Negative Regulation of Protein Translation by Mitogen-Activated Protein Kinase-Interacting Kinases 1 and 2

Abstract

Eukaryotic initiation factor 4E (eIF4E) is a key component of the translational machinery and an important modulator of cell growth and proliferation. The activity of eIF4E is thought to be regulated by interaction with inhibitory binding proteins (4E-BPs) and phosphorylation by mitogen-activated protein (MAP) kinase-interacting kinase (MNK) on Ser209 in response to mitogens and cellular stress. Here we demonstrate that phosphorylation of eIF4E via MNK1 is mediated via the activation of either the Erk or p38 pathway. We further show that expression of active mutants of MNK1 and MNK2 in 293 cells diminishes cap-dependent translation relative to cap-independent translation in a transient reporter assay. The same effect on cap-dependent translation was observed when MNK1 was activated by the Erk or p38 pathway. In line with these findings, addition of recombinant active MNK1 to rabbit reticulocyte lysate resulted in a reduced protein synthesis in vitro, and overexpression of MNK2 caused a decreased rate of protein synthesis in 293 cells. By using CGP 57380, a novel low-molecular-weight kinase inhibitor of MNK1, we demonstrate that eIF4E phosphorylation is not crucial to the formation of the initiation complex, mitogen-stimulated increase in cap-dependent translation, and cell proliferation. Our results imply that activation of MNK by MAP kinase pathways does not constitute a positive regulatory mechanism to cap-dependent translation. Instead, we propose that the kinase activity of MNKs, eventually through phosphorylation of eIF4E, may serve to limit cap-dependent translation under physiological conditions.

Mnk kinases in cytokine signaling and regulation of cytokine responses

“…Many of the studies cited in this review used the Mnk inhibitor CGP57380 which targets both Mnk1 and Mnk2. CGP57380 is a low weight molecular compound that was identified from the Novartis Pharma compound collection by in vitro kinase assays [127] and the IC50 against Mnk1 is seen at a concentration of 2.2 μM [9]. Additionally CGP57380 inhibits casein kinase, MAP2K1 and BR serine/threonine-protein kinase 2 at concentrations comparable to those required for Mnk inhibition [128].…”

“…Moreover, Mnk1/2 −/− mice do not have impaired rate of protein synthesis or cap dependent translation [8]. In another study, the expression of constitutively active Mnk1 and Mnk2 mutants or the activation of Mnk1 by stimulation of either p38 or Erk was found to diminish the rate of cap dependent translation relative to the internal ribosome entry site (IRES) mediated translation [9]. Given the reduced affinity of capped RNA for phosphorylated eIF4E it has been suggested that phosphorylation on S209 may result in the disassociation of eIF4E from the initiation complex allowing the 40S ribosome to scan for the initiation codon [10,11].…”

The serine-threonine kinase MNK1 is post-translationally stabilized by PML-RARα and regulates differentiation of hematopoietic cells

“…Figure 2d depicts a Western blot of MNK1 overexpression in the MNK1 stable cell line compared to cells containing pcDNA3 as the empty control vector. To gain insight into a potential role of MNK1 in eIF4E phosphorylation, we employed a potent small molecular weight inhibitor (CGP57380), which was a kind gift of Dr Gram (Novartis, Knauf et al, 2001). Experiments performed in the presence and absence of the MNK1 inhibitor CGP57380 showed that the eIF4E translation factor was consistently detectable and its expression levels were not altered by MNK1 overexpression.…”

“…Empty vector (pcDNA3)-transfected 32D cells served as control. MNK1-MA is a kinasedeficient mutant and MNK1-AA contains a mutation at a phosphorylation site (Knauf et al, 2001). Both MNK1 mutants are inactive in MNK1 function and could potentially function as dominant negative proteins.…”

“…32D cells were treated with 5 mM CGP57380 (MNK1 inhibitor, Novartis) for different time points (Knauf et al, 2001). After preparation of cell lysates, Western blot analyses were performed for eIF4E, phospho-eIF4E, MNK1 and actin.…”