The cellular 68,000-Mr protein kinase is highly autophosphorylated and activated yet significantly degraded during poliovirus infection: implications for translational regulation

TLDR

The study discusses how P68 degradation and increased eIF‑2α phosphorylation may affect protein synthesis regulation during poliovirus infection. The authors aimed to investigate whether P68 and its substrate eIF‑2 play translational regulatory roles during poliovirus infection and whether the observed increase in kinase activity is due to P68 synthesis. They showed that dsRNA produced during infection induces P68 autophosphorylation and developed an in‑vitro assay to confirm P68 degradation, while measuring protein levels to assess synthesis. P68 is highly autophosphorylated and activated during poliovirus infection, yet its protein levels decline sharply, with concurrent increases in eIF‑2α phosphorylation, indicating active degradation.

Abstract

We investigated the possible translational regulatory roles played by the interferon-induced, double-stranded-RNA-activated protein kinase (P68) and its natural substrate, eucaryotic initiation factor 2 (eIF-2), in poliovirus-infected cells. We demonstrated that protein kinase P68 was both highly autophosphorylated and activated during poliovirus infection. In accordance with these results, immunoprecipitation analysis revealed that phosphorylation of the endogenous eIF-2 alpha subunit also increased in poliovirus-infected cells. We found that double-stranded RNA synthesized during infection likely induced the high levels of P68 autophosphorylation. To determine whether the increase in kinase activity also could be attributed to induction of P68 synthesis, physical levels of protein kinase were measured. It was unexpectedly found that P68 protein levels did not increase but rather dramatically declined in poliovirus-infected cells. Pulse-chase experiments confirmed that the protein kinase was significantly degraded during virus infection. We corroborated our in vivo observations by developing an in vitro assay for P68 degradation using cell extracts. The possible consequences of P68 degradation and increased eIF-2 alpha phosphorylation for protein synthesis regulation in poliovirus-infected cells are discussed.