Interferon-Dependent Engagement of Eukaryotic Initiation Factor 4B via S6 Kinase (S6K)- and Ribosomal Protein S6K-Mediated Signals

Abstract

Although the roles of Jak-Stat pathways in type I and II interferon (IFN)-dependent transcriptional regulation are well established, the precise mechanisms of mRNA translation for IFN-sensitive genes remain to be defined. We examined the effects of IFNs on the phosphorylation/activation of eukaryotic translation initiation factor 4B (eIF4B). Our data show that eIF4B is phosphorylated on Ser422 during treatment of sensitive cells with alpha IFN (IFN-␣) or IFN-␥. Such phosphorylation is regulated, in a cell type-specific manner, by either the p70 S6 kinase (S6K) or the p90 ribosomal protein S6K (RSK) and results in enhanced interaction of the protein with eIF3A (p170/eIF3A) and increased associated ATPase activity. Our data also demonstrate that IFN-inducible eIF4B activity and IFN-stimulated gene 15 protein (ISG15) or IFN-␥-inducible chemokine CXCL-10 protein expression are diminished in S6k1/S6k2 double-knockout mouse embryonic fibroblasts. In addition, IFN-␣-inducible ISG15 protein expression is blocked by eIF4B or eIF3A knockdown, establishing a requirement for these proteins in mRNA translation/protein expression by IFNs. Importantly, the generation of IFN-dependent growth inhibitory effects on primitive leukemic progenitors is dependent on activation of the S6K/eIF4B or RSK/eIF4B pathway. Taken together, our findings establish critical roles for S6K and RSK in the induction of IFN-dependent biological effects and define a key regulatory role for eIF4B as a common mediator and integrator of IFN-generated signals from these kinases.Extensive work over the years has established that the control of initiation of mRNA translation occurs primarily at the step at which the 40S ribosomal subunit is recruited to mRNA, to be positioned at the initiation codon (15). Most eukaryotic mRNAs contain a 5Ј cap structure (m7GpppN) to which the cap-binding protein complex eukaryotic initiation factor 4F (eIF4F) is attached. eIF4F is composed of three subunits: eIF4E, the cap-binding subunit; eIF4A, a protein with RNA helicase activity that unwinds the mRNA 5Ј secondary structure; and eIF4G, a scaffolding protein that associates with other IFs (15,18,20,37,54). The unphosphorylated/activated form of the translational repressor 4E-BP1 (eIF4E-binding protein 1) competes with eIF4G for binding to eIF4E and blocks cap-dependent mRNA translation (37), while such 4E-BP1-eIF4E interactions are decreased when phosphorylation of 4E-BP1 occurs by the mammalian target of rapamycin (mTOR) kinase (15,18,20,37,54). Beyond phosphorylation of 4E-BP1, mTOR regulates activation of the p70 S6 kinase (S6K), which in turn phosphorylates several substrates, including the S6 ribosomal protein (rpS6), eIF4B, and the tumor suppressor PDCD4 (13,18,20,54).Although much is known about the roles of mTOR-generated signals in the control of mRNA translation for cytokines and growth factors, the roles of various mTOR effectors in the initiation of mRNA translation in response to interferons (IFNs) remain to be precisely defined. Type I (␣, ␤, ε, , and ) and I...

Autophagy Is a Critical Mechanism for the Induction of the Antileukemic Effects of Arsenic Trioxide

“…Immunoblotting using an ECL methodology was performed as in our previous studies (15)(16)(17). In the experiments in which pharmacological inhibitors of ERK1/2 and JNK were used, the cells were pretreated for 60 min with the indicated concentrations of the inhibitors prior to the addition of As 2 O 3 to the cultures.…”

“…siRNAs against Mek1, Mek2, and a different siRNA against beclin 1 (beclin 1 siRNA-2) were obtained from Ambion, Inc (Foster City, CA). To assess the effects of the siRNA-mediated knockdown of beclin 1 or Atg7, cells were cultured in a methylcellulose assay system as in previous studies (17).…”

Autophagic degradation of the BCR-ABL oncoprotein and generation of antileukemic responses by arsenic trioxide

“…22,[24][25][26] Immunoblotting using an enhanced chemiluminescence (ECL) methodology was performed as in our previous studies. [24][25][26][27] In the experiments in which the pharmacologic inhibitors pepstatin A and E-64d were used, the cells were pretreated for 60 minutes with the indicated concentrations of the inhibitors before the addition of AS 2 O 3 to the cultures. siRNA-mediated knockdown of Beclin 1 or Atg7 in human leukemic cells was performed using Nucleofector kits from Amaxa Biosystems.…”

“…The effects of AS 2 O 3 on leukemic progenitor colony formation were assessed by clonogenic assays in methylcellulose, as previously described. 22,24,25,27 Acridine orange staining and flow cytometric analysis Formation of acidic vesicular organelles (AVOs), a morphologic characteristic of autophagy was quantitated by acridine orange staining. 28 Acridine orange (0.5 mg/mL; Invitrogen) was added 15 minutes before collection and after washing with phosphate buffered saline, cells were analyzed by flow cytometry.…”

Mnk kinases in cytokine signaling and regulation of cytokine responses

“…Interestingly, in the case of Type I IFNs, engagement of Erk is important for transcription-independent IFNα induced apoptosis [90], while activation of Erk can negatively regulate the anti-proliferative effects of IFNα on CD4+ T cells [91] as well as in human myeloma cell lines [92]. Additionally a downstream Erk target, RSK1 has been shown to regulate eIF4B phosphorylation in certain hematopoietic cell types and plays an important role in the control of IFN-induced mRNA translation and IFNα antileukemic responses [93]. In the case of IFNγ, Erk regulates IFNγ-dependent proteosomal degradatation of PPARδ [94] and enhances IFNγ regulated transcription by CCAAT-enhancer binding protein-β (C/CEBP-β) [88].…”