Choice of PP1 catalytic subunit affects neither requirement for G-actin nor insensitivity to Sephin1 of PPP1R15A-regulated eIF2αP dephosphorylation

Abstract

The integrated stress response (ISR) is regulated by kinases that phosphorylate the subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2P-directed holophosphatase - a therapeutic target in diseases of protein misfolding - is comprised of a regulatory, PPP1R15, and a catalytic, Protein Phosphatase 1 (PP1) subunit. In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in promoting the essential function of eIF2P dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific de-phosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the PPP1R15A-containing PP1 holoenzyme to the [(ochlorobenzylidene)amino]guanidines (Sephin1 or Guanabenz), small molecule proteostasis modulators. It has been suggested that the source and method of purification of the PP1 catalytic subunit and the presence or absence of an N-terminal repeat-containing region in the PPP1R15A regulatory subunit might influence both the requirement for G-actin by the eIF2P-directed holophosphatase and its sensitivity to inhibitors. Here we report that in the absence of G-actin, PPP1R15A regulatory subunits were unable to accelerate eIF2P dephosphorylation beyond that affected by a catalytic subunit alone, whether PPP1R15A regulatory subunit had or lacked the N-terminal repeat-containing region and whether paired with native PP1 purified from rabbit muscle, or recombinant PP1 expressed in and purified from bacteria. Furthermore, none of the PPP1R15A-containing PP1c holophosphatases were inhibited by Sephin1 or Guanabenz.