npg Fused dimerization transduces Hedgehog signal 1444 many kinases, such as receptor tyrosine kinases, form a dimer, and then show kinase activity upon ligand bind- ing [29]. To examine whether dimerized Fu has kinase activity and to determine its phosphorylation target(s), we performed immunoprecipitation-based phosphoryla- tion experiments in the two dimerization systems. We found that dimerized Fv2-Fu or Fv2-FuK* but not Fv2- FuG13V immunoprecipitated products from S2 cells that were auto-phosphorylated after dimerization in im- munoprecipitated product (Figure 5A and Supplemen- tary information, Figure S8A). The dimerized Fu, based on Div-dimerization system, was auto-phosphorylated (Supplementary information, Figure S8B). To determine whether Cos2 can be phosphorylated by dimerized Fu, we used similar kinase assays. In an in vitro kinase assay, dimerized Fu (Fv2-Fu and Div-Fu), but not Fu G13V, phosphorylated Cos2 (Figure 5B1 and Supplementary information, Figure S8C). FuK and FuK* also displayed phosphorylation activity towards Cos2 after dimerization (Figure 5B2-5B3). Therefore, Fu following dimerization is auto-phosphorylated, and can phosphorylate Cos2. Su(fu) binds Fu in the absence of Hh, while, in the presence of Hh, the function of Su(fu) is inhibited by activated Fu [7]. Although Su(fu) has been shown to be Figure 5 The dimerization of Fu promotes Fu auto-phosphorylation and Cos2 and Su(fu) phosphorylation. (A) Phosphoryla- tion mobility shift of dimerized Fv2-Fu as induced by a synthetic agent (AP20187) in vitro with ATP. Fv2-FuG13V served as a control. Phosphatase was used to dephosphorylate the phosphorylated protein. (B) Phosphorylation of Cos2 by dimerized Fu. In Div-based dimerization system, dimerized Fu can phosphorylate Cos2 in vitro. S2 cells were transfected with Flag- Cos2, Div-FuG13V, Div-Fu, Div-FuKG13V, Div-FuK*G13V or Div-FuK*, respectively. Then, Cos2 or different Div-Fu fusion proteins were concentrated by immunoprecipitation, respectively. The in vitro kinase assays were followed. Phosphatase was used to dephosphorylate the phosphorylated proteins. Div-Fu (B1), Div-FuK (B2) and Div-FuK* (B3) can phosphorylate Cos2 but not the G13V mutants. (C) Phosphorylation of Flag-Su(fu) by activated Fu in an in vitro Div-Fu system. Su(fu) can be phosphorylated by Div-Fu and Div-FuK* but not Div-FuK. In (A-C), the mobility shift band of target proteins were labeled by stars. (D) Smo-CFPC (green), Cos2WT/SA-YFPC (red) and Myc-FuWT/G13V (blue) were co-transfected in S2 cells in the ab- sence/presence of Hh, to detect the effect of Cos2 phosphorylation or Fu kinase activity on co-localization between Smo and Cos2/Fu complex. Cell Research | Vol 21 No 10 | October 2011