Role of Phosphorylation and Basic Residues in the Catalytic Domain of Cytosolic Phospholipase A2α in Regulating Interfacial Kinetics and Binding and Cellular Function

Dawn E Tucker,Moumita Ghosh,Farideh Ghomashchi,Robyn Loper,Saritha Suram,Bonnie St John,Milena Girotti,James G Bollinger,Michael H Gelb,Christina C Leslie

doi:10.1074/jbc.m807299200

Abstract

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) is regulated by phosphorylation and calcium-induced translocation to membranes. Immortalized mouse lung fibroblasts lacking endogenous cPLA(2)alpha (IMLF(-/-)) were reconstituted with wild type and cPLA(2)alpha mutants to investigate how calcium, phosphorylation, and the putative phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding site regulate translocation and arachidonic acid (AA) release. Agonists that elicit distinct modes of calcium mobilization were used. Serum induced cPLA(2)alpha translocation to Golgi within seconds that temporally paralleled the initial calcium transient. However, the subsequent influx of extracellular calcium was essential for stable binding of cPLA(2)alpha to Golgi and AA release. In contrast, phorbol 12-myristate 13-acetate induced low amplitude calcium oscillations, slower translocation of cPLA(2)alpha to Golgi, and much less AA release, which were blocked by chelating extracellular calcium. AA release from IMLF(-/-) expressing phosphorylation site (S505A) and PIP(2) binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA(2)alpha, but calcium-induced translocation was not impaired. Consistent with these results, Ser-505 phosphorylation did not change the calcium requirement for interfacial binding and catalysis in vitro but increased activity by 2-fold. Mutations in basic residues in the catalytic domain of cPLA(2)alpha reduced activation by PIP(2) but did not affect the concentration of calcium required for interfacial binding or phospholipid hydrolysis. The results demonstrate that Ser-505 phosphorylation and basic residues in the catalytic domain principally act to regulate cPLA(2)alpha hydrolytic activity.

Highlights

Expressing phosphorylation site (S505A) and PIP2 binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA2␣, but calcium-induced translocation was not impaired
By expressing wild type and mutant forms of cPLA2␣ in lung fibroblasts lacking cPLA2␣, we investigated the functional role of phosphorylation and the PIP2 binding site in regulating calcium-dependent cPLA2␣ translocation and arachidonic acid (AA) release without interference of endogenous wild type enzyme
Effect of PIP2 on the Interfacial Properties of cPLA2␣ in Vitro—As a correlate to the cellular experiments, we studied the effect of PIP2 on interfacial binding and kinetics of wild type cPLA2␣ and cPLA2␣ containing mutations in basic residues in the catalytic domain

Summary

Introduction

Expressing phosphorylation site (S505A) and PIP2 binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA2␣, but calcium-induced translocation was not impaired Consistent with these results, Ser-505 phosphorylation did not change the calcium requirement for interfacial binding and catalysis in vitro but increased activity by 2-fold. Phosphorylation of cPLA2␣ on Ser505 increases its catalytic activity [14, 15, 24]; the role of phosphorylation in regulating calcium-induced translocation in cells has not been resolved. It has been reported that phosphorylation of cPLA2␣ on Ser-505 enhances the phospholipid binding affinity at low physiological calcium levels in vitro and in cells [25] This is consistent with another study showing that the inability of cPLA2␣ phosphorylation site mutants to release AA is overcome by inducing supraphysiological [Ca2ϩ]i [17]. The role of these basic residues in regulating translocation in cells stimulated with physiological agonists has not been investigated

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