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The role of adenosine 5'-triphosphate (ATP) in the activation mechanism of α-Ca2+/calmodulin- dependent protein kinase II (αCaMKII) was investigated using the T286A non-autophosphorylatable mutant of αCaMKII. Characterization of the T286A-αCaMKII mutant revealed kcat = 0.06 ± 0.02 s-1 for the T286A mutant, a 6 (± 2)-fold lower value compared to wild-type αCaMKII with 100 µM smooth muscle myosin light chain (MLC) as substrate. MLC phosphorylation by the T286A mutant and wild-type αCaMKII was cooperative, with Hill coefficients 2.3 ± 0.1 and 2.4 ± 0.3, respectively. Km values for MLC were 96 ± 28 µM with T286A-αCaMKII and 49 ± 29 µM for wild-type αCaMKII. Thus, while the activity of αCaMKII was sensitive to mutation of the Thr286 residue to Ala, the mechanisms of the wild-type and T286A mutant enzyme appeared similar. Kd for Ca2+/calmodulin was 2-fold reduced to 40 nM compared to that of wild-type αCaMKII (75 nM). ATP induced a 9-fold stabilization of Ca2+/calmodulin binding to the T286A mutant enzyme. Fluorescence stopped-flow kinetic experiments revealed that two Ca2+/ calmodulin-enzyme complexes were formed, the first, unaffected by ATP, with association and dissociation rate constants of 2 × 107 M-1 s-1 and 5 s-1, respectively, containing calmodulin in extended conformation. The second complex, in which calmodulin adopted a compact conformation, was formed with association rate constant 3 × 106 M-1 s-1 and dissociation at 0.15 s-1 in the absence and 0.015 s-1 in the presence of ATP. These data show that ATP is involved in the activation mechanism by forming two classes of Ca2+/calmodulin·αCaMKII-ATP complex. It is likely that only one of the complexes is on the activation pathway. [ABSTRACT FROM AUTHOR]

Thr(286) autophosphorylation is important for the role of alphaCaMKII in learning and memory. Phospho-Thr(286)-alphaCaMKII has been described to have two types of activity: Ca(2+)-independent partial activity and Ca(2+)/calmodulin-activated full activity. We investigated the mechanism of switching between the two activities in order to relate them to the physiological functioning of alphaCaMKII. Using a fluorometric coupled enzyme assay and smooth muscle myosin light chain (MLC) as substrate, we found that (1) Ca(2+)-independent activity of phospho-Thr(286)-alphaCaMKII represents 5.0 (+/-3.7)% of the activity measured in the presence of optimal concentrations of Ca(2+) and calmodulin and (2) Ca(2+) in the presence of calmodulin activates the enzyme with a K(m) of 137 (+/-56) nM and a Hill coefficient n = 1.8 (+/-0.3). In contrast, unphosphorylated alphaCaMKII has a K(m) for Ca(2+) in the presence of calmodulin of 425 (+/-119) nM and a Hill coefficient n = 5.4 (+/-0.4). Thus, the activity of phospho-Thr(286)-alphaCaMKII is essentially Ca(2+)/calmodulin dependent with MLC as substrate. In physiological terms, our data suggest that alphaCaMKII is only activated in stimulated neurones whereas Ca(2+)/calmodulin activation of phospho-Thr(286)-alphaCaMKII can occur in resting cells (approximately 100 nM [Ca(2+)]). Stopped-flow experiments using Ca(2+)/TA-cal [Ca(2+)/2-chloro-(epsilon-amino-Lys(75))-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin] showed that at 100 nM [Ca(2+)] partially Ca(2+)-saturated Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII complexes existed. These are likely to account for the activity of the phospho-Thr(286)-alphaCaMKII enzyme at resting [Ca(2+)]. Ca(2+) dissociation measurements by a fluorescent Ca(2+) chelator revealed that the limiting Ca(2+) dissociation rate constants were 1.5 s(-1) from the Ca(2+)/cal.alphaCaMKII and 0.023 s(-1) from the Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII complex, accounting for the differences in the Ca(2+) sensitivities of the Ca(2+)/cal.alphaCaMKII and Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII enzymes.