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Abstract The EPHA3 protein tyrosine kinase, a member of the ephrin receptor family, regulates cell fate, cell motility, and cell–cell interaction. These cellular events are critical for tissue development, immunological responses, and the processes of tumorigenesis. Earlier studies revealed that signaling via the STK4 -encoded MST1 serine-threonine protein kinase, a core component of the Hippo pathway, attenuated EPHA3 expression. Here, we investigated the mechanism by which MST1 regulates EPHA3. Our findings have revealed that the transcriptional regulators YAP1 and TEAD1 are crucial activators of EPHA3 transcription. Silencing YAP1 and TEAD1 suppressed the EPHA3 protein and mRNA levels. In addition, we identified putative TEAD enhancers in the distal EPHA3 promoter, where YAP1 and TEAD1 bind and promote EPHA3 expression. Furthermore, EPHA3 knockout by CRISPR/Cas9 technology reduced cell–cell interaction and cell motility. These findings demonstrate that EPHA3 is transcriptionally regulated by YAP1/TEAD1 of the Hippo pathway, suggesting that it is sensitive to cell contact-dependent interactions. 

RNA binding proteins (RBPs) regulate all aspects of RNA biogenesis from transcription, splicing, and translation to degradation, and they have a critical role in cellular homeostasis and functional diversity. Recent studies have indicated that altered expressions of RBPs are associated with many human diseases ranging from neurologic disorders to cancer. The transcriptional coregulator yes-associated protein 1 (YAP1), a critical nuclear effector of the mammalian Hippo pathway, regulates cell fate, cell contact, metabolism, and developmental processes. This study demonstrates a link between YAP1 and nucleophosmin1 (NPM1) protein. NPM1 is an RNA-binding protein that regulates many cellular activities, including ribosome biogenesis, RNA processing, chromatin remodeling, DNA repair, and genomic stability. We identified NPM1 from YAP1 protein complexes of androgen-responsive human cancer cells using proteomics approaches. Our proximity ligation assay demonstrated that YAP1 and NPM1 physically interacted with each other. The interaction between YAP1 and NPM1 occurred in cell nuclei and was regulated by androgen hormone signaling. In addition, our GST-pulldown assay demonstrated that NPM1 formed a protein complex with the proline-rich domain of YAP1. Furthermore, our enhanced RNA interactome capture (eRIC) assay showed that androgen also regulated the interaction of RBPs to polyA+ mRNA within the cell. Consistent with this observation, our eRIC assay combined with the mass spectrometry method enabled us to identify distinct RBP patterns in human cancer cells that are genetically related but phenotypically different. These observations indicate that global alterations of RBPs under changing environmental conditions may have essential roles in cellular physiology and disease biology. 

Sex comb on midleg-like-2 (SCML2), a conserved polycomb group protein, functions as a transcriptional repressor. SCML2 binds monomethylated lysine residues on histones and regulates homeotic gene expression during development in mammals and the fly. Using proteomic approaches, we have identified SCML2 as a binding partner of the YAP1 protein complexes isolated from nuclei of prostate cancer cell lines. Both SCML2 and YAP1 are known to regulate basic cellular biology, including stem cell maintenance and carcinogenesis. Our western blot analysis showed that, unlike androgen receptor (AR)-negative cancerous and non-cancerous prostate epithelium, AR-positive cell lines express the high levels of SCML2, suggesting a possible link between androgen hormonal signaling and SCML2. In addition, our immunofluorescence imaging revealed that androgen hormone signaling promoted the subcellular localization of SCML2 and YAP1 proteins compared with mock control. Enzalutamide, a potent pharmacological inhibitor of AR, significantly prevented the subcellular distribution ofYAP1 and SCML2. Consistent with this observation, our proximity ligation assay demonstrated that androgen also regulated the physical interaction between SCML2 and YAP1proteins that occurred primarily in cell nuclei. Enzalutamide also prevented protein-protein interaction between YAP and SCML2. Besides, our GST-pulldown assay revealed that SCML2 and proteins physically interact with each other in the test tube. Furthermore, our promoter-reporter assay showed that transfection of two different SCML2 siRNA enhanced the activation of the YAP-responsive promoter-reporter gene four-fold compared to mock siRNA control. These observations suggest that the interaction between SCML2 and YAP1 is biologically functional and crucial in human physiology and disease.