Stress-Induced Phosphorylation of PACT Reduces Its Interaction with TRBP and Leads to PKR Activation

Abstract

PACT is a stress-modulated activator of interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR) and is an important regulator of PKR-dependent signaling pathways. Stress-induced phosphorylation of PACT is essential for PACT's association with PKR leading to PKR activation. PKR activation by PACT leads to phosphorylation of translation initiation factor eIF2α, inhibition of protein synthesis, and apoptosis. In addition to positive regulation by PACT, PKR activity in cells is also negatively regulated by TRBP. In this study, we demonstrate for the first time that stress-induced phosphorylation at serine 287 significantly increases PACT's ability to activate PKR by weakening PACT's interaction with TRBP. A non-phosphorylatable alanine substitution mutant at this position causes enhanced interaction of PACT with TRBP and leads to a loss of PKR activation. Furthermore, TRBP overexpression in cells is unable to block apoptosis induced by a phospho-mimetic, constitutively active PACT mutant. These results demonstrate for the first time that stress-induced PACT phosphorylation functions to free PACT from the inhibitory interaction with TRBP and also to enhance its interaction with PKR.

Altered Activation of Protein Kinase PKR and Enhanced Apoptosis in Dystonia Cells Carrying a Mutation in PKR Activator Protein PACT

“…The full-length P222L mutant was subcloned into mammalian two-hybrid system vectors and pET15b (Novagen). TRBP constructs were as described before (21).…”

“…PKR Kinase Activity Assays-PKR kinase activity assays were performed using HeLa M cell extracts as described before (11,21). One g/ml of poly(I)⅐poly(C) was used as the standard activator.…”

“…Recently we showed that cellular stress signals cause PACT to dissociate from TRBP leading to PACT-mediated PKR activation. TRBP-PACT heterodimers present in unstressed cells dissociate, as PACT is phosphorylated on Ser-287 in M3 in response to oxidative stress, serum starvation, and endoplasmic reticulum (ER) stress (20,21) by a protein kinase yet to be identified. Stress-induced phosphorylation at serine 287 has a dual role in PACT-mediated PKR activation as it causes dissociation of the PACT-TRBP complex and at the same time increases PACT affinity for PKR (21).…”

“…TRBP-PACT heterodimers present in unstressed cells dissociate, as PACT is phosphorylated on Ser-287 in M3 in response to oxidative stress, serum starvation, and endoplasmic reticulum (ER) stress (20,21) by a protein kinase yet to be identified. Stress-induced phosphorylation at serine 287 has a dual role in PACT-mediated PKR activation as it causes dissociation of the PACT-TRBP complex and at the same time increases PACT affinity for PKR (21). Two PACT molecules can also interact via the conserved dsRBMs, and phosphorylation of serine 287 enhances PACT-PACT interactions (22).…”

Recent Advances in the Molecular Pathogenesis of Dystonia-Plus Syndromes and Heredodegenerative Dystonias

“…In the unstressed state, TRBP sequesters PACT and so prevents it from activating PKR. Under stress, PACT is released from TRBP [47], allowing it to stably dimerize [48] and bind PKR thereby facilitating PKR autophosphorylation and catalytic activation [49]. The exact mechanism by which the proven and putative mutations linked to DYT16 cause the observed clinical phenotype is unclear.…”