Dissociation of calmodulin-target peptide complexes by the lipid mediator sphingosylphosphorylcholine: implications in calcium signaling.
Previously we have identified the lipid mediator sphingosylphosphorylcholine (SPC) as the first potentially endogenous inhibitor of the ubiquitous Ca2+ sensor calmodulin (CaM) (Kovacs, E., and Liliom, K. (2008) Biochem. J. 410, 427-437). Here we give mechanistic insight into CaM inhibition by SPC, based on fluorescence stopped-flow studies with the model CaM-binding domain melittin. We demonstrate that both the peptide and SPC micelles bind to CaM in a rapid and reversible manner with comparable affinities. Furthermore, we present kinetic evidence that both species compete for the same target site on CaM, and thus SPC can be considered as a competitive inhibitor of CaM-target peptide interactions. We also show that SPC disrupts the complex of CaM and the CaM-binding domain of ryanodine receptor type 1, inositol 1,4,5-trisphosphate receptor type 1, and the plasma membrane Ca2+ pump. By interfering with these interactions, thus inhibiting the negative feedback that CaM has on Ca2+ signaling, we hypothesize that SPC could lead to Ca2+ mobilization in vivo. Hence, we suggest that the action of the sphingolipid on CaM might explain the previously recognized phenomenon that SPC liberates Ca2+ from intracellular stores. Moreover, we demonstrate that unlike traditional synthetic CaM inhibitors, SPC disrupts the complex between not only the Ca2+-saturated but also the apo form of the protein and the target peptide, suggesting a completely novel regulation for target proteins that constitutively bind CaM, such as ryanodine receptors.
Kovacs
Erika
E
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest H-1113, Hungary. kovacs@enzim.hu
Tóth
Judit
J
Vértessy
Beáta G
BG
Liliom
Károly
K
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008555:Melitten / Q000737:chemistry / Q000378:metabolism; D008823:Micelles; D008969:Molecular Sequence Data; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
D001059:Apoproteins; D002147:Calmodulin; D008823:Micelles; D019837:Ryanodine Receptor Calcium Release Channel; C005356:sphingosine phosphorylcholine; D010767:Phosphorylcholine; D008555:Melitten; D013110:Sphingosine; D002118:Calcium