G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases
- University of Cambridge, United Kingdom
Figures
Figure 1 with 2 supplements
A core functional domain in the C-terminus of PPP1R15 family members inhibits the integrated stress response (ISR).
(A) Cartoon depicting the domain organization of mammalian and viral PPP1R15 proteins. The N-terminal membrane-interaction domain is depicted by a broad line, the repeats, found in the …
Figure 1—figure supplement 1
An extended alignment of PPP1R15 regulatory subunit family members reveals that the conservation of amino acid sequence is confined to a ∼70 residue segment.
Alignment of C-terminal sequences of PPP1R15B from mouse (Q8BFW3), rat (D3ZP67), human (Q5SWA1) and cow (E1BKX2), PPP1R15A from frog (Q9W1E4), mouse (P17564), rat (Q6IN02), cow (Q2KI51), human …
Figure 1—figure supplement 2
Disordered and ordered regions predicted in PPP1R15 regulatory subunits.
Disorder prediction (based on DISOPRED [Ward et al., 2004]) along the primary sequence of mouse PPP1R15A (UNIPROT P17564, ‘A’) and human PPP1R15B (UNIPROT Q5SWA1, ‘B’), superimposed on a schema of …
Figure 2 with 1 supplement
PPP1R15 engages the PP1 catalytic subunit through multiple contacts.
(A) UV protein absorbance trace of a PPP1R15B(630–701)-PP1G(7–300) binary complex expressed in bacteria and resolved by size-exclusion chromatography. The indicated fractions from the chromatogram …
Figure 2—figure supplement 1
Variable occupancy of the M1 metal binding site in the PP1G-PPP1R15B binary complexes.
(A) Detail of the PP1G active site in the binary complexes constituted with different PPP1R15B chain lengths: 631–660 (magenta), 631–669 (cyan), 631–684 (green). Note an identically-positioned metal …
Figure 3 with 2 supplements
The PPP1R15B-PP1 binary complex lacks substrate specificity.
(A) UV protein absorbance trace of a PPP1R15B-PP1G complex co-expressed in bacteria as a GST-PPP1R15B(631–701)-MBP fusion protein alongside untagged PP1G(7–300), purified by glutathione affinity …
Figure 3—figure supplement 1
The PPP1R15A-PP1 binary complex also lacks selectivity towards the specific (eIF2aP) substrate over the non-specific (GSTP) substrate.
Images of Coomassie-stained Phos-Tag SDS-PAGE in which phosphorylated and de-phosphorylated non-specific substrate and product (GSTP and GST0, upper panel) and the specific substrate and product …
Figure 3—figure supplement 2
Phosphatase activity of the purified bacterially-expressed complexes is dependent on the presence of both the regulatory (PPP1R15A) and catalytic (PP1G) subunits.
(A) Image of Coomassie-stained Phos-Tag SDS-PAGE in which phosphorylated (GSTP) and non-phosphorylated (GST0) GST have been resolved. Escalating concentrations of the indicated complexes purified …
Figure 4 with 3 supplements
An activity in tissue lysate that endows the PPP1R15B-PP1G binary complex with specificity towards eIF2aP can be mimicked by pure G-actin.
(A) Images of Coomassie-stained Phos-Tag SDS-PAGE in which a phosphorylated non-specific substrate and de-phosphorylated product (GSTP and GST0, upper panel) and the specific substrate and product …
Figure 4—figure supplement 1
Substrate concentration dependence of the velocity of eIF2aP dephosphorylation by the PPP1R15B-PP1G-Actin ternary complex.
(A) Image of Coomassie stained Phos-Tag gels of eIF2aP dephosphorylation reactions by a ternary complex constituted of PPP1R15B-PP1G (5 nM) and G-actin (1 µM). Time points of 3, 5, 12, and 25 min …
Figure 4—figure supplement 2
Estimation of the concentration of eIF2a by quantitative immunoblotting of HEK293T cell lysates.
(A) Plot of the relationship between fluorescent intensity of the eIF2a signal in immunoblot to the mass of recombinant eIF2a (in pico moles) applied to the blot. An image of the immunoblot is …
Figure 4—figure supplement 3
Incorporation of G-actin inhibits PPP1R15B-PP1G phosphatase activity directed against non-specific substrates.
(A) Images of Coomassie stained Phos-Tag gels of a time course (2.5–40 min) of dephosphorylation reactions of phosphorylase A (PYGMP, upper panel) or GSTP (lower panel) (both at 1 µM) by a complex …
Figure 5 with 1 supplement
G-actin activates the eIF2aP-directed phosphatase activity of both PPP1R15A and PPP1R15B-containing binary complexes with an EC50 in the submicromolar range.
(A) Trace of the velocity of eIF2aP dephosphorylation by 16 nM PPP1R15A-PP1G (in red) and 68 nM PPP1R15B-PP1G (in blue) binary complexes in the presence of the indicated concentrations of G-actin …
Figure 5—figure supplement 1
An irrelevant protein, bovine serum albumin, has no effect on the dephosphorylation of eIF2a by the PPP1R15B-PP1G binary complex.
Trace of the velocity of eIF2aP dephosphorylation by 68 nM PPP1R15B-PP1G binary complexes in the presence of the indicated concentrations of G-actin (grey) or bovine serum albumin (BSA, red) both …
Figure 6 with 1 supplement
Mutations in conserved residues of the PPP1R15 core functional domain enfeeble its activation by actin.
(A) Traces of the velocity of eIF2aP dephosphorylation by wildtype and the indicated mutant PPP1R15B-PP1G complexes in the presence of the indicated concentrations of actin. (B) As in ‘A’ but with …
Figure 6—figure supplement 1
Mutations in conserved residues of PPP1R15A compromise function in vivo.
(A) Dual channel flow cytometric analysis of CHOP::GFP cells transfected with mCherry, fusion of wildtype PPP1R15A to mCherry or fusion of the indicated mutant derivatives of PPP1R15A to mCherry. …
Figure 7 with 2 supplements
G-Actin joins PPP1R15B-PP1G binary complexes to extend its active site-facing surface.
(A) UV protein absorbance trace of a PPP1R15B(631–701)-PP1G(7–323)-G-actin complex assembled from the bacterially-expressed binary complex and rabbit muscle G-actin and resolved by size-exclusion …
Figure 7—figure supplement 1
G-Actin also joins PPP1R15 A-PP1G binary complexes to form a stable ternary complex.
UV protein absorbance trace of a PPP1R15A(539–614)-PP1G(7–323)-G-actin complex assembled from the bacterially-expressed binary complex and rabbit muscle G-actin and resolved by size-exclusion …
Figure 7—figure supplement 2
A ternary complex of DNase I, G-actin, PP1G and PPP1R15A retains its eIF2aP-directed phosphatase activity.
(A) UV protein absorbance trace of a PPP1R15A (539–614)-PP1G(7–323)-G-actin and DNase I complex assembled from the bacterially-expressed binary complex, rabbit muscle G-actin and bovine pancreatic …
Figure 8 with 2 supplements
Mutation of residues predicted to affect substrate-enzyme binding enfeeble dephosphorylation by the selective ternary complex.
(A) High ambiguity driven protein docking by HADDOCK (Dominguez et al., 2003) model of yeast eIF2a's regulatory N-terminus (PDB: 1Q46) with substrate phospho-S51 docked at the active site of the …
Figure 8—figure supplement 1
The wildtype and mutant eIF2aP substrates have indistinguishable retention profiles on size exclusion chromatography.
Shown are plots of UV protein absorbance traces of the indicated eIF2aP substrates discussed in the results section and utilized in the experiment shown in Figure 8. The bacterially-expressed …
Figure 8—figure supplement 2
Kinetic analysis of dephosphorylation reactions shown in Figure 8B.
(A) Plot of time-dependent dephosphorylation of the indicated substrates by the PPP1R15B-MBP-PP1-actin ternary complex (TC) and the PPP1R15B-MBP-PP1 binary complex (BC). (B) Plot of the logarithm of …
Author response image 1
Tables
Table 1
Structure parameters
| PP1G-PPP1R15B(631–660) | PP1G-PPP1R15B(631–669) | PP1G-PPP1R15B(631–684) | PP1G-PPP1R15B(631–701)-actin | |
|---|---|---|---|---|
| Data collection | ||||
| Synchrotron beamline | Diamond I02 | Diamond I02 | Diamond I03 | Diamond I04-1 |
| Space group | P21212 | P21212 | P41212 | C121 |
| Cell dimensions | ||||
| a,b,c; Å | 66.8, 67.89, 156.38 | 67.01, 67.86, 156.75 | 67.54, 67.54, 158.01 | 103.9, 149.9, 318.7 |
| α, β, γ; ⁰ | 90, 90, 90 | 90, 90, 90 | 90, 90, 90 | 90, 91.03, 90 |
| Resolution, Å | 51.26-1.61 (1.65-1.61) | 33.94-1.55 (1.58-1.55) | 51.34-1.85 (1.89-1.85) | 82.79-7.88 (8.08-7.88) |
| Rmerge | 0.084 (0.807) | 0.097 (0.737) | 0.094 (0.982) | 0.142 (0.680) |
| Rmeas | 0.101 (0.956) | 0.12 (0.927) | 0.107 (1.118) | 0.199 (0.953) |
| <I/σ (I)> | 12.5 (3.0) | 7.4 (1.8) | 9.7 (1.5) | 7.2 (1.6) |
| CC1/2 | 0.997 (0.746) | 0.995 (0.685) | 0.998 (0.914) | 0.980 (0.627) |
| Completeness, % | 99.8 (100) | 92.6 (99.9) | 100 (100) | 98 (99.2) |
| Redundancy | 6.3 (6.7) | 5.1 (4.9) | 7.8 (8.2) | 3.4 (3.5) |
| Refinement | ||||
| Rwork | 0.176 | 0.172 | 0.176 | 0.370 |
| Rfree | 0.203 | 0.203 | 0.222 | 0.400 |
| No. of reflections | 92584 | 96347 | 32078 | 5111 |
| No. of atoms | 5493 | 5701 | 2662 | 28185 |
| Average B-factors | 24.4 | 25.2 | 45.2 | 334 |
| Metal ion occupancies | Chain A: M2 0.95 | Chain A: M2 0.79 | Chain A: M2 0.76 | n/a |
| M1 0.25 | ||||
| Chain C: M2 0.99 | Chain C: M2 0.90 | |||
| M1 0.22 | ||||
| rms deviations | ||||
| Bond lengths (Å) | 0.006 | 0.006 | 0.011 | 0.0097 |
| Bond angles (⁰) | 1.044 | 1.054 | 1.221 | 1.271 |
| Ramachandran favoured region, % | 96.6 | 96.4 | 96.8 | 97.2 |
| Ramachandran outliers, % | 0 | 0 | 0 | 0 |
| MolProbity score(percentile) | 1.23 (98%) | 1.2 (98%) | 1.22 (98%) | 1.58 (100%) |
| PDB code | 4V0V | 4V0W | 4V0X | 4V0U |
Additional files
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Supplementary file 1
List of the plasmids used in this study, their unique lab identifier, lab name, description, PMID of the relevant reference (if available), figure in which they first appear and cognate label in figure legend.
- https://doi.org/10.7554/eLife.04871.026
