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. 2016 Dec 15;18(1):72–86. doi: 10.15252/embr.201642681

Figure 4. Osmotic stress induces YAP Ser128 phosphorylation and inhibits its 14‐3‐3 binding.

Figure 4

  1. NLK induces YAP Ser128 phosphorylation. Flag‐YAP WT or S128A mutant was co‐transfected with or without Myc‐NLK. Phosphorylation was determined by Western blot using YAP S128 phosphospecific antibody.
  2. NLK phosphorylates YAP Ser128 in vitro. NLK‐WT or NLK‐KN was transfected into HEK293A cells and immunoprecipitated. An in vitro NLK kinase assay was performed using recombinant GST‐YAP as a substrate. Phosphorylation of YAP Ser128 was determined by immunoblotting with phospho‐YAP (S128) antibody.
  3. Osmotic stress induces YAP phosphorylation at Ser128. In the left panel, HEK293A cells were treated with sorbitol and endogenous YAP was immunoprecipitated. In the right panel, Flag‐YAP was transfected into HEK293A cells, and Flag‐YAP was immunoprecipitated. YAP Ser128 phosphorylation was detected by a phosphospecific antibody. Data are presented as mean ± SEM. *P < 0.05 (two‐tailed Student's t‐test, n = 3).
  4. NLK deficiency reduces YAP S128 phosphorylation. Two CRISPR/Cas9 gRNA plasmids targeting different sites of NLK were transfected into HEK293A. WT cells and two pools of NLK CRISPR/Cas9‐transfected cells were treated with 0.2 M sorbitol for the indicated time points. Data are presented as mean ± SEM. *P < 0.05 (two‐tailed Student's t‐test, n = 4).
  5. The S128D phosphomimetic mutant abolishes YAP interaction with 14‐3‐3. Flag‐YAP WT and mutant constructs were co‐transfected with Myc‐14‐3‐3 into HEK293A cells. Cells were serum starved for 16 h. 14‐3‐3 was immunoprecipitated with Myc antibody, and the associated YAP was detected with Flag antibody.
  6. YAP Ser128 phosphorylation is required for disruption of YAP‐14‐3‐3 interaction by osmotic stress. Flag‐YAP WT or Flag‐YAP S128A constructs were co‐transfected with Myc‐14‐3‐3 into HEK293A cells. Cells were serum starved for 16 h, and treated with 0.2 M sorbitol for the indicated time points or refreshed with serum containing medium for 1 h. Refreshing medium served as a control to disrupt YAP and 14‐3‐3 binding. 14‐3‐3 was immunoprecipitated with Myc antibody, and the associated YAP was detected with Flag antibody. Data are presented as mean ± SEM. *P < 0.05 (two‐tailed Student's t‐test, n = 3).

Source data are available online for this figure.