Figure 1. Hippo Signaling Phosphorylates Yki S168 and Promotes Its Cytoplasmic Localization.

(A) S2 cells expressing HA-Yki (left) or HA-Yki plus Hpo, Sav, and Wts plasmids (right) were stained with α-HA antibody. Nuclear HA-Yki signal is detected in 100% of cells expressing HA-Yki alone, but in only 9% of cells coexpressing Hpo-Sav-Wts.

(B) Cells were transfected as in (A) and analyzed by subcellular fractionation. Note the decrease of nuclear Yki under active Hippo signaling (compare lanes 2 and 4). dSREBP (nuclear) and tubulin (cytosolic) were used as quality control for fractionation. C, cytoplasmic; N, nuclear.

(C) Cells were transfected as in (A), and α-HA immunoprecipitates were probed with α-14-3-3 (top gel). Cell lysates were also probed with the indicated antibodies (middle and bottom gels). Yki, but not Yki^S168A, immunoprecipitated 14-3-3 under active Hippo signaling.

(D) Cells were transfected as in (A), and α-HA immunoprecipitates were probed with α-P-Yki(S168) and α-HA antibodies (top two gels). Cell lysates were also probed with the indicated antibodies (bottom three gels). Yki, but not Yki^S168A, showed S168 phosphorylation, which was further increased under active Hippo signaling. Also note that Yki, but not Yki^S168A, showed mobility shift under active Hippo signaling (compare lanes 2 and 4; indicated by white and black dots, respectively).

(E) Wts phosphorylates Yki at S168 in vitro. V5-tagged Wts (or kinase-dead Wts^KD) was expressed alone or together with Hpo-Sav in S2 cells, immunoprecipitated, and incubated with GST-Yki (or GST-Yki^S168A), and the reaction products were probed with α-P-Yki(S168). The input kinase and substrate are also shown (bottom two gels). Note that our GST-Yki preparation contains two Yki-related bands (Huang et al., 2005). Strong S168 phosphorylation was detected when Wts (lane 3), but not Wts^KD (lane 4), was coexpressed with Hpo-Sav. No S168 phosphorylation was detected using GST-Yki^S168A as a substrate (lane 5).

(F) Subcellular fractionation of Yki^S168A mutant. The relative proportion of cytoplasmic and nuclear YkiS^168A was not changed under active Hippo signaling.

(G) S2 cells expressing HA-Yki or HA-Yki^S168A were treated with or without insulin. α-HA immunoprecipitates were probed with the indicated antibodies (top two gels). Cell lysates were also probed with the P-S6K or P-Akt antibodies (bottom two gels). Arrowheads mark the phospho-Akt signals. Insulin stimulates the phosphorylation of Akt and S6K, but not that of Yki (compare lanes 1 and 2).

(H) S2 cells expressing HA-Yki or HA-Yki^S168A plus a constitutively active Akt mutant (myr-Akt) were analyzed as in (G). Similar P-Yki(S168) levels were seen in the presence or absence of myr-Akt (compare lanes 1 and 2).