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Mitotic phosphorylation of eukaryotic initiation factor 4G1 (eIF4G1) at Ser1232 by Cdk1:cyclin B inhibits eIF4A helicase complex binding with RNA.

ABSTRACT: During mitosis, global translation is suppressed, while synthesis of proteins with vital mitotic roles must go on. Prior evidence suggests that the mitotic translation shift involves control of initiation. Yet, no signals specifically targeting translation initiation factors during mitosis have been identified. We used phosphoproteomics to investigate the central translation initiation scaffold and "ribosome adaptor," eukaryotic initiation factor 4G1 (eIF4G1) in interphase or nocodazole-arrested mitotic cells. This approach and kinase inhibition assays, in vitro phosphorylation with recombinant kinase, and kinase depletion-reconstitution experiments revealed that Ser1232 in eIF4G1 is phosphorylated by cyclin-dependent kinase 1 (Cdk1):cyclin B during mitosis. Ser1232 is located in an unstructured region of the C-terminal portion of eIF4G1 that coordinates assembly of the eIF4G/-4A/-4B helicase complex and binding of the mitogen-activated protein kinase (MAPK) signal-integrating kinase, Mnk. Intense phosphorylation of Ser1232 in mitosis strongly enhanced the interactions of eIF4A with HEAT domain 2 of eIF4G and decreased association of eIF4G/-4A with RNA. Our findings implicate phosphorylation of eIF4G1(Ser1232) by Cdk1:cyclin B and its inhibitory effects on eIF4A helicase activity in the mitotic translation initiation shift.

SUBMITTER: Dobrikov MI

PROVIDER: S-EPMC3911517 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Mitotic phosphorylation of eukaryotic initiation factor 4G1 (eIF4G1) at Ser1232 by Cdk1:cyclin B inhibits eIF4A helicase complex binding with RNA.

Dobrikov Mikhail I MI Shveygert Mayya M Brown Michael C MC Gromeier Matthias M

Molecular and cellular biology 20131118 3

During mitosis, global translation is suppressed, while synthesis of proteins with vital mitotic roles must go on. Prior evidence suggests that the mitotic translation shift involves control of initiation. Yet, no signals specifically targeting translation initiation factors during mitosis have been identified. We used phosphoproteomics to investigate the central translation initiation scaffold and "ribosome adaptor," eukaryotic initiation factor 4G1 (eIF4G1) in interphase or nocodazole-arrested ...[more]

PMID: 24248602

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Project description:Ischemic strokes are caused by a reduction in cerebral blood flow and both the ischemic period and subsequent reperfusion induce brain injury, with different tissue damage depending on the severity of the ischemic insult, its duration, and the particular areas of the brain affected. In those areas vulnerable to cerebral ischemia, the inhibition of protein translation is an essential process of the cellular response leading to delayed neuronal death. In particular, translation initiation is rate-limiting for protein synthesis and the eukaryotic initiation factor (eIF) 4F complex is indispensable for cap-dependent protein translation. In the eIF4F complex, eIF4G is a scaffolding protein that provides docking sites for the assembly of eIF4A and eIF4E, binding to the cap structure of the mRNA and stabilizing all proteins of the complex. The eIF4F complex constituents, eIF4A, eIF4E, and eIF4G, participate in translation regulation by their phosphorylation at specific sites under cellular stress conditions, modulating the activity of the cap-binding complex and protein translation. This work investigates the phosphorylation of eIF4G1 involved in the eIF4E/eIF4G1 association complex, and their regulation in ischemia-reperfusion (IR) as a stress-inducing condition. IR was induced in an animal model of transient cerebral ischemia and the results were studied in the resistant cortical region and in the vulnerable hippocampal CA1 region. The presented data demonstrate the phosphorylation of eIF4G1 at Ser1147, Ser1185, and Ser1231 in both brain regions and in control and ischemic conditions, being the phosphorylation of eIF4G1 at Ser1147 the only one found in the eIF4E/eIF4G association complex from the cap-containing matrix (m7GTP-Sepharose). In addition, our work reveals the specific modulation of the phosphorylation of eIF4G1 at Ser1147 in the vulnerable region, with increased levels and colocalization with eIF4E in response to IR. These findings contribute to elucidate the molecular mechanism of protein translation regulation that underlies in the balance of cell survival/death during pathophysiological stress, such as cerebral ischemia.

Project description:Background:Cap-dependent mRNA translation is essential for the translation of key oncogenic proteins at optimal levels and is highly regulated by the rate limiting, initiation step in protein synthesis. Eukaryotic Translation Initiation Factor 4 Gamma 1 (EIF4G1) serves as a scaffold for assembly of cap-dependent translation components in EIF4F complex formation. In the current study, we analyzed the role and expression of EIF4G1 in Pan human cancer panels through various approaches. Methods:Immunohistochemistry analysis of EIF4G1 protein was done on high-density multi-organ Human Cancer tissue microarray (TMA) derived from the patient samples from different cancers. We used multiple clinical cohorts to analyze the EIF4G1 mRNA expression across human cancers. TCGA data analysis of EIF4G1 was done through Ualcan and c-bioportal web servers. Western blots for EIF4G1 protein was done for different cell lines in representing the multiple cancer types. Dependency score was calculated through Cancer Dependency Map. Clonogenic, tumorosphere assay and cell invasion assay were done with EIF4G complex inhibitor. Association of EIF4G1 mRNA and Kaplan-Meier survival analysis was done on available TCGA datasets. Results:We observed an increase in EIF4G1 protein levels in tissue sections from different cancers as compared to their respective normal tissue. Our analysis of the TCGA data revealed that EIF4G1 mRNA expression is significantly increased in tumor tissues compared to respective control tissues across human cancers and variable expression was observed among different datasets. We discovered that alteration frequency in EIF4G1 is prevalent in human cancers e.g. prostate cancer (~?25%), ovarian cancer (~?15%), Head and Neck cancer (~?13%) and cervical cancer (~?12.5%). EIF4G1 mRNA and protein levels were high across cancer cell lines from multiple organs. Our analysis of DepMap datasets utilizing depletion assays revealed that EIF4G1 is critical for cancer cell survival. Treatment with EIF4G complex inhibitor impaired clonogenic, tumorosphere formation potential and inhibited cell invasion. Moreover, higher EIF4G1 mRNA level was associated with a lower median survival of patients in multiple tumor types. Conclusions:These studies show that EIF4G1 is amplified/over-expressed in multiple cancers and plays an essential role in cancer cell survival, as such EIF4G1 could serve as a novel potential target for therapeutic intervention across many cancers.

Dataset Information

Mitotic phosphorylation of eukaryotic initiation factor 4G1 (eIF4G1) at Ser1232 by Cdk1:cyclin B inhibits eIF4A helicase complex binding with RNA.

Publications

Mitotic phosphorylation of eukaryotic initiation factor 4G1 (eIF4G1) at Ser1232 by Cdk1:cyclin B inhibits eIF4A helicase complex binding with RNA.

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