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. 2019 Dec 3;116(51):25756–25763. doi: 10.1073/pnas.1912320116

Fig. 1.

Fig. 1.

psMEK effects in vivo. (A) RAF phosphorylates 2 serine residues (red, S218 and S222) in the activation loop of MEK. When MEK is unphosphorylated, its inactive conformation is favored, shown by the larger arrowhead facing to the left. When RAF phosphorylates MEK, the active conformation is favored. (B) psMEK contains S > D mutations in the activation loop that mimic the effects of RAF phosphorylation. The phosphomimetic substitutions maintain psMEK in the active conformation. Constitutively active psMEK is controlled by photoswitchable Dronpa domains (green) that block the active site when dimerized (in 400 nm light) and expose the active site when dissociated (in 500 nm light). (C) ERK is only active (dpERK) at the poles in the 3-h-old Drosophila embryo. Ectopic ERK signaling in the middle of the embryo disrupts formation of the denticle belts in the larvae and is lethal (>36 h p. f.). Embryos expressing psMEK (black bars) exhibited significant lethality when exposed to 500 nm light. Illumination of 400 nm was not lethal. Wavelength of light illumination had no effect on lethality in the WT control embryos (gray bars). Error bars represent means and SDs for 3 replicates. Pooled N numbers are as follows: NOreR, 400 nm = 291, NOreR, 500 nm = 543, NpsMEK, 400 nm = 1166, NpsMEK, 500 nm = 2,179, NpsMEK, dark = 621, and NpsMEK, room light = 607. (D) In zebrafish embryos, ERK is endogenously active in the blastoderm margin at ∼6 h.p.f. ERK signaling in nonmarginal cells causes defective morphogenetic movements that elongate the embryo, measured as the aspect ratio (major axis “a”/minor axis “b”) of the yolk at the tailbud stage (10 h.p.f.). Embryos expressing psMEK via mRNA injection (black bars) do not show a significant wavelength-dependent increase in aspect ratio. Uninjected embryos (gray bars) remained spherical in both 400 and 500 nm light. The yellow line represents the maximum aspect ratio reported in SI Appendix, Fig. S2 and ref. 28. Pooled numbers of measured embryos are as follows: NpsMEK, 400 nm =, 112 NpsMEK, 500 nm = 112, Nuninjected, 400 nm = 114, and Nuninjected, 500 nm = 97. In C and D, *P < 0.05.