Ligation of the alpha M-2* signaling receptor regulates synthesis of cytosolic phospholipase A(2)

We have studied the regulation of cytosolic phospholipase A(2) (cPLA(2)) sy nthesis in macrophages stimulated with receptor-recognized forms of alpha ( 2)-macroglobulin (alpha M-2*). [S-35]methionine-labeled cells were stimulat ed with alpha M-2* and [S-35]cPLA(2) was immunoprecipitated with a monoclon al antibody directed against cPLA(2), The precipitates were electrophoresed , immunoblotted, cPLA(2) detected by Enhanced Chemifluorescence, and its ra dioactivity determined. Stimulation of cells with alpha M-2* caused a two- to threefold increase in cPLA(2) synthesis compared to buffer-treated cells which was consistently maximal at 200 pM of alpha M-2*. Actinomycin D or c ycloheximide treatment of cells drastically reduced alpha M-2*-induced cPLA (2) synthesis. Likewise, inhibition of protein kinase C with chelerythrin, farnesyl transferase with manumycin A, MEK kinase with U0126, Erk1/2 kinase s with PD98059, p38MAPK with SB203580, PI 3-kinase with wortmannin or LY294 002, p(70s6k) with rapamycin, or depletion of [Ca2+](i) with either BAPTA/A M or EGTA drastically reduced alpha M-2* induction of cPLA(2), Inhibition o f NF kappaB activation with BAY11-7182 or PGA(1) also abolished alpha M-2* induction of cPLA(2). We conclude that alpha M-2*-induced cPLA(2) synthesis is controlled by [Ca2+](i) levels, tyrosine kinase activity, the p21(ras)- dependent MAPK and PI 3-kinase downstream signaling pathways, and regulatio n of NF kappaB. (C) 2001 Academic Press.