Protein kinase A antagonizes Hedgehog signaling by regulating both the activator and repressor forms of Cubitus interruptus

Abstract

The Hedgehog (Hh) family of secreted proteins controls many aspects of animal development. In Drosophila, Hh transduces its signal via Cubitus interruptus (Ci), a transcription factor present in two forms: a full-length activator and a carboxy-terminally truncated repressor that is derived from the full-length form by proteolytic processing. The proteolytic processing of Ci is promoted by the activities of protein kinase A (PKA) and Slimb, whereas it is inhibited by Hh. Here we show that PKA inhibits the activity of the full-length Ci in addition to its role in regulating Ci proteolysis. Whereas Ci processing is blocked in both PKA and slimb mutant cells, the accumulated full-length Ci becomes activated only in PKA but not in slimb mutant cells. Moreover, PKA inhibits an uncleavable activator form of Ci. These observations suggest that PKA regulates the activity of the full-length Ci independent of its proteolytic processing. We also provide evidence that PKA regulates both the proteolytic processing and transcriptional activity of Ci by directly phosphorylating Ci. We propose that phosphorylation of Ci by PKA has two separable roles: (1) It blocks the transcription activity of the full-length activator form of Ci, and (2) it targets Ci for Slimb-mediated proteolytic processing to generate the truncated form that functions as a repressor.

Figure 1

High levels of constitutive PKA activity do not promote Ci processing in slimb mutant cells. (A) A wild-type late third-instar wing disc stained with an anti-Ci antibody (2A1) that recognizes the full-length form of Ci. In this and subsequent figures, all of the wing discs are shown with anterior to the left and ventral up. The arrow indicates the accumulation of full-length Ci in A compartment cells near the A/P compartment boundary. (B) A late third-instar wing disc expressing UAS–mC* with MS1096 and stained with 2A1. High levels of constitutive PKA activity block the accumulation of full-length Ci in A compartment cells near the A/P compartment boundary. (C) A late third-instar wing disc expressing UAS–GFP with MS1096. GFP is expressed almost uniformly in the wing pouch region with slightly higher levels in dorsal compartment cells. The GFP expression reflects the pattern of Gal4 expression driven by the MS1096 Gal4 line. (D–F) A late third-instar wing disc containing slimb clones and expressing UAS–mC* with MS1096 and doubly stained with 2A1 (red in D and F) and a Myc antibody (green in E and F). slimb mutant cells (marked by the lack of Myc–GFP expression and indicated by the arrows) accumulate high levels of full-length Ci even though they express high levels of mC*.

Figure 2

Different phenotypes induced by PKA and slimb mutant clones. (A) A wild-type adult wing with its longitudinal veins indicated by numbers. (B–D) Supernumerary wings organized by a clone of PKA mutant cells (B) or a clone of slimb mutant cells (C,D). Both PKA and slimb mutant cells are marked by y⁻ and are situated between two arrows along the wing margin. In the supernumerary wings, PKA mutant cells form intervein tissues flanked by ectopic vein3; slimb mutant cells form ectopic vein3. The supernumerary wings organized by slimb clones are smaller than that induced by the PKA clone and contain incomplete veins (C,D). (E,F) A late third-instar wing disc carrying slimb clones and showing the accumulation of full-length Ci (E) and dpp–lacZ expression (F). Anteriorly situated slimb mutant clones (indicated by the arrows) accumulate high levels of full-length Ci and express dpp–lacZ at levels lower than those of the endogenous dpp–lacZ at the A/P compartment boundary (arrowhead). (G,H) A late third-instar wing disc carrying PKA mutant clones and showing Ci accumulation (G) and dpp–lacZ expression (H). Anteriorly situated PKA mutant clones (arrows) accumulate high levels of full-length Ci and express dpp–lacZ at levels comparable to those of the endogenous dpp–lacZ (arrowhead).

Figure 3

PKA but not slimb mutant cells ectopically express ptc–lacZ. (A,B) Late third-instar wing discs carrying PKA (A) or slimb (B) mutant clones and doubly stained for the marker gene (CD2) expression (green in left and right panels) and ptc–lacZ expression (red in middle and right panels). Both PKA and slimb mutant clones are marked by the lack of CD2 expression. Anteriorly situated PKA mutant cells ectopically express ptc–lacZ (arrow in A); slimb mutant cells do not (arrow in B). (C) A late third-instar wing disc carrying slimb mutant clones and expressing MS1096/UAS–R*. The disc was doubly stained for GFP expression (green in left and right panels) and ptc–lacZ expression (red in middle and right panels). slimb mutant cells are marked by the lack of GFP expression (arrows). Anteriorly situated slimb mutant cells with reduced PKA activity express ptc–lacZ. The levels of ectopic ptc–lacZ expression are lower than those at the compartment boundary because expressing UAS–R* only partially eliminates PKA activity.

Figure 4

PKA inhibits the activity of an uncleavable activator form of Ci. Wing discs in all panels were stained for ptc–lacZ expression. (A) A late third-instar wing disc showing the wild-type ptc–lacZ expression. (B) A wing disc expressing a constitutively active form of PKA catalytic subunit (mC*) under the control of the MS1096 Gal4 line. ptc–lacZ expression is suppressed in the wing pouch region (arrow). (C) A wing disc expressing an uncleavable Ci (HACi^U) with MS1096. ptc–lacZ is ectopically expressed in P but not in A compartment cells away from the compartment boundary. (D) A wing disc expressing UAS–HACi^U and UAS–mC* under the control of the MS1096 Gal4 line. Ectopic ptc–lacZ expression induced by HACi^U is partially suppressed by coexpression of mC*. The arrow indicates a gap in the ptc–lacZ expression domain. The suppression of ptc–lacZ expression is more evident in A compartment cells near the A/P compartment boundary (indicated by the arrow).

Figure 5

Effects of mutating the PKA phosphorylation sites in Ci on its processing and activity. (A) Cell extracts from wing discs expressing either HACi or HACi^−3P with MS1096 were probed with an anti-HA antibody. HACi is processed to form the truncated form (Ci-75) whereas HACi^−3P is not. (B,C) Wing discs expressing HACi (B) or HACi^−3P (C) under the control of tub>CD2>GV. The discs were doubly labeled to show ptc–lacZ expression (left) and HA staining (right). Only P (small arrow) but not A (big arrow) compartment cells that express HACi induce ectopic expression of ptc–lacZ (B). In contrast, both A and P compartment cells that express HACi^−3P ectopically activate ptc–lacZ, although the levels of ptc–lacZ expression in A compartment cells (big arrow in C) are lower than those in P compartment cells (small arrow in C) or at the compartment boundary (arrowhead).

Figure 6

Mutating PKA phosphorylation sites in Ci^U alters its activity. Wing discs expressing UAS–HACi^U (A) or UAS–HACi^U−3P (B) under the control of the MS1096 Gal4 line were doubly stained for ptc–lacZ expression (green) and HA expression (red). HACi^U induces ectopic ptc–lacZ expression only in P but not in A compartment cells (A). In contrast, HACi^U−3P activates ptc–lacZ in both A and P compartment cells (B). Note that the levels of ptc–lacZ in A compartment cells are lower than those in P compartment cells (B).

Figure 7

PKA inhibits Ci activity in slimb Su(fu) double mutant cells. Late third-instar wing discs carrying slimb Su(fu) double mutant clones with (B,D) or without (A,C) the expression of a mutant form of PKA regulatory subunit (R*). The wing discs were doubly stained for GFP expression (green) and ptc–lacZ expression (red in A and B) or En expression (red in C and D). slimb Su(fu) double mutant clones are marked by the lack of GFP expression (big arrows); the twin clones, which are homozygous for the wild-type slimb and Su(fu) genes, are marked by high levels of GFP expression (small arrows in B and D). slimb Su(fu) double mutant cells situated in the A compartment ectopically express low levels of ptc–lacZ (arrow in A) and do not express En (arrow in C). slimb Su(fu) mutant cells expressing MS1096/UAS–R* ectopically activate ptc–lacZ at high levels (big arrow in B) as well as En (big arrow in D). Cells expressing MS1096/UAS–R* but homozygous for the wild-type slimb and Su(fu) genes activate ptc–lacZ at low levels (small arrow in B) and do not express En (small arrow in D). Arrowheads in the middle panels indicate the A/P compartment boundary.

Figure 8

Model for dual regulation of Ci by PKA. Phosphorylation of Ci in its carboxy-terminal region by PKA keeps the full-length Ci (Ci155) in an inactive form and targets it for Slimb-mediated proteolytic processing to generate the truncated repressor form (Ci75). Su(fu) and Cos2 also negatively regulate Ci by forming a complex with Ci (see text for details).