Faculty, Staff and Student Publications
Publication Date
10-7-2008
Journal
Biochemistry
Abstract
Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.
Keywords
Adenosine Diphosphate, Animals, Binding Sites, Calcium, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Calmodulin, Calorimetry, Fluorometry, Models, Molecular, Nucleotides, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Rats, Temperature
DOI
10.1021/bi8007033
PMID
18795794
PMCID
PMC2665295
PubMedCentral® Posted Date
October 2009
PubMedCentral® Full Text Version
Author MSS
Published Open-Access
yes