Abstract WMP110: Cerebral Ischemia-Reperfusion Alters Postsynaptic Kinase Activities Through Ubiquitination

Background: Aberrant activity of the postsynaptic density (PSD)-associated kinases CaMKII, PKC, Pyk2 and Cdk5 contributes to post-ischemic neuronal death. However, the mechanism(s) leading to altered activity after ischemia have not been discovered. We investigated the role of ubiquitination in post-ischemic PSD kinase activity regulation. Methods: Cerebral ischemia was induced in male C57BL6 mice by middle cerebral artery occlusion (MCAO), followed by 1-hour reperfusion. Ubiquitinated and phosphorylated proteins in the ischemic neocortex were identified by nanoLC-MS/MS and analyzed for GO enrichment. PSD isolation was carried out by detergent extraction. Kinase ubiquitination was confirmed by co-immunoprecipitation with ubiquitin. Protein phosphorylation was assessed with phospho-specific antibodies and kinase activities were measured by transfer of γ 32 P to peptide substrates. Removal of ubiquitin from PSD lysates was achieved by addition of USP2 deubiquitinase. Results: MS analysis revealed that MCAO increases ubiquitination of proteins associated with the PSD (p=5.14x10 -33 , n=60/group). This is also evidenced by biochemical detection of ubiquitin at the synapse and PSD but not cytosol of cortical MCAO extracts (p<0.001, n=6/group). Interestingly, a significant number of ubiquitinated PSD proteins exhibit kinase activity (p=1.02x10 -23 , n=60/group) and include the kinases CaMKII, PKC, Pyk2 and Cdk5, which were biochemically verified (n=3/group). Concurrent with ubiquitination, MCAO also changes the phosphorylation state of PSD-associated proteins (p=3.66x10 -13 , n=60/group). Pyk2 and Cdk5 target sites are hyper-phosphorylated in post-ischemic PSD extracts, while CaMKII and PKC targets show decreased phosphorylation (p<0.05, n=5/group). In line with this, post-ischemic CaMKII (-69±6% of sham, p<0.001, n=9/group) and PKC (-63±8% of sham, p<0.001, n=6/group) activities are severely suppressed at the PSD, which is restored by de-ubiquitination (CaMKII: 105.8±23% of sham, n=6/group; PKC: 101.4±11% of sham, n=4/group). Conclusions: Ubiquitination is a potent and reversible modulator of kinase activity at the PSD that could impact tissue outcome and, as such, may open new avenues for the treatment of ischemic stroke.