Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase

Abstract

The attenuation of protein synthesis via the phosphorylation of eIF2α is a major stress response of all eukaryotic cells. The growth-arrest- and DNA-damage-induced transcript 34 (GADD34) bound to the serine/threonine protein phosphatase 1 (PP1) is the necessary eIF2α phosphatase complex that returns mammalian cells to normal protein synthesis following stress. The molecular basis by which GADD34 recruits PP1 and its substrate eIF2α are not fully understood, hindering our understanding of the remarkable selectivity of the GADD34:PP1 phosphatase for eIF2α. Here, we report detailed structural and functional analyses of the GADD34:PP1 holoenzyme and its recruitment of eIF2α. The data highlight independent interactions of PP1 and eIF2α with GADD34, demonstrating that GADD34 functions as a scaffold both in vitro and in cells. This work greatly enhances our molecular understanding of a major cellular eIF2α phosphatase and establishes the foundation for future translational work.

Figure 1. The GADD34 PP1-binding domain is intrinsically disordered

(a) GADD34 domain structure; ER localization domain, PEST domain (4 PEST repeats; labeled 1 – 4; each ~40 aa) and a PP1-binding domain (includes canonical PP1-binding motif RVxF [KVRF]). GADD34_241-674, GADD34_513-631 and GADD34_552-621 are constructs used. (b) Annotated 2D [¹H,¹⁵N] HSQC of GADD34_552-621. The narrow ¹H^N chemical shift dispersion is a hallmark of IDPs. (c) Secondary Structure Propensity (SSP) analysis of GADD34_552-621 reveals two preferred α-helical secondary structures (helices α1 and α2). (d) Helices α1 and α2 have also reduced fast timescale motions.

Figure 2. The 3D structure of the GADD34:PP1 holoenzyme

(a) GADD34 (residues 553-568, light blue, electron density 2F_o-F_c contoured at 1σ) and PP1α_7-300 (gray, surface) form a complex. Two Mn²⁺ ions (pink spheres) are bound at the PP1 active site; bound phosphate is shown as sticks. No electron density was observed for GADD34 residues 552 or 569-591. (b) GADD34 PP1-binding domain with the two primary interaction sites (RVxF and ΦΦ residues are shown as sticks) highlighted. (c) SDS-PAGE of ~20 GADD34_552-591:PP1α_7-300 crystals; comparable migration of GADD34 from crystals and control (GADD34_552-591 alone) shows that no proteolytic degradation of GADD34_552-591 occurred during crystallization. (d) ITC of GADD34_552-567 and PP1α_7-330.

Figure 3. Recruitment of eIF2α is mediated by the GADD34 PEST domain

(a) Immuno-precipitation (IP) experiment using WT- and KARA-GADD34. GADD34 recruits eIF2α from the HEK293T cell lysate independent of PP1 binding. (b) IP experiment using His₆-GADD34_241-674 and His₆-GADD34_513-631. Only His₆-GADD34_241-674 binds eIF2α (HEK293T cells lysate), while both His₆-GADD34_241-674 and GADD34_513-631 bind PP1. (c) FLAG vector or FLAG-GADD34 proteins were expressed in HEK293 cells and IP using anti-FLAG-conjugated to agarose beads. The IP and whole cell lysates (WCL) were subjected to immunoblotting (IB) with indicated antibodies. Molecular weight markers (kD) are shown.

Figure 4. Bimolecular Fluorescence Complementation (BiFC) assays highlight the role of GADD34 as a scaffolding protein

(a) Flag-tagged GADD34 enhances the BiFC signal that results from the complementation of YFP from N-YFP-PP1α and C-YFP-eIF2α. Over-expression of neurabin-1, a neuronal PP1 regulatory protein (negative control) does not assemble the BiFC complex. (b) Localization of GFP-tagged GADD34 and neurabin-1 (COS-7 cell overexpression). (c) Model: GADD34 domain structure and the proposed PP1α and eIF2α binding sites (PP1 and eIF2α structures are shown).