FY2011 Annual Report

Cell Signal Unit

Professor Tadashi Yamamoto

Abstract

Protein-tyrosine kinases are important not only for the development of malignant tumors but also for the regulation of growth and function of normal cells. We are interested in cell signaling downstream of protein-tyrosine kinases that are relevant to physiological properties of mammals such as neuronal function, cancer development and diabetes. Currently we are studying the biological role of Tob, which becomes phosphorylated by MAP kinase upon EGF receptor activation, in DNA damage-induced apoptosis and in adipocyte differentiation. As Tob interacts with the CCR4-NOT deadenylase complex, we are also studying the role of Tob in mRNA deadenylaton. Furthermore, the physiological significance of the CCR4-NOT complex has been characterized using mouse lines in which components of the CCR4-NOT complex are genetically mutated. We are also interested in the function of basal ganglia and are analyzing the molecular and physiological functions of cell adhesion molecules, kinases and adaptor proteins that appeared to be important in neuronal synaptic regulation.

1. Staff

  • Dr.Naosuke Hoshina, Researcehr
  • Dr.Yo-taro Shirai, Researcehr
  • Ms.Miyuki Hoshina,Technical Staff
  • Ms.Chisato Kikuguchi, Technical Staff
  • Ms.Kanako Yamauchi,Staff
  • Ms.Kaori Yamashiro,Research Administrator

2. Collaborations

  • Theme: Physiological studies of the CCR4-NOT complex
    • Type of collaboration: Joint research
    • Researchers:
      • Kuba K and Imai Y. Department of Physiology, Graduate School of Medicine, Akita University
  • Theme: Structural analysis of the CCR4-NOT complex
    • Type of collaboration:Joint research
    • Researchers:
      • Bartlam M and Rao Z. College of Life Sciences, Nankai University, China
  • Theme: Molecular mechanism of mRNA degradation mediated by the CCR4-NOT deadenylase
    • Type of collaboration: Joint research
    • Researchers:
      • Morita M, Fabian M, and Sonenberg N. Department of Biochemistry and Goodman Cancer Research Center, McGill University, Canada
  • Theme: Bioinformatics of gene expression affected by impairment of mRNA degradation machinery
    • Type of collaboration: Joint research
    • Researchers:
      • Nagashima T and Okada M. Laboratory for Cellular System Modeling, RIKEN Research Center for Allergy and Immunology
  • Theme: Identification of signaling proteins downstream of protein tyrosine kinases by mass spectrometry
    • Type of collaboration: Joint research
    • Researchers:
      • Oyama M and Tsumoto K. Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo
  • Theme: Roles of the Fyn tyrosine kinase and its substrates in the central nervous system
    • Type of collaboration: Joint research
    • Researchers:
      • Nakazawa T and Kano M. Department of Neurophysiology, Graduate School of Medicine, University of Tokyo

3. Activities and Findings

Protein-tyrosine kinases are important not only for the development of malignant tumors but also for the regulation of growth and function of normal cells. Our current interest is to characterize cell signaling downstream of protein-tyrosine kinases that are relevant to physiological properties of mammals such as neuronal function cancer development and diabetes.

1. The biological role of Tob family proteins and CCR4-NOT complex.

(i) Studies on the Tob family of proteins.

To dissect signaling pathway downstream of ErbB/EGF receptor family tyrosine kinases, we screened a cDNA expression library with c-ErbB-2 protein and identified tob that encoded a 45kDa protein. Tob displayed homology with the growth suppressing proteins, Btg1 and Btg2/Pc3. Successively we identified and characterized Tob2 and ANA that were homologous to Tob. These proteins composed a functionally related anti-proliferative protein family, called the Tob/Btg family. Upon EGF stimulation MAP kinase became activated downstream of the EGF receptor. We found that MAP kinase in turn phosphorylated Tob, resulting in suppression of the antiproliferative activity of Tob. Further analyses of Tob family proteins with gene manipulated mouse lines revealed followings. #1: Mice lacking Tob were prone to develop cancer. #2: Mice lacking Tob showed osteopetrosis-like phenotype due to enhanced differentiation and proliferation of osteoblasts. In contrast, #3, mice lacking Tob2 had decreased bone mass due to the increment of osteoclasts. #4: Tob2-deficinet mice exhibit increased adiposity with augmented mass of the epididymal WAT. #5: Mice lacking ana, which was specifically expressed in type II alveolar epithelial cells, developed spontaneous lung adenocarcinoma. The underlying molecular mechanisms of these phenotypes have been addressed to reveal that Tob negatively regulates cyclin D expression. Tob was also shown to negatively regulate BMP (bone morphogenetic protein)-mediated signaling. Tob2 was shown to negatively regulate formation of osteoclasts by suppressing RANKL expression through its interaction with Vitamin D3 receptor. In addition, Tob2 could negatively regulate adipogenesis by inhibiting PPARg2 expression.

  Apart from these anti-proliferative and/or anti-differentiation characters, we found that Tob exhibited anti-apoptotic properties in response to DNA damage. UV (high dose)-induced stress promoted proteasome-dependent degradation of Tob, triggering an apoptotic signal. Exogenous expression of Tob that was engineered to be degradation-resistant suppressed UV-induced apoptosis. Interestingly, suppression of Tob by small-interfering RNAs resulted in frequent induction of apoptosis in response to low-doses of UV that do not promote Tob degradation but rather stabilize Tob. This induction of apoptosis occurred irrespectively of the presence or absence of functional p53. Taken together, clearance of Tob provides a novel p53-independent pathway for UV-induced apoptosis. We recently identified several molecules which were responsible for a low dose of UV-induced Tob stabilization. These molecules included Cdc7 and HMGB1 and appeared to contribute to suppression of a low dose of UV-induced apoptosis.

(ii) Studies on the CCR4-NOT deadenylase complex.

  Apparently Tob exhibited multiple functions. To address the molecular mechanisms that would explain the multi-functionality, we carried out mass spectrometric analysis of the Tob-interacting proteins prepared from the lysates of HeLa cells and identified CNOT proteins that formed a large (2.0 MDa and 1.2 MDa) complex called CCR4-NOT. The CCR4-NOT complex was conserved from yeast to human and was associated with the mRNA deadenylase activity. In yeast two components of the complex, Ccr4p and Caf1p, possessed the deadenylase activity. The mammalian orthologs of Ccr4p were CNOT6 and CNOT6L, and those of Caf1p were CNOT7 and CNOT8. Our recent structural analysis of the CNOT6L complexed with nucleotides revealed a deadenylation mechanism involving a pentacovalent phosphate transition. We further found that Tob suppressed the deadenylase activity of CNOT complex at least in vitro.

To help understand the physiological significance of the CCR4-NOT deadenylase complex in mammals, we have knocked-down the expression of each subunit by treating the cultured cells with shRNA and/or siRNA. Depletion of CNOT1 or CNOT2 induced protein overexpression due to the stabilization of many mRNA species, resulting in the ER stress-induced apoptotic death. Depletion of CNOT6L from NIH3T3 cells specifically augmented the stability of p27kip1 mRNA, causing cell cycle arrest at the G1 phase. CNOT3 depletion in cultured cells induced elevation of the mad1 mRNA level and impaired the M-phase checkpoint response. It remains to be elucidated how the CNOT deadenylase complex recognize the specific mRNAs.

To examine the in vivo functions of CCR4-NOT deadenylase complex, we generated gene-engineered mouse lines each lacking individual cnot genes. Recent analysis of cnot3-depleted mice revealed that the CNOT3 subunit of the CCR4-NOT deadenylase complex was critical to metabolic regulation. cnot3+/- mice were lean with hepatic and adipose tissues containing reduced levels of lipids, and showed increased metabolic rates and enhanced glucose tolerance. cnot3+/- mice mice remained lean and sensitive to insulin even on a high-fat diet. Furthermore, introduction of cnot3 haplodeficiency in ob/ob mice ameliorated the obese phenotype. Hepatic expression of most mRNAs was not altered in cnot3+/- vis-à-vis wild-type mice. However, the levels of specific mRNAs, such as those coding for energy metabolism-related PDK4 and IGFBP1, were increased in cnot3+/- hepatocytes, having poly(A) tails that were longer than those seen in control cells. We provided evidence that CNOT3 was involved in recruitment of the CCR4-NOT deadenylase to the 3' end of specific mRNAs. Finally, as CNOT3 levels in the liver and white adipose tissues decreased upon fasting, we have proposed that CNOT3 responds to feeding conditions to regulate deadenylation-specific mRNAs and energy metabolism. Similarly, change of expression of various mRNAs in mice lacking other subunits has been monitored by employing expression microarray analysis, Real-time PCR, and Northern blot analysis. So far a subset (not all) of mRNA species was shown to be increased in each gene-engineered mouse, which is consistent with the idea that the CNOT complex targets specific group of mRNA for deadenylation. Efforts to elucidate the molecular mechanism by which the CCR4-NOT complex recognizes specific mRNAs are ongoing.

2. Roles of protein kinases in the central nervous system.

The Src-family protein-tyrosine kinases are implicated in various neural functions such as formation of neural network, myelination, and synaptic plasticity. To analyze the roles of Src and Fyn, we have been focusing on various substrates of these kinases, including N-methyl-D-aspartate (NMDA) type of ionotropic glutamate receptors. Our own studies showed that GluN2A and GluN2B subunits of NMDA receptors, which play important roles in learning, memory formation, and emotional behavior, were the major substrates of Fyn and Src. We identified Tyr-1472 phosphorylation on GluN2B and Tyr-1325 phosphorylation on GluN2A as the major tyrosine phosphorylation site of GluN2B and GluN2A, respectively. Using the knock-in mouse lines expressing mutant GluN2B with a Tyr-1472-Phe (Y1472F) mutation or expressing mutant GluN2A with a Tyr-1325-Phe (Y1325F) mutation, we showed that NR2B Tyr-1472 phosphorylation was important for fear-related learning in the amygdala and that NR2A Tyr-1325 regulated depression-related behavior. We also found that Tyr-1472 phosphorylation was involved in thermal nociception in mice.

In parallel of the studies on NMDAR phosphorylation, we have been trying to identify binding partners and substrates of Fyn in the brain using solid-phase phosphorylation screening, yeast two-hybrid screening, and proteomic approaches. As a result, we have identified a number of putative mediators of Fyn-mediated signaling, including NYAP, FAK, p250GAP, TCGAP, Nogo-A, and RhoGEFs. Recently, we demonstrated that NYAP family proteins (NYAP1, NYAP2, and NYAP3) were the most heavily tyrosine-phosphorylated proteins in the developing neuron. Upon stimulation with Contactin5, the NYAPs were tyrosine phosphorylated by Fyn. Phosphorylated NYAPs interacted with PI3K p85 and activated PI3K, Akt, and Rac1. Moreover, the NYAPs interacted with the WAVE1 complex which mediated remodelling of the actin cytoskeleton after activation by PI3K-produced PIP(3) and Rac1. By simultaneously interacting with PI3K and the WAVE1 complex, the NYAPs bridged a PI3K-WAVE1 association. Disruption of the NYAP genes in mice affected brain size and neurite elongation. In conclusion, the NYAPs activated PI3K and concomitantly recruited the downstream effector WAVE complex to the close vicinity of PI3K and regulate neuronal morphogenesis.

4. Publications

4.1 Journals

  1. Takahashi A, Kikuguchi C, Morita M, Shimodaira T, Tokai-Nishizumi N, Yokoyama K, Ohsugi M, Suzuki T, Yamamoto T．Involvement of CNOT3 in mitotic progression through inhibition of MAD1 expression. Biochem Biophys Res Commun.  419,268-73,2012 (March)
  2. Yang K, Trepanier C, Sidhu B, Xie YF, Li H, Lei G, Salter MW, Orser BA, Nakazawa T, Yamamoto T, Jackson MF, Macdonald JF.　Metaplasticity gated through differential regulation of GluN2A versus GluN2B receptors by Src family kinases. EMBO J. 31,805-16,2011 (December)
  3. Katano T, Nakazawa T, Nakatsuka T, Watanabe M, Yamamoto T, & Ito S. Involvement of spinal phosphorylation cascade of Tyr1472-NR2B, Thr286-CaMKII, and Ser831-GluR1 in neuropathic pain. Neuropharmacol. 60:609-16, 2011 (December)
  4. Fabian MR, Cieplak MK, Frank F, Morita1 M, Green J, Srikumar T, Nagar B, Yamamoto T, Raught B, Duchaine TF and Sonenberg N. miRNA-mediated deadenylation is orchestrated by GW182 through two conserved motifs that interact with CCR4-NOT. Nat Struct and Mol Biol, 18,1211-7,2011 (October)
  5. Masuda N, Shimodaira T, Shiu SJ, Tokai-Nishizumi N, Yamamoto T, Ohsugi M. Microtubule stabilization triggers the plus-end accumulation accumulation of Kif18/kinesin-8. Cell Struct Funct, 2, 261-267, 2011 (November)
  6. Ito　K, Takahashi A, Morita M, Suzuki T, and Yamamoto T, The role of the CNOT1 subunit of the CCR4-NOT complex in mRNA deadenylation and cell viability. Protein & Cell, 2, 755-763, 2011 (October)
  7. Yokoyama K, Tezuka T, Kotani M, Nakazawa T, Hoshina N, Shimoda Y, Kakuta Y, Sudo K, Watanabe K, Iwakur Y and Yamamoto T, NYAP: a phosphoprotein family that links PI3K to WAVE1 signaling in neurons. EMBO J, 30, 4739-4754, 2011 (September)
  8. Morita M, Oike Y, Nagashima T, Kadomatsu T, Tabata M, Suzuki T, Nakamura T, Yoshida N, Okada M, and Yamamoto T, Obesity resistance and increased hepatic expression of catabolism-related mRNAs in Cnot3+/- mice. EMBO J, 30, 4678-4691, 2011 (September)
  9. Chen C, Ito K, Takahashi A, Wang G, Suzuki T, Nakazawa T, Yamamoto T, Yokoyama K. Distinct expression patterns of the subunits of the CCR4-NOT deadenylase complex during neural development. Biochem Biophys Res Commun.411:360-4. 2011 (June)
  10. Suzuki T, Kim M, Kozuka-Hata H, Watanabe M, Oyama M, Tsumoto K, Yamamoto T. Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination. Biochem Biophys Res Commun. 409:70-4. 2011 (April)
  11. Ito K, Inoue T, Yokoyama K, Morita M, Suzuki T, and Yamamoto T. CNOT2-depletion disrupts and inhibits the CCR4-NOT deadenylase complex and induces apoptotic cell death. Genes to Cells 4: 368-79, 2011 (April)

4.2 Books and other one-time publications

  1. Saito M., Kumazawa K, Doi A, Takebe S, Amari T, Oyama M, Semba K, Yamamoto T. ErbB2/HER2, its contribution to basic cancer biology and development of molecular targeted therapy. In "Breast Cancer Cells, Book 1" (ed. Gunduz M), INTECH, in press

4.3 Oral and Poster Presentations

  1. Miho Ohsugi, Kaori Yamada, Kazuo Yamagata, Tadashi Yamamoto.  Female pronuclear formation in mouse oocytes requires Kid-mediated telophase chromosome clustering 6th UK-Japan Cell Cycle Workshop　　Lake Windermere, England　　Apr. 13, 2011
  2. Miho Ohsugi, Tetsuhiro Shimodaira, Natsuko Masuda, Tadashi Yamamoto. Localization of Kif18A/kinesin-8 depends on its tail domain and microtubule stability, International symposium “Cell Division”　　Hakone, Japan　　June 30, 2011
  3. Toru Suzuki, Hisao Masai and Tadashi Yamamoto. CDC7-dependent inhibition of Tob degradation maintains cell survival after DNA damage, 70th Annual Meeting of the Japanese Cancer Association, Nagoya, Japan, Oct. 3-5, 2011
  4. Akinori Takahashi, Masahiro Morita, Toru Suzuki, Tadashi Yamamoto. Tob2 inhibits adipocyte differentiation, Todai Forum 2011, Lyon, France, Oct. 20-21, 2011
  5. Tadashi Yamamoto.  The CCR4-NOT complex, a deadenylase that controla decay for specific sets of mRNAs in a context-dependent manner, Todai Forum 2011, Lyon, France, Oct. 20-21, 2011
  6. Toru Suzuki, Junko Tsuzuku, Akiyo Hayashi, Yasushi Shiomi, Hiroko Iwanari, Yasuhiro Mochizuki, Takao Hamakubo, Tasuhiko Kodama, Hideo Nishitani, Hisao Masai, and Tadashi Yamamoto. Competition of Cdc7/Tob with Cul4-DDB1-Cdt2 E3 complex in DNA damage-induced apoptosis, The 34th Annual Meeting of the Molecular Biology Society of Japan, Yokohama, Japan 　Dec. 13-16, 201
  7. Akinori Takahashi, Masahiro Morita, Toru Suzuki, and Tadashi Yamamoto. Tob2 inhibits adipogenesis, The 5th global COE retreat Integrative Life Sciencebased on the Study of Biosignaling Mechanism 2011, Yatsugatake, Japan, Mar. 3-4, 2010
  8. Kenji Iemura, Naoki Oshimori, Tadashi Yamamoto, Miho Ohsugi. AMPKγ is required for correct kinetochore-microtubule attachments,  The 63rd Annual Meeting of the Japan Society for Cell Biology、Sapporo, Japan,　May  27-29, 2011
  9. Kenji Iemura, Naoki Oshimori, Tadashi Yamamoto, Miho Ohsugi. AMPKγ is required for correct kinetochore-microtubule attachments,  UCSF Granlibakken Tetrad Retreat,  California, USA  Sep. 8-9. 2011
  10. Noriko Tokai-Nishizumi, Miho Ohsugi, Tadashi Yamamoto. Tropomyosin 4, actin binding protein, contributes to the proper spindle orientation during mitosis and meiosis,  the 2011 Annual Meeting of the American Society for Cell Biology, Denver, CO, USA , Dec. 5, 2011
  11. Toru Suzuki, Junko Tsuzuku, Akiyo Hayashi, Yasushi Shiomi, Hiroko Iwanari, Yasuhiro Mochizuki, Takao Hamakubo, Tasuhiko Kodama, Hideo Nishitani, Hisao Masai, and Tadashi Yamamoto. Competition of Cdc7/Tob with Cul4-DDB1-Cdt2 E3 complex in DNA damage-induced apoptosis,  The 34th Annual Meeting of the Molecular Biology Society of Japan,  Yokohama, Japan,　Dec. 13-16, 2011
  12. Akinori Takahashi, Masahiro Morita, Toru Suzuki, Tadashi Yamamoto. Tob2 inhibits adipocyte differentiation,  The 34th Annual Meeting of the Molecular Biology Society of Japan , Yokohama , Japan, Dec. 13-16, 2011
  13. Tetsuhiro Shimodaira，Natsuko Masuda，Tadashi Yamamoto，Miho Ohsugi. The C-terminal region of Kif18A is essential for its localization and function in chromosome alignment during mitosis, The 34th Annual Meeting of the Molecular Biology Society of Japan、Kanagawa, Japan, Dec, 2011
  14. Tetsuhiro Shimodaira, Tadashi Yamamoto, Miho Ohsugi.  Kif18A/Kinesin-8 regulates chromosome alignment through its tail domain,  The 29th Chromosome Workshop, Miyagi, Japan,  Jan. 25-27, 2012      
  15. Kenji Iemura、Tadashi Yamamoto, Miho Ohsugi.  Misalignment of mitotic chromosomes induces spindle pole misintegrity,  The 29th Chromosome Workshop, Miyagi, Japan,  Jan. 25-27, 2012
  16. Sho Soeda, Kaori Yamada, Tadashi Yamamoto, Miho Ohsugi. Mechanisms underlying the Kid/kinesin-10-mediated anaphase chromosome compaction in mouse zygote,  The 29th Chromosome Workshop, Miyagi, Japan,  Jan. 25-27, 2012
  17. Kenji Iemura, Tadashi Yamamoto, Miho Ohsugi.  Correction error　of　kinetochore-microtubule attachment induces mitotic centrosome disruption,  The 5th global COE retreat Integrative Life Sciencebased on the Study of Biosignaling Mechanism 2011, Yatsugatake, Japan, Mar. 3-4, 2010

5. Intellectual Property Rights and Other Specific Achievements

Nothing to report

6. Meetings and Events