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

Abstract

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 calciumdependent 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 a 2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [ 3 H]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 [ 3 H]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.

Perinuclear localization of cytosolic phospholipase A2α is important but not obligatory for coupling with cyclooxygenases

“…1). This is in line with a previous report that a chimera of cPLA 2 α constitutively targeted to the plasma membrane by the N‐terminal targeting sequence of the tyrosine kinase Lck leads to increased AA release [19]. Even though the phospholipid compositions of the plasma membrane and the perinuclear membrane may differ significantly, the ability of cPLA 2 α to hydrolyze phospholipids without showing appreciable head group selectivity [20] may permit its equivalent action on both membranes.…”

Identification of cis-acting determinants mediating the unconventional secretion of tau

“…Taking this into consideration, we hypothesized that redirecting tau to distant cellular compartments would inhibit its release from cells, whereas the redirection to the plasma membrane could potentially enhance its secretion. To address this, we generated artificial tau E14 constructs that re-routed the protein to the nucleus or the plasma membrane through nuclear localization (NLS) or plasma membrane targeting signals 51 , respectively (Fig. 4 d).…”

Intracellular phospholipase A2 expression and location in human macrophages: Influence of synthetic material surface chemistry

“…This would allow access to cell membrane phospholipids for AA release as has been extensively reported on (Dennis, 2000; Balsinde et al, 2002; Dinnes et al, 2005). Previous studies assessing various forms of cell activation have indicated localization of cPLA 2 to the plasma membrane (Durstin et al, 1994; Schievella et al, 1995; Klapisz et al, 1999), nuclear envelope (Fatima et al, 2005), endoplasmic reticulum (Schievella et al, 1995), in addition to the golgi apparatus (Evans and Leslie, 2004).…”