Analysis of RACK1 Redistribution and Phosphorylation in Cortical Neuron Compartments in Response to NMDA Receptor Activation

RACK1 (Receptor for Activated C Kinase 1) is a ribosomal 40S subunit protein that also functions as a signaling scaffold, playing key roles in synaptic plasticity, cytoskeletal regulation, and neurotransmitter responses in neurons. To investigate RACK1 behavior upon activation of local translation, we examined changes in RACK1 expression and phosphorylation levels in distinct compartments of cortical neurons following NMDA receptor stimulation. Using a porous membrane culture system to physically separate soma and neurites, we performed Phos-tag electrophoresis and Western blotting. RACK1 levels were lower in the neurite fraction compared to the soma. Phos-tag electrophoresis revealed slowly migrating bands corresponding to phosphorylated RACK1 isoforms. NMDA receptor activation reduced the proportion of these phosphorylated forms and increased the non‑phosphorylated RACK1 fraction. These data indicate that NMDA stimulation induces not only RACK1 redistribution but also changes in its post‑translational status within the neurite compartment. The observed electrophoretic mobility shifts likely reflect a transition of RACK1 from a phosphorylated, functionally dimer‑associated state to a predominantly non‑phosphorylated monomeric form, which may regulate its signaling and ribosomal functions.