A protein module called the WW domain recognizes and binds to a short oligopeptide called the PY motif, PPxY, to mediate protein-protein interactions. The PY motif is present in the transcription activation domains of a wide range of transcription factors including c-Jun, AP-2, NF-E2, C/EBPalpha and PEBP2/CBF, suggesting that it plays an important role in transcriptional activation. Mutation of the PY motif in the subregion of the activation domain of the DNA-binding subunit of PEBP2, PEBP2alpha, abolishes its transactivation function. Using yeast two-hybrid screening, it has been demonstrated that Yes-associated protein (YAP) binds to the PY motif of PEBP2alpha through its WW domain. The C-terminal region of YAP fused to the DNA-binding domain of GAL4 shows transactivation as strong as that of GAL4-VP16. Exogenously expressed YAP confers transcription-stimulating activity on the PY motif fused to the GAL4 DNA-binding domain as well as to native PEBP2alpha. The osteocalcin promoter is stimulated by exogenous PEBP2alphaA and a dominant negative form of YAP strongly inhibits this activity, suggesting YAP involvement in this promoter activity in vivo. These results indicate that the PY motif is a novel transcription activation domain that functions by recruiting YAP as a strong transcription activator to target genes (Yagi, 1999).
Specific protein-protein interactions are involved in a large number of cellular processes and are mainly mediated by structurally and functionally defined domains. The nuclear phosphoprotein p73 can engage in a physical association with the Yes-associated protein (YAP). This association occurs under physiological conditions as shown by reciprocal co-immunoprecipitation of complexes from lysates of P19 cells. The WW domain of YAP and the PPPPY motif of p73 are directly involved in the association. Furthermore, as required for ligands to group I WW domains, the terminal tyrosine (Y) of the PPPPY motif of p73 is essential for the association with YAP. Unlike p73alpha, p73beta, and p63alpha, which bind to YAP, the endogenous as well as exogenously expressed wild-type p53 (wt-p53) and the p73gamma isoform do not interact with YAP. Indeed, YAP interacts only with those members of the p53 family that have a well conserved PPXY motif, a target sequence for WW domains. Overexpression of YAP causes an increase of p73alpha transcriptional activity. Differential interaction of YAP with members of the p53 family may provide a molecular explanation for their functional divergence in signaling (Strano, 2001).
The transcriptional coactivator Yes-associated protein (YAP) has been shown to interact with and to enhance p73-dependent apoptosis in response to DNA damage. YAP requires the promyelocytic leukemia gene (PML) and nuclear body localization to coactivate p73. YAP imparts selectivity to p73 by promoting the activation of a subset of p53 and/or p73 target promoters. Endogenous p73, YAP, and p300 proteins are concomitantly recruited onto the regulatory regions of the apoptotic target gene p53AIP1 only when cells are exposed to apoptotic conditions. Silencing of YAP by specific siRNA impairs p300 recruitment and reduces histone acetylation on the p53AIP1 target gene, resulting in delayed or reduced apoptosis mediated by p73. YAP contributes to the DNA damage-induced accumulation of p73 and potentiates the p300-mediated acetylation of p73. Altogether, these findings identify YAP as a key determinant of p73 gene targeting in response to DNA damage (Strano, 2005).
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, the expression has been documented 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, 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. These 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 (Komuro, 2003).
It has been proposed that ligand-dependent Regulated Intramembrane Proteolysis (RIP) of ErbB-4 receptors generates 80 kDa Intra-Cellular Domains (E4.ICDs) that relocate to the nuclear compartments where they implement the signaling abilities of the ErbB-4 receptors. The E4.ICD may directly regulate gene transcription or, in an alternative scenario, the tyrosine kinase activity of E4.ICDs may target proteins involved in transcriptional regulation upon its relocation into the nucleus. The transcriptional coactivator YAP65, here referred to as YAP (Yes Associated Protein), has been identified as binding partner of ErbB-4 in a two hybrid screening in yeast. Interaction between YAP and ErbB-4 occurs via the WW domain of YAP and the PPPPY at positions 1297-1301 and the PPPAY at positions 1052-1056 of the amino acid sequence of the Cyt-1 isoform of ErbB-4. Stechiometry of binding is regulated by the ligand-dependent phosphorylation of Tyr 1056 in the PPPAYTPM module that functions as a 'biochemical switch' to decrease the association of YAP to ErbB-4. In principle, this novel interaction highlights new mechanisms of signaling propagation from the ErbB-4 receptors, offering supporting evidences that the E4.ICDs forms released following ligand-receptor engagement may recruit YAP and relocate to the nucleus to implement or regulate transcription (Omerovic, 2004).
The WW domain-containing oxidoreductase, WWOX, is a tumor suppressor that is deleted or altered in several cancer types. WWOX interacts with p73 and AP-2gamma and suppresses their transcriptional activity. Yes-associated protein (YAP), also containing WW domains, associates with p73 and enhances its transcriptional activity. In addition, YAP interacts with ErbB-4 receptor tyrosine kinase and acts as transcriptional coactivator of the COOH-terminal fragment (CTF) of ErbB-4. Stimulation of ErbB-4-expressing cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) results in the proteolytic cleavage of its cytoplasmic domain and translocation of this domain to the nucleus. WWOX physically associates with the full-length ErbB-4 via its first WW domain. Coexpression of WWOX and ErbB-4 in HeLa cells followed by treatment with TPA results in the retention of ErbB-4 in the cytoplasm. Moreover, in MCF-7 breast carcinoma cells, expressing high levels of endogenous WWOX, endogenous ErbB-4 is also retained in the cytoplasm. In addition, the results show that interaction of WWOX and ErbB-4 suppresses transcriptional coactivation of CTF by YAP in a dose-dependent manner. A mutant form of WWOX lacking interaction with ErbB-4 has no effect on this coactivation of ErbB-4. Furthermore, WWOX is able to inhibit coactivation of p73 by YAP. In summary, these data indicate that WWOX antagonizes the function of YAP by competing for interaction with ErbB-4 and other targets and thus affect its transcriptional activity (Aqeilan, 2005).
Mammals express four highly conserved TEAD/TEF transcription factors (see Drosophila Scalloped) that bind the same DNA sequence, but serve different functions during development. TEAD-2/TEF-4 protein purified from mouse cells is associated predominantly with a novel TEAD-binding domain at the amino terminus of YAP65, a powerful transcriptional coactivator. YAP65 interacts specifically with the carboxyl terminus of all four TEAD proteins. Both this interaction and sequence-specific DNA binding by TEAD are required for transcriptional activation in mouse cells. Expression of YAP in lymphocytic cells that normally do not support TEAD-dependent transcription (e.g., MPC11) result in up to 300-fold induction of TEAD activity. Conversely, TEAD overexpression squelches YAP activity. Therefore, the carboxy-terminal acidic activation domain in YAP is the transcriptional activation domain for TEAD transcription factors. However, whereas TEAD was concentrated in the nucleus, excess YAP65 accumulates in the cytoplasm as a complex with the cytoplasmic localization protein, 14-3-3. Because TEAD-dependent transcription is limited by YAP65, and YAP65 also binds Src/Yes protein tyrosine kinases, it is proposed that YAP65 regulates TEAD-dependent transcription in response to mitogenic signals (Vassilev, 2001).
The Yes-associated protein (YAP) transcriptional coactivator is a key regulator of organ size and a candidate human oncogene inhibited by the Hippo tumor suppressor pathway. The TEAD family of transcription factors binds directly to and mediates YAP-induced gene expression. This study reports the three-dimensional structure of the YAP (residues 50-171)-TEAD1 (residues 194-411) complex, in which YAP wraps around the globular structure of TEAD1 and forms extensive interactions via three highly conserved interfaces. Interface 3, including YAP residues 86-100, is most critical for complex formation. This study reveals the biochemical nature of the YAP-TEAD interaction, and provides a basis for pharmacological intervention of YAP-TEAD hyperactivation in human diseases (Li, 2010).
The Hippo signaling pathway controls cell growth, proliferation, and apoptosis by regulating the expression of target genes that execute these processes. Acting downstream from this pathway is the YAP transcriptional coactivator, whose biological function is mediated by the conserved TEAD family transcription factors. The interaction of YAP with TEADs is critical to regulate Hippo pathway-responsive genes. This study describe the crystal structure of the YAP-interacting C-terminal domain of TEAD4 in complex with the TEAD-interacting N-terminal domain of YAP. The structure reveals that the N-terminal region of YAP is folded into two short helices with an extended loop containing the PXXPhiP motif in between, while the C-terminal domain of TEAD4 has an immunoglobulin-like fold. YAP interacts with TEAD4 mainly through the two short helices. Point mutations of TEAD4 indicate that the residues important for YAP interaction are required for its transforming activity. Mutagenesis reveals that the PXXPhiP motif of YAP, although making few contacts with TEAD4, is important for TEAD4 interaction as well as for the transforming activity (Chen, 2010).
Outside cells of the preimplantation mouse embryo form the trophectoderm (TE), a process requiring the transcription factor Tead4. This study shows that transcriptionally active Tead4 can induce Cdx2 and other trophoblast genes in parallel in embryonic stem cells. In embryos, the Tead4 coactivator protein Yap localizes to nuclei of outside cells, and modulation of Tead4 or Yap activity leads to changes in Cdx2 expression. In inside cells, Yap is phosphorylated and cytoplasmic, and this involves the Hippo signaling pathway component Lats. It is proposed that active Tead4 promotes TE development in outside cells, whereas Tead4 activity is suppressed in inside cells by cell contact- and Lats-mediated inhibition of nuclear Yap localization. Thus, differential signaling between inside and outside cell populations leads to changes in cell fate specification during TE formation (Nishioka, 2009).
During mouse development, the first lineage specified is the trophoblast/placenta lineage, set aside during blastocyst formation. In the blastocyst, the trophoblast, or trophectoderm (TE), surrounds the inner cell mass (ICM), which will give rise to the fetus and other extraembryonic tissues. The homeodomain transcription factor Cdx2 is expressed in the TE at the blastocyst stage. Cdx2 is required for TE development and is sufficient to promote trophoblast fate in ICM-derived embryonic stem (ES) cells, including suppression of ICM and induction of TE genes. Conversely, ICM fates are regulated by a distinct set of transcription factors, including the POU family transcription factor Oct3/4 (encoded by Pou5f1). Prior to blastocyst formation, Cdx2 and Oct3/4 are initially coexpressed throughout the embryo. Mutual antagonism between these two factors may contribute to the eventual segregation of their expression domains, with Cdx2 in outside cells of the TE and Oct3/4 in inside cells of the ICM. However, molecular mechanisms that initially interpret inside/outside positional information within the embryo to establish this pattern are not known (Nishioka, 2009 and references therein).
The TEAD/TEF family transcription factor Tead4 is essential for TE development and Cdx2 expression prior to the blastocyst stage. This observation provided the first clue about molecular mechanisms acting upstream of the TE/ICM lineage distinction. However, whether Tead4 acts permissively or instructively in this process has been unclear, since Tead4 itself is not restricted to outside cells (Nishioka, 2009).
This study sought to identify cofactors and signaling components that could impart positional information to spatially regulate Tead4 activity in the embryo. Many lines of evidence have suggested that TEAD-mediated transcription is regulated by the Ser/Thr kinase Hippo in Drosophila, or Stk3 (Mst) in mice. In Drosophila, Hippo inhibits the Yorkie (Yki) coactivator and suppresses cell proliferation. These activities are mediated by a Tead protein, Scalloped. In mammals, Hippo signaling comprises a growth-regulating pathway, which controls cell contact-mediated inhibition of proliferation. In this context, cell-cell contact regulates nuclear accumulation of a Yki homolog, Yes-associated protein 1 (Yap1, Yap hereafter), through Hippo signaling and controls cell proliferation by regulating transcriptional activity of Tead proteins. In the mouse embryo, Tead1-/-; Tead2-/- mutants die soon after implantation due to reduced cell proliferation and increased apoptosis. Tead1/2 interact genetically with Yap, suggesting that the roles of these genes in Hippo signaling are conserved in mice (Nishioka, 2009).
This study examined the role of Tead4 in TE development using both ES cells and preimplantation embryos. Active Tead4 can promote multiple trophoblast genes in parallel, including Cdx2. In the embryo, the Tead coactivator Yap localizes to nuclei only in outside cells and is excluded from inside cell nuclei by the Hippo signaling pathway component Lats. These observations suggest that Tead4/Yap interpret positional information along the inside/outside axis of the embryo to restrict expression of Cdx2 and TE fates to outside cells (Nishioka, 2009).
Yes-associated protein (YAP) is a potent transcription coactivator acting via binding to the TEAD transcription factor, and plays a critical role in organ size regulation. YAP is phosphorylated and inhibited by the Lats kinase, a key component of the Hippo tumor suppressor pathway. Elevated YAP protein levels and gene amplification have been implicated in human cancer. This study reports that YAP is inactivated during embryonic stem (ES) cell differentiation, as indicated by decreased protein levels and increased phosphorylation. Consistently, YAP is elevated during induced pluripotent stem (iPS) cell reprogramming. YAP knockdown leads to a loss of ES cell pluripotency, while ectopic expression of YAP prevents ES cell differentiation in vitro and maintains stem cell phenotypes even under differentiation conditions. Moreover, YAP binds directly to promoters of a large number of genes known to be important for stem cells and stimulates their expression. These observations establish a critical role of YAP in maintaining stem cell pluripotency (Lian, 2010).
The Hippo pathway defines a novel signaling cascade regulating cell proliferation and survival in Drosophila, which involves the negative regulation of the transcriptional coactivator Yorkie by the kinases Hippo and Warts. The human ortholog of Yorkie, YAP, maps to a minimal amplification locus in mouse and human cancers, and it mediates dramatic transforming activity in MCF10A primary mammary epithelial cells. This study shows that LATS proteins (mammalian orthologs of Warts) interact directly with YAP in mammalian cells and that ectopic expression of LATS1, but not LATS2, effectively suppresses the YAP phenotypes. Furthermore, shRNA-mediated knockdown of LATS1 phenocopies YAP overexpression. Because this effect can be suppressed by simultaneous YAP knockdown, it suggests that YAP is the primary target of LATS1 in mammalian cells. Expression profiling of genes induced by ectopic expression of YAP or by knockdown of LATS1 reveals a subset of potential Hippo pathway targets implicated in epithelial-to-mesenchymal transition, suggesting that this is a key feature of YAP signaling in mammalian cells (Zhang, 2009).
Several developmental pathways contribute to processes that regulate tissue growth and organ size. The Hippo pathway has emerged as one such critical regulator. However, how Hippo signaling is integrated with other pathways to coordinate these processes remains unclear. This study shows that the Hippo pathway restricts Wnt/β-Catenin signaling by promoting an interaction between TAZ (a yorkie homolog, sharing approximately 50% sequence identity with YAp, with a similar topology, containing two central WW domains and a C-terminal transactivation domain) and DVL in the cytoplasm. TAZ inhibits the CK1δ/epsilon-mediated phosphorylation of DVL, thereby inhibiting Wnt/β-Catenin signaling. Abrogation of TAZ levels or Hippo signaling enhances Wnt3A-stimulated DVL phosphorylation, nuclear β-Catenin, and Wnt target gene expression. Mice lacking Taz develop polycystic kidneys with enhanced cytoplasmic and nuclear β-Catenin. Moreover, in Drosophila, Hippo signaling modulates Wg target gene expression. These results uncover a cytoplasmic function of TAZ in regulating Wnt signaling and highlight the role of the Hippo pathway in coordinating morphogenetic signaling with growth control (Varelas, 2010).
The Hpo/Wts kinase, which in vertebrates is represented by MST and LATS, respectively, converges on the transcriptional regulator Yorkie in Drosophila, or the vertebrate homologs, TAZ and YAP. Recent studies have focused on the transcriptional roles for Yorkie, TAZ, and YAP in controlling growth and cell fate choice. However, these proteins are also found in the cytoplasm, where TAZ has been shown to associate with the plasma membrane and actin cytoskeleton. TAZ cytoplasmic localization is mediated by LATS-dependent phosphorylation of serine 89, which drives binding to the cytoplasmic retention factor, 14-3-3. Altogether, these findings raised the possibility that TAZ has cytoplasmic functions. This study demonstrates that one such function is to constrain Wnt signaling by inhibiting CK1δ/epsilon-mediated DVL phosphorylation (see model). Although the ability of CK1δ/epsilon to phosphorylate DVL has been well established, the precise role of this event in Wnt signaling remains to be fully understood. This study has observed that CK1δ/epsilon-independent signaling can contribute to Wnt-induced β-Catenin stabilization. Nevertheless, the data clearly show that DVL phosphorylation and Wnt pathway activation caused by the loss of TAZ expression requires CK1δ/epsilon. Abrogation of LATS expression, which leads to the nuclear accumulation of TAZ, reduced TAZ-DVL interaction, enhanced DVL phosphorylation, and promoted Wnt pathway activation in cells. Furthermore, it was shown that loss of hpo or wts function or enhanced expression of yki in Drosophila wing imaginal discs also modulates Wnt signaling. Thus, by promoting the cytoplasmic localization of TAZ, the Hippo pathway not only prevents nuclear TAZ activity, but also leads to inhibition of Wnt signaling by antagonizing DVL activation. This study has focused on TAZ, but it will be interesting to determine whether the related protein, YAP, has a similar activity in the Wnt pathway. Indeed, functional redundancy is suggested by the Taz/Yap double-knockout mice, which display early embryonic lethality (Varelas, 2010).
Wnts are known to act throughout mammalian kidney development, and hyperactivation of Wnt/β-Catenin signaling causes severe kidney defects. For example, loss of Apc function results in nuclear β-Catenin accumulation that leads to cystic kidneys, and transgenic mice expressing an activated mutant of β-Catenin also develop cysts. Taz mutant mice also develop polycystic kidneys, and increased cytoplasmic and nuclear β-Catenin was found in both cystic and precystic regions of Tazgt/gt kidneys, indicating Wnt pathway activation. Moreover, cysts formed in humans with ADPKD, caused by mutations in PC1 or PC2, display a gene expression profile consistent with Wnt pathway activation. β-Catenin associates with PC1 (Lal, 2008), and TAZ has been proposed to indirectly regulate the activity of PC2 via the SCF ubiquitin ligase β-TrCP. It was confirmed that TAZ and TAZ (1-393, ΔWW), the DVL-binding mutant that cannot rescue the effect of loss of TAZ on Wnt signaling, both interact with β-TrCP. Thus, it is concluded that TAZ modulates Wnt signaling via direct regulation of DVL; however, TAZ may also function indirectly to regulate the PC complex via β-TrCP. Whether TAZ may have other additional roles in Wnt signaling remains to be determined. Altogether, these results suggest that, in the kidney, TAZ restricts canonical Wnt signaling that otherwise would disrupt tubule morphogenesis and the formation of cysts (Varelas, 2010).
Defects in cilia are also known to be involved in the pathogenesis of polycystic kidney disease. Indeed, recent analyses of Taz mutant kidneys have revealed that Taz localizes to cilia, and that cystic regions in Taz −/− kidneys have underdeveloped cilia. Interestingly, primary cilia have recently been implicated in restraining Wnt signaling (Corbit, 2008), and DVL has been reported in cilia (Park, 2008). These findings raise the intriguing possibility that TAZ may link suppression of DVL activation in the kidney to ciliogenesis. Of note, no evidence was found that the MDA-MB-231 breast cancer cells used in this study possess cilia, indicating that cilia, per se, are not required for TAZ inhibition of Wnt signaling. Nevertheless, it will be interesting to examine the relationship between TAZ regulation of ciliogenesis and DVL modulation in kidney epithelia (Varelas, 2010).
The development of multicellular organisms requires cell-cell communication and the coordinated action of distinct signaling pathways for proper patterning of the body and organs. In the Drosophila wing imaginal disc, the Wg morphogen gradient coordinates the expression of an array of developmentally important genes. These studies reveal that the Hippo pathway functions to restrain Wg signaling, and that loss of hpo or wts increases the levels of Arm (β-Catenin) outside the normal expression domain and enhances expression of Wg target genes, particularly at a distance from the Wg source. Thus, Hippo pathway activation may, in effect, serve to restrain the sensitivity of cells to Wg signals, thus sharpening the Wg response gradients to promote appropriate tissue patterning events. How the Hippo kinase cascade is regulated by upstream pathways remains unclear. However, genetic studies place a protocadherin, Fat, and an ERM domain protein, Expanded, upstream of Hpo/Wts kinases. Furthermore, and in accordance with analysis of hpo and wts mutants, fat and expanded mutants display enhanced Wg target gene expression independent of Wg ligand production. The results suggest that this occurs via a direct molecular link. In this regard, it is intriguing to note that mice null for Fat4, a mammalian homolog of Drosophila fat, also develop polycystic kidneys. Direct communication between the Hippo and Wnt pathways may thus be an ancient mechanism that coordinates morphogen signaling with tissue growth control in animals (Varelas, 2010).
This study shows that TAZ inhibits Wnt signaling via DVL, providing for a direct molecular link between the Hippo and Wnt signaling pathways. Other studies in Drosophila have implicated the Hippo pathway as a positive regulator of Notch signaling, and an essential role for TAZ in mediating TGFβ-dependent Smad signaling has been shown. Furthermore, TAZ is induced by BMP signaling to regulate mesenchymal cell differentiation. Altogether, these studies suggest a critical and direct role for TAZ in integrating signals from numerous morphogen signaling pathways (Varelas, 2010).
The Hippo signaling pathway plays an important role in regulation of cell proliferation. Cell density regulates the Hippo pathway in cultured cells; however, the mechanism by which cells detect density remains unclear. This study demonstrates that changes in cell morphology are a key factor. Morphological manipulation of single cells without cell-cell contact resulted in flat spread or round compact cells with nuclear or cytoplasmic Yap, respectively. Stress fibers increased in response to expanded cell areas, and F-actin regulated Yap downstream of cell morphology. Cell morphology- and F-actin-regulated phosphorylation of Yap, and the effects of F-actin were suppressed by modulation of Lats (Warts in Drosophila). These results suggest that cell morphology is an important factor in the regulation of the Hippo pathway, which is mediated by stress fibers consisting of F-actin acting upstream of, or on Lats, and that cells can detect density through their resulting morphology. This cell morphology (stress-fiber)-mediated mechanism probably cooperates with a cell-cell contact (adhesion)-mediated mechanism involving the Hippo pathway to achieve density-dependent control of cell proliferation (Wada, 2011).
To understand the role of the Yes-associated protein (YAP), binding partners of its WW1 domain were isolated by a yeast two-hybrid screen. One of the interacting proteins was identified as p53-binding protein-2 (p53BP-2). YAP and p53BP-2 interact in vitro and in vivo using their WW1 and SH3 domains, respectively. The YAP WW1 domain binds to the YPPPPY motif of p53BP-2, whereas the p53BP-2 SH3 domain interacts with the VPMRLR sequence of YAP, which is different from other known SH3 domain-binding motifs. By mutagenesis, this unusual SH3 domain interaction was shown to be due to the presence of three consecutive tryptophans located within the betaC strand of the SH3 domain. A point mutation within this triplet, W976R, restores the binding selectivity to the general consensus sequence for SH3 domains, the PXXP motif. A constitutively active form of c-Yes was shown to decrease the binding affinity between YAP and p53BP-2 using chloramphenicol acetyltransferase/enzyme-linked immunosorbent assay, whereas the overexpression of c-Yes does not modify this interaction. Since overexpression of an activated form of c-Yes results in tyrosine phosphorylation of p53BP-2, it is proposed that the p53BP-2 phosphorylation, possibly in the WW1 domain-binding motif, might negatively regulate the YAP.p53BP-2 complex (Espanel, 2001).
Members of the TGF-beta family of growth factors signal from the cell surface through serine/threonine kinase receptors. Intracellular propagation of the signal occurs by phosphorylation of intracellular proteins of the Smad family. Smad7 belongs to the subclass of inhibitory Smads that function as antagonists of TGF-beta signaling. A yeast two-hybrid screen of a human placental cDNA expression library using full-length mouse Smad7 as bait identified Yes-Associated Protein (YAP65) as a novel Smad7-interacting protein. The association of Smad7 with YAP65 was confirmed using co-expressed tagged proteins in COS-7 cells. Deletion of the PY motif of Smad7 reduced but did not abolish YAP65-Smad7 association, suggesting the existence of several interacting domains. YAP65 potentiates the inhibitory activity of Smad7 against TGF-beta-induced, Smad3/4-dependent, gene transactivation. Furthermore, YAP65 augments the association of Smad7 to activated TGF-beta receptor type I (TbetaRI), whereas YAP65(1-301), which exerts a dominant-negative effect against Smad7-driven inhibition of TGF-beta signaling, reduces these interactions. Together, these data provide the first evidence that YAP65 is a Smad7 partner that facilitates the recruitment of the latter to activated TbetaRI, and enhances the inhibitory activity of Smad7 against TGF-beta signaling (Ferrigno, 2002).
Although initially described as a cytosolic scaffolding protein, YAP (Yes-associated protein of 65 kDa) is known to associate with multiple transcription factors in the nucleus. Using affinity chromatography and mass spectrometry, YAP was shown to interact with heterogeneous nuclear ribonuclear protein U (hnRNP U), an RNA- and DNA-binding protein enriched in the nuclear matrix that also plays a role in the regulation of gene expression. hnRNP U interacts specifically with the proline-rich amino terminus of YAP, a region of YAP that is not found in the related protein TAZ. Although hnRNP U and YAP localize to both the nucleus and the cytoplasm, YAP does not translocate to the nucleus in an hnRNP U-dependent manner. Furthermore, hnRNP U and YAP interact only in the nucleus, suggesting that the association between the two proteins is regulated. Co-expression of hnRNP U attenuates the ability of YAP to increase the activity of a p73-driven Bax-luciferase reporter plasmid. In contrast, hnRNP U has no effect when co-expressed with a truncated YAP protein lacking the hnRNP U-binding site. Because YAP is distinguished from the homologue TAZ by its proline-rich amino terminus, the YAP-hnRNP U interaction may uniquely regulate the nuclear function(s) of YAP. The YAP-hnRNP U interaction provides another mechanism of YAP transcriptional regulation (Howell, 2004).
Src/Yes tyrosine kinase signaling contributes to the regulation of bone homeostasis and inhibits osteoblast activity. The endogenous Yes-associated protein (YAP), a mediator of Src/Yes signaling, interacts with the native Runx2 protein, an osteoblast-related transcription factor, and suppresses Runx2 transcriptional activity in a dose-dependent manner. Runx2, through its PY motif, recruits YAP to subnuclear domains in situ and to the osteocalcin (OC) gene promoter in vivo. Inhibition of Src/Yes kinase blocks tyrosine phosphorylation of YAP and dissociates endogenous Runx2-YAP complexes. Consequently, recruitment of the YAP co-repressor to subnuclear domains is abrogated and expression of the endogenous OC gene is induced. These results suggest that Src/Yes signals are integrated through organization of Runx2-YAP transcriptional complexes at subnuclear sites to attenuate skeletal gene expression (Zaidi, 2004).
The size, composition and functioning of the spinal cord is likely to depend on appropriate numbers of progenitor and differentiated cells of a particular class, but little is known about how cell numbers are controlled in specific cell cohorts along the dorsoventral axis of the neural tube. This study shows that FatJ cadherin, identified in a large-scale RNA interference (RNAi) screen of cadherin genes expressed in the neural tube, is localised to progenitors in intermediate regions of the neural tube. Loss of function of FatJ promotes an increase in dp4-vp1 progenitors and a concomitant increase in differentiated Lim1(+)/Lim2(+) neurons. These studies reveal that FatJ mediates its action via the Hippo pathway mediator Yap1: loss of downstream Hippo components can rescue the defect caused by loss of FatJ. Together, these data demonstrate that RNAi screens are feasible in the chick embryonic neural tube, and show that FatJ acts through the Hippo pathway to regulate cell numbers in specific subsets of neural progenitor pools and their differentiated progeny (Van Hateren, 2011).
An affinity purification method has been used to identify substrates of protein kinase B/Akt (see Drosophila Akt). One protein that associates with 14-3-3 in an Akt-dependent manner is shown to be the Yes-associated protein (YAP), which is phosphorylated by Akt at serine 127, leading to binding to 14-3-3. Akt promotes YAP localization to the cytoplasm, resulting in loss from the nucleus where it functions as a coactivator of transcription factors including p73. p73-mediated induction of Bax expression following DNA damage requires YAP function and is attenuated by Akt phosphorylation of YAP. YAP overexpression increases, while YAP depletion decreases, p73-mediated apoptosis following DNA damage, in an Akt inhibitable manner. Akt phosphorylation of YAP may thus suppress the induction of the proapoptotic gene expression response following cellular damage (Basu, 2003).
YAP is a 65 kDa protein (sometimes termed YAP65 or YAP1) that was originally identified due to its interaction with the Src family tyrosine kinase Yes. YAP contains either one or two WW domains depending on alternative splicing and also a PDZ interaction motif, an SH3 binding motif, and a coiled-coil domain. YAP has been reported to interact with p53 binding protein-2, an important regulator of the apoptotic activity of p53. Through its carboxyl terminus, YAP binds to the PDZ-containing protein EBP50, a submembranous scaffolding protein. YAP is a transcriptional coactivator that binds and activates Runx transcription factors and the four TEAD/TEF transcription factors. YAP is homologous to TAZ (45% identity), a transcriptional coactivator that is regulated by interaction with 14-3-3 and PDZ domain-containing proteins. YAP also interacts with the p53 family member p73, resulting in an enhancement of p73's transcriptional activity. YAP phosphorylation by Akt suppresses its ability to promote p73-mediated transcription of proapoptotic genes in response to DNA damaging agents and the resulting cell death. This extends the range of mechanisms whereby Akt can promote cellular survival in the face of apoptotic stimuli (Basu, 2003).
The conserved Hippo signaling pathway regulates organ size in Drosophila and mammals. While a core kinase cascade leading from the protein kinase Hippo (Hpo) (Mst1 and Mst2 in mammals) to the transcription coactivator Yorkie (Yki) (YAP in mammals) has been established, upstream regulators of the Hippo kinase cascade are less well defined, especially in mammals. Using conditional knockout mice, it was demonstrated that the Merlin/NF2 tumor suppressor and the YAP oncoprotein function antagonistically to regulate liver development. While inactivation of Yap led to loss of hepatocytes and biliary epithelial cells, inactivation of Nf2 led to hepatocellular carcinoma and bile duct hamartoma. Strikingly, the Nf2-deficient phenotypes in multiple tissues were largely suppressed by heterozygous deletion of Yap, suggesting that YAP is a major effector of Merlin/NF2 in growth regulation. These studies link Merlin/NF2 to mammalian Hippo signaling and implicate YAP activation as a mediator of pathologies relevant to Neurofibromatosis 2 (Zhang, 2010).
In a screen for gene copy-number changes in mouse mammary tumors, a tumor was identified with a small 350-kb amplicon from a region that is syntenic to a much larger locus amplified in human cancers at chromosome 11q22. The mouse amplicon contains only one known gene, Yap, encoding the mammalian ortholog of Drosophila Yorkie (Yki), a downstream effector of the Hippo(Hpo)–Salvador(Sav)–Warts(Wts) signaling cascade, identified in flies as a critical regulator of cellular proliferation and apoptosis. In nontransformed mammary epithelial cells, overexpression of human YAP induces epithelial-to-mesenchymal transition, suppression of apoptosis, growth factor-independent proliferation, and anchorage-independent growth in soft agar. Together, these observations point to a potential oncogenic role for YAP in 11q22-amplified human cancers, and they suggest that this highly conserved signaling pathway identified in Drosophila regulates both cellular proliferation and apoptosis in mammalian epithelial cells (Overholtzer, 2006; full text of article).
TAZ is a WW domain containing a transcription coactivator that modulates mesenchymal differentiation and development of multiple organs. The TAZ transcription coactivator is closely related to YAP, which is the mammalian ortholog of the Drosophila Yki, a key component in the Hippo pathway. This study shows that TAZ is phosphorylated by the Lats tumor suppressor kinase, a key component of the Hippo pathway, whose alterations result in organ and tissue hypertrophy in Drosophila and contribute to tumorigenesis in humans. Lats phosphorylates TAZ on several serine residues in the conserved HXRXXS motif and creates 14-3-3 binding sites, leading to cytoplasmic retention and functional inactivation of TAZ. Ectopic expression of TAZ stimulates cell proliferation, reduces cell contact inhibition, and promotes epithelial-mesenchymal transition (EMT). Elimination of the Lats phosphorylation sites results in a constitutively active TAZ, enhancing the activity of TAZ in promoting cell proliferation and EMT. The results elucidate a molecular mechanism for TAZ regulation and indicate a potential function of TAZ as an important target of the Hippo pathway in regulating cell proliferation tumorigenesis (Lei, 2008).
Regulation of organ size is important for development and tissue homeostasis. In Drosophila, Hippo signaling controls organ size by regulating the activity of a TEAD transcription factor, Scalloped, through modulation of its co-activator protein Yki. This study shows that mouse Tead proteins regulate cell proliferation by mediating Hippo signaling. In NIH3T3 cells, cell density and Hippo signaling regulated the activity of endogenous Tead proteins by modulating nuclear localization of a Yki homolog, Yap1, and the resulting change in Tead activity altered cell proliferation. Tead2-VP16 mimicked Yap1 overexpression, including increased cell proliferation, reduced cell death, promotion of EMT, lack of cell contact inhibition and promotion of tumor formation. Growth-promoting activities of various Yap1 mutants correlated with their Tead-co-activator activities. Tead2-VP16 and Yap1 regulated largely overlapping sets of genes. However, only a few of the Tead/Yap1-regulated genes in NIH3T3 cells were affected in Tead1-/-;Tead2-/- or Yap1-/- embryos. Most of the previously identified Yap1-regulated genes were not affected in NIH3T3 cells or mutant mice. In embryos, levels of nuclear Yap1 and Tead1 varied depending on cell type. Strong nuclear accumulation of Yap1 and Tead1 were seen in myocardium, correlating with requirements of Tead1 for proliferation. However, their distribution did not always correlate with proliferation. Taken together, mammalian Tead proteins regulate cell proliferation and contact inhibition as a transcriptional mediator of Hippo signaling, but the mechanisms by which Tead/Yap1 regulate cell proliferation differ depending on the cell type, and Tead, Yap1 and Hippo signaling may play multiple roles in mouse embryos (Ota, 2009).
TGF-beta and BMP receptor kinases activate Smad transcription factors by C-terminal phosphorylation. This study identified a subsequent agonist-induced phosphorylation that plays a central dual role in Smad transcriptional activation and turnover. As receptor-activated Smads form transcriptional complexes, they are phosphorylated at an interdomain linker region by CDK8 and CDK9, which are components of transcriptional mediator and elongation complexes. These phosphorylations promote Smad transcriptional action, which in the case of Smad1 is mediated by the recruitment of YAP (Drosophila homolog: Yorkie) to the phosphorylated linker sites. An effector of the highly conserved Hippo organ size control pathway, YAP supports Smad1-dependent transcription and is required for BMP suppression of neural differentiation of mouse embryonic stem cells. The phosphorylated linker is ultimately recognized by specific ubiquitin ligases, leading to proteasome-mediated turnover of activated Smad proteins. Thus, nuclear CDK8/9 drive a cycle of Smad utilization and disposal that is an integral part of canonical BMP and TGF-beta pathways (Alarcon, 2009).
The present findings reveal a remarkable integration of Smad regulatory functions by agonist-induced, CDK8/9-mediated phosphorylation of the linker region and highlight this event as an integral feature of the transcriptional action and turnover of receptor-activated Smad proteins. Agonist-induced linker phosphorylation of R-Smads is a general feature of BMP and TGF-β pathways, occurring in all the responsive cell types examined, shortly after Smad tail phosphorylation. The evidence identifies CDK9 as the kinases involved and does not support a major role for MAPKs or cell-cycle-regulatory CDKs in this process. CDK8 and cyclinC are components of the Mediator complex that couples enhancer-binding transcriptional activators to RNAP II for transcription initiation. CDK9 and cyclinT1 constitute the P-TEFb complex, which promotes transcriptional elongation. CDK8 and CDK9 phosphorylate overlapping serine clusters in the C-terminal domain of RNAP II, a region which integrates regulatory inputs by binding proteins involved in mRNA biogenesis. Thus, CDK8 and CDK9 may provide coordinated regulation of Smad transcriptional activators and RNAP II (Alarcon, 2009).
Precedent exists for the ability of CDK8 to phosphorylate enhancer-binding transcription factors. The CDK8 ortholog Srb10 in budding yeast phosphorylates Gcn4 marking this transcriptional activator of amino acid biosynthesis for recognition by the SCF(Cdc4) ubiquitin ligase. In mammalian cells, CDK8 phosphorylates the ICD signal transduction component of Notch, targeting it to the Fbw7/Sel10 ubiquitin ligase. However, whereas CDK8-mediated phosphorylation inhibits Gcn4 and Notch activity, this study shows that phosphorylation of agonist-activated Smads by CDK8/9 enables Smad-dependent transcription before triggering Smad turnover (Alarcon, 2009).
Activated Smads undergo proteasome-mediated degradation as well as phosphatase-mediated tail dephosphorylation to keep signal transduction closely tied to receptor activation. This study shows that BMP-induced Smad1-ALP generates binding sites for Smurf1, accomplishing in the nucleus what MAPK-mediated phosphorylation of basal-state Smad1 accomplishes in the cytoplasm. Similarly, TGF-β-induced linker phosphorylation of Smad2/3 provides a binding site for Nedd4L (Alarcon, 2009).
The results also reveal a positive role for ALP in Smad-dependent transcription. Smad proteins with phosphorylation-resistant linker mutations are more stable as receptor-activated signal transducers than their wild-type counterparts, yet they are transcriptionally less active. Indeed, mutation of Smad1 linker phosphorylation sites (in a wild-type Smad5 background) does not result in a straight BMP gain-of-function phenotype but rather in an unforeseen gastric epithelial phenotype. Although the interpretation of this phenotype is confounded by the contribution of MAPK signaling to linker phosphorylation, it is consistent with the present evidence that Smad1 linker phosphorylation plays an active role in BMP signaling (Alarcon, 2009).
Focusing on Smad1 to define this dual role, it was found that the phosphorylated linker sites, together with a neighboring PY motif, are recognized also by the transcriptional coactivator YAP. Smurf1 and YAP present closely related WW domains with a similar selectivity toward linker-phosphorylated Smad1. YAP is recruited with Smad1 to BMP responsive enhancers and knockdown of YAP inhibits BMP-induced Id gene responses in mouse embryonic stem cells. Both BMP and YAP act as suppressors of neural differentiation in specific contexts. This study shows that YAP supports the ability of BMP to block neural lineage commitment through the induction of Id family members, creating a link between YAP-dependent BMP transcriptional output and ES cell fate determination (Alarcon, 2009).
Thus, a common structure fulfills two opposite functions -- Smad1 transcriptional action and turnover -- by recruiting different proteins, YAP and Smurf1, at different stages of the signal transduction cycle. The cyclic recruitment and continuous turnover of transcription factors on target enhancers are required for the proper response of cells to developmental and homeostatic cues. It is proposed that Smad activation by TGF-β family agonists accomplishes this important requirement through linker phosphorylation that triggers transcriptional action and messenger turnover in one stroke (Alarcon, 2009).
Activation of the Hippo pathway by cell density cues triggers a kinase cascade that culminates in the inactivation of YAP (Yorkie in Drosophila), a transcriptional coactivator that acts through interactions with enhancer-binding factors, including TEAD/scalloped, Runx, p73, and others. Yorkie/YAP promotes cell proliferation and survival and organ growth, whereas the upstream components of the inhibitory kinase cascade constrain organ size and act as tumor suppressors. Elucidating the links between the Hippo pathway and other signaling cascades is an important open question. The evidence that YAP is recruited to BMP-activated Smad1 reveals a link between the BMP and the Hippo pathways. Both these signaling cascades have the capacity to control organ size and do so in a manner suggestive of interactions with other patterned signals. An example is the regulation of imaginal disc growth by Dpp via cell competition, a process by which slow proliferating cells are eliminated in favor of their higher-proliferating neighbors. A genetic screen for negative regulators of Dpp signaling that protect cells from being outcompeted identified upstream components of the Hippo pathway. Inactivation of these factors elevated Dpp target gene expression, presumably by failing to inhibit Yorkie, and allowed cells to outcompete their neighbors, suggesting a functional convergence of the Hippo and BMP pathways that foreshadowed these findings (Alarcon, 2009).
Although ALP is a general event in Smad activation, YAP may not be a universal partner of linker-phosphorylated Smad1. Smad ALP likely plays a wider role potentially acting to recruit coactivators other than YAP, depending on the cellular context or the target gene. Also of interest is the identity of factors that may play an analogous role in linker-phosphorylated Smad2/3 in the TGF-β pathway. The linker phosphorylation sites and PY motifs of Smad1 and Smad2/3 are conserved in the otherwise divergent linker regions of the Drosophila orthologs Mad/dSmad1 and dSmad2, respectively. Although the contribution of the MAPK pathway in linker phosphorylation precludes a clearcut genetic investigation of these functions, they are probably conserved across metazoans. A concerted search for Smad phospholinker interacting factors would answer many of these questions and would fully elucidate the role of the Smad linker region as a centerpiece in the function, regulation, and connectivity of Smad transcription factors (Alarcon, 2009).
Medulloblastoma is the most common solid malignancy of childhood, with treatment side effects reducing survivors' quality of life and lethality being associated with tumor recurrence. Activation of the Sonic hedgehog (Shh) signaling pathway is implicated in human medulloblastomas. Cerebellar granule neuron precursors (CGNPs) depend on signaling by the morphogen Shh for expansion during development, and have been suggested as a cell of origin for certain medulloblastomas. Mechanisms contributing to Shh pathway-mediated proliferation and transformation remain poorly understood. This study investigated interactions between Shh signaling and the recently described tumor-suppressive Hippo pathway in the developing brain and medulloblastomas. Up-regulation is reported of the oncogenic transcriptional coactivator yes-associated protein 1 (YAP1; homolog of Drosophila Yorkie), which is negatively regulated by the Hippo pathway, in human medulloblastomas with aberrant Shh signaling. Consistent with conserved mechanisms between brain tumorigenesis and development, Shh induces YAP1 expression in CGNPs. Shh also promotes YAP1 nuclear localization in CGNPs, and YAP1 can drive CGNP proliferation. Furthermore, YAP1 is found in cells of the perivascular niche, where proposed tumor-repopulating cells reside. Post-irradiation, YAP1 was found in newly growing tumor cells. These findings implicate YAP1 as a new Shh effector that may be targeted by medulloblastoma therapies aimed at eliminating medulloblastoma recurrence (Fernandez-L, 2009).
This study demonstrates that YAP1 and its transcriptional partner, TEAD1, are highly expressed in Shh-driven medulloblastomas in both humans and mice. YAP1 is amplified in a subset of human medulloblastomas -- specifically, SHH-associated medulloblastomas. Moreover, YAP1 expression is up-regulated by the Shh pathway in proliferating CGNPs. Shh signaling regulates YAP1 nuclear localization through its binding to IRS1, and YAP1 activity promotes CGNP proliferation, at least in part through interactions with TEAD1. In mouse medulloblastomas, YAP1 protein localized to the cells occupying the perivascular niche (PVN) that have been proposed to have cancer stem cell properties. Indeed, YAP1-positive cells remain alive and disseminated through the tumor after the tumor bulk cells have been eradicated by radiation. These findings mark YAP1 as a mediator of normal proliferation in the developing cerebellum, and as a potential target for medulloblastoma therapies aimed at eliminating tumor-reinitiating cells (Fernandez-L, 2009).
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