PPM1G dephosphorylates eIF4E to reduce selective mRNA translation.

doi:10.21203/rs.3.rs-2847028/v1

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PPM1G dephosphorylates eIF4E to reduce selective mRNA translation.

https://doi.org/10.21203/rs.3.rs-2847028/v1

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The mRNA 5’cap-binding eukaryotic translation initiation factor 4E (eIF4E) plays a major role in control of mRNA translation in health and disease. One mechanism of regulation of eIF4E activity is via phosphorylation of eIF4E by MNK kinases, which promotes the translation of a subset of mRNAs encoding pro-tumorigenic and pro-inflammatory proteins. Work on eIF4E phosphatases has been paltry. Here, we show that PPM1G is the phosphatase that dephosphorylates eIF4E. We describe the binding motif in PPM1G that shares an eIF4E binding site with eIF4G and 4E-binding proteins (4E-BPs). We show that PPM1G is a nucleocytoplasmic shuttling protein that dephosphorylates eIF4E in the cytoplasm. We demonstrate that PPM1G inhibits cell proliferation and the phospho-eIF4E-dependent synthesis of several proteins involved in cell proliferation, survival, and inflammatory response.

Biological sciences/Molecular biology/Translation

Biological sciences/Cell biology/Post-translational modifications/Phosphorylation

Biological sciences/Biochemistry/Proteins/Tumour-suppressor proteins

Canonical eukaryotic mRNA translation commences with the recognition of the mRNA 5’-m7GpppN (where m is a methyl group and N is any nucleotide) cap structure by the heterotrimeric eukaryotic translation initiation factor 4F (eIF4F) complex ¹. eIF4F is comprised of a cap-binding subunit eIF4E, a large scaffolding protein eIF4G and an ATP-dependent RNA helicase eIF4A. eIF4G recruits the 43S pre-initiation complex (PIC) to the mRNA, while eIF4A unwinds the secondary structure of the mRNA 5’UTR to facilitate PIC loading and subsequent scanning until the initiation codon is engaged ^2–6.

eIF4E is the least abundant translation initiation factor and thus plays a pivotal role in control of translation initiation ^{7, 8}. eIF4E can be sequestered from eIF4G by 4E-BPs (eIF4E-binding proteins) which constitute a family of 3 proteins (4E-BP1, 2 and 3) in mammals ^9–12. In response to energy, nutrition and growth factors, the mammalian (mechanistic) target of rapamycin complex1(mTORC1) sequentially phosphorylates 4E-BP at multiple sites in a hierarchical order (T37, T46, T70 and S65) to cause the release of eIF4E, thereby allowing eIF4E-eIF4G association to reconstitute the eIF4F complex and subsequent cap-dependent mRNA translation initiation ^{13, 14}.

eIF4E is phosphorylated by the mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (MNKs)^15–19. eIF4G serves as a docking site for MNKs which phosphorylate eIF4E at a single site, serine 209 (S209) ^16–18. MNK2 exhibits a high basal activity and maintains eIF4E phosphorylation under physiological conditions while MNK1 has low basal activity and phosphorylates eIF4E in response to the Ras pathway activation or under stress ^{16–18, 20}. eIF4E phosphorylation promotes the synthesis of a subset of proteins (e.g., MMP-3, MMP-9, SNAIL, Mcl1, Bcl-2, Cyclin D1, MYC, Ccl2, Ccl7, PD-L1, IκBα and RANTES) involved in cell invasion, survival, oncogenesis and inflammatory response ^21–30.

mTOR and MNKs activities are frequently upregulated in cancer to promote the synthesis of pro-tumorigenic proteins by elevating 4E-BPs and eIF4E phosphorylation respectively. Thus, preventing their phosphorylation by mTOR and/or MNK inhibition can potentially lead to cancer therapies ^{2, 31–34}. While there are several mTOR inhibitors (rapamycin analogs; rapalogs) in the clinic for cancer and others are in clinical trials, there is only one MNK inhibitor (Tomivosertib) which is tested in clinical trials for solid and hematological cancers ³⁵. mTOR inhibitors have shown only limited benefit in the clinic³⁶.

PPM1G (Protein Phosphatase, Mg2+/Mn2 + Dependent 1G; also called PP2C_γ) is a member of the metal-ion dependent serine/threonine protein phosphatase PPM (or PP2C) family. PPM1G is distinct from other protein phosphatases in that it contains a long acidic region which is important for its binding to substrates and regulators ³⁷. PPM1G is essential for mouse embryonic viability³⁸. Low expression of PPM1G is associated with alcohol use disorder^{39, 40} while induction of PPM1G causes neuronal death in Parkinson disease⁴¹. PPM1G promotes the activity of viral and cellular transcription factors such as HIV Tat and NF-κB ^42–44. PPM1G regulates alternative splicing ^45–47, inflammatory responses and cancer development ^{42, 43, 47–53}. PPM1G was reported to dephosphorylate 4E-BP1⁵⁴, which is the predominant eIF4E binding protein in most tissues, with brain as a notable exception⁵⁵. We therefore investigated whether PPM1G will prevent eIF4E-dependent mRNA translation by dephosphorylating 4E-BP1. Unexpectedly, we found that PPM1G dephosphorylates eIF4E but not 4E-BP1. We showed the importance of PPM1G-phospho-eIF4E pathway in the control of the expression of several proteins involved in cell proliferation, survival, and inflammation.

PPM1G dephosphorylates eIF4E but not 4E-BP1.

We studied the tumor suppressor activity of the phosphatase PPM1G as it was reported that PPM1G dephosphorylates 4E-BP1 ⁵⁴. We expected that ectopic expression of PPM1G would reduce the phosphorylation 4E-BP1 and prevent cap-dependent protein synthesis. Surprisingly, in HEK293H cells, PPM1G expression strongly diminished the phosphorylation of eIF4E but surprisingly not the phosphorylation of 4E-BP1 (Fig. 1A). Corroborating the latter results, knockout (KO) of PPM1G promoted the phosphorylation of eIF4E without altering 4E-BP1 phosphorylation (Fig. 1B). Moreover, the increase in eIF4E phosphorylation in PPM1G KO cells was a specific outcome of PPM1G deletion since ectopic expression of PPM1G in PPM1G KO cells ameliorated eIF4E phosphorylation (Fig. 1C). Notably, both PPM1G overexpression and KO did not affect MNKs’ phosphorylation at sites which govern their activities^{15, 56, 57} (Fig. 1D and 1E), indicating that PPM1G directly dephosphorylates eIF4E. In strong support of this conclusion, HA-tagged eIF4E, but not HA-tagged 4E-BP1, became dephosphorylated in an in vitro phosphatase assay (Fig. 1F).

We next investigated whether PPM1G associates with eIF4E in cells. Immunoprecipitated FLAG-tagged PPM1G co-purified with endogenous eIF4E, but not 4E-BP1 (Fig. 1G). In addition, the eIF4E homologous protein-4EHP, which is 30% identical to eIF4E⁵⁸, did not co-purify with FLAG-tagged PPM1G (Fig. 1G). Furthermore, FLAG-tagged wildtype eIF4E, as well as its non-phosphorylatable (eIF4E_S209A) and phospho-mimetic mutants (eIF4E_{S208D; S209D}), equally associated with endogenous PPM1G (Fig. 1H). Thus, PPM1G associates with eIF4E regardless of its phosphorylation status.

It was reported that the protein phosphatase 2A catalytic subunit (PP2A_Cα) dephosphorylates eIF4E⁵⁹. However, eIF4E did not bind to PP2Ac_α (Figure S1A). Moreover, overexpression of the PP2Ac_α supressed the phosphorylation of eIF4E kinase MNK2 and its upstream kinase ERK activity (Figure S1B), while deletion of PP2Ac_α promoted their phosphorylation (Figure S1C). Thus, PP2A_Cα may promote eIF4E dephosphorylation indirectly. Taken together, the results indicate that PPM1G directly dephosphorylates eIF4E but not 4E-BP1, while PP2A decreases eIF4E phosphorylation by targeting its upstream kinases.

PPM1G contains two potential 4E-BMs, but only one of which mediates eIF4E binding.

To delineate the region of PPM1G that interacts with eIF4E, we sought a potential 4E binding motif (4E-BM) in the PPM1G sequence. eIF4E binding proteins such as eIF4G, 4E-BPs and 4E-T contain a canonical 4E-BM: YxxxxLΦ, where Y is tyrosine, L is leucine, x represents any amino acid and Φ is a hydrophobic residue ^60–63. PPM1G contains two closely spaced YxxxxL consensus sequences without the Φ (Fig. 2A), like the 4E-BM (YxxxxMK) identified in Drosophila 4E-BP ⁶⁴. The first Y₇₀xxxxL₇₅ sequence (hereafter referred to as 4E-BM1) of PPM1G is conserved in several PPM family phosphatases while the second Y₈₅xxxxL₉₀ sequence (4E-BM2) is unique to PPM1G (Fig. 2A). We next investigated the effect of wild-type PPM1G, its catalytically inactive mutant (PPM1G_D496A) ⁴⁵, a 4E-BM1 mutant (PPM1G_Y70A) and a 4E-BM2 mutant (PPM1G_Y85A) on eIF4E phosphorylation. PPM1G and the 4E-BM1 mutant, but neither the inactive mutant nor the 4E-BM2 mutant, caused the dephosphorylation of eIF4E (Fig. 2B), demonstrating that the binding site for eIF4E resides in 4E-BM2. Moreover, although the homologous family member PPM1A contains the 4E-BM1 (Fig. 2A), it did not associate with eIF4E in cells (Fig. 2C). According to the predicted PPM1G structure from the web server RaptorX ⁶⁵, 4E-BM2 is surface exposed while 4E-BM1 is buried in the protein core (Figure S2A), which explains the finding that only 4E-BM2 interacts with eIF4E. Importantly, 4E-BM2 is conserved in all PPM1G orthologues (Figure S2B). Mutation of both Y85 and L90 residues of the 4E-BM2 into alanines (hereinafter referred to as YL_mut) caused a dramatic decrease in PPM1G’s binding to eIF4E and its capacity to dephosphorylate eIF4E in HEK293H cells (Fig. 2D). We next investigated whether PPM1G directly binds to eIF4E through its 4E-BM2 by using bacterially purified GST-tagged eIF4E to pull down bacterially purified HIS-tagged GFP, PPM1G and PPM1G-YL_mut which were analyzed by SDS/polyacrylamide gel electrophoresis (Figure S2C). Only His-PPM1G associated with GST-eIF4E, demonstrating that the 4E-BM2 is required for direct PPM1G binding to eIF4E (Fig. 2E).

The 4E-BM2 of PPM1G is highly specific for eIF4E binding.

To examine whether 4E-BM2 is required only for the interaction with eIF4E as opposed to other PPM1G interacting proteins, we examined the binding of PPM1G and PPM1G-YL_mut to two established PPM1G interacting proteins: B56δ and α-catenin ⁴⁸. YL_mut reduced ~ 85% PPM1G’s binding to eIF4E without affecting its binding to B56δ and α-catenin (Fig. 3A). We next examined whether the YL_mut affects PPM1G’s catalytic activity. PPM1G was reported to promote the dephosphorylation of α-catenin in breast cancer cell lines ⁴⁸, which was not reproduced in HEK293H cells (Fig. 3A) Therefore, FLAG tagged GFP, PPM1G and PPM1G-YL_mut were purified from HEK293H cells (Figure S3A) and a phosphatase assay was performed. PPM1G-YL_mut retained ~ 50% of the dephosphorylation activity of wild type PPM1G towards eIF4E in vitro, because of its reduced binding to eIF4E (Figure S3B). Additionally, both PPM1G and PPM1G-YL_mut could dephosphorylate α-catenin, supporting the conclusion that the YL_mut does not abolish PPM1G activity (Figure S3C).

eIF4E residues V69, W73 and L135 are required for PPM1G binding.

We next investigated which residues of eIF4E are required for PPM1G binding. We first modeled the eIF4E and PPM1G docking site using the programme ClusPro 2.0 and noted three residues (V69, W73 and L135) that are predicted to interact with the Y85 and L90 residues of 4E-BM2 of PPM1G via hydrogen bonds (Figure S3D). Strikingly, the three residues on eIF4E are the same to those contacted by other eIF4E partners :4E-BP1, eIF4G and 4E-T ^{62, 64, 66}. Mutation of the three residues into alanines (hereinafter referred to as VWL_mut) resulted in ~ 80% decrease in eIF4E expression (Fig. 3B, left panel) most probably due to enhanced degradation ⁶⁷. We thus increased the input protein (6mg) to saturate the anti-FLAG agarose-affinity gel (15ul) in the immunoprecipitation experiment to obtain a comparable amount of immunoprecipitated FLAG-eIF4E and FLAG-eIF4E-VWL_mut (Fig. 3B, right panel). In contrast to FLAG-eIF4E, FLAG-eIF4E-VWL_mut failed to associate with either PPM1G or 4E-BP1 (Fig. 3B), indicating that the VWL residues are essential for the interaction of eIF4E with its partners. To rule out the possibility that the VWL_mut did not disrupt the binding of eIF4E with its partners because of denaturation, we investigated the binding of FLAG-eIF4E-VWL_mut to Angel1, which is a member of the CCR4 deadenylase family and eIF4E interactor ⁶⁸. Angel1 contains a canonical 4E-BM which is required for eIF4E binding but does not compete with 4E-BP1 for the same residues on eIF4E ⁶⁸. Angel1 associated with FLAG-eIF4E and FLAG-eIF4E-VWL_mut to the same extent (Fig. 3B, left panel), indicating that the VWL residues are only required for a subset of 4E-BM containing eIF4E interactors. As both 4E-BP1 and PPM1G interact with the VWL residues of eIF4E, we investigated whether 4E-BP1 activation precludes eIF4E-PPM1G interaction. FLAG-tagged PPM1G was purified from either PP242 ⁶⁹ treated or untreated HEK293H cells. PP242 treatment led to the dephosphorylation of 4E-BP1 which disrupted ~ 90% PPM1G and eIF4E interaction without affecting the binding of PPM1G with B56δ and α-catenin (Fig. 3C), demonstrating that 4E-BP1 activation by PP242 specifically prevents the PPM1G-eIF4E interaction. As cap-bound eIF4E is associated with eIF4G, it is anticipated that PPM1G could not associate with the cap-bound eIF4E. As expected, in a m7GDP (cap analog)-agarose pull-down assay experiment, eIF4G, but not PPM1G, were pulled down with cap-bound eIF4E (Figure S3E). Taken together, PPM1G shares eIF4E binding residues with eIF4G and 4E-BP1.

PPM1G is a nucleocytoplasmic shuttling protein that dephosphorylates eIF4E in the cytoplasm.

eIF4E is mainly cytoplasmic and partially (~ 30%) nuclear ⁷⁰ while PPM1G is predominantly nuclear with sparse cytoplasmic localization ⁴⁸. Thus, we investigated in which cellular compartment PPM1G dephosphorylates eIF4E. PPM1G contains a bipartite nuclear localization signal (NLS) at its C-terminal end ⁷¹. By mutating the first two residues of the NLS, lysine 519 and arginine 520, into stop codons (Figure S4A), we generated a truncated PPM1G (PPM1G_ΔNLS) which predominantly localized to the cytoplasm (Figure S4B). As compared to wildtype PPM1G, PPM1G_ΔNLS expression further decreased ~ 50% eIF4E phosphorylation in HEK293H cells (Fig. 4A), indicating that the cytoplasmic PPM1G promotes the dephosphorylation of eIF4E. In addition, PPM1G contains a canonical nuclear export signal (NES) consensus sequence (Φ–X2,3–Φ–X2,3–Φ–X–Φ, where Φ represents L, I, V, M, or F and X2,3 represents any two or three amino acids) ⁷²: MDGRVNGGLNL (Figure S4C). It is thus highly likely that PPM1G is shuttling between the nucleus and the cytoplasm. To examine this, a Fluorescence Loss in Photobleaching (FLIP) assay was performed to compare the nucleocytoplasmic mobility of PPM1G-GFP with the PPM1G_NESmut-GFP. Using a laser to bleach the GFP signal in a relatively small region of interest (ROI) in the cytoplasm, we observed a progressive decay of the fluorescence of nuclear PPM1G-GFP over time, indicating that PPM1G- GFP is continuously transported into the cytoplasm where the GFP signal is bleached (Fig. 4B). Compared to the PPM1G-GFP, PPM1G_NESmut-GFP fluorescence decay in the nucleus is significantly delayed during the FLIP assay, indicating that the predicted NES is required for the efficient nuclear export of PPM1G (Fig. 4B). The NES mutant form of PPM1G showed a ~ 50% reduction in its ability to dephosphorylate eIF4E compared to wild type PPM1G (Fig. 4C), bolstering the conclusion that the nuclear export of PPM1G into the cytoplasm is required for eIF4E dephosphorylation. In summary, our results demonstrate that PPM1G nuclear export and cytoplasmic localization is required for efficient dephosphorylation of eIF4E.

PPM1G inhibits translation of mRNAs encoding pro-proliferation, pro-survival, and pro-inflammation proteins.

We next investigated whether PPM1G interdicts mRNA translation. Ectopic expression of PPM1G had no effect on the polysome profile in HEK293H cells (Fig. 5A), indicating that PPM1G does not affect global mRNA translation. However, we expected that PPM1G will decrease phospho-eIF4E-dependent mRNA translation by suppressing eIF4E phosphorylation. Indeed, Mcl-1 and Cyclin D1 expression was reduced by PPM1G, but not PPM1G-YL_mut, while in contrast MMP3 and Bcl-2 synthesis was enhanced in PPM1G KO cells (Fig. 5B and 5C).

The MNKs/phospho-eIF4E pathway bolsters immune responses ^{27, 29, 30}. PPM1G is expected to ameliorate the synthesis of pro-inflammatory factors by suppressing eIF4E phosphorylation. We used poly (I:C) to transfect GFP, PPM1G and PPM1G-YL_mut expressing HEK293H cells to mimic viral infection and subsequently performed a human cytokine array analysis. RANTES (regulated upon activation, normal T cell expressed and secreted (also known as CCL5)) production was reduced ~ 80% in PPM1G expressing cells compared to GFP or PPM1G-YL_mut expressing cells (Fig. 5D), indicating that RANTES expression is PPM1G-phospho-eIF4E dependent. Notably, phospho-eIF4E-dependent production of RANTES but not MIF and IL-8 synthesis has been reported in asthmatic airway smooth muscle cells, breast cancer cells and murine melanomas^{27, 29, 30}.

We next examined the physiological consequence of the reduction of mRNA translation by PPM1G. eIF4E phosphorylation promotes cell proliferation ^73–75. Thus, PPM1G is expected to impair cell proliferation. In comparison to GFP ectopic expression, PPM1G ectopic expression caused a reduction of ~ 60% in the proliferation of HEK293H cells while PPM1G-YL_mut expression decreased ~ 30% cell proliferation (Fig. 5E). PPM1G but not PPM1G-YL_mut expression impaired ~ 46% proliferation in another cell line, A549, a non-small-cell lung cancer (NSCLC) (Fig. 5F). Consistent with the anti-proliferation results, PPM1G, but not PPM1G-YL_mut, promoted the dephosphorylation of eIF4E and decreased phospho-eIF4E dependent Mcl-1 and Bcl-2 protein synthesis in A549 cells (Figure S5A and S5B).

Taken together, our results reveal that the PPM1G-phospho-eIF4E pathway plays an important role in cell proliferation and suppresses the expression of proteins involved in the cell cycle, cell survival and inflammatory responses.

Phosphorylation of eIF4E is linked to cancer development and progression ⁷⁶. MNKs-eIF4E phosphorylation targeted therapy is in clinical trials ⁷⁷. However, kinase inhibition alone inescapably leads to drug resistance ⁷⁸ and thus it is necessary to implement multiple synergistic strategies, such as phosphatase activation ^{79, 80}.

PPM1G associates with and dephosphorylates eIF4E but not 4E-BP1 (Fig. 1A, 1B, 1E, 1F and 1G) contrary to a published report ⁵⁴. SDS-PAGE of 4E-BP1 usually displays three main isoforms named α, β and γ (Fig. 1A) based on ascending phosphorylation levels ^{14, 81}. Dephosphorylation of 4E-BP1 results in a dramatic reduction of the hyperphosphorylated β and γ isoforms and an accumulation of hypo- or unphosphorylated α isoform ¹³ (Fig. 3C). However, the published phosphatase experiment of Liu et al.⁵⁴, showed that PPM1G progressively dephosphorylated 4E-BP1 without changing the ratio among the isoforms. In addition, the latter study demonstrated that PPM1G associated only with the hyper-phosphorylated 4E-BP1 but not the unphosphorylated 4E-BP1, whereas under mTOR inhibitor treatment, PPM1G strongly associated with the unphosphorylated 4E-BP1 ⁵⁴. Because of this unexplained behaviour and our results, we conclude that PPM1G is not the major phosphatase targeting 4E-BP1.

The catalytic subunit of protein phosphatase 2A (PP2A_Cα) was reported to dephosphorylate both 4E-BP1 and eIF4E ^{59, 82–84}. PP2A comprises a family of heterotrimeric phosphatases consisting of a catalytic subunit (2 isoforms: α and β), a scaffold subunit (2 isoforms: α and β) and a variable regulatory subunit (17 isoforms), which gives rise to more than 70 different holoenzymes with divers functions ⁸⁵. Therefore, changes in the amount of PP2A_Cα unsurprisingly lead to perturbation of many signaling pathways, such as MEK, ERK, AKT, mTORC1, TGFβ and Wnt ^86–92, among which ERK and mTORC1 are the established eIF4E and 4E-BP1 upstream kinases, respectively. Indeed, we showed that PP2A_Cα expression or knockdown dramatically affects MNK2 and its upstream kinase ERK activity (Figure S1B and S1C). In addition, a physical interaction between eIF4E and specific PP2A holoenzyme has never been reported and we failed to detect an interaction between PP2A_Cα with eIF4E either (Figure S2A). Therefore, PP2A is most probably an indirect repressor of eIF4E phosphorylation.

We showed that the 4E-BM2 of PPM1G contains the YxxxxL consensus sequence but with an arginine in place of Φ (a hydrophobic amino acid) following the leucine at the end (Fig. 2A). The 4E-BM(YxxxxMK) of drosophila 4E-BP does not possess a hydrophobic amino acid either and contains instead a lysine (K) which has aliphatic side chain ⁶⁴. We further showed that the 4E-BM2 is required for PPM1G to interact with eIF4E and dephosphorylate eIF4E in cells and test tube (Fig. 2B, 2C, 2D and 2E). Notably, we demonstrated that the 4E-BM2 functions specifically for eIF4E binding (Fig. 3A, S3B and S3C).

eIF4E residues V69, W73 and L135 that are required for 4E-BP1 binding^{64, 66} are also essential for PPM1G binding (Fig. 3B and S3D). Indeed, dephosphorylated 4E-BP1 prevented PPM1G-eIF4E binding (Fig. 3C), because both 4E-BP1 and eIF4G also contain an auxiliary eIF4E binding sequence ^{64, 93} which is absent in PPM1G. PPM1G does not associate with the cap-bound-eIF4E (Figure S3E), since eIF4E can bind to the cap with strong affinity only as part of the eIF4F complex ⁹⁴.

We showed that PPM1G altered the phospho-eIF4E-dependent synthesis of a subset of proteins (Fig. 5B, 5C, 5D and S5B). Notably, all the proteins including Mcl-1, Cyclin D1, Bcl-2, MMP3 and RANTES are validated phospho-eIF4E targets^{21–23, 25, 95}.

We further demonstrated that PPM1G suppresses eIF4E phosphorylation to inhibit the proliferation of both HEK293H cells and A549 cells (Fig. 5E and 5F), suggesting a conserved function.

In conclusion, our study documents a detailed molecular mechanism of eIF4E dephosphorylation by PPM1G and reveals the importance of PPM1G-phospho-eIF4E pathway in the restriction of cell proliferation and the expression of proteins involved in the cell cycle, survival, and inflammation (Figure S5C). We wish that our study will help to develop a better eIF4E phosphorylation targeted therapeutic strategy.

Limitations of the study

It is important to obtain an atomic resolution of the PPM1G-eIF4E complex which would explain how PPM1G selectively dephosphorylate eIF4E. We have not studied the interaction of endogenous PPM1G with eIF4E because of the poor yield of either protein.

Mutagenesis and Plasmids

DNA mutations were generated with the Quick Change Lightning Site-Directed Mutagenesis Kit (Agilent, Cat#21508) according to the manufacturer’s instructions. Plasmids pHA-eIF4E(Cat#17343) and pLN-HA-4E-BP1(Cat#79438) were from Addgene. The pCMV3-FLAG-PPM1G (Cat#HG-11245-NF) was from Sino biological inc. and pcDNA3-FLAG-PP2A_Cα was a gift from Dr Anne-Claude Gingras. Other mammalian cell and bacteria expression vectors were generated in the Gateway system (Invitrogen). The pDONR221λ or 223 entry vector containing the coding sequence of the gene of interest was recombined with the expression vector to generate the following plasmids: pDEST-CMV-PPM1G-GFP, pDEST-CMV-PPM1G_NESmut-GFP, pDEST-527-6xHIS-PPM1G, pDEST-527-6xHIS-PPM1G-YL_mut, pDEST-527-6xHIS-GFP, pcDNA3-FLAG-GFP, pcDNA3-FLAG-PPM1A, pcDNA3-FLAG-PPM1H, pcDNA3-FLAG-eIF4E, pcDNA3-FLAG-eIF4E_Capmut, pcDNA3-FLAG-eIF4E_VWLmut, pMH-HA-GFP, pMH-HA-PPM1G, pMH-HA-α-catenin.

Cells

The HEK293H cell line was a gift from Joseph Marcotrigiano and the A549 cell line was provided by Sidong Huang. Cell lines were maintained in complete DMEM media with 10% FBS and 100 units/ml penicillin-streptomycin, at 37⁰C with 5% CO₂ and were confirmed to be mycoplasma-free by PlasmoTest (Invivogen #rep-pt1).

Transient transfection

2x10⁶ cells were seeded in complete medium in a 10cm dish for overnight growth. 30µl Lipofectamine 2000 and 5–10µg DNA (5µg for HEK293H cells and 10µg for A549 cells) were separately diluted in 250µl Opti-MEM medium. After 5 minutes DNA and Lipofectamine were combined for 15 minutes at room-temperature and added onto cells in a dropwise manner. For A549 cells, cell medium was replaced by OPTI-MEM before the transfection. After addition of the Lipofectamine-DNA mixture, cells were maintained at 37⁰C for 6 hours in a CO₂ incubator. The OPTI-MEM was subsequently replaced by 10 ml fresh medium and 24 hours later, cells were collected for Western blot analysis or treated for other experiments.

Generation of cell lines

For the PPM1G knockout cell lines, 5ug of pCLIP-Cas9-hCMV-tRFP and 5ug of pCLIP-Dual-SFFV-ZsGreen containing two Cas9 guide RNAs (sgRNA) targeting PPM1G, or a scrambled sequence were co-transfected into HEK293H cells. 48 hours after transfection, cells were resuspended and sequentially diluted to one cell per 100µl of medium. Cells were transferred into a 96-well plate with 100µl medium (one cell) per well. Upon reaching confluency, cells were transferred into a 48-well plate. Cells that lost expression of PPM1G, GFP (sgRNA vector), and RFP (Cas9 vector) were kept for further experiments.

For generation of stable cell lines, 7µg of DNA or shRNA with 7µg of each lentiviral packaging plasmid (PLP1, PLP2, and VSVG) were co-transfected into HEK293T cells using 50µl of Lipofectamine 2000. 48 hours after transfection, cell culture medium was collected and passed through a 0.45µm filter. 1x10⁶ HEK293H cells were incubated with the lentivirus-containing medium supplied with 5mg/ml polybrene. After 48 hours, virus-containing medium was discarded, and the cells were cultured in medium supplied with selection antibiotic (200ug/ml G418 for PPM1G-GFP and PPM1G_NESmut-GFP expressing cells and 4µg/ml puromycin for shRNA expressing cells).

Immunoblotting, immunoprecipitation and m⁷GDP-pull-down.

Cells were resuspended using a cell scraper. After a centrifugation at 1000 rpm for 5 minutes at 4°C, the cell medium was removed, and cells were resuspended in 1x ice-cold PBS (Phosphate-Buffered Saline). PBS was discarded following a centrifugation at 1000 rpm for 5 minutes at 4°C and ice-cold lysis buffer (50mM HEPES-KOH pH 7.5, 50mM KCl, 2mM MgCl₂, 2mM EGTA, 5% glycerol, 0.5% NP40, and EDTA-free protease inhibitor cocktail (Roche) in double-distilled water) was added. Next, cell lysates were centrifuged at 15000 rpm for 5 minutes at 4°C and the supernatant was collected. Total protein concentration was determined using the Bio-Rad Protein Assay Dye Reagent Concentrate (Cat#5000006). For immunoblotting, collected supernatant was mixed with an equal volume of 2x Laemmli sample buffer (Bio-Rad Cat# 1610737) and heated for 5 minutes at 95°C. After centrifugation, the sample was resolved on a 10–12% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane, blocked with 5% milk in TBST (Tris-buffered saline + 0.1%Tween), washed with TBST and incubated overnight with antibodies in 5% BSA in TBST on a shaker at 4°C. The next day, the membrane was washed with TBST, then incubated with HRP (horseradish peroxidase) conjugated secondary antibodies in 5% milk in TBST for 1 hour at room temperature. After three washes with TBST, the membrane was incubated with ECL (Enhanced Chemiluminescence) for one minute and exposed against an X-ray film. Antibodies were removed with stripping buffer (2mM glycine pH 2.0, 0.1% SDS,) for re-probing with different antibodies. For immunoprecipitation, 1-2mg of total protein was incubated with pre-washed 25µl of settled anti-FLAG affinity gel or 25µl of anti-HA magnetic beads in a tube rotator at 4°C for 1 hour. After a 5-minute centrifugation at 2000rpm at 4°C, the supernatant was removed, and the gel or beads were washed three times with cell lysis buffer. Proteins were eluted from the gel or beads by adding 30µl of 2x Laemmli buffer (Bio-Rad). For m⁷GDP pulldown assay, 0.5mg of total protein was incubated with pre-washed 15µl of settled m7GDP-agarose beads (Jena Bioscience) in a tube rotator for 30 minutes at 4°C. Supernatant was removed after a 5-minute centrifugation at 2000rpm at 4°C. After three washes with cell lysis buffer, proteins were eluted by adding 30µl of 2x Laemmli buffer.

Phosphatase assay

FLAG-tagged GFP, PPM1G, and PPM1G-YL_mut proteins were immunoprecipitated from HEK293H cells using 30 µl of settled anti-FLAG affinity gel. Proteins were eluted three times with 30 µl of 0.1 mg/ml FLAG peptide dissolved in phosphatase assay buffer (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 0.25 mg/ml BSA)⁹⁶. HA-tagged 4E-BP1, eIF4E, and α-catenin proteins were purified from the HEK293H cells using 20 µl of settled anti-HA magnetic beads and eluted three times with 30 µl of 1 mg/ml HA peptide dissolved in phosphatase assay buffer. Elution of each protein were combined into an Eppendorf tube and protein concentration and purity were estimated using SDS/Polyacrylamide gel electrophoresis and Coomassie blue staining. Eluted proteins were stored at -80°C. For the phosphatase assay, 500 ng of FLAG-tagged GFP, PPM1G, and PPM1G-YL_mut were separately incubated with 50 ng of HA-tagged 4E-BP1, eIF4E, or α-catenin in phosphatase buffer containing 2 mM MnCl₂⁵⁴(45 µL total volume) in an Eppendorf Thermos-mixer (30°C, 1000 rpm) for 30 minutes. The assay was stopped by adding 15 µl of 4x Laemmli protein sample buffer (Bio-Rad), and the protein dephosphorylation was analyzed by Western blot using specific antibodies and phosphorylation site-specific antibodies.

Recombinant protein production and GST-pulldown assay

BL21 competent cells were transformed with 100 ng of pDEST-527-6xHIS-GFP, PPM1G, or PPM1G-YL_mut and spread on LB agar plates containing 100 µg/ml ampicillin. After overnight incubation at 37°C, a single colony was picked and incubated in 50 ml LB buffer containing 100 µg/ml ampicillin for overnight growth in a bacterial incubator. The following day, 20 ml of bacteria in LB were transferred to 1000 ml of fresh LB buffer with ampicillin and grown in a bacterial incubator until the OD at 600 nm reached between 0.4 and 0.6. IPTG (isopropyl β-d-1-thiogalactopyranoside) was subsequently added with a final concentration of 1 mM and the bacteria were cultured overnight at 18°C. The next day, the bacteria were collected by centrifugation, lysed in 20 ml lysis buffer (20 mM Tris-HCl pH 8, 500 mM NaCl, 10 mM imidazole, 10% glycerol, 0.1% Triton, and 0.1% sodium lauroyl sarcosinate), sonicated, and centrifuged. The supernatant was retained, and 0.5 ml of settled Ni-NTA resin was added and incubated on a wheel at 4°C for one hour. After three washes with 1x PBS containing 25 mM imidazole, proteins were eluted with 250 mM imidazole in 1x PBS. Protein concentration and purity were analyzed using SDS/Polyacrylamide gel electrophoresis and Coomassie blue staining. For the GST pull-down assay, 1 µg of GST-eIF4E (LSBio, Cat# LS-G39814) was incubated with 0.5 µl of settled glutathione magnetic agarose beads (Thermo Scientific, Cat# 78601) in equilibration buffer (according to manufacturer's instructions) on a rotator at 4°C for 30 minutes. After three washes with equilibration buffer, GST-eIF4E was separately incubated with 100 ng of 6xHIS-tagged GFP, PPM1G, and PPM1G-YL_mut in equilibration buffer on a rotator at 4°C for 30 minutes. Beads were washed three times with equilibration buffer and the proteins were eluted by adding 30 µl of 2x Laemmli buffer (Bio-Rad).

Immunofluorescence and confocal microscopy

For immunofluorescence, 2 x 10⁶ cells were spread on coverslips in a 10 cm plate the day before transfection. The following day, the cells were transfected with 5 µg of plasmid. 24 hours later, the cells were fixed with 4% formaldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES-KOH pH 6.9, 10 mM EGTA, 4 mM MgSO₄,) for 30 minutes at room temperature. Cells were then washed three times for 5 minutes with fresh TBST (Tris-buffered saline with Tween-20), incubated in blocking buffer (2% BSA and 0.1% Triton in PHEM buffer) and overnight with primary antibodies (anti-FLAG, anti-HA, and anti-α-tubulin, 1:200) in PHEM buffer with 2% BSA. After three washes with TBST, cells were incubated with secondary antibodies (Alexa 488, Alexa 546, 1:200) for 1 hour at room temperature. The cells were washed three times with TBST and the coverslip was placed on a drop of mounting medium with DAPI on a slide. The edge of the coverslip was sealed with nail polish, and the slide with coverslip was placed in a dark slide box at 4°C. The images of fixed and live cells were taken using confocal microscopy (LSM710) with a 63x oil immersion lens.

For the FLIP (fluorescence loss in photobleaching) experiment, cells were spread on 14 mm glass microwell dishes (MATTEK, Cat# P35G-1.5-14c) the day before the experiment. In each round of the FLIP assay, two adjacent GFP-expressing cells were chosen. The fluorescence in a small region of interest (ROI, indicated by a green square) in the cytoplasm of one cell was bleached with maximum laser (488 nm) intensity and analyzed, while the fluorescence in a ROI (red square) in the nucleus of the same cell and in a ROI (blue square) in the nucleus of the neighboring cell were also analyzed. After the first image acquisition, 60 repetitions of one second bleaching and image acquisitions were performed. The fluorescence loss in the nucleus of the bleached cell was calculated and normalized using the formula: (F(A2tn) / F(A2t0)) * (F(A3t0) / F(A3tn)), where F(A2tn) and F(A2t0) are the mean fluorescence of the ROI red square at time n and at time 0 (prior to bleaching), respectively, and F(A3tn) and F(A3t0) are the mean fluorescence of the ROI blue square at time n and at time 0, respectively.

IncuCyte analysis

24 hours after transfection, cells were resuspended with trypsin and viable cell number was determined using a cell counter (Bio-Rad) by excluding Trypan blue-stained dead cells. 0.1 x 10⁶ cells were seeded in a 6-well plate. Once attached to the bottom, cell proliferation was monitored using the IncuCyte ZOOM system (Essen BioScience, MI, US).

Cytokine array assay

24 hours after DNA transfection, cells were incubated in 4 ml of fresh cell medium and then transfected with 15 µg of poly(I:C). After overnight incubation, cell medium was collected and centrifuged. The supernatant was transferred to a 1.5 ml Eppendorf tube, and the human cytokine array assay was performed according to the manufacturer's instructions (R&D, Cat#ARY005B).

Polysome profiling

Polysome profiling was performed according to published protocol⁹⁷. HEK293H cells were transfected with FLAG-GFP, FLAG-PPM1G, and FLAG-PPM1G-YL_mut in a 15 cm plate. 24 hours later, cells (90% confluency) were treated with 100 µg/ml cycloheximide (CHX) for 5 minutes at 37°C. Cells were washed twice with ice-cold PBS containing CHX and collected by scraping. Cells were centrifuged at 1000 rpm for 5 minutes at 4°C and lysed in hypotonic buffer (5 mM Tris-HCl, pH 7.4, 1.5 mM KCl, 2.5 mM MgCl₂, 200 U/ml RNase inhibitor, 2 mM DTT, 1 x protease inhibitor, 100 µg/ml CHX, 0.5% Triton X-100, and 0.5% sodium deoxycholate). After centrifugation at 15,000 rpm for 5 minutes at 4°C, the supernatant was retained, and the RNA concentration was determined using a Nanodrop 2000 (Thermo Fisher). Equal amounts of each sample were loaded onto a 10%-50% sucrose gradient and then centrifuged at 36,000 rpm for 2 hours at 4°C using an SW 40 Ti rotor in an Optima L-80 XP ultracentrifuge (Beckman). Polysome fractions were collected using a Teledyne ISCO fractionator, and the optical density at 254 nm was measured using TracerDAQ.

ACKNOWLEDGMENTS

This work was supported by Cancer Research Society (CRS) operating grant from 2020 to 2022. We thank professor Gergely Lukacs at McGill University and Professor Vincent Archambault at University of Montreal for their instructive advice and suggestions.

AUTHOR CONTRIBUTIONS

P.W. designed and performed the experiments, wrote the manuscript. Z.X.L., H.H. and S.K. helped with the structure analysis. H.X. performed the mycoplasma test, C.T.Y, A.S., H.X., S.K. and S.B. helped with some immunoblotting experiments. J.C. and J.R. helped with microscopy. M.N.B., N.F., L.G., R.S., V.S., J.K. and all others helped with the writing and English correction. B.N. and N.S. supervised this research and wrote the manuscript.

DECLARATION OF INTERESTS

We declare no competing interests.

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PPM1G dephosphorylates eIF4E to reduce selective mRNA translation.

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