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. 2011 Jun 14;20(6):802-14.
doi: 10.1016/j.devcel.2011.04.020.

Hedgehog activates fused through phosphorylation to elicit a full spectrum of pathway responses

Qianhe Zhou  1 , Daniel Kalderon
Affiliations

Hedgehog activates fused through phosphorylation to elicit a full spectrum of pathway responses

Qianhe Zhou et al. Dev Cell. .

Abstract

In flies and mammals, extracellular Hedgehog (Hh) molecules alter cell fates and proliferation by regulating the levels and activities of Ci/Gli family transcription factors. How Hh-induced activation of transmembrane Smoothened (Smo) proteins reverses Ci/Gli inhibition by Suppressor of Fused (SuFu) and kinesin family protein (Cos2/Kif7) binding partners is a major unanswered question. Here we show that the Fused (Fu) protein kinase is activated by Smo and Cos2 via Fu- and CK1-dependent phosphorylation. Activated Fu can recapitulate a full Hh response, stabilizing full-length Ci via Cos2 phosphorylation and activating full-length Ci by antagonizing Su(fu) and by other mechanisms. We propose that Smo/Cos2 interactions stimulate Fu autoactivation by concentrating Fu at the membrane. Autoactivation primes Fu for additional CK1-dependent phosphorylation, which further enhances kinase activity. In this model, Smo acts like many transmembrane receptors associated with cytoplasmic kinases, such that pathway activation is mediated by kinase oligomerization and trans-phosphorylation.

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Figures

Figure 1
Figure 1. CK1α acts downstream of Smo
(A) The indicated transgenes were expressed evenly throughout wing discs using C765-GAL4 at 25C, followed by staining for expression of the Hh target genes ptc-lacZ (red) and En (green). The posterior (right) edge of ptc-lacZ expression (arrows) marks the boundary between posterior Hh-producing cells and anterior (left) Hh-responsive cells. Constitutively active SmoD1–3 induced strong ectopic anterior ptc-lacZ and En expression, which was largely prevented by an RNAi transgene directed towards CK1α and restored by co-expression of excess CK1α. (B) Extracts of wild-type or Su(fu)LP mutant wing discs expressing the indicated transgenes under the control of C765-GAL4 at 29C were subjected to Western Blot analysis using antibodies to Fu and Su(fu). Ci-5m induces Hh target genes but does not activate Fu ectopically (Price and Kalderon, 1999). SmoD1–3-induced upward shifts, indicative of phosphorylation of Fu and Su(fu), which were reduced by inhibition of CK1α.
Figure 2
Figure 2. Fu kinase activation requires Cos2 and Fu activation loop phosphorylation
(A) Clones lacking Cos2 activity (arrows, no green GFP) failed to induce En expression (red) in response to Hh at the AP border (left panel) or in response to SmoD1–3 (middle panel) unless GAP-Fu was expressed together with SmoD1–3 (right panel) throughout the wing disc using C765-GAL4 at 29C (B) Sequence alignment of kinase activation loops bordered by conserved (bold) DFG and APE motifs. Potential Fu phosphorylation sites are colored, with green arrows representing consensus primed CK1 sites. For the mammalian kinases shown, phosphorylation sites contributing to activation are colored, with red highlighting residues equidistant from the conserved APE motif. (C) Wild-type (WT) or single residue variants of Flag-tagged Fu were expressed under the control of C765-GAL4 at 18C in fumH63 wing discs and stained for both ptc-lacZ (red) and En (green) expression. Arrows indicate the posterior borders of ptc-lacZ expression, providing a landmark for assessing anterior, Hh-dependent, En expression (to the left of the arrows). Full (+++), strong (++), poor (+) and zero (-) rescue of ptc-lacZ and En induction by Hh are indicated. (D) Flag-tagged Fu with T151E and T154E substitutions (Fu-EE) expressed under the control of C765-GAL4 at 29C induced (left panels) strong ectopic ptc-lacZ (red) and En (green) in anterior cells (left panels; arrows indicate the AP boundary) and increased Cos2 S572P phospho-epitope staining (red) in anterior smo mutant clones (marked by green Flag staining and arrows; right panels). (E) Western blot of extracts of wing discs expressing Fu transgenes using C765-GAL4 at 29C, showing gel mobility shifts (arrows) of tagged Fu-EE and endogenous Su(fu), indicative of increased phosphorylation promoted by Fu-EE but not by wild-type Fu.
Figure 3
Figure 3. Regulation of Fu activity by CK1 and Hh
(A) Co-expressing SmoD1–3 with Fu-EE or Fu-EEAAV using C765-GAL4 at 29C in fumH63 wing discs induced strong ectopic anterior En (green) expression (left of arrows), which was largely prevented by CK1 RNAi transgene expression. (B) Extracts of wing discs expressing the indicated transgenes under the control of C765-GAL4 at 29C were subjected to Western Blot analysis using antibodies against Su(fu). CK1 RNAi reduced upward shifts, indicative of phosphorylation, induced by SmoD1–3, Fu-EE, Fu-EE 1–473 and Fu-EEAAV. (C) Flag-tagged Fu variants were expressed using C765-GAL4 at the indicated temperatures in fumH63 wing discs and stained for both ptc-lacZ (red) and En (green) expression to reveal zero (-) or full (+++) rescue, or full rescue accompanied by ectopic pathway activity (right panels). Arrows indicate the posterior borders of ptc-lacZ expression.
Figure 4
Figure 4. Fu-stimulated Fu phosphorylation activates Fu
(A) Western blots with HA or Flag antibody of extracts of Kc cells expressing high levels of tagged Fu-EE variants and Cos2. G13V and S159A substitutions prevented a robust gel mobility shift. (B) Western blot with Flag antibody of extracts of Kc cells co-expressing high levels of Cos2 together with the indicated Flag-tagged Fu and HA-tagged Fu variants (using a 3:1 ratio of HA-Fu to Flag-Fu). HA-FuEE, but not kinase-defective HA-FuEE G13V S159A, induced upward shifts of Flag-Fu variants, indicating Fu intermolecular phosphorylation. (C) Western blot with Flag antibody of extracts of Kc cells co-expressing low levels of Cos2 and the indicated Flag-tagged Fu variants, with or without SmoD1–3 and Hh, showing a Hh-stimulated mobility shift for wild-type Fu but not for kinase-defective Fu G13V. (D) Western blot with Fu antibody of wild-type (WT) or fumH63 wing disc extracts expressing SmoD1–3, showing Fu mobility shift depends on Fu kinase activity. (E) Western blot with Flag antibody of extracts of Kc cells co-expressing low levels of Cos2 and the indicated Flag-tagged Fu variants. Kinase-defective Flag-Fu proteins (due to S159A or G13V substitutions) showed mobility shifts only when co-expressed with SmoD1–3, Hh and low levels of HA-FuEE. (F) Fu variants were expressed under the control of C765-GAL4 at 29C in wing discs either alone (top row) or together with Fu-EE Δ271–344 (bottom row). Ptc (red) was ectopically induced in anterior cells only when Fu-WT, Fu-EEE or Fu1–473 were co-expressed with Fu-EE Δ271–344. Arrows indicate the AP boundary.
Figure 5
Figure 5. Fu stabilizes Ci-155 via Cos2 S572 phosphorylation
(A) Strongly elevated anterior full-length Ci-155 (red; 2A1 antibody) and ptc-lacZ (green) were induced by expressing Fu-EE but not wild-type Fu using C765-GAL4 at 29C, and were substantially reduced by co-expressing activated mouse PKA catalytic subunit (mC*). (B) smo cos2 double mutant clones, marked by GFP (green, arrows) showed elevated full length Ci-155 (red). (C) smo clones expressing Flag-Fu-EE, marked by Flag staining (green, arrows), in Su(fu)LP discs at 29C, showed elevated Ci-155 (red) levels. (D-G) smo cos2 clones expressing Cos2 WT or Cos2 S572A, marked by GFP (green, arrows), showed normal or reduced anterior Ci-155 (red) staining (D, F), while co-expressing Fu-EE increased Ci-155 levels in clones expressing Cos2 WT (E) but not in clones expressing Cos2 S572A (F). Transgene expression in (C-F) was limited to smo cos2 clones using MARCM and C765-GAL4 at 29C.
Figure 6
Figure 6. Fu antagonizes mouse SUFU and activates the Hh pathway independent of Su(fu)
(A) Normal AP border ptc-lacZ (red) expression in fumH63 Su(fu)LP wing discs (top left) was inhibited by expression of the indicated fly Su(fu) variants or mouse Su(fu) (mSUFU) using C765-GAL4 at 29C (top row). Arrows indicate the posterior edge of ptc-lacZ expression in the wing pouch region, where responses to Hh are clearest. The same Su(fu) variants permitted normal induction of ptc-lacZ (red) and En (green) by Hh in the wing pouch region of the AP border in Su(fu)LP wing discs (middle and bottom row). (B) Fu-EE co-expressed with Su(fu)-5A or mSUFU using C765-GAL4 at 29C induced strong ectopic ptc-lacZ (red) and En (green) in Su(fu)LP mutant wing discs. (C) AP border En (green, arrows) staining was reduced in fumH63 Su(fu)LP wing discs (right panels) compared to normal (not shown) or Su(fu)LP wing discs (left panels), while ptc-lacZ (red) expression was normal in each case. (D) Wing discs expressing SmoD1–3 using C765-GAL4 at 29C showed strong ectopic anterior En (green) expression for wild-type (left) and Su(fu)LP discs (right), no ectopic En in fumH63 discs and only very little ectopic En in fumH63 Su(fu)LP discs (middle panels). Arrows indicate the posterior border of ptc-lacZ in (A-D). (E) Expressing GAP-Fu using C765-GAL4 at 29C (right panels of each pair) in slimb1 Su(fu)LP clones (lacking green GFP, arrows) did not increase Ci-155 levels (red, left) but increased ectopic expression of both Ptc (red, middle) and En (red, right) in the mutant clones.
Figure 7
Figure 7. Summary model of Fu activation mechanism and activities
We postulate that a low affinity interaction between the catalytic (light blue and dark blue for upper and lower Fu molecules) and non-catalytic domains of two Fu molecules (indicated by green diagonal lines) promotes cross-phosphorylation (directly or perhaps via an unknown intermediate kinase) only if association with a Hh-activated Smo-Cos2 complex, or artificially high local Fu concentration, augments this association. Primary, reciprocal cross-phosphorylation in the activation loop and non-catalytic domain (red arrows, illustrated only for activity of the light blue kinase domain) promotes secondary phosphorylation (attributed to CK1; curved arrows) and leads to full Fu activation and phosphorylation of downstream substrates (red arrows, shown only for dark blue kinase domain). Cos2 phosphorylation on S572 inhibits Ci-155 processing, leading to higher levels of Ci-155, while Su(fu) phosphorylation on S321 and S324 is unimportant. Phosphorylation of additional unidentified substrates (indicated by dashed lines) leads to Su(fu)-independent activation of Ci-155 and, in collaboration with CK1, inhibition of antagonism of Ci-155 by Su(fu).
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