TEAD Activity Research Articles

NOS1AP Functionally Associates with YAP To Regulate Hippo Signaling.

Deregulation of cellular polarity proteins and their associated complexes leads to changes in cell migration and proliferation. The nitric oxide synthase 1 adaptor protein (NOS1AP) associates with the tumor suppressor protein Scribble to control cell migration and oncogenic transformation. However, how NOS1AP is linked to the cell signaling events that curb oncogenic progression has remained elusive. Here we identify several novel NOS1AP isoforms, NOS1APd, NOS1APe, and NOS1APf, with distinct cellular localizations. We show that isoforms with a membrane-interacting phosphotyrosine binding (PTB) domain can associate with Scribble and recognize acidic phospholipids. In a screen to identify novel binding proteins, we have discovered a complex consisting of NOS1AP and the transcriptional coactivator YAP linking NOS1AP to the Hippo signaling pathway. Silencing of NOS1AP reduces the phosphorylation of YAP and of the upstream kinase Lats1. Conversely, expression of NOS1AP promotes YAP and Lats1 phosphorylation, which correlates with reduced TEAD activity and restricted cell proliferation. Together, these data implicate a role for NOS1AP in the regulation of core Hippo signaling and are consistent with the idea that NOS1AP functions as a tumor suppressor.

Cell competition in mouse NIH3T3 embryonic fibroblasts is controlled by the activity of Tead family proteins and Myc

Cell competition is a short-range communication originally observed in Drosophila. Relatively little is known about cell competition in mammals or in non-epithelial cells. Hippo signaling and its downstream transcription factors of the Tead family, control cell proliferation and apoptosis. Here, we established an in vitro model system that shows cell competition in mouse NIH3T3 embryo fibroblast cells. Co-culture of Tead-activity-manipulated cells with normal (wild-type) cells caused cell competition. Cells with reduced Tead activity became losers, whereas cells with increased Tead activity became super-competitors. Tead directly regulated Myc RNA expression, and cells with increased Myc expression also became super-competitors. At low cell density, cell proliferation required both Tead activity and Myc. At high cell density, however, reduction of either Tead activity or Myc was compensated for by an increase in the other, and this increase was sufficient to confer 'winner' activity. Collectively, NIH3T3 cells have cell competition mechanisms similar to those regulated by Yki and Myc in Drosophila. Establishment of this in vitro model system should be useful for analyses of the mechanisms of cell competition in mammals and in fibroblasts.

Cell competition in mouse NIH3T3 embryonic fibroblasts is controlled by the activity of Tead family proteins and Myc.

Cell competition is a short-range communication originally observed in Drosophila. Relatively little is known about cell competition in mammals or in non-epithelial cells. Hippo signaling and its downstream transcription factors of the Tead family, control cell proliferation and apoptosis. Here, we established an in vitro model system that shows cell competition in mouse NIH3T3 embryo fibroblast cells. Co-culture of Tead-activity-manipulated cells with normal (wild-type) cells caused cell competition. Cells with reduced Tead activity became losers, whereas cells with increased Tead activity became super-competitors. Tead directly regulated Myc RNA expression, and cells with increased Myc expression also became super-competitors. At low cell density, cell proliferation required both Tead activity and Myc. At high cell density, however, reduction of either Tead activity or Myc was compensated for by an increase in the other, and this increase was sufficient to confer 'winner' activity. Collectively, NIH3T3 cells have cell competition mechanisms similar to those regulated by Yki and Myc in Drosophila. Establishment of this in vitro model system should be useful for analyses of the mechanisms of cell competition in mammals and in fibroblasts.

Mammalian Tead proteins regulate cell proliferation and contact inhibition as transcriptional mediators of Hippo signaling

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. Here, we show 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.

Tead proteins activate theFoxa2enhancer in the node in cooperation with a second factor

The cell population and the activity of the organizer change during the course of development. We addressed the mechanism of mouse node development via an analysis of the node/notochord enhancer (NE) of Foxa2. We first identified the core element (CE) of the enhancer, which in multimeric form drives gene expression in the node. The CE was activated in Wnt/beta-catenin-treated P19 cells with a time lag, and this activation was dependent on two separate sequence motifs within the CE. These same motifs were also required for enhancer activity in transgenic embryos. We identified the Tead family of transcription factors as binding proteins for the 3' motif. Teads and their co-factor YAP65 activated the CE in P19 cells, and binding of Tead to CE was essential for enhancer activity. Inhibition of Tead activity by repressor-modified Tead compromised NE enhancer activation and notochord development in transgenic mouse embryos. Furthermore, manipulation of Tead activity in zebrafish embryos led to altered expression of foxa2 in the embryonic shield. These results suggest that Tead activates the Foxa2 enhancer core element in the mouse node in cooperation with a second factor that binds to the 5' element, and that a similar mechanism also operates in the zebrafish shield.

Inhibition of the TEF/TEAD transcription factor activity by nuclear calcium and distinct kinase pathways

Transcription enhancer factor (TEF/TEAD) is a family of four transcription factors that share a common TEA-DNA binding domain and are involved in similar cellular functions, such as cell differentiation and proliferation. All adult tissues express at least one of the four TEAD genes, so this family of transcription factors may be of widespread importance, yet little is known about their regulation. Here we examine the factors that regulate TEAD activity in CHO cells. RT-PCR indicated the presence of TEAD-1, TEAD-3, and both isoforms of TEAD-4, but not TEAD-2. Quantitative measurements showed that TEAD-4 is most abundant, followed by TEAD-3, then TEAD-1. We examined the relative effects of nuclear and cytosolic Ca 2+ on TEAD activity, since TEAD proteins are localized to the nucleus and since free Ca 2+ within the nucleus selectively regulates transcription in some systems. Chelation of nuclear but not cytosolic Ca 2+ increased TEAD activity two times above control. Inhibition of mitogen-activated protein kinase (MAPK) also increased TEAD activity, while cAMP decreased TEAD activity, and protein kinase C had no effect. Together, these results show that nuclear Ca 2+, MAPK, and cAMP each negatively regulate the activity of the TEAD transcription factor.

TEAD/TEF transcription factors utilize the activation domain of YAP65, a Src/Yes-associated protein localized in the cytoplasm.

Mammals express four highly conserved TEAD/TEF transcription factors that bind the same DNA sequence, but serve different functions during development. TEAD-2/TEF-4 protein purified from mouse cells was associated predominantly with a novel TEAD-binding domain at the amino terminus of YAP65, a powerful transcriptional coactivator. YAP65 interacted specifically with the carboxyl terminus of all four TEAD proteins. Both this interaction and sequence-specific DNA binding by TEAD were required for transcriptional activation in mouse cells. Expression of YAP in lymphocytic cells that normally do not support TEAD-dependent transcription (e.g., MPC11) resulted in up to 300-fold induction of TEAD activity. Conversely, TEAD overexpression squelched 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 accumulated in the cytoplasm as a complex with the cytoplasmic localization protein, 14-3-3. Because TEAD-dependent transcription was limited by YAP65, and YAP65 also binds Src/Yes protein tyrosine kinases, we propose that YAP65 regulates TEAD-dependent transcription in response to mitogenic signals.