PKCβII Activation Promotes Membrane-Proximal Enrichment of Ribosome-Bound RACK1

The scaffold protein RACK1 (Receptor for Activated C Kinase 1) integrates signaling and translation, acting as a core component of the 40S ribosomal subunit. It binds activated Protein Kinase C (PKC) isoforms and membrane receptors. We used an auxin-inducible degron (AID2) system in human HAP1 cells to selectively deplete the free (cytoplasmic) pool of RACK1. The engineered RACK1-mAID-mClover3 fusion was rapidly degraded in the cytoplasm upon addition of 5-phenyl-indole-3-acetic acid (5-Ph-IAA), while the ribosome-bound pool remained detectable in ribosomal fractions, indicating that ribosome association makes RACK1 relatively less accessible to AID2-mediated proteolysis. Upon activation of PKCβII with phorbol-12-myristate-13-acetate (PMA), imaging at defined time points revealed closely matched kinetics of PKCβII membrane recruitment and membrane-proximal enrichment of ribosome-bound RACK1, peaking at ~10 min. Our data support a model in which activated PKCβII engages ribosome-bound RACK1 at membrane-proximal sites, consistent with a diffusion-capture mechanism in which PKCβII first accumulates at the membrane and then captures ribosome-bound RACK1, thereby recruiting the translational machinery to sites of signal input for membrane-proximal translation. These findings provide new insights into the spatial organization of translation.