Phosphorylation of the Cap-Binding Protein Eukaryotic Translation Initiation Factor 4E by Protein Kinase Mnk1 In Vivo

Abstract

Eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA 5 cap and brings the mRNA into a complex with other protein synthesis initiation factors and ribosomes. The activity of mammalian eIF4E is important for the translation of capped mRNAs and is thought to be regulated by two mechanisms. First, eIF4E is sequestered by binding proteins, such as 4EBP1, in quiescent cells. Mitogens induce the release of eIF4E by stimulating the phosphorylation of 4EBP1. Second, mitogens and stresses induce the phosphorylation of eIF4E at Ser 209, increasing the affinity of eIF4E for capped mRNA and for an associated scaffolding protein, eIF4G. We previously showed that a mitogen-and stress-activated kinase, Mnk1, phosphorylates eIF4E in vitro at the physiological site. Here we show that Mnk1 regulates eIF4E phosphorylation in vivo. Mnk1 binds directly to eIF4G and copurifies with eIF4G and eIF4E. We identified activating phosphorylation sites in Mnk1 and developed dominant-negative and activated mutants. Expression of dominant-negative Mnk1 reduces mitogeninduced eIF4E phosphorylation, while expression of activated Mnk1 increases basal eIF4E phosphorylation. Activated mutant Mnk1 also induces extensive phosphorylation of eIF4E in cells overexpressing 4EBP1. This suggests that phosphorylation of eIF4E is catalyzed by Mnk1 or a very similar kinase in cells and is independent of other mitogenic signals that release eIF4E from 4EBP1.Mitogens stimulate protein and RNA synthesis (56, 65). The increase in protein synthesis is partly due to increased initiation on preexisting mRNAs, with the result that those mRNAs are recruited into larger polysomes. In addition to an increase in basal translation, specific mRNAs are preferentially upregulated, suggesting that mitogenic signal transduction pathways impinge on the components of the translation machinery that interact with the mRNA. mRNAs are brought to the ribosome by eukaryotic initiation factor (eIF4F) (for reviews, see references 58, 60, and 61). eIF4F is a multiprotein complex formed from 25-, 46-and 220-kDa subunits, called eIF4E, eIF4A, and eIF4G, respectively. eIF4E, also known as cap-binding protein, is responsible for binding the 5Ј-terminal 7-methyl-GTP (m 7 GTP) cap found on all eukaryotic mRNAs. eIF4A is a subunit of an RNA helicase that seems to unwind secondary structure in the mRNA. eIF4G is the scaffolding subunit, to which the other subunits bind. It also has a binding site for eIF3, which links the eIF4F-mRNA complex to the 40S ribosomal subunit. In yeast, eIF4G has an additional functional region, to which the poly(A)-binding protein and the 3Ј end of the mRNA bind (64). Besides serving as a passive scaffold, eIF4G plays a regulatory role, stimulating the binding of capped mRNA to eIF4E (24). Thus, the eIF4F complex promotes interactions between the 5Ј end of the mRNA, the ribosome, and an RNA helicase.As the main mRNA-binding component of the translation machinery, the eIF4F complex has the potential to distinguish between mRNAs for differential translat...

Inhibition of Myc-dependent apoptosis by eukaryotic translation initiation factor 4E requires cyclin D1

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The gene encoding the splicing factor SF2/ASF is a proto-oncogene

“…30). The MNK2 gene, MKNK2, is alternatively spliced, generating two catalytically active isoforms that are differentially regulated and localized 31 .…”

Starting the protein synthesis machine: eukaryotic translation initiation

“…(18,38) In mammals, this region of eIF4G binds the eIF4E-kinase Mnk-1. (46,47) Phosphorylation of eIF4E at serine 209 by Mnk-1 generally correlates with increased translation rates (48) and may serve as a marker of eIF4F integrity. It has long been thought that phosphorylation increases the capbinding affinity of eIF4E.…”