Role of phosphorylation and basic residues in the catalytic domain of cytosolic phospholipase A2α in regulating interfacial kinetics and binding and cellular function

Abstract

Group IVA cytosolic phospholipase A2 (cPLA2α) is regulated by phosphorylation and calcium-induced translocation to membranes. Immortalized mouse lung fibroblasts lacking endogenous cPLA2α (IMLF-/-) were reconstituted with wild type and cPLA2α mutants to investigate how calcium, phosphorylation, and the putative phosphatidylinositol 4,5-bisphosphate (PIP2) binding site regulate translocation and arachidonic acid (AA) release. Agonists that elicit distinct modes of calcium mobilization were used. Serum induced cPLA2α translocation to Golgi within seconds that temporally paralleled the initial calcium transient. However, the subsequent influx of extracellular calcium was essential for stable binding of cPLA2α to Golgi and AA release. In contrast, phorbol 12-myristate 13-acetate induced low amplitude calcium oscillations, slower translocation of cPLA2α to Golgi, and much less AA release, which were blocked by chelating extracellular calcium. AA release from IMLF-/- expressing phosphorylation site (S505A) and PIP2 binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA2α, but calcium-induced translocation was not impaired. Consistent with these results, Ser-505 phosphorylation did not change the calcium requirement for interfacial binding and catalysis in vitro but increased activity by 2-fold. Mutations in basic residues in the catalytic domain of cPLA2α reduced activation by PIP2 but did not affect the concentration of calcium required for interfacial binding or phospholipid hydrolysis. The results demonstrate that Ser-505 phosphorylation and basic residues in the catalytic domain principally act to regulate cPLA2α hydrolytic activity. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.