Dissociation of Calmodulin-Target Peptide Complexes by the Lipid Mediator Sphingosylphosphorylcholine: IMPLICATIONS IN CALCIUM SIGNALING

Erika Kovacs,Judit Tóth,Beáta G Vértessy,Károly Liliom

doi:10.1074/jbc.m109.053116

Erika Kovacs, Judit Tóth + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.053116

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Abstract

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.

Highlights

Calmodulin (CaM)3 is the ubiquitous Ca2ϩ sensor of eukaryotic cells [1]
We present kinetic evidence that both species compete for the same target site on CaM, and SPC can be considered as a competitive inhibitor of CaM-target peptide interactions
Our findings suggest a novel endogenous regulation for CaM and proposes that CaM might be an intracellular receptor for the sphingolipid

Summary

EXPERIMENTAL PROCEDURES

Preparation of Dansyl-labeled CaM—CaM was purified from bovine brain using phenyl-Sepharose affinity chromatography and dansylated according to Kovacs and Liliom [14]. Equilibrium Fluorescence Peptide-binding Assays—Fluorescence of dansyl-labeled CaM and the Trp residue of the RyR peptide was monitored on a Jobin Yvon Fluoromax-3 spectrofluorimeter at 25 °C in 10 mM HEPES, pH 7.4, 100 mM KCl, and 1 mM CaCl2. When screening with lipids SPC, S1P, LPC, LPA, and LT-SPC, dansyl-CaM, RyR peptide, and lipid concentrations were 0.2, 0.5, and 100 ␮M, respectively. When measuring dose response for SPC, dansyl-CaM and RyR peptide concentrations were 0.2 and 0.5 ␮M, respectively, and the SPC concentration varied between 10 and 100 ␮M. In the complimentary set of experiments, the Trp residue of the RyR peptide was excited at 295 nm, and spectra were recorded from 310 to 400 nm. Experiments with dansyl-labeled apoCaM were carried out to measurements with Ca2ϩ-saturated CaM, only in buffer containing 1 mM EGTA instead of 1 mM CaCl2. Each spectrum was corrected for corresponding lipid, protein, peptide, and buffer effects by subtracting a matching buffer scan

RESULTS

SPC chase ME chase

Kc ϫ KL

DISCUSSION