Erbin Inhibits RAF Activation by Disrupting the Sur-8-Ras-Raf Complex
2005; Elsevier BV; Volume: 281; Issue: 2 Linguagem: Inglês
10.1074/jbc.m507360200
ISSN1083-351X
AutoresPenggao Dai, Wen‐Cheng Xiong, Lin Mei,
Tópico(s)Tendon Structure and Treatment
ResumoErbin is a member of the LAP (leucine-rich repeat (LRR) and PDZ domain) family. It inhibits Ras-mediated activation of ERK in response to growth factors. In this study, we investigated the mechanisms by which Erbin regulates the Ras-Raf-MEK pathway. The N-terminal LRR domain was necessary and sufficient to inhibit neuregulin-activated expression of ϵ416-Luc, a reporter of ERK activation. On the other hand, Erbin had no effect on Ras activation, but it attenuated neuregulin-induced Raf activation, suggesting that Erbin may regulate Raf activation by Ras. Via the LRR domain, Erbin interacts with Sur-8, a scaffold protein necessary for the Ras-Raf complex. Expression of Erbin attenuated the interaction of Sur-8 with active Ras and Raf. Moreover, Erbin-shRNA, which suppressed Erbin expression at mRNA and protein levels, increased the interaction of Sur-8 with Ras and Raf, ERK activation, and neuregulin-induced expression of endogenous acetylcholine receptor ϵ-subunit mRNA. These results demonstrate a regulatory role of Erbin in the Ras-Raf-MEK pathway, suggesting that Erbin may inhibit ERK activation by disrupting the Sur-8-Ras/Raf interaction. Erbin is a member of the LAP (leucine-rich repeat (LRR) and PDZ domain) family. It inhibits Ras-mediated activation of ERK in response to growth factors. In this study, we investigated the mechanisms by which Erbin regulates the Ras-Raf-MEK pathway. The N-terminal LRR domain was necessary and sufficient to inhibit neuregulin-activated expression of ϵ416-Luc, a reporter of ERK activation. On the other hand, Erbin had no effect on Ras activation, but it attenuated neuregulin-induced Raf activation, suggesting that Erbin may regulate Raf activation by Ras. Via the LRR domain, Erbin interacts with Sur-8, a scaffold protein necessary for the Ras-Raf complex. Expression of Erbin attenuated the interaction of Sur-8 with active Ras and Raf. Moreover, Erbin-shRNA, which suppressed Erbin expression at mRNA and protein levels, increased the interaction of Sur-8 with Ras and Raf, ERK activation, and neuregulin-induced expression of endogenous acetylcholine receptor ϵ-subunit mRNA. These results demonstrate a regulatory role of Erbin in the Ras-Raf-MEK pathway, suggesting that Erbin may inhibit ERK activation by disrupting the Sur-8-Ras/Raf interaction. Neuregulin is a family of epidermal growth factor-containing polypeptides implicated in regulating neuron migration, neurite outgrowth, and expression of neurotransmitter receptors (1Lemke G. Mol. Cell. Neurosci. 1996; 7: 247-262Crossref PubMed Scopus (218) Google Scholar, 2Falls D.L. Exp. Cell Res. 2003; 284: 14-30Crossref PubMed Scopus (838) Google Scholar). Neuregulins act by activating their receptors, the 180-kDa ErbB receptor tyrosine kinases ErbB2, ErbB3, and ErbB4 (3Kirschbaum M.H. Yarden Y. J. Cell. Biochem. 2000; : 52-60Crossref Scopus (39) Google Scholar, 4Schaeffer L. de Kerchove d'Exaerde A. Changeux J.P. Neuron. 2001; 31: 15-22Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Upon activation, tyrosine residues in the carboxyl termini become phosphorylated and serve as docking sites for cytoplasmic signaling molecules such as Shc and Grb2 to activate various downstream signaling pathways. Grb2 brings guanyl nucleotide exchange factor (SOS) to the plasma membrane in proximity with Ras and expedites the exchange of GDP for GTP on Ras (5Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar). Activated Ras (GTP-bound) then directly binds to Raf and allows the latter to be activated (6Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2274) Google Scholar, 7Katz M.E. McCormick F. Curr. Opin. Genet. Dev. 1997; 7: 75-79Crossref PubMed Scopus (274) Google Scholar). Active Raf triggers sequential activation of MEK, 2The abbreviations used are: MEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseLRRleucine-rich repeatAChRacetylcholine receptorshRNAsmall hairpin RNAGSTglutathione S-transferaseRBDRas-binding domainLucluciferase. 2The abbreviations used are: MEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseLRRleucine-rich repeatAChRacetylcholine receptorshRNAsmall hairpin RNAGSTglutathione S-transferaseRBDRas-binding domainLucluciferase. a MAPK kinase, and ERK (8Kerkhoff E. Houben R. Loffler S. Troppmair J. Lee J.E. Rapp U.R. Oncogene. 1998; 16: 211-216Crossref PubMed Scopus (114) Google Scholar).Erbin is a intracellular protein of the LAP family (LRR and PDZ), containing a LRR domain and a PDZ domain (9Kolch W. Sci. STKE. 2003; 2003: pe37PubMed Google Scholar, 10Borg J.P. Marchetto S. Le Bivic A. Ollendorff V. Jaulin-Bastard F. Saito H. Fournier E. Adelaide J. Margolis B. Birnbaum D. Nat. Cell Biol. 2000; 2: 407-414Crossref PubMed Scopus (254) Google Scholar, 11Huang Y.Z. Wang Q. Xiong W.C. Mei L. J. Biol. Chem. 2001; 276: 19318-19326Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Besides Erbin, LAP proteins include Densin-180 (12Apperson M.L. Moon I.S. Kennedy M.B. J. Neurosci. 1996; 16: 6839-6852Crossref PubMed Google Scholar), Lano (13Saito H. Santoni M.J. Arsanto J.P. Jaulin-Bastard F. Le Bivic A. Marchetto S. Audebert S. Isnardon D. Adelaide J. Birnbaum D. Borg J.P. J. Biol. Chem. 2001; 276: 32051-32055Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), LET-413, an Erbin orthologue in Caenorhabditis elegans (14Legouis R. Gansmuller A. Sookhareea S. Bosher J.M. Baillie D.L. Labouesse M. Nat. Cell Biol. 2000; 2: 415-422Crossref PubMed Scopus (149) Google Scholar), and Scribble in Drosophila (15Bilder D. Perrimon N. Nature. 2000; 403: 676-680Crossref PubMed Scopus (547) Google Scholar). Densin-180 is a protein associated with the postsynaptic density in the rat brain, which may be involved in the localization of synaptic proteins (11Huang Y.Z. Wang Q. Xiong W.C. Mei L. J. Biol. Chem. 2001; 276: 19318-19326Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 16Kennedy M.B. Trends Neurosci. 1997; 20: 264-268Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). LET-413 and Scribble are junctional proteins involved in establishing and maintaining epithelial cell polarity (17Navarro C. Nola S. Audebert S. Santoni M.J. Arsanto J.P. Ginestier C. Marchetto S. Jacquemier J. Isnardon D. Le Bivic A. Birnbaum D. Borg J.P. Oncogene. 2005; 24: 4330-4339Crossref PubMed Scopus (169) Google Scholar). Erbin is believed to play a role in basolateral targeting in epithelial cells (18Dillon C. Creer A. Kerr K. Kumin A. Dickson C. Mol. Cell. Biol. 2002; 22: 6553-6563Crossref PubMed Scopus (33) Google Scholar, 19Legouis R. Jaulin-Bastard F. Schott S. Navarro C. Borg J.P. Labouesse M. EMBO Rep. 2003; 4: 1096-1102Crossref PubMed Scopus (90) Google Scholar). It is shown to interact with various proteins via the PDZ domain including ErbB2 (9Kolch W. Sci. STKE. 2003; 2003: pe37PubMed Google Scholar, 10Borg J.P. Marchetto S. Le Bivic A. Ollendorff V. Jaulin-Bastard F. Saito H. Fournier E. Adelaide J. Margolis B. Birnbaum D. Nat. Cell Biol. 2000; 2: 407-414Crossref PubMed Scopus (254) Google Scholar, 18Dillon C. Creer A. Kerr K. Kumin A. Dickson C. Mol. Cell. Biol. 2002; 22: 6553-6563Crossref PubMed Scopus (33) Google Scholar), p120 catenin (p0071 and δ-catenin) (20Izawa I. Nishizawa M. Tomono Y. Ohtakara K. Takahashi T. Inagaki M. Genes Cells. 2002; 7: 475-485Crossref PubMed Scopus (59) Google Scholar), ARVCF (21Jaulin-Bastard F. Arsanto J.P. Le Bivic A. Navarro C. Vely F. Saito H. Marchetto S. Hatzfeld M. Santoni M.J. Birnbaum D. Borg J.P. J. Biol. Chem. 2002; 277: 2869-2875Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 22Laura R.P. Witt A.S. Held H.A. Gerstner R. Deshayes K. Koehler M.F. Kosik K.S. Sidhu S.S. Lasky L.A. J. Biol. Chem. 2002; 277: 12906-12914Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), EBP0 (23Rangwala R. Banine F. Borg J.P. Sherman L.S. J. Biol. Chem. 2005; 280: 11790-11797Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), PAPIN (24Ohno H. Hirabayashi S. Iizuka T. Ohnishi H. Fujita T. Hata Y. Oncogene. 2002; 21: 7042-7049Crossref PubMed Scopus (38) Google Scholar), β4-integrin (25Favre B. Fontao L. Koster J. Shafaatian R. Jaunin F. Saurat J.H. Sonnenberg A. Borradori L. J. Biol. Chem. 2001; 276: 32427-32436Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and bullous pemphigoid antigen-1 (25Favre B. Fontao L. Koster J. Shafaatian R. Jaunin F. Saurat J.H. Sonnenberg A. Borradori L. J. Biol. Chem. 2001; 276: 32427-32436Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The unique region between the LRR and PDZ domains associates with PSD-95 (11Huang Y.Z. Wang Q. Xiong W.C. Mei L. J. Biol. Chem. 2001; 276: 19318-19326Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and Smad (26Warner D.R. Roberts E.A. Greene R.M. Pisano M.M. Biochem. Biophys. Res. Commun. 2003; 312: 1185-1190Crossref PubMed Scopus (27) Google Scholar). The LRR domain binds to active Ras (27Huang Y.Z. Zang M. Xiong W.C. Luo Z. Mei L. J. Biol. Chem. 2003; 278: 1108-1114Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). We showed that Erbin inhibits growth factor-induced activation of ERK (27Huang Y.Z. Zang M. Xiong W.C. Luo Z. Mei L. J. Biol. Chem. 2003; 278: 1108-1114Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Suppression of Erbin expression potentiates neuregulin-induced neuronal differentiation of PC12 cells and disrupts E-cadherin adherence junctions in Schwann cells (23Rangwala R. Banine F. Borg J.P. Sherman L.S. J. Biol. Chem. 2005; 280: 11790-11797Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). These results indicate that this protein may function as a signaling molecule in addition to being a scaffolding protein.In this study, we investigated mechanisms of Erbin in inhibiting ERK activation. Erbin bound to Sur-8 and inhibited the interaction of Sur-8 with Ras and Raf. Suppression of Erbin expression in cells increased the interaction of Sur-8 with active Ras and Raf, ERK activation, and neuregulin-induced expression of endogenous AChR ϵ-subunit mRNA. Taken together, these results demonstrate a regulatory role of Erbin in the Ras-Raf-MEK pathway, suggesting that Erbin may inhibit ERK activation by disrupting the Sur-8-Ras-Raf complex.EXPERIMENTAL PROCEDURESCell Culture and Transfection—HEK 293, COS-7, and C2C12 cells were cultured as described previously (27Huang Y.Z. Zang M. Xiong W.C. Luo Z. Mei L. J. Biol. Chem. 2003; 278: 1108-1114Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). COS-7 cells were using an adenovirus-aided DEAE-dextran transfection procedure (28Forsayeth J.R. Garcia P.D. BioTechniques. 1994; 17: 354-358PubMed Google Scholar, 29Wang Z.Z. Mathias A. Gautam M. Hall Z.W. J. Neurosci. 1999; 19: 1998-2007Crossref PubMed Google Scholar). Cells in 10-cm dishes (70–90% confluence) were incubated with 5.0 ml of Dulbecco's modified Eagle's medium, 1.0 ml of E1-defective adenovirus lysates, 0.48 mg of DEAE-dextran, and 5 μg of plasmid DNA for 2 h at 37 °C. The cells were rinsed with phosphate-buffered saline and incubated with Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.Immunoprecipitation and Immunoblotting—Cell lysates (∼400 μg of protein) were incubated without or with indicated antibodies 1 h at 4 °C and subsequently with protein A- or protein G-agarose beads overnight at 4 °C on a rotating platform. After centrifugation, beads were washed five times with the modified radioimmune precipitation assay buffer. Bound proteins were eluted with the SDS sample buffer, resolved by SDS-PAGE, and transferred onto nitrocellulose membranes (Schleicher and Schuell). Nitrocellulose membranes were incubated at room temperature for 1 h in blocking buffer containing Tris-buffered saline with 0.1% Tween (TBS-T) and 5% milk or 5% bovine serum albumin, followed by incubation with indicated antibodies in the blocking buffer. After being washed three times for 5 min each with TBS-T, the membrane was incubated with horseradish peroxidase-conjugated donkey anti-mouse or anti-rabbit IgG (Amersham Biosciences) followed by washing. Immunoreactive bands were visualized with enhanced chemiluminescence substrate (Pierce). In some experiments, the nitrocellulose filter was incubated in a buffer containing 62.5 mm Tris-HCl, pH 6.7, 100 mm β-mercaptoethanol, and 2% SDS at 50 °C for 30 min and then washed with 0.1% Tween 20 in 50 mm Tris-buffered saline at room temperature for 1 h and reblotted with different antibodies. The following antibodies were used: FLAG (M2, Sigma), Myc (9E10, Santa Cruz Biotechnology), phospho-Raf-1 (Ser-338, Upstate), phospho-MAPK (Promega), anti-Ras (clone RAS10, Upstate), GST (Biocompare), and Erbin (28Forsayeth J.R. Garcia P.D. BioTechniques. 1994; 17: 354-358PubMed Google Scholar). For quantitative analysis, autoradiographic films were scanned with Epson Expression 1680, and the captured image was analyzed with NIH Image software.Luciferase Assay—C2C12 myoblasts were cotransfected with or without Myc-Erbin, plus the ϵ-subunit promoter-luciferase transgene that contains 416 nucleotides of the 5′-untranslated region of the ϵ-subunit gene (30Si J. Luo Z. Mei L. J. Biol. Chem. 1996; 271: 19752-19759Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). 24 h after transfection, myoblasts were incubated in the differentiation medium to induce myotube formation. Myotube formation was completed 48 h after the switch to differentiation medium. C2C12 myotubes were stimulated with neuregulin at a final concentration of 10 nm at 37 °C for 24 h. pRL-SV40, which expresses Renilla luciferase under the control of the SV40 promoter (Promega), was cotransfected as a control to monitor the transfection efficiency. 48 h after transfection, cells were lysed, and the activities of the two different luciferases were assayed with their respective substrates with a dual luciferase assay kit (Promega).Ras Assay—Active GTP-bound Ras was precipitated by the minimal Ras-binding domain (RBD) (amino acids 51–131) of Raf1 (31de Rooij J. Bos J.L. Oncogene. 1997; 14: 623-625Crossref PubMed Scopus (420) Google Scholar, 32Tanowitz M. Si J. Yu D.H. Feng G.S. Mei L. J. Neurosci. 1999; 19: 9426-9435Crossref PubMed Google Scholar). Briefly, serum-starved HEK 293 cells were treated with neuregulin (10 nm) for 10 min and lysed in Ras-binding buffer (15% glycerol, 50 mm Tris/HCl, pH 7.4, 1% Nonidet P-40, 200 mm NaCl, 10 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 1 μg/ml leupeptin, and 2 μg/ml aprotinin). Cleared lysates were incubated for 1 h with recombinant GST-RBD (15 μg/sample) that had been precoupled to glutathione-agarose beads. After four washes in the Ras-binding buffer, bead complexes were separated on a 12% SDS-PAGE and transferred to nitrocellulose membrane for Western blotting. Ras was detected using the rat monoclonal antibody Y13-259 followed by horseradish peroxidase-coupled goat anti-rat antiserum (Santa Cruz Biotechnology).Reverse Transcription-PCR—Total RNA was extracted by TRIzol (Invitrogen) and first-strand cDNA prepared using the SuperScript III First-strand Synthesis Kit (Invitrogen) according to the manufacturer's instruction. PCR parameters were 25 cycles (94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s) followed by a final extension cycle at 72 °C for 7 min. PCR product was resolved on 6% acrylamide gels and visualized by ethidium bromide. Primers were as follows: 5′-TGA TGC TGA AAG TGG CCC ACC AGC C-3′ and 5′-TGA AGA AAC TTC TCG TAC AAT GAT G-3′ for Erbin; 5′-CCA TGT CCC CGC GGC TGC GC-3′ and 5′-GAG CCC ACG CTG AAG AGC AC-3′ for the AChR ϵ-subunit; and 5′-GCT CGT CGTCGA CAA CGG CTC-3′ and 5′-CAAACATGATCT GGG TCA TCT TCT C-3′ for β-actin.Inhibition of Erbin Expression by Small Hairpin RNA (shRNA)—shRNA virus was generated by the BLOCK-iT Lentiviral RNAi Expression System (Invitrogen) according to the manufacturer's instruction. Briefly, analysis of the Erbin sequence by a program provided by Invitrogen, three sequences were picked and cloned into the pENTR/U6 entry construct to yield pENTR/U6-Erbin. They were transfected in HEK 293 cells for their ability to suppress endogenous Erbin expression assayed by Western blot. Two clones, 2583 and 4049, were found effective in suppressing Erbin expression and were used for viral production. 2583 encodes 5′-CAC CGC TGA TGA CAC TCA CAA ATT ACG AAT AAT TTG TGA GTG TCA TCA GC-3′, whereas 4049 encodes 5′-CAC CGC ATC CCT CTA GAG AAC AAC TCG AAA GTT GTT CTC TAG AGG GAT GC-3′. pLenti6-Erbin was generated by left-right recombination between the pENTR/U6 entry construct and pLenti6/BLOCK-iT-DEST. 293FT producer cells were cotransfected with pLenti6-Erbin and a packaging mix to produce lentivirus.RESULTS AND DISCUSSIONErbin Inhibits Activation of Raf but Not Ras—In an earlier report, we showed that Erbin inhibits ERK activation (27Huang Y.Z. Zang M. Xiong W.C. Luo Z. Mei L. J. Biol. Chem. 2003; 278: 1108-1114Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The site of action was mapped to be between Raf and Ras using phospho-ERK as the readout, because Erbin inhibits ERK activation by active Ras but not by active Raf. Raf is a serine-threonine kinase that phosphorylates and activates the dual specificity kinase, MEK (MAPKK) (33Luo Z. Tzivion G. Belshaw P.J. Vavvas D. Marshall M. Avruch J. Nature. 1996; 383: 181-185Crossref PubMed Scopus (201) Google Scholar) which, in turn, phosphorylates and activates ERK. Although these observations suggest that Erbin may inhibit Raf activation, it is possible that Ras activation may also be a target of regulation. To address these questions, we studied the effects of Erbin on Ras and Raf activation in HEK 293 cells. Tagged Ras and Raf were cotransfected with Erbin or LRR. After stimulation with neuregulin, active Ras was purified by the Ras-binding domain of Raf immobilized on beads. The RBD binds to active Ras with an affinity 3 orders of magnitude higher than inactive GDP-bound Ras and has been used widely to purify active Ras (31de Rooij J. Bos J.L. Oncogene. 1997; 14: 623-625Crossref PubMed Scopus (420) Google Scholar, 32Tanowitz M. Si J. Yu D.H. Feng G.S. Mei L. J. Neurosci. 1999; 19: 9426-9435Crossref PubMed Google Scholar, 34Herrmann C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Purified active Ras was revealed by immunoblotting with anti-Ras antibody. As shown in Fig. 1, A and B, active Ras was increased by neuregulin. However, the increase was not altered by Erbin or LRR, suggesting that Erbin may have little effect on Ras activation.In light of the inability of Erbin to inhibit Ras activation and active Raf-mediated ERK activation, we determined whether Erbin inhibits Raf activation. FLAG-Raf1 was cotransfected with Myc-Erbin or LRR in HEK 293 cells. FLAG-Raf1 was immunoprecipitated with anti-FLAG antibody and blotted by anti-phospho-Raf antibody. Neuregulin elicited Raf1 activation in control HEK 293 cells (4 ± 0.2-fold above basal, mean ± S.E., n = 3) (Fig. 1, C and D). In cells co-expressing Erbin and LRR, Raf1 activation was inhibited by 40 and 45% (2.4 ± 0.2 and 2.2 ± 0.2, respectively; mean ± S.E., n = 3). These observations indicate that Erbin inhibits Raf activation but has no apparent effect on Ras activation.The LRR Domain Is Necessary and Sufficient to Inhibit ϵ416-Luc Expression—Erbin has three domains: the N-terminal LRR domain, an LAP-specific domain that is C-terminal to the LRR, and the C-terminal PDZ domain (13Saito H. Santoni M.J. Arsanto J.P. Jaulin-Bastard F. Le Bivic A. Marchetto S. Audebert S. Isnardon D. Adelaide J. Birnbaum D. Borg J.P. J. Biol. Chem. 2001; 276: 32051-32055Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The LRR domain has been shown to be sufficient to inhibit ERK activation (27Huang Y.Z. Zang M. Xiong W.C. Luo Z. Mei L. J. Biol. Chem. 2003; 278: 1108-1114Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). To exclude the possible involvement of other domains, it was important to determine whether the LRR domain is required for the inhibitory effect. To this end, we characterized the effects of ΔLRR, a deletion mutant without the LRR domain, on neuregulin-induced expression of ϵ416-Luc (Fig. 2B). ϵ416-Luc is a reporter transgene containing a 416-bp 5′-flanking region of the AChR ϵ-subunit gene and the luciferase gene, which responds to neuregulin stimulation in skeletal muscle cells (30Si J. Luo Z. Mei L. J. Biol. Chem. 1996; 271: 19752-19759Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 32Tanowitz M. Si J. Yu D.H. Feng G.S. Mei L. J. Neurosci. 1999; 19: 9426-9435Crossref PubMed Google Scholar, 35Si J. Mei L. Brain Res. Mol. Brain Res. 1999; 67: 18-27Crossref PubMed Scopus (29) Google Scholar). Expression of constitutively active Ras (V12), Raf (BXB), or Mek1 (ddMek1) increased the promoter activity of ϵ416-Luc. Expression of constitutively active Ras and Raf increases expression of theϵ416 transgenes (30Si J. Luo Z. Mei L. J. Biol. Chem. 1996; 271: 19752-19759Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). As shown in Fig. 2A, co-expression of Erbin suppressed RasV12-induced activation of ϵ416-Luc. In contrast, however, Erbin appeared to be unable to inhibit RafBXB- or ddMek1-induced ϵ416-Luc expression. These results confirm our early report that Erbin may function downstream or at the level of Ras and, at the same time, validate the reporter for a reliable readout. Erbin, LRR, or ΔLRR was cotransfected with ϵ416-Luc in C2C12 myoblasts, and the resulting myotubes were stimulated with neuregulin and assayed for luciferase activity as described under "Experimental Procedures." Although Erbin and LRR attenuated neuregulin-induced expression of ϵ416-Luc, the deletion of LRR (ΔLRR) prevented Erbin from inhibiting neuregulin-induced expression. These results suggest that the LRR domain is necessary and sufficient for inhibition of ϵ416-Luc expression.FIGURE 2The LRR domain is required for Erbin inhibition of neuregulin induction of ϵ416-Luc. A, Erbin inhibition of ϵ416-Luc expression by active Ras but not active Raf or MEK1. C2C12 myoblasts were transfected with ϵ416-Luc with various amounts of expression constructs of active Ras, Raf, or MEK1. A Renilla luciferase plasmid, pRL-TK, was cotransfected as a control to monitor the transfection efficiency and sample handling. After differentiation, cells were lysed, and firefly and Renilla luciferase activities were assayed. Cells transfected with an empty vector were taken as 100%. Shown is a representative experiment in duplicates, which was repeated three times with similar results. B, the LRR domain is necessary and sufficient to inhibit neuregulin-induced ϵ416-Luc expression. C2C12 cells were transfected with ϵ416-Luc, pRL-TK, and the indicated Erbin constructs (structures are shown below the histogram). Luciferase activities were assayed as in A. *, p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Erbin Interaction with Sur-8—Sur-8 is believed to be a positive regulator of the Ras-Raf-MEK pathway, functioning as a scaffold that enhances ERK activation by facilitating the interaction between Ras and Raf (33Luo Z. Tzivion G. Belshaw P.J. Vavvas D. Marshall M. Avruch J. Nature. 1996; 383: 181-185Crossref PubMed Scopus (201) Google Scholar, 36Li W. Han M. Guan K.L. Genes Dev. 2000; 14: 895-900PubMed Google Scholar). Sur-8 is composed almost entirely of 18 LRRs that show homology to the LRR domain of Erbin. Interestingly, like Erbin, Sur-8 interacts with active RasV12 but not with inactive RasN17 (36Li W. Han M. Guan K.L. Genes Dev. 2000; 14: 895-900PubMed Google Scholar). We reasoned that Erbin may interact with Sur-8 and thus disrupt its interaction with Ras to inhibit ERK activation. First we tested whether Erbin and Sur-8 interact in cells. GST-Sur-8 and Myc-Erbin were cotransfected into HEK 293 cells. Cell lysates were incubated with glutathione-conjugated agarose beads to pull down Sur-8, and the resulting precipitates were blotted with anti-Myc antibody. As shown in the top panels of Fig. 3A, Myc-Erbin was detected in the precipitates of GST-Sur-8. In a reciprocal experiment, GST-Sur-8 was present in immunoprecipitates of Myc-Erbin (Fig. 3A, bottom panels). These results suggest that Erbin interacts with Sur-8 in cells. Further analyses showed that Erbin interaction with Sur-8 requires the LRR domain. Deletion of this domain prevented Erbin from interacting with Sur-8 (Fig. 3B). To determine whether the interaction occurs between the two proteins at endogenous levels, HEK 293 cell lysates were incubated with anti-Sur-8 serum, and the resulting precipitates were probed with anti-Erbin antibodies (Fig. 3C). Erbin was detected in Sur-8 precipitates, suggesting that endogenous Erbin and Sur-8 may interact in cells.FIGURE 3Interaction between Erbin and Sur-8. A, co-immunoprecipitation of Sur-8 and Erbin. COS-7 cells were transfected with mammalian expression constructs of GST-Sur-8 or/and Myc-Erbin. Top panels, cell lysates were incubated with glutathione-conjugated beads to purify GST-Sur-8. The resulting complex was probed with anti-Myc antibody. Lysates were also blotted with anti-GST or anti-Myc antibodies to show expression of transfected proteins. Bottom panels, cell lysates were incubated with anti-Myc antibody and subsequently protein G beads to precipitate Myc-Erbin. The complex was probed with anti-GST antibody. The presence of transfected proteins in lysates was revealed by immunoblotting (IB) with indicated antibodies. IP, immunoprecipitate. PD, pull down. B, the LRR domain in Erbin is necessary and sufficient to interact with Sur-8. COS-7 cells were transfected with mammalian expression constructs of GST-Sur-8 together with Myc-Erbin, Myc-LRR, or Myc-ΔLRR. Cell lysates were incubated with glutathione-conjugated beads to purify GST-Sur-8, and the resulting complex was probed with anti-Myc antibody. Expression of GST-Sur-8 was shown in the lower panel. C, interaction of endogenous Erbin and Sur-8. COS-7 lysates were incubated with anti-Sur-8 antibody or control serum and subsequently with protein A beads to precipitate Sur-8. The resulting complex was probed with anti-Erbin antibody (upper panel) or anti-Sur-8 antibody (lower panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Disruption of the Sur-8-Ras and -Raf Interaction by Erbin—To determine whether Erbin regulates the Sur-8-Ras interaction, HEK 293 cells were transfected with GST-Sur-8, FLAG-RasV12, and Myc-Erbin. Cell lysates were incubated with glutathione-conjugated agarose beads to pull down Sur-8, and the resulting precipitates were blotted for RasV12 with anti-FLAG antibody. Sur-8 and RasV12 co-precipitated (Fig. 4A), in agreement with earlier studies (36Li W. Han M. Guan K.L. Genes Dev. 2000; 14: 895-900PubMed Google Scholar). However, co-expression blocked the interaction of Sur-8 with RasV12. This effect was dose-dependent (Fig. 4B). The amount of RasV12 interacting with Sur-8 decreased as the concentration of Erbin increased. These results demonstrated that Erbin may be able to disrupt the Sur-8-Ras complex. In addition, Erbin expression appeared to inhibit the interaction between Sur-8 and Raf (Fig. 4C). These observations are in agreement with the notion that Erbin inhibits ERK activation by disrupting Sur-8 interaction with Ras and Raf.FIGURE 4Disruption of the Sur-8-Ras interaction by Erbin. A and B, HEK 293 cells were transfected with mammalian expression constructs of FLAG-Ras (V12) and GST-Sur-8 without or with Myc-Erbin at a single concentration (A) or at various concentrations (B). GST-Sur-8 was purified as described for Fig. 3, and the resulting complex was probed with anti-Ras antibody. Expression of transfected proteins was revealed by immunoblotting (IB) with the indicated antibodies. C, disruption of the Sur-8-Raf interaction by Erbin. Experiments were the same as in A and B, except FLAG-Raf was transfected. PD, pull down.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Increased Sur-8-Ras and -Raf Interaction in Cells in Which Expression of Erbin Was Suppressed—To further study the role of Erbin in regulating ERK activation, we explored the consequences of suppression of Erbin expression by using a small interfering RNA approach, which diminishes the expression of a specific gene in cells. RNA interference has been shown recently to specifically suppress the expression of endogenous and heterologous genes in mammalian cell lines (37Dillin A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6289-6291Crossref PubMed Scopus (78) Google Scholar, 38Novina C.D. Sharp P.A. Nature. 2004; 430: 161-164Crossref PubMed Scopus (625) Google Scholar, 39Scherer L.J. Rossi J.J. Nat. Biotechnol. 2003; 21: 1457-1465Crossref PubMed Scopus (399) Google Scholar). The Erbin DNA sequence was analyzed by a Web-based program (BLOCK-iT™ RNAi Designer) for putative siRNA sequences. Three dis
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