Ceramide 1-Phosphate Acts as a Positive Allosteric Activator of Group IVA Cytosolic Phospholipase A2α and Enhances the Interaction of the Enzyme with Phosphatidylcholine*

Previous findings from our laboratory have demonstrated that cPLA2α 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 cPLA2α (>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 (K As) 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.

• Publication year

  2005

• Venue

  Journal of Biological Chemistry

• Publication date

  2005-05-06

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1074/JBC.M414173200PMID 15743759
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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