Fig. 4

4E-BP contributes to the inhibition of MAD phosphorylation. a Microarray analysis of transcriptional changes caused by expression of crc in S2 cells. Volcano plot of transcriptional profiles of HA-crcA.pMT-Puro S2 stable cells relative to HA.pMT-Puro S2 stable cells, each treated with 0.7 mM copper sulphate for 3 h (red symbols) or 6 h (blue symbols). Vertical broken lines indicate 2^{−/+ 0.7}-fold change. Horizontal broken line indicates P = 0.05 threshold. d4E-BP is indicated at 3 h (red) and 6 h (blue). b Immunoblot of cell lysates expressing myc-Tkv in the absence or presence of crc. c Quantification of b, samples normalised to no Cu²⁺ for each cell line. n = 3. P value calculated using ANOVA with Bonferroni post hoc testing. d Immunoblot of S2 cell lysates to assess the effect of d4E-BP small interfering RNA (RNAi) on MAD phosphorylation caused by 0.5 nM Dpp concentrations. e Quantification of d. n = 3. P value calculated using ANOVA with Bonferroni post hoc testing. f Representative fluorescence micrographs of pupal wings of the indicated genotypes at 30 h after pupariation stained red for pMAD. Scale bars = 100 μm. g Representative photomicrographs (5× objective) of adult wings of the indicated genotypes. Scale bars = 200 μm. h Quantification of animals from g. Left graph indicates proportion of animals failing to eclose by 14. Right graph indicates frequency of wing vein phenotype if eclosing adults. P values calculated using X² statistics with Bonferroni correction for multiple comparisons. i Schematic of interaction between integrated stress response (ISR) and BMP signalling. eIF2α is phosphorylated by GCN2 to P-eIF2α; PPP1R15 (R15) dephosphorylates P-eIF2α. P-eIF2α directly inhibits most cap-dependent translation of mRNAs, but induces expression of crc (Drosophila ATF4). Targets of crc further affect translation, e.g. 4E-BP antagonises translation of some mRNAs. Ongoing translation is necessary for efficient BMP signalling, and so repression of protein synthesis by the ISR inhibits BMP signalling