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. 2009 Feb 10;106(6):1832-7.
doi: 10.1073/pnas.0809632106. Epub 2009 Jan 30.

Ppp1r15 gene knockout reveals an essential role for translation initiation factor 2 alpha (eIF2alpha) dephosphorylation in mammalian development

Heather P Harding  1 , Yuhong Zhang, Donalyn Scheuner, Jane-Jane Chen, Randal J Kaufman, David Ron
Affiliations

Ppp1r15 gene knockout reveals an essential role for translation initiation factor 2 alpha (eIF2alpha) dephosphorylation in mammalian development

Heather P Harding et al. Proc Natl Acad Sci U S A. .

Abstract

Diverse cellular stress responses are linked to phosphorylation of serine 51 on the alpha subunit of translation initiation factor 2. The resultant attenuation of protein synthesis and activation of gene expression figure heavily in the adaptive response to stress, but dephosphorylation of eIF2(alphaP), which terminates signaling in this pathway, is less well understood. GADD34 and CReP, the products of the related mammalian genes Ppp1r15a and Ppp1r15b, can recruit phosphatase catalytic subunits of the PPP1 class to eIF2(alphaP), but the significance of their contribution to its dephosphorylation has not been explored systematically. Here we report that unlike Ppp1r15a mutant mice, which are superficially indistinguishable from wild type, Ppp1r15b(-/-) mouse embryos survive gestation but exhibit severe growth retardation and impaired erythropoiesis, and loss of both Ppp1r15 genes leads to early embryonic lethality. These loss-of-function phenotypes are rescued by a mutation, Eif2a(S51A), that prevents regulated phosphorylation of eIF2alpha. These findings reveal that the essential process of eIF2(alphaP) dephosphorylation is the predominant role of PPP1R15 proteins in mammalian development.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Targeted deletion of Ppp1r15b, encoding CReP, increases basal and stress-induced eIF2α phosphorylation. (A) Cartoon depicting the strategy for targeted deletion of the promoter and portion of exon 1, encoding amino acids 1–416 of Ppp1r15b, by replacement with a pGK-Neo cassette. The targeting vector, wild-type (Ppp1r15b+), the targeted allele (Ppp1r15b−), and the PCR primers used to detect both alleles are depicted. (B) Immunoblots of the products of the Ppp1r15a or Ppp1r15b genes (GADD34 and CReP) immunopurified from wild-type or Ppp1r15b−/− cells that were untreated or treated with tunicamycin (Tm, 2 μg/ml) for 6 h to induce ER stress. (C) Immunoblots of phospho-eIf2α [eIF2(αP)], total eIF2α, or GADD34 from cells of the indicated Ppp1r15a (15a+/+, 15aΔC/ΔC, or 15a+/ΔC) and Ppp1r15b genotypes (15b+/+, 15b−/−, or 15b+/−) following exposure to thapsigargin (Tg 400 nM) to induce ER stress.
Fig. 2.
Fig. 2.
Stunted growth and perinatal lethality of Ppp1r15b−/− mice. (A) Graph depicting the distribution of genotypes found in 10-day-old pups born of Ppp1r15b+/− parents. (*Probability of finding no Ppp1r15b−/− progeny among 99 individuals by chance; P = 2.5 × 10−60.) (B) Graph depicting the mean weights + SD of e18.5 embryos conceived by Ppp1r15+/− parents. Paired two-tailed t-test results with P values are indicted above brackets. (C) Photograph of newborn Ppp1r15b+/+, Ppp1r15b+/−, and Ppp1r15b−/− mice. (D) Immunoblot of eIF2(αP), total eIF2α, and GADD34 from cytoplasmic extracts prepared from the liver of newborn mice of the indicated genotype.
Fig. 3.
Fig. 3.
Impaired erythropoeisis in Ppp1r15b−/− mice. (A) Hematological profile of e18.5 embryos. The mean ± SD for the following parameters are displayed: HCT, hematocrit (the ratio of volume of all blood cells to whole blood); HGB, hemoglobin concentration in whole blood; RBC, red blood cell count; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; RDW, red blood cell distribution width (a measure of size heterogeneity); PLT, platelet count; MPV, mean platelet volume. P values for paired Student's t test comparing both genotypes are displayed. Ppp1r15b+/− (n = 10), Ppp1r15b−/− mice (n = 9). (B) Photograph of Wright's stained blood smears from e18.5 wild-type and Ppp1r15b−/− mice. Barbed arrows indicate nucleated cells; plain arrows indicate echinocytic (deformed) cells. (C) Photomicrograph of hematoxylin- and eosin-stained liver sections of e16.5 wild-type and Ppp1r15b−/− mice. (Magnification: Upper, 5×; Lower, 20×.) (D) Dual-color FACS analysis of freshly isolated fetal liver cells from Ppp1r15b+/+ and Ppp1r15b−/− e15.5 and e16.5 embryos stained with antisera to the erythroid-specific marker Ter119 and CD71 (the transferin receptor).
Fig. 4.
Fig. 4.
Rescue of Ppp1r15b−/− by an eIF2aS51A mutation that eliminates the phosphorylation site on eIF2α. (A) Photo micrograph of e18.5 embryos isolated from intercrosses of eIF2a+/S51A; Ppp1r15b+/− mice (e+/A; 15b+/−). Genotypes are indicated below each embryo with bold type to highlight those embryos with homozygous mutations in one or both genes. The arrowheads indicate eIF2a+/A; 15b+/−; the narrow arrows indicate eIF2a+/A; Ppp1r15b−/− or eIF2a+/+; Ppp1r15b−/−; and the bold arrow indicates the double eIF2aA/A; Ppp1r15b−/− individuals from one litter. (B) Mean weights + SD at e18.5 observed among progeny of 10 litters of e+/A; 15b+/− intercrosses. The number of individuals of each genotype scored (left to right) is n = 2, 12, 3, 3, 12, 5, 12, 19, 15. The P value for the one-sided t test assessing the rescue of the Ppp1r15b−/− weight defect by the eIF2aA/A genotype is indicated above the bracket. (C) Mean red blood cell counts + SD observed from the embryos shown in (B). The P value for the one-sided t test assessing the rescue of the Ppp1r15b−/− RBC defect by the eIF2aA/A genotype is indicated above the bracket.
Fig. 5.
Fig. 5.
Compound homozygous Ppp1r15aΔC/ΔC; Ppp1r15b−/− embryos fail to develop past the preimplantation period. (A) Genotypes of embryos isolated at day e12.5 from matings of Ppp1r15a+/ΔC; Ppp1r15b+/− mice with the number and percent of expected (#Ex, %Ex) and observed (#Ob, %Ob) genotypes indicated. (*Probability of finding no compound Ppp1r15aΔC/ΔC; Ppp1r15b−/− progeny among 97 individuals by chance; P = 5.78 × 10−25.) (B) Photograph of e3.5 blastocysts isolated from matings of Ppp1r15aΔC/ΔC; Ppp1r15b+/− mice cultured for 2.5 days before genotyping.
Fig. 6.
Fig. 6.
Rescue of the early lethality of compound mutant Ppp1r15aΔC/ΔC; Ppp1r15b−/− embryos by the S51A mutation that eliminates the phosphorylation site on eIF2α. (A) Table of genotypes observed in e17.5 embryos isolated from intercrosses of Ppp1r15aΔC/ΔC; eIF2a+/S51A; Ppp1r15b+/− parents with the number and percent of expected (#Ex, %Ex) and observed (#Ob, %Ob) indicated. (*Probability of all 7 Ppp1r15aΔC/ΔC; Ppp1r15b−/− embryos inheriting an eIF2aA/A genotype by chance; P = 6.1 × 10−05.) (B) Photomicrograph of e17.5 embryos isolated from intercrosses of Ppp1r15aΔC/ΔC; eIF2a+/S51A; Ppp1r15b+/− parents (abbreviated 15aΔC/ΔC; e+/A; 15b+/−). Genotypes are indicated below each embryo. (C) Photograph of crystal violet-stained mouse embryonic fibroblasts of the indicated genotype 10 days after transduction with a Puror-marked retrovirus expressing either a wild-type or a S51A mutant allele of human eIF2α and selection with puromycin.
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