PMA up-regulates 5-LO phosphorylation by MK2 in MM6 cells: inhibition by calphostin C.
Phosphorylation of 5-LO by immunoprecipitates of MK2 from MM6 cells was determined by in vitro kinase assays. Differentiated MM6 cells (2.5 × 107 in 1 mL PGC buffer) were incubated with the indicated concentrations of calphostin C for 30 minutes at 37°C under ordinary fluorescent light. Then PMA (100 nM) was added for 10 more minutes before stimulation with 5 μM A23187 for 3 minutes at 37°C. Incubations were stopped by the addition of 2 vol ice-cold stop-buffer. MK2 was immunoprecipitated, and aliquots of the MK2-IPs corresponding to the same cell numbers were subjected to in vitro kinase assay with 5-LO (3 μg) as substrate. Results are representative of at least 3 separate experiments.
