Ribosomal Protein RACK1 as a Potential Substrate of PKR Protein Kinase: In Vitro Analysis
TL;DR
Although the protein kinase PKR binds to the ribosome, it is not the kinase that directly phosphorylates RACK1 and causes its dissociation from the ribosome.
Abstract
The double-stranded RNA-activated protein kinase PKR is a key mediator of the cellular stress response. PKR is known to associate with the ribosome upon activation, while the RACK1 ribosomal protein dissociates from the ribosome upon phosphorylation under stress conditions. It was assumed that these two processes may be interconnected, with PKR directly phosphorylating RACK1, causing its dissociation. Here, we experimentally tested the hypothesis regarding the kinase activity of PKR towards RACK1. PKR kinase activity towards the canonical substrate eIF2α, as well as autophosphorylation activity in the presence of 40S ribosomal subunits, were confirmed in vitro. However, phosphorylation of either recombinant RACK1 or native RACK1 within 40S ribosomal subunits was not detected in the presence of PKR. Thus, although the protein kinase PKR binds to the ribosome, it is not the kinase that directly phosphorylates RACK1 and causes its dissociation from the ribosome. Alternative PKR signaling pathways mediated by other kinases appear to be involved in the stress-induced mechanism of RACK1 dissociation from the ribosome.