Yanyan Zhang et al. npg 1443 the plasma membrane by inducing Fu dimerization and leading to pathway activation, we examined whether FKBP- or Div-induced Fu dimerization could activate downstream gene expression independently of Smo and Cos2. We found that dimerized FuFL or FuK* induced ptc reporter expression in the absence of Smo or Cos2, while the activity of dimerized FuK was partially depen- dent on Smo (Figure 4D-4E). Dimerization of Fu triggers its kinase activity and pro- motes auto-phosphorylation and the phosphorylation of Cos2 and Su(fu) Fu is the Hh pathway-specific Ser/Thr kinase and is a major member of the Cos2/Fu complex. Previous studies have suggested that Cos2 might be a phosphorylation tar- get of Fu [7, 20, 21, 28], but the mechanism by which Fu kinase activity is regulated remains unknown. Notably, Figure 4 Induced Fu dimerization triggers the Hh pathway, which is independent of Smo and Cos2. (A-E) The ptc-luc reporter assay shows the pathway activity changes in S2 cells transfected with Ci155 and Su(fu). Error bars, 1 sd (triplicate wells). S2 cells were co-transfected with the indicated Div-based Fu expression constructs (A), or FKBP-based Fu constructs and treat- ed with or without AP20187 (B). (C) S2 cells were co-transfected with Fv2-FuG13V or Fv2-Fu, and treated with the graded AP20187. Along with the concentration change of the synthetic agent (AP20187), the ptc-luc reporter activity increased from low to high when the cells were transfected with Fv2-Fu (red line, Fv2-Fu; blue line, Fv2-FuG13V). (D, E) With Smo or Cos2 RNAi to knockdown endogenous protein expression, in both Fu dimerization system based on FKBP or Div, dimerized Fu could trigger ptc-luciferase reporter activity bypassing Smo and Cos2. www.cell-research.com | Cell Research