FIG. 8.

TTP is a substrate of MAPKAPK2. (A) Purified recombinant kinases MKK6, p38, and MAPKAPK2 (MK2) were mixed with the substrate GST-TTP (TTP) or hsp27 in the combinations indicated. Phosphorylation reactions were allowed to proceed for 30 min, and then the phosphorylated products were separated by SDS-PAGE and visualized by phosphorimaging. Lane 1, TTP alone; lane 2, MK2 and TTP; lane 3, p38 and TTP; lane 4, MKK6 and TTP; lane 5, MKK, p38, and TTP; lane 6, p38, MK2, and TTP; lane 7, MKK6, MK2, and TTP; lane 8, MKK6, p38, MK2, and TTP; lane 9, MKK6, p38, MK2, and hsp27. (B) RAW264.7 cells were left untreated (−) or stimulated with 10 ng of LPS per ml for 2 h (+), and then lysates were prepared, p38 or MAPKAPK2 was immunoprecipitated, and immune-complex kinase assays were performed using hsp27, His₆-tagged MAPKAPK2 or GST-TTP as substrate (Subst). Phosphorylated products were separated by SDS-PAGE and visualized by phosphorimaging. (C) RAW264.7 cells were stimulated with 10 ng of LPS per ml for the times indicated, and then immune-complex assays of MAPKAPK2 activity were performed as described for panel B, using recombinant hsp27 as the substrate. Phosphorylated products were separated by SDS-PAGE and visualized by phosphorimaging.