FIGURE 7.

The phosphorylation of IscU by MK2 also occurs in mammalian cells. A and B, FLAG-hMK2 was coexpressed with HA-hIscU2 in 293T cells. FLAG-hMK2 was immunoprecipitated using FLAG M2 beads 36 h after transfection, and HA-hIscU2 was immunoprecipitated using HA beads. The cell lysates and immunoprecipitates were analyzed by Western blot using antibodies against FLAG and HA. C, FLAG-hIscU2 was overexpressed in 293T cells and subsequently immunoprecipitated (IP) using FLAG M2 beads 36 h after transfection. The protein samples were treated with or without calf intestinal alkaline phosphatase (CIAP). The expression and phosphorylation levels of hIscU2 were determined by Western blot. D, FLAG-hIscU2 and FLAG-hIscU2-S29A were overexpressed in 293T cells, respectively. The expression and phosphorylation of hIscU2 were determined by Western blot. E, GST-mIscU was incubated with or without dMK2EE in kinase buffer. Phosphorylation of mIscU was detected by anti-phospho-mIscU antibody. Proteins of dMK2EE and mIscU were determined by Coomassie Blue staining. F, HeLa cells were infected with lentivirus-expressing shGFP or MK2 shRNAs for 48 h. Protein level of MK2 and phosphorylation level of hIscU2 were determined by Western blot. G, The mitochondrial aconitase (m-aconitase) activity was measured using MK2^−/− and MK2^+/+ MEF cells. mOD, milli optical density. H, the cytoplasmic aconitase (c-aconitase) activity was measured using MK2^−/− and MK2^+/+ MEF cells. I, the complex I activity was measured using MK2^−/− and MK2^+/+ MEF cells. Error bars reflect S.E.; star signifies p < 0.05.