Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase that is highly enriched in neurons, particularly at the post-synaptic density (PSD), and has been demonstrated to play a critical role in mediating synaptic plasticity. In particular, much work has been done in this lab investigating the dynamic translocation of GFP- labeled CaMKII to the PSD following stimulation. CaMKII has been shown to exist in various phosphorylated states that can function as a type of molecular memory, but this phenomena remains poorly understood. In the proposed experiments, I will use rat hippocampal neurons to transfect with CaMKII-GFP and image with confocal microscopy to elucidate how stimulation frequency coding controls transition between these localization and phosphorylation states. Also, preliminary data from our lab suggests that protein phosphatases regulate the CaMKH phosphorylation state, and that GFP-labeled protein phosphatase 1 (PP1) also translocates to the PSD following stimulation. To better understand this important regulation, PPIUFP transfection experiments using various PP1 mutations and pharmacological manipulations will be done to elucidate the mechanism of PP1 behavior. Simultaneously monitoring of CaMKII and PP1 will be done with dual-colored GFPs, and the crosstalk between the CaMKII and PP1 signaling pathways will be explored.This work should help clarify the cellular mechanisms underlying synaptic plasticity.
