Abstract
The interaction of calmodulin with its target proteins is known to affect the kinetics and affinity of Ca(2+) binding to calmodulin. Based on thermodynamic principles, proteins that bind to Ca(2+)-calmodulin should increase the affinity of calmodulin for Ca(2+), while proteins that bind to apo-calmodulin should decrease its affinity for Ca(2+). We quantified the effects on Ca(2+)-calmodulin interaction of two neuronal calmodulin targets: RC3, which binds both Ca(2+)- and apo-calmodulin, and alphaCaM kinase II, which binds selectively to Ca(2+)-calmodulin. RC3 was found to decrease the affinity of calmodulin for Ca(2+), whereas CaM kinase II increases the calmodulin affinity for Ca(2+). Specifically, RC3 increases the rate of Ca(2+) dissociation from the C-terminal sites of calmodulin up to 60-fold while having little effect on the rate of Ca(2+) association. Conversely, CaM kinase II decreases the rates of dissociation of Ca(2+) from both lobes of calmodulin and autophosphorylation of CaM kinase II at Thr(286) induces a further decrease in the rates of Ca(2+) dissociation. RC3 dampens the effects of CaM kinase II on Ca(2+) dissociation by increasing the rate of dissociation from the C-terminal lobe of calmodulin when in the presence of CaM kinase II. This effect is not seen with phosphorylated CaM kinase II. The results are interpreted according to a kinetic scheme in which there are competing pathways for dissociation of the Ca(2+)-calmodulin target complex. This work indicates that the Ca(2+) binding properties of calmodulin are highly regulated and reveals a role for RC3 in accelerating the dissociation of Ca(2+)-calmodulin target complexes at the end of a Ca(2+) signal.
Highlights
Calmodulin (CaM)1 is a small (16.8 kDa) ubiquitous Ca2ϩbinding protein that has been shown to play a central role in Ca2ϩ signaling in a wide variety of cell types
We considered the fact that both CaM kinase II and RC3 are known to be present at high concentrations in neurons, and both reside in the same compartments: soma, dendrites, and dendritic spines [35, 36]
We previously showed that PEP-19 can increase the rates of Ca2ϩ dissociation from CaM bound to CaM kinase II [14]
Summary
Calmodulin (CaM) is a small (16.8 kDa) ubiquitous Ca2ϩbinding protein that has been shown to play a central role in Ca2ϩ signaling in a wide variety of cell types. CaM leads to a conformational change in the protein, allowing it to bind to and activate a large number of intracellular target proteins, including enzymes [1,2,3], ion channels (4 – 6), cytoskeletal elements [7, 8], and transcriptional and translational machinery [9, 10] In this way, CaM is poised as a critical intermediate in numerous cell processes. Using CaM fragments, the high affinity sites were mapped to the C-terminal lobe and the low affinity sites to the N-terminal lobe [12] These differences in Ca2ϩ binding kinetics of the two lobes provide CaM with the potential for lobe-specific tuning of its interactions with target proteins in response to rises and falls in Ca2ϩ levels. We examine the effects of a member of two distinct classes of neuronal CaM targets, RC3, and CaM kinase II, on the interaction of Ca2ϩ with CaM
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