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Fig. 3. Activities of Cos2 are affected by mutations in Ser572. (A) Hh-expressing cl8 cells were transfected with increasing amounts of smo and with different amounts of cos2-WT. Ci reporter activity increased in the presence of low levels of transfected cos2-WT. (B-D) smo was transfected at a fixed concentration (10 ng) together with fixed amounts of fu-WT or fu-KD and with different amounts (0-100 ng) of cos2-WT (B), cos2-572A (C) or cos2-572D (D). Notice that Fu-WT potentiates Ci activity, whereas Fu-KD abolishes the positive effect of Cos2. (E) Fixed amounts of ci and ptc-luc vectors were co-transfected with cos2 constructs into S2 cells. At high, but not at low, concentrations, the various Cos2 proteins similarly repressed Ci transactivation. (F) The levels of the three different Cos2 proteins (500 ng) and the Ci proteins were similar in each pool of transfected cells. (G-I) At fixed quantities of ci and ptc-luc vectors, different amounts of the cos2-WT (G), cos2-572A (H) and cos2-572D (I) constructs were co-transfected into S2 cells, together with fixed amounts of fu-WT or fu-KD and in the presence or absence (control) of wild-type Hh. In Hh-stimulated cells, the repressive activity of Cos2-WT is regulated by Fu kinase activity, but it is not in the cos2 mutants in which the phosphosite at position 572 has been affected.

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