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. 2007 Oct;13(4):481-95.
doi: 10.1016/j.devcel.2007.09.006.

Regulation of Ci-SCFSlimb binding, Ci proteolysis, and hedgehog pathway activity by Ci phosphorylation

Margery G Smelkinson  1 , Qianhe Zhou, Daniel Kalderon
Affiliations

Regulation of Ci-SCFSlimb binding, Ci proteolysis, and hedgehog pathway activity by Ci phosphorylation

Margery G Smelkinson et al. Dev Cell. 2007 Oct.

Abstract

Hedgehog (Hh) proteins signal by inhibiting the proteolytic processing of Ci/Gli family transcription factors and by increasing Ci/Gli-specific activity. When Hh is absent, phosphorylation of Ci/Gli triggers binding to SCF ubiquitin ligase complexes and consequent proteolysis. Here we show that multiple successively phosphorylated CK1 sites on Ci create an atypical extended binding site for the SCF substrate recognition component Slimb. GSK3 enhances binding primarily through a nearby region of Ci, which might contact an SCF component other than Slimb. Studies of Ci variants with altered CK1 and GSK3 sites suggest that the large number of phosphorylation sites that direct SCF(Slimb) binding confers a sensitive and graded proteolytic response to Hh, which collaborates with changes in Ci-specific activity to elicit a morphogenetic response. We also show that when Ci proteolysis is compromised, its specific activity is limited principally by Su(fu), and not by Cos2 cytoplasmic tethering or PKA phosphorylation.

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Figures

Figure 1
Figure 1. Identification of a Slimb binding site on Ci between PKA sites 1 and 2
(A) Schematic showing the Zinc finger (Zn) DNA-binding domain shared by Ci-155 and Ci-75, the phosphorylated region (Phos) depicted in B and C, and the region of Ci deleted in the Ci-U transgene. (B) Amino acids 834−899 of Ci showing the three (lightly boxed) groups of PKA sites (red, P1-P3), PKA-primed GSK3 sites (green, G2 and G3) and PKA-primed CK1 sites (blue, C1-C3). Motifs resembling a consensus (DSGxxS) Slimb binding site are underlined in purple. The four Ala substitutions of Ci-Ds2 and Ci-Us3 (bracketed black dots) and residues deleted in Ci-Δ862−883 are indicated. (C) Single Ala substituents tested in this study are indicated by residue number. The extended Slimb binding motif defined here is underlined (purple) and arrows indicate critical consecutive CK1 (blue) and GSK3 (green) phosphorylations. Dashed underlining indicates residues of a potential weak Slimb binding motif. (D) GST-Ci proteins with the named substitutions were phosphorylated by the indicated protein kinases and incubated with extracts of Kc cells transfected with an HA/Flag-Slimb-Myc expression vector. Proteins brought down with glutathione beads were visualized on Western blots with HA antibody. In this and other Figures, thick red boxes group single experiments that test GST-Ci variants relative to each other and a wild-type (WT) control strictly in parallel. HA-Slimb band intensities were measured, corrected for background and expressed relative to GST-Ci-WT with no phosphorylation (fixed at 1.0) for that experiment. GST blots (not shown) confirm similar protein levels and similarly efficient phosphorylation, judged by mobility shifts.
Figure 2
Figure 2. The primary Slimb binding site of Ci includes S852 (G2)
(A) Sequences of named GST-Ci variants (left) between PKA sites 1 and 2 (orange), highlighting inserted residues in pink, GSK3 sites primed by PKA site 2 in green and S852 phosphorylated only by CK1 in blue. The core Slimb binding motif (black line), S852 and PKA site 2 (arrows) are highlighted. (A, D) Flag/HA-Slimb-Myc binding to GST-Ci variants, as in Figure 1. (B) Ci-SL has altered PKA sites 1 and 3 (P1A, P3A). PKA site 2 (red) primes phosphorylation of three GSK3 sites (green) to create the Slimb/β-TRCP binding consensus of β-catenin. (C) Binding of phosphorylated GST-Ci-WT and GST-Ci-SL to Flag/HA-Slimb-Myc-WT (“Slimb-WT”) or Flag/HA-Slimb(R333A/R353A)-Myc (“Slimb-RA”). Numbers show binding relative to Slimb-WT (top row) or Slimb-RA (bottom row). 5% of the extract used for each binding reaction is also shown.
Figure 3
Figure 3. GSK3 enhancement of Ci-Slimb binding requires GSK3 sites primed by PKA site 3 and SCF complex formation
(A) GST-Ci variants binding to wild-type Flag/HA-Slimb-Myc probed with HA antibody. Also shown are relevant Ci and Gli-2/3 sequences. (B) Western blots for the indicated antigens (right) of extracts of Kc cells expressing transfected Myc-Slimb and the indicated Flag/HA-Slimb derivatives (top line) before (“Input”) or after immune precipitation with Flag antibody (“IP: Flag”). (C) Schematic of domains of Slimb and associated SCF complex proteins as a dimer bound to E2 enzymes. Folding of the two SCF complexes around the D-D axis toward each other likely gives a more accurate representation (Tang et al., 2007). (D, E) Binding of Flag/HA-Slimb-ΔD (D) or Flag/HA-Slimb-ΔF (E) expressed alone (upper panels) or together with Flag/HA-Slimb-WT to GST-Ci and GST-Ci-SL assayed with Flag antibody (D) or HA antibody (E). Corrected band intensities are shown relative to SL-WT binding to Ci-WT, and also for the lower panels relative to Sl-ΔD or SL-ΔF binding to Ci-WT (bottom line).
Figure 4
Figure 4. Hh only fully blocks Ci-155 processing if CK1 regulates Slimb binding
(A-D) Conversion of Ci-155 to Ci-75 repressor was assayed in positively marked wing disc clones that lack GAL80 and therefore express the indicated Ci variants together with GFP (green). Reduced expression of hh-lacZ (red) in posterior cells (right), indicating Ci-75 repressor, was seen for Ci-SL (C, arrow), especially outside the central wing blade primordia (C, arrowhead), but not (open arrowheads) for Ci-WT (A), Ci-G2,3E (B) or Ci-Y846G (D).
Figure 5
Figure 5. Su(fu) and Fu kinase regulate the activity of proteolysis-resistant Ci-155
(A) Ectopic induction of ptc-lacZ (blue; assayed by β-galactosidase activity) by ci transgenes driven by C765-GAL4 was seen only in posterior wing disc cells (right) for Ci-WT at 29C and 20C but was also seen in anterior cells for Ci-S849A at 29C. At 20C, Ci-S849A induced anterior ptc-lacZ only when Su(fu) was absent. (B) Selective induction of ptc-lacZ (red; detected by antibody to β-galactosidase) in posterior cells (right) by Ci-S849A and Ci-P1−3A expressed at 20C using C765-GAL4 was eliminated in fumH63 wing discs lacking Fu kinase activity but was only reduced in discs expressing Ci-WT (here at 20C) or Ci-U (here at 29C).
Figure 6
Figure 6. Specific activity of proteolysis-resistant Ci-155 is not significantly reduced by PKA or by Cos2
(A-I) Activation of the ptc-lacZ reporter (red) by Ci-WT (A-C), Ci-S849A (D-F) and Ci-P1−3A (G-I) expressed using C765-GAL4 at 20C was compared in posterior clones (arrowheads), marked by loss of GFP (green), that lacked activity of Smo alone (A, D, G) or Smo together with either PKA (B, E, H) or Cos2 (C, F, I). ptc-lacZ induction was increased by the additional loss of PKA and Cos2 for Ci-WT (B, C vs A) but not for Ci-S849A (D-F) or Ci-P1−3A (G-I).
Figure 7
Figure 7. Ectopic Hh-target gene induction due to inefficient Ci-155 proteolysis
C-terminal Myc epitope staining (red) of Ci variants expressed at 29C using C765-GAL4 was highest in posterior cells (right) only for Ci-Y846G and some Ci-Us3 and Ci-Ds2 lines. The inferred inefficient proteolysis of Ci-155 in other lines was accompanied in every case except for one Ci-Us3 line by ectopic induction of anterior ptc-lacZ (blue), assayed by β-galactosidase activity. “G2,3E” is S852E/S888E and “SRRAS” is S855A/S891A. Results of Ci-75 repressor assays (“hh-lacZ repression”) and Slimb binding assays (expressed as a percentage of wild-type Ci binding after phosphorylation with PKA, CK1 and GSK3) are also tabulated for each Ci variant.
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