β-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development

Abstract

β-Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. In this study, we report a new in vivo function of the Drosophila β-arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF-κB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. Our findings uncover a new in vivo function of β-arrestins and present a new mechanism of ERK inhibition by the Drosophila β-arrestin Krz.

Figure 1

Maternal krz¹ loss of function phenotypes. (A, C, E) General morphology of stage 15 embryos visualized using anti-Elav antibody which stains all neurons. In all embryo panels, anterior is to the left, dorsal is up. (A) FRT control, (C, E) krz¹ maternal mutant embryos. (B, D, F) Cuticular preparations of (B) FRT control and (D, F) krz¹ maternal mutants. Anterior-ventral and posterior-ventral holes in the mutants (D) are indicated with asterisks. (G) The Torso (pink) and Toll (blue) signalling pathways intersect at the blastoderm stage. (H) Quantification of the phenotypic classes observed in the cuticles of krz¹ maternal mutant embryos (72 cuticles scored). ^*One hole either in the anterior or posterior; ^**two holes at both anterior and posterior ends. (I) A western blot on extracts from staged FRT and krz¹ maternal mutant embryos. IB: immunoblot. HSP70 antibody was used as a loading control. (J) Hatch rates of FRT control embryos, krz¹ maternal mutant embryos, and krz¹ maternal mutant embryos obtained from females carrying one copy of the genomic rescue construct, krz 5.7.

Figure 2

Loss of krz results in an expansion of expression domains of Torso target genes. (A–D) In situ hybridization with antisense (A, C) tll and (B, D) hkb probes in (A, B) stage-4 FRT control and (C, D) krz¹ maternal mutant embryos. (E, F) Quantification of anterior and posterior tll and hkb expression domains. Mean values for the widths of the expression domains are plotted as percent embryo length (% EL, posterior is 0%). Error bars represent one standard deviation. Numbers of embryos quantified: tll in FRT, 19; tll in krz, 18; hkb in FRT, 17; hkb in krz, 19. The P-values for the comparisons between the FRT and mutant embryos were calculated separately for the anterior and posterior domains, and are shown under the graphs.

Figure 3

Loss of krz results in increased levels of dpERK. (A, B) Expression of doubly phosphorylated activated ERK (dpERK) in (A) stage-4 control and (B) krz¹ maternal mutant embryos. Embryos were stained with an antibody specific for the activated form of ERK. (C) Average intensity of dpERK expression plotted around the sagittal circumference of the embryos (D, dorsal; A, anterior; V, ventral; P, posterior). (D, F) Quantification of the average dpERK intensity (D) and average width of dpERK expression (F) in 28 Hist-GFP control and 28 krz¹ maternal mutant embryos (au, arbitrary signal intensity units). (E) Western blot of protein expression in staged krz¹ maternal mutant embryos and FRT controls. HSP70 antibody was used as loading control.

Figure 4

Krz preferentially interacts with inactive ERK. (A) S2 cells were transfected with HA–Krz, HA–Krz-K336A, HA–Krz-R209E or HA–Krz-R209E-K336A together with Drosophila ERK–Flag. Samples were immunoprecipitated with anti-HA beads and analysed by western blotting. (B) S2 cells were transfected with Drosophila ERK–Flag alone, or together with HA–Krz-R209E or HA–Krz. Cells were treated with insulin at a final concentration of 20 μM. Samples were immunoprecipitated with anti-Flag or anti-HA beads. Immunoprecipitates were analysed by western blot with anti-HA antibody, anti-total ERK antibody and anti-dpERK antibody. (C) S2 cells were transfected with HA–Krz or HA–Krz-R209E together with the indicated Drosophila ERK–Flag versions. Samples were immunoprecipitated with anti-HA beads and analysed by western blotting. (D) S2 cells were transfected with HA–Krz and either Drosophila wild-type ERK–Flag or ERK-D334N–Flag. Lysates were immunoprecipitated with anti-HA beads and immunoblotted. (E) S2 cells were transfected with HA–Krz together with the indicated Drosophila ERK–Flag versions. Samples were immunoprecipitated with anti-HA beads and analysed by western blotting. IP, immunoprecipitated samples; IB, immunoblots.

Figure 5

Krz inhibits ERK phosphorylation by MEK. (A) S2 cells were transfected with Drosophila ERK–Flag alone or in combination with HA–Krz-R209E, and immunoprecipitated with either anti-Flag or anti-HA beads. 320 ng of purified human MEK2 was added to samples as indicated, and extent of phosphorylation of Drosophila ERK–Flag was then assayed with anti-dpERK antibody by western blot analysis. (B) S2 cells were transfected with Drosophila ERK–Flag alone or in the combinations indicated with GFP–Krz, Drosophila MEK–V5 and/or Drosophila HA–Raf. Cells were treated with insulin at a final concentration of 20 μM as indicated. Samples were immunoprecipitated with anti-Flag beads, and extent of phosphorylation of Drosophila ERK–Flag was then assayed with anti-dpERK antibody by western blot analysis. IP, immunoprecipitated samples; IB, immunoblots. (C) A model of Krz regulation of RTK signalling. Krz limits the activity of receptor tyrosine kinases (RTKs) by preferentially binding and sequestering an inactive form of ERK. This sequestration prevents ERK from being phosphorylated by the upstream kinase, MEK, and therefore reduces an overall output of signalling downstream of RTKs.

Figure 6

Krz inhibits RTK signalling in different tissues and at different developmental stages. (A, B) Expression of doubly phosphorylated activated ERK (dpERK) in (A) stage-10.5 FRT and (B) krz¹ maternal mutant embryos. An expansion of the dpERK pattern in the mutants is consistent with an overall increase in dpERK levels observed at this stage on western blots (see Figure 3E, 4–8 h). (C–K) Genetic interactions between krz and an activating mutation in ERK, rl^Sem. (C–F) Wing phenotypes. (G–J) Eye phenotypes. The sizes of the wings can be directly compared because same magnification was used in (C–F). Magnification was also constant for (G–J). The genotypes were, (C, G) da-GAL4, (D, H) UAS-rl^Sem/da-GAL4, (E, I) UAS-rl^Sem/da-GAL4 krz¹, (F, J) UAS-NTAP-Krz/+; UAS-rl^Sem/da-GAL4 krz¹. UNK is an abbreviation for UAS-NTAP-Krz. (K) Survival to adult reported as percentage of all progeny for the UAS-rl^Sem/da-GAL4, UAS-rl^Sem/da-GAL4 krz¹, and UAS-NTAP-Krz/+; UAS-rl^Sem/da-GAL4 krz¹ genotypes.

Figure 7

Loss of krz results in increased nuclear Dorsal as well as expanded Toll target gene expression domains. (A, B) In situ hybridization with antisense twist (twi) probe in (A) stage-4 FRT control and (B) krz¹ maternal mutant embryos. (C, D) In situ hybridization with antisense rhomboid (rho) probe in (C) stage-4 FRT control and (D) krz¹ maternal mutant embryos. (E–H) Visualization of Dorsal protein localization with anti-Dorsal antibody viewed (E, F) ventrally and (G, H) in a transverse optical cross section in (E, G) stage-4 control FRT and (F, H) krz¹ maternal mutant embryos. (I) Western blot of protein expression in staged krz¹ maternal mutant embryos and FRT controls. HSP70 antibody was used as loading control. (J) S2 cells were transfected with HA–Krz or HA–Krz-R209E together with Cactus-V5. Samples were immunoprecipitated with anti-HA beads and analysed by western blotting. IP, immunoprecipitated samples; IB, immunoblots.

Figure 8

Krz regulates Torso and Toll shared target gene zen. (A, B) In situ hybridization with antisense zen probe in (A) stage-4 FRT control and (B) krz¹ maternal mutant embryos. (C, D) A diagram explaining the effects of loss of krz on Torso and Toll mutual regulation of zen. A boxed area indicates the same position in (C) the wild-type and (D) krz¹ maternal mutant embryos. (C) In the wild-type embryo, Dorsal nuclear translocation represses zen transcription in the cells inside the boxed area. By limiting Torso activity in these cells, Krz ensures that zen is repressed. (D) In krz maternal mutant embryos, loss of krz results in an upregulation of Torso activity, which overcomes Dorsal-mediated repression and leads to an abnormal de-repression of zen in the boxed area. Dashed line in the embryo schematic in (D) indicates a boundary of the wild-type zen expression pattern.