Anionic lipids activate group IVA cytosolic phospholipase A2 via distinct and separate mechanisms

Preeti Subramanian,Mohsin Vora,Luciana B Gentile,Robert V Stahelin,Charles E Chalfant

doi:10.1194/jlr.m700356-jlr200

Abstract

Previously, ceramide-1-phosphate (C1P) and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] were demonstrated to be potent and specific activators of group IVA cytosolic phospholipase A2 (cPLA2alpha). In this study, we hypothesized that these anionic lipids functionally activated the enzyme by distinctly different mechanisms. Indeed, surface plasmon resonance and surface dilution kinetics demonstrated that C1P was a more potent effector than PI(4,5)P2 in decreasing the dissociation constant of the cPLA2alpha-phosphatidylcholine (PC) interaction and increasing the residence time of the enzyme on the vesicles/micelles. PI(4,5)P2, in contrast to C1P, decreased the Michaelis-Menten constant, increasing the catalytic efficiency of the enzyme. Furthermore, PI(4,5)P2 activated cPLA2alpha with a stoichiometry of 1:1 versus C1P at 2.4:1. Lastly, PI(4,5)P2, but not C1P, increased the penetration ability of cPLA2alpha into PC-rich membranes. Therefore, this study demonstrates two distinct mechanisms for the activation of cPLA2alpha by anionic lipids. First, C1P activates cPLA2alpha by increasing the residence time of the enzyme on membranes. Second, PI(4,5)P2 activates the enzyme by increasing catalytic efficiency through increased membrane penetration.

Highlights

Ceramide-1-phosphate (C1P) and phosphatidylinositol-4,5-bisphosphate [PI[4,5]P2] were demonstrated to be potent and specific activators of group IVA cytosolic phospholipase A2
Using surface dilution kinetics coupled with surface plasmon resonance (SPR) technology, we previously demonstrated the role of C1P in regulating the association of group IVA cytosolic phospholipase A2 (cPLA2a) with PC-rich micelles/vesicles via basic amino acids in the b-groove of the C2 domain that were shown to be critical for the C1P-cPLA2a interaction
For the first time, we have explored the mechanistic differences in the activation of cPLA2a by the anionic lipids C1P and PI[4,5]P2

Summary

Introduction

Ceramide-1-phosphate (C1P) and phosphatidylinositol-4,5-bisphosphate [PI[4,5]P2] were demonstrated to be potent and specific activators of group IVA cytosolic phospholipase A2 (cPLA2a). We first determined the number of molecules of the anionic lipids required for maximal activation of the enzyme in the mixed-micelle system. To demonstrate the effects of PI[4,5]P2 and C1P on the dissociation rate constant or membrane residence time, we measured the kinetics and affinity of cPLA2a binding with lipid vesicles using SPR.

Results

Conclusion