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.
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.
Subramanian
Preeti
P
Department of Biochemistry, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298-0614, USA.
Stahelin
Robert V
RV
Szulc
Zdzislaw
Z
Bielawska
Alicja
A
Cho
Wonhwa
W
Chalfant
Charles E
CE
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
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