N-terminal and C-terminal plasma membrane anchoring modulate differently agonist-induced activation of cytosolic phospholipase A2.
The 85 kDa cytosolic phospholipase A2 (cPLA2) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA2 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 cPLA2 activation, we constructed chimeras of cPLA2 constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA2) or the C-terminal targeting signal of K-Ras4B (cPLA2-Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the alpha2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [3H]arachidonic acid, indicating that constitutive association of cPLA2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA2 increased [3H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA2-Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts of recombinant wild-type cPLA2. The lack of stimulation of cPLA2-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.
Klapisz
E
E
UPRES-A CNRS, Paris, France.
Ziari
M
M
Wendum
D
D
Koumanov
K
K
Brachet-Ducos
C
C
Olivier
J L
JL
Béréziat
G
G
Trugnan
G
G
Masliah
J
J
D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology; D017124:Conserved Sequence; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D016718:Arachidonic Acid; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D010741:Phospholipases A; D054467:Phospholipases A2; D018631:ras Proteins