Ca²⁺/Calmodulin-Dependent Activation and Inactivation Mechanisms of αCaMKII and Phospho-Thr₂₈₆-αCaMKII

Abstract

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.

Distinct Mechanisms of Calmodulin Binding and Regulation of Adenylyl Cyclases 1 and 8

“…52 The N-terminus of AC8, however, contains an amphipathic 1-8-14 motif, in which CaM binding is dependent on Ca 2+ as shown by GST pull-down assays (Figure 2) and offline nano-ESI-MS (Figure 3E,F), and the association with the N-lobe mutant of CaM also requires Ca 2+ (Figure 4C). As suggested for a phosphorylated form of CaMKII, 53 AC8 may be capable of associating with partially occupied CaM at a resting [Ca 2+ ] i , which supports the inhibition of AC8 activity by CaM 12 . 2,14 AC8-C2b exerts a dramatic effect on the ability of the N-lobe of CaM to interact with Ca 2+ .…”

Calmodulin Association with Connexin32-derived Peptides Suggests trans-Domain Interaction in Chemical Gating of Gap Junction Channels

“…Peptides-Cx32 (Gjb1) peptides were synthesized at the University of Rochester (Rochester, NY) and were purified to homogeneity by HPLC using previously described procedures (17 SpectroscopiesStopped flow kinetic measurements of Ca 2ϩ dissociation were carried out using quin 2 (Molecular Probes) and a Hi-Tech Scientific SF-61DX2 stopped flow system as previously described (26). Briefly, fluorescence excitation was set to 320 nm with 1-nm slit width and fluorescence emission from quin 2 was collected using a 530-nm cutoff filter.…”

Ca2+-dependent and -independent mechanisms of calmodulin nuclear translocation

“…Excitation was set to 278 nm and emission was measured at 305 nm. [Ca 2+ ] concentrations were calculated as described before (Tzortzopoulos et al, 2004).…”