Par-1 Regulates Tissue Growth by Influencing Hippo Phosphorylation Status and Hippo-Salvador Association
Figure 5
Par-1 interacts with Hpo-Sav and regulates the phosphorylation of Hpo at Ser30.
(A–B) Par-1 interacts with Hpo and Sav in vitro. S2 cells were transfected with HA-tagged full-length or truncated Par-1 and Hpo (A) or Sav (B) constructs. The cell lysates were immunoprecipitated, followed by Western blot analysis with the indicated antibodies. Note that weak binding (asterisk indicated) between full-length Par-1/Par-1-C and Hpo and Sav were detected, whereas the N-terminal truncation of Par-1, which contained the kinase domain, showed a much stronger interaction signal. (C) Par-1 induces phosphorylation shift of Hpo-KD in vitro. S2 cells were transfected with the indicated constructs. The cell lysates were subjected to phosphorylation mobility shift assays. Note the phosphorylation shift of Hpo-KD in the presence of Par-1. Phos-tag was used to enhance the phosphorylation shift (see Materials and Methods for further details). (D) Par-1 regulates phosphorylation of Hpo-KD at Ser30 in S2 cells. S2 cells were transfected with the indicated constructs. The cell lysates were subjected to a phosphorylation mobility shift assay. The Hpo Ser30 site was mutated to an alanine. Note that the Hpo(S30A) mutant did not shift in the presence of Par-1. (E–F) Par-1 induces the phosphorylation of Hpo-KD at Ser30 in S2 cells. S2 cells were transfected with the indicated constructs. The cell lysates were subjected to Western blot analyses. Note that the phospho Hpo(Ser30) antibody could only detect Par-1-induced phosphorylation in the Hpo-KD samples but not in the Hpo(Ser30) mutant samples. The asterisks indicate non-specific bands. Lambda-PP indicates λ-phosphatase. (G) Par-1 inhibits Hpo(Thr195) phosphorylation. S2 cells were transfected with the indicated constructs. The cell lysates were immunoprecipitated, followed by Western blot analyses to detect p-Hpo(Thr195) levels. Note that Par-1 inhibited Hpo(Thr195) phosphorylation in a kinase-dependent manner, whereas the Hpo(S30A) mutant could not be inhibited. (H) Quantification of p-Hpo(Thr195) levels. p-Hpo(Thr195) levels were quantified using densitometry. The results were expressed as the mean ± SEM from three independent experiments. *p<0.05. (I) Hpo(S30A) results in a higher phosphorylation shift of Yki. S2 cells were transfected with the indicated constructs. The cell lysates were subjected to a phosphorylation mobility shift assay. Note that the phosphorylation shift of Yki was enhanced in the presence of Hpo(S30A) and that the Hpo(S30A) mutant was resistant to Par-1 induced Yki dephosphorylation. (J–K) Hpo(S30A) shows enhanced activity compared with wild-type Hpo in vivo. Control wings (J) or wings expressing UAS-Hpo (J′) or UAS-Hpo(S30A) (J″) with C765 were shown. The relative wing size was quantified using an unpaired t-test (K). The results represented the mean ± SEM.*p<0.05, **p<0.01, ***p<0.001 (n>6) for each genotype. Note that the Hpo(S30A) flies exhibited smaller wings than the Hpo flies.