Live imaging of endogenous Ca2+/calmodulin-dependent protein kinase II in neurons reveals that ischemia-related aggregation does not require kinase activity

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) forms 12meric holoenzymes. These holoenzymes cluster into larger aggregates within neurons under ischemic conditions and invitro when ischemic conditions are mimicked. This aggregation is thought to be mediated by interaction between the regulatory domain of one kinase subunit with the T-site of another kinase subunit in a different holoenzyme, an interaction that requires stimulation by Ca2+/CaM and nucleotide for its induction. This model makes several predictions that were verified here: Aggregation invitro was reduced by the CaMKII inhibitors tatCN21 and tatCN19o (which block the T-site) as well as by KN93 (which is CaM-competitive). Notably, these and previously tested manipulations that block CaMKII activation all reduced aggregation, suggesting an alternative mechanism that instead requires kinase activity. However, experiments with the nucleotide-competitive broad-spectrum kinase inhibitors staurosporin and H7 showed thatthis is not the case. In vitro, staurosporine and H7 enabled CaMKII aggregation even in the absence of nucleotide. Within rat hippocampal neurons, an intra-body enabled live monitoring of endogenous CaMKII aggregation. This aggregation was blocked by tatCN21, but not by staurosporine, even though both effectively inhibit CaMKII activity. These results support the mechanistic model for CaMKII aggregation and show that kinase activity is not required.