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. 2018 Mar 13;9:1061. doi: 10.1038/s41467-018-03278-w

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Establishment of a split luciferase LATS biosensor (LATS-BS). a Schematic diagram of YAP1 structure with LATS phosphorylation site (S127) and surrounding 15 amino acids (YAP15) indicated. b YAP15 is sufficient for interaction with 14-3-3. GST-tagged YAP (YAP-GST), YAP15 with wild-type sequence (YAP15-S127-GST), or LATS phosphorylation-mutant YAP15 (YAP15-S127A-GST) was purified. Five micrograms of GST fusion protein was incubated with recombinant LATS kinase. One hundred micrograms of cell lysate from HEK293 transiently expressing 14-3-3-FLAG was added. YAP, YAP15-S127, or YAP15-S127A/14-3-3 binding was assessed by GST pull-down assay, followed by western blotting. c Domain structure of the LATS-BS. For Nluc-YAP15, firefly luciferase amino acids 1-416 (Nluc) were fused to the N-terminal of YAP15 (120–134) separated by a glycine/alanine linker (5′-GGAGG-3′). For 14-3-3-Cluc, luciferase amino acids 394–550 (Cluc) were fused to the C-terminal of 14-3-3 separated by a glycine/serine linker (5′-GGSGGGGSGG-3′). d Mechanism of action for the LATS-BS. At baseline, there is no interaction between YAP15 and 14-3-3; thus, the LATS-BS shows minimal bioluminescence activity. However, LATS-dependent phosphorylation of YAP15-S127 leads to 14-3-3 binding, luciferase complementation, and high biosensor signal. e Validation of LATS-BS activity. LATS-BS was transfected alone or with LATS2 or/and MST2 into HEK293. Biosensor activity or NLuc-YAP15-S127 phosphorylation was determined 48 h after transfection. For lysophosphatidic acid (LPA) treatment, cells were stimulated with 10 μM LPA for 1 h (n = 3). f LATS-BS responds specifically to LATS kinase activity. Mutation of the LATS kinase consensus motif (HXRXXS/T; H, histidine; R, arginine; S, serine; T, threonine; X, any amino acid) in Nluc-YAP15 abolishes LATS-BS activation and Nluc-YAP15 S127-phosphorylation (n = 3). g LATS-BS activity is reduced by MST or LATS knockout. LATS-BS was transfected into CRISPR-Cas9-generated LATS1/2 or MST1/2 knockout HEK293A. Biosensor activity was determined 48 h after transfection (n = 3). h LATS-BS can be stably expressed to detect LATS activity. HEK293A with doxycycline (Dox)-inducible LATS2 overexpression and stable LATS-BS expression were treated with Dox for the indicated times. Biosensor activity and endogenous YAP (YAP-pS127) phosphorylation status and Nluc-YA15P-S127 (Nluc-YAP15-pS127) were determined by luciferase assay and western blotting, respectively (n = 3). Data are represented as mean ± SD