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. 2024 Mar 21;134(10):e174587. doi: 10.1172/JCI174587

Figure 2. AARS1 directly interacts with and lactylates YAP-TEAD.

Figure 2

(A) Lysine lactylome in lactate-treated HGC27 cells. A total of 1,182 lactylated proteins and 2,789 lactylated sites with q value (–log10) greater than 10 were identified. Histone sites modified through lactylation are shown in gray. Lactylation on Hippo pathway–associated components are shown in red. (B) KEGG pathway analysis of lactylated proteins identified using lactylation proteomics in HGC27 cells cultured in glucose-free medium supplemented with 25 mM lactate. (C) Mass spectra of lactylated sites of YAP (K90) and TEAD1 (K108). (D) Immunoblotting showing the lactylation of endogenous YAP and TEAD1 proteins using pan-Klac antibody. (E) Lactylation levels of exogenous YAP and TEAD1 in cells transfected with the indicated plasmids. (F and G) Lactylation levels of endogenous YAP (F) and TEAD1 (G) in lactate-treated HGC27 cells. Glc, glucose; Lac, lactate. (H) Coimmunoprecipitation analysis of the endogenous interaction between YAP (left) or TEAD1 (right) and AARS1 in lactate-treated HGC27 cells. (I) Pull-down assay showing the direct interaction between AARS1 and YAP-TEAD1. Top: MBP pull-down assay to detect interaction between AARS1 and MBP-YAP (1–291). Bottom: GST pull-down assay to detect interaction between GST-AARS1 and His-sumo-TEAD1 (HS-TEAD1). Asterisks represent the indicated proteins. (J) Mass spectrometry to determine the lactylation of the synthetic YAP K90 and TEAD1 K108 peptides catalyzed by AARS1 and its catalytic-dead mutant 5M in vitro. (K) Immunoblotting with pan-Klac antibody to detect AARS1-induced lactylation of TEAD1 in vitro. CBB staining showing the purified AARS1 and TEAD1 used in in vitro lactylation assay. Asterisks represent the AARS1 and TEAD1 proteins. The final concentrations of PPi in the reaction mixture were 2 mM (+) and 10 mM (++), respectively. (L) Lactylation levels of YAP (left) and TEAD1 (right) in cells overexpressing AARS1 or its 5M mutant. (M) Lactylation levels of YAP (left) and TEAD1 (right) in AARS1-knockdown HEK293FT cells.