pressor to block downstream gene expression. In the presence of Hh, however, the binding of Hh to Ptc allows Smo to be activated, resulting in the transduction of the Hh signal through the Cos2/Fu complex. In this case, Ci155 is stabilized and translocates to the nucleus, where it functions as a transcriptional activator, turning on downstream gene expression [3]. Therefore, a graded Hh signal could be translated into different transcriptional responses via Hh signaling components. However, more questions remain regarding Hh signaling cascade. For example, it is not known how the gradient information of a Hh signal is transduced through Smo and the Cos2/Fu complex to distinct downstream target genes. In addition, although many of the canonical Hh signal components are phosphorylated in Drosophila, including Smo, Cos2, Fu and Su(fu) [7], the specific role for these phosphory- lation events in the regulation of Hh signal transduction remains poorly understood. For instance, Smo is phos- phorylated by PKA and CKI in the presence of Hh [8-10] and transduces the Hh signal by interacting with Cos2/Fu complex [11-16]. However, it is poorly understood how the kinase activity of Fu is regulated and how activated Fu functions in this signaling cascade. In this study, we found that Smo activation, different levels of which are induced by different Smo mutants, recruits the Cos2/Fu complex to the membrane through the interaction between Smo and Cos2, which further induces Fu dimerization. Dimerized Fu is auto-phospho- rylated and transduces the Hh signal by phosphorylating Cos2 and Su(fu). This process promotes the dissociation of the full-length Ci and Cos2 or Su(fu), resulting in the translocation of Ci155 into the nucleus, which activates the expression of target genes. Our findings indicate that cells respond to Hh signaling through the regulation of Fu dimerization and its kinase activity. Results The recruitment of the Cos2/Fu complex by Smo to the plasma membrane is regulated by the Hh gradient Smo could potentially transduce Hh signaling by physically interacting with the Cos2/Fu complex [11- 14]. To investigate how the interaction between Smo and the Cos2/Fu complex is regulated by Hh, we first examined the co-localization of Smo and the Cos2/Fu complex in the presence or absence of Hh signaling. By co-transfecting Smo (tagged by CFP at its C terminus, Smo-CFPC), Cos2 (tagged by YFP at its C terminus, Cos2-YFPC) and Fu (tagged by Myc at its N terminus, Myc-Fu) in S2 cells, we found that Cos2 co-localized with Fu independent of Hh signaling. In the absence of Hh, however, the Cos2/Fu complex only partially co- www.cell-research.com | Cell Research Yanyan Zhang et al. npg 1437 localized with Smo in the cytoplasm. In the presence of Hh, the Cos2/Fu complex was highly co-localized with Smo at the cell membrane (Figure 1A). To investigate whether the accumulation of the Cos2/Fu complex at the plasma membrane, induced by Hh signaling, were dependent on Smo activity, we examined the co-local- ization of Cos2/Fu with two Smo mutants: SmoSA, a phosphorylation-defective Smo that fails to respond to Hh signaling, and SmoSD, a constitutively active Smo irrespective of Hh signaling [9, 17]. If the plasma mem- brane accumulation of the Cos2/Fu complex, induced by Hh, was independent of Smo activity, the Cos2/Fu complex co-transfected with SmoSA would be located at the cell membrane following Hh treatment. However, if the accumulation was dependent on Smo activity, the Cos2/Fu complex would be located at the cell membrane following co-transfection with SmoSD in the absence of Hh. We found that, the Cos2/Fu complex remained in the cytoplasm when co-transfected with SmoSA-CFPC, even after Hh treatment. In contrast, when SmoSD-CFPC was co-transfected, the Cos2/Fu complex was recruited to the cell membrane even without Hh treatment (Figure 1A). These results suggest that the cellular membrane accumulation of the Cos2/Fu complex is dependent on the activation of Smo. We next investigated how the Hh signal controls the interaction between Smo and Cos2/ Fu. We used SmoSA-CFPC, SmoWT-CFPC, SmoSD 1 - CFPC, SmoSD12-CFPC and SmoSD-CFPC to examine the effects of different Hh concentration inputs because the three Smo mutants (SmoSD1, SmoSD12 and SmoSD), which contain Ser to Asp substitution in one, two and three phosphorylation clusters, respectively, and exhibit progressively higher levels of constitutive signaling can mimic low to high activity, respectively [9]. We found that an increasing amount of Smo co-localized with the Cos2/Fu complex when Smo activation was increased, indicating that the interaction between Smo and Cos2/Fu might be regulated by the Hh gradient (Figure 1B). Fluorescence resonance energy transfer (FRET) has been broadly employed in the detection of protein-pro- tein interaction in living cells [18]. To investigate how Smo interacts with the Cos2/Fu complex, we measured FRET between the Smo C-terminal tail and Cos2 or Fu in both S2 cells and wing imaginal discs. The last 59 amino acids of the Smo C-terminal tail could interact with Fu directly [19]. However, no FRET activity was detected between Smo and Fu by using either Smo-CF- PC/Fu-YFPN (Fu tagged by YFP at N-terminus) or Smo- CFPC/Fu-YFPC (Fu tagged by YFP at C-terminus) in our experiments (data not shown). The last 59 amino acids of the Smo C-terminal tail interacts directly with Fu [19], but the CFP tag on the C-terminus of Smo might be too