N-terminal and C-terminal plasma membrane anchoring modulate differently agonist-induced activation of cytosolic phospholipase A(2)

The 85 kDa cytosolic phospholipase A(2) (cPLA(2)) plays a key role in liber ating arachidonic acid from the sn-2 position of membrane phospholipids. Wh en activated by extracellular stimuli, cPLA(2) undergoes calcium-dependent translocation from cytosol to membrane sites which are still a matter of de bate. In order to evaluate the effect of plasma membrane association on cPL A(2) activation, we constructed chimeras of cPLA(2) constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA(2)) or the C-terminal targeting signal of K- Ras4B (cPLA(2)-Ras). Constitutive expression of these chimeras in Chinese h amster ovary cells overproducing the alpha(2B) adrenergic receptor (CHO-2B cells) did not affect the basal release of [H-3]arachidonic acid, indicatin g that constitutive association of cPLA(2) with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA(2) incre ased [H-3]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA(2)-Ras inhibited it, compa red with parental CHO-2B cells and CHO-2B cells producing comparable amount s of recombinant wild-type cPLA(2). The lack of stimulation of cPLA(2)-Ras was not due to a decreased enzymatic activity as measured using an exogenou s substrate, or to a decreased phosphorylation of the protein. These result s show that the plasma membrane is a suitable site for cPLA2 activation whe n orientated correctly.