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Genes to Cells (2009) 14, 607-615. doi:10.1111/j.1365-2443.2009.01292.x
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Nuclear localization and pro-apoptotic signaling of YAP2 require intact PDZ-binding motif

 Tsutomu Oka1 and Marius Sudol1,2

1 Geisinger Clinic, Weis Center for Research, Danville, PA 17822-2608, USA
2 Mount Sinai School of Medicine, Department of Medicine, New York, NY 10029, USA


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 Endnote
 References
 
The Hippo signaling pathway regulates the intrinsic size of organs by controlling two opposing processes, proliferation and apoptosis. The nuclear effector of this pathway is Yes kinase-associated protein (YAP) which is a WW domain-containing transcriptional co-activator. In addition to WW domains, YAP2 has a Post-synaptic density, Discs large, Zonula occludens-1 (PDZ)-binding motif that is located at its COOH terminus. To determine whether the localization of YAP2 in cells is PDZ-binding motif dependent, we generated a delta C mutant of YAP2 lacking the five most COOH terminal amino acids, -FLTWL, which constitute a well-conserved PDZ-binding motif. We report here that the PDZ-binding motif is necessary for YAP2 localization in the nucleus, for the stabilization of p73, and for promoting apoptosis of HEK293 cells maintained at low concentration of serum. We suggest that an unknown PDZ domain-containing protein (or proteins) functions as a shuttle, facilitating YAP2 translocation from the cytoplasm to the nucleus. Since the Hippo pathway acts as a tumor suppressor pathway, the PDZ complex of YAP represents a potential target of cancer therapy.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 Endnote
 References
 
The Hippo signaling pathway controls the intrinsic size of organs by coordinating two opposing processes, proliferation and cell death (Harvey et al. 2007). One of the effectors of the Hippo pathway is Yes kinase-associated protein (YAP). Originally, the YAP gene was isolated by functional cloning, as Yes- and Src-kinase binding protein (Sudol 1994). Subsequently, YAP was shown to be a part of the tri-partite complex containing, in addition to Yes kinase, a scaffold protein known as EBP50 (Ezrin–Radixin–Moesin-binding phosphoprotein of 50 kDa). The complex was localized to the apical membrane of airway epithelia, and it was proposed that it regulates ion channels and transporters (Mohler et al. 1999). Recently, YAP was shown to act as a transcriptional co-activator downstream of various signaling complexes (Yagi et al. 1999; Espanel & Sudol 2001; Vassilev et al. 2001; Komuro et al. 2003) but most directly YAP was implicated in the Hippo signaling pathway (Huang et al. 2005).

There are two major isoforms of YAP, named YAP1 and YAP2, which are generated by differential splicing (Sudol et al. 1995). YAP1 contains one Tryptophan–Tryptophan domain (WW domain) and YAP2 contains two WW domains. In general, WW domains are small protein modules known to mediate protein complexes by interaction with linear, proline-rich peptide motifs in cognate proteins (Bork & Sudol 1994; Chen & Sudol 1995). Both YAP1 and YAP2 have an ability to translocate from the cytoplasm to the nucleus and this process is controlled in part by WW domain-mediated complexes with large tumor suppressor kinase 1 (Lats1) and Lats2 kinase.

Most recently, we reported that when human embryonic kidney cells (HEK293) or mouse fibroblasts (NIH3T3) were grown in media containing low concentrations of serum, YAP2 was able to bind to and stabilize the pro-apoptotic member of the p53 family, p73, and thus promote cell apoptosis (Oka et al. 2008). However, the YAP1 isoform did not bind p73 and did not induce apoptosis in our cellular model. Therefore, we focused our analysis on the YAP2 isoform. We also showed that the cytoplasmic localization of YAP2 was dependent on YAP2 phosphorylation at Serine 127 by Lats1 kinase. The phospho-Serine 127 is a part of peptide motif that binds to the 14–3-3 anchor and sequesters YAP2 in the cytoplasm.

In addition to WW domains, YAP2 has a Post-synaptic density, Discs large, Zonula occludens-1 (PDZ)-binding motif that is located at its COOH terminus. As PDZ domains are frequently present in sub-membrane region located scaffold proteins, we decided to examine if the localization of YAP2 in cells is PDZ motif dependent. To analyze the role of the PDZ binding motif in YAP2, we generated a delta C mutant of YAP2 lacking five most COOH terminal amino acids, which constitute a well-conserved PDZ-binding motif. Interestingly, we found that the PDZ-binding motif was necessary for YAP2 localization in the nucleus, as the delta C mutant of YAP was found only in the cytoplasm and was excluded from the nucleus. We also documented that the PDZ-binding motif is indispensable for the stabilization of p73, and for promoting apoptosis. We suggest that an unknown PDZ domain-containing protein (or proteins) functions as a shuttle, facilitating YAP2 translocation from the cytoplasm to the nucleus, and we indicate that this mechanism may not be conserved in Drosophila.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 Endnote
 References
 
Complex of YAP2 with Lats1 is intact in YAP2 delta C mutant

YAP2 physically interacts with Lats1 via a complex mediated by two WW domains of YAP2 and two PPxY motifs of Lats1 (Oka et al. 2008). To determine if the truncation of the PDZ-binding motif in YAP2 (delta C mutant, Fig. 1A) affects its ability to bind to Lats1, we transiently co-expressed both Lats1 and YAP2 wild type (YAP2 WT) or Lats1 and YAP2 mutants (delta C and S127A) in HEK293 cells. Immunoprecipitation of cell lysates with Flag antibody was performed prior to immunoblotting with YAP antibody (Fig. 1B). The YAP2 WT as well as delta C and S127A YAP mutants bound to Lats1 but not to Lats1 mutant whose two PPxY motifs were rendered inactive in terms of WW domain binding by replacing the conserved Tyrosine (Y) with Alanine (A) (Lats1 PY1*&2*).


Figure 1
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  		Figure 1  Binding of YAP2 to Lats1. (A) Schematic structure of YAP2 protein and its delta C mutant. YAP2 has two WW domains in its central region and a PDZ-binding motif at its C-terminal end. The last five amino acids (-FLTWL) are truncated in delta C mutant. TAD stands for transcription activation domain. S127 is a common phosphorylation site for Lats1 and possibly for Akt kinases. (B) YAP2 delta C mutant has an ability to bind to Lats1. YAP2 WT, delta C, or S127A in HisMAX B vector were individually transfected with Lats1 cDNA construct [either WT or double PPPY-PPPA mutant (PY1*&2*)] into HEK293 cells. Cell lysates were immunoprecipitated with Flag antibodies, resolved on SDS PAGE and immunoblotted with YAP antibody or Flag antibody. (C) YAP2 delta C mutant is still phosphorylated by Lats1. Either YAP2 WT or YAP2 delta C mutant was transiently co-expressed in HEK293 cells with Flag-tagged Lats1 and Mst2 or the control vector. Cell lysates were resolved on SDS-PAGE followed by immunoblotting with YAP antibody or Flag antibody.

 
We previously reported that S127 in YAP2 is phosphorylated by activated Lats1 kinase and that this phosphorylation could regulate the localization of YAP2 protein (Oka et al. 2008). To test whether the YAP2 delta C mutant is phosphorylated by Lats1 kinase, we performed a gel shift assay (Fig. 1C). Both, the protein band of YAP2 WT and that of YAP2 delta C mutant were shifted upwards when Lats1 and its activating kinase Mst2 were transfected together. From these data we conclude that the lack of the PDZ-binding motif in YAP2 did not impair the physical and functional complex between Lats1 and YAP2.

PDZ-binding motif of YAP2 is crucial to promote apoptosis

To study the role of PDZ-binding motif in YAP2 function, we used HEK293 cells that express YAP2 in an inducible fashion and undergo apoptosis and detachment when maintained in 1% serum. Since apoptotic markers were detected before cell detachment, the anoikis, as the cell detachment-induced apoptosis, was excluded. We also showed that the expression of YAP2 WT and YAP2 S127A mutant in HEK293 cells induced detachment of the cells (Oka et al. 2008). To determine if the PDZ-motif plays a role in this process, we generated cells that express YAP2 delta C mutant in inducible fashion and scored them for detachment. The expressions of control vector, YAP2 WT, YAP2 delta C and YAP2 S127A mutants were individually induced in HEK293 cells and maintained in DMEM containing 1% FBS for 96 h. The relative number of attached cells present on plates was determined for each of the induced YAP2 proteins (Fig. 2A). Interestingly, the expression of YAP2 delta C mutant had almost no influence on cell detachment, while YAP2 WT and YAPS127A mutant showed a significant effect. The ratio of attached cells with induced YAP2 WT to attached cells without induced YAP2 WT was 0.608 (Fig. 2A, 2nd bar) i.e., the number of attached cells decreased by 39.2% in YAP2 induced cultures. As documented previously, YAP2 S127A mutant was more potent as the number of attached cells was reduced by about 50% in induced cells.


Figure 2
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  		Figure 2  Delta C mutant of YAP2 was inactive in promoting cell detachment. (A) YAP2 WT, delta C mutant and S127A mutant were individually induced in HEK293 cells and maintained in DMEM containing 1% FBS for 96 h. After the removal of detached cells, attached cells were trypsinized and counted. The ratios of ‘number of induced cells to number of non-induced cells’ for each YAP2 protein variant are shown. The lower panel is the immunoblotting with YAP antibody to verify the expression of induced proteins. (B) The same experiment as in A was conducted by using NIH3T3 cells instead of HEK293 cells. DMEM contained 2.5% FBS instead of 1% of serum.

 
To rule out the possibility that this phenomenon is specific to HEK293 cells, we also established NIH3T3 cells expressing YAP2 delta C mutant in an inducible fashion and scored these cells in low serum for cell detachment. Overexpressed YAP2 WT also caused detachment of NIH3T3 cells, whereas YAP2 delta C mutant failed to reduce the number of attached NIH3T3 cells (Fig. 2B).

As the detachment of YAP2 expressing cells correlated with apoptosis (Oka et al. 2008), we suspected that the YAP2 delta C mutant would fail to promote apoptosis. To confirm this suspicion, we probed for apoptotic markers in YAP2 delta C mutant expressing cells. Two antibodies: one against poly-[ADP-ribose]-polymerase (PARP) and the other against activated Caspase-3 were used to determine if these cells undergo apoptosis (Fig. 3). We assayed the total population of cells that included both the attached cells and floating cells. In the presence of YAP2 WT or S127A, both the cleaved fragment of PARP and the activated Caspase-3 protein were detected, suggesting that these cells underwent apoptosis (Fig. 3 lane 4 and 8). However, in the case of YAP2 delta C, we could not detect the two markers of apoptosis (Fig. 3, lane 6). We suggest that the intact PDZ-binding motif of YAP2 is required for the pro-apoptotic activity of YAP.


Figure 3
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  		Figure 3  Delta C mutant of YAP2 fails to promote apoptosis. YAP2 WT, delta C mutant and S127A mutant were individually induced in HEK293 cells and maintained in DMEM containing 1% FBS for 96 h. The resulting cells were harvested, followed by immunoblotting using antibodies against PARP (upper panel), active Caspase-3 (middle panel), and GAPDH (lower panel).

 
PDZ-binding motif of YAP2 controls its nuclear localization

The C-terminal PDZ-binding motif of TAZ, a paralog of YAP, was shown to be required for nuclear localization of TAZ (Kanai et al. 2000), whereas the PDZ-binding motif of YAP2 was shown to be necessary for YAP localization to the apical membrane in lung epithelia (Mohler et al. 1999). Based on those observations, we considered that the localization of YAP2 delta C mutant in HEK293 cells would differ from that of YAP2 WT. To test this hypothesis, green fluorescent protein (GFP)-fused constructs of YAP2 WT, delta C and S127A mutants were individually transfected into HEK293 cells and the cells were observed by fluorescence microscopy (Fig. 4). The nuclei were demarcated by DAPI staining. If the silhouette of the nucleus was clearly discernible and the cytoplasm was bright, we defined it as ‘localized in the cytoplasm.’ In more than 66% of cells that expressed GFP YAP2 WT, the fluorescent YAP2 localized in the cytoplasm. As expected, GFP YAP2 S127A mutant localized preferentially in the nucleus. Intriguingly, GFP YAP2 delta C mutant was completely excluded from the nucleus and showed only cytoplasmic localization, suggesting that the PDZ-binding motif of YAP2 is required for the nuclear localization.


Figure 4
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  		Figure 4  Delta C mutant of YAP2 was excluded from the nucleus. (A) Localizations of YAP2 WT, delta C mutant and S127A mutant proteins. YAP2 WT as well as its mutants were fused to GFP and expressed in HEK293 cells. The cells were observed under a fluorescence microscope. Size bar = 20 µm. Note the bright cytoplasmic aggregate seen frequently in HEK293 cells stained with YAP antibody. (B) The percentage of cells showing cytoplasmic localization of GFP-YAP2 WT, delta C mutant and S127A mutant is indicated. This experiment was repeated three times independently with ca. 100 cells observed in each case.

 
PDZ-binding motif of YAP2 is indispensable for p73 stabilization

YAP2 is known to stabilize the protein level of pro-apoptotic protein, p73, and this process represents one of the mechanisms by which YAP2 promotes apoptosis (Strano et al. 2005; Levy et al. 2007; Oka et al. 2008). Therefore, we asked here if YAP2 delta C mutant could stabilize p73. Co-immunoprecipitation assays revealed that YAP2 delta C bound to the exogenous p73 (Fig. 5A, lane 2) as well as to the endogenous p73 (Fig. 5B, lane 2). The WW domains of YAP2 were required for the association with p73, because the double mutant of WW domains failed to bind p73 (Fig. 5A, lane 4 and Fig. 5B, lane 4). However, the YAP2 delta C mutant did not stabilize p73 (compare 6th lane with 5th lane in Fig. 5C upper panel) even though it was able to bind to p73. In the presence of YAP2 WT, the protein level of p73 was relatively increased (Fig. 5C, upper panel, lane 3 and 4). This was also the case for YAP2 S127A mutant (Fig. 5C, upper panel, lane 7 and 8). These results indicate that the PDZ-binding motif of YAP2 is required for the stabilization of p73. We also used cycloheximide treatment to inhibit de novo protein synthesis and showed that the amount of HA-p73 was clearly increased in the presence of YAP2 WT, but it was not increased in the presence of YAP2 delta C mutant (Fig. 5D).


Figure 5
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  		Figure 5  PDZ binding domain is required for YAP2 to stabilize p73. (A) Delta C mutant binds to p73. Flag-tagged YAP2 WT, delta C mutant, S127A mutant or YAP2–1&2 WW* mutant were individually transfected with HA-p73 into HEK293 cells. Cell lysates were immunoprecipitated with Flag antibodies, resolved on SDS PAGE and immunoblotted with HA antibody or Flag antibody. (B) Endogenous p73 binds to YAP2. Indicated Flag-tagged plasmids were individually transfected into HEK293 cells. Cell lysates were immunoprecipitated with YAP antibody, resolved on SDS PAGE and immunoblotted with p73 antibody or Flag antibody. (C) Delta C mutant did not stabilize p73. HEK293 cells that express control vector, YAP2 WT, YAP2 delta C or YAP2 S127A in an inducible fashion were transfected with HA-p73. 24 h later, the cells were plated in fresh DMEM containing 1% FBS, Blasticidin (5 µg/mL) and Zeocin (200 µg/mL). Tetracycline (1 µg/mL) was added to the medium to induce the expression of YAP2. 96 h after induction, the cells were harvested, followed by immunoblotting using antibodies to HA (upper panel), YAP (middle panel) and GAPDH (lower panel). (D) YAP2 needs PDZ binding motif to stabilize p73. HA-p73 and the indicated plasmids were transfected into HEK293 cells, and each plate was divided into two plates. 24 h later, one plate was harvested, and 300 µg/mL of cycloheximide (CHX; Sigma) was added to the other plate and harvested after additional 24 h. Cell lysates were immunoblotted using antibodies to HA (upper panel), YAP (middle panel) and GAPDH (lower panel).

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 Endnote
 References
 
The Hippo signaling pathway was recently proposed as an important string of molecular interactions, which controls the intrinsic size of organs by coordinating two opposing processes, proliferation and cell death. Therefore, the precise delineation of this pathway should help us better understand normal growth and its abnormal forms, including cancer. In this report, we elucidated a new aspect of the Hippo pathway, namely the critical role of the PDZ domain-binding motif of YAP in controlling its nuclear localization. Interestingly, this newly uncovered facet of YAP function is well-conserved throughout the animal kingdom, including simple Hydra, but it is not conserved in Drosophila, where the core of the Hippo pathway was originally elucidated by genetics.

We report here that the delta C mutant of YAP2 lacking five most COOH terminal amino acids, which constitute a bona fide PDZ-binding motif (Jemth & Gianni 2007), when expressed in HEK293 cells was found only in the cytoplasm and was excluded from the nucleus. We also documented that the PDZ-binding motif is indispensable for the stabilization of p73 in promoting apoptosis of HEK293 cells maintained in low concentration of serum, the cellular assay of the mammalian Hippo pathway (Oka et al. 2008). In sum, we conclude that an unknown PDZ domain-containing protein (or proteins) controls YAP2 translocation from the cytoplasm to the nucleus (Fig. 6).


Figure 6
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  		Figure 6  Schematic depiction of three YAP2-mediated complexes showing that YAP2 without PDZ binding motif is not able to promote apoptosis. Lats1 phosphorylates YAP2 at S127, which prevents YAP2 from entering the nucleus because of the 14-3-3 anchor (left complex). YAP2 that is not bound to 14-3-3, such us YAP2 S127A mutant, easily goes to the nucleus and promotes pro-apoptotic genes (right complex). YAP2 stabilizes p73. However, when the PDZ motif of YAP is mutated, p73 protein is not stabilized by YAP2 and cells do not undergo apoptosis (middle complex).

 
The following aspects of this study deserve further comment: (i) the mechanism by which a PDZ domain-containing protein controls YAP2 localization and its pro-apoptotic function; (ii) the list of putative candidate proteins that contain PDZ domains, interact with YAP2 and regulate its localization; (iii) the functional implications of the absence of the PDZ domain-binding region in Drosophila YAP, dYAP (yki); and (iv) YAP interacting and PDZ domain-containing proteins as potential targets of cancer therapy.

We envision two possibilities by which a PDZ domain-containing protein that interacts with YAP2 controls its nuclear localization. One possibility is that the unknown PDZ domain-containing protein serves as a shuttle transporting YAP2 from the cytoplasm into the nucleus. The second scenario stipulates that the putative PDZ domain-containing protein is expressed and concentrated in the nucleus and serves there as a strong anchor for YAP2, without which YAP2 would prefer cytoplasmic milieu. In the latter case the transport of YAP2 to the nucleus would be carried out by another unknown protein. Identification of the YAP2-interacting, PDZ domain-containing protein, or proteins, should clarify which of the two possibilities is true.

p73 is a pro-apoptotic member of the p53 family of transcription factors, and it is known to activate target genes that induce apoptosis. p73 is also known to be stabilized by YAP in response to DNA damage (Strano et al. 2001, 2005; Levy et al. 2007), and this stabilization depends on intact PPxY motifs in p73 and the functional WW domains in YAP2 (Oka et al. 2008). In our recent report, YAP2 mutants, whose ability to bind to p73 were impaired, could no longer stabilize p73 (Oka et al. 2008). Interestingly, in this study, we found that YAP2 delta C mutant still bound to p73 but failed to stabilize it. The simplest explanation could be that YAP2 stabilizes p73 by bringing it into the nucleus to protect it from degradation in the cytosol. We cannot exclude that the conformation of YAP2 delta C mutant protein might be changed compared to the conformation of the wild-type YAP2, and the protein with compromised fold may not be effective in stabilizing p73. A possible role of a PDZ domain-containing protein that interacts with YAP2, and helps in concert with YAP2 to stabilize p73, cannot be ignored either.

The most recent study on the regulatory circuit that controls the Itch ubiquitin ligase mediated degradation of p73 provides a more subtle explanation (Levy et al. 2008). Under normal conditions, YAP was shown to complex with Runx transcription factor, bind in concert to the promoter of Itch gene to up-regulate its transcription and p73 degradation. However, under stress conditions YAP1 is phosphorylated by c-Abl on Tyrosine 357 and the phosphorylated YAP1 does not co-activate Runx in promoting Itch transcription, resulting in the stabilized population of p73 (Levy et al. 2008). In view of these findings it would be important to determine whether the YAP2 WT and delta C YAP2 mutant differ in terms of phosphorylation on Tyrosine 391 (the equivalent of tyrosine 357 of YAP1), and if YAP2 Tyrosine 391 Alanine mutant is able to induce apoptosis in HEK293 cells stressed by low serum-containing media.

The C terminus of YAP has an obvious and well-conserved PDZ binding motif (Fig. 7) (Tonikian et al. 2008). The core of this motif, the –FLTWL sequence is similar to peptides that bind to PDZ domain 1 of TJP1, Tight Junction Protein 1 (Zhang et al. 2006). Since the first, amino terminally located PDZ domains of the three TJPs (TJP1, TJP2 and TJP3) are nearly identical, they are predicted to interact with YAP2. Although TJPs are basic components of tight junctions (Tsukita et al. 2001), TJP2 was shown to translocate to the nucleus (Traweger et al. 2003) and under some conditions, TJP1 was also reported in the nuclear compartment (Gottardi et al. 1996).


Figure 7
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  		Figure 7  Sequence alignment of human YAP carboxy-terminal 20 amino acids, which harbor a PDZ binding motif, with sequences of other YAP orthologues of selected vertebrate and invertebrate animals. The sequence of human YAP (NCBI— Accession Number: NP_006097 [GenBank] ) was used as a query in basic protein BLAST. Sequences of Hydra and Anemone YAP were provided by Dr. Robert Steele from University of California at Irvine. Multiple sequence alignment program: MUSCLE, version 3.7, created by Robert C. Edgar, was used through the ExPASy suite. Note that the carboxy-terminal sequence of the fruit fly YAP is quite divergent from the remaining sequences and does not seem to have a canonical (terminal or internal) PDZ binding motif.

 
The extensive and fine mapping of the PDZ domain specificity in the human proteome allowed us to predict several proteins as high affinity interactors with YAP2 C-terminal sequence (Tonikian et al. 2008). In addition to TJP proteins, MPDZ, INADL, SLC9A3R2 are predicted as likely candidates of YAP2, especially SLC9A3R2, which was shown to be localized in the nucleus, like TJP1 and 2 proteins (Poulat et al. 1997). Less likely partners of YAP2 are SCRIB, PDZK1, ERBB2iP, LRRC7, and MAGI3 because they scored as weaker binders in the phage display screen (Tonikian et al. 2008). Since these candidate proteins were predicted based on the in vitro screen of phage displayed peptides probed with individual PDZ domains, they need to be validated in an in vivo setting for biological relevance. However, the involvement of YAP2 in regulation of adhesion (Oka et al. 2008) and cell to cell junctions (Zhao et al. 2007) resonates well with the predicted proteins, such as TJPs (Tsukita et al. 2001), which could very well function as bone fide biological partners of YAP in the Hippo pathway.

In the case of Drosophila YAP, yki, the carboxy-terminal sequence does not contain a known PDZ domain ligand and there is no obvious internal ligand sequence either. In fact, it seems that the Drosophila YAP carboxy-terminal sequence was designed to not bind PDZ domains (Fig. 7). In view of this finding it is interesting to know how human YAP was able to complement dYAP (yki) in Drosophila (Huang et al. 2005), since nuclear localization is critical for the function of both the human YAP and fly YAP.

It is important to mention here that the naturally-occurring isoforms of YAP that lack a large portion of the carboxy-terminal region were reported in neurons undergoing atypical cell death induced by repression of transcription (Hoshino et al. 2006). These neuron-specific YAP delta C isoforms lacked both the transcription activation domain and the PDZ-binding motif, and were able to act as dominant negative mutants that attenuated neuronal death.

The loss of core components of the Hippo pathway can lead to uncontrolled cell growth, which indicates that this is a tumor suppressor pathway. Indeed, YAP was proposed as both, an oncogene in liver (Zender et al. 2006) and tumor suppressor in certain class of breast cancers (Yuan et al. 2008). Study of YAP expression in common solid tumors, including colon, lung, ovary and breast tumors, revealed relative changes in subcellular localization of YAP in neoplastic cells compared to the normal controls (Steinhardt et al. 2008). Increase in both, cytoplasmic and nuclear localization of YAP protein was generally observed in these tumors. Since YAP is a nuclear effector of the Hippo pathway (Huang et al. 2005; Oka et al. 2008), controlling its translocation to the nucleus by interfering with the PDZ domain-mediated complex(es) through small molecule inhibitors, or by up-regulating the expression of the PDZ domain-containing shuttle protein, one should be able to control growth of cancer cells and perhaps redirect them toward apoptotic death (Oka & Sudol 2008).


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
Endnote
 References
 
Cell culture and transfections

Human embryonic kidney cell line, known as HEK 293, was originally generated by transformation of cultures of normal human embryonic kidney cells with a sheared adenovirus 5 DNA (Graham et al. 1977). HEK293 and NIH3T3 cells were obtained from ATCC and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were transiently transfected using Lipofectamine (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions.

Plasmids

Delta C mutant of human YAP2 was generated as follows: the Kpn I–Hind III fragment was excised from p2xFlag-CMV2 YAP2 (Oka et al. 2008) and was ligated into pQE-TriSystem His-Strep 2 Vector (Qiagen, Valencia, CA). The Hind III site at its 3' end was blunted prior to self-ligation. The resulting sequence (gaaagctagctt) contains a stop codon, which leads to the truncation of the last 5 amino acids in YAP2. Thus generated cDNA was subcloned into pcDNA4His-MAX B vector (Invitrogen) and pcDNA4/TO/myc-His vector (Invitrogen) by using KpnI–NotI sites. Next, the KpnI–XbaI fragment was subcloned into pEGFP-C3 vector (Clontech, Palo Alto, CA) and p2xFlag-CMV2 vector. All other plasmids were generated as described previously (Oka et al. 2008).

Antibodies and immunoprecipitation

HA antibody (Y-11) was purchased from Santa Cruz Biotechnology. Anti-Poly-[ADP-Ribose]-Polymerase (PARP) was obtained from Roche. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was from Abcam. Active Caspase 3 antibody and Flag-M2 antibody were from Sigma. Polyclonal antibody against human YAP was generated in rabbits as described previously (Sudol et al. 1995; Oka et al. 2008). Immunoprecipitation was conducted as described before (Oka et al. 2008).

Establishment of cell lines that express YAP in an inducible system

pcDNA4/TO/myc-His YAP2 delta C was transfected into HEK293 cells which had been already transfected with pcDNA6/TR vector (Invitrogen) and selected by adding Blasticidin to a final concentration of 5 µg/mL. Second selection was performed by adding both Blasticidin (5 µg/mL) and Zeocin (400 µg/mL). Tetracycline was added to the medium to a final concentration of 1 µg/mL in order to induce the expression of YAP delta C. The same protocol was used for NIH3T3 cells, except a higher concentration of Blasticidin (10 µg/mL) was used in the selection of transformants. All other cell lines were generated as described previously (Oka et al. 2008).

Cell counting assay

HEK293 cells were distributed in DMEM containing 1% FBS, Blasticidin (5 µg/mL), and Zeocin (200 µg/mL), at an initial density of about 2%–3%. After 12 h, Tetracycline (1 µg/mL) was added to the medium to induce the expression of YAPs. After 96 h, floating cells were removed and attached cells were trypsinized and counted. For NIH3T3 cells, more FBS and Blasticidin were added to the medium at 2.5% and 10 µg/mL, respectively.

Fluorescence microscopy

After transfecting appropriate expression plasmids, HEK293 cells were cultured for 24 h. The nuclei were stained with DAPI (Invitrogen) according to manufacturer's instructions and the cells were observed by fluorescence microscopy.


   Endnote
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 Endnote
References
 
The recombinant plasmids reported in this article have been deposited in the Addgene plasmid repository (http://www.addgene.org).


   Acknowledgements
 
The authors thank Drs. Sadchev Sidhu, David Gfeller and Gary Bader (University of Toronto, Canada) for sharing with us unpublished data on the PDZ domain mapping and for helping us with the discussion of our data. Dr. Heather King is thanked for helping us with the generation of mutated YAP cDNA constructs. Dr. Robert Steele (University of California at Irvine) is acknowledged for the sequences of hydra and anemone YAP. Dr. William Schwindinger is thanked for valuable comments on the manuscript. Our research was supported by grants from the PA Department of Health and Breast Cancer Coalition. The authors declare no conflict of interest.


   Footnotes
 
Communicated by: Tadashi Yamamoto

Dedicated to the memory of Hidesaburo Hanafusa.

* Correspondence: msudol1{at}geisinger.edu


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 Endnote
 References
 
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Received: 6 November 2008
Accepted: 16 February 2009





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