FIGURE 4.

dMK2 phosphorylates dIscU at serine 20. A, GST-dIscU, GST-dIscU-S20A, GST-dIscU-S42A, GST-dIscU-T83A, and GST-dIscU-S104A were incubated with or without GST-dMK2 in kinase buffer containing [³²P]ATP. Phosphorylation of dIscU was detected by autoradiography. p indicates phospho form. B, GST-dIscU or GST-dIscU-S20A was incubated with or without GST-dMK2EE in a kinase buffer containing [³²P]ATP. Phosphorylation of dIscU was analyzed by autoradiography and not detected in dIscU-S20A mutants. Proteins of dMK2EE, dIscU, and dIscU-S20A were determined by Coomassie Blue staining. C, FLAG-tagged-dIscU was overexpressed in Drosophila S2 cells, and FLAG-tagged protein was subsequently immunoprecipitated using FLAG M2 beads 4 days after transfection. The band corresponding to dIscU was excised from Coomassie Blue-stained gels and subjected to in-gel digestion for mass spectra. D, GFP-dIscU was overexpressed in background of both yw and dMK2^−/− flies. Fly proteins were then precipitated with TCA/acetone, suspended in IEF sample buffer, and subjected to isoelectric focusing using a mini gel.