Figure 3. Fortilin prevents Mst1 from phosphorylating and deactivating PRX1.

(A). An in vitro phosphorylation assay shows the binding-dependent inhibitory effect of fortilin on PRX1 phosphorylation by Mst1. Abbreviations: IB, immunoblot; α-PRX1, anti-PRX1 antibody; α-p-Thr, anti-phosphothreonine antibody; α-Fortilin, anti-fortilin antibody. Increasing doses of recombinant human fortilin (lanes 3–6), but not its mutant (fortilin_ΔL7R, lanes 8–11), decreased phosphorylation of PRX1 by Mst1 in vitro. Densitometry was used to quantify the amount of threonine-phosphorylated PRX1. (B). Fortilin, but not fortilin_ΔL7R, protects PRX1 enzymatic activity from inhibition by Mst1. NADPH, nicotinamide adenine dinucleotide phosphate; A.U., arbitrary unit. Results are shown as the mean ± SD three independent experiments. (C). Fortilin preserves the enzymatic activity of PRX1 by preventing Mst1 from phosphorylating PRX1. IB, immunoblot; α-phosphothreonine, anti-phosphothreonine antibody; α-Fortilin, anti-fortilin antibody; A.U., arbitrary unit derived from the densitometric ratio of the phosphorylated PRX1 threonine band to the respective total PRX1 band. The means and errors ( ± SD) of the graph were calculated from three independent experiments. (D). The PRX1 dimer interacts with two fortilin molecules. Fortilin occludes the PRX1 Thr¹⁸³ phosphorylation site on the C-terminal tail of one subunit of the dimer and Thr⁹⁰ on the second subunit. (E). Interaction facet between fortilin and dimerized PRX1s. (F). A model of physical and functional interaction between dimerized PRX1s and fortilins. Without fortilin, PRX1 is accessible by Mst1 for the phosphorylation of Thr⁹⁰ and Thr¹⁸³, key activity-regulating residues of PRX1. See also Fig. S3.