Abstract
The integrated stress response (ISR) is regulated by kinases that phosphorylate the α subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2αP-directed holophosphatase, a therapeutic target in diseases of protein misfolding, is comprised of a regulatory subunit, PPP1R15, and a catalytic subunit, protein phosphatase 1 (PP1). In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in the essential function of eIF2αP dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific dephosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the holoenzyme to the [(o-chlorobenzylidene)amino]guanidines Sephin1 or guanabenz, putative 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 the requirement for G-actin and sensitivity of the holoenzyme to inhibitors. We found that eIF2αP dephosphorylation by PP1 was moderately stimulated by repeat-containing PPP1R15A in an unphysiological low ionic strength buffer, whereas stimulation imparted by the co-presence of PPP1R15A and G-actin was observed under a broad range of conditions, low and physiological ionic strength, regardless of whether the PPP1R15A regulatory subunit had or lacked the N-terminal repeat–containing region and whether it was paired with native PP1 purified from rabbit muscle or recombinant PP1 purified from bacteria. Furthermore, none of the PPP1R15A-containing holophosphatases tested were inhibited by Sephin1 or guanabenz.
Highlights
The integrated stress response (ISR) is regulated by kinases that phosphorylate the ␣ subunit of translation initiation factor 2 and phosphatases that dephosphorylate it
phosphatase 1 (PP1) produced in Escherichia coli may differ in its enzymatic activity from PP1 purified from animal tissues, both in its substrate specificity and in its sensitivity to regulatory subunits
This enabled a comparison of the catalytic subunit content of PP1N preparation with the bacterially expressed PP1␥, which served as a reference
Summary
The integrated stress response (ISR) is regulated by kinases that phosphorylate the ␣ subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. PPP1R15A or PPP1R15B form a complex with PP1 via a conserved region of ϳ70 amino acids (PPP1R15A residues 555– 624) located at their C termini (5, 8 –11) (Fig. 1A) This conserved C-terminal region of either PPP1R15 regulatory subunit is sufficient to promote eIF2␣P dephosphorylation and to inactivate the ISR [4, 5, 10, 11]. Inactivation of the PPP1R15A gene, which decelerates eIF2␣P dephosphorylation and prolongs the ISR, is protective in certain cellular and animal models of diseases associated with enhanced unfolded protein stress (16 –19) This has generated interest in targeting the PPP1R15A-containing holophosphatase for inhibition by small molecules (reviewed in Ref. 20), an endeavor that requires detailed knowledge of the enzymatic mode of action. Does the isotype of the PP1 catalytic subunit or its source (recombinant versus native) influence the requirement for G-actin by the eIF2␣P-directed holophosphatase? What role does the N-terminal repeat– containing region of PPP1R15A play in eIF2␣P dephosphorylation by the holophosphatase? Do these factors influence the sensitivity of eIF2␣P dephosphorylation to guanabenz and Sephin1?
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