HOME > Members > GCOE Organizing Members > Masaki Inagaki MD, PhD

Masaki Inagaki MD, PhDVisiting Professor, Department of Cellular Oncology, Nagoya University Graduate School of Medicine

Specialized field

Cell Biology (Cytoskeleton and Cell Cycle)

Career Summary

M.D. Faculty of Medicine, Mie University
Ph.D. Faculty of Medicine, Mie University
Researcher, Laboratory of Experimental Radiology,
Aichi Cancer Center Research Institute
Senior Researcher, Laboratory of Experimental Radiology,
Aichi Cancer Center Research Institute
Head (Chief), Department of Neurophysiology,
Tokyo Metropolitan Institute of Gerontology
Chief, Division of Biochemistry,
Aichi Cancer Center Research Institute
Visiting Professor, Department of Cellular Oncology,
Graduate School of Medicine, Nagoya University,

Research Theme

Mechanisms of the regulation of cell architecture and cell cycle

Research Summary

Mechanisms of the regulation of cell architecture and cell cycle

A. Aim
[1] Checkpoint kinase 1 (Chk1) is one of the most important contributors to genetic stability, disorders of which are often observed in cancer cells. In response to stalled replication and genotoxic stresses, Chk1 is phosphorylated at Ser317 and Ser345 by ataxia-telangiectasia mutated- and Rad3-related (ATR) and thereby activated. This prevents premature entry into mitosis through inhibition of cyclin-dependent kinases (Cdks). To further dissect Chk1 function, we have studied the roles of Chk1 phosphorylation.
[2] How cells coordinate proliferation and differentiation is a fundamental problem in cell and developmental biology, as well as in cancer biology. The keratin intermediate filament network is abundant in epithelial cells, but its function in the regulation of proliferation and differentiation is unclear. To address this issue, we have performed searches for keratin-binding proteins. In this study, we examined the function of trichoplein, a novel keratin filament-binding protein.

B. Results
[1] The ATR-Chk1 pathway is a sentinel of cell-cycle progression. On the other hand, the Ras-MAPK-p90 RSK pathway is a central node in cell signaling downstream of growth factors. These pathways are closely correlated in cell proliferation but their interaction is largely unknown. Here we have elucidated the consequences of Chk1 phosphorylation at Ser280 by p90 RSK (90 kDa ribosomal S6 kinase) downstream of growth factor stimulation. Following stimulation of receptor tyrosine kinase with growth factor, p90 RSK is activated downstream of the MAPK (mitogen-activated protein kinase) cascade and then phosphorylates Chk1 specifically at Ser280. Although Chk1 constantly shuttles between the cytoplasm and nucleus, Ser280 phosphorylation promotes nuclear retention of Chk1. Since Chk1 is activated in the nucleus, such nuclear accumulation is likely to be of great use in the preparation for DNA damage checkpoint. Together with Chk1 phosphorylation at Ser345 by ATR and its autophosphorylation at Ser296, which are critical for checkpoint signaling, Chk1-Ser280 phosphorylation is elevated in a p90RSK-dependent manner after UV irradiation. In addition, Chk1 phosphorylation at Ser345 and Ser296 after UV irradiation is attenuated by the treatment with p90 RSK inhibitor or by Ser280 mutation to Ala. These results suggest that p90 RSK facilitates nuclear Chk1 accumulation through Chk1-Ser280 phosphorylation and that this pathway plays an important role in the preparation for monitoring genetic instability during cell proliferation. Since the Ras-MAPK pathway is up-regulated in a wide spectrum of human cancers, our observations point to a possibility that the p90 RSK-Chk1 pathway may serve as a barrier to protect genomic integrity in the case of Ras-MAPK up-regulation (Fig.1).
Our studies have shown that Chk1 regulation is more complex than previously considered. Further analyses are required to evaluate Chk1 as a molecular target for cancer therapy.
[2] The primary cilium is an antenna-like organelle which modulates differentiation, sensory functions and signal transduction. Once cilia are disassembled at G0/G1 transition, ciliary reproduction is strictly inhibited in proliferating cells. However, the mechanisms responsible for this inhibition remain largely unknown.
We previously reported that trichoplein, a novel keratin-binding protein, localizes to the centriole in proliferating cells, in addition to its localization on cell-cell contacts in polarized epithelial cells. Recently, we further found that the centriolar trichoplein disappears from ciliated mother centrioles in quiescent cells. Exogenous expression of trichoplein inhibited primary cilia assembly in serum-starved cells whereas RNAi-mediated depletion accelerated assembly under cultivation with serum. In vitro and in vivo kinase assays revealed that trichoplein controls Aurora A (AurA) activation at the centrioles predominantly in G1 phase. In vitro analyses further confirmed that trichoplein binds and activates AurA directly. Using trichoplein fragments and mutants, we could demonstrate that 1-130 a.a. of trichoplein is a minimum requirement for centriolar localization and blocking cilia assembly. However, the mutant with Ala52 and Trp54 to Asp, featuring weakened binding to AurA, failed to block cilia assembly despite its centriolar localization. Collectively, our results indicate that suppression of primary cilia assembly by trichoplein necessitates not only the ability to localize to centrioles but also binding and activation of AurA. With respect to cell cycle regulation, trichoplein- or AurA-knockdown also induced G1 arrest but this phenotype was reversed by simultaneous knockdown of IFT20, a treatment resulting in an inability to form cilia in cells. These data suggest that the trichoplein-AurA pathway is integral to continuous suppression of primary cilia assembly, which is required for G1 progression (Fig.2).

Principal Research Achievement

  1. Inoko A, Matsuyama M, Goto H, Ohmuro-Matsuyama Y, Hayashi Y, Enomoto M, Ibi M, Urano T, Yonemura S, Kiyono T, Izawa I, Inagaki M: Trichoplein and Aurora A block aberrant primary cilia assembly in proliferating cells. J Cell Biol, in press (2012)
  2. Li P, Goto H, Kasahara K, Matsuyama M, Wang Z, Yatabe Y, Kiyono T, Inagaki M: P90 RSK arranges Chk1 in the nucleus for monitoring of genomic integrity during cell proliferation. Mol Biol Cell, in press (2012)
  3. Goto H, Izawa I, Li P, Inagaki M: Novel regulation of checkpoint kinase 1 (Chk1): Is Chk1 a good candidate for anti-cancer therapy? Cancer Sci, in press (2012)
  4. Ohmuro-Matsuyama Y, Inagaki M, Ueda H. Detection of Protein Phosphorylation by Open-Sandwich Immunoassay. In Integrative Proteomics, Leung H-C, ed. (InTech), pp. 197-214 (2012)
  5. Ibi M, Zou P, Inoko A, Shiromizu T, Matsuyama M, Hayashi Y, Enomoto M, Mori D, Hirotsune S, Kiyono T, Tsukita S, Goto H, Inagaki M: Trichoplein controls microtubule anchoring at the centrosome by binding to Odf2 and ninein. J Cell Sci, 124, 857-864 (2011)
  6. Helfand BT, Mendez MG, Murthy SN, Shumaker DK, Grin B, Mahammad S, Aebi U, Wedig T, Wu YI, Hahn KM, Inagaki M, Herrmann H, Goldman RD: Vimentin organization modulates the formation of lamellipodia. Mol Biol Cell, 22, 1274-1289 (2011)
  7. Matsuyama M, Goto H, Kasahara K, Kawakami Y, Nakanishi M, Kiyono T, Goshima N, Inagaki M: Nuclear Chk1 prevents premature mitotic entry. J Cell Sci, 124, 2113-2119 (2011)
  8. Ichijima Y, Yoshioka K, Yoshioka Y, Shinohe K, Fujimori H, Unno J, Takagi M, Goto H, Inagaki M, Mizutani S, Teraoka H: DNA lesions induced by replication stress trigger mitotic aberration and tetraploidy development. PLoS One, 5, e8821 (2010)
  9. Kasahara K, Goto H, Enomoto M, Tomono Y, Kiyono T, Inagaki M. 14-3-3γ mediates Cdc25A proteolysis to induce S and G2 arrest after DNA damage. EMBO J, 29, 2802-2812 (2010)
  10. Kawase T, Matsuo K, Suzuki T, Hirose K, Hosono S, Watanabe M, Inagaki M, Iwata H, Tanaka H, Tajima K. Association between vitamin D and calcium intake and breast cancer risk according to menopausal status and receptor status in Japan. Cancer Sci, 101, 1234-1240 (2010)
  11. Bargagna-Mohan P, Paranthan RR, Hamza A, Dimova N, Trucchi B, Srinivasan C, Elliott GI, Zhan CG, Lau DL, Zhu H, Kasahara K, Inagaki M, Cambi F, Mohan R. Withaferin A targets intermediate filaments GFAP and vimentin in A model of retinal gliosis. J Biol Chem, 285, 7657-7669 (2010)
  12. Ichijima Y, Yoshioka K, Yoshioka Y, Shinohe K, Fujimori H, Unno J, Takagi M, Goto H, Inagaki M, Mizutani S, Teraoka H. DNA lesions induced by replication stress trigger mitotic aberration and tetraploidy development. PLoS ONE, 5, e8821 (2010)
  13. Enomoto M, Goto H, Tomono Y, Kasahara K, Tsujimura K, Kiyono T, Inagaki M. Novel positive feedback loop between Cdk1 and Chk1 in the nucleus during the G2/M transition. J Biol Chem, 284, 34223-30 (2009)
  14. Li ZF, Wu X, Jiang Y, Liu J, Wu C, Inagaki M, Izawa I, Mizisin AP, Engvall E, Shelton GD. Non-pathogenic protein aggregates in skeletal muscle in MLF1 transgenic mice. J Neurol Sci, 264, 77-86 (2008)
  15. Toyo-oka K, Mori D, Yano Y, Shiota M, Iwao H, Goto H, Inagaki M, Hiraiwa N, Muramatsu M, Wynshaw-Boris A, Yoshiki A, Hirotsune S. Protein phosphatase 4 catalytic subunit regulates Cdk1 activity and microtubule organization via NDEL1 dephosphorylation. J Cell Biol, 180, 1133-1147 (2008)
  16. Izawa I, Nishizawa M, Hayashi Y, Inagaki M. Palmitoylation of ERBIN is required for its plasma membrane localization. Genes Cells, 13, 691-701 (2008)
  17. Lin YM, Chen YR, Lin JR, Wang WJ, Inoko A, Inagaki M, Wu YC, Chen RH. eIF3k regulates apoptosis in epithelial cells by releasing caspase 3 from keratin-containing inclusions. J Cell Sci, 121, 2382-2393 (2008)
  18. Sugimoto M, Inoko A, Shiromizu T, Nakayama M, Zou P, Yonemura S, Hayashi Y, Izawa I, Sasoh M, Uji Y, Kaibuchi K, Kiyono T, Inagaki M. The keratin-binding protein Albatross regulates polarization of epithelial cells. J Cell Biol,183, 19-28 (2008)
  19. Ikegami Y, Goto H, Kiyono T, Enomoto M, Kasahara K, Tomono Y, Tozawa K, Morita A, Kohri K, Inagaki M. Chk1 phosphorylation at Ser286 and Ser301 occurs with both stalled DNA replication and damage checkpoint stimulation. Biochem Biophys Res Commun, 377, 1227-1231 (2008)
  20. Goto H et al. Production of a site- and phosphorylation state-specific antibody. Nature Protocols 2: 2574-2581 (2007)
  21. Goto H et al. Complex formation of Plk1 and INCENP required for Metaphase-anaphase transition. Nature Cell Biol. 8: 180-187 (2006)
  22. Yamaguchi T et al. Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis. J. Cell Biol. 171: 431-436 (2005)
  23. Ivaska J et al. PKCepsilon-mediated phosphorylation of vimentin controls integrin recycling and motility. EMBO J. 24: 3834-3845 (2005)
  24. Minoshima Y et al. Aurora B Phosphorylates MgcRacGAP and Induces PhoGAP Activity during M Phase : Identification of a RhoGAP Indispensable for Cytokinesis. Dev. Cell 4: 549-560 (2003)
  25. Inada H et al. Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J. Cell Biol. 155: 415-425 (2001)
  26. Hirota T et al. Zyxin, a regulator of actin filament assembly, targets the mitotic apparatus by interacting with h-warts/LATS1 tumor suppressor. J Cell Biol. 149: 1073-1086 (2000)
  27. Kawano Y et al. Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo. J. Cell Biol. 147: 1023-1038 (1999)
  28. Yasui Y et al. Roles of Rho-associated kinase in cytokinesis; Mutations in Rho-associated kinase phosphorylation sites impair cytokinetic segregation of glial filaments. J. Cell Biol. 143: 1249-1258 (1998)
  29. Sekimata M et al. Detection of protein kinase activity specifically activated at metaphase-anaphase transition. J. Cell Biol. 132: 635-641 (1996)
  30. Ogawara M et al. Differential targeting of protein kinase C and CaM kinase II signalings to vimentin J. Cell Biol. 131: 1055-1066 (1995)
  31. Matsuoka Y et al. Two different protein kinases act on a different time schedule as glial filament kinase during mitosis. EMBO J. 11: 2895-2902 (1992)
  32. Fukami K et al. Requirement of phosphatidylinositol 4.5-bisphosphate for α-actinin function. Nature 359: 150-152 (1992)
  33. Nishizawa K et al. Specific localization of phospho-intermediate filament protein in the constricted area of dividing cells. J. Biol. Chem. 266: 3074-3079 (1991)
  34. Okamoto T et al. Identification of Gs activator region of the β2-adrenergic receptor that is autoregulated via PKA-dependent phosphorylation. Cell 67: 723-730 (1991)
  35. Ohno S et al.Tissue-specific expression of three distinct types of rabbit protein kinase C. Nature 325: 161-166 (1987)
  36. Inagaki M et al. Site-specific phosphorylation induces disassembly of vimentin filaments in vitro. Nature 328: 649-652 (1987)


1987: Award of San-ikai; Biopharmacological study of protein kinase C
1994: Incitement Award of the Japanese Cancer Association; Biological significance of protein phosphorylation on the cytoskeleton disruption in malignant cells
2001: Award of Inoue Foundation for Science; Establishment of a site- and phosphorylation state-specific antibody, and its applicative study.