Rie Watanabe-Fukunaga

Mnk2 and Mnk1 Are Essential for Constitutive and Inducible Phosphorylation of Eukaryotic Initiation Factor 4E but Not for Cell Growth or Development

ABSTRACT Mnk1 and Mnk2 are protein kinases that are directly phosphorylated and activated by extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein (MAP) kinases and implicated in the regulation of protein synthesis through their phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) at Ser209. To investigate their physiological functions, we generated mice lacking the Mnk1 or Mnk2 gene or both; the resulting KO mice were viable, fertile, and developed normally. In embryonic fibroblasts prepared from Mnk1-Mnk2 DKO mice, eIF4E was not detectably phosphorylated at Ser209, even when the ERK and/or p38 MAP kinases were activated. Analysis of embryonic fibroblasts from single KO mice revealed that Mnk1 is responsible for the inducible phosphorylation of eIF4E in response to MAP kinase activation, whereas Mnk2 mainly contributes to eIF4Es basal, constitutive phosphorylation. Lipopolysaccharide (LPS)- or insulin-induced upregulation of eIF4E phosphorylation in the spleen, liver, or skeletal muscle was abolished in Mnk1−/− mice, whereas the basal eIF4E phosphorylation levels were decreased in Mnk2−/− mice. In Mnk1-Mnk2 DKO mice, no phosphorylated eIF4E was detected in any tissue studied, even after LPS or insulin injection. However, neither general protein synthesis nor cap-dependent translation, as assayed by a bicistronic reporter assay system, was affected in Mnk-deficient embryonic fibroblasts, despite the absence of phosphorylated eIF4E. Thus, Mnk1 and Mnk2 are exclusive eIF4E kinases both in cultured fibroblasts and adult tissues, and they regulate inducible and constitutive eIF4E phosphorylation, respectively. These results strongly suggest that eIF4E phosphorylation at Ser209 is not essential for cell growth during development.

Nuclear cataract caused by a lack of DNA degradation in the mouse eye lens

The eye lens is composed of fibre cells, which develop from the epithelial cells on the anterior surface of the lens. Differentiation into a lens fibre cell is accompanied by changes in cell shape, the expression of crystallins and the degradation of cellular organelles. The loss of organelles is believed to ensure the transparency of the lens, but the molecular mechanism behind this process is not known. Here we show that DLAD (‘DNase II-like acid DNase’, also called DNase IIβ) is expressed in human and murine lens cells, and that mice deficient in the DLAD gene are incapable of degrading DNA during lens cell differentiation—the undigested DNA accumulates in the fibre cells. The DLAD-/- mice develop cataracts of the nucleus lentis, and their response to light on electroretinograms is severely reduced. These results indicate that DLAD is responsible for the degradation of nuclear DNA during lens cell differentiation, and that if DNA is left undigested in the lens, it causes cataracts of the nucleus lentis, blocking the light path.

Human and mouse Fas (APO-1/CD95) death receptor genes each contain a p53-responsive element that is activated by p53 mutants unable to induce apoptosis

p53 is a tumor suppressor protein that induces apoptosis at least in part through its ability to act as a sequence-specific transactivator. This work reports that intron 1 of the mouse Fas death receptor gene contains a p53-responsive element (p53RE) that matches the p53 consensus sequence and that is located between nucleotides +1704 and +1723 from the transcription initiation site. This element is specifically bound by p53 and functions as a p53-dependent enhancer in mammalian or in yeast reporter gene assays. Contrary to bax, another known pro-apoptotic p53-target gene, both mouse and human FASp53REs are still activated by the discriminatory p53 mutants Pro-175 and Ala-143, a class of mutants unable to induce apoptosis. We propose that p53-dependent up-regulation of Fas does not induce apoptosis per se but sensitizes the cell to other pro-apoptotic signal(s). The functional conservation of p53-dependent Fas up-regulation argues strongly in favor of its biological importance and suggests that murine models may be used to study further the in vivo role of Fas in the p53 response.

Inhibition of Mammalian Target of Rapamycin Induces Phosphatidylinositol 3-Kinase-Dependent and Mnk-Mediated Eukaryotic Translation Initiation Factor 4E Phosphorylation

ABSTRACT The initiation factor eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in initiating translation of mRNAs, including those encoding oncogenic proteins. Therefore, eIF4E is considered a survival protein involved in cell cycle progression, cell transformation, and apoptotic resistance. Phosphorylation of eIF4E (usually at Ser209) increases its binding affinity for the cap of mRNA and may also favor its entry into initiation complexes. Mammalian target of rapamycin (mTOR) inhibitors suppress cap-dependent translation through inhibition of the phosphorylation of eIF4E-binding protein 1. Paradoxically, we have shown that inhibition of mTOR signaling increases eIF4E phosphorylation in human cancer cells. In this study, we focused on revealing the mechanism by which mTOR inhibition increases eIF4E phosphorylation. Silencing of either mTOR or raptor could mimic mTOR inhibitors’ effects to increase eIF4E phosphorylation. Moreover, knockdown of mTOR, but not rictor or p70S6K, abrogated rapamycins ability to increase eIF4E phosphorylation. These results indicate that mTOR inhibitor-induced eIF4E phosphorylation is secondary to mTOR/raptor inhibition and independent of p70S6K. Importantly, mTOR inhibitors lost their ability to increase eIF4E phosphorylation only in cells where both Mnk1 and Mnk2 were knocked out, indicating that mTOR inhibitors increase eIF4E phosphorylation through a Mnk-dependent mechanism. Given that mTOR inhibitors failed to increase Mnk and eIF4E phosphorylation in phosphatidylinositol 3-kinase (PI3K)-deficient cells, we conclude that mTOR inhibition increases eIF4E phosphorylation through a PI3K-dependent and Mnk-mediated mechanism. In addition, we also suggest an effective therapeutic strategy for enhancing mTOR-targeted cancer therapy by cotargeting mTOR signaling and Mnk/eIF4E phosphorylation.

Eukaryotic Translation Initiation Factor 4F Architectural Alterations Accompany Translation Initiation Factor Redistribution in Poxvirus-Infected Cells

ABSTRACT Despite their self-sufficient ability to generate capped mRNAs from cytosolic DNA genomes, poxviruses must commandeer the critical eukaryotic translation initiation factor 4F (eIF4F) to recruit ribosomes. While eIF4F integrates signals to control translation, precisely how poxviruses manipulate the multisubunit eIF4F, composed of the cap-binding eIF4E and the RNA helicase eIF4A assembled onto an eIF4G platform, remains obscure. Here, we establish that the poxvirus infection of normal, primary human cells destroys the translational repressor eIF4E binding protein (4E-BP) and promotes eIF4E assembly into an active eIF4F complex bound to the cellular polyadenylate-binding protein (PABP). Stimulation of the eIF4G-associated kinase Mnk1 promotes eIF4E phosphorylation and enhances viral replication and protein synthesis. Remarkably, these eIF4F architectural alterations are accompanied by the concentration of eIF4E and eIF4G within cytosolic viral replication compartments surrounded by PABP. This demonstrates that poxvirus infection redistributes, assembles, and modifies core and associated components of eIF4F and concentrates them within discrete subcellular compartments. Furthermore, it suggests that the subcellular distribution of eIF4F components may potentiate the complex assembly.

Type I interferon (IFN)-dependent activation of Mnk1 and its role in the generation of growth inhibitory responses

We provide evidence for the existence of an IFN-regulated cellular pathway involving the mitogen-activated protein kinase (MAPK)-integrating kinase (Mnk) 1. Our data demonstrate that type I (α, β) IFNs induce phosphorylation/activation of Mnk1, which, in turn, regulates phosphorylation of the eukaryotic initiation factor 4E (eIF4E) on Ser-209. Such Mnk activation depends on upstream engagement of Jak1, and requires downstream activation of the Mek/Erk MAPK pathway. In studies using double Mnk1−/−Mnk2−/− knockout mouse embryonic fibroblasts (MEFs), we found that engagement of Mnk kinases is essential for mRNA translation of the Isg15 and Isg54 genes, suggesting an important role for this pathway in mRNA translation of IFN-stimulated genes (ISGs). Importantly, our data demonstrate that pharmacological inhibition of Mnk kinases or siRNA-mediated knockdown of Mnk1 and Mnk2 results in partial reversal of the suppressive effects of IFNα on normal and leukemic hematopoietic progenitors, establishing a key role for this pathway in the generation of the growth inhibitory effects of type I IFNs. Together, our findings establish that the Mnk/eIF4E kinase pathway is activated in an IFN-inducible manner and plays important roles in mRNA translation for ISGs and generation of IFN-inducible anti-proliferative responses.

The fused protein kinase regulates Hedgehog-stimulated transcriptional activation in Drosophila Schneider 2 cells

The Drosophila segment polarity genefused encodes a putative protein-serine/threonine kinase, and plays a critical role in the signal transduction for Hedgehog (Hh)-dependent gene expression. We show that theDrosophila Schneider 2 (S2) cell line has the potential to transduce the Hh-triggered intracellular signals, leading to the activation of target gene expression, when a transcription factor, Cubitus interruptus (Ci), is provided exogenously. Using S2 cells transfected with the Ci-expressing plasmid and a patchedpromoter reporter construct, we demonstrate that the forced expression of Fused (Fu) stimulates Hh-triggered and Ci-dependent transcriptional activation. The N-terminal kinase domain of Fu is required for this activity, but the C-terminal domain is not. Two kinase-inactive Fu mutants fail to enhance the reporter activation, indicating that the kinase catalytic activity is essential for this function. Negative components of the Hh-signaling pathway, Costal-2 and Suppressor of Fused, strongly antagonize the Fu activity, irrespective of the presence or absence of the Fu C-terminal domain, suggesting an indirect mechanism for the inhibition of Fu by these proteins. Furthermore, mutational analyses of threonine 158 and serine 159, in the activation segment of the Fu protein kinase, indicate that threonine 158 is essential for Fu activity and that phosphorylation of this threonine residue may be involved in the activation of the kinase catalytic activity upon Hh stimulation.

Regulation of Arsenic Trioxide-induced Cellular Responses by Mnk1 and Mnk2

Arsenic trioxide (As2O3) is a potent inducer of apoptosis of malignant cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As2O3 induces phosphorylation/activation of the MAPK signal-integrating kinases (Mnks) 1 and 2 in leukemia cell lines. Such activation is defective in cells with targeted disruption of the p38α MAPK gene, indicating that it requires upstream engagement of the p38 MAPK pathway. Studies using Mnk1–/– or Mnk2–/–, or double Mnk1–/–Mnk2–/– knock-out cells, establish that activation of Mnk1 and Mnk2 by arsenic trioxide regulates downstream phosphorylation of the eukaryotic initiation factor 4E at Ser-209. Importantly, arsenic-induced apoptosis is enhanced in cells with targeted disruption of the Mnk1 and/or Mnk2 genes, suggesting that these kinases are activated in a negative-feedback regulatory manner, to control generation of arsenic trioxide responses. Consistent with this, pharmacological inhibition of Mnk activity enhances the suppressive effects of arsenic trioxide on primary leukemic progenitors from patients with acute leukemias. Taken together, these findings indicate an important role for Mnk kinases, acting as negative regulators for signals that control generation of arsenic trioxide-dependent apoptosis and antileukemic responses.

Loss of MNK function sensitizes fibroblasts to serum-withdrawal induced apoptosis

Map kinase‐interacting protein kinases 1 and 2 (MNK1, MNK2) function downstream of p38 and ERK MAP kinases, but there are large gaps in our knowledge of how MNKs are regulated and function. Mice deleted of both genes are apparently normal, suggesting that MNKs function in adaptive pathways during stress. Here, we show that mouse embryo fibroblasts (MEFs) obtained from mnk1 (–/–)/mnk2 (–/–) as well as mnk1 (–/–) and mnk2 (–/–) mice are sensitized to caspase‐3 activation upon withdrawal of serum in comparison to wild‐type cells. Caspase‐3 cleavage occurs with all cells in the panel, but most rapidly and robustly in cells derived from mice lacking both MNK genes. Treatment of wild‐type MEFs in the panel with a compound (CGP57380) that inhibits MNK1 and MNK2 sensitizes wild‐type cells for serum‐withdrawal induced apoptosis, suggesting that sensitization is due to loss of MNK function and not to a secondary event. Reintroduction of wild‐type MNK1 in the double knockout MEFs results in decreased sensitivity to serum withdrawal that is not observed for wild‐type MNK2, or the kinase dead variant. Our work identifies MNKs as kinases involved in anti‐apoptotic signaling in response to serum withdrawal.

Critical role of the p400/mDomino chromatin‐remodeling ATPase in embryonic hematopoiesis

The SWI2/SNF2 family ATPase, p400/mDomino, is a core subunit of a large chromatin‐remodeling complex, and is currently suggested to play a unique function in histone variant exchange, a process by which chromatin structure is altered. Here, we investigated the role of p400/mDomino in mammalian development by generating mutant mice with a targeted deletion of the N‐terminal domain of p400/mDomino (referred to as mDomΔN/ΔN). The mDomΔN/ΔN mice died on embryonic day 11.5 (E11.5), and displayed an anemic appearance and slight deformity of the neural tube. DNA microarray and quantitative RT‐PCR analyses revealed that all of the embryonic globin genes and a globin chaperone gene were poorly expressed in the mDomΔN/ΔN embryo and yolk sac on E8.5, indicating that primitive erythropoiesis was impaired. A hematopoietic colony assay indicated that the hematopoietic activity of the yolk sac was significantly blocked in the mutant mice. We also found that the expression of a limited set of Hox genes, including Hoxa7, Hoxa9 and Hoxb9, was drastically enhanced in the mDomΔN/ΔN yolk sacs. These results suggest that p400/mDomino plays a critical role in embryonic hematopoiesis by regulating the expression of developmentally essential genes such as those in the Hox gene cluster.