Fig. 4: WAKL4 phosphorylates NRAMP1 at the Tyr488 residue. | Nature Communications

Fig. 4: WAKL4 phosphorylates NRAMP1 at the Tyr488 residue.

From: The Arabidopsis receptor-like kinase WAKL4 limits cadmium uptake via phosphorylation and degradation of NRAMP1 transporter

Fig. 4

a Multiple amino acid sequence alignment of AtNRAMP1 with other NRAMPs. Identical and similar residues are boxed in red highlight/red font. b Analysis of the phosphorylation of NRAMP1 C-terminal (NRAMP1(3)) by WAKL4 using an in vitro kinase assay. Top: phosphorylated proteins were detected by immunoblotting using an Anti-thiophosphate ester antibody. Bottom: recombinant NRAMP1(3) and WAKL4 were detected by CBB staining. c–e Analysis of the phosphorylation of NRAMP1 by WAKL4 in vivo. WAKL4 phosphorylates the Tyr488 site of NRAMP1 in a Cd-induced way (c). Cd-induced phosphorylation of NRAMP1 is dependent on WAKL4 (d). NRAMP1 Tyr488 residue phosphorylation responds to Cd treatment (e). Total proteins were extracted from 10-day-old seedlings treated with 1/5Hoagland plus the indicated concentrations of metal stresses (70 μM CdCl2, 1.5 mM MnCl2, 400 μM ZnSO4, 70 μM NiSO4, 70 μM CoCl2, 30 μM CuSO4 or 400 μM FeEDTA) for 4 h. NRAMP1-GFP protein was immunoprecipitated (IP) by incubating with anti-GFP magnetic beads. Western blot (WB) was performed using anti-phosphotyrosine (Anti-P-Tyr) and anti-GFP antibodies. The relative intensity by ImageJ of phosphorylated NRAMP1-GFP bands (p-NRAMP1) was as shown. The experiments were repeated at least three times with similar results.

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