Cysteine residues are essential for dimerization of Hippo pathway components YAP2L and TAZ

www.nature.com/scientificreports OPEN Received: 30 November 2017 Accepted: 12 February 2018 Published: xx xx xxxx Cysteine residues are essential for dimerization of Hippo pathway components YAP2L and TAZ Prem Khanal1, Zongchao Jia 2 & Xiaolong Yang1 Hippo signalling pathway is an emerging signalling pathway that plays important roles in organ size control, tumorigenesis, metastasis, stress response, apoptosis, stem cell differentiation and renewal during development and tissue homeostasis. Recent studies reported that human serine/threonine protein kinase, Mst1, a core component of the Hippo pathway can be activated through formation of homodimer. However, it is still unclear whether or not other components of the Hippo pathway are also regulated through dimerization. Here we provide the first evidence that Hippo components and oncoprotein YAP2L and TAZ can form homodimer in vitro and in vivo by forming disulphide bond through cysteine residue(s). We have also shown that the homodimers of YAP2L/TAZ are more stable and showed more oncogenic behaviour than their corresponding monomers as revealed by colony formation and cell transformation assay. Since cysteine post-translational regulation plays important roles in redox signalling, tumorigenesis and drug resistance, further studies on the functional effect of this dimerization through post-translational modulation of cysteine residues in YAP2L/TAZ will provide a significant contribution to our understanding of the roles of YAP2L/TAZ in cancer development and therapy. One of the common properties of the proteins is that they can form dimer or high-order oligomers through self-association1. The formation of either intermolecular or intramolecular dimers or oligomers is very important process in protein folding2. Proteins usually form dimers or oligomers through specific motifs such as leucine Zipper3,4, helix-loop-helix5, Ankyrin6 and PAS-domain7 or disulphide bond formation between cysteines (C)8. The dimeric or oligomeric forms of proteins have several impacts on their roles in cellular process1. For example, they can increase the activity and stability of proteins9,10, help to transport of molecules across cell membranes11,12, increase protein binding affinity to DNA and transcriptional activity13,14, cause increased cell proliferation, transformation and drug resistance13,15, and inhibit cell transformation16, Therefore, identification and characterization of novel dimeric/multimeric proteins are very important for fully understanding their functions in different cellular process. The Hippo signaling pathway, which was initially identified in Drosophila, is highly conserved in mammals17. This novel signalling pathway is involved in regulation of tissue growth, organ size, cell proliferation, apoptosis, tumorigenesis, mechanotransduction, drug resistance, stem cell renewal and differentiation18–28. Serine/threonine (S/T) kinases MST1/2 and LAT1/2, adaptor/scaffold proteins MOB1 and WW45, and two WW domain containing transcriptional co-activators YAP and its paralog TAZ are the main components of the Hippo pathway24,29–35. YAP and TAZ are the main downstream effector of Hippo pathway and are involved in cell proliferation, drug resistance and many other tumorigenic processes by transactivation of downstream genes (CTGF, Cy61, BMP4, etc.) in the nucleus through transcription factor TEAD or Smad23,35–39. Activation of LATS1/2 by upstream members (MST1/2, MOB1, and WW45) of the Hippo signalling cascade phosphorylates and inactivates YAP and TAZ by promoting either their cytoplasmic retention or degradation, preventing them from transactivating downstream genes in the nucleus40,41. Many studies have already shown the different mechanisms which leads to either increased or decreased levels or activity of YAP/TAZ and their importance in cancer biology. For example, while SCFβ-TRCP E3 ubiquitin ligase decreases the stability of YAP and TAZ by ubiquitin-mediated degradation27,42, PP1A and ASPP2 increases the TAZ activity by antagonizing its negative regulator LATS kinase through TAZ dephosphorization43. In addition, 1 Department of Pathology and Molecular Medicine, Queen’s University, Kingston, Canada. 2Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Canada. Correspondence and requests for materials should be addressed to X.Y. (email: ) SCientifiC REPOrts | (2018) 8:3485 | DOI:10.1038/s41598-018-21828-6 1 www.nature.com/scientificreports/ Figure 1. In vitro and in vivo dimerization of YAP2L isoform. (A) Schematic representation of YAP1, YAP2 and YAP2L isoforms. (B) YAP2L isoform form dimer in vivo, HEK293 cells were transfected with HA or FLAGtagged-YAP1, -YAP2 and -YAP2L plasmids alone or together. The cells were harvested in 1%NP-40 lysis buffer. After checking the expression level of HA or FALG- tagged-YAP1,-YAP2 and-YAP2L, equal amount of cell lysates were subjected to co-immunoprecipitation assays using anti-HA antibody and immublotting analysis were performed using anti-FLAG or anti-HA antibody respectively. (C) YAP2L isoform form dimer in vitro, 200 µg of cell lysates from FLAG-tagged-YAP1,-YAP2 and -YAP2L plasmids transfected HEK293 cells were precleared with 50% GSB beads overnight at 4◦C. After then, supernatants were mixed with 5 µg of GST or YAPGST and incubated for 2 hrs followed by addition of 20 μl of 50% GSB beads for another 1 hr. The beads were then washed, eluted by 2XSDS sample buffer and subjected to western blotting against anti-FLAG antibody. Ponceau-S staining shows the equal amount of fusion protein used for pull down. 1/10 input (20 µg) represents 1/10 of protein lysate used for pull down. it has been reported that one of the major components of the Hippo pathway, MST1 undergoes dimerization, which increases its kinase activity44,45. However, whether and how the levels or activities of other components of the Hippo pathway are also regulated through dimerization are largely unknown. In this study, we have shown that both YAP and TAZ undergo dimerization through cysteine residues, resulting in increase in their stability which leads to enhanced tumorigenesity of H460 lung cancer cells. This finding provides novel mechanism by which YAP and TAZ are regulated and have significant implication in our understanding the roles of YAP and TAZ in cancer. Results YAP2L isoform forms dimer in vitro and in vivo. The YAP (Yes associated protein 1) oncogene, a major component of hippo signalling pathway, is a transcriptional coactivator of target genes and involved in cell proliferation and survival28. Since YAP protein has eight isoforms in human46 and there is no antibody to distinguish each isoform, we chose three most commonly studied isoforms of YAP (YAP1, YAP2 and YAP2L) (Fig. 1A) and tested them individually by expressing each isoform in cells. We first carried out a co-immunoprecipitation assay among the three different isoforms of YAP. HA or FLAG-tagged YAP1, YA (...truncated)