A MAPK docking site is critical for downregulation of Capicua by Torso and EGFR RTK signaling

Abstract

Early Drosophila development requires two receptor tyrosine kinase (RTK) pathways: the Torso and the Epidermal growth factor receptor (EGFR) pathways, which regulate terminal and dorsal-ventral patterning, respectively. Previous studies have shown that these pathways, either directly or indirectly, lead to post-transcriptional downregulation of the Capicua repressor in the early embryo and in the ovary. Here, we show that both regulatory effects are direct and depend on a MAPK docking site in Capicua that physically interacts with the MAPK Rolled. Capicua derivatives lacking this docking site cause dominant phenotypes similar to those resulting from loss of Torso and EGFR activities. Such phenotypes arise from inappropriate repression of genes normally expressed in response to Torso and EGFR signaling. Our results are consistent with a model whereby Capicua is the main nuclear effector of the Torso pathway, but only one of different effectors responding to EGFR signaling. Finally, we describe differences in the modes of Capicua downregulation by Torso and EGFR signaling, raising the possibility that such differences contribute to the tissue specificity of both signals.

Figure 1

Different motifs in Cic mediate repressor function and downregulation by Torso. (A) Diagram of Cic protein and evolutionary conservation of the C1 and C2 motifs. The Drosophila sequences shown correspond to residues 1345–1355 (C1) and 1052–1071 (C2); the total length of Cic is 1403 amino acids. Identical and similar residues are boxed in black and gray, respectively. Ag, Anopheles gambiae; Am, Apis mellifera; Ce, Caenorhabditis elegans; Hs, Homo sapiens; Mm, Mus musculus; Tn, Tetraodon nigroviridis. The distantly related C2-like sequences from vertebrate orthologs are present in approximately equivalent positions between the HMG box and the C-terminus of those proteins. (B) Diagram of Cic derivatives expressed under the control of cic regulatory sequences. The HA tag is represented by a gray oval. The rescue activities and regulation of each construct are indicated on the right. The rescue activity of Cic^ΔC2 was assayed using weak cic^ΔC2 insertions that do not cause embryonic lethality in one copy (asterisk). (C) Embryonic cuticle of wild-type embryo. (D) cic¹ mutant embryo with strongly suppressed trunk and abdomen. (E) cic¹ mutant embryo rescued by maternal expression of Cic^mini. (F) cic¹ embryo showing only partial rescue by Cic^ΔC1. (G–L) Distribution of different Cic derivatives at the posterior of blastoderm embryos. Embryos were stained with anti-HA antibody (green) and rhodamine–phalloidin (red) to visualize the cortical actin associated with the plasma membrane. Wild-type Cic (G), Cic^mini (H), Cic^miniNLS (J) and Cic^ΔC1 (K) proteins exhibit significant downregulation at the pole. In contrast, Cic^ΔC2 accumulates ectopically at the pole and in germ cells (L). Note the cytoplasmic localization of Cic^ΔHMG and its accumulation at the pole (I). Identical results were obtained at the anterior of the embryo (Supplementary Figure 1 and data not shown).

Figure 2

Cic^ΔC2 interferes with embryonic terminal development. (A, B) Cuticle phenotypes resulting from maternal expression of Cic^ΔC2. Shown are examples of intermediate phenotypes characterized by the lack of posterior terminal structures including the A8 segment (A, arrowhead), and stronger effects where additional segments (typically A6 and A7, arrowheads) are affected (B). (C–H) mRNA expression patterns of tll (C, D), hkb (E, F) and hb (G, H) in wild-type (C, E, G) and cic^ΔC2 (D, F, H) embryos. The mutant embryos exhibit marked repression of tll and hkb at the posterior pole, and shifted expression of the posterior hb stripe. At the anterior region, hkb expression appears markedly reduced (arrowhead in F). We also note a slight anterior shift (of approximately 6% egg length) of the dorsal tll stripe (arrowhead in D).

Figure 3

The C2 motif is a MAPK docking site. (A) Diagram of Cic protein showing the different fragments assayed for binding in yeast and in vitro. The limits of each Cic fragment are as follows: Cic¹ (1050–1115), Cic² (1307–1379), Cic³ (1050–1079) and Cic⁴ (942–1116). The C2 amino-acid sequences deleted in Cic^1ΔC2 and Cic^4ΔC2 are 1054–1064 and 1052–1072, respectively. (B) Yeast two-hybrid assay using different LexA-Cic baits and B42 fusion preys. Positive interactions (visualized by lacZ reporter activation) are observed between C2-containing fragments and Rolled or human Erk2. Negative controls are Rolled^ΔCD, a form of Rolled lacking the C-terminal 52 amino acids that should be unable to bind substrates, and Hairy. (C) Pull-down assay using the indicated GST fusions and in vitro translated (IVT) Rolled or Luciferase (Luc) labeled with ³⁵S-methionine. Cic⁴ binds Rolled in a C2-dependent manner. Negative controls show little or no interaction between Hairy and Rolled, or between Cic and Luciferase. Input lanes were loaded with 10% of the protein used in the binding reactions. (D) In vitro phosphorylation assay using the indicated GST fusions and either Erk2 or Jun kinase (JNK) protein immunopurified from HeLa cells unstimulated (−) or stimulated (+) for kinase activation. Positive control substrates for Erk2 and JNK were myelin basic protein (MBP) and Jun, respectively. Erk2 specifically phosphorylates Cic⁴ but not Cic^4ΔC2. The gels shown in (C) and (D) were stained with Coomassie to ensure equal loading and integrity of all GST fusions (not shown).

Figure 4

EGFR signaling induces nucleocytoplasmic redistribution of Cic via the C2 motif. (A) Partial view of a stage-10 cic-HA egg chamber costained with anti-HA (green, A) and anti-Mirror (Mirr; blue, A′) antibodies, and with rhodamine–phalloidin (red, A′) to label the cortical actin. The merged image is shown in (A″). (B) Detail of the dorsal-anterior region of the above egg chamber; expression of Mirr is not shown. The merged image is shown in (B′). The preferential nuclear accumulation of Cic in some cells (asterisk in B) probably results from regional differences in the levels of Ras/MAPK activation within the dorsal-anterior patch at this stage (Peri et al, 1999). (C, D) High magnifications of two areas from (B′) corresponding to dorsal-anterior (C, 1) and lateral (D, 2) follicle cells. Cic accumulates in the cytoplasm of dorsal-anterior but not lateral follicle cells (arrows). The central areas of nuclei devoid of staining probably correspond to nucleoli (empty arrowheads). (E) Dorsal view of a stage-11 cic-HA egg chamber stained with anti-HA (green, E) and anti-Mirr (red, E′) antibodies. The merged image is shown in (E″). Note the nuclear accumulation of Cic in cells that express Mirr (arrowheads) and the preferential nuclear accumulation of Cic in a row of cells in the presumptive midline (open arrowhead). (F) Detail of the dorsal-anterior region of the egg chamber shown in (E). (G) Partial view of a stage-10 cic^ΔHMG-HA egg chamber stained as in (E). (H) Detail of the boxed area shown in (G). Cic^ΔHMG is excluded from the dorsal-anterior nuclei (arrow), whereas only the nucleoli remain devoid of protein in more posterior cells (open arrowhead). (I) Partial view of a stage-10 cic^ΔC2-HA egg chamber stained as in (E). In the merge panels, colocalization of the green and red channels appears in yellow–orange, whereas colocalization of green and blue channels appears in cyan. Stages of egg chambers are indicated. nc, nurse cells.

Figure 5

Cic^ΔC2 causes DV patterning defects in the egg. (A) Dorsal-anterior region of a wild-type eggshell showing the two symmetrical respiratory appendages. (B, C) Equivalent views of cic^ΔC2 eggshells showing moderate (B) and severe (C) fusions of appendages. The frequencies of phenotypic classes resulting from strong cic^ΔC2 combinations are as follows: wild-type, 40%; moderate, 25%; severe, 30%. (D–G) mRNA expression patterns of twi (D, E) and zen (F, G) in wild-type (D, F) and cic^ΔC2 (E, G) embryos.

Figure 6

Cic^ΔC2 interferes with the regulatory network controlling DV polarity in the ovary. (A–F) mRNA expression patterns of pipe (A, B), rho (C, D) and argos (E, F) in wild-type (A, C, E) and cic^ΔC2 (B, D, F) egg chambers. Lateral views are shown in (A) and (B); dorsal views in (C–F). The cic^ΔC2 egg chambers exhibit moderate expansion of pipe expression (arrowhead in panel B) and reduced or absent expression of both rho and argos in the dorsal-anterior region (panels D and F, respectively). Note that the expression of argos at the posterior end of the egg chamber is unaffected by Cic^ΔC2 (F). (G) Dorsal view of a stage-11 wild-type egg chamber doubly stained for activated MAPK (MAPK^act, green) and actin (red). Two patches of activated MAPK (empty arrowheads) are separated by a row of cells devoid of staining (solid arrowhead). (H, I) Dorsal views of wild-type (H) and cic^ΔC2 (I) egg chambers at stage 12 stained as in G. The two patches of activated MAPK staining appear fused in the mutant chamber (empty arrowhead in I). Stages of egg chambers are indicated. nc, nurse cells.