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J. Biol. Chem., Vol. 278, Issue 35, 33334-33341, August 29, 2003
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From the Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029
Received for publication, May 28, 2003 , and in revised form, June 9, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 (4, 5). 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 (6). ErbB-3 (7) and fibroblast growth factor receptor I (8, 9) 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
(10,
11). 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-
-converting enzyme, followed by a
subsequent cleavage by 
-secretase that release the ErbB-4 intracellular
domain fragment from the membrane. The processing by -secretase
facilitates the translocation of the COOH-terminal fragment of ErbB-4
(CTF)1 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)
(12–14).
This process is called regulated intramembrane proteolysis and it represents a
relatively new paradigm of signal transduction
(15,
16). 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
(12). In the case of APP, its
intracellular fragment is produced by -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
(14). 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 (14,
17).
Isoforms of the ErbB-4 receptor generated by alternative splicing have been described (Ref. 3 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 (18). The other isoform contains a sequence change in the phosphatidylinositol 3-kinase (PI3K) binding region within the cytoplasmic domain (19, 20). 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 (21, 22). Within the ErbB-4 sequence we identified several proline-rich motifs that represent potential binding sites for WW domains.
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The WW domain is a protein-protein interaction module composed of 35–40 amino acids (23, 24). The domain binds ligands containing proline-rich sequences (25). 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 (26), the TEAD/TEF family of transcription factors (27), and p73 (28, 29). 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 (29, 30). 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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
(11,
21) and pEF6-
E2ErbB-4
constructs have been described elsewhere
(11). To prepare expression
vector for the GAL4 DNA binding domain fused with ErbB-4 CTF and
CTF(K), 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 pEF6E2ErbB-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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-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 (32, 33) and on the understanding of the WW domain molecular structure (34) 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.
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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 (29). 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 (35).
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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 (10).
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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
(10), 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 
kinase: 17-fold;
YAP2-CTF, 10-fold, and YAP2-CTF 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 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.
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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 (29). 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 (29). 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 -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
-secretase cleavage.
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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 BC014733 [GenBank] and the human cDNA corresponds to this transcript has been also deposited to the data base with the accession number BC038235 [GenBank] . The 700-bp bands were observed in many tissues including skeletal muscle, testis, ovary, kidney, liver, lung, and bladder. Interestingly, the relative expression of the 600-bp band was detected almost equally to the 700-bp band in the diencephalons-medulla oblongata and cerebrum. In the cerebellum, only the 600-bp band was detected indicating that the YAP2 isoform could be expressed at higher levels in the neural tissues compared with the other tissues. The PCR YAP products from heart and spleen were not amplified under our PCR conditions, although YAP expression has been documented in these tissues by Northern blot analysis (36).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-secretase cleavage,
translocates together with YAP to the nucleus upon ligand or TPA stimulation.
YAP may act not only as a transcriptional co-activator for the CTF, but also
may act as a carrier protein for the translocation of the CTF from the
membrane to the nucleus. The following aspects of the work deserve further
comments: (i) the localization of YAP and the networking with other signaling
pathways; (ii) the role of various isoforms of YAP and isoforms of ErbB4 in
signaling; and (iii) the emerging models of YAP-dependent transcriptional
activation of the CTF-ErbB4 and its nuclear function. It has been reported that YAP localizes to the apical membrane in epithelial cells associating with the EBP50 PDZ domain through its COOH-terminal PDZ domain binding sequence (37). It is therefore possible to assume that YAP and ErbB-4 colocalize at the plasma membrane in epithelial cells. The localization of YAP is regulated by the binding of 14-3-3 to the phosphorylation site and the phosphorylated YAP does not enter the nucleus (29, 30). It follows that co-transcriptional activity of the CTF·YAP complex may be regulated by YAP phosphorylation and the ultimate level of YAP protein in the cell nucleus. Similar to the YAP-mediated transactivation of p73 and the YAP-related protein TAZ (29, 30), the co-transcriptional activity for the ErbB4-CTF is also increased by the mutation of the phosphorylation site of YAP (Fig. 5a). Through its ability to phosphorylate YAP, Akt may inhibit a number of transcription factors by blocking rate-limiting transcriptional co-activation. Neither the Carpenter laboratory (10) nor our laboratory were able to detect transcriptional activity of the ErbB4-CTF itself using the Gal4 system in COS-7 cells, in which the endogenous YAP is present. This may because of the absence of YAP in the nucleus caused by the relatively high activity of Akt in COS-7 cells (29); however, the exogenously expressed YAP strongly stimulated the transcription. Although the unphosphorylatable mutation (S127A) of the crucial Ser-127 in YAP increases the localization of YAP in the nucleus, this mutation was not sufficient to allow the translocation of all available YAP to the nucleus (data not shown). Among possible mechanisms that regulate translocation of YAP to the nucleus by retaining the protein in the cytoplasm or at the plasma membrane, one may propose the anchoring role of PDZ domain-containing proteins such as EBP50 (37) and MUPP1 (27, 38). Further research should uncover the conditions and factors that stimulate the translocation of YAP to the nucleus.
In this study, we have used the alternatively spliced CYT-2 isoform of
ErbB-4 that does not activate the PI3K pathway because of the lack of PI3K
binding site (19). We have
chosen this isoform in our experiments arguing that the COOH-terminal fragment
produced from the CYT-1 isoform (with PI3K site) might not associate with YAP
in the nucleus under physiological conditions. In cells expressing the CYT-1
isoform, upon the ligand stimulation in which the CTF should translocate to
the nucleus, Akt is activated through the PDK-PI3K pathway
(39,
40), but Akt is
"silent" in the CYT-2 isoform-expressing cells because of the
absence of the activation of the PI3K pathway
(20). We presume that after
ligand stimulation, the cytoplasmic fragment of the CYT-2 isoform is cleaved
from the membrane by -secretase and forms a complex with YAP in the
nucleus. However, in the case of the CYT1 isoform, the PI3K pathway-activated
Akt would phosphorylate YAP and retain it in the cytoplasm for other
functions. Alternatively, because of the PI3K binding site in CYT-1 and its
flanking sequence contain the PPXY (PY2) sequence, this region might
work as a binding site for YAP to inhibit the PI3K pathway so that YAP could
escape from phosphorylation by Akt. These interesting but putative scenarios
of signaling switches are being now investigated.
The expression of ErbB-4 is ubiquitous in several tissues. The CYT-1 isoform is predominant in heart, breast, and abdominal aorta, whereas the expression of the CYT-2 is abundant in neural tissues including cerebellum, cerebral cortex, spinal cord, and medulla oblongata (19). The expression of YAP is also ubiquitous and similar to the CYT-2 isoform, YAP2 isoform is predominantly expressed in neural tissues, suggesting a possible CYT-2·YAP2 complex functioning in neural tissues. Furthermore, the expression of YAP in heart could not be detected in the RT-PCR analysis and this correlates with the absence of expression in the CYT-2 but not the CYT-1 in the same assay (19). We also have found a new transcript of YAP2 with insertion of 16 amino acids. This transcript is widely expressed in various tissues but not detected in cerebellum. The inserted sequence of the 16-amino acid "QAIRNINPSTAN-APKC" does not have any homology (or sequence similarity) to known motifs or domains. Interestingly, we could not identify the YAP1 homologue in mouse tissues using the RT-PCR approach. Furthermore, we could not find YAP1 homologue in the mouse and even in the human EST data base using human YAP1 sequence, although many of YAP2 and YAP2L sequences are present in the EST data bases. Altogether the data showing that YAP1 is not detected in mouse tissues and the absence of YAP1 in the human and mouse EST data base suggest that YAP2 (or YAP2L) is the major isoform and YAP1 is a minor isoform expressed in human tissues. Alternatively, it is possible that YAP1 is only expressed under special conditions or during limited stages of development. In the kidney of a HIV-1 transgenic mouse that has severe renal failure (41), in addition to the transcript of YAP2 and YAP2 with the 16-amino acid insertion, we uncovered a different transcript in which the WW domain is deleted in half.2 This unusual isoform may act as a dominant negative form of YAP in renal disease. In summary, we have found three variants, most likely splicing variants, that encode YAP1, YAP2, and YAP2 16-amino acid insertions in human and two variants YAP2 and YAP2 16-amino acid insertion in mouse. We documented that YAP2 is a stronger transcriptional co-activator than YAP. The difference between YAP1 and YAP2 in the structure is the number of WW domains and the insertion of four amino acids in the transcription activation region in YAP1. The presence of the additional WW domain might have a positive effect and the insertion might have an inhibitory effect on transcription. In fact, the mutation of the second WW domain slightly affected the transcription activity by an unknown mechanism. In this context the identification of the protein that associates with the second WW domain of YAP2 might be important. The precise signaling differences among YAP splicing variants remain unclear.
The YAP-dependent transcriptional activation of the COOH-terminal fragment
of ErbB-4 described here invites working models for this new signaling route.
Two simple models for the role of YAP·CTF complex in the nucleus are
proposed. The CTF might translocate to the nucleus together with YAP and
associate with several transcription factors on the promoter to activate
transcription (Fig. 8,
left). In this case, the CTF might work as a carrier of YAP for the
specific transcription factor. This is quite similar to the COOH-terminal
fragment of APP and a WW domain protein FE65 complex described by Cao and
Sudhof (14). The COOH-terminal
fragment of APP produced by -secretase itself does not have any
transcriptional activity and requires FE65 as a co-activator. Furthermore, the
transcription factor that recruits the complex to the specific promoter has
not been identified in APP-FE65-Tip60 nor ErbB-4CTF-YAP so far. Identification
of the transcription factors as binding partners for ErbB-4 or FE65 in the
nucleus is of importance.
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Another possible scenario stipulates that the CTF alone might translocate to the nucleus where YAP already exists in complex with transcription factors, such as p73 (28, 29), Runx2 (26), or TEAD/TEF (27) (Fig. 8, right). In the case of p73 and Runx2, the complex could be of an inhibitory nature, because the CTF may compete with these transcription factors for binding to the WW domain of YAP. In contrast in the case of TEAD/TEF, the translocation of the CTF to the nucleus might result in a ternary protein complex composed of YAP-TEAD/TEF-CTF because the binding is not through the WW domain-PPXY link and its translocation might affect the activity of the YAP-mediated transcription by TEAD/TEF. Further experiments using those transcription factors directly may elucidate downstream molecular targets of YAP·ErbB4 complex.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant AR45626 and
Human Frontier Science Program Organization Grant RG0234. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence should be addressed. Tel.: 212-241-9431; Fax:
212-987-0389; E-mail:
Marius.Sudol{at}mssm.edu.
1 The 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.
2 A. Komuro and M. Sudol, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 F. Zeng, M.-Z. Zhang, A. B. Singh, R. Zent, and R. C. Harris ErbB4 Isoforms Selectively Regulate Growth Factor induced Madin-Darby Canine Kidney Cell Tubulogenesis Mol. Biol. Cell, November 1, 2007; 18(11): 4446 - 4456. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. I. Aqeilan, V. Donati, E. Gaudio, M. S. Nicoloso, M. Sundvall, A. Korhonen, J. Lundin, J. Isola, M. Sudol, H. Joensuu, et al. Association of Wwox with ErbB4 in Breast Cancer Cancer Res., October 1, 2007; 67(19): 9330 - 9336. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Omerovic, L. Santangelo, E. M.-R. Puggioni, J. Marrocco, C. Dall'Armi, C. Palumbo, F. Belleudi, L. Di Marcotullio, L. Frati, M.-R. Torrisi, et al. The E3 ligase Aip4/Itch ubiquitinates and targets ErbB-4 for degradation FASEB J, September 1, 2007; 21(11): 2849 - 2862. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-M. Feng, C. I. Sartor, D. Hunter, H. Zhou, X. Yang, L. S. Caskey, R. Dy, R. S. Muraoka-Cook, and H. S. Earp III The HER4 Cytoplasmic Domain, But Not Its C Terminus, Inhibits Mammary Cell Proliferation Mol. Endocrinol., August 1, 2007; 21(8): 1861 - 1876. [Abstract] |
