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. 2011 Jul 5;4(180):ra43.
doi: 10.1126/scisignal.2001747.

Sequential phosphorylation of smoothened transduces graded hedgehog signaling

Ying Su  1 , Jason K Ospina, Junzheng Zhang, Andrew P Michelson, Adam M Schoen, Alan Jian Zhu
Affiliations

Sequential phosphorylation of smoothened transduces graded hedgehog signaling

Ying Su et al. Sci Signal. .

Abstract

The correct interpretation of a gradient of the morphogen Hedgehog (Hh) during development requires phosphorylation of the Hh signaling activator Smoothened (Smo); however, the molecular mechanism by which Smo transduces graded Hh signaling is not well understood. We show that regulation of the phosphorylation status of Smo by distinct phosphatases at specific phosphorylated residues creates differential thresholds of Hh signaling. Phosphorylation of Smo was initiated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) and further enhanced by casein kinase I (CKI). We found that protein phosphatase 1 (PP1) directly dephosphorylated PKA-phosphorylated Smo to reduce signaling mediated by intermediate concentrations of Hh, whereas PP2A specifically dephosphorylated PKA-primed, CKI-phosphorylated Smo to restrict signaling by high concentrations of Hh. We also established a functional link between sequentially phosphorylated Smo species and graded Hh activity. Thus, we propose a sequential phosphorylation model in which precise interpretation of morphogen concentration can be achieved upon versatile phosphatase-mediated regulation of the phosphorylation status of an essential activator in developmental signaling.

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Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
PP1 and Wdb-PP2A differentially regulate the phosphorylation and localization of Smo. (A to L) GFP-Smo localization in cl-8–gsmo cells transfected with the indicated dsRNAs in the absence of exogenous Hh. (A) gal80 dsRNA was used as a negative control because gal80 is not present in the fly genome, whereas (B) ptc dsRNA was a positive control, which resulted in the localization of Smo to the cell surface (arrows). dsRNAs targeting the PP2A catalytic subunit Mts (C), the scaffolding subunit CG17291 (D), or one of the regulatory B subunits Wdb (E) promoted the surface localization of Smo. dsRNAs for the other PP2A B subunits (F to H) or any of four PP1c subunits (I to L) had no effect on the localization of Smo. Scale bar, 15 μm. (M and N) Western blotting (WB) analysis of cl-8–gsmo cells transfected with dsRNAs in the absence of exogenous Hh. Slow-migrating, phosphorylated forms of GFP-Smo (GFP-pSmo) were detected with an antibody against GFP. β-Tubulin served as a loading control. (O) Western blots of cl-8–gsmo cells treated with either Hh or TC or transfected with the nipp1 plasmid in the absence of Hh. (P) Luciferase assays for cl-8–gsmo cells transfected with the ptc-luciferase plasmid. Cells were either treated with Hh for 24 hours or transfected with the indicated dsRNAs and cultured in the absence of exogenous Hh for 4 days. SDs are shown (n = 3 experiments). *P < 0.005.
Fig. 2
Fig. 2
Smo forms complexes with PP1c and Wdb-PP2A. (A to C) Immunocomplexes formed in (A) cl-8–gfp, (B) cl-8–gsmo, or (C) cl-8–gsmo cells transfected with individual pp1c-V5 plasmids. GFP or GFP-Smo was immunoprecipitated (IP) with an antibody against GFP conjugated to agarose. GFP-Smo formed complexes with PP2A catalytic (Mts) and regulatory (Wdb) subunits as well as with PP1c subunits. GAPDH served as a negative control. (D and E) Three versions of recombinant GST-Smo cytoplasmic tail fusions (D), all of which contain three PKA-CKI consensus clusters (circles), were generated in bacteria (E) for in vitro direct binding assays with PP1 or PP2A. Only GST-Smo 557–1036 (asterisk) contains a putative type II PP1-binding site (F-xx-R/K-x-R/K), which is marked by a solid square at amino acid positions 597 to 602 (81). (F to H) A direct interaction between GST-Smo and PP2A or PP1c. GST-Smo bound on glutathione beads was incubated with (F) the purified PP2A A:C (Mts) dimer, (G) MBP-Wdb, or (H) recombinant PP1c. Compared with the shorter GST fusions, GST-Smo 557–1036 showed stronger binding to Mts (panel F, top). The binding between GST-Smo and the PP2A A scaffolding subunit was detectable only in a low-stringency wash condition (panel F, bottom). (G) MBP-Wdb efficiently pulled down GST-Smo 600–800 and GST-Smo 557–1036 (asterisk). (H) Only GST-Smo 557–1036, which contains the PP1-binding site, pulled down recombinant PP1c. GST served as a negative control.
Fig. 3
Fig. 3
PP1 and Wdb-PP2A regulate distinct thresholds of Hh signaling. Stabilization of CiFL, induction of dpp, and induction of ptc and production of Col protein in wing discs correlate with low-, intermediate-, and high-threshold Hh signaling, respectively. Immunofluorescence was used to visualize CiFL, Col, Smo, and Ptc proteins, whereas in situ hybridization was used to detect dpp and ptc mRNA, as indicated in the panels. (A to E) Normal Hh-responsive gene expression patterns. (A) MS1096-Gal4–driven mCD8-gfp was expressed to a greater extent in the dorsal (d) as compared to the ventral (v) compartment of the wing disc. The dorsal-ventral (d/v) boundary is marked by a dashed line in panel A. (F to T) Distinct effects on Hh-responsive gene expressions in wing discs overexpressing (F to J) the PP1 inhibitor NIPP1, (K to O) WdbDN, or (P to T) wild-type Wdb driven by MS1096-Gal4. wdbDN encodes a dominant-negative mutant that inhibits the function of Wdb-PP2A (28). (U to Y) Clonal analyses of wdb function in wing discs. Ectopic wdb expression in clones (U to W, marked by mCD8-GFP; arrows) abolished Ptc protein production (W, right image), but stabilized CiFL (U, right image) and induced ectopic dpp transcription (V, right image) in anterior clones located away from the AP boundary. Both CiFL and Smo were stabilized in wdbIP loss-of-function clones (X and Y, identified by the absence of GFP; arrows). Magnified images of the areas boxed in (U to W, left panels) and (X) are shown in (U to W, right panels) and (Y), respectively. wdb-overexpressing clones were generated by the flip-out technique (54), as described in Materials and Methods.
Fig. 4
Fig. 4
PP1 and PP2A dephosphorylate distinct species of pSmo. (A and B) PP1 or PP2A alone incompletely dephosphorylated GFP-pSmo. Hh-induced cl-8–gsmo lysates were treated with increasing amounts of (A) PP1 or (B) PP2A. Western blotting analysis with an antibody against GFP (top panels) detected all forms of GFP-pSmo (box brackets), which were completely removed by CIP. The α-Smo-pS667 antibody (middle panels) specifically detected PKA-phosphorylated GFP-Smo (GFP-PKA-pSmo). PP1 efficiently dephosphorylated GFP-PKA-pSmo, whereas PP2A treatment enriched for the α-Smo-pS667 antibody–specific GFP-PKA-pSmo. GFP-PKA-pSmo species treated with PP2A were greatly reduced in abundance by additional PP1 activity (panel B, compare lane 6 with lanes 2 to 4). The Western blot analyzed with the α-Smo-pS667 antibody (A, middle panel) was overexposed to reveal PKA-pSmo in lysates containing enhanced amounts of pSmo. β-Tubulin served as the loading control. (C and D) PP1 dephosphorylates PKA-phosphorylated Smo. GST-PKA-pSmo labeled with cold ATP was dephosphorylated by PP1, but not by PP2A, as indicated by the decrease in the extent of detection of GST-PKA-pSmo with α-Smo-pS667 (C). This PP1 response was inhibited by PP1-I2, a specific PP1 inhibitor. Radiolabeled PKA–phosphorylated GST-Smo (GST-PKA-32pSmo) was dephosphorylated by PP1, but not by PP2A (D). λ-Phosphatase (λPpase) was used as a positive control in panels C to F. (E) GST-Smo (lane 1) was phosphorylated with PKA alone (lanes 2 and 3) or PKA and CKI (lanes 4 to 8) in the presence of cold ATP. Treating GST-CKI-pSmo with PP2A (lane 5), but not PP1 (lane 6), enriched for the α-Smo-pS667–reactive GST-PKA-pSmo, which was reversed by additional PP1 activity (lane 7). (F) PP2A specifically targets CKI consensus modifications. Individual PKA-CKI clusters in GST-Smo were phosphorylated by PKA in the presence of ATP followed by CKI-mediated phosphorylation in the presence of [γ-32P]ATP (lanes 1, 3, and 5), resulting in selective incorporation of [γ-32P]ATP only at CKI consensus serines within a single cluster, as schematically shown. PP2A removed CKI phosphorylation–specific 32P-phosphates from individual PKA-CKI clusters (lanes 2, 4, and 6).
Fig. 5
Fig. 5
Distinct effects of phosphorylation-defective Smo variants on Hh signaling. (A to D) ap-Gal4–driven mCD8-gfp in the dorsal compartment (B) did not affect Hh signaling (C and D). The dorsal-ventral boundary is marked by a dashed line in (B). (E to T) Wild-type (WT) wing discs expressing (E to H) smo (smoWT), (I to L) GSK (smoGSK-SA), (M to P) CKI (smoCKI-SA), or (Q to T) PKA (smoPKA-SA) phosphorylation–defective smo variants driven by ap-Gal4 in the dorsal compartment (box brackets). Overexpressed SmoCKI-SA (M, O, and P) and SmoPKA-SA (Q, S, and T) reduced high-threshold Hh signaling (including ptc mRNA expression and Col production, and CiFL at the AP boundary, as indicated by arrows). In contrast, these two mutants had distinct effects on intermediate-threshold Hh signaling (N and R). SmoPKA-SA substantially reduced dpp transcription (R). Overexpressed smoCKI-SA did not result in obvious expansion of the area in which dpp was expressed.
Fig. 6
Fig. 6
Sequential phosphorylation of Smo transduces graded Hh signaling. (A to O) PKA-mediated phosphorylation of Smo (SmoCKI-SA) rescues dpp transcription in smo2 loss-of-function clones. Magnified images of the areas boxed in panels A, F, and K are shown in panels B to E, G to J, and L to O, respectively. dpp mRNA (red) was detected by in situ hybridization (D, I, and N), followed by incubation with antibodies against Myc (green) and GFP (blue). Cells in smo2 clones (C, H, and M) did not express Myc, whereas cells expressing smo phosphorylation variants (E, J, and O) did not express CFP, respectively. Cells in a smo2 clone with transgene expression (that is, Myc-negative and CFP-negative) are circled with dashed lines (B, G, and L). dpp mRNA was not expressed in cells of a smo2 clone lacking the smo transgene (that is, Myc-negative but CFP-positive, solid line in panel D). SmoCKI-SA (D) and SmoGSK-SA (I), but not SmoPKA-SA (N), rescued dpp transcription in smo2 clones. (P) Model for the transduction of graded Hh signaling by sequential phosphorylation of Smo. Shown are four different states of Smo phosphorylation in response to Hh signaling: unphosphorylated Smo, basal level of Smo phosphorylation (Basal-pSmo), moderately phosphorylated Smo (Moderate-pSmo), and enhanced phosphorylation of Smo (Enhanced-pSmo). Stabilization of the Hh signaling transcription factor CiFL, which is indicative of low-threshold Hh signaling, requires a basal amount of Smo phosphorylation. PKA- and PP1-mediated, moderately phosphorylated Smo is sufficient to induce the transcription of dpp, an intermediate-threshold Hh signaling target. Enhanced phosphorylation of Smo, mediated by CKI and Wdb-PP2A, is required to maintain the expression of high-threshold Hh targets, including ptc and Col. (Q and R) cl-8–gsmo cells were incubated with increasing amounts of Hh-conditioned medium (Hh-CM, 10 to 75% in fresh medium) for 8 hours. An antibody against GFP was used to detect Hh-induced progressive phosphorylation of Smo (top panel). The maximal extent of Smo phosphorylation was observed when cells were treated with 50 to 75% Hh-CM (lanes 4 and 5); however, the abundance of GFP-PKA-pSmo, detected by α-Smo-pS667 (middle panel), increased initially (lanes 1 to 3), reached a maximal intensity with 50% Hh-CM treatment (lane 4), and then declined sharply at 75% Hh-CM (lane 5). The amounts of Hh protein present in Hh-CM were detected with an antibody against Hh (bottom panel). Quantification of the migration and intensity of pSmo species in panel Q, as described in Materials and Methods, is shown in panel R.
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