Sphingomyelinase and TNF- stimulate p38 MAPK and MAPKAP kinase-2 activity. (A) U937 cells were stimulated with 10 ng/mL TNF- (T) for 5 minutes or 100 mU/mL bacterial sphingomyelinase for the indicated duration. Detergent-solubilized cell lysates were incubated with -p38 MAPK and protein A-Sepharose beads. Activity (top) of the washed immunoprecipitates were determined by³²P incorporation into a peptide corresponding to amino acids 1-96 of ATF-2. Immunoblot analysis was performed to detect phosphotyrosine (4G10; middle) and with -p38 MAPK (bottom) to confirm equal amounts of p38 MAPK in the immunoprecipitates. (B) U937 cells were preincubated with SB203580 (1 μmol/L) or vehicle alone for 20 minutes and then stimulated with either TNF- (10 ng/mL; T) or bacterial sphingomyelinase (100 mU/mL) for the indicated duration and detergent-solubilized. Lysates were incubated with 1 μg -MAPKAP kinase-2 antibody and protein G-Sepharose beads, and kinase activity was determined from the washed immunoprecipitates by measuring the³²P transferred to Hsp25.