Abstract
The integrated stress response is regulated by kinases that phosphorylate translation initiation factor 2α and phosphatases that dephosphorylate it. Genetic and biochemical data indicate that the eIF2α-directed holophosphatase - a therapeutic target in diseases of protein misfolding - is comprised of a regulatory, PPP1R15, and a catalytic, Protein Phosphatase 1 (PP1), subunit. However, differing reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific de-phosphorylation1-3. An additional concern relates to the sensitivity of this PP1 holoenzyme to the [(o-chlorobenzylidene)amino]guanidines (Sephin1 or Guanabenz) small molecule proteostasis modulators3,4. We find that in the absence of G-actin, PPP1R15A regulatory subunit fragments were unable to accelerate eIF2α dephosphorylation beyond that affected by a catalytic subunit alone, whether PP1 was purified from rabbit muscle or from bacteria. Furthermore, we did not observe Sephin1 or Guanabenz inhibition of eIF2α dephosphorylation by any PPP1R15A-containing holophosphatase.