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. 2015 Oct 5;6:8357. doi: 10.1038/ncomms9357

Figure 3. MAP4K4 acts through LATS to phosphorylate and inhibit YAP.

Figure 3

(a) A sequential kinase assay demonstrates that MAP4K4 activates LATS2 by in vitro phosphorylation. The hypophosphorylated and inactive GST-tagged full-length LATS2 purified from the HEK293A cells treated with fresh FBS for 45 min was incubated with immunoprecipitated Flag-MST2 or Flag-MAP4K4 in vitro in the presence of ATP. Recombinant GST-YAP was thereafter added to the reaction to measure GST-LATS2 kinase activity. (b) MAP4K4 overexpression induces YAP phosphorylation in a LATS1/2-dependent manner in HEK293A cells. (c) Doxycycline (Dox) inducible expression shows that the kinase activity of MAP4K4 is required to increase YAP and LATS phosphorylation in HEK293A cells. (d) MAP4K4 reduces YAP-TEAD interaction in HEK293A cells. HA-YAP, Myc-TEAD4, and MAP4K4 or MST2 constructs were co-transfected into HEK293A cells. TEAD4 was thereafter immunoprecipitated with anti-Myc antibody followed by immunoblotting with anti-HA antibody. (e) MAP4K4 suppresses TEAD reporter activity. The constructs expressing the indicated genes were co-transfected with a TEAD firefly luciferase reporter into HEK293A cells. 3 replicates are included in this experiment. The error bars represent s.d. ***P<0.001; student's t-test was applied. (f) MAP4K4 suppresses the expression of YAP target genes CTGF and CYR61. The cells with tetracycline-inducible MAP4K4 overexpression were incubated with doxycycline for 24 h, and then their CTGF and CYR61 expression were measured by quantitative real-time PCRs. Two replicates are included in this experiment. The error bars represent s.d. *P<0.05; **P<0.01; Student's t-test was applied. (g) MAP4K4 inhibits cell growth in a manner dependent on LATS1/2. WT and LATS1/2-dKO HEK293A cells were transfected with MAP4K4 and its KR mutant constructs. The cell number was counted every 24 h. 3 replicates are included in this experiment. The error bars represent s.e.m. *P<0.05; **P<0.01; MAP4K4-KR versus control. #P<0.05; ##P<0.01; MAP4K4-WT vs. control. Student's t-test was applied.