Phosphorylation of Specific Serine Residues in the PKR Activation Domain of PACT Is Essential for Its Ability to Mediate Apoptosis

Abstract

Activation of the latent protein kinase, PKR, by extracellular stresses and triggering of resultant cellular apoptosis are mediated by the protein, PACT, which itself gets phosphorylated in stressed cells. We have analyzed the underlying biochemical mechanism by carrying out alanine-scanning mutagenesis of the PKR activation domain of PACT. Among the indispensable residues identified were two serine residues, whose phosphorylation was essential for the cellular actions of PACT. Two-dimensional gel analysis, Western analysis using phosphoamino acid-specific antiserum, and in vivo 32 P labeling of PACT demonstrated that constitutive phosphorylation of one of the two residues, Ser 246 , was required for stress-induced phosphorylation of the other, Ser 287 . Substitution of either of them by threonine or aspartic acid, but not alanine, was tolerated. Substitution of both residues with the phosphoserine mimetic, aspartic acid, produced a mutant PACT that, unlike the wild-type protein, caused PKR activation and apoptosis, even in unstressed cells. These results indicate that phosphorylation of specific serine residues in the activation domain of PACT is the major mode of transmission of cellular stress response to PKR.

Aptamer-Mediated Blockade of IL4Rα Triggers Apoptosis of MDSCs and Limits Tumor Progression

“…[24][25][26][27][28] or activation of RNA-dependent protein kinase (PKR; refs. [29][30][31][32][33]. Alternatively, aptamer binding may modify some signaling pathways in MDSCs affecting their survival.…”

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

“…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).…”

The double-stranded RNA-binding protein, PACT, is required for postnatal anterior pituitary proliferation

“…A hallmark of PACT function is its ability to activate PKR in the absence of dsRNA, suggesting PACT has virus-independent cellular roles (6). The dsRBMs of PACT, domains 1 and 2, mediate strong interaction with the 2 dsRBMs of PKR, but the ability to activate PKR is imparted by a third domain of PACT that binds to the PKR kinase domain (7)(8)(9). Binding of domain 3 causes PKR autophosphorylation by disrupting an intramolecular interaction within PKR that is responsible for keeping PKR in an inactive conformation (10).…”

“…Binding of domain 3 causes PKR autophosphorylation by disrupting an intramolecular interaction within PKR that is responsible for keeping PKR in an inactive conformation (10). In mammalian cells, PACT itself is phosphorylated by treatment with various chemical stresses or growth factor withdrawal, allowing it to associate with PKR with increased affinity (7,9,11,12).…”