Fig. 3: Effect of phosphorylation on the activity of rMrp2.
From: Structural basis for the modulation of MRP2 activity by phosphorylation and drugs
a The phosphorylation state of rMrp2 was analysed by Pro-Q Diamond Phosphoprotein Stain (top panel) and Coomassie blue (bottom panel) of the SDS-PAGE. Densitometry analysis shows a 48% enhancement of phosphorylation upon in vitro treatment with the HEK293 kinase extract; white and black bars correspond to the top and bottom panels of the SDS-PAGE, respectively. Phosphorylation state abbreviations: rMrp2R-* (partially phosphorylated), rMrp2R-de (fully dephosphorylated) and rMrp2R-pho (fully phosphorylated). Representative SDS-PAGE from three repeats. b (left panel) Mass spectrometry-based phosphorylation mapping of rMrp2 protein showing presence or absence of detection across different conditions. The phosphorylated residues have been mapped onto the rMrp2 structure (right panel). The phosphorylated residues that are present in both the rMrp2R-* and rMRP2R-pho samples are shown as blue spheres. The sites that are unique to the rMrp2R-pho sample are shown as red spheres; the additionally phosphorylated R-___domain residues are labelled for clarity (a star indicates a phosphorylated R-___domain residue that has not been built in the structure). c Effect of probenecid on the ATPase activity of rMrp2 in different phosphorylation states. Dephosphorylation by λ-protein phosphatase does not affect the basal ATPase (white bars), whereas full phosphorylation increases the basal ATPase by 2-fold. Probenecid can stimulate the basal ATPase activity of rMrp2R-* and rMRP2R-de but it has no additional effect on rMrp2R-pho (grey bars). Results are represented as means from three independent experiments. Significantly different significantly different as estimated by one way ANOVA (p = 2 ×10−7) followed by Tukey test p < 0.025 (red, yellow and green triangles:not significant). d rMrp2-dependent uptake of CDF in proteoliposomes. Transport was initiated by adding (empty bars and light grey bars) or not (dark grey bars) ATP-MgCl2, to the samples containing (light grey) or not (empty and dark grey) 1 mM probenecid. rMrp2R-pho displays significantly increased transport activity relative to the rMrp2R-* and rMrp2R-de proteins. Probenecid inhibits CDF transport. Results are represented as means from three independent experiments. Significantly different significantly different as estimated by one way ANOVA (p = 1.6 ×10−19) followed by Tukey test p < 0.025 (red, yellow and green triangles: not significant). Source data are provided as a Source Data file.
