Phosphorylation of MRP14 by p38 MAPK in monocytes. (A) [³²P]-Phosphate-labeled monocytes were stimulated with arsenite (ARS, 0.5 mM) for 15 minutes with or without preincubation with pharmacologic p38 inhibitor SB202190 (ARS + SB, 7 μM) for 30 minutes. MRP14 was immunoprecipitated and analyzed by SDS-PAGE and autoradiography (upper panel). All experiments presented in this figure were performed at least 3 times. Equal amounts of immunoprecipitated MRP14 were confirmed by Western blotting using a mAb to MRP14 (lower panel). (B) [³²P]-Phosphate-labeled monocytes were incubated with medium as control (CON), 100 nM PMA, 0.5 mM arsenite (ARS), or 5 μg/mL anisomycin (ANI) for 15 minutes as indicated. In some experiments, monocytes were preincubated with SB202190 (SB, 7 μM) or PD98059 (PD, 20 μM) for 30 minutes. Data are shown as percent change of [³²P]-phosphate incorporation into MRP14 as compared to nonstimulated cells (CON). Phosphorylation was analyzed by phosphorimaging of MRP14 bands after SDS-PAGE. Protein loads were confirmed by Western blotting using a mAb against MRP14 (lower panel). (C) Monocytes were stimulated as described in panel B. Activation of p38 MAPK was studied indirectly by determining the activity of the downstream kinase MAPKAP-K2/3 using Hsp27 as substrate (upper panel). Protein loads were confirmed by Western blotting using an antiserum to MAPKAPK-2/3 (lower panel). (D) Purified MRP14 (5 μg) was incubated with active recombinant p38 MAPK and 5 μCi (0.185 MBq) γ[³²P]-ATP for 15 minutes in the presence of increasing amounts of MRP8 (0-10 μg) in the absence or presence of 60 μM calcium as indicated. When MRP14 was replaced by 3pK (K > M) as substrate, no inhibitory effect of MRP8 was observed. Incorporation of [³²P]-phosphate into MRP14 and 3pK (K > M) was visualized by SDS-PAGE and autoradiography. Similar results were obtained when immunoprecipitating preactivated p38 from transfected embryonic kidney 293 cells (data not shown). (E) Purified MRP14 or 3pK (K > M) was incubated with preactivated p38 and 5 μCi (0.185 MBq) γ[³²P]-ATP in the absence or presence of 60 μM calcium and studied as described. Addition of calcium had no influence on p38 activity, as indicated by the lack of effects on phosphorylation of MRP14 or 3pk (K > M). (F) MRP14 and phospho-MRP14 were purified from human granulocytes and MRP14-T113A from transfected E coli BL21 cells and incubated with preactivated p38 and γ[³²P]-ATP and processed as described in panel A. Only the nonphosphorylated isoform of MRP14 shows incorporation of [³²P]-phosphate in the presence of preactivated p38. Data shown are representative for at least 3 independent experiments. (G) Purified MRP8/MRP14 (▪) and MRP8/phospho-MRP14 (•) were exposed to increasing concentrations of calcium, and conformational changes of the protein complex were detected by changes of the intrinsic fluorescence maximum (protein concentration, 5 μg/mL). Phosphorylation of MRP14 shifted conformational changes to higher calcium concentrations. Data represent means of 4 independent experiments (maximal deviation between individual experiments < 0.7 nm).