Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases

Commun Biol. 2024 Mar 13;7(1):321. doi: 10.1038/s42003-024-06009-8.

Abstract

Ubiquitin modifications alter protein function and stability, thereby regulating cell homeostasis and viability, particularly under stress. Ischemic stroke induces protein ubiquitination at the ischemic periphery, wherein cells remain viable, however the identity of ubiquitinated proteins is unknown. Here, we employed a proteomics approach to identify these proteins in mice undergoing ischemic stroke. The data are available in a searchable web interface ( https://hochrainerlab.shinyapps.io/StrokeUbiOmics/ ). We detected increased ubiquitination of 198 proteins, many of which localize to the postsynaptic density (PSD) of glutamatergic neurons. Among these were proteins essential for maintaining PSD architecture, such as PSD95, as well as NMDA and AMPA receptor subunits. The largest enzymatic group at the PSD with elevated post-ischemic ubiquitination were kinases, such as CaMKII, PKC, Cdk5, and Pyk2, whose aberrant activities are well-known to contribute to post-ischemic neuronal death. Concurrent phospho-proteomics revealed altered PSD-associated phosphorylation patterns, indicative of modified kinase activities following stroke. PSD-located CaMKII, PKC, and Cdk5 activities were decreased while Pyk2 activity was increased after stroke. Removal of ubiquitin restored kinase activities to pre-stroke levels, identifying ubiquitination as the responsible molecular mechanism for post-ischemic kinase regulation. These findings unveil a previously unrecognized role of ubiquitination in the regulation of essential kinases involved in ischemic injury.

MeSH terms

  • Animals
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Disks Large Homolog 4 Protein
  • Focal Adhesion Kinase 2
  • Ischemia
  • Ischemic Stroke*
  • Mice
  • Phosphotransferases
  • Post-Synaptic Density
  • Stroke*
  • Ubiquitin
  • Ubiquitination

Substances

  • Disks Large Homolog 4 Protein
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Focal Adhesion Kinase 2
  • Phosphotransferases
  • Ubiquitin