100 ~tM H 2 0 2 for 0 to 30 min and then used for extraction of protein.

2.5.2. Exposure to temperature shock Cells were transferred into seawater of a temperature of 26°C (9°C above the ambient temperature).

2.5.3. Exposure to ultraviolet light The light source used in this study was a 20 W UVB lamp (UV-B TL 12RS Philips, Hamburg; radiation flux • e(W): UVC 0.01, UVB 0.22, UVA 0.14 and visible light 0.1) with a peak at the mercury emission line at 312 nm. The dosage used within the ultraviolet B range was 30 J / m 2. After irradiation of the cells they were left for 0 to 30 min in the dark and then analyzed.

2.6. Gel electrophoresis and Western blotting Total tissue extracts (20 pg/lane) were subjected to electrophoresis in 12% polyacrylamide gels containing 0.1% NaDodSO 4 as described by Laemmli (1970). For Western blotting experiments the proteins were electrotransferred to PVDF-Immobilon P membranes (Millipore) using a semi-dry blotting apparatus (Wiens et al., 1998). The membranes were incubated with rabbit anti-human p38 antibodies (1:1000 dilution). This antibody specifically recognizes the active form of h u m a n p38 MAPK, dual phosphorylated on residues T180 and Y182; it cross-reacts with D. melanogaster p38 as well as with murine p38 but not with inactive p38 or other

B6hm et al.

98 MAPKs (e.g., JNK and ERK). In one series of experiments the p38 MAP kinase antibody (which recognizes the total p38 kinase) was applied at a 1:500 dilution. After an incubation period of 1 h at room temperature, and subsequent washing steps, the blots were incubated with goat anti rabbit IgG, peroxidase-coupled (1:5000 dilution). Detection of the immunocomplex was carried out using the BM Chemoluminescence Blotting Substrate kit from Boehringer Mannheim.

2.7. Immunoprecipitation and protein kinase assay Tissue samples were treated for 30 min at an osmolarity of 1 M with respect to NaC1. Then the samples were solubilized with the above mentioned lysis-buffer, supplemented with 25 mM [3-glycerophosphate, 0.5 mM DTT, 0.5 mM Na3VO 4 and 2 mM Na-pyrophosphate, centrifuged (20 000 g; 15 min; 4°C) and 100 btg (with respect to protein) were subjected to immunoprecipitation. This step was performed using the p38 MAP kinase antibody (which recognizes the total p38 kinase) and protein G-Sepharose (Pharmacia-LKB) as described (Han et al., 1998). In a control assay the antibody was omitted from the immunoprecipitation. The immunoprecipitates were washed twice with the same buffer and subsequently twice with the kinase buffer (25 mM HEPES, pH 7.5, 25 mM ]3-glycerophosphate, 25 mM MgC12, 0.1 mM Na3VO 4 and 0.5 mM DTT). Finally the material was suspended in 30 btL of kinase buffer. Protein kinase reactions were performed in 40 pL assays using kinase buffer with the immunoprecipitates (30 btL) and initiated by the addition of the myelin basic protein (2 btg) and 50 btM [T-32p]ATP (8 Ci/mmol). Where indicated the specific inhibitor for p38 kinase SB 203580 (Lee et al., 1994) was added to the reaction. The reaction was terminated after 30 min (30°C) by addition of Laemmli buffer (Laemmli, 1970). Phosphorylation of myelin basic protein was detected by 12% polyacrylamide gel electrophoresis containing 0.1% NaDodSO 4 (as described above), followed by autoradiography and phosphoimager analysis using a GS-525 Molecular Imager (Bio-Rad).

2.8. Protein determination Protein content was determined with tire Lowry method (Lowry et al., 1951).

3. RESULTS AND DISCUSSION 3.1. Cloning of p38 stress activated kinase from S. domuncula The sequence of S. domuncula cDNA of 1324 nucleotides has been deposited in the EMBL databank under acces-

p38 of the SAPK2 subfamily from the sponge S. domuncula

Biology of the Cell 92 (2000) 95-104 sion number Y18861. This cDNA encodes a 366 aa long protein (calculated M r 42 209) which shows highest similarity with p38 stress activated protein kinases (SAPK2). Like in all other MAPKs from the p38 (SAPK2) subfamily the dual phosphorylation site in the kinase subdomain VIII reads TGY. The protein was therefore named p38_SD. Northern blot analysis was performed with the sponge SDp38 clone as a probe yielding one prominent band of approximately 1.5 kb, confirming that a full length cDNA was isolated (not shown). The estimated instability index for the p 3 8 S D polypeptide is 53.5 and it is hence classified to the unstable proteins (PC/GENE, 1995). Sequences of seven representatives of p38 proteins were aligned with p38_SD as shown in figure 1. Two signature sequences characteristic for the SAPK2 subfamily of protein kinases (Kfiltz, 1998) are underlined: LLKHM(x)HEN(IV)I(x)LLD(IV)F(TS)P, located in the kinase subdomains III-IV and AVNEDCEL(KR)ILDF in subdomains VIb-VII. Note that yeast HOG1, a member of YSAPK subfamily, does not have the same signature. A third conserved global signature sequence discriminating MAPKs from all other eukaryotic protein kinases (Kfiltz, 1998) located in subdomain VIII is also underlined (figure 1); it includes the dual phosphorylation site TxY. Only this motif is not perfectly conserved in p38 SD: a tryptophan (W) residue is found at the position of the invariant tyrosine (Y, marked with *). An indicative residue in the amino-terminal part in the SAPK2 subfamily is serine at position 62, which is however replaced by threonine in p38_SD. Threonine at this position is characteristic of YAPKS (HOG1). Additional unique residues in SAPK2 proteins are AI58, E179 and T204. T204 is replaced by lysine (K204) in p38_SD. Glutamate at position 179 is in close proximity to the dual phosphorylation site and may be important for conferring activator specificity (K61tz, 1998). Another important residue is T106, because this threonine interacts with the 4-phenyl ring of highly specific pyridinyl-imidazole inhibitor of human p38c~ (CSBP2) MAP kinase (Tong et al., 1997). T106 is replaced by methionine (M106) in p 3 8 S D and the same was found also in SAPK27 and SAPK25 subfamilies, which are not sensitive to this class of inhibitors. It is therefore possible that the sponge p38 is also not sensitive to the pyridinyl-imidazole inhibitor. This inhibitor of p38c~ kinases acts as an anti-inflammatory agent, because it prevents the production of inflammatory cytokines in response to bacterial infections (Lee et al., 1994), which can induce lethal toxic shock in humans, p38 kinases are therefore important targets for novel anti-inflammatory drugs. To test this assumption p38 was immunoprecipitated using the antibody which recognizes the total p38 kinase. Subsequently the protein kinase reaction was carried out using the myelin basic protein as substrate and [7-32p]ATP as described in Materials and

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Figure 1. Multiple alignment of the aa sequences of p38 from S. domuncula (p38 (Sd), Y18861), S. cerevisiae HOG1 (Hog1 (Sc), L06279), p38 from C. elegans (p38 (Ce), U58752), p38a from D. melanogaster (p38 (Din), U86867), p38 from common carp Cyprinus carpio (p38 (Cc), D83274), p38 from Xenopus laevis (p38 (XI), X80751) and human p38/CSBP2 (p38 (Hs), L35264). Borders of the XI subdomains in the protein kinase domain are indicated by arrowheads. Amino acid residues specific for SAPK2 are indicated. TGY dual phosphorylation site is marked by * * *. Characteristic signature sequences are underlined (see text) and the aa change Y to W in S. domuncula p38 is shown by * and arrowhead. Identical aa in all seven sequences are shown in inverted type and residues conserved in at least four sequences are shaded.

methods and in Lee et al. (1994). The data show that the specific inhibitor for p38 kinase, the pyridinyl-imidazole c o m p o u n d SB 203580, did not inhibit the kinase p38 of the SAPK2 subfamily from the sponge S. domuncula

reaction at the concentration of 10 pM, confirming the suggestions obtained from the sequence data. Infigure 2 it is shown that in the absence of the p38 antibody no B6hm et al.

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Figure 2. Influence of the pyridinyl-imidazole compound SB 203580 on the phosphorylation of the substrate myelin basic protein by immunoprecipitated p38. Immunoprecipitation was performed with extracts from samples incubated at an osmolarity of 1 M NaCI either in the absence (-, antibody; lane a) or the presence of the p38 antibody (+, lanes b and c). The assays were performed in the absence of the potential inhibitor (-, SB 203580; lanes a and b) or it was added at the concentration of 10 pM (+, lane c) to the kinase reaction. The inhibitor was added before the substrate to the assay. The proteins in the reaction assays were resolved by NaDodSO4-polyacrylamide gel electrophoresis, and the autoradiogram is shown. The protein size markers are indicated on the left.

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Figure 3. Rooted phylogenetic tree of complete aa sequences of MAPKs from SAPK2 and YSAPK subfamilies. The seven MAPKs, used for the alignment (figure 1), have been included in this analysis; S. domuncula (p38 Sd), S. cerevisiae HOG1 (Hog1 Sc), p38 from C. elegans (p38 Ce), p38a from D. melanogaster (p38 Dm), p38 from common carp Cyprinus carpJo (p38 Cc), p38 from Xenopus laevis (p38 XI) and human p38/CSBP2 (p38 Hs). In addition, the following kinases have been added for the construction of the tree: p38 Mm (mouse, U10871), p38b Mm (mouse, D83073), p38 Rn (rat, U73142), p38 Cf (dog, AF003597), p38B Hs (human, U53443), p38b Dm (D. melanogaster, AF035548), ERK6 Hs (human, X79483), SAPK3 Mm (mouse, Y13439), pSAPK3 Dr (zebrafish, Danio rerio, Y15075), HOG1 Ca (yeast Candida albicans, X90586) and STY1 Sp (yeast Schizosaccharomyces pombe, X89262). The numbers at the nodes refer to the level of confidence as determined by bootstrap analysis. Scale bar indicates an evolutionary distance of O. 1 aa substitution per position in the sequence. MAPK1 Pf from protozoan Plasmodium falciparum (U36377) was used as the outgroup protein. B6hm et al.

Biology of the Cell 92 (2000) 95-104 phosphorylation of the substrate for p38, myelin basic protein (Han et al., 1998), occurred (lane a). However, if the antibody was added to the immunoprecipitation then a strong signal (corresponding to the p20 myelin basic protein (Roth et al., 1987)) is seen in the autoradiogram (lane b). If the pyridinyl-imidazole compound SB 203580 is added to the reaction no measurable difference in the strength of the signal is seen (lane c). This finding supports the suggestion concluded from the sequence data that the sponge kinase is lacking the binding site for this inhibitor.

3.2. Phylogenetic relationships of p38 from S. domuncula The overall homology of p38_SD (identity plus similarity) is highest with vertebrate p38cz MAP kinases (8183%; identity at least 62%). However, the homology among the protein kinase domains (subdomains I-XI) is still higher: 89-90%, with identity more than 71%. This is a very remarkable sequence conservation for phylogenetically extremely distant animal phyla. Lower overall homology was found with p38a from D. melanogaster (77%) and still lower with p38 from C. elegans (74%), which is an indication of higher evolutionary rates in protostomes. S. cerevisiae HOG1 shares 58% overall homology with p38_SD. The rooted phylogenetic tree of the entire aa sequences of SAPK2 and YSAPK family of MAPKs is shown in figure 3. YSAPK forms a separate branch, while the members of the SAPK27 kinases and p38a and b (o~,13) cluster together, p38_SD branches off first from this cluster, with the exception of C. elegans p38. Due to the apparently accelerated evolutionary rate in C. elegans genes, p38_Ce is less homologous to vertebrate p38 kinases than sponge p38 and the same was also found with several other C. elegans orthologs/homologs of sponge and human proteins (Gamulin et al., submitted).

3.3. Activation of p38 MAPK in S. domuncula after shock treatment Tissue samples were treated with seawater of a NaC1 molarity of i M. Immediately after transfer of the tissue into this medium, time 0 (control), or after an incubation for 2 to 30 min tissue aliquots were taken and extracted. In the controls no band corresponding to the activated p38 MAPK could be identified by Western blot analysis. An antibody was used which specifically recognizes the activated p38 MAP kinase (containing phosphorylated Thr180 and Tyr182 residues). It is evident that already after 2 min of incubation the activated p38 MAPK can be seen; the intensity of the band increases steadily during the 30 min incubation period (figure 4A, lane a). Then, the tissue samples

p38 of the SAPK2 subfamily from the sponge S. domuncula

101 were treated for 30 min at different osmolarity; 0.25 to 2 M (final concentration) of NaC1 were adjusted in the seawater medium. It becomes evident that only under conditions of hyperosmolarity with respect to the seawater (above 0.5 M NaC1) an activation of p38 can be measured (figure 4A, lane b). Additional experiments were performed with different stressors. The results revealed that activation of sponge p38 does not occur in the presence of 100 ~tM of h y d r o g e n peroxide, a concentration at which this c o m p o u n d modulates apoptosis in h u m a n cells (Lee and Shacter, 1999) (figure 4A, lane c). Furthermore, the two other extracellular stressors tested, elevated temperature (9°C above the ambient temperature) or ultraviolet light (30 J / m 2) displayed no marked activation of p38 (figure 4A, lane d, e). In one control experiment it was established that the polyclonal antibody, raised against the conserved regions of mammalian p38 which recognized the total p38 molecule, cross-reacted with the sponge protein also under conditions at which p38 is inactive (at NaC1 concentrations below 0.5 M) (figure 4B).

4. CONCLUSION P38 kinase kinase (MAPKK) and MAPKK kinases have not yet been completely identified in S. domuncula. However, a protein kinase with high homology to h u m a n stress responsive Krs kinases (Taylor et al., 1996) was recently identified in S. domuncula and was named Krs_SD (Kruse et al., 1997). Krs kinases are homologous to the Ste 20 family of protein kinases (Creasy and Chernoff, 1995) that function early in the pheromone responsive signal transduction cascade in yeast (Leberer et al., 1992). It was suggested that mammalian Krs kinases may also function early in a cell stress response p a t h w a y (Taylor et al., 1996) and that mammalian homologs of Ste 20 are upstream regulators of p38 (Kyriakis and Avruch, 1996). We found that, like the sponge p38 MAPK, also the S. domuncula Krs kinase is involved in osmosensing (Fafandjel et al., in preparation). It is therefore very likely that also the entire conserved p38 protein kinase cascade pathway exists in sponges. MAPKs were subject to evolutionary radiation resulting in many paralogous isoforms that are key elements of at least 12 parallel signal transduction cascade pathways which coexist in a single vertebrate cell (Kfiltz, 1998). SAPKs are not present in plants and probably evolved in the animal/fungi lineage after it diverged from plants. It would be therefore interesting to see what additional MAPKs (ERKs, SAPKs) exist in sponges. With the recent discovery that sponges utilize cytokine-like molecules which comprise high sequence similarity to mammalian cytokines (M~iller et al., 1999a) it

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Biology of the Cell 92 (2000) 95-104

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1

~1 osmotic stress 2 M

was pressing and now successful to search also in these animals for signal transduction pathways, ultimately causing modulation of gene expression. It has been established that the interaction between MADS-box protein serum-response factor (SRF), the ternary complex factor (TCF) and DNA response elements confer a highly specific regulation of gene expression of the stress-activated pathway (reviewed in Wasylyk et al., 1998), e.g., osmotic stress, ultraviolet radiation (Lim et al., 1998) and cytokines (Laursen et al., 1998). Two main extracellular signal-regulated kinase (ERK)-based signaling pathways are involved; i) the stress-activated protein kinase pathway SAPK/JNK; and ii) the p38 (also termed RK, CSBP and SAPK2) pathway (Molnar et al., 1997). First elements of the nuclear targets of these pathways have been identified in sponges: the SRF (Scheffer et al., 1997) and its response element CArG (Gamulin et al., 1997). Indications have also been elaborated that p38 is involved in cytokine-mediated signaling pathways (to be published). Here we show that the sponge S. domuncula utilizes the p38 kinase as a selective signaling pathway to respond to extracellular stress (figure 5). This important p38 of the SAPK2 subfamily from the sponge S. domuncula

Figure 4. Effect of different stressors on Tyr phosphorylation of p38 MAPK. A. The antibody, specific for activated p38 MAP kinase (which recognizes the phosphorylated Thr180 and Tyr182 residues) was used for the Western blot assays, a. Effect of osmotic stress. The sponge tissue was treated with seawater adjusted to 1 M with respect to NaCI. At time 0 and after 2 to 30 min tissue aliquots were taken and extracted. The soluble fraction (20 rtg/lane) was analyzed by Western blot analysisas described in Materials and methods. The arrow points to the phosphorylated form of the p38 MAPK. b. Tissue samples were exposed to seawater, containing 0.25 to 1 M (final concentrations) of NaCI. 30 rain after incubation the cells were assayed, c. Cells from S. domunculawere exposed to 100 pM of hydrogen peroxide for the indicated incubation periods, d. Response to elevated temperature. The cells were transferred to 26°C and then analyzed, e. Exposure of cells to ultraviolet light (UVB)for 0-30 min. B. The p38 MAP kinase antibody, which recognizes the total p38 kinase was applied for the Western blot. Tissue samples which had been exposed to 0.25 to i M of NaCIfor 30 min (parallelblot to the one shown in A, lane b) were analyzed. Further details are given in Materials and methods.

finding led us to postulate that the stress factors, e.g., osmotic stress (shown here) or cytokines cause, like in mammalian systems (Wasylyk et al., 1998), an activation of small G-proteins. As one example, the Cdc42 protein, should be mentioned which is present in S. domuncula (to be published) and known to activate downstream p38 MAPK (Bourdoulous et al., 1998). The intermediate steps, the two kinases, MAP kinase kinase kinase and MAP kinase kinase, are being cloned at present from S. domuncula. Finally, in analogy to the mammalian system (Wasylyk et al., 1998), it is assumed that p38 MAPK phosphorylates the two components of the TCF, Elk-1 and Sap-la, which in turn interact with the SRF, bound at the serum-response elements within the promoter of immediate early genes. Among them is the immediate early gene, erg-1, which is upregulated after environmental stress (Lim et al., 1998).

ACKNOWLEDGMENTS This work was supported by grants from the Deutsche Forschungsgemeinschaft (Mu 348/12-3) and from the B6hm et al.

Biology of the Cell 92 (2000) 95-104

103

Extracellular stress factors (osmotic stress, cytokines)

Cdc42

MAP kinase kinase kinase

MAP kinase kinase

p38/SAPK2 kinase

I Net-b

Elk-1

Sap-1 a I I

TCF

I promoter

Y

immediate early gene

Bundesministerium ffir Forschung und Technologie (under the coordination of the DLR-Bonn-Internationales Btiro).

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p38 of the SAPK2 subfamily from the sponge S. domuncula

Figure 5. Proposed hierarchy and pathway of immediate early gene expression mediated by the p38 MAPK system. It is outlined that extracellular stressors activate small G proteins (e.g., Cdc42), which stimulate the kinase cascade, with the target p38 MAPK. This kinase phosphorylates protein(s) of the ternary complex factor (TCF), a process which facilitates DNA binding of the protein serum-response factor (SRF) dimer in the promoter region of immediate early genes.

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