Calmodulin conformational changes in the activation of protein kinases.
The conformation of Ca2+/calmodulin changes from extended when free in solution to compact when bound in peptide complexes. The extent and kinetics of calmodulin compaction in association with Ca2+/calmodulin-dependent protein kinases (CaMKs), as well as target peptides, were investigated by fluorescence, resonance energy transfer and stopped-flow kinetics. Compaction of Ca2+/calmodulin labelled with resonance energy-transfer probes in association with target peptides was rapid (>350 s(-1)). With the target enzymes smooth-muscle myosin light-chain kinase, CaMKIV and CaMKII, the rates of calmodulin compaction were one-two orders of magnitude lower compared with those of the peptides and in the case of alphaCaMKII, ATP binding and Thr(286) auto-phosphorylation were required for calmodulin compaction. In the absence of nucleotides, Ca2+/calmodulin bound to alphaCaMKII in extended conformations, initially probably attached by one lobe only. Kinetic data suggest that in the activation process of Ca2+/calmodulin-dependent protein kinases, productive as well as unproductive complexes are formed. The formation of productive complexes with Ca2+/calmodulin thus may determine the rate of activation.
Török
K
K
Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, London, U.K. k.torok@sghms.ac.uk
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D009130:Muscle, Smooth / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase; D011487:Protein Conformation
D002147:Calmodulin; D046911:Macromolecular Substances; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase