Control of PERK eIF2alpha kinase activity by the endoplasmic reticulum stress-induced molecular chaperone P58IPK

Abstract

P58(IPK) is an Hsp40 family member known to inhibit the interferon (IFN)-induced, double-stranded RNA-activated, eukaryotic initiation factor 2alpha (eIF2alpha) protein kinase R (PKR) by binding to its kinase domain. We find that the stress of unfolded proteins in the endoplasmic reticulum (ER) activates P58(IPK) gene transcription through an ER stress-response element in its promoter region. P58(IPK) interacts with and inhibits the PKR-like ER-localized eIF2alpha kinase PERK, which is normally activated during the ER-stress response to protect cells from ER stress by attenuating protein synthesis and reducing ER client protein load. Levels of phosphorylated eIF2alpha were lower in ER-stressed P58(IPK)-overexpressing cells and were enhanced in P58(IPK) mutant cells. In the ER-stress response, PKR-like ER kinase (PERK)-mediated translational repression is transient and is followed by translational recovery and enhanced expression of genes that increase the capacity of the ER to process client proteins. The absence of P58(IPK) resulted in increased expression levels of two ER stress-inducible genes, BiP and Chop, consistent with the enhanced eIF2alpha phosphorylation in the P58(IPK) deletion cells. Our studies suggest that P58(IPK) induction during the ER-stress response represses PERK activity and plays a functional role in the expression of downstream markers of PERK activity in the later phase of the ER-stress response.

Fig 1.

P58^IPK contains an ERSE and is induced during the UPR. (A) Sequence alignment of the 5′-flanking region of human (GenBank accession no. NT009952) and mouse (GenBank accession no. ) P58^IPK. Conserved nucleotides are shaded. The ERSE is boxed, and the nucleotide changes introduced into the ERSE are shown in lowercase letters. The A of the ATG initiation codon (marked as M) is designated +1. (B) NIH 3T3 cells, transfected with the indicated constructs, were either untreated or treated with tunicamycin (2 μg/ml) for 16 h and subjected to a luciferase assay. Luciferase activity is depicted as relative light intensity. Gray bars, untreated cells; black bars, tunicamycin-treated cells; SV40, simian virus 40. (C) Northern blot analysis of poly(A)⁺ RNA (2 μg per lane) isolated from NIH 3T3 cells grown in the presence or absence of tunicamycin (Tm). (Upper) Probed with a 1,070-bp mouse P58^IPK fragment. (Lower) Probed with actin-specific probe. (D) NIH 3T3 cells were treated with tunicamycin for the indicated hours. An equal amount of cell lysate (100 μg) was loaded in each lane and subjected to immunoblot analysis by using indicated antibodies.

Fig 2.

P58^IPK interacts with and inhibits PERK. (A) Schematic diagram for PERK, PERKΔC, and PKR. The P58^IPK-interacting region of PKR, located in the kinase domain, was aligned with the corresponding region of PERK. Identical sequences are indicated in white. (B) His-P58^IPK pulls down PERK. His-tagged P58^IPK (lanes 2, 4, and 6) or His-tag alone (lanes 1, 3, and 5), bound on beads, was incubated with lysates from AR42J cells grown in the absence or presence of thapsigargin (Tg) or tunicamycin (Tm). The bead-bound protein complex was subjected to Western blotting (WB) by using the indicated antibodies. (C) HA-PERK pulls down P58^IPK. HA-PERK (lanes 1 and 3) or HA-PERKΔC (lanes 2 and 4) from mock-treated or tunicamycin-treated COS1 cells was used as bait to pull down purified GST-P58^IPK. The bead-bound protein complex was subjected to Western blotting by using indicated antibodies. (D) PERK coimmunoprecipitates with P58^IPK. COS1 cells cotransfected with P58^IPK and PERK (lanes 1–3) or PERKΔC (lanes 4 and 5), either treated with tunicamycin or untreated, were lysed and immunoprecipitated (IP) with anti-PERK antibody (lanes 2, 3, and 5) or normal rabbit serum (NRS, lanes 1 and 4). The precipitates then were subjected to Western blotting by using indicated antibodies. (E) P58^IPK inhibits PERK kinase activity. Purified GST-PERK was incubated with purified WT eIF2α (lanes 2–8) or S51A mutant eIF2α (lane 1) and [γ-³²P]ATP in the presence of purified GST-P58^IPK (lanes 5–8) or GST control (lanes 2–4). Hsp40 was included as a specific inhibitor to P58^IPK (lane 8). Reaction mixtures were subjected to SDS/PAGE and visualized by autoradiography.

Fig 3.

P58^IPK and PERK associate with the ER. HA-P58^IPK with (A–C) or without (D–F) PERK-myc was transfected into COS1 cells. Cells were fixed and costained with rat anti-HA antibody, followed by FITC-conjugated anti-rat serum and mouse anti-myc (mouse) (A–C) or mouse anticalnexin (D–F) antibodies, followed by Texas red-conjugated anti-mouse serum. The white arrows in A (P58^IPK, green), B (PERK, red), and C (merger of A and B) point to the PERK nontransfected cells. The white arrows in D (P58^IPK, green), E (calnexin, red), and F (merger of D and E) point to the P58^IPK nontransfected cells.

Fig 4.

Overexpression of P58^IPK attenuates PERK-mediated eIF2α phosphorylation. (A) P58^IPK Tet-Off-inducible cells were grown in the presence or absence of tetracycline (1 μg/ml) for 2 days. Then cells were treated with thapsigargin (Tg, 1 μM) for the indicated hours. Equal amounts of cell lysates from these cells were subjected to Western blotting by using the indicated antibodies. (B) Similar to A except that cells were treated with tunicamycin (Tm, 2 μg/ml) for the indicated period.

Fig 5.

Deletion of P58^IPK enhances UPR-mediated eIF2α phosphorylation and BiP/Chop induction. (A) P58^IPK+/+ (BL/6) and P58^IPK−/− mouse ES cells, treated with tunicamycin (Tm, 2 μg/ml) for the indicated hours, were lysed and subjected to Western blotting by using the indicated antibodies. Densitometry analysis was performed on the Western data. The ratio of the phosphor-eIF2α signal versus the total eIF2α signal is shown on the y axes by values in arbitrary units. Gray bars, P58^IPK+/+ cells; black bars, P58^IPK−/− cells. (B) Tunicamycin-treated cells (for the indicated hours) were immunoblotted with anti-BiP antibody and analyzed by densitometry. (C) Cells were treated with thapsigargin (Tg) for the indicated hours, immunoblotted with anti-Chop antibody, and analyzed by densitometry.