Fig. 1. Phosphorylation of eIF2Bε in vivo and in vitro. (A) HEK293 cells transfected with empty vector (lane 1) or a vector encoding HA-tagged rat eIF2Bε (lane 2) were metabolically labelled with [³²P]orthophosphate and then extracted as described in Materials and methods. Endogenous eIF2Bε (lane 1, 100 µg of lysate protein) or HA-tagged eIF2Bε (lane 2, equivalent to 5 µg of lysate protein) was immunoprecipitated and analysed by SDS–PAGE followed by autoradiography. Labelled arrows on the left show the migration positions of subunits of human eIF2B, apart from ‘X’ which denotes an additional polypeptide co-immunoprecipitated with eIF2B, probably ABC50. Labelled arrows on the right indicate subunits of eIF2. Arrowheads indicate radiolabelled bands. The position of HA-tagged eIF2Bε is also indicated. (B) Immunoblots of HEK293 cells transfected with empty vector (1) or vector for HA-eIF2Bε (2) developed with an antiserum for eIF2Bε. (C and D) Two-dimensional mapping of peptides from HA-eIF2Bε labelled in vivo. HA-tagged rat eIF2Bε was expressed in HEK293 cells that were metabolically labelled with [³²P]orthophosphate. eIF2Bε was isolated by immunoprecipitation and then digested with Asp-N (C) or trypsin (D), and the resulting phosphopeptides were analysed by two-dimensional mapping. The polarity of electrophoresis (+/–) and the direction of chromatography (arrow) are shown. The narrow arrowhead marks the origin and the diamond the position of the dinitrophenyllysine marker. (C) and (D) are annotated indicating the terminology used to refer to the peptides in the text.

Fig. 3. Analysis of peptides from eIF2Bε phosphorylated by GSK3. eIF2Bε made in insect cells was radiolabelled in vitro using GSK3. (A) This was then subjected to digestion by Asp-N and the resulting peptides were analysed by reverse-phase (C₁₈) HPLC. The dotted line indicates the percentage (v/v) of CH₃CN. (B and C) The material in fractions 65 (B) or 74 (C) from the HPLC was analysed by two-dimensional mapping. (D and E) These fractions (as indicated) were subjected to solid-phase sequencing, and release of radioactivity was determined at each cycle. The sequences of the peptides are also displayed (as shown by the release of radioactivity and the masses of the peptides determined by mass spectrometry, see text: Sp denotes phosphoserine). (F and G) Two-dimensional analysis of tryptic peptides from either bacterially expressed rat eIF2Bε phosphorylated in vitro by DYRK2 (F) or rat eIF2Bε expressed in insect cells and phosphorylated by GSK3 (G). Peptides t1 and t3 (see text) are indicated.

Fig. 5. Phosphorylation of eIF2Bε by CK2 and CK1. (A and B) GST–eIF2Bε made in E.coli was phosphorylated in vitro by CK2 and then subjected to cleavage with trypsin. The resulting peptides were analysed by reverse-phase chromatography (A) or two-dimensional mapping (B) (additional mapping data, not shown, identified the individual peptides with either the first or second peak observed on HPLC, as indicated). (C) The material in fraction 46 was subjected to one cycle of gas-phase Edman degradation followed by 10 cycles of solid-phase sequencing, release of radioactivity being monitored during the latter. (D) Full-length eIF2Bε (FL) and truncation mutants lacking the last 33 (Δ33) or 63 (Δ63) residues were expressed in E.coli and purified. A 0.5 µg aliquot of each was then incubated with [γ-³²P]ATP and CK2, as indicated. Samples were then analysed by SDS–PAGE and autoradiography. (E) HEK293 cells were transfected with vectors encoding wild-type eIF2Bε or a double point mutant, S712/713GA, each with HA tags. After metabolic labelling of cells with [³²P]orthophosphate, immunoprecipitation, SDS–PAGE and tryptic digestion, peptides were analysed by two-dimensional mapping. Only the region around the origin and including peptide t2 is shown. (F) Alignment of the extreme C-termini of eIF2Bε for a range of species (Dm, Drosophila melanogaster; Rn, Rattus norvegicus; Sc, Saccharomyces cerevisiae; At, Arabidopsis thaliana; Ce, Caenorhabditis elegans) showing the conservation of potential CK2 sites (S in bold) and the highly conserved WL motif (bold). Numbers are those of the final residue in the polypeptide. (G) GST–eIF2Bε was phosphorylated by CK1 in vitro and digested with Asp-N. Resulting peptides were displayed by two-dimensional mapping.

Fig. 7. Analysis of phosphorylation of endogenous eIF2Bε. (A and B) Endogenous eIF2Bε from metabolically labelled HEK293 cells was digested with Asp-N or trypsin, and peptides were resolved on two-dimensional maps. (C) Schematic summary of the peptides in (A) and (B). (D) Peptide A1 was digested with trypsin and the products were analysed by two-dimensional mapping. (E and F) Peptide a2 (Figure 1C) was digested with trypsin. Two-dimensional maps of the starting peptide (E) and the tryptic products (F) are shown. For details of the annotation see text.

Fig. 9. Effects of point mutations on the interaction of eIF2Bε with eIF2 in vivo and on eIF2Bε activity. (A and B) HEK293 cells were transfected with vectors encoding wild-type eIF2Bε, the indicated point mutants, or vector alone, as a negative control. eIF2Bε was then immunoprecipitated from cell lysates using anti-HA antibodies and analysed by SDS–PAGE followed by immunoblotting with antibodies to either eIF2α or HA (as a loading control). (C) eIF2Bε was immunoprecipitated from transfected HEK293 cells using anti-HA antibodies and then used in eIF2B activity assays, as described in Materials and methods. Immunoprecipitations were carried out in triplicate, two for the exchange assay and one for SDS–PAGE and immunoblotting using anti-HA antibodies to check for equal levels of eIF2Bε (upper panel). The results show the average of two independent experiments ± SD and are expressed as the percentage of [³H]GDP released from complexes with eIF2, corrected for the low activity seen with samples from cells transfected with the empty vector (lower panel). Similar data were obtained in four sets of experiments.

Fig. 2. Phosphorylation of eIF2Bε in vitro. (A) eIF2Bε expressed in E.coli (as a GST fusion protein) was incubated with CK1, CK2 or GSK3 for 20 min in the presence of [γ-³²P]ATP, and samples were analysed by SDS–PAGE and autoradiography. (B) eIF2Bε (0.5 µg) expressed in E.coli (as a GST fusion protein) or Sf21 cells (His6 tagged, as indicated) was incubated with GSK3 for the indicated times (min) in the presence of [γ-³²P]ATP, and samples were analysed by SDS–PAGE and autoradiography. (C) A 0.5 µg aliquot of eIF2Bε expressed in Sf21 cells (lane 1) or E.coli (as a GST fusion protein;lane 2), as indicated, was analysed by SDS–PAGE and immunoblotting using an antibody which recognizes eIF2Bε when phosphorylated at Ser539 (the arrow shows the position of eIF2Bε). The dotted arrow shows the migration position of GST–eIF2Bε.

Fig. 4. Phosphorylation of eIF2Bε(S535A) and eIF2Bε(S539N) in vivo. HEK293 cells were transfected with vectors encoding each of these mutant proteins or wild-type (wt) eIF2Bε. (A) Cells were extracted and analysed by SDS–PAGE and immunoblotting using phosphospecific antisera or anti-HA (loading control). (B and C) Transfected cells were metabolically labelled with [³²P]orthophosphate and eIF2Bε proteins were isolated by immunoprecipitation/SDS–PAGE. They were then digested with Asp-N and peptides displayed on two-dimensional maps. Labelled arrows in (B) mark the additional peptides termed a8/9 seen in this map, and the labelled rings show the expected positions of the absent peptides a2/3. In (C), the positions of the absent peptides a8/9 are indicated by rings.

Fig. 6. Analysis of in vivo phosphorylation of S46A, S461A and S464A mutants of eIF2Bε. HEK293 cells were transfected with vectors encoding wild-type eIF2Bε or point mutants as indicated. After in vivo labelling, isolation of eIF2Bε by immunoprecipitation (anti-HA) and SDS–PAGE, eIF2Bε polypeptides were digested with Asp-N and the resulting peptides were analysed on two-dimensional maps.

Fig. 8. A region near the C-terminus of eIF2Bε modulates phosphorylation of Ser535 by GSK3. (A) HEK293 cells were transfected with vectors as indicated (‘vector’ = empty vector). Cell lysates were analysed by SDS–PAGE and immunoblotting using phosphospecific antisera or anti-HA (loading control) as indicated. (B and C) Full-length (FL) GST–eIF2Bε and the two truncation mutants (Δ33 and Δ63) were expressed in E.coli and purified. They were incubated with DYRK2 and ATP-MgCl₂ for the times indicated before analysis by SDS–PAGE and either autoradiography (B; incubations included [γ-³²P]ATP) or immunoblotting using anti-(P)Ser539 (C). (D) FL and truncated GST–eIF2Bε proteins were pre-incubated with DYRK and unlabelled ATP for 30 min. GSK3 and[γ-³²P]ATP were then added and incubations continued for the times shown, after which samples were analysed by SDS–PAGE and autoradiography. (E) Alignment of sequences of the GSK3-interacting region of axin and part of the region between residues 665 and 679 from mammalian eIF2Bε. Identical residues are shown in bold while similar ones are underlined. Hs, Homo sapiens, Mm, Mus musculus, Rn, Rattus norvegicus. (F) HEK293 cells were transfected with vectors encoding FL HA-eIF2Bε, the Δ63 mutant or empty vector and, after immunoprecipitation of GSK3α and β, samples were subjected to SDS–PAGE followed by immunoblotting with either anti-HA or an antiserum which recognizes GSK3α and β (as a loading control).

Fig. 10. eIF2Bε only displays significant activity when incorporated into eIF2B complexes. Extracts from HEK293 cells transfected with the vector encoding HA-tagged eIF2Bε (wild-type) were subjected to ion-exchange chromatography on Mono-Q as described in the Supplementary material. (A) The absorbance and conductivity traces from a typical chromatogram (representative of >15 runs performed). The absorbance (A₂₈₀, AU = absorbance units) and conductivity (salt gradient) traces are shown. (B) Selected fractions (numbers shown) were subjected to SDS–PAGE and western blotting using the indicated antisera. The elution positions of eIF2, eIF2B complexes and HA-eIF2Bε free of other eIF2B subunits are shown. (C) Selected fractions were subjected to immunoprecipitation with anti-HA and immunoprecipitates were analysed by SDS–PAGE and western blotting using anti-HA, anti-eIF2Bγ and anti-eIF2Bδ antibodies as indicated. Note that these data are from a different run from those in (A) and (B), so fraction numbers are shifted ‘rightwards’ by about half of one fraction. (D) Selected fractions were subjected to immunoprecipitation with anti-HA followed by a standard eIF2B activity assay using eIF2⋅[³H]GDP as substrate. Data are shown as the percentage of total bound [³H]GDP released during the 15 min assay and are typical of six sets of data for 293 cells expressing wild-type HA-eIF2Bε.