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Fig. 6. PP2A downregulates Ci phosphorylation and blocks Ci proteolytic
processing. (A) CiFL phosphorylation is upregulated by
PP2A RNAi. S2 cells were transfected with Flag-Ci and treated with OA or
indicated dsRNA. Cell extracts were subjected to direct western blot with
anti-Flag antibody. Arrow indicates hyperphosphorylated forms of Ci and
arrowhead indicates the hypophosphorylated or unphosphorylated forms.
β-Tubulin serves as loading control. The knockdown efficiency of
individual phosphatase was estimated by the method used for
Fig. 2A. (B) PP2A
downregulates CiFL phosphorylation. S2 cells were transfected with
Flag-Ci alone or along with indicated HA-tagged phosphatase and treated with
or without OA. Cell lysates were probed with anti-Flag or anti-HA antibodies.
(C) A disc shows the wild-type CiFL staining. (D) A
wing disc shows the CiFL stabilization by the treatment of
proteasome inhibitor MG132. (E,F) Wing discs expressing
UAS-WdbRNAi by ap-Gal4 were treated with or without MG132
and stained to show CiFL. Arrowhead in E indicates the destabilized
CiFL by Wdb RNAi. Arrowhead in F indicates that the destabilized
CiFL by Wdb RNAi was restored by MG132 treatment.
(G-H') Wing discs bearing smo3 clones and
expressing UAS-HA-CiFL alone or along with
UAS-Wdb by MS1096 Gal4 were stained to show the expression
of GFP (green) and hh-lacZ (red). Arrowheads in G and H indicate
smo3 clones that are marked by the lack of GFP expression.
Arrowheads in G' and H' indicate the hh-lacZ expression
in smo3 cells. (I) Western blot analysis of protein
extracts from wing discs expressing UAS-HA-CiFL or
co-expressing UAS-HA-CiFL with UAS-Wdb using the
MS1096 Gal4. Protein extracts were prepared from 400 wing discs,
immunoprecipitated and blotted with HA antibody. (J) A model for the
involvement of PP4 and PP2A in Hh signaling. PP4 negatively regulates Hh
signal transduction by antagonizing the phosphorylation of Smo. PP2A
positively regulates Hh pathway by counteracting kinases to downregulate
CiFL phosphorylation and attenuate its proteasome-mediated
processing.
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