Abstract
Maintaining synaptic protein levels that support both functional stability and plasticity is crucial for the proper function of the nervous system. Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a pivotal role in synaptic signaling, with its synaptic levels regulated by both activity-dependent translocation from the cytosol and local translation. However, the mechanisms governing the removal of CaMKII from synapses and its degradation remain poorly understood. In this study, we develop a HEK293T cell culture system to investigate what properties of CaMKII regulate its degradation. By isolating distinct conformational states of both Drosophila and rat CaMKII, we demonstrate that locking CaMKII in its active form via a phosphomimetic mutation at the autonomous activation site significantly reduces the half-life for both homologs. We identify ubiquitination as the primary degradation pathway for persistently active CaMKII mutants. Moreover, our findings suggest that the decreased half-life is primarily due to the exposure of the catalytic and regulatory domains, rather than changes in the oligomerization state of the holoenzyme or regulation of degradation pathways by the kinase's enzymatic activity. These results provide a template for understanding CaMKII degradation in neurons.
Keywords:
Ca2+/calmodulin-dependent protein kinase II (CaMKII); Drosophila; HEK293T cells; autophosphorylation; protein degradation; ubiquitin.