MULTIPLE C-TERMINAL SERINE PHOSPHORYLATION ACCOMPANIES BOTH PROTEIN-KINASE-C-DEPENDENT AND PROTEIN-KINASE-C-INDEPENDENT ACTIVATION OF CYTOSOLIC-85 KDA PHOSPHOLIPASE A(2) IN MACROPHAGES

Exposure of mouse macrophages to either phorbol ester or certain bacte ria was previously shown to cause increased phosphorylation of the cyt osolic 85 kDa phospholipase A(2) as well as a stable increase in its c atalytic activity. We have now attempted to map the major phosphorylat ion sites on the enzyme in such cells. Phosphorylation occurred on ser ine residues without a detectable increase in either phosphothreonine or phosphotyrosine. After CNBr cleavage five fragments showed increase d P-32 labelling. Among those the most heavily labelled fragment was i dentified as the most C-terminal (residues 698-749), containing six se rine residues. This was true whether phorbol ester or bacteria, causin g protein kinase C-independent phospholipase A(2) activation, was used as stimulus. The heavy phosphorylation of the most C-terminal fragmen t and an analysis of tryptic peptides derived from it suggested that m ore than one of the six serine residues became phosphorylated. Smaller increases also occurred in other CNBr-cleaved fragments from the C-te rminal part of the protein, including that carrying Ser-505, a known t arget of the mitogen-activated protein kinase ERK-2 (extracellular-sig nal regulated kinase). Dexamethasone treatment (1-100 nM for 20 h), wh ich was earlier shown to dose-dependently down-regulate the 85 kDa pho spholipase A(2) and its activation by phorbol ester and zymosan, was h ere shown also to counteract the protein kinase C-independent activati on and arachidonate release elicited by bacteria. It remains to be det ermined whether all phosphorylation sites are equally affected under t hose conditions.