Rikiro Fukunaga

MNK1, a new MAP kinase-activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates

We have developed a novel expression screening method for identifying protein kinase substrates. In this method, a λ phage cDNA expression library is screened by in situ, solid‐phase phosphorylation using purified protein kinase and [γ‐32P]ATP. Screening a HeLa cDNA library with ERK1 MAP kinase yielded cDNAs of previously characterized ERK substrates, c‐Myc and p90RSK, demonstrating the utility of this method for identifying physiological protein kinase substrates. A novel clone isolated in this screen, designated MNK1, encodes a protein‐serine/threonine kinase, which is most similar to MAP kinase‐activated protein kinase 2 (MAPKAP‐K2), 3pK/MAPKAP‐K3 and p90RSK. Bacterially expressed MNK1 was phosphorylated and activated in vitro by ERK1 and p38 MAP kinases but not by JNK/SAPK. Further, MNK1 was activated upon stimulation of HeLa cells with 12‐O‐tetradecanoylphorbol‐13‐acetate, fetal calf serum, anisomycin, UV irradiation, tumor necrosis factor‐α, interleukin‐1β or osmotic shock, and the activation by these stimuli was differentially inhibited by the MEK inhibitor PD098059 or the p38 MAP kinase inhibitor SB202190. Together, these results indicate that MNK1 is a novel class of protein kinase that is activated through both the ERK and p38 MAP kinase signaling pathways.

Human eukaryotic translation initiation factor 4G (eIF4G) recruits Mnk1 to phosphorylate eIF4E

Human eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA cap structure and interacts with eIF4G, which serves as a scaffold protein for the assembly of eIF4E and eIF4A to form the eIF4F complex. eIF4E is an important modulator of cell growth and proliferation. It is the least abundant component of the translation initiation machinery and its activity is modulated by phosphorylation and interaction with eIF4E‐binding proteins (4E‐BPs). One strong candidate for the eIF4E kinase is the recently cloned MAPK‐activated protein kinase, Mnk1, which phosphorylates eIF4E on its physiological site Ser209 in vitro. Here we report that Mnk1 is associated with the eIF4F complex via its interaction with the C‐terminal region of eIF4G. Moreover, the phosphorylation of an eIF4E mutant lacking eIF4G‐binding capability is severely impaired in cells. We propose a model whereby, in addition to its role in eIF4F assembly, eIF4G provides a docking site for Mnk1 to phosphorylate eIF4E. We also show that Mnk1 interacts with the C‐terminal region of the translational inhibitor p97, an eIF4G‐related protein that does not bind eIF4E, raising the possibility that p97 can block phosphorylation of eIF4E by sequestering Mnk1.

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.

Expression cloning of a receptor for murine granulocyte colony-stimulating factor

Two cDNAs encoding the receptor for murine granulocyte colony-stimulating factor (G-CSF) were isolated from a CDM8 expression library of mouse myeloid leukemia NFS-60 cells, and their nucleotide sequences were determined. Murine G-CSF receptor expressed in COS cells could bind G-CSF with an affinity and specificity similar to that of the native receptor expressed by mouse NFS-60 cells. The amino acid sequence encoded by the cDNAs has demonstrated that murine G-CSF receptor is an 812 amino acid polypeptide (Mr, 90,814) with a single transmembrane domain. The extracellular domain consists of 601 amino acids with a region of 220 amino acids that shows a remarkable similarity to rat prolactin receptor. The cytoplasmic domain of the G-CSF receptor shows a significant similarity with parts of the cytoplasmic domain of murine interleukin-4 receptor. A 3.7 kb mRNA coding for the G-CSF receptor could be detected in mouse myeloid leukemia NFS-60 and WEHI-3B D+ cells as well as in bone marrow cells.

Functional domains of the granulocyte colony-stimulating factor receptor.

The granulocyte colony‐stimulating factor (G‐CSF) receptor has a composite structure consisting of an immunoglobulin(Ig)‐like domain, a cytokine receptor‐homologous (CRH) domain and three fibronectin type III (FNIII) domains in the extracellular region. Introduction of G‐CSF receptor cDNA into IL‐3‐dependent murine myeloid cell line FDC‐P1 and pro‐B cell line BAF‐B03, which normally do not respond to G‐CSF, enabled them to proliferate in response to G‐CSF. On the other hand, expression of the G‐CSF receptor cDNA in the IL‐2‐dependent T cell line CTLL‐2 did not enable it to grow in response to G‐CSF, although G‐CSF could transiently stimulate DNA synthesis. Mutational analyses of the G‐CSF receptor in FDC‐P1 cells indicated that the N‐terminal half of the CRH domain was essential for the recognition of G‐CSF, but the Ig‐like, FNIII and cytoplasmic domains were not. The CRH domain and a portion of the cytoplasmic domain of about 100 amino acids in length were indispensable for transduction of the G‐CSF‐triggered growth signal.

PRC1: A Human Mitotic Spindle–Associated CDK Substrate Protein Required for Cytokinesis

We have identified a novel human protein, PRC1, that is involved in cytokinesis. PRC1 is a good substrate for several CDKs in vitro and is phosphorylated in vivo at sites that are phosphorylated by CDK in vitro, strongly suggesting that PRC1 is an in vivo CDK substrate. PRC1 has sequence homology to the budding yeast anaphase spindle elongation factor Ase1p. Like Ase1p, PRC1 protein levels are high during S and G2/M and drop dramatically after cells exit mitosis and enter G1. PRC1 is a nuclear protein in interphase, becomes associated with mitotic spindles in a highly dynamic manner during mitosis, and localizes to the cell mid-body during cytokinesis. Microinjection of anti-PRC1 antibodies into HeLa cells blocked cellular cleavage, but not nuclear division, indicating a functional role for PRC1 in the process of cytokinesis.

Growth and differentiation signals mediated by different regions in the cytoplasmic domain of granulocyte colony-stimulating factor receptor

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that regulates the proliferation and differentiation of neutrophils. The G-CSF receptor (G-CSFR) is a member of the hemopoietic growth factor receptor family. A G-CSFR expression plasmid was introduced into interleukin-3 (IL-3)-dependent mouse myeloid precursor FDC-P1 cells that normally do not respond to G-CSF. G-CSF stimulated proliferation of the transformants, down-regulated Thy-1 and F4/80 antigens on the cell surface, and induced expression of neutrophil-specific genes such as myeloperoxidase (MPO) and leukocyte elastase. On the other hand, neither granulocyte/macrophage colony-stimulating factor (GM-CSF) nor IL-3 induced MPO gene expression, but they inhibited G-CSFR-mediated MPO gene expression. These results suggested that the G-CSFR, but not the IL-3/GM-CSF receptors, transduced the neutrophilic differentiation signal into cells. Mutational analysis of the G-CSFR indicated that the N-terminal region of its cytoplasmic domain is sufficient to transduce the proliferation signal into cells, while the C-terminal region plays an essential role in transducing the differentiation signal.

Combined deficiency for MAP kinase-interacting kinase 1 and 2 (Mnk1 and Mnk2) delays tumor development

MAP kinase-interacting kinase 1 and 2 (Mnk1 and Mnk2) are protein-serine/threonine kinases that are activated by ERK or p38 and phosphorylate eIF4E, which is involved in cap-dependent translation initiation. However, Mnk1/2 double knockout (Mnk-DKO) mice show normal cell growth and development despite an absence of eIF4E phosphorylation. Here we show that the tumorigenesis occurring in the Lck-Pten mouse model (referred to here as tPten−/− mice) can be suppressed by the loss of Mnk1/2. Phosphorylation of eIF4E was greatly enhanced in lymphomas of parental tPten−/− mice compared with lymphoid tissues of wild-type mice, but was totally absent in lymphomas of tPten−/−; Mnk-DKO mice. Notably, stable knockdown of Mnk1 in the human glioma cell line U87MG resulted in dramatically decreased tumor formation when these cells were injected into athymic nude mice. Our data demonstrate an oncogenic role for Mnk1/2 in tumor development, and highlight these molecules as potential anticancer drug targets that could be inactivated with minimal side effects.

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.