MAPK Interacting Protein Kinase 1 and 2 (Mnk1 and Mnk2)
A kinase domain consists of an activation loop containing residues that need to be phosphorylated by upstream kinases and a catalytic loop containing an ATP-binding pocket that can phosphorylate the target kinase (Hanks et al. 1988). The activation loop of the Mnk kinases contains three threonine followed by a proline indicating potential MAPK phosphorylation sites (Johnson et al. 1996). Mutation of two threonine to alanine was found to abolish the kinase activity of the Mnk kinases (Waskiewicz et al. 1997; Scheper et al. 2001). The kinase domain of the Mnk kinases is distinguished by the presence of an Asp-Phe-Asp (DFD) in place of the canonical magnesium-binding Asp-Phe-Gly (DFG) motif in the ATP-binding pocket (Jauch et al. 2005). The phenylalanine residue sticks into the ATP-binding site of the non-phosphorylated and inactive kinase requiring a conformational change to activate the kinase activity (Jauch et al. 2005). Additionally the catalytic domain of Mnk1 and Mnk2 contain two inserts relative to other family kinases; the first insert lies between the DFD motif and the activation loop, while the second insert is located C-terminal to the activation loop (Jauch et al. 2005, 2006). The crystal structure of the catalytic domain of Mnk1 and Mnk2 indicates that the distinguishing features of the Mnk catalytic domain may lead to a lowered affinity for ATP as compared to other protein kinases (Jauch et al. 2005, 2006). The C-terminal MAPK-binding domain also plays an important role in the regulation of kinase activity as seen by increase in basal kinase activity on deletion of the C-terminal domain (Goto et al. 2009).
Mnk1 and Mnk2 are now known to undergo alternative splicing resulting in a and b isoforms (Scheper et al. 2003; O’Loghlen et al. 2004) (Fig. 1). The a and b isoforms differ in the last exon, and the b isoforms are characterized by lack of the C-terminal MAPK-binding domain. It is important to note that both the a and b isoforms possess kinase activity but exhibit significant differences in basal activity. The b isoforms lack a MAPK-binding site and hence are not activated in response to stimuli that activate the Erk and/or the p38 MAPK pathways (O’Loghlen et al. 2004, 2007). Additionally the lack of the suppressive C-terminal domain of the longer a isoforms results in higher basal activity of the b isoforms (O’Loghlen et al. 2004, 2007). Mnk1a has low basal activity that can be enhanced by activation of the Erk and or p38 MAPK pathway (Wang et al. 1998; Waskiewicz et al. 1999). In contrast Mnk2a has high basal activity, and its activity is not affected by the activation or inhibition of the Erk and/or p38 MAPK pathway (Parra et al. 2005). The differences in basal activity between Mnk1a and Mnk2a may be partly explained by the observation that while Mnk1a does not stably bind to phosphorylated Erk, Mnk2a is stably bound to phosphorylate Erk (Parra et al. 2005).
The different isoforms also exhibit differences in cellular localization. All Mnk isoforms contain a polybasic N-terminal region that can interact with importin a, a nuclear import protein (Scheper et al. 2003). Mnk1a and Mnk2a are mainly observed in the cytoplasm (Parra-Palau et al. 2003; Scheper et al. 2003). The C-terminal domain of Mnk1a contains a functional nuclear export signal (NES) accounting for its cytoplasmic localization (Scheper et al. 2003). Mnk2a on the other hand lacks a NES, but its C-terminal domain interferes with the interaction between the nuclear export proteins and its N-terminal NLS (Scheper et al. 2003). The Mnk1b and Mnk2b isoforms have been shown to exhibit a nuclear as well as cytoplasmic localization (Scheper et al. 2003; O’Loghlen et al. 2004).
The biological function of proteins is generally assessed by targeted gene deletion of the protein(s) of interest in mice or other animals. Deletion of Mnk1 and/or Mnk2 expression in mice demonstrated no significant phenotype (Ueda et al. 2004). The Mnk1−/−, Mnk2−/−, and Mnk1/2−/− mice were viable, were fertile, and did not exhibit any developmental defects (Ueda et al. 2004). In Mnk1 and Mnk2 knockout mice phosphorylation of eIF4E (eukaryotic initiation factor 4E, a cap-binding protein), a well-characterized substrate of the Mnk kinases was completely abolished despite stimulation with Erk and/or p38 MAPK activators (Ueda et al. 2004). Analysis of eIF4E phosphorylation in Mnk1−/− and Mnk2−/− showed that while Mnk2 expression is important for maintaining the basal phosphorylation of eIF4E, Mnk1 expression is required for eIF4E phosphorylation in response to stimuli that activate the Erk and/or p38 MAPK pathway (Ueda et al. 2004). Interestingly lack of Mnk1 and/or Mnk2 expression in mice had no effect on global protein synthesis or cap-dependent synthesis (Ueda et al. 2004). Collectively these results suggest the expression of Mnk1 and Mnk2 is not critical for survival.
Regulation of Mnk Activity
Besides the p38 and Erk MAPKs, Mnk activity can be positively or negatively regulated by other pathways. MAPK-mediated activation of human Mnk1 results in phosphorylation of Thr 209 and Thr 214 located in the activation domain, whereas mouse Mnk1 is phosphorylated on Thr 197 and Thr 202 (Shveygert et al. 2010). Phosphorylation of Mnk1 enhances its binding to the scaffolding protein eIF4G enabling its interaction with its substrate eIF4E (Pyronnet et al. 1999).
Mnk kinase activity is negatively regulated by p97, an eIF4G-related translational repressor, by the p21-activated kinase 2 (Pak2/γ-Pak) as well as by protein phosphatase A2 (PPA2). P97 shares a 28% homology with the C-terminal domain of eIF4G and can interact with multiple translation initiation factors, and its interaction with the Mnk kinases limits Mnk interaction with eIF4G resulting in decreased activation of its substrate eIF4E (Imataka et al. 1997; Pyronnet et al. 1999). Caspase 3-mediated cleavage of the serine/threonine kinase Pak2 was found to phosphorylate Mnk1 on Thr 22 and Ser 27 in the N-terminal domain resulting in reduced affinity to eIF4G (Orton et al. 2004). Pak2 also phosphorylates eIF4G, preventing its interaction with eIF4E ultimately resulting in decreased Mnk1-mediated phosphorylation of eIF4E (Ling et al. 2005). PPA2 is a phosphatase, and its inhibition results in increased phosphorylation of Mnk1 and its target eIF4E (Li et al. 2010).
Mnk Kinases and Regulation of mRNA Translation
Mnk kinases are sole kinases known to phosphorylate eIF4E (Ueda et al. 2004). eIF4E is a protein that binds to the 7-methyl-guanosine cap structure found at the 5′ end of eukaryotic mRNAs; the 5′ cap plays a role in regulating mRNA stability, translational efficiency, as well as RNA nuclear export (Sonenberg et al. 1979). The Mnk kinases have been shown to phosphorylate eIF4E on Ser 209 (Waskiewicz et al. 1999). Mnk kinases as well as eIF4E can bind the scaffolding protein eIF4G, and this interaction is required for Mnk-mediated phosphorylation of eIF4E (Shveygert et al. 2010). Multiple studies have shown the stimuli that result in an increase in protein synthesis often augment phosphorylation of eIF4E on Ser 209 (Scheper and Proud 2002). These observations suggest the Mnk kinases may play a role in regulating cap-dependent mRNA translation.
Multiple studies have shown an important role in promoting tumorigenesis by increased translation of oncogenic mRNAs via phosphorylation of eIF4E (Proud 2015). Phosphorylation of eIF4E has been shown to enhance the translation of mRNAs with highly structured (high GC content) 5′UTRs (untranslated regions) (Koromilas et al. 1992) such as c-myc, ornithine decarboxylase (ODC), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), etc. (van der Velden and Thomas 1999). Genetic studies in Mnk1/2 knockout mice have shown that lack of Mnk1/2 attenuates Pten loss induced lymphomas (Ueda et al. 2010). Inhibition of Mnk activity has been shown to suppress oncogenesis and/or metastasis in breast cancer (Ramalingam et al. 2014), acute myeloid leukemia (AML) (Kosciuczuk et al. 2016), prostate cancer (Kwegyir-Afful et al. 2016), glioblastoma (Bell et al. 2016), etc. Inhibition of the Mnk kinases has also been shown to improve response to traditional chemotherapy (Altman et al. 2010) as well as targeted therapy (Grzmil et al. 2016). Based on the lack of a phenotype in mice lacking both Mnk1 and Mnk2, inhibition of Mnk activity is a promising anticancer approach with minimal side effects.
Other studies have also shown a role for the Mnk kinases in the regulation of cap-independent translation. The Mnk kinases have been reported to promote the internal ribosome entry site (IRES)-mediated translation of c-myc in multiple myeloma (Shi et al. 2013). Mnk activity also facilitates IRES-mediated cap-independent translation of picornavirus RNA by attenuating the activity of the Ser/Arg (SR)-rich protein kinase (SRPK) (Brown et al. 2014).
The Mnk kinases have also been reported to play a role in the regulating the translation of mRNAs with AU-rich elements in the 3′UTR. T cell activation was reported to result in Mnk-mediated phosphorylation of A-rich element (ARE)-binding protein hnRNPA1 (human ribonuclear protein A1) resulting in attenuated binding of hnRNPA1 to the tumor necrosis factor α (TNFα) 3′UTR, ultimately promoting the translation of the TNFα mRNA (Buxade et al. 2005). The Mnk kinases have also been reported to phosphorylate the ARE-binding protein PSF (polypyrimidine tract-binding (PTB) protein)-associated splicing factor) resulting in enhanced binding to the TNFα 3′UTR, but its effect on the translation of TNFα mRNA was unclear (Buxade et al. 2008).
Mnk Kinases in Inflammation
Multiple studies have suggested an important role for the Mnk kinases in cytokine production and the regulation of cytokine responses (reviewed in Joshi and Platanias (2012)). Inhibition of Mnk activity was shown to result in attenuated production of TNF, IL-6, and monocyte chemoattractant protein-1 and increased IL-10 production in macrophages activated by multiple Toll-like receptor agonists (Rowlett et al. 2008). Additionally the Mnk kinases are now known to regulate the expression of multiple pro-inflammatory cytokines such as TNFα (Buxade et al. 2005), IL-17 (Noubade et al. 2011), as well as CCL5 (Chemokine (C-C motif) ligand 5) (Maruoka et al. 2000). Additionally the Mnk kinases also play an important role in mediating cytokine responses on stimulation with type I and type II IFNs (interferon) (Joshi et al. 2009, 2011), IL-2, IL-15 (Grund et al. 2005) as well as IL-17 (Laan et al. 2001).
Genetic studies in Mnk1 and Mnk2 knockout mice have shown that while the Mnk kinases are not essential for normal T cell development and function, induction of experimental autoimmune encephalomyelitis in mice was found to attenuate IFNγ and IL-17 production by CD4 T cells and decreased differentiation of Th1 and Th17 cells (Gorentla et al. 2013). As dysregulation of pro-inflammatory cytokine production is an integral event in multiple auto-immune diseases, development of Mnk inhibitors may have diverse translational applications.
The Mnk kinases are serine/threonine kinases that are activated by both mitogenic (Erk MAPK) and stress-induced pathways (p38 MAPK) processes. Mnk activity is also regulated by other proteins such as the translational repressor p97, Pak2, and PPA2. Based on their ability to phosphorylate the cap-binding protein eIF4E, Mnk kinases play an import role in regulating protein translation. Additionally Mnk kinases can also regulate cap-independent translation. Mnk kinases also play a central role in regulating pro-inflammatory cytokine production as well as regulating cellular responses on cytokine stimulation.
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