Autophosphorylation of CaMKII in Neural Signal Transduction

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DESCRIPTION (provided by applicant): The goal of the project is to build a quantitative, kinetic model for calcium-dependent signaling events during synaptic transmission in glutamatergic spines. Calcium/calmodulin-dependent protein kinase II (CaMKII) is a major target of the Ca 2+flux through NMDA-type glutamate receptors and is known to be a crucial component of several important neural protein phosphorylation pathways beneath the post-synaptic membrane of excitatory synapses. Activation of CaMKII involves the binding of four Ca 2+ ions to individual calmodulin (CaM) molecules and the association of CaM with a binding site on each CaMKII subunit that leads to activation of the catalytic domain. Of particular interest is the kinetics of activation of CaMKII by Ca 2+ and CaM. I will perform biochemical assays to determine binding constants for Ca 2+ to CaM and for Ca 2+ CaM to CaMKII, and determine whether cooperativity is enhanced in the presence of CaMKII. I will determine the intrinsic rate of autophosphorylation of CaMKII using concentrations of Ca, CaM, CaMKII that will be likely to occur at synapses. I will use mutant forms of CaM that cannot bind Ca at particular sites, and tryptic fragments of CaM containing either the amino or carboxyl EF hands, in order to directly measure the binding affinity for CaMKII of these separate sites in their Ca 2+ bound form. Using the kinetic parameters that I obtain, I will simulate the initial level of autophosphorylation when the Ca 2+ level changes, and compare predictions with experiments using a quench flow apparatus. I will then collaborate with investigators at the Salk Institute to simulate activation of CaMKII in the post-synaptic density in spines using the program MCell. CaMKII is involved in complex signaling pathways that lead to strengthening of synaptic strength (LTP), or, under different circumstances, weakening of synaptic strength (LTD). The proposed work will help us to understand how a Ca 2+ signal in a spine achieves the encoding of these changes with such high specificity.