Ceramide 1-phosphate acts as a positive allosteric activator of group IVA cytosolic phospholipase A2α and enhances the interaction of the enzyme with phosphatidylcholine

Preeti Subramanian, Robert V. Stahelin, Zdzislaw Szulc, Alicja Bielawska, Wonhwa Cho, Charles E. Chalfant

Research output: Contribution to journalArticle

100 Scopus citations

Abstract

Previous findings from our laboratory have demonstrated that cPLA 2α is directly activated by the emerging bioactive sphingolipid, ceramide 1-phosphate (C-1-P) (1). In this study, a Triton X-100/phosphatidylcholine (PC) mixed micelle assay was utilized to determine the kinetics and specificity of this lipid-enzyme interaction. Using this assay, the addition of C-1-P induced a dramatic increase in the activity of cPLA 2α (> 15-fold) with a Ka of 2.4 mol % C-1-P/Triton X-100 micelle. This activation was highly specific as the addition of other lipids had insignificant effects on cPLA2α activity. Studies using surface-dilution kinetics revealed that C-1-P had no effect on the Michaelis-Menten constant, (KmB), but decreased the dissociation constant (K8A) value by 87%. Thus, C-1-P not only increases the membrane affinity of cPLA2α but also may act as an allosteric activator of the enzyme. Surface plasmon resonance analysis of the C-1-P/cPLA2α interaction verified a decrease in the dissociation constant, demonstrating that CPLA2α bound PC vesicles containing C-1-P with increased affinity (5-fold) compared with PC vesicles alone. The effect on the dissociation rate of cPLA2α was also found to be lipid-specific with the exception of phosphatidylinositol 4,5-bisphosphate, which caused a modest increase in vesicle affinity (2-fold). Lastly, the binding site for C-1-P was determined to be within the C2-domain of cPLA2α, unlike phosphatidylinositol 4,5-bisphosphate. These data demonstrate a novel interaction site for C-1-P and suggest that C-1-P may function to recruit cPLA2α to intracellular membranes as well as allosterically activate the membrane-associated enzyme.

Original languageEnglish (US)
Pages (from-to)17601-17607
Number of pages7
JournalJournal of Biological Chemistry
Volume280
Issue number18
DOIs
StatePublished - May 6 2005
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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