2 Institute of Biology, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands; 3 Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apartado 553, E-41071 Sevilla, Spain; 4 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Received on May 8, 2003; revised on July 8, 2003; accepted on July 15, 2003
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Abstract |
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Key words: chitin oligosaccharides / ERK / zebrafish cells
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Introduction |
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Mammalian cells respond to a variety of extracellular stimuli via activation of specific mitogen-activated protein kinases (MAPKs) that orchestrate the transduction of the signal from receptors at the cell surface to the nucleus and play a major role in the integration of multiple biological responses. Three main classes of MAPK are recognized: the classic MAPK, also known as extracellular-regulated kinases, ERK1 and ERK2, and c-Jun N-terminal kinase (JNK) and p38 MAPK, which are activated by dual phosphorylation at neighboring threonine and tyrosine residues in the activation loop. Dephosphorylation of either residue results in MAPK inactivation (Johnson and Lapadat, 2002). The general scheme of ERK activation involves a cascade of phosphorylation events initiated by stimulation of the Ras proto-oncogene following activation of growth factor receptors. The cascade starts with the activation of one or more Raf family kinases, which phosphorylate and activate the MAPK kinases (MEK1/2). MEK in turn catalyzes dual phosphorylation and activation of ERKs (Chang and Karin, 2001
). Once activated, ERKs accumulate in the nucleus by as-yet undefined mechanisms that seem to involve the dissociation of ERK from MEK (Fukuda et al., 1997
) and/or a blockade of its export from the nucleus by neosynthesized nuclear anchors (Lenormand et al., 1998
).
The ERK pathway has been implicated in diverse cellular processes, including cell growth, proliferation, differentiation, and survival (Marshall, 1995; Ballif and Blenis, 2001
). Hence, finely tuned regulation of ERK activation is essential in conveying appropriate signals. The intensity, duration, and subcellular localization of ERK activation are well regulated. Scaffolding proteins and docking sites provide the means to avoid cross-activation between MAPK signaling pathways and permit precise and even cell-specific subcellular localization of ERKs (Pouysségur et al., 2002
). The variable responses elicited by this cascade in different cell types are also presumably determined by the cell-specific combination of downstream substrates.
Activated ERK phosphorylates numerous substrates in all cellular compartments (Lewis et al., 1998). More than 50 different ERK substrates have been identified so far. These include ubiquitous or lineage-restricted transcribed factors, the kinases RSK and MNK and proteins involved in nucleotide biosynthesis, cytoskeleton organization, ribosomal transcription, and membrane traffic (Sturgill et al., 1988
; Marais et al., 1993
; Treisman, 1996
; Fukunaga and Hunter, 1997
; Lewis et al., 2000
; Stefanovsky et al., 2001
). RSKs, 90-kDa ribosomal S6 serine/threonine kinases family (also known as MAPK-activated protein kinase-1, MAPKAP-K1) are activated by ERKs in vitro and in vivo via phosphorylation (Sturgill et al., 1988
; Dalby et al., 1998
).
The presence and function of MAPK pathways in zebrafish has been recently described. The insulin-like growth factor stimulates zebrafish cell proliferation by activating MAPK and PI3-kinase-signaling pathways (Pozios et al., 2001), while Fgf/MAPK signaling is required for development of the subpallial telencephalon and somite boundary formation in zebrafish embryos (Shinya et al., 2001
; Sawada et al., 2001
). Hence components upstream and downstream of the specific MAPK cascades in zebrafish are not known yet.
Here we show that chitin tetrasaccharides rapidly and specifically activate the ERK pathway in embryonic zebrafish cells. These findings provide strong evidence that CO signaling is initiated on the plasma membrane via activation of high-affinity oligosaccharide receptor system, which transmits the signal to nucleus probably using the Ras, Raf, MEK, ERK cascade.
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Results |
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Zebrafish cells were serum starved for 24 h, stimulated with 5 x 10-6 M chitin tetrasaccharides and EGF for the indicated times, lysed, and processed for western blotting. The activated/phosphorylated form of MAPKs, ERKs was monitored by specific dual phospho-p44/42 MAP kinase antibodies (dp ERK), which detect p44 and p42 MAP kinase (ERK1 and ERK2) only when catalytically activated by phosphorlation at Thr202 and Tyr204. The antibody does not appreciably cross-react with the corresponding phosphorylated threonine and tyrosine residues of either JNK/SAPK or p38 MAPK homologs. It does not cross-react with up to 2 µg nonphosphorylated p44/42 MAPK. The total protein level in all performed experiments was control by ERK1 antibody, which reacts with ERK1 (p44) and, to a lesser extent ERK2 (p42) by western blotting. Bands intensities were quantified by densitometry, and ERK activity was compared to the basal level at t = 0.
In vivo stimulation of ZF13 and ZF29 with chitin tetrasaccharide and EGF transiently induced the 815-fold activation/phosphorylation of ERKs, with a maximum after 15 min (Figure 1). The half maximal response was obtained at 10-9 M concentration (Figure 2). Although dp ERK antibodies are able to recognize both phosphorylated ERK1 and ERK2 in all experiments, one band of 44 kDa was preferentially detected, suggesting that ERK1 is mainly activated by COs and EGF. The anti-phospho-p38 MAPK and anti-phospho-SAPK/JNK antibodies showed absence of cross-reaction in the CO activation process observed with phospho-ERK1/2 antibody, ensuring the specificity in the western blots (Figure 1C).
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Discussion |
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First, we found that chitin tetrasaccharides transiently induced phosphorylation of ERKs, probably ERK1, with a maximum after 15 min stimulation, at 10-9 M concentration. This response was very specific for the structure of the glycan backbone. The replacement of one or two GlcNac residues by glucose and fucosylation by the NodZ protein resulted in a loss of their biological activity. In contrast, an analog of CO, thioCO, which could not be enzymatically hydrolyzed, was as potent an inducer as COs, excluding a role of possible degradation products. Furthermore, we also examined the signaling pathway to ERK activation by COs and concluded that a high-affinity oligosaccharide receptor system exists that transduces the signal to Raf, MEK, and ERK. Once activated/phosphorylated ERK phosphorylates p90RSK kinase, known as MAPK-activated protein kinase-1, MAPKAP-K1. Phosphorylated RSKs in turn activate transcription factors, such as CREB, and are involved in cell cycle control, memory formation, and suppression of apoptotic cell death (Nebreda and Gavin, 1999). Moreover, inactivation mutations in the gene encoding human RSK2 are associated with Coffin-Lowry syndrome, a disease that results in severe mental retardation as well as progressive skeletal deformation (Treisman, 1996
). The recently discovered RSK4 is deleted in patients with X-linked mental retardation (XLMR) and may be a candidate XLMR gene (Yntema et al., 1999
).
Our results strongly support the model shown in Figure 5, but final genetic identification of all components of CO signaling remain to be determined. Several lines of evidence in different cell types indicate that p90RSKs are activated by MAPKs in vivo via a Ras-dependent protein kinase cascade that is triggered by growth factors or tumor-promoting phorbol esters (Alessie et al., 1995). Moreover, other physiological substrates of p90RSK have been identified with different effects in development, cell cycle, suppression of apoptotic cell death, and progressive skeletal deformation (Treisman, 1996
; Nebreda and Gavin, 1999
). These findings promote future studies of p90RSK and its physiological substrates in zebrafish cells and embryos.
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In future research we aim to identify CO receptors and link them to downstream pathways, such as the ERK pathway, to fully understand CO function in vertebrate development.
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Materials and methods |
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Chitin tetrasaccharides were purchased from Seikagaku (Tokyo). Replacement of one or two central GlcNac residues of the chitin tetrasaccharide by Glc unit was received as described (Wang and Lee, 1995; Robina et al., 2002
; Southwick et al., 2002
). Fucosylation of COs by NodZ protein was performed as described previously (Bakkers et al., 1997
).
The syntheses of thioCO
The syntheses of di-N-acetyl-4-thiochitobiose (TG2), -methyl di-N-acetyl-4-thiochitobioside (MTG2),
-methyl tri-N-acetyl-4,4'-dithiochitotrioside (MTG3),
-methyl tetra-N-acetyl-4,4',4''-trithiochitotetraoside (MTG4), and ß-methyl tri-N-acetyl-4,4'-dithiochitotrioside (ß-MTG3) were reported in Wang and Lee (1995)
. The reducing thiochito-oligomers, tri-N-acetyl-4,4'-dithiochitotriose (TG3) and tetra-N-acetyl-4,4',4''-trithiochitotetraose (TG4), were prepared as follows.
TG3
The peracetylated 4,4'-dithiochitotriose (20 mg) (Wang and Lee, 1995) was de-O-acetylated with sodium methoxide in methanol (10 mM, 5 ml) at room temperature for 6 h. Water (1 ml) was added and the solution was neutralized with Dowex 50w-x8 resin (H+ form). After removal of the resin by filtration, the filtrate was concentrated. The trisaccharide was dissolved in a small amount of water and applied to a Sephadex G-10 column (1.5 x 90 cm), which was preequilibrated and eluted with 50 mM acetic acid. Fractions containing the trisaccharide were pooled and lyophilized to give 15.5 mg TG3 (yield: 81%). 1Hnuclear magnetic resonance (NMR) of TG3 (500 MHz, D2O, 60°C):
5.248 (d, J = 3.4 Hz, 0.35 H, H-1
), 4.7764.732 (two pairs of d, J = 10.2 Hz, 2 H, H-1',1''), 4.695 (d, J = 8.4 Hz, 0.65 H, H-1ß), 4.0573.470 (m, 16 H, sugar protons), 2.9572.856 (two pairs of t, 2 H, J = 10.4 Hz, H-4,4'), 2.0592.049 (singlets, 9 H, 3 N-acetyl).
TG4
The -methyl 4,4',4''-trithiochitotetraoside (4.5 mg) (Wang and Lee, 1995
) was per-O-acetylated with acetic anhydridepyridine (1:1, 1.5 ml) at room temperature for 5 h. Ice water (2 ml) was added, and the solution was evaporated to dryness. Trace of volatiles was removed by coevaporation of the residue with toluene (3 x 2 ml) to give 7.5 mg of the peracetylated
-methyl 4,4',4''-trithiochitotetraoside. The compound obtained was subjected to acetolysis with Ac2OAcOHH2SO4 (8:2:0.1, 2 ml) to selectively remove the 1-O-methyl group. After acetolysis at room temperature for 12 h, the solution was cooled to 0°C, and a cold solution of aqueous sodium acetate (0.2 M, 3 ml) was added. The mixture was evaporated to dryness and the residue was partitioned between CHCl3 (10 ml) and water (2 ml). The organic layer was separated and successively washed with 5% hydrochloric acid (1 ml), saturated sodium bicarbonate (1 ml), and water (2 x 1 ml), dried with anhydrous sodium sulfate, and filtered. The filtrate was evaporated to afford the peracetylated 4,4',4''-trithiochitotetraose as a white solid, which was subsequently de-O-acetylated with sodium methoxide in methanol (10-2 M, 5 ml). The product was purified by gel filtration with a Sephadex G-10 column the same way as described for the preparation of TG3 to give 3.5 mg TG4 (overall yield in three steps: 79%). 1H-NMR of TG4 (500 MHz, D2O, 60°C):
5.246 (d, J = 3.4 Hz, 0.39 H, H-1
), 4.7694.725 (3 pairs of d, J = 10.3 Hz, 3 H, H-1',1'',1'''), 4.693 (d, J = 8.0 Hz, 0.61 H, H-1ß), 4.0593.467 (m, 21 H, sugar protons), 2.9452.872 (three pairs of t, 3 H, J = 10.3 Hz, H-4,4',4''), 2.0502.047 (singlets, 12 H, 4 N-acetyl).
Cell lines
Cell lines ZF13 and ZF29c-1 were obtained from Hubrecht laboratory. These cells have been derived from dechorionated, disaggregated, 20-h-old zebrafish embryos. They have a fibroblast-like morphology (Peppelenbosch et al., 1995). For our experiments cells were grown at 25°C in 2 ml 67% L-15 medium supplemented with 10% fetal calf serum in 24-well plates (Greiner, Alphen aan de Rijn, The Netherlands) until the plates became 50% confluent. Before stimulation experiments, the medium was changed to serum-free medium (SFM) for 1824 h to reduce the basal level of phosphorylation. This SFM was then replaced with 1 ml SFM plus indicated stimulus for various times. Stimulation was terminated by quick replacement of SFM for 600 µl, 65°C sodium dodecyl sulfate (SDS) sample buffer (62.5 mM TrisHCl, 3% w/v SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% w/v bromophenol blue). The cells were scraped off the dish, and the cell lysates were transferred to a new tube, boiled for 3 min, and separated on 10% polyacrylamide slab gels (10 µg protein per lane).
Western immunoblotting
SDSpolyacrylamide gel electrophoresis was performed as described by Laemmli (1970). After electrophoresis, proteins were transferred to nitrocellulose membrane (Schleicher & Schuell, Den Bosch, The Netherlands) by western blotting. The membranes were blocked in 5% w/v nonfat dry milk in Tris-buffered saline-Tween 20 (TBST). The blots were incubated with a 1:1000 to 1:2000 dilution of the indicated antibody in TBST with 3% bovine serum albumin (Sigma, St. Louis, MO) for 1 h at room temperature or overnight at 4°C. Signal was detected using a 1:5000 to 1:10,000 dilution of horseradish peroxidaseconjugated anti-rabbit antibodies and the enhanced chemiluminescence method (Amersham).
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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