Phosphorylation state, solubility, and activity of calcium/calmodulin-dependent protein kinase II alpha in transient focal ischemia in mouse brain

Copyright KLUWER ACADEMIC/PLENUM PUBLDuring and after middle cerebral artery occlusion in mice, CaMKIIalpha protein was irreversibly translocated from the soluble to the Triton X-100-nonsoluble fraction. This decrease in solubility had a strong effect on activity: CaMKIIalpha was almost completely inactivated after being translocated. Results from solubilization experiments suggest that different mechanisms underlie the conversion of CaMKIIalpha protein from a soluble to a detergent nonsoluble form in ischemic as opposite to nonischemic tissue. Analysis of the phosphorylation state of CaMKIIalpha revealed that in the total homogenate and the Triton X-100-nonsoluble fraction, CaMKIIalpha phosphorylated at only one site was the dominant phosphorylated form, whereas in the soluble fraction CaMKII phosphorylated at two sites was the predominant phosphorylated species. Investigation of the mechanisms underlying ischemia-induced changes in the solubility of CaMKIIalpha could help to elucidate processes triggered by transient focal cerebral ischemia that lead to neuronal cell injury