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).
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).
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).
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).
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