title: Sub-cellular localisation studies may spuriously detect the Yes-associated protein, YAP, in nucleoli leading to potentially invalid conclusions of its function.
abstract: The Yes-associated protein (YAP) is a potent transcriptional co-activator that functions as a nuclear effector of the Hippo signaling pathway. YAP is oncogenic and its activity is linked to its cellular abundance and nuclear localisation. Activation of the Hippo pathway restricts YAP nuclear entry via its phosphorylation by Lats kinases and consequent cytoplasmic retention bound to 14-3-3 proteins. We examined YAP expression in liver progenitor cells (LPCs) and surprisingly found that transformed LPCs did not show an increase in YAP abundance compared to the non-transformed LPCs from which they were derived. We then sought to ascertain whether nuclear YAP was more abundant in transformed LPCs. We used an antibody that we confirmed was specific for YAP by immunoblotting to determine YAP's sub-cellular localisation by immunofluorescence. This antibody showed diffuse staining for YAP within the cytosol and nuclei, but, noticeably, it showed intense staining of the nucleoli of LPCs. This staining was non-specific, as shRNA treatment of cells abolished YAP expression to undetectable levels by Western blot yet the nucleolar staining remained. Similar spurious YAP nucleolar staining was also seen in mouse embryonic fibroblasts and mouse liver tissue, indicating that this antibody is unsuitable for immunological applications to determine YAP sub-cellular localisation in mouse cells or tissues. Interestingly nucleolar staining was not evident in D645 cells suggesting the antibody may be suitable for use in human cells. Given the large body of published work on YAP in recent years, many of which utilise this antibody, this study raises concerns regarding its use for determining sub-cellular localisation. From a broader perspective, it serves as a timely reminder of the need to perform appropriate controls to ensure the validity of published data.
authors: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, 6009, Australia.
authors: Megan L
authors: ML
authors: Finch
authors: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, 6009, Australia.
authors: Adam M
authors: AM
authors: Passman
authors: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, 6009, Australia.
authors: Robyn P
authors: RP
authors: Strauss
authors: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, 6009, Australia; Harry Perkins Institute of Medical Research, QEII Medical Centre, Nedlands and Centre for Medical Research, the University of Western Australia, Crawley, Western Australia, 6009, Australia.
authors: George C
authors: GC
authors: Yeoh
authors: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, 6009, Australia.
authors: Bernard A
authors: BA
authors: Callus
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D018797:Cell Cycle Proteins; D002460:Cell Line; D002466:Cell Nucleolus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D014157:Transcription Factors; D000091102:YAP-Signaling Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D010750:Phosphoproteins; D014157:Transcription Factors; D000091102:YAP-Signaling Proteins; C088374:YAP1 protein, human; C486223:Yap1 protein, mouse