The Hippo pathway effectors Yes-associated protein 1 (YAP) and its homolog TAZ are transcriptional coactivators that control gene expression by binding to TEA domain (TEAD) family transcription factors. The YAP/TAZ–TEAD complex is a key regulator of cancer-specific transcriptional programs, which promote tumor progression in diverse types of cancer, including breast cancer. Despite intensive efforts, the YAP/TAZ–TEAD complex in cancer has remained largely undruggable due to an incomplete mechanistic understanding. Here, we report that nuclear phosphoinositides function as cofactors that mediate the binding of YAP/TAZ to TEADs. The enzymatic products of phosphoinositide kinases PIPKIα and IPMK, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (P(I3,4,5)P3), bridge the binding of YAP/TAZ to TEAD. Inhibiting these kinases or the association of YAP/TAZ with PI(4,5)P2and PI(3,4,5)P3attenuates YAP/TAZ interaction with the TEADs, the expression of YAP/TAZ target genes, and breast cancer cell motility. Although we could not conclusively exclude the possibility that other enzymatic products of IPMK such as inositol phosphates play a role in the mechanism, our results point to a previously unrecognized role of nuclear phosphoinositide signaling in control of YAP/TAZ activity and implicate this pathway as a potential therapeutic target in YAP/TAZ-driven breast cancer.
Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, using advanced tools for real-time visualization of native YAP and target gene transcription dynamics, we show that a cycle of fast exodus of nuclear YAP to the cytoplasm followed by fast reentry to the nucleus (“localization-resets”) activates YAP target genes. These “resets” are induced by calcium signaling, modulation of actomyosin contractility, or mitosis. Using nascent-transcription reporter knock-ins of YAP target genes, we show a strict association between these resets and downstream transcription. Oncogenically-transformed cell lines lack localization-resets and instead show dramatically elevated rates of nucleocytoplasmic shuttling of YAP, suggesting an escape from compartmentalization-based control. The single-cell localization and transcription traces suggest that YAP activity is not a simple linear function of nuclear enrichment and point to a model of transcriptional activation based on nucleocytoplasmic exchange properties of YAP.
more » « less- PAR ID:
- 10191493
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Optogenetic tools are created to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activator ARHGEF11, is fused to BcLOV4, a photoreceptor that can be dynamically recruited to the plasma membrane by a light‐regulated protein‐lipid electrostatic interaction with the inner leaflet. Direct membrane recruitment of these proteins induces potent contractile signaling sufficient to separate adherens junctions with as little as one pulse of blue light. Induced cytoskeletal morphology changes are dependent on the alignment of the spatially patterned stimulation with the underlying cell polarization. RhoA‐mediated cytoskeletal activation drives yes‐associated protein (YAP) nuclear localization within minutes and consequent mechanotransduction verified by YAP‐transcriptional enhanced associate domain transcriptional activity. These single‐transgene tools do not require protein binding partners for dynamic membrane localization and permit spatiotemporally precise control over RhoA signaling to advance the study of its diverse regulatory roles in cell migration, morphogenesis, and cell cycle maintenance.
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YAP/TAZ is a master regulator of mechanotransduction whose functions rely on translocation from the cytoplasm to the nucleus in response to diverse physical cues. Substrate stiffness, substrate dimensionality, and cell shape are all input signals for YAP/TAZ, and through this pathway, regulate critical cellular functions and tissue homeostasis. Yet, the relative contributions of each biophysical signal and the mechanisms by which they synergistically regulate YAP/TAZ in realistic tissue microenvironments that provide multiplexed input signals remain unclear. For example, in simple two-dimensional culture, YAP/TAZ nuclear localization correlates strongly with substrate stiffness, while in three-dimensional (3D) environments, YAP/TAZ translocation can increase with stiffness, decrease with stiffness, or remain unchanged. Here, we develop a spatial model of YAP/TAZ translocation to enable quantitative analysis of the relationships between substrate stiffness, substrate dimensionality, and cell shape. Our model couples cytosolic stiffness to nuclear mechanics to replicate existing experimental trends, and extends beyond current data to predict that increasing substrate activation area through changes in culture dimensionality, while conserving cell volume, forces distinct shape changes that result in nonlinear effect on YAP/TAZ nuclear localization. Moreover, differences in substrate activation area versus total membrane area can account for counterintuitive trends in YAP/TAZ nuclear localization in 3D culture. Based on this multiscale investigation of the different system features of YAP/TAZ nuclear translocation, we predict that how a cell reads its environment is a complex information transfer function of multiple mechanical and biochemical factors. These predictions reveal a few design principles of cellular and tissue engineering for YAP/TAZ mechanotransduction.
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null (Ed.)The transcriptional co-activator YAP1 (yes-associated protein 1) is a critical nuclear effector of the Hippo pathway. The Hippo pathway regulates cell growth, cell motility, cell migration, and carcinogenesis, but poorly defined mechanism. We investigated biochemical and functional interactions between YAP1 and the nuclear factor (NF)-kappa B/RELA subunit in prostate cancer cell models. We demonstrated that endogenous YAP1 and RELA form protein complexes in the cell, as revealed by co-immunoprecipitation and western blotting. Compared with control, we found that combined treatment of cells with androgen and SDF-1a (stromal cell-derived factor-1 alpha) or RANKL (receptor activator of NF-kappa B ligand) enhanced the protein-protein interaction between YAP1 and RELA, as showed by proximity ligation assay. Our confocal microscopy experiment further showed that combined SDF-1aand androgen treatment promoted the YAP1 and RELA colocalization instead of single-agent treatment. Moreover, our promoter-reporter and RNAi experiments showed that knockdown of YAP1 or TEAD, a key mediator of the YAP transcription, significantly reduced the NF-Kappa B promoter-reporter gene activity. Also, disruption of YAP1 activity attenuated the TEAD-RELA interaction. Furthermore, the controlled expression of MST1/STK4, a potent inhibitor of YAP1, attenuated the NF-Kappa B-promoter reporter activity. Additionally, our unbiased bioinformatics analysis of the exiting ChIP-seq (chromatin immunoprecipitation-sequencing) data sets identified several genes that are likely co-regulated by the YAP1/TEAD and NF-Kappa B/RELA transcription factors. These findings suggest that cooperative androgen and cytokine signaling regulates Hippo/YAP and NF-Kappa B interaction. Thus, the YAP1/TEAD and NF-Kappa B/RELA interaction may have critical roles in cellular biology and human diseases.more » « less
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