Mechanism of group IVA cytosolic phospholipase A(2) activation by phosphorylation.
Group IVA cytosolic phospholipase A2 (cPLA2) has been shown to play a critical role in the agonist-induced release of arachidonic acid. To understand the mechanism by which phosphorylation of Ser505 and Ser727 activates cPLA2, we systematically analyzed the effects of S505A, S505E, S727A, S727E, S505A/S727A, S505A/S727E, and S505E/S727E mutations on its enzyme activity and membrane affinity. In vitro membrane binding measurements showed that S505A has lower affinity than the wild type or S505E for phosphatidylcholine membranes, which is exclusively due to faster desorption of the membrane-bound S505A. In contrast, neither S727A nor S727E mutation had a significant effect on the phosphatidylcholine vesicle binding affinity of cPLA2. The difference in in vitro membrane affinity between wild type (or S505E) and S505A increased with the decrease in Ca2+ concentration, reaching >60-fold at 2.5 microm Ca2+. When HEK293 cells transfected with cPLA2 and mutants were stimulated with ionomycin, the wild type and S505E translocated to the perinuclear region and caused the arachidonic acid release at 0.4 microm Ca2+, whereas S505A showed no membrane translocation and little activity to release arachidonic acid. Further mutational analysis of hydrophobic residues in the active site rim (Ile399, Leu400, and Leu552) indicate that a main role of the Ser505 phosphorylation is to promote membrane penetration of these residues, presumably by inducing a conformational change of the protein. These enhanced hydrophobic interactions allow the sustained membrane interaction of cPLA2 in response to transient calcium increases. On the basis of these results, we propose a mechanism for cPLA2 activation by calcium and phosphorylation.
Das
Sudipto
S
Department of Chemistry, University of Illinois, Chicago, Illinois 60607, USA.
Rafter
John D
JD
Kim
Kwang Pyo
KP
Gygi
Steven P
SP
Cho
Wonhwa
W
D016718:Arachidonic Acid / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins; D054513:Group IV Phospholipases A2; D006801:Humans; D015151:Immunoblotting; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008164:Luminescent Proteins / Q000378:metabolism; D013058:Mass Spectrometry; D008954:Models, Biological; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014162:Transfection
D008164:Luminescent Proteins; D010455:Peptides; D049452:Green Fluorescent Proteins; D016718:Arachidonic Acid; D015759:Ionomycin; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; C516389:PLA2G4A protein, human; D054467:Phospholipases A2; D007930:Leucine; D002118:Calcium