Fig. 1 | Nature Communications

Fig. 1

From: A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis

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

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