Kato-Homma, M. et al. CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression. Proc. Natl Acad. Sci. USA 102, 15688-15693

Department of Biomolecular Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2005; 102(43):15688-93. DOI: 10.1073/pnas.0506791102
Source: PubMed


Casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser/Thr protein kinase that plays an important role in cell cycle progression. Although its function in this process remains unclear, it is known to be required for the G(1) and G(2)/M phase transitions in yeast. Here, we show that CK2 activity changes notably during cell cycle progression and is increased within 3 h of serum stimulation of quiescent cells. During the time period in which it exhibits high enzymatic activity, CK2 associates with and phosphorylates a key molecule for translation initiation, eukaryotic translation initiation factor (eIF) 5. Using MS, we show that Ser-389 and -390 of eIF5 are major sites of phosphorylation by CK2. This is confirmed using eIF5 mutants that lack CK2 sites; the phosphorylation levels of mutant eIF5 proteins are significantly reduced, relative to WT eIF5, both in vitro and in vivo. Expression of these mutants reveals that they have a dominant-negative effect on phosphorylation of endogenous eIF5, and that they perturb synchronous progression of cells through S to M phase, resulting in a significant reduction in growth rate. Furthermore, the formation of mature eIF5/eIF2/eIF3 complex is reduced in these cells, and, in fact, restricted diffusional motion of WT eIF5 was almost abolished in a GFP-tagged eIF5 mutant lacking CK2 phosphorylation sites, as measured by fluorescence correlation spectroscopy. These results suggest that CK2 may be involved in the regulation of cell cycle progression by associating with and phosphorylating a key molecule for translation initiation.

Download full-text


Available from: Miwako K Homma, Sep 02, 2014
9 Reads
  • Source
    • "c-Src proteins recovered on an anti-FLAG M2 affinity gel from T98G cells expressing C-FLAG-tagged CA-c-Src were incubated with or without synthetic peptides (200 μg/ml) in kinase buffer consisting of 20 mM Hepes/NaOH, pH 7.4, 10 mM MgCl2 50 μM ATP and 92.5 kBq of γ-[32P]ATP in a total volume of 50 μl at 30°C for 20 min as described previously [22]. The synthetic peptides used were INDNYYEDLTAK [(NDUFV2 WT (wild-type)], INDNYFEDLTAK (NDUFV2 Y193F mutant), RYDTSYFVE (SDHA WT) and RYDTSFFVE (SDHA Y215F mutant). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr193 of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr215 of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability.
    Biochemical Journal 07/2012; 447(2):281-9. DOI:10.1042/BJ20120509 · 4.40 Impact Factor
    • "Interestingly, MPME significantly increased cofilin-1 and FBP aldolase 1, respectively involved in cells morphology and in cells energy metabolism (Esposito et al., 2004; Bai et al., 2011). Furthermore, MPME maintained the expression levels of Eif-5a-1 and CK-2e, respectively involved in cell cycle progression and cell proliferation, maintaining or improving the healthcare status of the cells (Collin et al., 1992; Homma et al., 2005). Therefore it is possible that MPME is able to modulate the physiologic cellular status thanks to the ability to induce key proteins involved in " cellular homeostasis " . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mucuna pruriens (Fabaceae) is an established herbal drug used for the management of male infertility, nervous disorders, and also as an aphrodisiac. It has been shown that its seeds are potentially of substantial medicinal importance. The ancient Indian medical system, Ayurveda, traditionally used M. pruriens, even to treat such things as Parkinson's disease. M. pruriens has been shown to have anti-parkinson and neuroprotective effects, which may be related to its anti-oxidant activity. In addition, anti-oxidant activity of M. pruriens has been also demonstrated in vitro by its ability to scavenge DPPH radicals and reactive oxygen species. In this review the medicinal properties of M. pruriens are summarized, taking in consideration the studies that have used the seeds extracts and the leaves extracts.
    Journal of Traditional and Complementary Medicine 03/2012; 2(4):331-339.
  • Source
    • "FL-5MP1 and its mutants were purified from H2557 transformants carrying pEMBL-FL-5MP1 and its derivatives, that had been grown in synthetic complete galactose medium lacking uracil (SCGal-ura), as described (22). Recombinant eIF5 was purified by G50 column fractionation after thrombin treatment of GST-human eIF5 (23). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The translation factor eIF5 is an important partner of eIF2, directly modulating its function in several critical steps. First, eIF5 binds eIF2/GTP/Met-tRNA(i)(Met) ternary complex (TC), promoting its recruitment to 40S ribosomal subunits. Secondly, its GTPase activating function promotes eIF2 dissociation for ribosomal subunit joining. Finally, eIF5 GDP dissociation inhibition (GDI) activity can antagonize eIF2 reactivation by competing with the eIF2 guanine exchange factor (GEF), eIF2B. The C-terminal domain (CTD) of eIF5, a W2-type HEAT domain, mediates its interaction with eIF2. Here, we characterize a related human protein containing MA3- and W2-type HEAT domains, previously termed BZW2 and renamed here as eIF5-mimic protein 1 (5MP1). Human 5MP1 interacts with eIF2 and eIF3 and inhibits general and gene-specific translation in mammalian systems. We further test whether 5MP1 is a mimic or competitor of the GEF catalytic subunit eIF2Bε or eIF5, using yeast as a model. Our results suggest that 5MP1 interacts with yeast eIF2 and promotes TC formation, but inhibits TC binding to the ribosome. Moreover, 5MP1 is not a GEF but a weak GDI for yeast eIF2. We propose that 5MP1 is a partial mimic and competitor of eIF5, interfering with the key steps by which eIF5 regulates eIF2 function.
    Nucleic Acids Research 07/2011; 39(19):8314-28. DOI:10.1093/nar/gkr339 · 9.11 Impact Factor
Show more