Supplementary Figure 4: Identification of AMPK phosphorylation sites in YAP.

(a) AMPK induces mobility shift of YAP-5SA. GST-YAP 5SA was used as substrates for in vitro AMPK phosphorylation. GST- YAP mobility was examined by western blot (b) Mass spectrometry analyses of AMPK-phosphorylated YAP. GST-YAP-5SA was purified from E.coli and was used as a substrate for an in vitro AMPK assay. After electrophoresis, the phosphorylated GST-YAP 5SA band was cut and digested with trypsin/thermolysine followed by mass spectrometry analyses. The green coloured bars (top) and amino acid residues (lower part) are sequences that were detected by mass spectrometry. Phosphorylation sites identified by mass spectrometry are indicated on top of the amino acid sequence. (c) S94 is the major AMPK phosphorylation site in the YAP (51-121) fragment. Experiments were similar to panel a. (d) All three indicated residues of YAP were mutated to alanine. A luciferase reporter controlled by multiple TEAD binding sequences was transfected into HEK293T cells together with 5 × UAS-luciferase reporter for Gal4-TEAD4 and Renilla constructs and indicated plasmids. After 48 h, the firefly luciferase activity was measured and normalized to the co-transfected Renilla luciferase internal control. (d) Two different substrates of AMPK, TSC2 and ULK1, and YAP truncations were expressed, purified, and used as substrates for in vitro AMPK phosphorylation in the presence of ³²P-ATP Phosphorylation was detected by autoradiography. GST-ULK1 (279-425), TSC2 (1300-1367) and YAP fragment proteins were detected by Coomassie staining. (e) Experiments are same as in panel c. except that a GST-YAP truncated form and UKL1 were used as substrates for in vitro AMPK phosphorylation in the presence of ³²P-ATP for the indicated times. (f) Experiments are the same as in panel d. exception GST-YAP WT and UKL1 used as substrate for the indicated times. (g) AMPK has no effect on the YAP and RUNX2 interaction. HEK293A cells were transfected with the indicated plasmids. Flag-RUNX2 was immunoprecipitated and the co-precipitated HA-YAP was detected by Western blot.