title: Ceramide 1-phosphate acts as a positive allosteric activator of group IVA cytosolic phospholipase A2 alpha and enhances the interaction of the enzyme with phosphatidylcholine.
abstract: Previous findings from our laboratory have demonstrated that cPLA(2)alpha 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(2)alpha (>15-fold) with a K(a) 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 cPLA(2)alpha activity. Studies using surface-dilution kinetics revealed that C-1-P had no effect on the Michaelis-Menten constant, K(m)(B), but decreased the dissociation constant (K (A)(s)) value by 87%. Thus, C-1-P not only increases the membrane affinity of cPLA(2)alpha but also may act as an allosteric activator of the enzyme. Surface plasmon resonance analysis of the C-1-P/cPLA(2)alpha interaction verified a decrease in the dissociation constant, demonstrating that cPLA(2)alpha bound PC vesicles containing C-1-P with increased affinity (5-fold) compared with PC vesicles alone. The effect on the dissociation rate of cPLA(2)alpha 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 cPLA(2)alpha, 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 cPLA(2)alpha to intracellular membranes as well as allosterically activate the membrane-associated enzyme.
authors.affiliation: Department of Biochemistry, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298-0614, USA.
authors.forename: Preeti
authors.initials: P
authors.lastname: Subramanian
authors.forename: Robert V
authors.initials: RV
authors.lastname: Stahelin
authors.forename: Zdzislaw
authors.initials: Z
authors.lastname: Szulc
authors.forename: Alicja
authors.initials: A
authors.lastname: Bielawska
authors.forename: Wonhwa
authors.initials: W
authors.lastname: Cho
authors.forename: Charles E
authors.initials: CE
authors.lastname: Chalfant
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D002518:Ceramides / Q000502:physiology*; D004357:Drug Synergism; D004789:Enzyme Activation / Q000502:physiology; D054513:Group IV Phospholipases A2; D006801:Humans; D007313:Insecta; D007527:Isoenzymes / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding / Q000502:physiology
chemical_list: D002518:Ceramides; D007527:Isoenzymes; D010713:Phosphatidylcholines; C065576:ceramide 1-phosphate; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; C516389:PLA2G4A protein, human; D054467:Phospholipases A2