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. 2010 Nov 26;285(48):37218-26.
doi: 10.1074/jbc.M110.174565. Epub 2010 Sep 27.

Casein kinase 2 promotes Hedgehog signaling by regulating both smoothened and Cubitus interruptus

Hongge Jia  1 , Yajuan Liu, Ruohan Xia, Chao Tong, Tao Yue, Jin Jiang, Jianhang Jia
Affiliations

Casein kinase 2 promotes Hedgehog signaling by regulating both smoothened and Cubitus interruptus

Hongge Jia et al. J Biol Chem. .

Abstract

Casein kinase 2 (CK2) is a typical serine/threonine kinase consisting of α and β subunits and has been implicated in many cellular and developmental processes. In this study, we demonstrate that CK2 is a positive regulator of the Hedgehog (Hh) signal transduction pathway. We found that inactivation of CK2 by CK2β RNAi enhances the loss-of-Hh wing phenotype induced by a dominant negative form of Smoothened (Smo). CK2β RNAi attenuates Hh-induced Smo accumulation and down-regulates Hh target gene expression, whereas increasing CK2 activity by coexpressing CK2α and CK2β increases Smo accumulation and induces ectopic Hh target gene expression. We identified the serine residues in Smo that can be phosphorylated by CK2 in vitro. Mutating these serine residues attenuates the ability of Smo to transduce high level Hh signaling activity in vivo. Furthermore, we found that CK2 plays an additional positive role downstream of Smo by regulating the stability of full-length Cubitus interruptus (Ci). CK2β RNAi promotes Ci degradation whereas coexpressing CK2α and CK2β increases the half-life of Ci. We showed that CK2 prevents Ci ubiquitination and degradation by the proteasome. Thus, CK2 promotes Hh signaling activity by regulating multiple pathway components.

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Figures

FIGURE 1.
FIGURE 1.
Inactivation of CK2β produces loss of Hh phenotype in fly adult wing. A, A wild-type (WT) adult wing showing interveins 1–5. B, C765-Gal4 fly with a WT wing. C, wing expressing CK2βRNAi by C765-Gal4. The phenotype assembles the effects of CK2βRNAi on multiple developmental pathways. D, wing from flies expressing Smo-PKA12 by C765-Gal4. E, wing from flies expressing Smo-PKA12 by C765-Gal4 in the background of CK2αTik/+. F, wing coexpressing Smo-PKA12 with CK2βRNAi by C765-Gal4. G, wing coexpressing Smo-PKA12 with CK2βRNAi and CK2β. H, ptc-Gal4 flies with WT wings. I, adult wing expressing CK2βRNAi by ptc-Gal4. J–M, wing discs expressing Smo-PKA12 alone (K), along with CK2αTik/+ (L), or along with CK2βRNAi (M) by C765-Gal4 stained for Ptc.
FIGURE 2.
FIGURE 2.
CK2 stabilizes Smo and Ci and up-regulates Hh target gene expression. A, wild-type (WT) wing disc was stained with anti-En antibody. B, wing disc expressing UAS-CK2βRNAi by ptc-Gal4 was stained for En. C and C′, wing disc coexpressing UAS-CK2βRNAi with UAS-CK2β by ptc-Gal4 was stained for En (C) and FLAG (C′). White lines in A–C indicate the A/P boundaries that are defined by Ci staining (data not shown). D–F, wing discs coexpressing UAS-CK2βRNAi with UAS-GFP by ap-Gal4 were stained for Smo (D), Ci (D′), Ptc-lacZ (E), or En (F). GFP expression in D″ marks the RNAi cells. G and G′, wild-type wing imaginal disc was immunostained for endogenous Smo and Ci. H–K, wing discs from flies expressing UAS-FLAG-CK2α (H), UAS-FLAG-CK2β (I), UAS-FLAG-CK2αKM (J), or UAS-FLAG-CK2αKM plus UAS-CK2β (K) by MS1096 Gal4 were stained for Ci. L–L″, wing disc coexpressing UAS-FLAG-CK2α with UAS-CK2β by MS1096 Gal4 was stained for Ci, Smo, and dpp-lacZ. M–N′, wing discs coexpressing UAS-FLAG-CK2α with UAS-CK2β by act>CD2>Gal4 were stained for CD2 (green), Ci (red), Dpp-lacZ (blue), or Smo (gray). Clones were marked by the lack of CD2 staining (M) or by the elevation of Ci (N). Arrows in M′ and N indicate the stabilization of Ci, arrows in M″ indicate the elevated dpp-lacZ expression, and arrows in N′ indicate the accumulation of Smo in both A- and P-compartment cells that was induced by overexpressing CK2α with CK2β. All wing imaginal discs shown in this study were oriented with anterior on the left and ventral on the top.
FIGURE 3.
FIGURE 3.
Smo is phosphorylated by CK2. A, CK2 consensus sequence and Smo sequence indicating putative CK2 phosphorylation sites. B, schematic drawing of GST-Smo constructs that were expressed in bacteria. C, CK2 phosphorylates Smo in vitro. Shown here is an in vitro kinase assay by using the purified GST-Smo fusion proteins and the commercial CK2 kinase. D, S2 cells transfected with Myc-Smo and treated with OA or with OA and CK2β dsRNA. Cell extracts were immunoprecipitated and blotted with anti-Myc antibody to detect Smo phosphorylation indicated by its mobility shift on the SDS gel. Arrow indicates hyperphosphorylated forms of Smo, and arrowhead indicates hypophosphorylated and unphosphorylated forms. GFP served as transfection control. Special lysis buffer containing NaF and Na3VO4 was used to examine Smo phosphorylation. The efficiency of CK2β RNAi is shown in the right panel where β-tubulin served as loading control. E, Myc-Smo transfected into S2 cells either with FLAG-CK2α and FLAG-CK2β or with the treatment of CK2β dsRNA. Cell extracts were immunoprecipitated (IP) with anti-Myc and blotted (WB) with anti-Myc to examine the levels of Smo. Regular lysis buffer was used to examine Smo levels. GFP served as transfection control. F–G′, wing discs carrying smo mutant clones immunostained for Ptc (red), En (red), or GFP (green). H–I′, wing discs containing smo mutant clones and expressing VK5-Myc-Smo by tubulinα promoter immunostained to show Ptc, En, and GFP. J–K′, wing discs containing smo mutant clones and expressing VK5-Myc-SmoCK2SA by tubulinα promoter immunostained to show Ptc, En, and GFP. smo mutant clones are recognized by the lack of GFP expression.
FIGURE 4.
FIGURE 4.
CK2 has an additional role downstream of Smo in Hh pathway. A–A″, wing disc bearing smo clones and expressing FLAG-CK2α and FLAG-CK2β by MS1096 Gal4 stained to show the expression of GFP and Ci. Arrow in A′ indicates a smo clone that is marked by the lack of GFP expression. B–C″, wing discs expressing VK5-Myc-SmoSD123CK2SD alone or along with CK2βRNAi by MS1096 Gal4 immunostained for ptc-lacZ and En. D–E″, wing discs expressing UAS-HA-Ci-3P alone or along with UAS-Dicer and UAS-CK2βRNAi by MS1096 Gal4 immunostained for Ptc and HA.
FIGURE 5.
FIGURE 5.
CK2 up-regulates Ci by blocking Ci degradation. A–A″, wing disc coexpressing FLAG-CK2α and FLAG-CK2β immunostained for Ci and Ci-lacZ. Of note, Ci, but not Ci-lacZ, was elevated by CK2. B, CK2 kinase activity required for Ci stabilization. Myc-Ci was transfected into S2 cells with either the indicated CK2 constructs or the treatment of CK2β dsRNA. Cell extracts were subjected to direct Western blotting (WB) with anti-Myc antibody to detect the levels of Myc-Ci, with anti-CK2β antibody to detect the exogenous CK2β expression and the endogenous CK2β level that indicates the efficiency of CK2β RNAi, with anti-FLAG antibody to detect the FLAG-tagged CK2α, with anti-β-tubulin antibody to detect β-tubulin that served as loading control. C, quantification of Myc-Ci relative levels. The level of Ci from cells transfecting Myc-Ci alone was set as 1. **, p < 0.01 (Student's t test). D, S2 cells cotransfected with Myc-Ci and GFP, or with Myc-Ci, GFP, and FLAG-CK2α plus FLAG-CK2β, followed by treatment with cycloheximide for the indicated times. GFP expression served as transfection control. E, quantification of Ci levels from Western blot analysis performed in D. CHX, cycloheximide.
FIGURE 6.
FIGURE 6.
CK2 down-regulates Ci ubiquitination and prevents the proteasome-mediated Ci degradation. A–B′, wing discs expressing UAS-CK2βRNAi by ap-Gal4 were treated with or without MG132 and immunostained for Ci. The treatment of MG132 restored Ci that was down-regulated by CK2β RNAi. GFP marks the RNAi cells. C, CK2 down-regulates Ci ubiquitination. S2 cells were transfected with Myc-Ci and incubated with CK2β dsRNA, or TBB, or cotransfected with FLAG-CK2α and FLAG-CK2β, followed by the treatment with or without MG132. Cell extracts were immunoprecipitated (IP) with anti-Myc antibody and blotted (WB) with anti-Myc antibody to determine the levels of Ci, or blotted with anti-ubiquitin antibody to examine the Ci-bound ubiquitin. IgG served as loading control. D, quantification analysis shows the ratio of ubiquitinated Ci to total Ci in C. Myc-Ci in lane 1 of C was set as 1. *, p < 0.05 (Student's t test). E, Cim1–6, with HIB-interacting sites mutated, is still regulated by CK2. S2 cells were cotransfected with HA-Cim1–6 and GFP, with either CK2α+CK2β or CK2β dsRNA treatment. Cell lysates were subjected to direct Western blotting with anti-HA antibody. F, quantification of HA-Cim1–6 relative levels from E is shown. The level of Cim1–6 from cells transfecting HA-Cim1–6 alone was set as 1. *, p < 0.05 (Student's t test). G, knockdown of the known E3s does not affect Ci ubiquitination that is induced by CK2 inactivation. S2 cells were transfected with Myc-Ci and treated with HIB dsRNA, Debra dsRNA, or Hyd dsRNA, with or without CK2β dsRNA. Cell extracts were immunoprecipitated with anti-Myc antibody and blotted with anti-Myc or anti-ubiquitin antibodies. IgG served as loading control.
FIGURE 7.
FIGURE 7.
CK2 stabilizes Gli proteins in Drosophila wing imaginal discs. A, Western blot (WB) analysis of protein extracts from wing discs expressing UAS-Myc-Gli1 or UAS-Myc-Gli2, either with UAS-CK2α and UAS-CK2β or with UAS-CK2βRNAi, under the MS1096 Gal4 driver. 25 wing discs were used for each sample. CK2β levels detected by anti-CK2β antibody indicated CK2β RNAi efficiency. β-Tubulin served as loading control. B, quantification analysis of the levels Gli protein from the experiment in A. The levels of Gli1 and Gli2 in lanes 1 and 4 were set as 1, respectively. *, p < 0.05 (Student's t test). C, model for the involvement of CK2 in Hh signaling. CK2 is positively involved in Hh signal transduction by phosphorylating and activating Smo and by preventing Ci ubiquitination thus attenuating its degradation by the proteasome.
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