A general strategy to characterize calmodulin-calcium complexes involved in CaM-target recognition: DAPK and EGFR calmodulin binding domains interact with different calmodulin-calcium complexes.
Calmodulin (CaM) is a ubiquitous Ca(2+) sensor regulating many biochemical processes in eukaryotic cells. Its interaction with a great variety of different target proteins has led to the fundamental question of its mechanism of action. CaM exhibits four "EF hand" type Ca(2+) binding sites. One way to explain CaM functioning is to consider that the protein interacts differently with its target proteins depending on the number of Ca(2+) ions bound to it. To test this hypothesis, the binding properties of three entities known to interact with CaM (a fluorescent probe and two peptide analogs to the CaM binding sites of death associated protein kinase (DAPK) and of EGFR) were investigated using a quantitative approach based on fluorescence polarization (FP). Probe and peptide interactions with CaM were studied using a titration matrix in which both CaM and calcium concentrations were varied. Experiments were performed with SynCaM, a hybrid CaM able to activate CaM dependent enzymes from mammalian and plant cells. Results show that the interaction between CaM and its targets is regulated by the number of calcium ions bound to the protein, namely one for the DAPK peptide, two for the probe and four for the EGFR peptide. The approach used provides a new tool to elaborate a typology of CaM-targets, based on their recognition by the various CaM-Ca(n) (n=0-4) complexes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
Therapeutic Innovation Laboratory, UMR CNRS 7200, University Strasbourg, Faculty of Pharmacy, 74, route de Rhin, 67401 Illkirch Cedrex, France.
Rania
R
Dagher
Shan
S
Peng
Sophie
S
Gioria
Marie
M
Fève
Maria
M
Zeniou
Michael
M
Zimmermann
Claire
C
Pigault
Jacques
J
Haiech
Marie-Claude
MC
Kilhoffer
D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry* / Q000378:metabolism*; D001671:Biochemistry / Q000379:methods*; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D064550:Death-Associated Protein Kinases; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013816:Thermodynamics
D051017:Apoptosis Regulatory Proteins; D002147:Calmodulin; D005456:Fluorescent Dyes; D050505:Mutant Proteins; D010455:Peptides; D011994:Recombinant Proteins; D066246:ErbB Receptors; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium