FIG. 2.
Lats2 inhibits the function of TAZ via phosphorylation. (A) TAZ contains four HXRXXS motifs as the putative Lats phosphorylation sites. The yeast Dbf2 optimal target sequence was aligned with the four HXRXXS motifs of human TAZ. (B) The putative HXRXXS motifs in TAZ are required for a mobility shift by Lats2 and Mst2. The wild type (WT) or the mutant Flag-TAZ was cotransfected with HA-Mst2 and HA-Last2, as indicated. The TAZ mobility shift was examined by Western blotting. The 4SA mutation denotes the quadruple mutation of all four putative phosphorylation sites. As indicated, treatment with lambda phosphatase abolished the mobility shift by Lats2 and Mst2 cotransfection, indicating that the mobility shift is due to phosphorylation. (C) The S89A and S4A TAZ phosphorylation mutants are resistant to inhibition by Mst2 and Lats2. The S89A mutation and the phosphorylation mimic S89D mutant made no difference in the inhibition by Mst2 and Lats2. The reporter assay was performed similar to that shown in panel A. The inhibition change (n-fold) for each mutant is shown at the top of this panel. (D) The coexpression of Mst2 and Lats2 increases TAZ Ser89 phosphorylation. Lats2K/R dramatically decreases TAZ Ser89 phosphorylation. Flag-TAZ was cotransfected with Lats2, Mst2, or kinase-dead Lats2K/R into 293T cells, as indicated. Flag-TAZ was immunoprecipitated, and phosphorylation of Ser89 was detected by pYAP(Ser127) antibody, which can recognize the Ser89 phosphorylation site because of sequence conservation. Lambda phosphatase treatment and the TAZS89A mutant abolished the recognition of phosphoTAZ by the anti-phosphoYAP antibody. This result confirms the specificity of the phosphoYAP antibody. (E) In vitro phosphorylation of TAZ by Lats2. HA-Lats2 or Flag-Mst2 was immunoprecipitated from transfected 293T cells. An in vitro kinase assay was performed using purified His-TAZ as a substrate. Phosphorylation of TAZ was detected by pYAP(S127) antibody. His-TAZ input was shown by Coomassie blue staining. (F) A knockdown of Lats decreases TAZ Ser89 phosphorylation. HeLa cells cotransfected HA-TAZ with small interfering (siRNA) for Lats1 and Lats2, as indicated. Phosphorylation and protein levels of TAZ were determined by Western blotting. A knockdown of Lats was verified by quantitative PCR (data not shown).