FIG 4.

YAP phosphorylation and dephosphorylation. (A) MT affects YAP mobility. Upon induction of EGFP or MT in NIH 3T3 cells by exposure to doxycycline for 24 h, whole-cell extracts were subjected to extended SDS-PAGE and Western blotting to detect YAP, MT, and p85 (loading control). (B) Phosphorylation controls YAP mobility. Upon induction of EGFP or MT in NIH 3T3 cells by exposure to doxycycline for 24 h, YAP immunoprecipitates were subjected to combined calf intestinal phosphatase (CIP) and lambda phosphatase treatment and subsequent extended SDS-PAGE and Western blotting to detect YAP. The WT MT and MT-103A samples were overloaded to correct for the lower levels of YAP seen in these cells. In doing this, we overloaded them compared to controls. (C) MT induces YAP phosphorylation at S397 and S127. NIH 3T3 cells were induced to express EGFP or MT by exposure to doxycycline for 24 h. After SDS-PAGE, phosphospecific Western blotting was conducted to detect YAP phosphorylation at S397 and S127 as well as total YAP, MT, and GAPDH (loading control) levels. In the bottom three blots, three times the amount of the R103A MT extract was loaded to normalize total YAP. (D) YAP destabilization is a consequence of MT signaling. NIH 3T3 cells were induced to express EGFP or MT by exposure to doxycycline for 24 h, and the Src inhibitor SU6656 was added for the last 16 h of induction. After SDS-PAGE, Western blotting was conducted to detect YAP phosphorylation at S397 as well as total YAP, MT, and p85 (a loading control). The bar graph represents quantification of Western blot data and shows the averages of data from two independent experiments. **, P < 0.01 versus MT-103A with SU6656 or the wild type without SU6656. (E) NIH 3T3 cells were prepared to express the T203E MT (Src-minus) mutant that is defective in binding Src and inducible by doxycycline. Extracts of these cells were compared to those of cells expressing wild-type MT for YAP S397 phosphorylation, total YAP, and MT by Western blotting. (F) Wild-type MT increases the association of YAP with PP2A. 293T cells were transiently transfected with vectors encoding MT and/or FLAG-YAP. After ∼40 h, YAP was immunoprecipitated with antibodies to the tag. After SDS-PAGE, Western blotting was carried out to detect YAP, PP2A, and MT. (G) MT-bound YAP is phosphorylated differently than unbound YAP in cells expressing wild-type MT. NIH 3T3 cells were induced to express MT by exposure to doxycycline for 24 h. MT was immunoprecipitated by using the MT-1 antibody. After extended SDS-PAGE, Western blotting of MT immunoprecipitates and the total cell extract was carried out to detect YAP and MT. The two bands marked “MT bound” represent identical samples loaded onto both sides of MT-bound YAP to show alignment compared to unbound YAP.