N-terminal and C-terminal plasma membrane anchoring modulate differently agonist-induced activation of cytosolic phospholipase A2

The 85 kDa cytosolic phospholipase A(2) (cPLA(2)) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA(2) undergoes calcium-dependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA(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 hamster ovary cells overproducing the alpha(2B) adrenergic receptor (CHO-2B cells) did not affect the basal release of [H-3]arachidonic acid, indicating that constitutive association of cPLA(2) with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA(2) increased [H-3]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA(2)-Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts 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 exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.