Substrate recognition determinants of human eIF2α phosphatases

Phosphorylation of the translation initiation factor eIF2α is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2α phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Attempts at reconstituting recombinant holophosphatases have generated two models, one proposing that substrate recruitment requires the addition of actin, whilst the second proposes that this function is encoded by R15s. The biological relevance of actin in substrate recruitment has not been evaluated. Here we generated a series of truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A 325-554 and R15B 340-639 . Actin does not bind these regions establishing that it is not required for substrate recruitment. Activity assays in cells showed that R15A 325-674 and R15B 340-713 , encompassing the substrate-binding region and the PP1 binding-region, exhibit wild-type activity. This study identifies essential regions of R15s and demonstrates they function as substrate receptors. This work will guide the design of future structural studies with biological significance.