The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity

Abstract

The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner. Here we demonstrate that the p90 ribosomal protein S6 kinase (RSK) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3.

Figure 1

Rapamycin-resistant eIF4B Ser422 phosphorylation is mediated by ERK1/2 MAPK signaling. (A) HeLa cells were deprived of serum in the presence or absence of 20 nM rapamycin for 16–18 h. Cells were pretreated with 10 μM of U0126 for 2 h, and then stimulated with either 20% serum or insulin (100 nM) for 30 min. Total cell extracts were subjected to SDS–PAGE followed by immunoblotting with phospho-eIF4B S422, phospho-S6K1 T389, and phospho-ERK1/2 T202/Y204 antibodies and the membrane was reprobed with anti-eIF4B antiserum. (B) HeLa cells were starved for serum as in (A) and stimulated for the indicated times with either 20% serum or insulin (100 nM). Cell extracts were resolved by SDS–PAGE and immunoblotted with phospho-eIF4G S1108, phospho-eIF4B S422, phospho-S6K1 T389, phospho-ERK1/2 T202/Y204, phospho-S6 S240/244 antibodies and the indicated total proteins. (C) HeLa cells were deprived of serum in the presence or absence of 20 nM rapamycin for 16–18 h. Cells were pretreated with 10 μM of U0126 for 2 h, and then stimulated with 20% serum for the indicated times. Total cell extracts were resolved by SDS–PAGE, immunoblotted with phospho-eIF4G S1108, phospho-eIF4B S422, phospho-S6K1 T389, phospho-ERK1/2 T202/Y204, and phospho-S6 S240/244 antibodies and reprobed for the indicated proteins with pan-specific antibodies. (D) Sequential activation of signaling pathways involved in eIF4B Ser422 phosphorylation. HeLa cells were deprived of serum for 16–18 h. Cells were then stimulated with 20% serum for the indicated amounts of time. Protein extracts were resolved by SDS–PAGE and probed for phospho-eIF4B S422, phospho-ERK1/2 T202/Y204, and phospho-S6K T389.

Figure 2

(A) eIF4B Ser422 phosphorylation persists in cells lacking S6K1 and S6K2. Hepatocytes derived from wt and S6K1/2 DKO animals were starved for nutrients and serum and stimulated with 1 μM insulin or 10% serum in the presence or absence of 20 nM rapamycin. Total cell lysates were immunoblotted with phospho-eIF4B S422, phospho-S6 S235/236, phospho-S6K1 T389, and rpL7 antibodies. (B) Substrate consensus sequences of S6K and RSK as compared to the eIF4B fragment encompassing the Ser422 phosphorylation site.

Figure 3

eIF4B Ser422 is dephosphorylated in PDK1 null and PDK1 PIF pocket mutant ES cells. Wt and PDK1^−/− knockout (A) or PDK1 PIF pocket mutant (B) ES cells were starved for 16–18 h in the presence or absence of 20 nM rapamycin and then stimulated with 20% serum for 15 min. Total cell extracts were resolved by SDS–PAGE and proteins were detected by immunoblotting using phospho-eIF4B S422, phospho-S6K1 T389, phospho-S6 S240/244, and phospho-ERK1/2 T202/Y204 antibodies. Membranes were reprobed with antibodies against the indicated total proteins and against PDK1 (arrows on the right indicate nonspecific bands).

Figure 4

Catalytically active RSK variants phosphorylate eIF4B in vitro and in vivo. (A) Wt and kinase-dead HA-RSK- and wt HA-S6K1-transfected HeLa cells were serum starved for 16–18 h, pretreated with either U0126 (10 μM; U0) or rapamycin (20 nM; RAP) as indicated, and stimulated with either serum or insulin for 15 min. An aliquot of the total cell lysate was immunoblotted for ERK1/2. Another aliquot was used to immunoprecipitate RSK variants and S6K1 using anti-HA antibody. Immunoprecipitates were split. Half was subjected to SDS–PAGE and probed for HA and the remaining half was assayed for in vitro kinase activity by using recombinant eIF4B as substrate. Samples were resolved by SDS–PAGE, stained with Coomassie brilliant blue, and exposed to an X-ray film. ³²P incorporation was quantified using a phosphorimager. A representative autoradiogram is shown. (B, C) HeLa cells cotransfected with Flag-tagged eIF4B together with wt, kinase-dead, and constitutively active RSK variants were serum starved for 16–18 h in the presence or absence of 10 μM U0126 (B) or 20 nM rapamycin (C) before serum stimulation for 15 min (B) or 90 min (C). Cell lysates were used to immunoprecipitate exogenous Flag-tagged eIF4B using anti-Flag (M2) antibody. Immune complexes were subjected to SDS–PAGE and probed with antibodies directed against phosphorylated eIF4B Ser422. Membranes were reprobed with anti-Flag antibody. Aliquots of total cell lysates were run on gel and probed with indicated antibodies. (D) HeLa cells were transfected with Flag-eIF4B. After 24 h, cells were deprived of serum in the presence or absence of increasing concentrations of RSK1/2 inhibitor fmk for 16–18 h. Cells were stimulated with 20% serum for 15 min. eIF4B was immunoprecipitated using anti-Flag antibody. Immune complexes were subjected to SDS–PAGE and Western blotting with phospho-eIF4B S422 antibody. The membrane was stripped and reprobed with Flag antibody. (E) HeLa cells were deprived of serum in the presence or absence of 10 μM RSK1/2 inhibitor fmk for 16–18 h. Cells were stimulated with 20% serum for 15 min. Total cell extracts were subjected to SDS–PAGE followed by immunoblotting with phospho-eIF4B S422, phospho-ERK1/2 T202/Y204, phospho-RSK S380, and phospho-S6K1 T389 antibodies and then reprobed for total eIF4B and ERK1/2.

Figure 5

RNAi-mediated silencing of RSK1 and RSK2 isoforms expression leads to reduced eIF4B Ser422 phosphorylation and inhibition of cap-dependent translation. (A) HeLa cells were subjected to RNAi using synthetic oligos nonspecific (Mock) or specific to RSK1 and RSK2 isoforms. At 24 h post-transfection, cells were serum starved for 16–18 h in the presence or absence of rapamycin, then indicated samples were treated with U0126 and stimulated with serum or insulin as shown. Total cell extracts were immunoblotted with phospho-eIF4B S422 and phospho-ERK1/2 T202/Y204 antibodies followed by reprobing for the corresponding total proteins. RSK1 and RSK2 Western blots were also carried out to demonstrate the efficiency of the knockdown. (B) HEK293 cells were transfected with the bicistronic luciferase construct and indicated siRNAs. After 48 h, cells were harvested and assayed for Renilla (RL) and firefly (FL) luminescence. Results are presented as average of RL/FL ratio±standard error from three independent experiments carried out in triplicate.

Figure 6

eIF4B Ser422 phosphorylation results in enhanced interaction between eIF4B and a complex containing eIF3. (A) HeLa cells cotransfected with Flag-tagged eIF4B and wt, kinase-dead, and constitutively active RSK variants were starved for 16–18 h in the presence or absence of 10 μM U0126 before serum stimulation for 15 min. Immunoprecipitation of Flag-tagged eIF4B was carried out using anti-Flag (M2) antibody. Immune complexes were subjected to SDS–PAGE and probed with a phosphospecific eIF4B S422 antibody and an eIF3a (p170) antibody. Membranes were reprobed with anti-Flag antibody. (B) HeLa cells were transfected with Flag-tagged wt eIF4B and Ser422 point mutants: Ser422Ala and Ser422Glu. After 16–18 h of serum starvation, cells were stimulated with serum for 15 min, and samples were processed as in (A).

Figure 7

Signaling pathways involved in eIF4B Ser422 phosphorylation. Growth factor-activated MAPK and PI3K cascades activate RSK and S6K proteins correspondingly and converge at the level of eIF4B phosphorylation. In systems where insulin is a marginal activator (dashed arrow) of MAPK cascade, insulin-induced eIF4B phosphorylation is absolutely sensitive to rapamycin. PDK1 protein plays a central role in activation of both RSK and S6K proteins and is indispensable for eIF4B phosphorylation.