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. 2011 Apr 15;286(15):13502-11.
doi: 10.1074/jbc.M110.217604. Epub 2011 Feb 11.

Dual Phosphorylation of suppressor of fused (Sufu) by PKA and GSK3beta regulates its stability and localization in the primary cilium

Yan Chen  1 , Shen Yue, Lu Xie, Xiao-hong Pu, Tian Jin, Steven Y Cheng
Affiliations

Dual Phosphorylation of suppressor of fused (Sufu) by PKA and GSK3beta regulates its stability and localization in the primary cilium

Yan Chen et al. J Biol Chem. .

Abstract

Suppressor of fused (Sufu) is an essential negative regulator of the sonic hedgehog (Shh) pathway, but little is known about how Sufu itself is normally regulated. Here, we report that Sufu is phosphorylated at Ser-342 and Ser-346 by GSK3β and cAMP-dependent protein kinase A (PKA), respectively, and phosphorylation at this dual site stabilizes Sufu against Shh signaling-induced degradation. We further show that localization of Sufu in the primary cilium is induced by Shh signaling and is required for the turnover of both phosphorylated and total Sufu. Perturbing Sufu phosphorylation with PKA inhibitors or replacing Ser-346 with alanine reduced the stay and replacing Ser-342 and Ser-346 with aspartic acid prolonged the stay of Sufu in the cilia. Finally, ciliary localization of Gli2/3 also required Smo and was similarly influenced by perturbations of PKA activity or mutations at the dual Sufu phosphorylation site. Thus, Shh likely induced trafficking of phospho-Sufu into the primary cilium in a complex with Gli2/3, and dephosphorylation triggered a retrograde export, allowing Sufu to be degraded by the ubiquitin-proteasome system.

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Figures

FIGURE 1.
FIGURE 1.
PKA stabilizes Sufu through controlling phosphorylation of Ser-342 and Ser-346. A, Sufu sequences surrounding the four PKA consensus sites. Mass spectrometry (mass spec) analysis shows that Ser-301/Thr-305 and Ser-342/Ser-346 are the actual phosphorylation sites by PKA in vivo. B, Western analysis showing that co-expression of PKAc reduces Sufu turnover after cycloheximide (CHX) treatment. Vect, vector. C, autoradiogram of in vitro kinase assays reveals Sufu as a substrate of PKA but not casein kinase I, casein kinase II, or GSK3β. Input GST and GST-Sufu are shown in the right panel. D, upper panel, autoradiogram of in vitro PKA kinase assay as in C showing that replacing any one of the four PKA consensus sites with alanine is not sufficient to affect Sufu phosphorylation but replacing all four sites abolished it. Lower panel, Western analysis of Myc-Sufu and mutants expressed by in vitro translation. IB, immunoblot. Western blot analysis (E) and quantification (F) of the turnover rate of Sufu and its mutants in transfected Sufu−/− MEFs. The data presented in F were derived from three repeated experiments. G, luciferase reporter assay for various Sufu mutants co-transfected with Gli1 and the 8×GliBS construct in Sufu−/− cells with Renilla luciferase as an internal control. Each data point represents results from triplicate wells. Error bars are standard deviations. CHX, cycloheximide. *, p < 0.01.
FIGURE 2.
FIGURE 2.
Shh signaling regulates Sufu phosphorylation in vivo. A, sequential recognition of Sufu at Ser-346 and Ser-342 by PKA and GSK3β, respectively. Myc-tagged wild type and mutant Sufu were individually force-expressed in HEK293 cells. The proteins were isolated by immunoprecipitation (IP) with anti-Myc antibodies and analyzed by Western blot with Ab342P and Ab346P for phosphorylated and anti-Sufu for total Sufu. IB, immunoblot. B, synergistic phosphorylation of Sufu by co-expression of PKAc and GSK3β. The loading in each lane was adjusted according to total Sufu to reveal changes in the level of phospho-Sufu and the Western analysis was carried out as in A. Vec, vector. C, activation of PKA with forskolin and isobutylmethylxanthine treatment dramatically increased, whereas in D, blocking GSK3β with SB216763 abolished phosphorylation of the endogenous Sufu. C and D, freshly isolated normal MEFs were treated with above compounds for the time as indicated, and the whole cell lysates were used in Western analysis. Note the exposure level of the Ab342P blot in C was intentionally set lower than that in D to avoid saturation of the film. E, inactivation of the Smo allele increased the endogenous levels of the total and phosphorylated Sufu in SmoFlox/Flox MEFs. Ad-Cre infection was carried out for 12 h before the proteins in the whole cell lysates were analyzed. This experiment was repeated, but only one representative Western blot was quantified in F.
FIGURE 3.
FIGURE 3.
Smo dependence of Shh signaling-induced localization of Sufu in primary cilia. A, representative immunofluorescent staining of Sufu (red) at the tips of primary cilia or B, quantification thereof in SmoFlox/Flox MEFs infected with mock solution or Ad-cre viruses. Viral infection was for 12 h, and thereafter the cells were treated with either ShhN ligand or purmorphamine (Purm) for 24 h as indicated. “n” denotes the total number of primary cilia counted at each data point. Primary cilia were marked with anti-acetylated α-tubulin staining (green). C, representative immunofluorescent staining of Sufu at the tip of the primary cilium in Ptch−/− cells. Cyclopamine (CPA) treatment was carried out after the cell culture reached confluence. D, quantification of Sufu-positive primary cilia; E, intensity of Sufu in primary cilia affected by cyclopamine treatment. F, effect of MG132 and cyclopamine on the intensity of Sufu in primary cilia in Ptch−/− cells. n/s, not statistically significant (p > 0.1).
FIGURE 4.
FIGURE 4.
Primary cilium is required for Sufu degradation. A, immunofluorescent staining of phospho-Sufu in primary cilia with Ab342P phospho-specific antibody. Sufu−/− cells were stained as negative controls. Western blot analysis (B) and quantification thereof (C) showing stabilization of Sufu in Kif3a−/− cells was carried out after blocking protein synthesis with cycloheximide (CHX) treatment as indicated. The data in the graph were derived from three repeated experiments. D, Western blot showing elevated phospho-Sufu in Kif3a−/− cells after anti-Sufu immunoprecipitation (IP) or in whole cell lysates (WCL). MG132 treatment for 6 h restored the level of total but not phosphorylated Sufu.
FIGURE 5.
FIGURE 5.
Phosphorylation promotes ciliary retention of Sufu. In Ptch−/− cells, immunofluorescent staining indicated that KT5720 treatment led to a rapid decrease of the percentage of Sufu-positive primary cilia (A) and the intensity of Sufu in primary cilia (B). Sufu was typically found in ∼95% of cilia, but this number decreased to ∼65% after KT5720 treatment for 24 h. C, representative autofluorescence images of Sufu-GFP and Sufu mutants in transfected Sufu−/− cells. Replacing Ser-346 with alanine decreased while replacing Ser-346 or both Ser-342 and Ser-346 with aspartic acid ciliary localization. Cilia were visualized by staining with anti-acetylated α-tubulin (red). The number of cilia counted for each data point was between 17 and 24. D, quantification of C. *, p < 0.01; **, p < 0.001. E, representative immunofluorescent staining of phospho-specific and total Sufu at ciliary tips in wild type or IFT122 null MEFs. Purmorphamine (Purm) treatment induced immunofluorescent staining of p-Sufu and total Sufu at the tips of primary cilia. In IFT122 null MEFs, p-sufu was not detected whereas total Sufu accumulated at the tip of cilia with or without purmorphamine treatment. F, quantification of E. G, live cell imaging of photoactivatable Sufu and S342D/S346D mutant force-expressed from PA-mCherry1-N1-Sufu and PA-mCherry1-Sufu-S342D/S346D, respectively, in Ptch−/− cells. Somatostatin receptor-3-GFP was co-transfected to mark for cilia. A type area to be photoactivated was marked, and images of merged green and red channels taken pre- and post-photoactivation were shown. H, relative fluorescence of PA-mCherry-Sufu or PA-mCherry1-Sufu-S342D/S346D was calculated at each time point after photoactivation by correcting for photoextinction of mCherry fluorescence from the total intensity recorded in the red channel at ciliary tips.
FIGURE 6.
FIGURE 6.
Phosphorylation promotes Shh signaling-induced co-localization of Sufu and Gli3 in primary cilia. Representative immunofluorescent staining of Gli3 (A) and quantification thereof (B) show Shh signaling-induced localization at the tip of the primary cilia in SmoFlox/Flox MEFs and curtailment by Ad-cre infection as in Fig. 3, A and B. C and D, effects of cyclopamine and KT5720 treatment on the percentage of Gli3 positive primary cilia and the intensity of Gli3, respectively. E, representative immunofluorescent staining and quantification of Gli3 (F) restored by Sufu and its phosphorylation site mutants that were force-expressed in Sufu−/− cells. *, p < 0.01. G, model for the regulation of Shh-induced co-localization of Sufu and Gli2/3 in primary cilia by phosphorylation. Purm, purmorphamine.
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