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. 2016 Dec 15;18(1):61–71. doi: 10.15252/embr.201642683

Figure EV3. Phosphorylations of Ser127 and Ser128 of YAP may be mutually exclusive.

Figure EV3

  1. Inhibition of NLK induces phosphorylation at Ser127 of YAP. HEK293T cells were treated with either 6‐bromoindirubin‐3′‐oxime (BIO, 4 μM; inhibitor of GSK3β), or lithium chloride (50 mM; inhibitor of GSK3β and NLK). MeBIO (4 μM) or KCl (50 mM) were used as negative controls for BIO or LiCl, respectively.
  2. Knockdown of GSK3β has no effect on YAP phosphorylation at Ser127. Lysates from HEK293T cells transfected with either siControl or siGSK3β were immunoblotted with antibodies indicated in the figure.
  3. YAP phosphorylation at Ser128 by NLK. Cell lysates from HEK293 cells transfected with empty vector (−), EGFP‐YAP, EGFP‐YAP‐S128A, and Flag‐NLK‐WT were immunoblotted with antibodies shown in the figure.
  4. Ectopic expression of wild‐type NLK, but not kinase‐negative NLK, enhanced the level of endogenous phospho‐YAP(Ser128). Cell lysates from HEK293T cells transfected with empty vector (−), Flag‐NLK‐WT, or Flag‐NLK‐KM were immunoblotted with antibodies indicated in the figure.
  5. Treatment with LiCl attenuated NLK‐mediated phosphorylation of YAP Ser128. Cell lysates from HEK293 cells transfected with empty vector (−), Flag‐NLK‐WT, or Flag‐NLK‐KM and treated KCl or LiCl. Lysates were immunoblotted with antibodies shown in the figure.
  6. NLK specifically phosphorylates YAP‐S128 in vitro. GST‐YAP or GST‐YAP(S128A) was incubated with immunoprecipitated Flag‐NLK‐WT or Flag‐NLK‐KM from lysates of HEK293T cells transfected with Flag‐NLK‐WT or Flag‐NLK‐KM as indicated in the figure. The level of phosphorylation at S128 was detected with pYAP‐S128 antibody.
  7. Phosphorylation of S128 by NLK inhibits phosphorylation of YAP‐S127 by LATS1. Immunoprecipitated Flag‐LATS1 from lysates of HEK293T cells transfected with Flag‐LATS1 was incubated with various mutant forms of GST‐YAP as shown in the figure. The level of phosphorylation at S127 was detected with pYAP‐S127 antibody.