Mitogens stimulate protein and RNA synthesis (56
). 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
, 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 5′-terminal 7-methyl-GTP (m7
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 3′ 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 5′ 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 translation in mitogen-treated cells. The eIF4E subunit is thought to be the main regulatory component since it is present in limiting molar amounts (11
), and eIF4E availability in quiescent cells is further restricted by eIF4E-binding proteins, including 4EBP1 or PHAS-I (61
). 4EBPs prevent eIF4E binding to eIF4G without altering mRNA binding to eIF4E (23
). Mitogenic stimuli induce the phosphorylation of 4EBPs and the release of eIF4E, allowing eIF4E to associate with eIF4G and participate in translation. In addition, mitogens stimulate the phosphorylation of ribosomal protein S6 and several translation initiation factors, including eIF4E. Phosphorylation of 40S ribosomal protein S6 specifically stimulates the translation of a class of mRNAs that have pyrimidine-rich 5′ ends (29
). However, cells lacking the S6 protein kinase still exhibit mitogen-induced increases in basal translation (32
), indicating that other mechanisms, such as 4EBP1 and eIF4E phosphorylation, also mediate mitogenic stimulation of translation.
Several lines of evidence suggest that phosphorylation of eIF4E stimulates translation initiation. Mitogen-enhanced eIF4E phosphorylation usually correlates with increased protein synthesis (36
), and phosphorylation increases the binding of eIF4E to capped mRNA and to eIF4G in vitro (5
). The location of Ser 209 adjacent to the cap-binding pocket is consistent with an effect of phosphorylation on mRNA binding (45
). Significantly, overexpression of wild-type eIF4E, but not of a substitution mutant that is not phosphorylated, leads to malignant transformation and high rates of protein synthesis (10
). Studies of cells transformed by eIF4E overexpression show an increased ability to translate mRNAs with increased cap-proximal secondary structure (37
), consistent with increased binding of the eIF4F complex and its associated helicase activity. While some other reports indicate that changes in eIF4E phosphorylation do not invariably correlate with increased translation (34
), it seems likely that eIF4E phosphorylation modulates translation initiation in cells.
Different signal transduction pathways control the phosphorylation of 4EBP1, S6, and eIF4E. In fibroblasts, the phosphorylation of 4EBP1 and S6 occurs by an extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase-independent signaling pathway that is sensitive to a specific inhibitor, rapamycin (4
). In contrast, mitogen-stimulated eIF4E phosphorylation occurs via a rapamycin-insensitive, Ras- and ERK/MAP kinase-dependent pathway (14
). This means that phosphorylation of eIF4E is independent of phosphorylation of 4EBPs. eIF4E is also phosphorylated in response to stresses, including anisomycin and hypertonicity, dependent on the p38 MAP kinase (49
). This suggests that the MAP kinases ERK and p38, or a protein kinase activated by ERK or p38, may phosphorylate eIF4E in cells. However, neither ERK nor p38 is a candidate to phosphorylate eIF4E directly, because the residue phosphorylated, Ser 209 (13
), lacks proline residues required for MAP kinase recognition (1
). Therefore, eIF4E is more likely to be phosphorylated by one or more MAP kinase-dependent protein kinases.
A number of MAP kinase-dependent kinases are now known, and of these, Mnk1, MAPKAPK-3 (3pK), and Msk1 are activated by both ERK and p38 (9
). Direct in vitro phosphorylation assays suggest that MAPKAPK-3 and Mnk1 can both phosphorylate eIF4E directly, while certain other MAP kinase-dependent protein kinases, including Rsk and MAPKAPK-2, cannot (51
). Under a variety of stimulation and inhibitor conditions, the in vivo phosphorylation of eIF4E correlates with the activity of Mnk1 (70
). This correlative evidence suggests that Mnk1, or a similarly activated protein kinase, phosphorylates eIF4E in cells. In view of the potential importance of eIF4E phosphorylation in regulating translation initiation, we have investigated whether Mnk1 phosphorylates eIF4E in vivo. In this report, we demonstrate that Mnk1 is a member of the eIF4F complex, binding directly to eIF4G. We have identified activating phosphorylation sites in Mnk1 and created activated and dominant negative mutants. Coexpression of a dominant negative mutant Mnk1 inhibits mitogen-induced and basal phosphorylation of eIF4E, while expression of an activated mutant Mnk1 results in constitutive phosphorylation of eIF4E. Phosphorylation of eIF4E by Mnk1 also occurs in the presence of excess 4EBP1. We suggest that Mnk1 or a protein kinase with similar binding properties and enzymatic specificity phosphorylates eIF4E in mitogen- and stress-stimulated cells.