- Email: [email protected]
Molecular cloning of mammalian Spred-3 which suppresses tyrosine kinase-mediated Erk activation
BBRC Biochemical and Biophysical Research Communications 302 (2003) 767–772 www.elsevier.com/locate/ybbrc
Molecular cloning of mammalian Spred-3 which suppresses tyrosine kinase-mediated Erk activation Reiko Kato,a Atsushi Nonami,a Takaharu Taketomi,a Toru Wakioka,a Asato Kuroiwa,b Yoichi Matsuda,b,c,d and Akihiko Yoshimuraa,* a
c
Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan b Laboratory of Animal Cytogenetics, Center for Advanced Science and Technology, Hokkaido University, Sapporo 060-0812, Japan Laboratory of Cytogenetics, Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0812, Japan d Chromosome Research Unit, Faculty of Science, Hokkaido University, Sapporo 060-0812, Japan Received 28 January 2003
Abstract We have reported on Spred-1 and Spred-2, which inhibit MAP kinase activation by interacting with c-kit and ras/raf. Here, we report the cloning of a third member in this family, Spred-3. Spred-3 is expressed exclusively in the brain and its gene locates in chromosome 19q13.13 in human. Like Spred-1 and -2, Spred-3 contains an EVH1 domain in the N-terminus and a Sprouty-related cysteine-rich region (SPR domain) in the C-terminus that is necessary for membrane localization. However, Spred-3 does not possess a functional c-kit binding domain (KBD), since the critical amino acid Arg residue in this region was replaced with Gly in Spred-3. Although Spred-3 suppressed growth factor-induced MAP kinase (Erk) activation, inhibitory activity of Spred-3 was lower than that of Spred-1 or Spred-2. By the analysis of chimeric molecules between Spred-3 and Spred-1, we found that the SPR domain, rather than KBD, is responsible for efficient Erk suppression. The finding of Spred-3 revealed the presence of a novel family of regulators for the Ras/MAP kinase pathway, each member of which may have different specificities for extracellular signals. Ó 2003 Elsevier Science (USA). All rights reserved.
Extracellular stimuli trigger a number of intracellular signal transduction cascades, among which extracellular stimulus-activated protein (Erk) kinases play important roles in many facets of cellular regulation [1–3]. Activation of the Erk cascade by receptor tyrosine kinases (RTKs), such as the epidermal growth factor (EGF) receptor, is initiated by binding of Shc and Grb2 to the phosphorylated tyrosine residues of the receptor. The complex of Grb2 and SOS activates Ras by GTP loading. Ras-GTP recruits Raf1 to the plasma membrane [3,4]. Then, Raf1 is phosphorylated and activated by not well-defined kinases with complex regulatory mechanisms [5–7]. Activated Raf then phosphorylates and activates the dual-specific kinase MEK, which phosphorylates and activates Erk. In addition, the Ras-independent Raf1-Erk activation mechanism has been recently demonstrated. For example, members of the * Corresponding author. Fax: +81-92-642-6825. E-mail address: [email protected] (A. Yoshimura).
protein kinase C (PKC) family of serine/threonine kinases have been implicated as potential activators of Raf [8]. In contrast to the activation mechanisms for the Raf1-Erk cascade, the regulatory mechanisms of this pathway remain to be investigated. Recently, we cloned a family of novel membrane-bound molecules, Spreds, which are related to Sprouty [9]. Drosophila Sprouty was identified as a negative regulator for several types of growth factor-induced Erk activation, including the fibroblast growth factor (FGF) and EGF [10,11]. Four Sprouty homologs are found in mammals. Vertebrate Sproutys have also been implicated in the negativefeedback regulation of FGF-signaling in embryogenesis [12,13] and angiogenesis [14], although their inhibitory mechanisms are still controversial [15–18]. Spred-1 and Spred-2 have a Sprouty-related C-terminal cysteine-rich (SPR) domain in addition to the N-terminal Ena/VASP homology (EVH) 1 domain, thus named after Sproutyrelated EVH1 domain-containing protein. The SPR
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00259-6
768
R. Kato et al. / Biochemical and Biophysical Research Communications 302 (2003) 767–772
domain of Sprouty and probably that of Spred are palmitoylated, thereby Sprouty and Spred localize in the membrane fraction [19]. Like Sprouty, Spred-1 and Spred-2 also down-regulate Ras/Erk signaling. We have shown that Spred interacts with Ras and inhibits Raf kinase activation without reducing Ras activation, and, interestingly, rather stabilizes EGF-induced Raf-1 translocation from cytosol to the plasma membrane [9]. As Spred inhibits active-Ras-induced Erk activation, Spred might modulate the unidentified activation steps of Raf by a novel mechanism. We have shown that Spred-1 and Spred-2 suppress several types of growth factor-mediated Erk activation, including EGF, FGF, VEGF (vascular endothelial cell growth factor), SCF (stem cell factor), and serum. In contrast, mammalian Sproutys have growth-factor selectivity; Sprouty-2 and Sprouty-4 inhibit FGF-induced Erk activation but do not affect EGF-induced Erk activation [20]. The mechanisms of the difference between Spred and Sprouty remain to be elucidated. In the present study, we describe a new member of the Spred family, Spred-3. The overall structure of Spred-3 is related to those of Spred-1 and Spred-2, but it cannot bind to c-kit, since a critical amino acid residue for the binding to c-kit is missing in this region. Spred-3 possessed less inhibitory activity against growth factor-induced Erk kinase activation. Interestingly, the SPR domain is responsible for effective suppression. Our finding suggests that each member of the Spred family may have a selective function for the regulation of the MAP kinase cascade.
designated as S3 þ K1, and that containing codon 1–250 of Spred-3 and codon 296–444 of Spred-1 was designated as S3 þ S1. Cells. Human EGF, SCF, and bFGF were purchased from PeproTech EC. 293 cells were grown on tissue-culture plates in DMEM (SIGMA) containing 10% calf serum. Mouse myoblast C2C12 cells were cultured in DMEM supplemented with 20% FBS. Luciferase assay. The Elk-1 activation was measured by the GAL4 DNA-binding domain (DB)/Elk-1 fusion system according to the manufacturerÕs instructions (PathDetect in vivo signal transduction pathway trans-reporting system, Stratagene). Briefly, 293 cells were transfected with 0.05 lg Elk-1 consisting of GAL4 DB and Elk-1, 0.1 lg pFR-Luc carrying the GAL4 UAS-fused luciferase gene, 0.2 lg pCH110 encoding the b-galactosidase gene under the control of the SV40 promoter, and Flag- or Myc-tagged Spred expression vectors. For SCF stimulation, c-kit cDNA (Wakioka et al., 2001) was included. After 24 h, cells were treated with 50 ng/ml EGF, bFGF, and SCF or 10% FCS for 6 h and then corrected and lysed with a PicaGene Reporter lysis buffer (TOYO Ink, Japan). The activity of luciferase and b-galactosidase was analyzed by using beetle luciferin (Promega) and o-nitrophenyl b-galactopilanoside (Nacalai Tesque, Japan) as substrates. In all reporter assays, 2 105 of 293 cells was plated on 6-well dishes and transfected by the calcium-phosphate method. Immunochemical analysis. Immunoprecipitation and immunoblotting were performed using anti-Myc (9E10), anti-Flag (M2), anti-Erk2 (Santa Cruz Biotechnology), and anti-phosphorylated Erk1/2 (Promega) antibodies as described [9,20–22]. Immunofluorescence microscopic analysis was done as described [9]. GFP-Erk was a gift from Dr. Y. Goto (Tokyo University). Northern hybridization. Total RNAs from various mouse tissues (10 lg per lane) were hybridized with the isolated Spred full-length cDNAs. Briefly, the probes were radiolabeled with [P32 ]dCTP by using the Amersham Rediprime random primer-labeling kit (product RPN1633). Prehybridization and hybridizations were performed at 65 °C in 0.5 M Na2 HPO4 /7% SDS/0.5 M EDTA, pH 8.0. All stringency washes were conducted at 65 °C with two initial washes in 2 SSC/ 0.1% SDS for 40 min, followed by a subsequent wash in 0.1 SSC/0.1% SDS for 20 min. Membranes were then exposed at )70 °C to X-ray film (Kodak) in the presence of intensifying screens.
Materials and methods Results and discussion Cloning of murine Spred-3 cDNA. BLAST searches in the public human genome database revealed the presence of a Spred-like sequence in chromosome 19p13 (Hs19_11266). The identified region was used for further search using the EST database. The full-length sequence of murine Spred-3 was obtained by EST sequences and by extending the 50 sequence by PCR on murine brain Marathon-Ready library cDNA (Clontech, Palo Alto, CA). A multiple alignment of Spred sequences was created using Geneworks and refined by eye. Conserved alignment patterns were drawn by Consensus (http:// www.bork.embl-heidelberg.de/Alignment/consensus.html). Chromosome mapping and fluorescence in situ hybridization. Mouse chromosomal mapping was conducted by the method of fluorescence in situ hybridization (FISH) as described [21] using various full-length or partial cDNA clones as probes. A search for human syndromes (or mouse defects in syntenic loci) associated with the mapped Spred genes was conducted in the Dysmorphic Human–Mouse Homology Database by an Internet server (http://www.hgmp.mrc.ac.uk/DHMHD/ hum_chrome1.html). Plasmid construction. cDNAs of c-kit mutants were subcloned into KpnI/NotI sites of pCDNA4/TO (Invitrogen). Chimeric mutants between Spred-1 and Spred-3 were generated by the standard polymerase chain reaction (PCR) method and subcloned into pCDNA3 for the 6 Myc epitope or pCMV2 for the Flag epitope as described [20]. The chimeric molecule containing EVH-1 domain of Spred-3 (codon 1– 191) and KBD and SPR domains of Spred-1 (codon 223–444) was
Cloning of a new member of the Spred family gene The nucleotide and predicted amino acid sequences of mouse and human Spred-1 and Spred-2 cDNAs were compared to mouse and human EST databases as well as a genomic database using the BLASTN algorithms [23]. We found the presence of a novel member of the Spred family, Spred-3, in the 19.p13.13 region of the human genome database. The exons were predicted from the Spred-1 and Spred-2 cDNA sequences. The human and mouse full-length cDNA was obtained by PCR and the 50 -end was determined by RACE-PCR. The cDNA contains a C-terminal Sprouty-like cysteinerich domain (SPR-domain) and an N-terminal Ena/ Vasodilator-stimulated phosphoprotein (VASP) homology-1 (EVH-1) domain (Fig. 1). The overall structure was similar to those of Spred-1 and Spred-2; thus, we named this gene Spred-3. The EVH1 domain and the SPR domain share 55% and 60% identity, respectively, between Spred-1 and Spred-3. Little similarity was
R. Kato et al. / Biochemical and Biophysical Research Communications 302 (2003) 767–772
769
Fig. 1. Comparison of the amino acid sequence of Spred family genes. The putative EVH1 domain, KBD, and the SPR domain are indicated. An asterisk indicates the Arg (R) residue, which has been shown to be necessary for Spred-1 and c-kit interaction.
observed in the intermediate region between the EVH1 and SPR domains. We have determined a region of about 50 amino acids of Spred-1 and Spred-3 for the ckit-binding domain (KBD) [9]. Although several amino acids were conserved in the KBD region, critical amino acids for the interaction between c-kit and Spred1(Arg247) were absent (Gly in Spred-3) (Fig. 1). The expression of each Spred isoform was determined by Northern hybridization with various tissue mRNAs. As shown in Fig. 2, in mice, Spred-2 was ubiquitously expressed and Spred-1 was expressed in the brain, kidney, and colon. On the other hand, Spred-3 was exclusively expressed in the brain. The chromosomal localization of Spreds is summarized in Table 1. Human genome localization was determined by NCBI database search and the murine position was determined by in situ hybridization. Each gene locates in different chromosomes. The distal regions of mouse chromosomes share a region of homology with the human chromosomes.
Fig. 2. Expression of Spreds in mouse tissues. Membranes with indicated mouse tissue total RNA (10 lg) were hybridized with mouse Spred cDNA probes. 28S and 16S ribosomal RNAs were visualized with EtBr staining.
Table 1 Chromosomal localization
Spred-1 Spred-2 Spred-3
Mouse
Human
2E5 11A3-A4 7A3
15q13.2 2p14 19q13.13
Chromosome localization was identified by NCBI database search for human and in situ hybridization for mouse.
Membrane localization of Spred-3 and inihibition of Erk activation Next, the function of Spred-3 was examined. First, we detected the plasma membrane localization of transfected Spred-3 as well as Spred-2 by immunofluorescence microscopy in C2C12 cells (Fig. 3). The C-terminal SPR domain was essential for plasma membrane localization, since a deletion mutant lacking the SPR domain localized in the cytoplasm (data not shown). These data indicate that the SPR domain of Spred-3 functions as a membrane-targeting domain as in other Spreds.
Fig. 3. Membrane localization of Spred-3. Flag-tagged Spred-1 and Spred-3 were transiently expressed in C2C12 cells, and cells were fixed and immunostained with a-Flag monoclonal antibody. Magnification: 100.
770
R. Kato et al. / Biochemical and Biophysical Research Communications 302 (2003) 767–772
Fig. 5. Binding to active c-kit and tyrosine phosphorylation of Spreds. (A) Schematic diagram of chimeras used in this study. (B) 293 cells were transfected with indicated Myc-tagged Spred constructs and constitutively activated c-kitD814V . After immunoprecipitation with an anti-Myc antibody, c-kit, and Spred-1 were blotted with anti-phosphotyrosine (anti-PY) and anti-Myc antibodies, respectively. Fig. 4. Comparison of the inhibitory activity of Spred family proteins for growth factor-induced Erk activation. 293 cells transfected with GFP-Erk cDNA were stimulated with 50 ng/ml EGF (A), FGF (B), and 100 ng/ml SCF or 10%FCS (C) for indicated periods. Wild type c-kit plasmid was included in (C) for transfection. Cell extracts were immunoblotted with phospho-Erk1/2 (P-Erk-1/2) or anti-Erk2 antibodies. Representative data of three independent experiments are shown.
Then, we examined whether Spred-3 can inhibit MAP kinase activation in response to growth factors like Spred-1 or Spred-2 (Fig. 4). Erk activation was assayed by measuring the phosphorylation of the TEY motif within the activation loop of co-transfected Erk (GFPErk) using a phospho-Erk-specific antibody [9]. As shown in Fig. 4, overexpression of Spred-1 strongly inhibited EGF-, FGF- as well as SCF-induced Erk activation. Spred-1 also suppressed FCS-induced Erk activation (Fig. 4D). Spred-3 also suppressed this growth factor-induced Erk activation. However, the suppression was incomplete, especially during the early phase of Erk activation, compared with that of Spred-1. Lower efficiency of Erk suppression by Spred-3 was confirmed by Elk-1 reporter gene assay (see Fig. 6A). These data suggest that the function of Spred-3 is slightly different from that of Spred-1. SPR domain is responsible for efficient suppression of Erk activation by Spreds Previously, we have shown that a region of about 50 amino acids called KBD is necessary for the binding between Spred-1 and c-kit and efficient suppression for MAP kinase activation [9]. To investigate whether
Spred-3 can interact with activated c-kit, we co-expressed Spred-3 and a constitutively activated c-kit mutant (c-kitD814V ) in 293 cells [24]. As shown in Fig. 5B, Spred-1 and Spred-2, but not Spred-3, were tyrosinephosphorylated and co-precipitated with c-kitD814V . To define the role of KBD for c-kit and Spred interaction, we created chimeric molecules between Spred-1 and Spred-3 (Fig. 5A). S3 þ K1, which contains EVH-1, an internal region of Spred-3 and KBD, and the SPR domain of Spred-1, was phosphorylated by and interacted well with c-kitD814V , while S3 þ S1, which contained EVH-1, an internal region and KBD of Spred-3, and the SPR domain of Spred-1, was neither phosphorylated nor bound to c-kitD814V . These data suggest that the KBD region of Spred-3 did not function as a c-kit binding domain and therefore lost the ability to be phosphorylated by c-kit. This is consistent with the lack of critical Arg residue for the interaction between c-kit and Spred-1 (Gly in Spred-3) as shown in Fig. 1. Next, to determine which domain is responsible for efficient suppression, we compared the effect of chimeric molecules between Spred-1 and Spred-3 on Erk suppression. As one of the nuclear targets of Erk is Elk-1, a transcription factor of the Ets family, FGF- or EGFinduced activation of Erk can be monitored by measuring the rate of Elk-1-dependent transcription. As shown in Figs. 6A and B, S3 þ K1 as well as S3 þ S1 inhibited Erk activation as effectively as Spred-1 and Spred-2. This was confirmed by monitoring the suppression of phosphorylation of Erk by Spred constructs (Fig. 6C). These data suggested that the SPR domain of
R. Kato et al. / Biochemical and Biophysical Research Communications 302 (2003) 767–772
771
Fig. 6. Suppression of Erk activation by Spred-3. (A, B) Effect of Spred-3 and chimeras on FGF- or EGF-induced Elk-1 reporter activity. 293 cells were transfected with the Elk-1 reporter plasmid and indicated amounts of cDNAs and stimulated with FGF (A) or EGF (B) (50 ng/ml) for 6 h. Then the Elk-1 reporter activity was measured. (C) Effect of Spred-3 and chimeras on Erk phosphorylation. GFP-ERK2 cDNA was transfected into 293 cells with indicated plasmids. Cells were treated with 100 ng/ml FGF for indicted periods and then cell extracts were immunoblotted with phosphoErk1/2 (P-Erk-1/2) or anti-Erk2 antibodies.
Spred-1 is responsible for the effective suppression of FGF- and EGF-induced Erk activation. It is interesting that binding to c-kit and phosphorylation of Spred are dependent on the KBD domain but that efficient suppression of Erk activation requires the SPR domain of Spred-1. Further analysis is underway for identifying the differences in the function of SPR domains between Spred-1 and Spred-3. We recently found that Spred-1 and Spred-2 bind to Raf-1 through the SPR domains (Taketomi et al., unpublished data). The binding affinity of the SPR domain of Spred-3 to Raf-1 may be weaker than those of Spred-1 and Spred-2. We reported that mammalian Sproutys selectively inhibit FGF-induced Erk activation but do not inhibit EGF-induced Erk activation [20]. In contrast, so far, we observed that Erk activation by any stimulation including EGF, FGF, VEGF, PDGF, SCF, serum, and LPA is efficiently suppressed by overexpression of Spred-1 or Spred-2. However, Spred-3 suppressed Erk activation partially by these stimuli. It is of interest to determine whether Spred-3 can inhibit Erk activation with high efficiency by a specific stimulation. Finding such stimulation may facilitate the determination of the physiological function of Spred-3. We have also shown that Spreds down-regulate Erk activation with a mechanism similar to but distinct from that of Sproutys. It is significant that both Spred and Sprouty localize in the
plasma membrane, thus regulating the early steps of Erk activation. However, Sproutys cannot inhibit activeRas-induced Erk activation, but Spreds can. Spreds, but not Sproutys, bind to Ras, while both can interact with Raf-1 through the SPR domain (Sasaki et al. unpublished data). These observations will facilitate clarifying the molecular mechanism whereby Spreds/Sproutys suppress Erk activation. The Ras/MAP kinase pathway has been shown to be regulated in many ways. Sprouty and MAP kinase phosphatases have been shown to be negative-feedback regulators of the Ras/MAP kinase pathway, since their expression is dependent on growth factors or MAP kinase activity. A Raf-1 binding protein, RKIP, which was previously identified as a phosphatidylethanolamine-binding protein, has been shown to down-modulate the activation of the Raf/MEK/Erk pathway [25]. Raf-1 activity was also regulated by phosphorylation through PKA and Akt. Therefore, multiple regulation steps exist to ensure the tight regulation of this pathway. We suspect that Spreds also play an important regulatory role in this pathway. Spred and Sprouty are evolutionary conserved, and four and three isoforms, respectively, have been identified in mammals to date. Thus, Spred and Sprouty may regulate the Ras/MAP kinase pathway in response to different stimulations in different tissues or organs. Fine
772
R. Kato et al. / Biochemical and Biophysical Research Communications 302 (2003) 767–772
tuning of this signal transduction pathway is necessary for the development and right response to external stimuli, since MAP kinases have been shown to possess multiple substrates and functions.
[14]
[15]
Acknowledgments We thank Dr. E. Nishida for the ERK2 plasmid and Dr. T. Sato and Dr. Y. Kaziro for the Ras plasmids. Part of this work was supported by grants from the Ministry of Education, Science, Technology, Sports and Culture of Japan, the Japan Health Science Foundation, the Human Frontier Science Program, the Japan Research Foundation for Clinical Pharmacology, and the Uehara Memorial Foundation.
[16]
[17]
[18]
References [1] J.E. Ferrell Jr., MAP kinases in mitogenesis and development, Curr. Top. Dev. Biol. 33 (1996) 1–60. [2] L. Chang, M. Karin, Mammalian MAP kinase signalling cascades, Nature 410 (2001) 37–40. [3] M.J. Robinson, M.H. Cobb, Mitogen-activated protein kinase pathways, Curr. Opin. Cell. Biol. 9 (1997) 180–186. [4] D.R. Lowy, B.M. Willumsen, Function and regulation of ras, Annu. Rev. Biochem. 62 (1993) 851–891. [5] D.K. Morrison, R.E. Cutler, The complexity of Raf-1 regulation, Curr. Opin. Cell. Biol. 9 (1997) 174–179. [6] E. Kerkhoff, U.R. Rapp, The Ras–Raf relationship: an unfinished puzzle, Adv. Enzyme Regul. 41 (2001) 261–267. [7] P.W. Sternberg, J. Alberola-Ila, Conspiracy theory: RAS and RAF do not act alone, Cell 95 (1998) 447–450. [8] C. Liebmann, Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity, Cell Signal. 13 (2001) 777–785. [9] T. Wakioka, A. Sasaki, R. Kato, T. Shouda, A. Matsumoto, K. Miyoshi, M. Tsuneoka, S. Komiya, R. Baron, A. Yoshimura, Spred is a Sprouty-related suppressor of Ras signalling, Nature 412 (2001) 647–651. [10] N. Hacohen, S. Kramer, D. Sutherland, Y. Hiromi, M.A. Krasnow, Sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways, Cell 92 (1998) 253–263. [11] T. Casci, J. Vinos, M. Freeman, Sprouty, an intracellular inhibitor of Ras signaling, Cell 96 (1999) 655–665. [12] A.A. de Maximy, Y. Nakatake, S. Moncada, N. Itoh, J.P. Thiery, S. Bellusci, Cloning and expression pattern of a mouse homologue of Drosophila sprouty in the mouse embryo, Mech. Dev. 81 (1999) 213–216. [13] J.D. Tefft, M. Lee, S. Smith, M. Leinwand, J. Zhao, P. Bringas Jr., D.L. Crowe, D. Warburton, Conserved function of mSpry-2, a
[19]
[20]
[21]
[22]
[23]
[24]
[25]
murine homolog of Drosophila sprouty, which negatively modulates respiratory organogenesis, Curr. Biol. 9 (1999) 219–222. G. Minowada, L.A. Jarvis, C.L. Chi, A. Neubuser, X. Sun, N. Hacohen, M.A. Krasnow, G.R. Martin, Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed, Development 126 (1999) 4465–4475. Y. Yigzaw, H.M. Poppleton, N. Sreejayan, A. Hassid, T.B. Patel, Protein-tyrosine phosphatase-1B (PTP1B) mediates the antimigratory actions of sprouty, J. Biol. Chem. 278 (2003) 284–288. H. Hanafusa, S. Torii, T. Yasunaga, E. Nishida, Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway, Nat. Cell Biol. 4 (2002) 850–858. J. Lim, P. Yusoff, E.S. Wong, S. Chandramouli, D.H. Lao, C.W. Fong, G.R. Guy, The cysteine-rich sprouty translocation domain targets mitogen-activated protein kinase inhibitory proteins to phosphatidylinositol 4,5-bisphosphate in plasma membranes, Mol. Cell. Biol. 22 (2002) 7953–7966. E.S. Wong, C.W. Fong, J. Lim, P. Yusoff, B.C. Low, W.Y. Langdon, G.R. Guy, Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signalling, EMBO J. 21 (2002) 4796–4808. M.A. Impagnatiello, S. Weitzer, G. Gannon, A. Compagni, M. Cotten, G. Christofori, Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells, J. Cell Biol. 152 (2001) 1087– 1098. A. Sasaki, T. Taketomi, T. Wakioka, R. Kato, A. Yoshimura, Identification of a dominant negative mutant of Sprouty that potentiates fibroblast growth factor- but not epidermal growth factor-induced ERK activation, J. Biol. Chem. 276 (2001) 36804– 36808. K. Yamada, K. Nishida, M. Hibi, T. Hirano, Y. Matsuda, Comparative FISH mapping of Gab1 and Gab2 genes in human, mouse and rat, Cytogenet. Cell Genet. 94 (2001) 39–42. T. Hanada, T. Yoshida, I. Kinjyo, S. Minoguchi, H. Yasukawa, S. Kato, H. Mimata, Y. Nomura, Y. Seki, M. Kubo, A. Yoshimura, A mutant form of JAB/SOCS1 augments the cytokine-induced JAK/STAT pathway by accelerating degradation of wild-type JAB/CIS family proteins through the SOCS-box, J. Biol. Chem. 276 (2001) 40746–40754. S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (1997) 3389–3402. T. Tsujimura, T. Furitsu, M. Morimoto, K. Isozaki, S. Nomura, Y. Matsuzawa, Y. Kitamura, Y. Kanakura, Ligand-independent activation of c-kit receptor tyrosine kinase in a murine mastocytoma cell line P-815 generated by a point mutation, Blood 83 (1994) 2619–2626. K. Yeung, T. Seitz, S. Li, P. Janosch, B. McFerran, C. Kaiser, F. Fee, K.D. Katsanakis, D.W. Rose, H. Mischak, J.M. Sedivy, W. Kolch, Suppression of Raf kinase activity and MAP kinase signalling by RKIP, Nature 401 (1999) 173–177.
Molecular cloning of mammalian Spred-3 which suppresses tyrosine kinase-mediated Erk activation Reiko Kato,a Atsushi Nonami,a Takaharu Taketomi,a Toru Wakioka,a Asato Kuroiwa,b Yoichi Matsuda,b,c,d and Akihiko Yoshimuraa,* a
c
Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan b Laboratory of Animal Cytogenetics, Center for Advanced Science and Technology, Hokkaido University, Sapporo 060-0812, Japan Laboratory of Cytogenetics, Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0812, Japan d Chromosome Research Unit, Faculty of Science, Hokkaido University, Sapporo 060-0812, Japan Received 28 January 2003
Abstract We have reported on Spred-1 and Spred-2, which inhibit MAP kinase activation by interacting with c-kit and ras/raf. Here, we report the cloning of a third member in this family, Spred-3. Spred-3 is expressed exclusively in the brain and its gene locates in chromosome 19q13.13 in human. Like Spred-1 and -2, Spred-3 contains an EVH1 domain in the N-terminus and a Sprouty-related cysteine-rich region (SPR domain) in the C-terminus that is necessary for membrane localization. However, Spred-3 does not possess a functional c-kit binding domain (KBD), since the critical amino acid Arg residue in this region was replaced with Gly in Spred-3. Although Spred-3 suppressed growth factor-induced MAP kinase (Erk) activation, inhibitory activity of Spred-3 was lower than that of Spred-1 or Spred-2. By the analysis of chimeric molecules between Spred-3 and Spred-1, we found that the SPR domain, rather than KBD, is responsible for efficient Erk suppression. The finding of Spred-3 revealed the presence of a novel family of regulators for the Ras/MAP kinase pathway, each member of which may have different specificities for extracellular signals. Ó 2003 Elsevier Science (USA). All rights reserved.
Extracellular stimuli trigger a number of intracellular signal transduction cascades, among which extracellular stimulus-activated protein (Erk) kinases play important roles in many facets of cellular regulation [1–3]. Activation of the Erk cascade by receptor tyrosine kinases (RTKs), such as the epidermal growth factor (EGF) receptor, is initiated by binding of Shc and Grb2 to the phosphorylated tyrosine residues of the receptor. The complex of Grb2 and SOS activates Ras by GTP loading. Ras-GTP recruits Raf1 to the plasma membrane [3,4]. Then, Raf1 is phosphorylated and activated by not well-defined kinases with complex regulatory mechanisms [5–7]. Activated Raf then phosphorylates and activates the dual-specific kinase MEK, which phosphorylates and activates Erk. In addition, the Ras-independent Raf1-Erk activation mechanism has been recently demonstrated. For example, members of the * Corresponding author. Fax: +81-92-642-6825. E-mail address: [email protected] (A. Yoshimura).
protein kinase C (PKC) family of serine/threonine kinases have been implicated as potential activators of Raf [8]. In contrast to the activation mechanisms for the Raf1-Erk cascade, the regulatory mechanisms of this pathway remain to be investigated. Recently, we cloned a family of novel membrane-bound molecules, Spreds, which are related to Sprouty [9]. Drosophila Sprouty was identified as a negative regulator for several types of growth factor-induced Erk activation, including the fibroblast growth factor (FGF) and EGF [10,11]. Four Sprouty homologs are found in mammals. Vertebrate Sproutys have also been implicated in the negativefeedback regulation of FGF-signaling in embryogenesis [12,13] and angiogenesis [14], although their inhibitory mechanisms are still controversial [15–18]. Spred-1 and Spred-2 have a Sprouty-related C-terminal cysteine-rich (SPR) domain in addition to the N-terminal Ena/VASP homology (EVH) 1 domain, thus named after Sproutyrelated EVH1 domain-containing protein. The SPR
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00259-6
768
R. Kato et al. / Biochemical and Biophysical Research Communications 302 (2003) 767–772
domain of Sprouty and probably that of Spred are palmitoylated, thereby Sprouty and Spred localize in the membrane fraction [19]. Like Sprouty, Spred-1 and Spred-2 also down-regulate Ras/Erk signaling. We have shown that Spred interacts with Ras and inhibits Raf kinase activation without reducing Ras activation, and, interestingly, rather stabilizes EGF-induced Raf-1 translocation from cytosol to the plasma membrane [9]. As Spred inhibits active-Ras-induced Erk activation, Spred might modulate the unidentified activation steps of Raf by a novel mechanism. We have shown that Spred-1 and Spred-2 suppress several types of growth factor-mediated Erk activation, including EGF, FGF, VEGF (vascular endothelial cell growth factor), SCF (stem cell factor), and serum. In contrast, mammalian Sproutys have growth-factor selectivity; Sprouty-2 and Sprouty-4 inhibit FGF-induced Erk activation but do not affect EGF-induced Erk activation [20]. The mechanisms of the difference between Spred and Sprouty remain to be elucidated. In the present study, we describe a new member of the Spred family, Spred-3. The overall structure of Spred-3 is related to those of Spred-1 and Spred-2, but it cannot bind to c-kit, since a critical amino acid residue for the binding to c-kit is missing in this region. Spred-3 possessed less inhibitory activity against growth factor-induced Erk kinase activation. Interestingly, the SPR domain is responsible for effective suppression. Our finding suggests that each member of the Spred family may have a selective function for the regulation of the MAP kinase cascade.
designated as S3 þ K1, and that containing codon 1–250 of Spred-3 and codon 296–444 of Spred-1 was designated as S3 þ S1. Cells. Human EGF, SCF, and bFGF were purchased from PeproTech EC. 293 cells were grown on tissue-culture plates in DMEM (SIGMA) containing 10% calf serum. Mouse myoblast C2C12 cells were cultured in DMEM supplemented with 20% FBS. Luciferase assay. The Elk-1 activation was measured by the GAL4 DNA-binding domain (DB)/Elk-1 fusion system according to the manufacturerÕs instructions (PathDetect in vivo signal transduction pathway trans-reporting system, Stratagene). Briefly, 293 cells were transfected with 0.05 lg Elk-1 consisting of GAL4 DB and Elk-1, 0.1 lg pFR-Luc carrying the GAL4 UAS-fused luciferase gene, 0.2 lg pCH110 encoding the b-galactosidase gene under the control of the SV40 promoter, and Flag- or Myc-tagged Spred expression vectors. For SCF stimulation, c-kit cDNA (Wakioka et al., 2001) was included. After 24 h, cells were treated with 50 ng/ml EGF, bFGF, and SCF or 10% FCS for 6 h and then corrected and lysed with a PicaGene Reporter lysis buffer (TOYO Ink, Japan). The activity of luciferase and b-galactosidase was analyzed by using beetle luciferin (Promega) and o-nitrophenyl b-galactopilanoside (Nacalai Tesque, Japan) as substrates. In all reporter assays, 2 105 of 293 cells was plated on 6-well dishes and transfected by the calcium-phosphate method. Immunochemical analysis. Immunoprecipitation and immunoblotting were performed using anti-Myc (9E10), anti-Flag (M2), anti-Erk2 (Santa Cruz Biotechnology), and anti-phosphorylated Erk1/2 (Promega) antibodies as described [9,20–22]. Immunofluorescence microscopic analysis was done as described [9]. GFP-Erk was a gift from Dr. Y. Goto (Tokyo University). Northern hybridization. Total RNAs from various mouse tissues (10 lg per lane) were hybridized with the isolated Spred full-length cDNAs. Briefly, the probes were radiolabeled with [P32 ]dCTP by using the Amersham Rediprime random primer-labeling kit (product RPN1633). Prehybridization and hybridizations were performed at 65 °C in 0.5 M Na2 HPO4 /7% SDS/0.5 M EDTA, pH 8.0. All stringency washes were conducted at 65 °C with two initial washes in 2 SSC/ 0.1% SDS for 40 min, followed by a subsequent wash in 0.1 SSC/0.1% SDS for 20 min. Membranes were then exposed at )70 °C to X-ray film (Kodak) in the presence of intensifying screens.
Materials and methods Results and discussion Cloning of murine Spred-3 cDNA. BLAST searches in the public human genome database revealed the presence of a Spred-like sequence in chromosome 19p13 (Hs19_11266). The identified region was used for further search using the EST database. The full-length sequence of murine Spred-3 was obtained by EST sequences and by extending the 50 sequence by PCR on murine brain Marathon-Ready library cDNA (Clontech, Palo Alto, CA). A multiple alignment of Spred sequences was created using Geneworks and refined by eye. Conserved alignment patterns were drawn by Consensus (http:// www.bork.embl-heidelberg.de/Alignment/consensus.html). Chromosome mapping and fluorescence in situ hybridization. Mouse chromosomal mapping was conducted by the method of fluorescence in situ hybridization (FISH) as described [21] using various full-length or partial cDNA clones as probes. A search for human syndromes (or mouse defects in syntenic loci) associated with the mapped Spred genes was conducted in the Dysmorphic Human–Mouse Homology Database by an Internet server (http://www.hgmp.mrc.ac.uk/DHMHD/ hum_chrome1.html). Plasmid construction. cDNAs of c-kit mutants were subcloned into KpnI/NotI sites of pCDNA4/TO (Invitrogen). Chimeric mutants between Spred-1 and Spred-3 were generated by the standard polymerase chain reaction (PCR) method and subcloned into pCDNA3 for the 6 Myc epitope or pCMV2 for the Flag epitope as described [20]. The chimeric molecule containing EVH-1 domain of Spred-3 (codon 1– 191) and KBD and SPR domains of Spred-1 (codon 223–444) was
Cloning of a new member of the Spred family gene The nucleotide and predicted amino acid sequences of mouse and human Spred-1 and Spred-2 cDNAs were compared to mouse and human EST databases as well as a genomic database using the BLASTN algorithms [23]. We found the presence of a novel member of the Spred family, Spred-3, in the 19.p13.13 region of the human genome database. The exons were predicted from the Spred-1 and Spred-2 cDNA sequences. The human and mouse full-length cDNA was obtained by PCR and the 50 -end was determined by RACE-PCR. The cDNA contains a C-terminal Sprouty-like cysteinerich domain (SPR-domain) and an N-terminal Ena/ Vasodilator-stimulated phosphoprotein (VASP) homology-1 (EVH-1) domain (Fig. 1). The overall structure was similar to those of Spred-1 and Spred-2; thus, we named this gene Spred-3. The EVH1 domain and the SPR domain share 55% and 60% identity, respectively, between Spred-1 and Spred-3. Little similarity was
R. Kato et al. / Biochemical and Biophysical Research Communications 302 (2003) 767–772
769
Fig. 1. Comparison of the amino acid sequence of Spred family genes. The putative EVH1 domain, KBD, and the SPR domain are indicated. An asterisk indicates the Arg (R) residue, which has been shown to be necessary for Spred-1 and c-kit interaction.
observed in the intermediate region between the EVH1 and SPR domains. We have determined a region of about 50 amino acids of Spred-1 and Spred-3 for the ckit-binding domain (KBD) [9]. Although several amino acids were conserved in the KBD region, critical amino acids for the interaction between c-kit and Spred1(Arg247) were absent (Gly in Spred-3) (Fig. 1). The expression of each Spred isoform was determined by Northern hybridization with various tissue mRNAs. As shown in Fig. 2, in mice, Spred-2 was ubiquitously expressed and Spred-1 was expressed in the brain, kidney, and colon. On the other hand, Spred-3 was exclusively expressed in the brain. The chromosomal localization of Spreds is summarized in Table 1. Human genome localization was determined by NCBI database search and the murine position was determined by in situ hybridization. Each gene locates in different chromosomes. The distal regions of mouse chromosomes share a region of homology with the human chromosomes.
Fig. 2. Expression of Spreds in mouse tissues. Membranes with indicated mouse tissue total RNA (10 lg) were hybridized with mouse Spred cDNA probes. 28S and 16S ribosomal RNAs were visualized with EtBr staining.
Table 1 Chromosomal localization
Spred-1 Spred-2 Spred-3
Mouse
Human
2E5 11A3-A4 7A3
15q13.2 2p14 19q13.13
Chromosome localization was identified by NCBI database search for human and in situ hybridization for mouse.
Membrane localization of Spred-3 and inihibition of Erk activation Next, the function of Spred-3 was examined. First, we detected the plasma membrane localization of transfected Spred-3 as well as Spred-2 by immunofluorescence microscopy in C2C12 cells (Fig. 3). The C-terminal SPR domain was essential for plasma membrane localization, since a deletion mutant lacking the SPR domain localized in the cytoplasm (data not shown). These data indicate that the SPR domain of Spred-3 functions as a membrane-targeting domain as in other Spreds.
Fig. 3. Membrane localization of Spred-3. Flag-tagged Spred-1 and Spred-3 were transiently expressed in C2C12 cells, and cells were fixed and immunostained with a-Flag monoclonal antibody. Magnification: 100.
770
R. Kato et al. / Biochemical and Biophysical Research Communications 302 (2003) 767–772
Fig. 5. Binding to active c-kit and tyrosine phosphorylation of Spreds. (A) Schematic diagram of chimeras used in this study. (B) 293 cells were transfected with indicated Myc-tagged Spred constructs and constitutively activated c-kitD814V . After immunoprecipitation with an anti-Myc antibody, c-kit, and Spred-1 were blotted with anti-phosphotyrosine (anti-PY) and anti-Myc antibodies, respectively. Fig. 4. Comparison of the inhibitory activity of Spred family proteins for growth factor-induced Erk activation. 293 cells transfected with GFP-Erk cDNA were stimulated with 50 ng/ml EGF (A), FGF (B), and 100 ng/ml SCF or 10%FCS (C) for indicated periods. Wild type c-kit plasmid was included in (C) for transfection. Cell extracts were immunoblotted with phospho-Erk1/2 (P-Erk-1/2) or anti-Erk2 antibodies. Representative data of three independent experiments are shown.
Then, we examined whether Spred-3 can inhibit MAP kinase activation in response to growth factors like Spred-1 or Spred-2 (Fig. 4). Erk activation was assayed by measuring the phosphorylation of the TEY motif within the activation loop of co-transfected Erk (GFPErk) using a phospho-Erk-specific antibody [9]. As shown in Fig. 4, overexpression of Spred-1 strongly inhibited EGF-, FGF- as well as SCF-induced Erk activation. Spred-1 also suppressed FCS-induced Erk activation (Fig. 4D). Spred-3 also suppressed this growth factor-induced Erk activation. However, the suppression was incomplete, especially during the early phase of Erk activation, compared with that of Spred-1. Lower efficiency of Erk suppression by Spred-3 was confirmed by Elk-1 reporter gene assay (see Fig. 6A). These data suggest that the function of Spred-3 is slightly different from that of Spred-1. SPR domain is responsible for efficient suppression of Erk activation by Spreds Previously, we have shown that a region of about 50 amino acids called KBD is necessary for the binding between Spred-1 and c-kit and efficient suppression for MAP kinase activation [9]. To investigate whether
Spred-3 can interact with activated c-kit, we co-expressed Spred-3 and a constitutively activated c-kit mutant (c-kitD814V ) in 293 cells [24]. As shown in Fig. 5B, Spred-1 and Spred-2, but not Spred-3, were tyrosinephosphorylated and co-precipitated with c-kitD814V . To define the role of KBD for c-kit and Spred interaction, we created chimeric molecules between Spred-1 and Spred-3 (Fig. 5A). S3 þ K1, which contains EVH-1, an internal region of Spred-3 and KBD, and the SPR domain of Spred-1, was phosphorylated by and interacted well with c-kitD814V , while S3 þ S1, which contained EVH-1, an internal region and KBD of Spred-3, and the SPR domain of Spred-1, was neither phosphorylated nor bound to c-kitD814V . These data suggest that the KBD region of Spred-3 did not function as a c-kit binding domain and therefore lost the ability to be phosphorylated by c-kit. This is consistent with the lack of critical Arg residue for the interaction between c-kit and Spred-1 (Gly in Spred-3) as shown in Fig. 1. Next, to determine which domain is responsible for efficient suppression, we compared the effect of chimeric molecules between Spred-1 and Spred-3 on Erk suppression. As one of the nuclear targets of Erk is Elk-1, a transcription factor of the Ets family, FGF- or EGFinduced activation of Erk can be monitored by measuring the rate of Elk-1-dependent transcription. As shown in Figs. 6A and B, S3 þ K1 as well as S3 þ S1 inhibited Erk activation as effectively as Spred-1 and Spred-2. This was confirmed by monitoring the suppression of phosphorylation of Erk by Spred constructs (Fig. 6C). These data suggested that the SPR domain of
R. Kato et al. / Biochemical and Biophysical Research Communications 302 (2003) 767–772
771
Fig. 6. Suppression of Erk activation by Spred-3. (A, B) Effect of Spred-3 and chimeras on FGF- or EGF-induced Elk-1 reporter activity. 293 cells were transfected with the Elk-1 reporter plasmid and indicated amounts of cDNAs and stimulated with FGF (A) or EGF (B) (50 ng/ml) for 6 h. Then the Elk-1 reporter activity was measured. (C) Effect of Spred-3 and chimeras on Erk phosphorylation. GFP-ERK2 cDNA was transfected into 293 cells with indicated plasmids. Cells were treated with 100 ng/ml FGF for indicted periods and then cell extracts were immunoblotted with phosphoErk1/2 (P-Erk-1/2) or anti-Erk2 antibodies.
Spred-1 is responsible for the effective suppression of FGF- and EGF-induced Erk activation. It is interesting that binding to c-kit and phosphorylation of Spred are dependent on the KBD domain but that efficient suppression of Erk activation requires the SPR domain of Spred-1. Further analysis is underway for identifying the differences in the function of SPR domains between Spred-1 and Spred-3. We recently found that Spred-1 and Spred-2 bind to Raf-1 through the SPR domains (Taketomi et al., unpublished data). The binding affinity of the SPR domain of Spred-3 to Raf-1 may be weaker than those of Spred-1 and Spred-2. We reported that mammalian Sproutys selectively inhibit FGF-induced Erk activation but do not inhibit EGF-induced Erk activation [20]. In contrast, so far, we observed that Erk activation by any stimulation including EGF, FGF, VEGF, PDGF, SCF, serum, and LPA is efficiently suppressed by overexpression of Spred-1 or Spred-2. However, Spred-3 suppressed Erk activation partially by these stimuli. It is of interest to determine whether Spred-3 can inhibit Erk activation with high efficiency by a specific stimulation. Finding such stimulation may facilitate the determination of the physiological function of Spred-3. We have also shown that Spreds down-regulate Erk activation with a mechanism similar to but distinct from that of Sproutys. It is significant that both Spred and Sprouty localize in the
plasma membrane, thus regulating the early steps of Erk activation. However, Sproutys cannot inhibit activeRas-induced Erk activation, but Spreds can. Spreds, but not Sproutys, bind to Ras, while both can interact with Raf-1 through the SPR domain (Sasaki et al. unpublished data). These observations will facilitate clarifying the molecular mechanism whereby Spreds/Sproutys suppress Erk activation. The Ras/MAP kinase pathway has been shown to be regulated in many ways. Sprouty and MAP kinase phosphatases have been shown to be negative-feedback regulators of the Ras/MAP kinase pathway, since their expression is dependent on growth factors or MAP kinase activity. A Raf-1 binding protein, RKIP, which was previously identified as a phosphatidylethanolamine-binding protein, has been shown to down-modulate the activation of the Raf/MEK/Erk pathway [25]. Raf-1 activity was also regulated by phosphorylation through PKA and Akt. Therefore, multiple regulation steps exist to ensure the tight regulation of this pathway. We suspect that Spreds also play an important regulatory role in this pathway. Spred and Sprouty are evolutionary conserved, and four and three isoforms, respectively, have been identified in mammals to date. Thus, Spred and Sprouty may regulate the Ras/MAP kinase pathway in response to different stimulations in different tissues or organs. Fine
772
R. Kato et al. / Biochemical and Biophysical Research Communications 302 (2003) 767–772
tuning of this signal transduction pathway is necessary for the development and right response to external stimuli, since MAP kinases have been shown to possess multiple substrates and functions.
[14]
[15]
Acknowledgments We thank Dr. E. Nishida for the ERK2 plasmid and Dr. T. Sato and Dr. Y. Kaziro for the Ras plasmids. Part of this work was supported by grants from the Ministry of Education, Science, Technology, Sports and Culture of Japan, the Japan Health Science Foundation, the Human Frontier Science Program, the Japan Research Foundation for Clinical Pharmacology, and the Uehara Memorial Foundation.
[16]
[17]
[18]
References [1] J.E. Ferrell Jr., MAP kinases in mitogenesis and development, Curr. Top. Dev. Biol. 33 (1996) 1–60. [2] L. Chang, M. Karin, Mammalian MAP kinase signalling cascades, Nature 410 (2001) 37–40. [3] M.J. Robinson, M.H. Cobb, Mitogen-activated protein kinase pathways, Curr. Opin. Cell. Biol. 9 (1997) 180–186. [4] D.R. Lowy, B.M. Willumsen, Function and regulation of ras, Annu. Rev. Biochem. 62 (1993) 851–891. [5] D.K. Morrison, R.E. Cutler, The complexity of Raf-1 regulation, Curr. Opin. Cell. Biol. 9 (1997) 174–179. [6] E. Kerkhoff, U.R. Rapp, The Ras–Raf relationship: an unfinished puzzle, Adv. Enzyme Regul. 41 (2001) 261–267. [7] P.W. Sternberg, J. Alberola-Ila, Conspiracy theory: RAS and RAF do not act alone, Cell 95 (1998) 447–450. [8] C. Liebmann, Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity, Cell Signal. 13 (2001) 777–785. [9] T. Wakioka, A. Sasaki, R. Kato, T. Shouda, A. Matsumoto, K. Miyoshi, M. Tsuneoka, S. Komiya, R. Baron, A. Yoshimura, Spred is a Sprouty-related suppressor of Ras signalling, Nature 412 (2001) 647–651. [10] N. Hacohen, S. Kramer, D. Sutherland, Y. Hiromi, M.A. Krasnow, Sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways, Cell 92 (1998) 253–263. [11] T. Casci, J. Vinos, M. Freeman, Sprouty, an intracellular inhibitor of Ras signaling, Cell 96 (1999) 655–665. [12] A.A. de Maximy, Y. Nakatake, S. Moncada, N. Itoh, J.P. Thiery, S. Bellusci, Cloning and expression pattern of a mouse homologue of Drosophila sprouty in the mouse embryo, Mech. Dev. 81 (1999) 213–216. [13] J.D. Tefft, M. Lee, S. Smith, M. Leinwand, J. Zhao, P. Bringas Jr., D.L. Crowe, D. Warburton, Conserved function of mSpry-2, a
[19]
[20]
[21]
[22]
[23]
[24]
[25]
murine homolog of Drosophila sprouty, which negatively modulates respiratory organogenesis, Curr. Biol. 9 (1999) 219–222. G. Minowada, L.A. Jarvis, C.L. Chi, A. Neubuser, X. Sun, N. Hacohen, M.A. Krasnow, G.R. Martin, Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed, Development 126 (1999) 4465–4475. Y. Yigzaw, H.M. Poppleton, N. Sreejayan, A. Hassid, T.B. Patel, Protein-tyrosine phosphatase-1B (PTP1B) mediates the antimigratory actions of sprouty, J. Biol. Chem. 278 (2003) 284–288. H. Hanafusa, S. Torii, T. Yasunaga, E. Nishida, Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway, Nat. Cell Biol. 4 (2002) 850–858. J. Lim, P. Yusoff, E.S. Wong, S. Chandramouli, D.H. Lao, C.W. Fong, G.R. Guy, The cysteine-rich sprouty translocation domain targets mitogen-activated protein kinase inhibitory proteins to phosphatidylinositol 4,5-bisphosphate in plasma membranes, Mol. Cell. Biol. 22 (2002) 7953–7966. E.S. Wong, C.W. Fong, J. Lim, P. Yusoff, B.C. Low, W.Y. Langdon, G.R. Guy, Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signalling, EMBO J. 21 (2002) 4796–4808. M.A. Impagnatiello, S. Weitzer, G. Gannon, A. Compagni, M. Cotten, G. Christofori, Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells, J. Cell Biol. 152 (2001) 1087– 1098. A. Sasaki, T. Taketomi, T. Wakioka, R. Kato, A. Yoshimura, Identification of a dominant negative mutant of Sprouty that potentiates fibroblast growth factor- but not epidermal growth factor-induced ERK activation, J. Biol. Chem. 276 (2001) 36804– 36808. K. Yamada, K. Nishida, M. Hibi, T. Hirano, Y. Matsuda, Comparative FISH mapping of Gab1 and Gab2 genes in human, mouse and rat, Cytogenet. Cell Genet. 94 (2001) 39–42. T. Hanada, T. Yoshida, I. Kinjyo, S. Minoguchi, H. Yasukawa, S. Kato, H. Mimata, Y. Nomura, Y. Seki, M. Kubo, A. Yoshimura, A mutant form of JAB/SOCS1 augments the cytokine-induced JAK/STAT pathway by accelerating degradation of wild-type JAB/CIS family proteins through the SOCS-box, J. Biol. Chem. 276 (2001) 40746–40754. S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (1997) 3389–3402. T. Tsujimura, T. Furitsu, M. Morimoto, K. Isozaki, S. Nomura, Y. Matsuzawa, Y. Kitamura, Y. Kanakura, Ligand-independent activation of c-kit receptor tyrosine kinase in a murine mastocytoma cell line P-815 generated by a point mutation, Blood 83 (1994) 2619–2626. K. Yeung, T. Seitz, S. Li, P. Janosch, B. McFerran, C. Kaiser, F. Fee, K.D. Katsanakis, D.W. Rose, H. Mischak, J.M. Sedivy, W. Kolch, Suppression of Raf kinase activity and MAP kinase signalling by RKIP, Nature 401 (1999) 173–177.