Stimulation of the Erk1/2 or p38 MAPK signaling pathways produces Mnk1 activation and eIF4E phosphorylation at Ser209. Since eIF4E phosphorylation is dependent on Mnk1 binding to eIF4G1 (14
), we investigated whether the upstream MAPK signal controls Mnk1-eIF4G interaction. To this end, we generated a stable HEK-293 cell line (HEK-293eIF4G
) that upon induction with tetracycline (Tet) expresses an N-terminally (myc) and C-terminally (Flag) tagged form of eIF4G1 (referred to as eIF4G from here on) (Fig. A). Treatment of these cells with 12-O
-tetradecanoylphorbol-13-acetate (TPA) produced robust activation of Erk1/2, which led to the phosphorylation of Thr209/Thr214 in the Mnk1 active site and eIF4E phosphorylation (Fig. ).
FIG. 1. Stimulation of cells with TPA results in increased binding of Mnk1 to eIF4G. (A) HEK-293eIF4G cells were uninduced (−) or Tet induced (+), serum starved for 18 h, and treated for 15 min with DMSO (−) or 100 nM TPA (+). (more ...)
We used HEK-293eIF4G lysates for anti-Flag immunoprecipitation (IP) of overexpressed eIF4G and analyzed eIF4G binding partners by immunoblotting. TPA treatment increased coimmunoprecipitation (co-IP) of Mnk1 with eIF4G substantially (Fig. ), while binding of other known interaction partners, such as the poly(A) binding protein (PABP), eIF4A, and eIF4E, was not affected (Fig. ). We also observed increased phospho-eIF4E coprecipitating with eIF4G, in step with enhanced eIF4E Ser209 phosphorylation as a consequence of TPA treatment. Next, we tested if a similar effect would be evident with overexpressed Mnk1 binding to endogenous eIF4G (Fig. ). We generated stable HEK-293 cells (HEK-293Mnk1) expressing hemagglutinin (HA)-tagged human Mnk1a in a Tet-inducible fashion. Lysates from HEK-293Mnk1 cells upon TPA treatment exhibited enhanced binding of endogenous eIF4G to HA-Mnk1 (Fig. ). To test if MAPK activation enhances physical proximity of Mnk1 and its substrate, we overexpressed myc-eIF4E and performed anti-myc IP (Fig. ). TPA exposure resulted in enhanced co-IP of Mnk1 with eIF4E, while eIF4E-eIF4G interaction was unchanged (Fig. ). Our results suggest that MAPK activation stimulates Mnk1 binding to eIF4G and, thus, interaction with its substrate eIF4E.
There are two isoforms of Mnk1a that have been reported to arise from alternate use of AUGs 12 codons apart (Mnk1a and -1aΔ12; Fig. A), and they may exhibit intrinsically distinct eIF4G binding activities (13
). Using anti-Mnk1 antibody, we consistently detected two distinct bands in total cell lysates, both of which decreased in mobility upon TPA stimulation (data not shown). Using electrophoresis conditions that achieve higher resolution, we observed two bands in Flag-eIF4G precipitates, likely representing both isoforms (Fig. ). When overexpressed, the two Mnk1a isoforms exhibited similar eIF4G binding responses upon TPA treatment of cells (Fig. ). We also investigated if Mnk2-eIF4G interaction is regulated by MAPK signals. Mnk2 shares 71% sequence identity with Mnk1 and phosphorylates eIF4E in vivo
). Since we lack reliable anti-Mnk2 antibodies, we overexpressed HA-tagged Mnk2. Binding of HA-Mnk2 to eIF4G did not increase upon the addition of TPA but, instead, was slightly diminished (Fig. ). We attribute this to competition of HA-Mnk2 with endogenous Mnk1, which upon activation with TPA binds more strongly to eIF4G in the same lysate (Fig. ). These data suggest that Mnk2-eIF4G binding is secondary to Mnk1-eIF4G interaction. Thus, Mnk2-eIF4G binding does not appear to be directly regulated by MAPK signal transduction but responds indirectly to competition with Mnk1.
FIG. 2. Differential regulation of Mnk1 and Mnk2 binding to eIF4G. (A) Schematic view of Mnk1 constructs used in this study. The HA tag, activation loop, truncation mutants, amino acids manipulated in this study, and the eIF4G and MAPK binding sites are indicated. (more ...)
Next, we tested if Mnk1-eIF4G interaction can be increased by a variety of treatments that stimulate eIF4E phosphorylation. Since both p38 and Erk1/2 MAPKs can activate eIF4E phosphorylation, we employed multiple treatments to induce the two signaling pathways (Fig. A). All conditions that stimulated Mnk1 phosphorylation markedly increased Mnk1-eIF4G association (Fig. ). Importantly, this effect was abrogated by the inhibition of MEK (immediately upstream of Erk1/2) with UO126 or p38 inhibition with SB203580 (Fig. ). Next, we verified if downregulation of Mnk1-eIF4G binding by inhibitors is due to decreased activity of their respective kinase targets. SB203580 and UO126 failed to inhibit Mnk1-eIF4G binding stimulated by Erk1/2 and p38 MAPK activation, respectively (Fig. ). This suggests that both inhibitors block Mnk1-eIF4G association through specific effects on their target kinases only.
FIG. 3. MAPK-mediated phosphorylation of Mnk1a at T209/T214 activates Mnk1-eIF4G interaction. (A) HEK-293eIF4G cells were uninduced or Tet induced, serum starved for 18 h, and treated for 15 min with DMSO, 20% fetal bovine serum (FBS), 100 nM TPA (Erk1/2 (more ...)
Since Erk1/2 and p38 MAPKs phosphorylate Mnk1, the most obvious explanation for increased eIF4G binding would be conformational changes triggered by Mnk1 phosphorylation. This has been predicted based on the crystal structure of the Mnk1 catalytic domain (6
). However, MAPK signals may also cause posttranslational modification of eIF4G (16
), which could conceivably modulate Mnk1 binding. To distinguish these possibilities, we created a mutant of Mnk1 in which Thr209 and Thr214 were mutated to Ala (Mnk1a T2A2) and a deletion variant which lacks the C-terminal 24 amino acids containing the MAPK binding site (Mnk1aΔ24) (Fig. ) (20
). Neither of these proteins can be phosphorylated by MAPKs. We used these expression constructs to transfect HEK-293eIF4G
cells and test binding to Flag-eIF4G (Fig. ). In both cases, binding to eIF4G was severely impaired. It is possible that the Mnk1a T2A2 mutant did not bind eIF4G due to misfolded conformation of the mutated protein. However, the T2A2 HA-Mnk1a protein was still capable of binding Erk1/2 to the same extent that WT HA-Mnk1 could (Fig. ). Thus, not only is phosphorylation of the Mnk1 active site required for kinase activity but it also determines binding to eIF4G.
Next, we investigated Mnk-eIF4G binding with the natural splice variant Mnk1b, which lacks 84 C-terminal amino acids relative to Mnk1a (Fig. ) (11
). Mnk1b had a substantially higher basal level of binding to eIF4G than HA-Mnk1a (Fig. A) and was nonresponsive to TPA stimulation (Fig. ). This suggests that a specific region within the Mnk1a C terminus, upstream of the terminal 24 amino acids, interferes with binding of eIF4G, and this interference is alleviated upon stimulation by MAPK. Our results are in agreement with previous data proposing that the C-terminal region of Mnk1a occludes access to the catalytic domain (5
FIG. 4. The C-terminal domain of unstimulated Mnk1a blocks binding to eIF4G. HEK-293eIF4G cells were uninduced or Tet induced and transfected with the following expression plasmids: control vector, HA-Mnk1a, or HA-Mnk1b (A), WT or A362P HA-Mnk1a (B), or WT or (more ...)
To test if the Mnk1a C terminus can indeed block binding to eIF4G, we utilized an alanine-to-proline mutation (A362P) that has been proposed to disrupt the α-helical conformation of the Mnk1 C terminus (5
). Indeed, A362P HA-Mnk1 exhibited significantly enhanced binding to eIF4G (which was unresponsive to TPA) (Fig. ), supporting the notion of an inhibitory effect of the Mnk1a C-terminal domain. The A362P mutation even rescued deficient binding of Mnk1aΔ24 (lacking the MAPK binding domain) to eIF4G (Fig. ). This suggests that destabilizing the inhibitory C-terminal region of Mnk1a bypasses the requirement for MAPK activation of Mnk1a to induce eIF4G binding.
Next, we investigated why phosphorylation of Thr209/Thr214 is important for eIF4G binding. Since Mnk1 may be capable of autophosphorylation (2
), the active kinase may assume an altered conformation favoring eIF4G binding upon phosphorylation of a secondary site(s). To investigate this possibility, we used a competitive inhibitor of ATP (CGP57380) to suppress Mnk1 kinase activity. Surprisingly, while this inhibitor diminished the levels of phospho-eIF4E, it strongly stimulated Mnk1 binding to eIF4G in the absence or presence of TPA (Fig. A). This finding indicates that catalytic activity of Mnk1 is not required for eIF4G binding and that inhibiting Mnk1 enzymatic activity increases eIF4G interaction.
FIG. 5. Mnk1 kinase activity limits its binding to eIF4G. (A) HEK-293eIF4G cells were uninduced or Tet induced, serum starved for 18 h, and incubated for 2 h with 10 μM CGP57380 (Mnk1 inhibitor) and then stimulated for 15 min with DMSO (−) or (more ...)
CGP57380 is a compound with a broad inhibitory spectrum. Thus, it could indirectly affect Mnk1-eIF4G interaction via secondary effects on kinases other than Mnk1. To exclude this possibility, we confirmed our findings by using a kinase-dead version of Mnk1. To this end, we established an Asp191→Ala (D191A) mutant which disrupts the metal-coordinating site in Mnk1 and abolishes kinase activity of the enzyme (8
). We confirmed that the D191A mutant was efficiently activated by TPA because it was readily recognized by the phosphospecific anti-Mnk1 antibody (Fig. ). However, the D191A mutant was unable to phosphorylate recombinant GST-eIF4E in an in vitro
kinase reaction as expected (Fig. ). Co-IP with eIF4G revealed that despite the expression level of the D191A mutant being significantly lower than that of WT HA-Mnk1a, the former exhibited substantially enhanced binding to eIF4G in the absence or presence of TPA (Fig. ). Thus, considering these two independent lines of investigation, we conclude that inhibiting Mnk1 catalytic activity results in elevated interactions between the kinase and the eIF4G scaffold. One plausible explanation for these findings could be that blocking phosphoryl transfer with CGP57380 or by the D191A mutation prevents Mnk1 from disassociating from eIF4G. Thus, Mnk1 may change its conformation after it completes phosphorylation of its substrate and subsequently dissociate from eIF4G.
Ultimately, it was critical to demonstrate what contribution Mnk1-eIF4G binding makes to eIF4E phosphorylation independently of Mnk1 catalytic activity. To address this problem, we created a deletion mutant of Mnk1 that lacked the eIF4G binding site (Mnk1aΔ4G) (Fig. ) and investigated whether it could phosphorylate eIF4E in vivo
. First, using a co-IP approach, we validated that HA-Mnk1aΔ4G did not bind eIF4G (Fig. A). Next, we examined if deletion of the eIF4G binding domain impairs the kinase activity of Mnk1. This is unlikely, given that the eIF4G binding site lies outside the catalytic domain (6
). We immunoprecipitated either WT or Δ4G Mnk1a proteins and tested their ability to phosphorylate recombinant eIF4E in an in vitro
kinase reaction. WT Mnk1a and mutant Mnk1a phosphorylated eIF4E equally well (Fig. ). Interestingly, we consistently detected significantly enhanced Mnk1a phosphorylation with the Δ4G mutant, either by immunoblotting with phospho-Mnk1-specific antibody or with an in vitro
kinase assay (Fig. ). Also, HA-Mnk1aΔ4G coimmunoprecipitated significantly higher levels of Erk1/2 than WT HA-Mnk1a, which probably explains the higher levels of phosphorylation (Fig. ). Since the MAPK binding site lies at the end of the C terminus of Mnk1a, this phenomenon further indicates that the N terminus of Mnk1a functionally interacts with the C terminus.
FIG. 6. Mnk1-eIF4G binding is required for efficient eIF4E phosphorylation. (A) HEK-293eIF4G cells were Tet induced, transfected for 24 h with either WT or Δ4G HA-Mnk1a, and stimulated for 15 min with 100 nM TPA. Cell extracts were subjected to IP with (more ...)
To investigate the inherent catalytic activity of Mnk1aΔ4G in vivo
, we used a system in which eIF4E phosphorylation is categorically absent. Immortalized mouse embryonic fibroblasts (MEF) from Mnk1/Mnk2 double-knockout cells (DKO MEFs) (21
) lack any activity that can phosphorylate eIF4E (Fig. ). We utilized these cells to transfect either the WT or HA-Mnk1aΔ4G. TPA stimulation of these cells produced efficient phosphorylation of both Mnk1a variants (Fig. ). To enrich phospho-eIF4E, we performed m7
G cap pulldowns (Fig. ). Since Mnk1/Mnk2 DKO MEFs lack inherent eIF4E phosphorylation (21
), weak signal with the phospho-eIF4E-specific antibody in the vector control lane likely represents cross-reactivity with unphosphorylated eIF4E. WT Mnk1 expression alone produced very modest eIF4E phosphorylation, which was substantially elevated after TPA stimulation of transfected cells. In contrast, HA-Mnk1aΔ4G failed to raise eIF4E phosphorylation levels significantly above the background, even after TPA stimulation (Fig. ). This indicates that Mnk1a-eIF4G binding is indeed required for efficient eIF4E phosphorylation in vivo