Positive Cooperativity and T286 Autophosphorylation is Observed in a Dimeric Mutant of Calcium-Calmodulin Dependent Protein Kinase II (CaMKII)

Abstract

CaMKII is a multimeric enzyme that regulates long-term potentiation in the hippocampus. The assembly and organization of 8-14 subunits in a holoenzyme is thought to be required for transduction of calcium spike frequencies, cooperative binding to calmodulin, persistent activation by T286 autophosphorylation, and translocation into synaptic spines. CaMKII assembles into a holoenzyme by virtue of its unique C-terminal association domain (AD). ADs form a central hub-like structure from which regulatory and catalytic domains project. Each AD interacts tightly with three other subunits, two laterally and one transversely, to form a stable core composed of two stacked rings. FRET and analytical centrifugation has indicated that individual catalytic domains can form pairs by virtue of low affinity binding sites. But catalytic domains of the CaMKII mutant lacking AD do not form pairs in physiological conditions. We hypothesize that catalytic domain pairing is the fundamental structure that allows positive cooperativity and T286 autophosphorylation. Furthermore we postulate that high affinity AD core interactions lead to a high local concentration of catalytic domains that allows low-affinity catalytic domain pairing. To test this hypothesis, we mutated the lateral surface of the CaMKIIα AD. When expressed in HEK293 cells, the mutation generated a paired dimeric CaMKIIα. In support of the hypothesis, our study shows that the dimeric enzyme has the same affinity for calmodulin, a similar Hill coefficient for enzymatic activity, and T286 autophosphorylation comparable to that observed in the wild type holoenzyme. In contrast, the monomeric mutant CaMKII lacking AD has lower affinity, no cooperativity, and reduced T286 autophosphorylation. While catalytic domain pairing was observed in the dimeric mutant, biophysical analysis suggests that the nature of the pairing might be altered.