Correspondence to Rüdiger Klein: rklein{at}neuro.mpg.de
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Introduction |
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The signaling events that are activated by Met have been well characterized and involve the concomitant activation of the Ras/MAPK and phosphatidyl-inositol 3 (PI3) kinase pathways, in addition to the recruitment and phosphorylation of the multiadaptor, GAB1 (Rosario and Birchmeier, 2003). It seems as if many of the downstream pathways are functionally redundant and extensively cross-wired. However, superimposed above a generic threshold signaling level, specific effectors seem to be required to achieve specific biologic functions (Maina et al., 2001).
Cell migration requires extensive remodeling of the cell cytoskeleton, which is mediated by members of the Rho family of small GTPases (for review see Labouesse, 2004). In their active GTP-bound state, they interact with several effector proteins. Many of these effectors share a common Cdc42-Rac interactive binding (CRIB) domain. CRIB domaincontaining effectors are structurally and functionally diverse and include protein kinases, actin binding proteins, and adaptor proteins, such as mitogen-inducible gene 6 (Mig6; see below next paragraph) (for review see Pirone et al., 2001).
Signaling by receptor tyrosine kinases requires a counterbalance by negative signaling events to ensure that appropriate thresholds of receptor signals are achieved and maintained for the right length of time. Irreversible inhibition is mediated most commonly by activation-dependent protein degradation through the ubiquitinproteasome pathway. Reversible inhibition can be achieved by protein tyrosine phosphatases, by dual-specificity MAPK phosphatases, and by phosphatase and tensin homologue proteins, which down-regulate the PI3 kinase pathway (Dikic and Giordano, 2003).
In a screen for HGF-induced changes in the transcriptome of cultured cells, we identified the Mig6 adaptor protein in a set of highly induced transcripts. Mig6 (also known as Gene 33 and receptor-associated late transducer) is considered an immediate early response gene that can be induced by a variety of external stimuli, including growth factors, cytokines, and stress factors (Wick et al., 1995; Makkinje et al., 2000; and references within). Overexpression and knock-down studies suggested that Mig6 was a selective inhibitor of EGF receptor family (also known as ErbB receptors)mediated mitogenesis and transformation (Fiorentino et al., 2000; Hackel et al., 2001; Fiorini et al., 2002; Anastasi et al., 2005; Xu et al., 2005). Its mechanism of action, receptor specificity, and influence on other cellular activities are poorly understood or unknown.
Here we show that Mig6 is a negative regulator of HGF/Met-induced cell migration. The effect was observed by Mig6 overexpression and was reversed by Mig6 small interfering RNA (siRNA) knock-down experiments, which indicates that endogenous Mig6 is part of a mechanism that inhibits Met signaling. The effect is observed in cells of hepatic origin as well as in primary neurons; this suggests that Mig6 functions across different cell lineages. Met lacks the sequence identified in EGF receptor as the Mig6 binding region (Hackel et al., 2001) and fails to bind Mig6 directly. Instead, Mig6 requires an intact CRIB domain to exert its inhibitory action, and suggests that Mig6 acts, at least in part, distal from the receptor, possibly by interacting with Rho family GTPases. Because Mig6 also is induced by HGF stimulation, our results suggest that Mig6 is part of a negative feedback loop that attenuates Met signaling in a variety of cellular functions.
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Results |
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Mig6 was chosen for further analysis because its expression was induced more strongly by HGF4.5- to 80-fold by 4 h of HGF stimulationthan by fibroblast growth factor 2 (FGF2) or PDGF (Fig. 1, A and B). Other transcripts did not show this preference for HGF (Fig. S1 B). The reduced response to FGF2 and PDGF was not due to lack of specific receptors or downstream transducers, because both growth factors induced robust phosphorylation of ERK/MAPKs in MLP29 cells (Fig. S2; available at http://www.jcb.org/cgi/content/full/jcb.200502013/DC1). Time courses of HGF stimulation revealed that Mig6 mRNA and protein were induced half maximally after 1 h and maintained for several hours (Fig. 1, C and D). The induction of Mig6 protein by EGF was more transient than that induced by HGF (Fig. 1 D). Induction of Mig6 protein by HGF in primary hepatocytes followed delayed kinetics (Fig. 1 E). We also confirmed the induction of endogenous Mig6 protein by immunostaining of MLP29 cells (Fig. 1, F and G). We next investigated the expression of Mig6 and Met in embryonic tissues. By in situ hybridization analysis, we found that both transcripts coexpressed in alveoli of embryonic (E13.5) lung, in liver parenchyma, and in intercostal and body wall muscles of wild-type embryos (Fig. 1, I, J, M, and N). Consistent with Met regulating mig6 transcript levels under physiologic conditions, we found reduced levels of mig6 mRNA in embryos that expressed a signaling-deficient Met receptor (metd/d) (Maina et al., 1996, 2001) (Fig. 1 K). Expression of Mig6 protein was confirmed in structures positive for mig6 mRNA, including intercostal and body wall muscles (Fig. S2).
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Mig6 is a physiologic suppressor of Met-mediated cell migration
We next investigated, using RNA interference, whether endogenous Mig6 suppressed HGF/Met-mediated cell migration by knocking down Mig6 protein levels (Elbashir et al., 2001). MLP29 cells were transfected with siRNAs specific for GFP or mig6, and the levels of Mig6 protein were analyzed by immunostaining and immunoblotting. Mig6 siRNA, but not control GFP siRNA, specifically knocked down endogenous Mig6 immunoreactivity 96 h after transfection (Fig. 3, A and B). Mig6 siRNA also suppressed HGF-stimulated induction of Mig6 (Fig. 3 C; compare 4-h time point in the presence and absence of mig6 siRNA). The reduction of protein levels was specific for mig6, because endogenous -tubulin and Met levels were unaffected (Fig. 3 C and not depicted). To investigate the role of Mig6 protein in cell migration, cells were transfected with mig6 siRNA oligonucleotides, and subjected to the Boyden chamber assay with different concentrations of HGF (Fig. 3). Representative images of Hoechst dyelabeled cells that migrated into the lower compartment are shown in Fig. 3 (DG). The induction of cell migration by HGF under these conditions was less strong, yet was still dose dependent (Fig. 3 H). Quantification of migrating cells revealed that under conditions of optimal HGF concentrations, knock down of Mig6 enhanced cell migration by approximately twofold (Fig. 3 H). Similar results were obtained with a separate set of siRNA oligonucleotides (unpublished data). These findings demonstrated that Mig6 is a physiologic inhibitor of HGF/Met-mediated cell migration of MLP29 cells.
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Next, we analyzed HGF-induced cell migration. Mig6FL inhibited cell migration twofold (Fig. 7 F). In contrast, Mig6 lacking the CRIB domain had no effect on HGF-induced cell migration (Fig. 7 F). The NH2-terminal fragment of Mig6 including the CRIB domain and, more importantly, the isolated CRIB domain inhibited HGF-induced cell migration to the same extent as did Mig6FL (Fig. 7 F). The effects were specific for HGF-stimulated cells, because cell migration that was induced by FBS was not affected by ectopic expression of Mig6 including the CRIB domain, but lacking the Ack homology domain, or CRIB (Fig. 7 E). If the mechanism of Mig6 action involved the binding and inhibition of Cdc42, the coexpression of dominant-active Cdc42 (Cdc42*) may rescue the antimigratory effect of Mig6. To test this, we cotransfected MLP29 cells with Mig6FL and GFP or with the same amounts of Mig6FL and Cdc42* expression plasmids, and performed cell migration assays. The combination of Mig6FL and GFP led to an efficient block of migration, whereas the coexpression of Mig6FL and Cdc42* completely rescued cell migration. Expression of Cdc42* alone had no significant effect in this assay (Fig. 7 H). These results suggest that the CRIB domain of Mig6 is necessary and sufficient for inhibition of HGF-induced cell migration. They further suggest that part of Mig6's mechanism of action involves the regulation of Rho GTPases, such as Cdc42.
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Discussion |
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Mig6 induction by external signals
We identified Mig6 in MLP29 cells as an RNA transcript that was induced highly by HGF and EGF and rather weakly by FGF2 and PDGF. Other investigators found that Mig6 was induced by serum, EGF and related ligands, and cellular stress factors (Wick et al., 1995; Makkinje et al., 2000; and references within). We have provided evidence that Met signaling is a major pathway for Mig6 expression in vivo by showing a reduction of mig6 transcript levels in embryonic liver and lung of mouse mutants expressing a severe signaling hypomorph of Met (Fig. 1). We conclude that in many circumstances, Mig6 is expressed at low levels, and its expression is induced by HGF/Met signaling to activate feedback inhibition with some delay after the initiation of Met signaling. Consequently, the cell responds robustly to Met signaling, until Mig6 levels are high enough to attenuate the Met response. Alternatively, other external signals may have induced the expression of Mig6 before the cells were exposed to HGF, thereby reducing the cell's ability to respond to Met signaling. Loss of the ability to induce the expression of Mig6 may be part of the multiple step process toward malignancy. Consistent with this model, a recent large-scale expression profiling study noted that Mig6 expression was down-regulated in patients who had breast cancer with short survival time (Amatschek et al., 2004). It seems as if loss of Mig6 provides a growth advantage and perhaps metastatic potential for breast cancer cells.
Mig6 modulates a variety of cellular responses
Previous work showed that Mig6 inhibits cell proliferation downstream of ErbB family receptors (Fiorentino et al., 2000; Hackel et al., 2001; Anastasi et al., 2003). This includes studies in which Mig6 was silenced by RNA interference, and provided first evidence for its role as an endogenous inhibitor of EGFR-mediated proliferation (Anastasi et al., 2005; Xu et al., 2005). Until now, the role of Mig6 in ErbB-mediated cell migration had not been addressed. However, this is an important question; numerous studies demonstrated that EGF and related ligands for EGFR/ErbB receptors stimulate chemotactic migration in vertebrate and invertebrate systems (Wells and Lillien, 2004). Our study has concentrated on the role of Mig6 in Met-mediated cell migration, a process that is implicated in several physiologic contexts, including myoblast migration during development, scattering and branching morphogenesis of epithelial cells, and neuronal migration in the developing forebrain (Powell et al., 2001; Birchmeier et al., 2003; Rosario and Birchmeier, 2003). Met signaling also is critical for neurite extension and branching of different neuronal subpopulations (Maina and Klein, 1999; Thompson et al., 2004), a process that has similarities with invasive growth of malignant cells (Trusolino and Comoglio, 2002). We found that Mig6 overexpression effectively and specifically reduced HGF-induced migration of a cell line of hepatic origin and of primary cortical neurons. Mig6 also effectively blocked HGF-induced neurite growth of primary sympathetic neurons. In converse experiments, mig6 knock-down effectively enhanced cell migration of hepatic progenitor cells and mildly, yet significantly, enhanced neurite growth of sympathetic neurons.
Mig6 acts distally from Met by way of interaction with Rho family GTPases
Mig6 is a multiadaptor molecule whose amino-terminal 38 amino acid residues show homologies with the conserved CRIB domain that is present in a variety of intracellular signaling molecules (Pirone et al., 2001). Mig6 contains putative binding sites for SH3-containing molecules such as Grb2, PI3K, and PLC (Fiorentino et al., 2000), and for 143-3 and PDZ-domain containing proteins. Notably, Mig6 includes a COOH-terminal region that is highly homologous to the noncatalytic portion of Ack1. A region within this Ack1 homology domain was identified as an EGFR-binding motif (Fiorentino et al., 2000; Anastasi et al., 2003) that was necessary and sufficient for the inhibition of EGFR signaling (Xu et al., 2005). Expression of Mig6/receptor-associated late transducer in tumor cells modestly reduced the levels of ErbB-mediated phospho-MAPK and phospho-Akt expression, which suggested some interference with mitogenic signaling pathways (Anastasi et al., 2005). Conversely, loss of Mig6 led to sustained MAPK phosphorylation in EGF-stimulated keratinocytes (Ballaro et al., 2005 and unpublished data). The association between Mig6 and Met by way of Grb2 interaction (Fig. 7) suggests the possibility that Mig6 inhibits Met signaling in a receptor-proximal fashion. However, we have been unable to detect changes in the levels of Met-mediated phospho-MAPK and phospho-Akt expression in cells that transiently overexpress Mig6 (Fig. S2). Therefore, we favor the view that the antimigratory effect of Mig6 in MLP29 cells involves other pathways.
Because the CRIB domain of Mig6 binds Cdc42 in a GTP-dependent manner (Makkinje et al., 2000 and this report), HGF stimulation activates Cdc42, and activated Cdc42 is required for HGF-induced lamellipodia formation and cell movement (Royal et al., 2000), we favor the possibility that Mig6 inhibits Cdc42-mediated cell movement by way of its CRIB domain. In support of this hypothesis, we show that (i) overexpression of a Mig6 construct lacking the CRIB domain, but retaining Grb2-binding capabilities, is unable to suppress HGF-induced migration; (ii) overexpression of the Mig6 CRIB domain alone is sufficient to inhibit HGF-induced migration; and (iii) coexpression of Mig6 with a dominant-active form of Cdc42 rescues the antimigratory effects of Mig6. Therefore, the mechanism of Mig6 inhibition of Met resembles the mechanism of Ack1 inhibition of EGFR. The Caenorhabditis elegans orthologue of Ack1, Ark, associates with EGFR by way of binding to Sem5, the C. elegans orthologue of Grb2 (for review see Worby and Margolis, 2000). Similar to Mig6, overexpression of the CRIB domain of Ack1 is sufficient to inhibit growth factorinduced activation of Cdc42 (Nur-E-Kamal et al., 1999). Furthermore, overexpression of the CRIB domain of another small GTPase-binding protein, PAK (an effector of Rac1), by way of its interaction with Rac1, inhibits Semaphorin 3A-induced growth cone collapse (Vastrik et al., 1999). Together, these results indicate that CRIB domains are sufficient to inhibit the biologic effects of specific GTPases.
We propose that Mig6 is part of a network of negative signaling molecules that fine tune and attenuate Met and ErbB signaling in development and disease. The analysis of mig6 mutant mice provided genetic evidence for a role of Mig6 in the maintenance of joints and cartilage (Zhang et al., 2005) and in skin morphogenesis and cancer (unpublished data). They will be an invaluable tool in our efforts to elucidate the inhibitory functions and molecular interactions of Mig6 in the context of an intact tissue.
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Materials and methods |
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Northern blots
Total RNA was extracted from different cell lines in various experimental conditions using the RNAClean Solution (Hybaid). 20 µg total RNA was electroporated and blotted onto Genescreen nylon membrane (NEN Life Science Products). Labeled probes were generated using random primers and hybridized (6x SSC, 5x Denhardt solution, and 100 µg salmon sperm DNA) with the membrane for 18 h at 65°C. cDNA inserts used as probes were obtained by NotI/SalI double restriction digestion of the NIA mouse 15k cDNA clones.
RNA interference
siRNA oligonucleotides (first set [AAGGUCAAGCUUGCCCCCUC-dTdT] and second set [GAGGAUCAAGUUAUGUGUGG-dTdT]) were designed and used for cell transfections as described (Elbashir et al., 2001). Sense and anti-sense siRNA oligonucleotides (DARMAKOM) were diluted in annealing buffer (100 mM K-Acetate, 30 mM Hepes-KOH, 2 mM Mg-Acetate) to the final concentration of 20 µM, denatured for 1 min at 90°C, and annealed by incubation for 1 h at 37°C. 6 µl siRNA duplex was transfected into 4 x 104 cells using oligofectamine (Invitrogen), according to the manufacturer's instructions. After transfection, cells were left in Dulbecco's minimum essential medium (DMEM) plus 0.1% FBS for 72 h and transfected a second time with the same siRNA duplex. The aphidicolin treatment was performed as described above. 24 h after the second transfection, cells were harvested and plated on the upper face of the Boyden chamber as described above.
Plasmids and expression vectors
Primer sequences are available upon request. The V5 COOH-terminal, GST NH2-terminal, and His COOH-terminal tagged Mig6 full-length and mutant proteins were generated using the Gateway Cloning technology according to the manufacturer's instructions (Invitrogen). In brief, Mig6 full-length and deletion mutants were amplified by PCR from the IRAK clone IRAKp961F0910 using oligonucleotides that contained the minimal recombination sequences (5'-ATTB1 and 3'-ATTB2). PCR amplified products were recombined into pDONOR201 vector by the use of a mixture of recombination proteins that was provided by the manufacturer. The pDONOR201 vectors containing Mig6 full-length or deletion mutants were shuttled into pCDNA 6.2 COOH-terminal-V5, pDEST 27 NH2-terminal-GST, and pDEST26 NH2-terminal-His plasmids (all Invitrogen) for mammalian expression or into pDest15 N-term-GST for bacterial expression. The LacZ-V5 expression plasmid was purchased (Invitrogen). GFP-Cdc42WT and dominant active expression plasmids were provided by M. Way. The CRIB domain of Ack was amplified by PCR from the I.M.A.G.E. Consortium cDNA Clones (Clone ID IMAGp998I1013902Q3) (Lennon et al., 1996) and was subcloned into pCRII-TOPO cloning vector (Invitrogen) following the manufacturer's instructions. The fragment was inserted into the pFAT2 vector for bacterial expression. The pGEX-NH2-terminal-GST-Mig6, containing the Mig6 COOH-terminal half (from aa 273 to 459), was provided by A. Ullrich and was used to produce the Mig6 antigen.
Primary cortical neuron culture, cell migration assays, and growth factors
MLP29 cells were cultured and transiently transfected as described (Muller et al., 2002). C2C12 were grown as described (Yaffe and Saxel, 1977). Primary hepatocytes were cultured as described (Maina et al., 2001). The transwell assay was performed as described previously (de Luca et al., 1999). In brief, 105 MLP29 cells were seeded on the upper face of the Boyden chamber membrane (8 µm pore; Costar), which was coated previously with 0.15 µg/cm2 of fibronectin (Sigma-Aldrich). The cells were stimulated with 40 ng/ml of hepatocyte growth factor (HGF) (R&D Systems), 10 ng/ml EGF, 30 ng/ml PDGF (Sigma-Aldrich), 25 ng/ml fibroblast growth factor 2 (FGF2; Sigma-Aldrich), and 100 ng/ml SDF-1 (Calbiochem). To assay cell migration in the absence of cell proliferation, cells were treated for 24 h before and during the migration assay with DMSO- (Fluka) or 1.6 µg/ml aphidicolin-containing media (Sigma-Aldrich).
Cortical neurons were obtained by digestion of E15.5 mouse telencephalon for 15 min at 37°C with trypsin-EDTA (GIBCO BRL). The neurons were washed twice with DMEM-F12 supplemented with 10% horse serum (GIBCO BRL), washed once in neurobasal medium containing B27 supplement (NB/B27, 50:1, GIBCO BRL) and were dissociated with a fire-polished glass Pasteur pipette. Cortical neurons were dissected and electroporated by mixing 6 x 105 cells with 24 µg expression plasmid, transferred into an electroporation cuvette (MBP Molecular Bioproducts), and electroporated (five pulses at 270 V of 3 msec separated by 1-s interval) using the Electrosquare porator (ECM830, BTX). The cells were incubated for 10 min at 4°C. 105 cells were seeded on the upper side of the Boyden chamber (5-µm pore, Costar), which was coated previously with poly-D-ornithin (Sigma-Aldrich). 3 h later, the appropriate growth factors were added to the lower compartment for 18 h. The cells were fixed with 4% PFA (Merck) for 30 min at room temperature and incubated for 10 min at 4°C. Boyden chamber filters were mounted onto glass coverslips in the presence of vectashield/DAPI (Vector Laboratories). The cells were analyzed directly under a Zeiss Axiophot fluorescent microscope.
In situ hybridization and biochemistry
In situ hybridizations and preparations of probes were performed as described (Helmbacher et al., 2003). Mig6 sense and anti-sense probes were obtained by NotI and SalI restriction digests of the NIA clone h3011f08. The met in situ probe was described previously (Helmbacher et al., 2003). Immunoblotting and immunocytochemistry were performed as described (Maina et al., 2001). Recombinant GST-Mig6 fusion proteins were purified according to standard procedures. MLP29 cell lysates were incubated for 1 h at 4°C with recombinant proteins that were immobilized on glutathione-sepharose. The glutathione-sepharose beads were washed several times in cell lysis buffer, eluted with sample buffer, and analyzed by immunoblotting.
Antibodies
The rabbit anti-Mig6 antibody was generated as described previously (Hackel et al., 2001). The anti-phospho-MAPK, anti-MAPK, and anti-phospho-Akt (New England Biolabs, Inc.) antibodies were used as described previously (Maina et al., 2001). Antibody dilutions: monoclonal anti-tubulin (Sigma-Aldrich) 1:2,000 for Western blot (WB) analysis; monoclonal anti-V5 (Invitrogen) 1:1,000 and 1:250 for WB and immunocytochemical (IC) analysis, respectively; rabbit polyclonal anti-Met (BIOMOL Research Laboratories) and anti-GST (Santa Cruz Biotechnology, Inc.) 1:500 for WB analysis; antimicrotubule-associated protein 2 (Sigma-Aldrich) 1:10,000 for IC analysis; monoclonal anti-Grb2 (Transduction Labs) 1:1,000 for WB analysis; donkey polyclonal antimouse Alexa488 (Molecular Probes) and antirabbit CY3 (Jackson ImmunoResearch Laboratories) 1:200 for IC analysis; and polyclonal goat antimouse and antirabbit HRP-conjugated (GE Healthcare) 1:2,000 for WB analysis.
Imaging
All cell images were obtained with an Axioplan-2 imaging fluorescent microscope (Carl Zeiss MicroImaging, Inc.) equipped with a RT Slider 2.3.1 digital color camera (Diagnostic Instruments). A 40x objective was used, except for the in situ hybridization images that were taken with a 20x lens (Carl Zeiss MicroImaging, Inc.).
Yeast two-hybrid system
The NH2-terminal half of Mig6 and the CRIB domain of PAK and Ack were inserted by way of BamHI/XhoI into the yeast two-hybrid prey vector pAct2. Cdc42WT and RacWT were inserted by way of BamHI/SalI into the pGBT9 bait vector. These plasmids were transformed in the indicated combination into the reporter strain PJ69-4A according to the CLONTECH Laboratories yeast protocol handbook. The double transformants were selected by plating onto synthetic media lacking leucine and tryptophane with 2% glucose as the carbon source (Leu/Trp). Double transformants were restreaked onto Leu/Trp or synthetic media lacking leucine, tryptophan, histidine, and adenine (quadruple drop out).
Recombinant protein purification and Cdc42 activation assay
Recombinant GST fusion proteins were purified according to standard procedures. MLP29 cell lysates were incubated for 1 h at 4°C with recombinant proteins immobilized on glutathione-sepharose. The glutathione-sepharose beads were washed several times in cell lysis buffer, eluted with sample buffer, and analyzed by immunoblotting. For the Cdc42 activation assay, MLP29 cells in various experimental conditions were lysed (50 mM Tris-Cl pH 7.4, 5 mM MgCl2, 200 mM NaCl, 1 mM sodium orthovanadate, 1% NP-40, 10% glycerol, and a mixture of protease inhibitors EDTA-free), and the lysates were incubated with the recombinant GST-Ack-CRIB protein bound to glutathione-sepharose beads for 1.5 h at 4°C. The proteins were eluted from the beads, and analyzed by SDS-PAGE and immunoblotting.
Paravertebral sympathetic neuron cultures
Superior cervical ganglia were dissected from postnatal day 40 (P40) CD1 mice. Neuron cultures were set up as described (Thompson et al., 2004). HGF stimulation and quantification of neuronal survival was done as described (Thompson et al., 2004). Sholl analysis was performed as described previously (Sholl, 1953). For protein extraction, primary cultures of dissociated P40 superior cervical ganglion neurons were grown for 36 h in culture before being stimulated for different times with HGF. Cells were harvested and homogenized as described above.
Online supplemental material
Fig. S1 shows Northern blot analysis of selected genes from the gene list. Fig. S2 shows the effects of Mig6 on canonical Met signaling, yeast two-hybrid interactions between Mig6 and Cdc42/Rac, and immunohistochemical analysis of overexpressed and endogenous Mig6. Table S1 and Table S2 list the genes that are regulated by HGF in MLP29 cells. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200502013/DC1.
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Acknowledgments |
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This work was supported by the Max-Planck Society and by additional grants from the Wellcome Trust (to A.M. Davies) and Boehringer Ingelheim (to R. Klein).
Submitted: 2 February 2005
Accepted: 19 September 2005
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