Multiple C-terminal serine phosphorylation accompanies both protein kinase C-dependent and -independent activation of cytosolic 85 kDa phospholipase A2 in macrophages.
Exposure of mouse macrophages to either phorbol ester or certain bacteria was previously shown to cause increased phosphorylation of the cytosolic 85 kDa phospholipase A2 as well as a stable increase in its catalytic activity. We have now attempted to map the major phosphorylation sites on the enzyme in such cells. Phosphorylation occurred on serine residues without a detectable increase in either phosphothreonine or phosphotyrosine. After CNBr cleavage five fragments showed increased 32P labelling. Among those the most heavily labelled fragment was identified as the most C-terminal (residues 698-749), containing six serine residues. This was true whether phorbol ester or bacteria, causing protein kinase C-independent phospholipase A2 activation, was used as stimulus. The heavy phosphorylation of the most C-terminal fragment and an analysis of tryptic peptides derived from it suggested that more than one of the six serine residues became phosphorylated. Smaller increases also occurred in other CNBr-cleaved fragments from the C-terminal part of the protein, including that carrying Ser-505, a known target of the mitogen-activated protein kinase ERK-2 (extracellular-signal regulated kinase). Dexamethasone treatment (1-100 nM for 20 h), which was earlier shown to dose-dependently down-regulate the 85 kDa phospholipase A2 and its activation by phorbol ester and zymosan, was here shown also to counteract the protein kinase C-independent activation and arachidonate release elicited by bacteria. It remains to be determined whether all phosphorylation sites are equally affected under those conditions.
Wijkander
J
J
Department of Cell and Molecular Biology, Lund University, P.O. Box 94, SE-221 00 Lund, Sweden.
Gewert
K
K
Svensson
U
U
Holst
E
E
Sundler
R
R
D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005260:Female; D016967:Fusobacterium nucleatum / Q000502:physiology; D006194:Gardnerella vaginalis / Q000502:physiology; D017737:Macrophages, Peritoneal / Q000201:enzymology*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D010748:Phosphopeptides; D016718:Arachidonic Acid; D012694:Serine; D003907:Dexamethasone; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate