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

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.