WW Domain-containing Protein YAP Associates with ErbB-4 and Acts as a Co-transcriptional Activator for the Carboxyl-terminal Fragment of ErbB-4 That Translocates to the Nucleus
2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês
10.1074/jbc.m305597200
ISSN1083-351X
AutoresAkihiko Komuro, Makoto Nagai, Nicholas E. Navin, Marius Sudol,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoThe ErbB-4 receptor protein-tyrosine kinase is proteolytically processed by membrane proteases in response to the ligand or 12-O-tetradecanoylphorbol-13-acetate stimulation resulting in the cytoplasmic fragment translocating to the cell nucleus. The WW domain-containing co-transcriptional activator Yes-associated protein (YAP) associates physically with the full-length ErbB-4 receptor and functionally with the ErbB-4 cytoplasmic fragment in the nucleus. The YAP·ErbB4 complex is mediated by the first WW domain of YAP and the most carboxyl-terminal PPXY motif of ErbB-4. In human tissues, we documented the expression of YAP1 with a single WW domain and YAP2 with two WW domains. It is known that the COOH-terminal fragment of ErbB4 does not have transcriptional activity by itself; however, we show here that in the presence of YAP its transcriptional activity is revealed. There is a difference in the extent of transactivation activity among YAP isoforms: YAP2 is the stronger activator compared with YAP1. This transactivation is abolished by mutations that abrogate the YAP·ErbB4 complex formation. The unphosphorylatable mutation that increases the nuclear localization of YAP increases transcription activity. The COOH-terminal fragment of ErbB-4 and full-length YAP2 overexpressed in cells partially co-localize to the nucleus. Our data indicate that YAP is a potential signaling partner of the full-length ErbB4 receptor at the membrane and of the COOH-terminal fragment of ErbB-4 that translocates to the nucleus to regulate transcription. The ErbB-4 receptor protein-tyrosine kinase is proteolytically processed by membrane proteases in response to the ligand or 12-O-tetradecanoylphorbol-13-acetate stimulation resulting in the cytoplasmic fragment translocating to the cell nucleus. The WW domain-containing co-transcriptional activator Yes-associated protein (YAP) associates physically with the full-length ErbB-4 receptor and functionally with the ErbB-4 cytoplasmic fragment in the nucleus. The YAP·ErbB4 complex is mediated by the first WW domain of YAP and the most carboxyl-terminal PPXY motif of ErbB-4. In human tissues, we documented the expression of YAP1 with a single WW domain and YAP2 with two WW domains. It is known that the COOH-terminal fragment of ErbB4 does not have transcriptional activity by itself; however, we show here that in the presence of YAP its transcriptional activity is revealed. There is a difference in the extent of transactivation activity among YAP isoforms: YAP2 is the stronger activator compared with YAP1. This transactivation is abolished by mutations that abrogate the YAP·ErbB4 complex formation. The unphosphorylatable mutation that increases the nuclear localization of YAP increases transcription activity. The COOH-terminal fragment of ErbB-4 and full-length YAP2 overexpressed in cells partially co-localize to the nucleus. Our data indicate that YAP is a potential signaling partner of the full-length ErbB4 receptor at the membrane and of the COOH-terminal fragment of ErbB-4 that translocates to the nucleus to regulate transcription. Cells are continuously exposed to diverse stimuli ranging from soluble paracrine and endocrine factors to signaling molecules on neighboring cells. These extracellular signals are transduced to cell nuclei to achieve an appropriate developmental or proliferative response. Receptor protein-tyrosine kinases play pivotal roles in this process. Upon binding of their cognate ligands, the intrinsic protein-tyrosine kinase activity of the receptor is significantly elevated and initiates a network of signaling pathways including the well characterized Ras/mitogen-activated protein kinase and the signal transducers and activators of transcription pathways (1Hunter T. Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2278) Google Scholar, 2Schlessinger J. Cell. 2000; 103: 193-200Abstract Full Text Full Text PDF PubMed Scopus (3555) Google Scholar, 3Carpenter G. Exp. Cell Res. 2003; 284: 66-77Crossref PubMed Scopus (206) Google Scholar). Whereas many cell surface receptors transmit signals to the nucleus through complex protein cascades, several examples of membrane receptors translocating itself to the nucleus have been described (4Carpenter G. Curr. Opin. Cell Biol. 2003; 15: 143-148Crossref PubMed Scopus (144) Google Scholar, 5Wells A. Marti U. Nat. Rev. 2002; 3: 1-6Crossref Scopus (120) Google Scholar). In the case of ErbB-1, the epidermal growth factor receptor, addition of its cognate ligand causes translocation of the ligand-receptor complex to the nucleus and the complex binds to the cyclin D1 promoter activating its transcription (6Lin S.Y. Makino K. Xia W. Matin A. Wen Y. Kwong K.Y. Bourguignon L. Hung M.C. Nat. Cell Biol. 2001; 3: 802-808Crossref PubMed Scopus (903) Google Scholar). ErbB-3 (7Offterdinger M. Schofer C. Weipoltshammer K. Grunt T.W. J. Cell Biol. 2002; 157: 929-940Crossref PubMed Scopus (177) Google Scholar) and fibroblast growth factor receptor I (8Reilly J.F. Maher P.A. J. Cell Biol. 2001; 152: 1307-1312Crossref PubMed Scopus (202) Google Scholar, 9Maher P.A. J. Cell Biol. 1996; 134: 529-536Crossref PubMed Scopus (200) Google Scholar) have been also reported to be present in nucleus. Recently, it has been suggested that ErbB-4, the newest member of the epidermal growth factor protein-tyrosine kinase receptor family, activates gene expression in a more direct manner (10Ni C.Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (759) Google Scholar, 11Lee H.J. Jung K.M. Huang Y.Z. Bennett L.B. Lee J.S. Mei L. Kim T.W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). The binding of its ligand, heregulin, or activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate provokes an ectodomain cleavage by a metalloprotease tumor necrosis factor-α-converting enzyme, followed by a subsequent cleavage by γ-secretase that release the ErbB-4 intracellular domain fragment from the membrane. The processing by γ-secretase facilitates the translocation of the COOH-terminal fragment of ErbB-4 (CTF) 1The abbreviations used are: CTF, COOH-terminal fragment of ErbB-4; HA, hemagglutinin; GFP, green fluorescent protein; PDZ, PSD-95/discs large/ZO-1; APP, amyloid precursor protein; WT, wild-type; PI3K, phosphatidylinositol 3-kinase; YAP, Yes-associated protein. to the nucleus where it may affect the transcription of target genes. Similar mechanisms have been described for the proteolytic processing of the Notch receptor and the Alzheimer's amyloid precursor protein (APP) (12Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4950) Google Scholar, 13Fortini M.E. Nat. Rev. 2002; 3: 673-684Crossref Scopus (345) Google Scholar, 14Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1058) Google Scholar). This process is called regulated intramembrane proteolysis and it represents a relatively new paradigm of signal transduction (15Heldin C.H. Ericsson J. Science. 2001; 294: 2111-2113Crossref PubMed Scopus (33) Google Scholar, 16Ebinu J.O. Yankner B.A. Neuron. 2002; 34: 499-502Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). In Notch-1 signaling, the ligand binding causes intra-membrane cleavage of Notch-1 and generation of a transcriptionally active fragment. The Notch signaling pathway plays an important role in the cell-fate specification process in multicellular organisms. The intracellular fragment of Notch is translocated to nucleus and binds directly to downstream transcription factors of the C promoter binding factor/suppressor of hairless/Lag-1 family, to control transcriptional repression and activation of Notch target genes (12Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4950) Google Scholar). In the case of APP, its intracellular fragment is produced by γ-secretase and translocates to the nucleus to form a multimeric complex with the nuclear adaptor protein Fe65 and the histone acetyltransferase Tip60. This multicomponent complex is able to activate transcription via Gal4 or LexA reporters (14Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1058) Google Scholar). In that experimental system, a robust transcriptional activation was observed only when the COOH-terminal fragment of APP was co-expressed with Fe65; the COOH-terminal fragment alone was not active. Fe65 is a typical adaptor protein composed of a WW domain and two PTB domains. The PTB1 domain binds to the histone acetyltransferase Tip60 and the PTB2 domain interacts with the cytoplasmic tail of APP (14Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1058) Google Scholar, 17Fiore F. Zambrano N. Minopoli G. Donini V. Duilio A. Russo T. J. Biol. Chem. 1995; 270: 30853-30856Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Isoforms of the ErbB-4 receptor generated by alternative splicing have been described (Ref. 3Carpenter G. Exp. Cell Res. 2003; 284: 66-77Crossref PubMed Scopus (206) Google Scholar and Fig. 1a) including the JM-a isoform that is sensitive to the cleavage, whereas the JM-b isoform is insensitive to the cleavage because of the sequence difference in the juxtamembrane region (18Elenius K. Corfas G. Paul S. Choi C.J. Rio C. Plowman G.D. Klagsbrun M. J. Biol. Chem. 1997; 272: 26761-26768Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The other isoform contains a sequence change in the phosphatidylinositol 3-kinase (PI3K) binding region within the cytoplasmic domain (19Elenius K. Choi C.J. Paul S. Santiestevan E. Nishi E. Klagsbrun M. Oncogene. 1999; 18: 2607-2615Crossref PubMed Scopus (145) Google Scholar, 20Kainulainen V. Sundvall M. Maatta J.A. Santiestevan E. Klagsbrun M. Elenius K. J. Biol. Chem. 2000; 275: 8641-8649Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). This site is deleted in an isoform designated CYT-2 and the other isoform that contains PI3K site is designated as CYT-1 form. The ErbB4-CTF that was shown to translocate to the nucleus contains a protein-tyrosine kinase domain, autophosphorylation sites, and the PDZ domain recognition site (21Huang Y.Z. Won S. Ali D.W. Wang Q. Tanowitz M. Du Q.S. Pelkey K.A. Yang D.J. Xiong W.C. Salter M.W. Mei L. Neuron. 2000; 26: 443-455Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 22Ni C.Y. Yuan H. Carpenter G. J. Biol. Chem. 2003; 278: 4561-4565Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Within the ErbB-4 sequence we identified several proline-rich motifs that represent potential binding sites for WW domains. The WW domain is a protein-protein interaction module composed of 35–40 amino acids (23Chen H.I. Sudol M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7819-7823Crossref PubMed Scopus (489) Google Scholar, 24Bork P. Sudol M. Trends Biochem. Sci. 1994; 19: 531-533Abstract Full Text PDF PubMed Scopus (348) Google Scholar). The domain binds ligands containing proline-rich sequences (25Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). The largest class of WW domains binds ligands containing PPXY motif. The three PPXY sequences in the COOH-terminal region of ErbB-4 completely match the consensus motif that is recognized by the Class I WW domains. Here we report that the WW domain-containing proteins, Yes-associated protein (YAP) and its isoforms associate with the cytoplasmic region of ErbB-4 and transactivate the COOH-terminal fragment of ErbB-4-dependent transcription in the Gal4 system. YAP has been characterized as a co-transactivator for several transcription factors including the Runx family proteins (26Yagi R. Chen L.F. Shigesada K. Murakami Y. Ito Y. EMBO J. 1999; 18: 2551-2562Crossref PubMed Scopus (453) Google Scholar), the TEAD/TEF family of transcription factors (27Vassilev A. Kaneko K.J. Shu H. Zhao Y. DePamphilis M.L. Genes Dev. 2001; 15: 1229-1241Crossref PubMed Scopus (542) Google Scholar), and p73 (28Strano S. Munarriz E. Rossi M. Castagnoli L. Shaul Y. Sacchi A. Oren M. Sudol M. Cesareni G. Blandino G. J. Biol. Chem. 2001; 276: 15164-15173Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 29Basu S. Totty N.F. Irwin M.S. Sudol M. Downward J. Mol. Cell. 2003; 11: 11-23Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar). Most recently, it has been reported that the localization of YAP in the nucleus is regulated by Akt kinase and subsequent binding to 14-3-3 protein (29Basu S. Totty N.F. Irwin M.S. Sudol M. Downward J. Mol. Cell. 2003; 11: 11-23Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 30Kanai F. Marignani P.A. Sarbassova D. Yagi R. Hall R.A. Donowitz M. Hisaminato A. Fujiwara T. Ito Y. Cantley L.C. Yaffe M.B. EMBO J. 2000; 19: 6778-6791Crossref PubMed Scopus (582) Google Scholar). However, none of the upstream or membrane signals that communicate with YAP have been previously described. This is the first report showing the stimulation of transcription by ErbB-4 in complex with YAP. Plasmids—The BamHI-EcoRI fragments that include coding region of human YAP1 and YAP2 were excised from pcDNA3-YAP and cDNA clone HYAP5 (31Sudol M. Bork P. Einbond A. Kastury K. Druck T. Negrini M. Huebner K. Lehman D. J. Biol. Chem. 1995; 270: 14733-14741Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), respectively, and ligated into pcDNA4HIS-MAX (Invitrogen). The KpnI-XbaI fragment was excised from the pcDNA4HIS-MAX and the fragments were ligated into the p2xFLAG-CMV2 (Sigma) vector to prepare expression vectors, p2xFLAG-YAP1 and p2xFLAG-YAP2. The expression construct encoding full-length ErbB-4 with COOH-terminal HA epitope tag (11Lee H.J. Jung K.M. Huang Y.Z. Bennett L.B. Lee J.S. Mei L. Kim T.W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 21Huang Y.Z. Won S. Ali D.W. Wang Q. Tanowitz M. Du Q.S. Pelkey K.A. Yang D.J. Xiong W.C. Salter M.W. Mei L. Neuron. 2000; 26: 443-455Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar) and pEF6-ΔE2ErbB-4 constructs have been described elsewhere (11Lee H.J. Jung K.M. Huang Y.Z. Bennett L.B. Lee J.S. Mei L. Kim T.W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). To prepare expression vector for the GAL4 DNA binding domain fused with ErbB-4 CTF and CTF(ΔK), PCR amplification was performed using the primer sets: 5′-gggtcgacttagaaggaagagcatcaaaaag; cctctagacaccacagtattccggtg-3′ and 5′-ccgtcgacttcagggtgatgatcgtatg-3′; 5′-cctctagacaccacagtattccggtg-3′, respectively, and subcloned into SalI-XbaI site of pM vector (Clontech). The YAP (S127A, 1WW*, and 2WW*) and ErbB-4 point mutants were constructed by using QuikChange site-directed mutagenesis kit (Stratagene). The reporter plasmid pG5luc that expresses firefly luciferase and pRL-SV40 that expresses Renilla luciferase as an internal control were purchased from Clontech and Promega, respectively. Cell Culture and Antibodies—Human embryo kidney 293T cells, COS-7 cells were cultured in Dulbecco's modified Eagle's medium, 10% fetal calf serum. The polyclonal antibody that recognizes the COOH-terminal region of ErbB-4 (C-18) was purchased from Santa Cruz. The M2-antibody and the HA antibody were purchased from Sigma and Roche Diagnostics, respectively. Immunoprecipitation and Immunoblotting—For analysis of the interaction between YAP and ErbB-4, 293T cells transfected with p2xFLAG-YAP1 or -YAP2 and pcDNA3.1-ErbB-4 or pEF6ΔE2ErbB-4 using FuGENE 6 (Roche) were lysed with RIPA buffer (10 mm Tris-HCl (pH 7.4), 5 mm EDTA, 300 mm NaCl, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) 36–48 h after transfection and immunoprecipitated using anti-FLAG M2 affinity gel (Sigma). The immunoprecipitates were washed with the RIPA buffer and bound proteins were separated on a SDS-PAGE and immunoblotted by HA antibody. Luciferase Assay—Cells in 12-well dishes were transfected with the plasmids indicated in the figure legends using FuGENE 6 (Roche Diagnostics) harvested 36–48 h later. Firefly and Renilla luciferase activities were assayed with the dual luciferase assay system (Promega) and firefly luciferase activity normalized with respect to Renilla luciferase activity. All experiments were performed at least three times. Immunofluorescence Microscopy—COS-7 cells on glass coverslips were transfected with p2xFLAG-YAP2 and/or pcDNA3-ErbB-4 (CTF-(676–1292)) using FuGENE 6 (Roche Diagnostics). After 36–48 h, cells were fixed with 4% formaldehyde and permeabilized in 0.25% Triton X-100 in phosphate-buffered saline, blocked in 10% goat serum. For the colocalization study of exogenously expressed YAP and the COOH-terminal fragment of ErbB-4, transfected cells were stained with FLAG M2 antibody (Sigma) and ErbB-4 antibody (Santa Cruz) followed by incubation with rhodamine-conjugated anti-mouse IgG and fluorescein isothiocyanate-conjugated anti-rabbit IgG. Images were acquired using Nikkon microscope Eclipse TE2000-S equipped with a CCD camera. RT-PCR—Total RNA was isolated from various tissues obtained from FVBN mice (8 weeks old) using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Total RNA (1 μg) was treated with DNase I (Amp grade, Invitrogen) to eliminate residual contamination of DNA. The DNase-treated RNA was subsequently transcribed to cDNA with Superscript II enzyme according to the manufacturer's instructions (Stratagene) using oligo-(dT)18 primer. The cDNA was subjected to PCR analysis and primers were designed to be able to distinguish mouse YAP1 and YAP2 isoforms with primer pairs to sandwich both WW domains: 5′-ccctgatgatgtaccactgcc-3′ (nucleotides 654–674 of mouse YAP2) and 5′-ccactgttaagaaagggatcgg-3′ (nucleotides 1271–1251 of mouse YAP2). Glyceraldehyde-3-phosphate dehydrogenase cDNA was amplified as a positive control with primers: 5′-accacagtccatgccatcac-3′ and 5′-tccaccaccctgttgctgta-3′. The PCR products were separated on a 2% agarose gel. Human YAP1 and YAP2—It has been reported that human YAP contains a single WW domain and mouse YAP contains two WW domains. We found a splicing variant that encodes human YAP with two WW domains. YAP with a single WW domain, known previously as human YAP (31Sudol M. Bork P. Einbond A. Kastury K. Druck T. Negrini M. Huebner K. Lehman D. J. Biol. Chem. 1995; 270: 14733-14741Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), is designated here as YAP1 and the new YAP with two WW domains is designated as YAP2. The difference in the nucleotide sequence between YAP1 and YAP2 is an insertion of the additional WW domain encoding region, its flanking sequence, and the deletion of the four amino acid sequence “QVRP” in YAP2 (Fig. 1b), suggesting that YAP2 is a splicing variant. The second WW domain of human YAP2 differs from the second WW domain of mouse YAP by one amino acid in the aromatic triplet located in the second β-strand of the domain. The amino acid sequence of human YAP2 shares 90.2% sequence homology with the mouse YAP, indicating that YAP2 is most likely the human homologue of mouse YAP. YAP1 and YAP2 Associate with ErbB-4 through the WW Domain—To assess interaction between human YAPs and ErbB-4, co-immunoprecipitation experiments were performed. Both the HA-tagged CYT-2 isoform of the full-length ErbB-4 and FLAG-tagged YAP1 or YAP2 were transiently expressed in 293T cells, and the cell lysate was immunoprecipitated with anti-FLAG M2 antibody. The immunoprecipitant was probed with HA antibody to detect the coprecipitation of ErbB-4. In the presence of YAP1 or YAP2, ErbB-4 was pulled-down (Fig. 2b, lanes 2 and 5) indicating that YAP1 or YAP2 associates with ErbB-4 in cells. YAP2 seemed consistently more efficient in co-precipitation of ErbB-4 than YAP1 (Fig. 2a, lane 2 and 5), suggesting that YAP2 associates with ErbB-4 stronger than YAP1 does. Next, to determine whether this association is mediated by the WW domain of YAP1, a WW domain mutant was created in which the second conserved tryptophan and the conserved proline were each substituted to alanine (Fig. 2a). Based on our previous studies (32Chen H.I. Einbond A. Kwak S.J. Linn H. Koepf E. Peterson S. Kelly J.W. Sudol M. J. Biol. Chem. 1997; 272: 17070-17077Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 33Ermekova K.S. Zambrano N. Linn H. Minopoli G. Gertler F. Russo T. Sudol M. J. Biol. Chem. 1997; 272: 32869-32877Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) and on the understanding of the WW domain molecular structure (34Macias M.J. Hyvonen M. Baraldi E. Schultz J. Sudol M. Saraste M. Oschkinat H. Nature. 1996; 382: 646-649Crossref PubMed Scopus (362) Google Scholar) such mutants should render the domain inactive in terms of ligand binding. The WW domain mutation in YAP1 completely abolished the binding to ErbB-4 (Fig. 2b, lane 4). Interestingly, the analogous mutation of the second WW domain in YAP2 did not have any effect on binding to ErbB-4 (Fig. 2c, lane 3). These results indicate that the first WW domain of YAPs is primarily responsible for the interaction with ErbB-4 but not the second WW domain in YAP2. A Mutation of the 14-3-3-binding/Akt Phosphorylation Site in YAP Affects the Binding to ErbB-4 —It has been reported that the serine 127 residue in YAP is phosphorylated by a protein kinase, Akt, and it is recognized by the 14-3-3 protein (29Basu S. Totty N.F. Irwin M.S. Sudol M. Downward J. Mol. Cell. 2003; 11: 11-23Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar). The binding by 14-3-3 causes translocation of YAP from the cytoplasm from the nucleus. To determine the importance of this site for the binding to ErbB-4, the serine residue at position 127 was substituted to alanine (Fig. 2a) and binding was examined by co-precipitation experiments. The mutation of serine residues at position 127 in both YAP1 and YAP2 attenuated the binding to ErbB-4 (Fig. 2b, lanes 3 and 6). YAPs Associate with ErbB-4 through the PPXY Motif Located in the Cytoplasmic Region—To make sure that YAPs bind to PPXY motifs in ErbB4, the tyrosine residues in the PPXY motifs were substituted to alanines. There are two PPXY motifs in the cytoplamic region of ErbB-4 at positions 1031–1040 (NIPPPIYTSR) and 1280–1288 (LPPPPYRHR). Two mutants were created, the first PPXY mutant was designated as PY1 and the second mutant was designated as PY3 (Fig. 3a). The co-immunoprecipitation experiments showed that the substitution of tyrosine residue in the most COOH-terminal PPXY region (PY3) completely abolished the binding to both YAP isoforms (Fig. 3b, lanes 6 and 9). On the contrary, the binding to YAPs was not affected by the mutation of the first PPXY sequence (Fig. 3b, lanes 5 and 8). All the above co-precipitation experiments indicated that the association between both YAPs and ErbB-4 is through the WW domain (the first WW domain in YAP2) in YAP and the most COOH-terminal PPXY sequence in ErbB-4. These data are in full agreement with the results of the optimal binding of PPXY-containing 10-mer peptides selected for YAP1 WW domain from phage display and SPOT membrane peptide repertoires (35Linn H. Ermekova K.S. Rentschler S. Sparks A.B. Kay B.K. Sudol M. Biol. Chem. 1997; 378: 531-537Crossref PubMed Scopus (65) Google Scholar). The CTF Requires YAP for Stimulation of Transcription via the Gal4 Transactivation System—To make sure that the CTF is localized in the nucleus, the CTF was expressed in COS-7 cells and stained with ErbB-4 polyclonal antibody. Overexpression of the CTF showed prominent nuclear localization and weak cytosolic/membrane localization in COS-7 cells (Fig. 4a, right panel). The localization is distinct compared with the control, although this antibody showed low background of nuclear staining in non-transfected cells (Fig. 4a, left panel). This data confirms a previous result from the Carpenter laboratory obtained with the CTF-GFP fusion and cell fractionation study (10Ni C.Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (759) Google Scholar). It was also shown that the CTF with the deleted kinase domain has an ability to transactivate transcription in the Gal4 system in contrast to the intact CTF. Here we have used the same Gal4 transactivation system (10Ni C.Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (759) Google Scholar), however, in the presence of YAP1 or YAP2. The Gal4 DNA binding domain (1–147) fused with the entire COOH-terminal fragment or with the deletion of the kinase domain of ErbB-4 was coexpressed with the Gal4-luciferase reporter in COS-7 cells and the luciferase activity was measured. As shown previously, only the COOH-terminal fragment with the kinase domain deletion could stimulate transcription 3–5-fold over the Gal4 control but the intact CTF fragment or the vector control were negative in the assay (Fig. 4, lanes 1–3). As we expected, coexpression of YAP1 or YAP2 significantly transactivated both the intact and kinase-deleted versions of CTF-mediated transcription: YAP1-CTF, 3-fold, and YAP1-CTF δ kinase: 17-fold; YAP2-CTF, 10-fold, and YAP2-CTF δ kinase, 78-fold over the Gal4 control. These results indicate that both YAPs co-activate transcription and that YAP2 is a stronger co-activator of transcription than YAP1. The Association of YAP with the ErbB-4 Cytoplasmic Fragment Is Essential for Transactivation—In the subsequent experiments, we mainly used the intact version of CTF in the Gal4 assay, because the kinase-deleted fragment has not been detected under physiological conditions. To confirm that the transactivation by YAP is because of the direct association with the CTF-ErbB-4 fragment, we used YAP2 with a mutation in the WW domain that does not associate with ErbB-4 in the Gal4 assay. Although YAP2 with the mutation in the second WW domain activated less than the wild type (WT) YAP2, YAP2 with the mutation in the first WW domain did not activate transcription at all (Fig. 5a). Furthermore, mutation of the PPXY sequence (PY3) of the CTF that is essential for binding to YAP also abolished the stimulation of transcription either on the entire COOH-terminal fragment or the δ kinase domain CTF mutant. As expected the PY1 mutant did not have any effect in the assay (Fig. 5, b and c). These data indicate that the transactivation is caused by the YAP·CTF complex mediated by the WW domain of YAP and the most COOH-terminal PPXY sequence of the ErbB-4CTF. It has been reported that YAP is mostly localized in the cytoplasm and its nuclear localization is inhibited by the binding of 14-3-3 upon Akt phosphorylation (29Basu S. Totty N.F. Irwin M.S. Sudol M. Downward J. Mol. Cell. 2003; 11: 11-23Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar). It has also been known that the mutation of serine 127 to alanine increases transactivation activity of YAP because of a greater nuclear localization of YAP and therefore decided to assess the nonphosphorylatable mutant (YAP-S127A) in the same Gal4 assay. Although, in the immunoprecipitation experiments using the full-length ErbB-4, the mutation attenuated the binding of YAP, this mutation caused a transactivation increase about 1.9-fold compared with the WT (Fig. 5a) in agreement with the previously reported data in the slightly different “read-out,” the p73 mediated transcription assay (29Basu S. Totty N.F. Irwin M.S. Sudol M. Downward J. Mol. Cell. 2003; 11: 11-23Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar). The attenuation of YAP association described here may be because of the change in localization of YAP to the nucleus and its inaccessibility to the membrane localized full-length receptor. The CTF Co-localizes Partially with YAP in Cells—To determine whether the CTF produced by the γ-secretase cleavage co-localizes with YAP in cells, COS-7 cells were co-transfected with pcDNA3HA-ErbB-4 CTF-(676–1292) and p2xFLAG-YAP2. The CTF showed prominent localization in the nucleus and FLAG-YAP2 showed mostly cytoplasmic distribution. In a clearly discernible population of cells, FLAG-YAP2 showed the co-localization with the CTF in the cell nuclei (Fig. 6), suggesting that YAP sublocalized in the nucleus cooperates with the native CTF derived from γ-secretase cleavage. Expression of YAP Transcripts in Several Tissues—Because the human YAP1 and YAP2 isoforms were described here for the first time, and the differences in their transcriptional activity was documented, we decided to investigate the expression pattern of each YAP transcript in various tissues. Because of the difficulties in obtaining normal human tissues, freshly dissected mouse tissues were used instead. Accordingly, we designed the mouse RT-PCR primers so that mouse YAP1 and YAP2 transcripts could be unequivocally amplified and distinguished. The sequence of the mouse YAP2 homologue and the topology of human YAP1 and YAP2 sequences were considered in the RT-PCR strategy (Fig. 7a). As we expected, doublet bands derived from two isoforms were observed, but the expected size of 500 bp for the mouse YAP1 homologue transcript was not detected (Fig. 7b). Instead, the slower migrating band with 700 bp was amplified in most mouse tissues together with the 600-bp band expected to be the mouse YAP2 homologue transcript. We confirmed the PCR product by direct sequence analysis and identified the 600-bp band as previously known YAP (mouse YAP2 homologue). The 700-bp band turned out to represent a new isoform with an insertion of 48 nucleotides; we designated this isoform as YAP2L. This sequence has been already submitted to the NCBI data base with the accession number of BC014
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