Ser515 phosphorylation-independent regulation of cytosolic phospholipase A2α (cPLA2α) by calmodulin-dependent protein kinase: Possible interaction with catalytic domain A of cPLA2α

Calmodulin (CaM)-dependent protein kinase (CaM kinase) is proposed to regulate the type α of cytosolic phospholipase A2 (cPLA2α), which has a dominant role in the release of arachidonic acid (AA), via phosphorylation of Ser515 of the enzyme. However, the exact role of CaM kinase in the activation of cPLA2α has not been well established. We investigated the effects induced by transfection with mutant cPLA2α and inhibitors for CaM and CaM kinase on the Ca2+-stimulated release of AA and translocation of cPLA2α. The mutation of Ser515 to Ala (S515A) did not change cPLA2α activity, although S228A and S505A completely and partially decreased the activity, respectively. Stimulation with hydrogen peroxide (H2O2, 1 mM) and A23187 (10 µM) markedly released AA in C12 cells expressing S515A and wild-type cPLA2α, but the responses in C12-S505A, C12-S727A, and C12-S505A/S515A/S727A (AAA) cells were reduced. In HEK293T cells expressing cPLA2α, A23187 caused the translocation of the wild-type, the every mutants, cPLA2α-C2 domain, and cPLA2α-Δ397-749 lacking proposed phosphorylation sites such as Ser505 and Ser515. Treatment with inhibitors of CaM (W-7) and CaM kinase (KN-93) at 10 µM significantly decreased the release of AA in C12-cPLA2α cells and C12-S515A cells. KN-93 inhibited the A23187-induced translocation of the wild-type, S515A, AAA and cPLA2α-Δ397-749, but not cPLA2α-C2 domain. Our findings show a possible effect of CaM kinase on cPLA2α in a catalytic domain A-dependent and Ser515-independent manner.