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. 2006 Jan 16;172(2):201-9.
doi: 10.1083/jcb.200508099.

Activation-dependent substrate recruitment by the eukaryotic translation initiation factor 2 kinase PERK

Stefan J Marciniak  1 , Lidia Garcia-Bonilla, Junjie Hu, Heather P Harding, David Ron
Affiliations

Activation-dependent substrate recruitment by the eukaryotic translation initiation factor 2 kinase PERK

Stefan J Marciniak et al. J Cell Biol. .

Abstract

Regulated phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) by the endoplasmic reticulum (ER) stress-activated protein kinase PERK modulates protein synthesis and couples the production of ER client proteins with the organelle's capacity to fold and process them. PERK activation by ER stress is known to involve transautophosphorylation, which decorates its unusually long kinase insert loop with multiple phosphoserine and phosphothreonine residues. We report that PERK activation and phosphorylation selectively enhance its affinity for the nonphosphorylated eIF2 complex. This switch correlates with a marked change to the protease sensitivity pattern, which is indicative of a major conformational change in the PERK kinase domain upon activation. Although it is dispensable for catalytic activity, PERK's kinase insert loop is required for substrate binding and for eIF2alpha phosphorylation in vivo. Our findings suggest a novel mechanism for eIF2 recruitment by activated PERK and for unidirectional substrate flow in the phosphorylation reaction.

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Figures

Figure 1.
Figure 1.
Activation-dependent conformational changes correlate with eIF2 recruitment by PERK. (A) Immunoblot of immunopurified PERK, phosphorylated eIF2α (P-eIF2α), and total eIF2α (T-eIF2α) from mouse fibroblasts exposed to 400 nM thapsigargin, an ER stress–inducing agent, for 1 h. (B) Immunoblots of FV2E-PERK, P-eIF2α, and T-eIF2α from Fv2E-PERK–expressing CHO cells exposed to 10 nM AP20187, the activating ligand, for the indicated period of time. (C) Coomassie-stained SDS-PAGE of purified bacterially expressed wild-type PERK kinase domain (WT), K618R mutant (KR), and purified wild-type PERK kinase domain that had been dephosphorylated in vitro with λ-phage phosphatase (Dephos WT) followed by partial tryptic digest. Tryptic fragments specific to the inactive conformation are indicated by asterisks. A fragment specific to the active conformation is indicated by an arrowhead. Small amounts of contaminating trypsin stable GST were also released from the glutathione–Sepharose beads affinity matrix after cleavage at the TEV site and are visible on the gel (GST).
Figure 2.
Figure 2.
Preferential binding of unphosphorylated eIF2α to activated PERK. (A) Immunoblots of bound and soluble proteins from cell lysate that had been incubated with the indicated GST-PERK proteins immobilized on a glutathione–Sepharose bead matrix. The bait proteins were stained with Coomassie (top). (B) Autoradiograph and corresponding Coomassie-stained gel of 32P-labeled proteins after incubation of bacterially expressed wild-type and S51D mutant eIF2α-NTD with wild-type or K618R mutant GST-PERK and γ-[32P]ATP. (C) Immunoblot of FV2E-PERK from untreated cells and cells treated with 10 nM AP20187 for 1 h. Lanes 1 and 2 are the lysate input, and lanes 3–6 are the material bound to Ni-NTA agarose beads that had not (mock) or had been coated with 6His-tagged eIF2α-NTD. (D) Coomassie-stained proteins (top three panels) and antiphospho-eIF2α blot (bottom) from bound and soluble fractions of an experiment in which purified eIF2α-NTD was added to immobilized GST-PERK in the presence of the indicated concentration of ATP. Note the selective elution of the phosphorylated product from the immobilized kinase. (E) Coomassie-stained SDS-PAGE (top and bottom) and autoradiograph of the top panel of the proteins eluted at the indicated salt concentrations from activated GST-PERK bound to a glutathione–Sepharose bead affinity matrix. The matrix of the top and middle panels was loaded with a mixture of unphosphorylated eIF2α-NTD and tracer amounts of radiolabeled phosphorylated P–eIF2α-NTD, whereas the bottom panel was loaded with purified eIF2α-NTD bearing a phosphomimetic mutation S51D. A graphical representation of the same data is also presented.
Figure 3.
Figure 3.
Deletion of PERK's kinase insert loop does not abolish the in vitro kinase activity. (A) The outline of protein kinase A and PERK's cytosolic domain based on a crystal structure (PKA) or a model (PERK). Proteins are drawn to scale, and PERK's large kinase insert loop is modeled as a compact globular structure. (B) ATP dependence of autokinase activity of wild-type PERK and PERK lacking the kinase insert loop (ΔL). Lineweaver-Burk plot inset; representative experiment shown. (C) Autoradiograph of eIF2α-NTD phosphorylation reaction performed with the indicated kinase in the presence of varying concentrations of substrate. (D) Graphical representation of P–eIF2α-NTD levels from C. Hill plot inset; representative experiment shown. (E) Immunoblots of phosphorylated and total eIF2α from reactions in which reticulocyte lysate had been incubated with the indicated PERK preparations; the latter are revealed by Coomassie staining (top).
Figure 4.
Figure 4.
PERK's kinase insert loop is required for efficient recruitment of substrate. (A) Coomassie-stained partial tryptic digest of TEV-cleaved recombinant Δ-loop PERK and dephosphorylated Δ-loop PERK as in Fig. 1 C. A fragment specific to the active conformation is indicated by an arrowhead. (B) Immunoblots of bound and soluble proteins from cell lysate that had been incubated with the indicated GST-PERK proteins immobilized on a glutathione–Sepharose bead matrix. The bait proteins were stained with Coomassie (top). (C) Autoradiography of eIF2α-NTD phosphorylated by the indicated PERK kinase preparations at increasing salt concentrations. (D) Graphical representation of P–eIF2α-NTD levels from C.
Figure 5.
Figure 5.
Phosphorylated residues on PERK interact with eIF2α. (A and B) Release of 32P from GST-PERK by λ-phage phosphatase in the absence (open square) and presence of eIF2α-NTD (A) or S51D eIF2α-NTD (B; 1.5 mg/ml, closed triangle; 3.75 mg/ml, closed diamond; 7.5 mg/ml, closed square). P values for the divergence of curves were determined by two-factor analysis of variance between groups: *, P < 0.05; **, P < 0.01. (C) Release of 32P from GST-PERK Δ-loop by λ-phage phosphatase in the absence (open square) and presence of eIF2α-NTD (1.5 mg/ml, closed triangle; 3.75 mg/ml, closed diamond; 7.5 mg/ml, closed square). (D) Coomassie-stained proteins from bound and soluble fractions of an experiment in which purified eIF2α-NTD was added to immobilized GST-PERK (WT), GST-PERKK618R (KR), and GST-PERKK618R;Σ(S/T→D) in which insert loop serine and threonine residues were mutated to aspartates (K618R;∑(S/T→D)) in the absence or presence of 10 μM ATP. Error bars represent SEM.
Figure 6.
Figure 6.
PERK's kinase insert loop is essential for in vivo recruitment and phosphorylation of eIF2α. (A) Anti-myc immunoblot of affinity-purified His6-FV2E-PERK-9E10, associated eIF2α, phosphorylated eIF2α (P-eIF2α), and total eIF2α (T-eIF2α) in the lysate of untreated and AP20187-treated (100 nM and 1 μM) 293T cells expressing the wild-type protein (WT), the K618R mutant (KR), and the Δ-loop version (ΔL). (B) Cartoon representing a hypothetical model of the interaction of activated PERK with its substrate. ER stress promotes oligomerization of PERK in the plane to the membrane, leading to transautophosphorylation. eIF2 complex is recruited to the highly phosphorylated insert loop before being passed on to the catalytic center. Phosphorylation of eIF2 (denoted as P) prevents further interaction with the insert loop and leads to product release.
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