1st International Symposium on Carcinogenic Spiral & 9th International Conference
on Protein Phosphatase
February 1 (Tue) – 3 (Thu), 2011 Tetsumon Memorial Hall, The University of Tokyo, Japan
Organizer
Masanori HATAKEYAMA, M.D. Ph.D.
1 Welcoming Address
Welcome to the Joint Meeting of the 1st International Symposium on Carcinogenic Spiral and the 9th International Conference on Protein Phosphatase. As the organizer, I am delighted to host many eminent scholars from Japan as well as overseas. During the Conference, you will be able to learn about exciting up-to-date discoveries in the field of protein phosphatase research. I also would like to emphasize that the second day of the conference will provide a unique scientific opportunity that admixes cutting-edge research on tyrosine phosphatases with cutting-edge research on infection, inflammation and cancer. This special session was made possible by the generous support from Scientific Research on Innovative Areas, a MEXT Grant-in Aid Project. I hope that all of the participants will benefit from valuable scientific information and fruitful discussions throughout the 3-day Conference.
I would like to again express a warm-hearted welcome to all of you gathered here. I also express my special thanks to those who have taken time out of their busy schedule to be here. I hope that all of the participants enjoy the Conference and also enjoy Tokyo, a city with lots of fun and excitement.
Best regards,
Masanori Hatakeyama, M.D., Ph.D. Organizer 1st International Symposium on Carcinogenic Spiral 9th International Conference on Protein Phosphatase
2 General Information
Congress 1st International Symposium on Carcinogenic Spiral & 9th International Conference on Protein Phosphatase
Date February 1 (Tue) – 3 (Thu), 2011
Venue Tetsumon Memorial Hall Hongo Campus, The University of Tokyo 14th floor of Faculty of Medicine Experimental Research Bldg. 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan TEL: 81-3-5841-3408 FAX: 81-3-5841-3406
Oral Presentation Please bring a CD, a USB memory stick, or your own computer with a mini D-sub 15-pin connector. Please upload your file before your session starts.
Poster Presentation A poster board, 200 cm in height and 90 cm in width, is available for each presenter. Pins are provided by organizers. Please set up your poster before the morning session and remove it after the poster session ends.
Group Photo A group photo is scheduled from 12:05 following the morning session on February 2.
Reception Reception will be held at Capo PELLICANO (13th floor of Faculty of Medicine Experimental Research Bldg.) from 18:30 on February 2.
3 Floor Map Faculty of Medicine Experimental Research Bldg., University of Tokyo
1st Floor Entrance
Disaster Control Tetsumon Center Cafe
EV EV EV EV
Faculty of Medicine Bldg. 1
14th Floor Oral Presentation Poster Presentation
Vaulted Ceiling (Capo PELLICANO)
Front Desk TetsumonTetsumon Memorial HallHall
EV EV EV EV
4 University of Tokyo, Hongo Campus Map
Ikenohata Gate
Faculty of Medicine Experimental Research Bldg. Asano South Gate
Asano Main Gate Yayoi Gate
Tatsuoka Gate
Gate to Faculty Main Gate Akamon of Agriculture (Red Gate)
0 50 100 200 300 (m)
Tetsumon Memorial Hall, Hongo Campus, University of Tokyo 14th floor of Faculty of Medicine Experimental Research Bldg. 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
5 Access Map to University of Tokyo
for Ohji for Toride
Gate to Faculty Nezu of Agriculture Todaimae Ikenohata Gate
Main The University of Tokyo Gate [Hongo Campus]
Ueno Akamon Ueno for Ikebukuro (Red Gate) Tatsuoka Gate Ueno-hirokoji UNIVERSITY BUS 01 Okachimachi for Kasuga Hongo-sanchome for Kuramae UNIVERSITY Yushima Hongo-sanchome BUS 07
Ochanomizu for Meguro Ochanomizu for Chiba for Yotsuya Ochanomizu for Shinjuku Shin-ochanomizu
Marunouchi Line TOKYO BUS 43 Oedo Line TOKYO
Chiyoda Line for Yoyogi-uehara Namboku Line JR Line for Shinjuku
Tetsumon Memorial Hall, Hongo Campus, University of Tokyo 14th floor of Faculty of Medicine Experimental Research Bldg. 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Nearest Stations Hongo-sanchome Station (Subway Marunouchi Line) 10 min. walk Hongo-sanchome Station (Subway Oedo Line) 10 min. walk Yushima Station or Nezu Station (Subway Chiyoda Line) 15 min. walk Todaimae Station (Subway Namboku Line) 20 min. walk
6 Program February 1 (Tue)
Opening Lecture Chair: !"#$#%"&!"'()*#+,$!!"#$%&'()*+,-./*0%./*)12/3*)456-78,%9
9:30-10:15 !"#($#&-,./$, !:828$#*(54;/3*<-7-59! “Regulation of Cellular Functions by PP2C Family Members”
Session 1-1. Ser/Thr Phosphatase Part 1 Chairs: !"#($#&-,./$,!!:828$#*(54;/3*<-7-59! 0,%,1)&23,1,&!04%*(54;/3*<-7-59!
10:15-10:35 !,1)$/&0)4"#5,*!=#>->8?8*(54;/3*<-7-59! “Reciprocal Inhibition System between PP2A and CDK” 10:35-10:50 67/1,$)&8,9#",$,!!=#>->8?8*(54;/3*<-7-59! “Protein Phosphatase 6 Regulates Apoptosis via CaMKII and p27” * 10:50-11:05 Coffee Break
11:05-11:20 :)%"#$)&;"/.,(!!@8$$-4.8*(54;/3*<-7-59! “Suppression of Breast Cancer Cells Proliferation by Novel Inhibitor for p53-Inducible Protein Phosphatase PPM1D” 11:20-11:40 6'#+)&!/3#/$,!!=45$4*(54;/3*<-7-59 “A Powerful Genetic Strategy to Screen for Inhibitors of MAP Kinase Signalling and Its Application to Genomic Drug Discovery”
Session 1-2. Dual Specific Phosphatase Chairs: <'(=!"'(3&>'(3!!(A)"3*()B9 ?#1)%"#&@,+,3,.,!!'-?C/*A-51%,*A%5?%,*D5E?/3*<-7-59!
11:40-12:00 ?#$)%"#&!"#.,!!04F-64*A-51%,*A%5?%,*G%E%-,12*D5E?/3*<-7-59! “Analysis of Protein Phosphatases in Human Glioma Patients” 12:00-12:15 !,5,1%/3/&2)+/.,!!04%*(54;/3*<-7-59! “Contribution of p38! in Blood Cells to the Regulation of Obesity and Blood Glucose Level”
12:15-13:30 Lunch / Organizing Committee
13:30-15:30 Poster Session
7
Session 1-3. Tyr Phosphatase Part 1 Chairs: A'(9,.#(&<&@''*!!H5?-,48*A-51%,*D5E?/3*A-5-.-9 -,+,%"#&0,1)B,+#&!=8I%*(54;/3*<-7-59*
15:30-16:05 0#4"'*&C&-$'.D*,7!!01+4CC*(54;/3*A-5-.-9 “Regulation and Oncogenic Activities of PTP1B” 16:05-16:20 8)%/+'&0,1%/)!!(A*"-;4E3*()B9* “Regulation of Brown Fat Adipogenesis by Protein Tyrosine Phosphatase 1B” 16:20-16:40 >,E,B&<&?,9!!(A*"-;4E3*()B9* “Differential Regulation of Endoplasmic Reticulum Stress by Protein Tyrosine Phosphatase 1B and T Cell Protein Tyrosine Phosphatase” 16:40-17:00 -B/=;"#(3&0'(3!!D5E?/*J48C8641-C*A2%>/*B1-.%>4-*)4541-3*:-4K-59 “S-Nitrosylation and Inactivation of PTPs Protects Cardiomyocytes Against Hypoxic Injury” 17:00-17:15 @,)1)&@,3,1,!!(A*"-;4E3*()B9! “Hepatic Src Homology Phosphatase 2 Regulates Energy Balance and Metabolism”
8 February 2 (Wed)
Keynote Lecture Chair: 0,%,()$#&?,1,+'7,.,!!(54;/*:8$F83*<-7-59
9:00-9:45 @#4")*,%&8&-)(+%*!A8C.*)7,456*@-,I8,*L-I/3*()B9! “Functional Analysis of Protein Tyrosine Phosphatases”
Session 2-1. Carcinogenic Spiral Part 1 Chairs: 0,%,)&0,1%/)+,!!=F8?8*(54;/3*<-7-59 8/(#1,5,&!"#.)1)"()!!A24I-*D5E?/*:%12/3*<-7-59!
9:45-10:20 0,%,()D/&2%"#.,!!=-5-M-K-*(54;/3*<-7-59*
“Prostaglandin E2-Associated Inflammation and Bacterial Infection in Gastric Tumorigenesis” 10:20-10:55 F,G#5&0&H#$%"/I!!"#$%&'()*+,-./*0%./*)12/3*)456-78,%9 “Posttranslational Regulation of the Wnt Signaling Pathway” * 10:55-11:10 Coffee Break
Session 2-2. Carcinogenic Spiral Part 2 Chairs: 8,B/(,$#&J+#7)%"#!!=F8?8*(54;/3*<-7-59 -,5,1%/3/&-,(#3/4"#!!(54;/*:8$F83*<-7-59!
11:10-11:45 8/(#1,5,&!"#.)1)"()!!A24I-*D5E?/*8N*:%1258C86F3*<-7-59 “Dys-regulation of Lipid Metabolism May Link to the Development of Hepatocellular Carcinoma” 11:45-12:20 0,%,)&0,1%/)+,*!=F8?8*(54;/3*<-7-59 “Immunological Dysregulation and Oncogenesis by Human T-Cell Leukemia Virus Type 1”
12:20-12:30 Group Photo
12:30-14:00 Lunch
9 Special Lecture Chair: F,G#5&0&H#$%"/I!!"#$%&'()*+,-./*0%./*)12/3*)456-78,%9
14:00-14:45 -,5,1%/3/&-,(#3/4"#*!(54;/*:8$F83*<-7-59 “Regulation of Immune Responses and Oncogenesis by the IRF Family of Transcription Factors and Their Partners”
Session 2-3. Carcinogenic Spiral part 3 Chairs: 0,%,()D/&2%"#.,*!=-5-M-K-*(54;/3*<-7-59 -,+'%"#&JB/.,!!=8I%*(54;/3*<-7-59!
14:45-15:20 -%/+,%,&!'7,!!@8$$-4.8*(54;/3*<-7-59 “Functional Divergence of RNA-Sensing Systems in Dendritic Cells for Induction of Antitumor Effectors” 15:20-15:55 8,B/(,$#&J+#7)%"#&!=F8?8*(54;/3*<-7-59 “Polysaccharide Nanogel DDS for Cancer Immunotherapy”
15:55-16:10 Coffee Break
Session 2-4. Carcinogenic Spiral Part 4 Chairs: @#4")*,%&8&-)(+%*!A8C.*)7,456*@-,I8,*L-I/3*()B9! -%/+,%,&!'7,!!@8$$-4.8*(54;/3*<-7-59*
16:10-16:45 A'(9,.#(&<&@''**!H5?-,48*A-51%,*D5E?/3*A-5-.-9 “Tyrosine Phosphatases in Health and Disease” 16:45-17:20 0,%,()$#&?,1,+'7,.,!!(54;/*:8$F83*<-7-59 “Role of SHP2 Tyrosine Phosphatase in Gastric Carcinogenesis” 17:20-17:55 <'(=!"'(3&>'(3!!(A)"3*()B9 “Identification of PTPN11/Shp2 as a Tumor Suppressor in Liver Cancer” 17:55-18:30 -,+'%"#&JB/.,*!=8I%*(54;/3*<-7-59 “Helicobacter pylori Infection and Gastric Cancer: Relationship between Gastric Cancer and Diversity of Helicobacter pylori”
18:30-20:30 Reception
10 February 3 (Thu)
Session 3-1.Tyr Phosphatase Part 2 Chairs: 0#4"'*&C&-$'.D*,7!!01+4CC*(54;/3*A-5-.-9! -,+'%"#&-%/D,1,!!:8$F8*0%.41-C*O*"%5?-C*(54;/3*<-7-59*
9:00-9:35 -,+,%"#&0,1)B,+#&!=8I%*(54;/3*<-7-59 “Regulation of Intestinal Immunity by SAP-1, a Microvillus-Specific Receptor-Type Protein Tyrosine Phosphatase” 9:35-10:10 K#&L",(3!!(A)"3*()B9 “Beyond Insulin Secretion: Substrate Identification of a Mitochondrial Phosphatase” 10:10-10:25 0/('.,%,&0)$#!!+#5>-*(54;/3*<-7-59 “Shear Stress Regulates Cellular Localization of Vascular Endothelial–Protein Tyrosine Phosphatase (VE-PTP)” 10:25-10:45 -)%"#)&M,1,(,D'!!'-,-*P8>%5QE*(54;/3*<-7-59 “Phospho-Paxillin Is an Oncogenic in Vivo Target of the Tumor Suppressor Receptor Protein Tyrosine Phosphatase T”
10:45-11:00 Coffee Break
Session 3-2. Ser/Thr Phosphatase Part 2 Chairs: -,1%/7,&0,'5,*!(54;/*:8$F83*<-7-59* ?#$)%"#&!"#.,!!04F-64*A-51%,*A%5?%,*G%E%-,12*D5E?/3*<-7-59!
11:00-11:20 8)"%/+'&-,+'5,!!(54;/*:8$F83*<-7-59! “Regulation of Cellular Stress Response by Mitochondrial Protein Phosphatase PGAM5” 11:20-11:55 !"#$#%"&!"'()*#+,$!!"#$%&'()*+,-./*0%./*)1288C3*)456-78,%9 “Translation Repression in the Management of Protein Misfolding Disorders” 11:55-12:15 ?#5'1,+,&:,+/$,!!(54;/*R-,4E*"4.%,8?3*S,-51%9! “What Is “Information” in Signal Transduction?”
12:15-12:25 Closing Remarks
11
Oral Presentation
12 Day-1| 9:30-10:15 Opening Lecture
Regulation of Cellular Functions by PP2C Family Members
Shinri Tamura, Yuko Nagaura, Toko Chida, Yusuke Kanto and Takayasu Kobayashi Department of Biochemistry, Institute of Development, Aging and Cancer, Tohoku Universiry, 4-1 Seiryo-machi, Aobaku, Sendai 980-8570, Japan
Protein phosphatase 2C (PP2C) is a family of protein Ser/Thr phosphatases. To date the mammalian PP2C gene family is known to be composed of 14 different genes. This is in contrast to the small number of genes encoding the catalytic subunits of other protein Ser/Thr phosphatase families, such as PP1, PP2A and PP2B (3, 2, and 2 genes, respectively). Four (PP2C!, PP2C", PP2C#, and PP2C$) of the 14 PP2C family members were initially cloned by our group. A homology search indicated that the PP2C family has a distinct evolutionary origin, while the catalytic proteins of PP1, PP2A and PP2B originated from a common molecular ancestor. The unique molecular evolution of PP2C raises the possibility that the PP2C family plays a unique physiologic role in the regulation of cellular functions. Previous works by us and others indeed suggested that PP2C as a family participates in the regulation of stress response of cells in various ways. Another important feature of PP2C family is that the family members are essentially monomeric enzymes, whereas other Ser/Thr phosphatase family members are oligomeric enzymes. Therefore, it is predicted that each PP2C molecule contains not only catalytic domain but also regulatory and/or subcellular localization determining domains. In fact, we have demonstrated the existence of unique functional domains in some PP2C family members. In this presentation, I would like to review our previous works briefly and talk about current works of novel functions of PP2C family members.
13 Shinri Tamura Department of Biochemiastry, Institute of Development, Aging and Cancer, Tohoku University E-mail: [email protected]
EDUCATIONS/TRAINING Tohoku University MD Medicine 1974 Tohoku University PhD Medicine 1978 University of Virginia School of Medicine Postdoc Biochemistry 1981-1984
POSITIONS 1978-1988 Assistant Professor , Research Institute for Tuberculosis and Cancer, TohokuUniversity 1988-1991 Associate Professor, Hirosaki University School of Medicine 1991-1993 Professor, Research Institute for Tuberculosis and Cancer, Tohoku University 1993-Present Professor, Institute of Development, Aging and Cancer, Tohoku University
SELECTED RECENT PUBLICATIONS 1. Henmi, T., Amano, K., Nagaura, Y., Matsumoto, K., Echigo, S., Tamura, S. and Kobayashi, T. A mechanism for the suppression of interleukin-1-induced NF%B activation by protein phosphatase 2C$-2. Biochem. J. 423, 71-78, 2009 2. Saito, S., Matsui, H., Kawano, M., Kumagai, K., Tomishige, N., Hanada, K., Echigo, S., Tamura, S. and Kobayashi, T. Protein phosphatase 2C" is an endoplasmic reticulum integral membrane protein that dephosphorylates the ceramide transport protein CERT to enhance its association with organelle membranes. J. Biol. Chem. 283, 6584-6593, 2008 3. Sasaki, M., Ohnishi, M., Tashiro, F., Niwa, H., Suzuki, A., Miyazaki, J., Kobayashi, T. and Tamura, S. Disruption of the mouse protein Ser/Thr phosphatase 2C& gene leads to early pre-implantation lethality. Mech. Dev. 124, 489-499, 2007 4. Akiyama, S., Yonezawa, T., Kudo, T., Li, M.G., Wang, H., Ito, M., Yoshioka, K., Ninomiya-Tsuji, J., Matsumoto, K., Kanamaru, R., Tamura, S. and Kobayashi, T. Activation mechanism of c-Jun amino-terminal kinase in the course of neural differentiation of P19 embryonic carcinoma cells. J. Biol. Chem. 279, 36616-36620, 2004 5. Li, M.G., Katsura, K., Nomiyama, H., Komaki, K., Ninomiya-Tsuji, J., Matsumoto, K., Kobayashi, T. and Tamura, S. Regulation of the interleukin-1-induced signaling pathways by a novel member of the protein phosphatase 2C family (PP2C"). J. Biol. Chem. 278, 12013-12021, 2003
14 Day-1| 10:15-10:35 Session 1-1
Reciprocal Inhibition System between PP2A and CDK
Satoru Mochida1,2, Tim Hunt1 1Cancer Research UK, Clare Hall Laboratories, U.K. 2Priority Organization for Innovation and Excellence, Kumamoto University, Japan
Proper control of the entry into and exit from mitosis, in which a mother cell divides its cellular component between two daughter cells, is crucial to ensure the accurate distribution of genomic information. A failure of this control can cause losses or gains of chromosomes, a phenomenon frequently observed in tumor cells. Entry into mitosis depends on the activity of cyclin-dependent kinases (CDKs). Conversely, exit from mitosis occurs when mitotic cyclins are degraded, thereby extinguishing CDK activity. Exit from mitosis must also require mitotic phosphoproteins to revert to their interphase hypophosphorylated forms, however, and there has long been a controversy about which phosphatase is responsible for dephosphorylating CDK substrates. We reported that PP2A associated with B55' regulatory subunit dephosphorylated a wide range of mitotic CDK substrates in Xenopus laevis egg extracts (Mochida et al., 2009, EMBO J.). The activity of this form of PP2A is regulated during cell cycle — high in interphase and suppressed during mitosis, exactly opposite to CDK activity. Our next question was about the regulatory mechanism of this phosphatase activity. Other groups have reported in 2009 that Greatwall, a protein kinase required for proper mitosis in flies and frogs, was somehow responsible for the mitotic suppression of PP2A activity. We report here that !-Endosulfine and its close relative ARPP-19 are substrates of Greatwall, and find that they specifically inhibit PP2A-B55' in vitro. !-Endosulfine was indeed required for mitotic entry in extracts. These results suggest that CDK directs an inactivation of the opposing phosphatase via a pathway involving Greatwall and a-Endosulfine (Mochida et al., 2010, Science).
15 Day-1| 10:35-10:50 Session 1-1
Protein Phosphatase 6 Regulates Apoptosis via CaMKII and p27
Ryutaro Kajihara1, Shota Fukushige1, Norifumi Shioda2, Kano Tanabe1, Kohji Fukunaga2, Seiji Inui1 1Department of Immunology and Hematology, Division of Health Sciences, Faculty of Life Sciences, Kumamoto University, Japan 2Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Japan
Protein phosphatase (PP) 6 is a serine threonine phosphatase which belongs to the PP2A subfamily of protein phosphatases. PP6 has been implicated in the control of apoptosis. A dominant negative form PP6 (DN-PP6) mutant cDNA was prepared and transfected into HeLa cells to investigate the regulation of apoptosis. HeLa cells expressing DN-PP6 showed increased resistance to apoptosis induced by TNF and cycloheximide. In addition, DN-PP6 induces the phosphorylation of CaMKII and the up-regulation of p27 and Bcl-xl. Overexpression or activation of CaMKII also increased the expression of p27. Furthermore, CaMKII phosphorylated serine 10 of p27, which induces the translocation of p27 from nucleus to cytoplasm and increases the stability of p27. Overexpression of wild type but not the S10A mutant p27 cDNA increased the expression of Bcl-xL and conferred apoptosis resistance to HeLa cells. These results indicated that PP6 and CaMKII regulated apoptosis by controlling the expression level of p27.
16 Day-1| 11:05-11:20 Session 1-1
Suppression of Breast Cancer Cells Proliferation by Novel Inhibitor for p53-Inducible Protein Phosphatase PPM1D
Yoshiro Chuman, Hiroaki Yagi, Yuki Kozakai, Kazuyasu Sakaguchi Laboratory of Biological Chemistry, Department of Chemistry, Faculty of Science, Hokkaido University, Japan
PPM1D (Wip1, PP2C') is a member of PPM1 (former PP2C) type Ser/Thr protein phosphatase family, and induced in response to DNA damage in p53 dependent manner. Recently it has been reported that gene amplifications and over-expressions of PPM1D are observed in several human cancers including breast cancers, and deficient of PPM1D gene causes a tumor resistant phenotype in mice. These findings suggest that PPM1D acts as an oncogene and that PPM1D is a viable anti-cancer target. Recently, we have identified a series of novel small molecule inhibitors of PPM1D by screening of compounds in our own chemical library. The strongest compound,
SPI-001 inhibited PPM1D activity in a noncompetitive manner with high affinity (Ki = 0.59 µM) and high selectivity. In this study, we analyzed the effects of the PPM1D inhibitor SPI-001 on cells proliferation of breast cancer cells. The effects of SPI-001 on cell viability were determined using MCF7 cells, breast cancer cell line in which the PPM1D gene is amplified. SPI-001 showed suppression of cell growth and induced cell death. The cell cycle analysis by flow cytometry showed increase of cells in G2/M and sub-G1 phases by treatment of SPI-001. Furthermore, SPI-001 induced phosphorylation of p53 at Ser15 and accumulation of p53 protein in MCF7 cells. These data suggested that SPI-001 suppressed proliferation of MCF7 cells by activation of p53 through the inhibition of endogenous PPM1D. Taken together, the PPM1D inhibitor can be a potent candidate for anti-cancer drug.
17 Day-1| 11:20-11:40 Session 1-1
A Powerful Genetic Strategy to Screen for Inhibitors of MAP Kinase Signalling and Its Application to Genomic Drug Discovery
Reiko Sugiura Laboratory of Molecular Pharmacogenomics, Kinki University School of Pharmaceutical Sciences, Japan
Mitogen-activated protein kinases (MAPKs), found in all eukaryotes, are signal-transducing enzymes playing a central role in a variety of biological processes. We have demonstrated that MAPK and calcineurin phosphatase act antagonistically in the Cl- homeostasis in fission yeast and developed a genetic screen that aims to identify negative regulators of the Pmk1 MAPK signalling. The first gene we identified encodes the MAPK phosphatase Pmp1, which dephosphorylates and inhibits MAPK (Sugiura et al., EMBO J. 1998). The second gene identified was pek1+, encoding MAPK kinase (MAPKK), which phosphorylates and activates Pmk1 in its phosphorylated form, thus acting as an upstream MAPKK for Pmk1. Surprisingly, Pek1 in its unphosphorylated state binds phosphorylated Pmk1, thereby inhibiting the Pmk1 MAPK signalling, and hence Pek1 acts as a phosphorylation-dependent molecular switch (Sugiura et al., Nature 1999). We also demonstrated that a novel RNA-binding protein Rnc1 plays a crucial role in negative feedback regulation of MAPK signalling, by binding and stabilizing the mRNA of a MAPK phosphatase at the post-transcriptional level (Sugiura et al., Nature 2003). Our discovery highlights a potential role and an emerging view of RNA-binding protein as a regulator of cell signaling and as a future target of drug discovery. As MAP kinase signal transduction pathways are one of the most attractive targets for cancer therapy, inhibitors that target this signaling appear to be promising drug candidates for the treatment of cancer. Here, I first give an overview of the use of yeast as a model system for drug discovery and then, I introduce our molecular genetic strategy to identify regulators of MAPK signaling and the application of this approach to drug discovery.
18 Day-1| 11:40-12:00 Session 1-2
Analysis of Protein Phosphatases in Human Glioma Patients
Hiroshi Shima Division of Cancer Chemotherapy Miyagi Cancer Center Research Institute 47-1 Nodayama, Medeshima-Shiode, Natori, Miyagi 981-1293, Japan
Glioblastomas are the most common and lethal type of malignant brain tumor. Glioblastoma is characterized by highly proliferative and invasive activity, and widespread infiltration of tumor cells into the surrounding brain tissues. Recent standard therapy for glioblastoma includes surgical resection, radiotherapy, and adjuvant temozolomide chemotherapy administrated both during and after radiotherapy. However, most patients develop tumor recurrence or progression after this multimodality treatment. There is clearly an urgent need to develop new classes of treatment modalities, such as molecular target-directed therapy. Most studies on the molecular targeted therapy in gliomas thus far had focused on receptor tyrosine kinases and intracellular serine/threonine kinases. In attempt to identify protein phosphatases as molecular targets of novel diagnostic and therapeutic strategies in the treatment of glioma patients, we have examined gene expression of protein phosphatase genes in human glioma samples, [1,2]. We found that Ki-67 labeling index in glioma tissues was significantly correlated with the expression of CDC25A [2], and down regulation of Dusp26 may contribute to malignant phenotypes of glioma [1].
References 1. Tanuma N. et al. Oncogene 13(12), 752-761, 2009 2. Yamashita Y. et al. J Neurooncol 100(1), 43-49, 2010
19 Hiroshi Shima Division of Cancer Chemotherapy, Miyagi Cancer Center Research Institute E-mail: [email protected]
EDUCATIONS/TRAINING Akita University MD Medicine 1982 Akita University PhD Medicine 1986 National Cancer Center Research Institute Postdoc Cancer Biology 1986-1987
POSITIONS 1987-1991 Staff Scientist, National Cancer Center Research Institute 1991-1995 Section Head, National Cancer Center Research Institute 1995-1998 Research Fellow, Friedrich Miescher Institute 1998-2000 Associate Professor, Institute for Immunological Science, Hokkaido University 2000-2005 Associate Professor, Institute for Genetic Medicine, Hokkaido University 2005-present Head, Division of Cancer Chemotherapy, Miyagi Cancer Center Research Institute 2007-present Professor, Division of Cancer Molecular Biology, Tohoku University School of Medicine
SELECTED RECENT PUBLICATIONS
1. Tanuma N, Nomura M, Ikeda M, Kasugai I, Tsubaki Y, Takagaki K, Kawamura T, Yamashita Y, Sato I, Sato M, Katakura R, Kikuchi K, Shima H. Protein phosphatase Dusp26 associates with KIF3 motor and promotes N-cadherin-mediated cell-cell adhesion. Oncogene 13(12), 752-761, 2009 2. Tanuma N, Kim S-E, Beullens M, Tsubaki Y, Mitsuhashi S, Nomura M, Kawamura T, Isono K, Koseki H, Sato M, Bollen M, Kikuchi K, Shima H. Nuclear inhibitor of protein phosphatase-1 (NIPP1) directs protein phosphatase-1(PP1) to dephosphorylate the U2 snRNP component, spliceosome-associated protein 155 (Sap155). J Biol Chem 283(51), 35805-35814, 2008 3. Katagiri C, Masuda K, Urano T, Yamashita K, Kikuchi K, Shima H. Phosphorylation of Ser-446 determines stability of MKP-7. J Biol Chem 280(15), 14716-14722, 2006
20 Day-1| 12:00-12:15 Session 1-2
Contribution of p38! in Blood Cells to the Regulation of Obesity and Blood Glucose Level
Sadatsugu Ookuma, Kazuto Sugimura, Masato Ogata Department of Biochemistry and Proteomics, Mie University Graduate School of Medicine 2-174, Edobashi, Tsu-city, Mie 514-8507, Japan
DSPs (dual specificity phosphatases) regulate activity of MAPKs (Mitogen-activated protein kinase) which control signal transduction in the eukaryotic cell. p38 is one of MAPK family protein kinases, which regulate a variety of intracellular signal transduction. p38 is known as a stress-activated protein kinase, and has four homologs such as !, ", # and $ in mammal. In four homologs, p38! is thought to be autholog, and affects inflammation, differentiation and cell senescence. Recent studies have revealed that chronic inflammation plays an important role in pathogenesis of metabolic syndrome. To examine that p38! is engaged in metabolic syndrome, we made conditional knockout mice of p38! in hematopoietic cells and vascular endotherium by using Cre-loxP system. Feeding with normal diet (ND), p38! conditional knockout mice showed no significant difference in body weight or blood glucose level compared with wild-type mice. However, feeding with High-Fat diet (HFD), body weight, blood glucose level and insulin level were significantly lower in p38! conditional knockout mice than in wild-type mice. Western blot analysis showed that p38! is activated with HFD-feeding in peripheral blood cells. Then we tested infiltration of macrophages into adipose tissues and expression of cytokines. In p38! conditional knockout mice, HFD-induced infiltration of CD11c-positive macrophages was suppressed, and expression of pro-inflammatory cytokines was reduced both in peripheral blood cells and in abdominal adipose tissues. Thus, p38! is involved in the regulation of obesity and blood glucose level when feeding with HFD, possibly through control of inflammatory cells in mice.
21 Day-1| 15:30-16:05 Session 1-3
Regulation and Oncogenic Activities of PTP1B
Michel L. Tremblay Goodman Cancer Research Centre and Dept. of Biochemistry McGill University 1160 Pine Avenue, Montreal, Quebec, H3A 1A1, Canada
PTP1B is the prototype for the superfamily of PTPs, not only because it was the first PTP identified, but also, it is as yet the only targeted PTP in clinical settings for metabolic diseases. Since a large number of PTKs were found to act as oncogenes in vitro and in vivo, it was expected that the counteracting PTPs, including PTP1B, would mainly function as tumor suppressors. During the past 10 years, others and our laboratory demonstrated that unexpectedly when over-expressed PTP1B behaves as an oncogene. Moreover, in a recent literature survey, we identified that there are as many human PTPs acting as oncogenes than those working as tumor suppressors (1). Hence, the preconception and simplistic idea linking PTP expression to cancer protection was incorrect. The role of PTP1B in diabetes and obesity has been well established with antisense base therapies being in advanced clinical trials. Several cancers have been positively associated with metabolic syndrome as risk factors (breast, prostate, colon, etc) and in part this association has maintained our interest in pursuing the study of PTP1B in metabolism with the intent of potentially connecting its function within the ontology of these major diseases and various cancers. Thus, we identified several novel PTP1B substrates that provide different mechanisms by which partly explained how PTP1B contributes to its metabolic as well as pro-oncogenic activities in mammals (i.e. cortactin, and Stam2). We also found that several important lipases are modulated by PTP1B, influencing the severity of metabolic syndromes and cancer outcomes. In this presentation we will describe novel findings on the function of PTP1B in breast and prostate cancers and its potential activity in influencing lipid metabolism.
Julien et al. Nat Rev Cancer (2011)
22 Michel L. Tremblay Goodman Cancer Research Centre, McGill University, Montreal, Canada E-mail: [email protected]
EDUCATIONS/TRAINING Université de Sherbrooke M.Sc. Microbiology 1982 McMaster University Ph.D. Virology 1988 National Institute of Health Postdoc Cancer Biology 1988-1992
POSITIONS AND HONORS 1992-1997 Assistant Professor, Dept. of Biochemistry, McGill University 1997-2001 Associate-Professor Dept. of Biochemistry, McGill University 2001-present Professor, Dept. of Biochemistry, McGill University
2000 to present: Director, Goodman Cancer Research Centre, McGill University 2004 to present: National Scientist, Quebec government 2005 to present: James McGill Professor 2005 to present: Jeanne and Jean-Louis Levesque Chair in Cancer Research 2006 to present: Fellow of the Royal Society of Canada
SELECTED RECENT PUBLICATIONS
1. Julien SG, Dubé N, Hardy S, Tremblay ML. Inside the human cancer tyrosine phosphatome (In press, Nature Cancer Review) Jan 2011. 2. Stuible M, Abella JV, Feldhammer M, Nossov M, Sangwan V, Blagoev B, Park M, Tremblay ML. PTP1B targets the endosomal sorting machinery: dephosphorylation of regulatory sites on the ESCRT component STAM2. J Biol Chem. 2010 Jul 30;285(31):23899-907. 3. Heinonen, K., Bourdeau, A., Doody, K.M., Tremblay, ML. Protein tyrosine phosphatases PTP-1B and TC-PTP play non-redundant roles in macrophage development and interferon-# signaling. PNAS 106(23):9368-72, 2009. 4. Doody, K.M., Bourdeau A, Tremblay, ML. T-cell protein tyrosine phosphatase is a key regulator in immune cell signaling: lessons from the knockout mouse model and implications in human disease. ML. Immunol Rev. 228(1):325-41, 2009. 5. Julien, S.G., Dubé, N., Read, M., Penney, J., Paquet, M., Han, Y., Kennedy, B.P., Muller, W.J. and Tremblay, ML. Protein tyrosine phosphatase 1B deficiency or inhibition delay ErbB2- induced mammary tumorigenesis and protects from lung metastasis. Nat. Genet. 39(3):338-46, 2007.
23 Day-1| 16:05-16:20 Session 1-3
Regulation of Brown Fat Adipogenesis by Protein Tyrosine Phosphatase 1B
Kosuke Matsuo, Ahmed Bettaieb, Naoto Nagata, Izumi Matsuo, and Fawaz G. Haj University of California Davis, Nutrition Department, Davis, CA 95616, USA
Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of insulin signaling and energy balance, but its role in brown fat adipogenesis requires additional investigation. To precisely determine the role of PTP1B in adipogenesis, we established preadipocyte cell lines from wild type and PTP1B knockout (KO) mice. In addition, we reconstituted KO cells with wild type, substrate-trapping (D/A) and sumoylation-resistant (K/R) PTP1B mutants, then characterized differentiation and signaling in these cells. KO, D/A- and WT-reconstituted cells fully differentiated into mature adipocytes with KO and D/A cells exhibiting a trend for enhanced differentiation. In contrast, K/R cells exhibited marked attenuation in differentiation and lipid accumulation compared with WT cells. Expression of adipogenic markers PPAR(, C/EBP!, C/EBP', and PGC1! mirrored the differentiation pattern. In addition, the differentiation deficit in K/R cells could be reversed completely by the PPAR( activator troglitazone. PTP1B deficiency enhanced insulin receptor (IR) and insulin receptor substrate 1 (IRS1) tyrosyl phosphorylation, while K/R cells exhibited attenuated insulin-induced IR and IRS1 phosphorylation and glucose uptake compared with WT cells. In addition, substrate-trapping studies revealed that IRS1 is a substrate for PTP1B in brown adipocytes. Moreover, KO, D/A and K/R cells exhibited elevated AMPK and ACC phosphorylation compared with WT cells. These data indicate that PTP1B is a modulator of brown fat adipogenesis and suggest that adipocyte differentiation requires regulated expression of PTP1B.
24 Day-1| 16:20-16:40 Session 1-3
Differential Regulation of Endoplasmic Reticulum Stress by Protein Tyrosine Phosphatase 1B and T Cell Protein Tyrosine Phosphatase
Ahmed Bettaieb1, Siming Liu1, Yannan Xi1, Naoto Nagata1, Kosuke Matsuo1, Izumi Matsuo1, Heike Keilhack2, Tony Tiganis3 and Fawaz G. Haj1 1Nutrition Department, University of California Davis, Davis, CA 95616, USA 2Merck Research Laboratories, Boston, MA 02115, USA 3Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia
Protein-tyrosine phosphatase 1B (PTP1B) and T cell protein-tyrosine phosphatase (TCPTP) are closely related phosphatases implicated in the control of glucose homeostasis. PTP1B and TCPTP can function coordinately to regulate protein tyrosine kinase signaling and PTP1B has been implicated previously in the regulation of endoplasmic reticulum (ER) stress. We assessed the roles of PTP1B and TCPTP in regulating ER stress in the endocrine pancreas. PTP1B and TCPTP expression was determined in pancreata from chow and high fat diet (HFD) fed mice and the impact of PTP1B and TCPTP over- or under-expression on palmitate- or tunicamycin-induced ER stress signaling assessed in MIN6 insulinoma " cells. PTP1B expression was increased and TCPTP expression decreased in pancreata of mice on HFD, as well as in palmitate-treated MIN6 cells. PTP1B over-expression or TCPTP knockdown in MIN6 cells mitigated palmitate-induced PERK/eIF2! signaling, while PTP1B deficiency enhanced ER stress. Moreover, PTP1B deficiency increased ER stress-induced cell death while TCPTP deficiency protected MIN6 cells from ER stress-induced death. ER stress coincided with the inhibition of Src family kinases (SFKs) which was exacerbated by PTP1B over-expression and prevented by TCPTP knockdown. Inhibition of SFKs ameliorated the protective effect of TCPTP deficiency on ER stress-induced cell death. We demonstrate that PTP1B and TCPTP play non-redundant roles in modulating ER stress in pancreatic " cells and suggest that changes in PTP1B and TCPTP expression may serve as an adaptive response for the mitigation of chronic ER stress.
25 Day-1| 16:40-17:00 Session 1-3
S-Nitrosylation and Inactivation of PTPs Protects Cardiomyocytes against Hypoxic Injury
Tzu-Ching Meng Institute of Biological Chemistry Academia Sinica 128 Academia Road, Section 2, Nankang, Taipei 115, Taiwan
We observed that, when co-cultured with endothelia, the hypoxia-induced injury of cardiomyocytes was significantly attenuated. One of cytoprotectors secreted from endothelia was identified as nitric oxide (NO). We have investigated the underlying mechanism through which NO provides the protective effects during cardiac ischemic injury. Rat myocardia H9c2 cells underwent a loss of F-actin cytoskeletal integrity and intercellular adherens, concomitant with a drastic decrease of protein phosphotyrosine (pTyr) signaling and activation of endogenous protein tyrosine phosphatases (PTPs) in response to hypoxia. Intriguingly, treatment of a physiological form of NO carrier S-nitrosoglutathione (GSNO) not only reversed the hypoxia-induced cytoskeletal changes but also sustained pTyr levels of cellular proteins. The stable isotope labeling in cell culture (SILAC) in conjunction with LC-MS/MS analyses identified a cluster of cytoskeletal regulators whose pTyr levels were dependent upon environmental oxygen, yet could be maintained in hypoxia with GSNO supply. Employing the Biotin Switch Method, we further showed that several endogenous PTPs were S-nitrosylated, therefore inactivated by GSNO treatment in cells under the hypoxic condition. Taken together, our data suggested that NO might regulate actin dynamics via inactivation of PTPs. This hypothesis was further confirmed by the direct application of a general PTP inhibitor to cells under hypoxia. Thus, suppression of PTP activity by NO or specific inhibitors may provide a novel therapeutic opportunity for ischemic heart.
26 Tzu-Ching Meng Institute of Biological Chemistry, Academia Sinica, Taiwan E-mail: [email protected]
EDUCATIONS/TRAINING National Taiwan University BS Agricultural Chem 1989 National Taiwan University MS Medical Toxicology 1994 University of Nebraska Medical Center PhD Biochem/Mol Biol 1999 Cold Spring Harbor Laboratory Postdoc Cell Signaling/PTPs 1999-2003
POSITIONS AND HONORS 2003-2008 Assistant Research Fellow, Institute of Biological Chemistry, Academia Sinica 2008-present Associate Research Fellow, Institute of Biological Chemistry, Academia Sinica 2003-2010 Assistant Professor, Institute of Biochemical Sciences, National Taiwan University 2010-present Associate Professor, Institute of Biochemical Sciences, National Taiwan University
2009: Research Award for Junior Investigators, Academia Sinica 2009: Dr. Ta-You Wu’s Memorial Award for Young Investigators, National Science Council, Taiwan 2009: Important Finding of the Year, pointed by National Science Council, Taiwan
SELECTED RECENT PUBLICATIONS 1. Hsu, M.F. and Meng, T.C. Enhancement of insulin responsiveness by nitric oxide-mediated inactivation of protein tyrosine phosphatases. J. Biol Chem. 285, 7919-7928, 2010
2. Ku, H.Y., Wu, C.L., Rabinow, L., Chen, G.C. and Meng, T.C. The organization of F-actin via the concerted regulation of Kette by PTP61F and dAbl. Mol. Cell. Biol. 29, 3623-3632, 2009
3. Chen, Y.Y., Chu, H.M., Pan, K.T., Teng, C.H., Wang, D.-L., Wang, A.H.-J., Khoo, K.H. and Meng, T.C. Cysteine S-nitrosylation protects protein tyrosine phosphstase 1B against oxidation-induced permanent inactivation. J. Biol. Chem. 283, 35265-35272, 2008
4. Chang, Y.C., Lin, S.Y., Liang, S.Y., Pan, K.T., Chou, C.C., Chen, C.H., Liao, C.L., Khoo, K.H. and Meng, T.C. Tyrosine phosphoproteomics and identification of substrates of protein tyrosine phosphatase dPTP61F in Drosophila S2 cells by mass spectrometry-based substrate trapping strategy. J. Proteome Res. 7, 1055-1066, 2008
5. Teng, C.H., Huang, W.N. and Meng, T.C. Several dual specificity phosphatases coordinate to control the magnitude and duration of JNK activation in signaling response to oxidative stress. J. Biol. Chem. 282, 28395-28407, 2007
27 Day-1| 17:00-17:15 Session 1-3
Hepatic Src Homology Phosphatase 2 Regulates Energy Balance and Metabolism
Naoto Nagata1, Kosuke Matsuo1, Mirela Delibegovic2, Izumi Matsuo1, James Graham1,3, Susan Gray4, Wentian Yang2, Jason Kim4,5, Peter Havel1,3, Benjamin G. Neel2 and Fawaz G. Haj1 1Nutrition Department, University of California Davis, Davis, CA 95616, USA 2Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA 3Department of Molecular Biosciences, University of California Davis, Davis, CA 95618, USA 4Program in Molecular Medicine and 5 Department of Medicine, Division of Endocrinology, Metabolism and Diabetes, University of Massachusetts Medical School, Worcester, MA 01605, USA
To address the metabolic role of hepatic Src homology phosphatase 2 (Shp2), we generated mice with liver specific Shp2 deletion (LSHKO) using tissue-specific Cre-LoxP system, and then we analyzed body mass, insulin sensitivity, insulin signaling and glucose tolerance. LSHKO mice fed chow diet exhibited improved insulin sensitivity and glucose tolerance compared with controls. These findings correlated with, and were most likely caused by, increased tyrosine phosphorylation of IRS1/IRS2 in the liver, accompanied by increased PI3K/Akt signaling. After 26 weeks of high-fat diet (HFD) feeding, LSHKO weighted 12% less than controls, and exhibited improved insulin sensitivity and glucose tolerance. In support of these, LSHKO exhibited significant increase of energy expenditure (EE) by 10%, and increased tyrosyl and serine phosphorylation of total and mitochondrial STAT3 that could lead to increased EE. Hyperinsulinemic euglycemic clamp revealed that insulin-suppressed hepatic glucose production and glucose infusion rate were elevated in LSHKO, by 4- and 6-fold, respectively. Moreover, in LSHKO, the expression of TNF-!, MCP1 and ER stress makers were suppressed, suggesting hepatic Shp2 deletion attenuated HFD-induced inflammation and ER stress. Our study indicates that hepatic Shp2 is a negative regulator of insulin action, and plays a regulatory role in energy balance under conditions of HFD feeding.
28 Day-2| 9:00-9:45 Keynote Lecture
Functional Analysis of Protein Tyrosine Phosphatases
Nicholas K. Tonks Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
The protein tyrosine phosphatases (PTPs) are now recognized as critical regulators of signal transduction under normal and pathophysiological conditions. These enzymes belong to a family of cysteine-dependent phosphatases, which are characterized by a conserved ‘C[X]5R’ motif and hydrolyze phosphoester bonds in proteins and non-protein substrates. Overall, the objective of the lab is to examine PTP regulation and function in cell and animal models, to define the role of PTPs in critical tyrosine phosphorylation-dependent signaling events in human disease. It is anticipated that these studies will lead to identification of novel therapeutic targets and biomarkers from among the PTPs themselves, or from the signaling pathways they regulate. Currently, there are four broad areas of research in the lab covering (i) functional analysis of members of the PTP family, (ii) regulation of PTP function, in particular by reversible oxidation, (iii) development of novel approaches to therapeutic intervention in PTP function, and (iv) characterization of a knockout mouse model to define the function of JSP1, a member of the PTP family that is a novel regulator of MAP kinase signaling. Projects from these areas will be described.
29 Nicholas K. Tonks PhD, FRS Biosketch
Dr Nick Tonks was awarded a BA in Biochemistry from the University of Oxford in 1981 and PhD in Biochemistry from the University of Dundee in 1985, where he worked in the laboratory of Prof. Sir Philip Cohen. From 1985-88 he performed postdoctoral studies in the laboratory of one of the pioneers in the field of protein phosphorylation, Prof Edmond Fischer (1992 Nobel Laureate), in the Department of Biochemistry at the University of Washington and in 1988 he accepted a faculty position there as Research Assistant Professor. In 1990 he joined the faculty of Cold Spring Harbor Laboratory and was promoted to Professor in 1995.
While at the University of Washington, Dr Tonks was the first to purify an enzyme termed a protein tyrosine phosphatase (PTP). With his collaborators he established the existence of receptor-like PTPs, showing for the first time that this family of enzymes had the potential to trigger directly a cell’s response to envirionmental stimuli. This led to a shift in emphasis in research in the area of signal transduction, so that now it is recognized that PTPs are critical regulators of signal transduction in their own right and that disruption of their function underlies several major human diseases. Dr Tonks takes a multidisciplinary approach to characterizing the structure, regulation and function of members of the PTP superfamily. Overall, the objective of the lab is to develop tools for analysis of PTP regulation and function and integrate them with state of the art cell and animal models, to define critical tyrosine phosphorylation-dependent signaling events in human disease and thereby identify new therapeutic targets.
Dr Tonks has published over 160 papers in the field and has been granted 10 patents. His research has been recognized by several awards, including the Colworth Medal for 1993, which is awarded annually by the British Biochemical Society to an outstanding British Biochemist under the age of 35, and he was a Pew Scholar in the Biomedical Sciences from 1991-95. In 2001, he was elected to Fellowship of the Royal Society, which is the National Academy of Sciences of the UK.
30 Day-2| 9:45-10:20 Session 2-1
Prostaglandin E2-Associated Inflammation and Bacterial Infection in Gastric Tumorigenesis
Masanobu Oshima Division of Genetics, Cancer Research Institute Kanazawa University Kakuma-machi, Kanazawa 920-1192, Japan
Activation of Wnt signaling is one of the important mechanisms for gastric cancer development. On the other hand, Helicobacter pylori infection induces prostaglandin
E2 (PGE2) signaling, which plays a key role in tumorigenesis. We have investigated an interaction of these two pathways in gastric tumorigenesis by construction of mouse models [1, 2]. Wnt activation increased self-renewal activity of gastric epithelial cell, while induction of PGE2 pathway resulted in inflammation and metaplastic hyperplasia in the stomach. Notably, inflammation-associated gastric cancer is developed in the mouse stomach when Wnt and PGE2 pathways are activated simultaneously. Moreover, macrophage-derived TNF-! promoted Wnt activity of gastric cancer cells, which may be one of the mechanisms of inflammation in tumorigenesis [3]. We next constructed germfree mouse colonies to examine the role of commensal bacteria in gastric tumorigenesis. Importantly, inflammation and tumorigenesis were significantly suppressed in the stomach of germfree mouse models [4]. Moreover, chemokine expression and macrophage infiltration were suppressed in the germfree mouse stomach. These results, taken together, indicate that PGE2 signaling and bacterial infection cooperatively induce inflammatory responses, which is required for tumor development. Thus, targeting PGE2-associated inflammatory responses will be an effective preventive strategy against gastric cancer.
References 1. Oshima H. et al. EMBO J, 23, 1669-1678, 2004. 2. Oshima H. et al. Gastroenterology, 131, 1086-1095, 2006. 3. Oguma K. et al. EMBO J, 27, 1671-1681, 2008. 4. Oshima H. et al. Gastroenterology, 2010 in press.
31 Masanobu Oshima Division of Genetics, Cancer Research Institute Kanazawa University E-mail: [email protected]
EDUCATIONS/TRAINING Hokkaido University DVM Veterinary Medicine 1988 Hokkaido University PhD Veterinary Medicine 1995 Merck Research Laboratories Postdoc Gene Therapy 1997-1999
POSITIONS AND HONORS 1988-1992 Research Scientist, Chugai Pharmaceutical Co. Ltd, Japan 1992-1999 Research Scientist, Banyu Tsukuba Research Institute (Merck), Japan 2000-2005 Associate Professor, Department of Pharmacology, Kyoto University Graduate School of Medicine, Japan 2005-present Professor, Division of Genetics, Cancer Research Institute, Kanazawa Univ., Japan
2005-present: Board member, Japanese Society of Veterinary Science 2009-present: Associate Editor, Cancer Science
SELECTED RECENT PUBLICATIONS
1. Oshima H, Hioki K, Popivanova BK, Oguma K, van Rooijen N, Ishikawa T, and Oshima M. PGE2 signaling and bacterial infection recruit tumor-promoting macrophages to mouse gastric tumors. Gastroenterology, 2010, in press.
2. Du YC, Oshima H, Oguma K, Kitamura T, Itadani H, Fujimura T, Piao YS, Yoshimoto T, Minamoto T, Taketo MM, and Oshima M. Induction and downregulation of Sox17 and its possible roles during the course of gastrointestinal tumorigenesis. Gastroenterology, 137, 1346-1357, 2009.
3. Oshima H, Itadani H, Kotani H, Taketo MM, and Oshima M. Induction of prostaglandin E2 pathway promotes gastric hamartoma development with suppression of bone morphogenetic protein signaling. Cancer Res, 69, 2729-2733, 2009.
4. Oguma K, Oshima H, Aoki M, Uchio R, Naka K, Nakamura S, Hirao A, Saya H, Taketo MM, and Oshima M. Activated macrophages promote Wnt signaling through tumor necrosis factor-! in gastric tumor cell. EMBO J 27, 1671-1681, 2008.
5. Guo X, Oshima H, Kitamura T, Taketo, MM, and Oshima M. Stromal fibroblasts activated by tumor cells promote angiogenesis in mouse gastric cancer. J Biol Chem, 283: 19864-19871, 2008.
6. Oshima H, Matsunaga A, Fujimura T, Tsukamoto T, Taketo MM, and Oshima M. Carcinogenesis
in mouse stomach by simultaneous activation of the Wnt signaling and prostaglandin E2 pathway. Gastroenterology 131: 1086-1095, 2006.2006.
7. Oshima M, Oshima H, Matsunaga A, and Taketo MM. Hyperplastic gastric tumors with spasmolytic polypeptide-expressing metaplasia (SPEM) caused by TNF-!-dependent inflammation in COX-2/mPGES-1transgenic mice. Cancer Res 65: 9147-9151, 2005.
8. Oshima H, Oshima M, Inaba K, and Taketo MM. Hyperplastic gastric tumors induced by activated macrophages in COX-2/mPGES-1 transgenic mice. EMBO J 23: 1669-1678, 2004.
32 Day-2| 10:20-10:55 Session 2-1
Posttranslational Regulation of the Wnt Signaling Pathway
David M. Virshup Program in Cancer and Stem Cell Biology Duke-NUS Graduate Medical School Singapore 8 College Road, Singapore 169857
Production of Wnts is critical for both cellular proliferation and development. Over-activity of multiple Wnts is found in cancer, and "-catenin is only one of many downstream signaling pathways activated by these Wnts. Targeting these diverse Wnt-stimulated pathways more broadly may be a useful approach to cancer. We use genetic and small molecule approaches to study specific steps in Wnt signaling and test if inhibition of Wnt secretion affects cancer proliferation. Recent findings demonstrate that the pathway can fruitfully be targeted and provide novel insights into basic mechanisms of Wnt activity.
References Coombs et al. WLS-dependent secretion of WNT3A requires Ser209 acylation and vacuolar acidification. J. Cell. Sci. (2010) 123: 3357-67 Virshup and Shenolikar. From promiscuity to precision: protein phosphatases get a makeover. Mol. Cell (2009) 33:537-45 Coombs et al. Wnt signaling in development, disease and translational medicine. Current drug targets (2008) 9:513-31
33 David M. Virshup Director, Program in Cancer and Stem Cell Biology; Professor of Pediatrics [email protected]
EDUCATIONS 1977 B.A., Chemistry, Beloit College 1981 M.D. Johns Hopkins U. School of Medicine
POSITIONS AND HONORS 1990 Joint Appointment, Department of Molecular Biology and Genetics, Johns Hopkins 1990-2007 Assistant, Associate, Full Professor of Pediatrics, with Adjunct in Oncological Sciences, University of Utah; tenure 1997 2007- Professor and Director, Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School Singapore 2007- Professor with tenure, National University of Singapore 2007- Professor of Pediatrics with tenure, Duke University School of Medicine
2000 American Society for Clinical Investigation 2000-05 Editorial Board, Journal of Biological Chemistry 2001-05 American Cancer Society Tumor Biology and Endocrinology Study Section 2002- Association of American Physicians 2006 Study Section Chair, American Cancer Society, Tumor Biology and Endocrinology 2006, 2008 Co-organizer, Organizer, FASEB Summer Conference on Protein Phosphatases 2008-2013 Singapore Translational Research Award
SELECTED RECENT PUBLICATIONS 1. Gallego M, Virshup DM. (2007) Post-translational modifications regulate the ticking of the circadian clock. Nature Reviews Mol Cell Biol. 8:139-48. 2. W Luo, A Peterson, B A Garcia, G Coombs, B Kofahl, R Heinrich, J Shabanowitz, D F. Hunt, H. J Yost, and DM Virshup. (2007) Protein phosphatase 1 regulates assembly and function of the !-catenin degradation complex. EMBO Journal. 26, 1511-1521.- 3. I-C Tsai, J Amack, Z-H Gao, Vi Band, H. J Yost, DM Virshup. (2007). A Wnt-CKI"-Rap1 Pathway Regulates Gastrulation by Modulating SIPA1L1, a Rap GTPase Activating Protein. Developmental Cell, 12, 335 4. Forester CM, Maddox J, Louis JV, Goris J, Virshup DM. (2007) Control of mitotic exit by PP2A regulation of Cdc25C and Cdk1. Proc Natl Acad Sci U S A. 104:19867-72. 5. Virshup DM and Shenolikar S. From promiscuity to precision: protein phosphatases get a makeover. Mol. Cell (2009) vol. 33 (5) pp. 537-45 6. GS Coombs, Jia Yu, C A Canning, C A Veltri, T M Covey, J K Cheong, V Utomo, N Banerjee, Z H Zhang, R C Jadulco, G P Concepcion, T S Bugni, MK Harper, I Mihalek, C. M Jones, C M Ireland, and David M Virshup. (2010) WLS/GPR177-dependent secretion of WNT3A requires serine 209 acylation and vacuolar acidification. Journal of Cell Science, 123(19): 3357-67
34 Day-2| 11:10-11:45 Session 2-2
Dys-regulation of Lipid Metabolism May Link to the Development of Hepatocellular Carcinoma
Kunitada Shimotohno Research Institute, Chiba Institute of Technology 2-17-1 Tsudanuma, Chiba 275-0016, Japan
Hepatitis C virus (HCV) is a major causative agent of serious liver diseases including chronic hepatitis, liver cirrhosis, end stage of liver failure and hepatocellular carcinoma. Patients with the C-type chronic hepatitis often develop steatosis, which is suggested to contribute to the progression of liver fibrosis. HCV affects hepatic lipid metabolism by accumulating the triglycerides level in the liver cells. In vitro HCV replication system of infectious HCV clone was used to clarify the role of accumulation of lipid as well as to search for any involvement of lipid metabolism for virus proliferation. Upon introduction of the infectious HCV genome RNA synthesized in vitro into Huh7, a human hepatoma-derived cell line, autonomous replication of the genome and release of infectious virus particles to culture medium occur. Analysis of the cells bearing HCV genome replication showed accumulation of lipid droplets (LDs). Accumulation of the LDs depends on expression of HCV Core, a capsid protein of virion. The Core not only enhances accumulation of LDs but also recruit other HCV proteins around the LDs, which is essential for productive infection. Analysis of virus particles released to culture medium showed the presence of two distinguishable viruses by buoyant density; those with smaller value are infectious and the others non-infectious. Moreover, we observed that inhibition of release of lipoprotein by MTP inhibitor resulted in suppression of virus release to culture medium. HCV in culture medium associates with apolipoproteins such as apolipoprotein-A and -B (Apo-A, -B). Depletion of Apo-E from HCV infected cells reduced the production of infectious HCV. Furthermore, we found the evidence that Apo-E receptor seems to be a HCV receptor. By taking these results together, we conclude that HCV hijacks lipid metabolism and transport pathway of lipid for its propagation. Abnormal lipid metabolism by HCV infection may be involved in exacerbation of liver diseases.
References 1. Hishiki T, Shimizu Y, Tobita R, Sugiyama K, Ogawa K, Funami K, Ohsaki Y, Fujimoto T, Takaku H, Wakita T, Baumert TF, Miyanari Y, Shimotohno K. Infectivity of hepatitis C virus is influenced by association with apolipoprotein E isoforms. J Virol. 84(22): 12048-12057, 2010. 2. Shimizu Y, Hishiki T, Sugiyama K, Ogawa K, Funami K, Kato A, Ohsaki Y, Fujimoto T, Takaku H, Shimotohno K. Lipoprotein lipase and hepatic triglyceride lipase reduce the infectivity of hepatitis C virus (HCV) through their catalytic activities on HCV-associated lipoproteins. Virology. 407(1): 152-915, 2010 3. Miyanari Y, Atsuzawa K, Usuda N, Watashi K, Hishiki T, Zayas M, Bartenschlager R, Wakita T, Hijikata M, Shimotohno K. The lipid droplet is an important organelle for hepatitis C virus production. Nat Cell Biol. 9(9): 1089-1097, 2007
35 Kunitada Shimotohno Research Institute, Chiba Institute of Technology E-mail: [email protected]
EDUCATIONS/TRAINING Hokkaido University PhD 1973 University of Wisconsin Post doc Viral Oncology 1978-1981
POSITIONS AND HONORS 1972-1983 Research Scientist, Department of Molecular Genetics, National Institute of Genetics, Japan 1983-1985 Section Head, Virology Division, National Cancer Center Research Institute 1985-1996 Chief, Virology Division, National Cancer Center Research Institute 1996-2007 Professor, Department of Viral Oncology, Institute for Virus Research, Kyoto University 2007-2009 Professor, Keio University School of Medicine 2009-present Professor, Chiba Institute of Technology
1985: Incentive Award of The Genetic Society of Japan 1994: Hideyo Noguchi Memorial Prize for Medicine 2006: 15th Yoshida Tomizo Award from Japanese Cancer Association
1994-present: Associate Editor of Cancer Science 1996-present: Editorial Board Member of Journal of Virology
SELECTED RECENT PUBLICATIONS 1. Hishiki T, Shimizu Y, Tobita R, Sugiyama K, Ogawa K, Funami K, Ohsaki Y, Fujimoto T, Takaku H, Wakita T, Baumert TF, Miyanari Y, Shimotohno K. Infectivity of hepatitis C virus is influenced by association with apolipoprotein E isoforms. J Virol. 84(22): 12048-12057, 2010. 2. Shimizu Y, Hishiki T, Sugiyama K, Ogawa K, Funami K, Kato A, Ohsaki Y, Fujimoto T, Takaku H, Shimotohno K. Lipoprotein lipase and hepatic triglyceride lipase reduce the infectivity of hepatitis C virus (HCV) through their catalytic activities on HCV-associated lipoproteins. Virology. 407(1): 152-915, 2010 3. Arimoto K, Funami K, Saeki Y, Tanaka K, Okawa K, Takeuchi O, Akira S, Murakami Y, Shimotohno K. Polyubiquitin conjugation to NEMO by triparite motif protein 23 (TRIM23) is critical in antiviral defense. Proc Natl Acad Sci USA. 107(36): 15856-15861, 2010 Aly HH, Qi Y, Atsuzawa K, Usuda N, Takada Y, Mizokami M, Shimotohno K, Hijikata M. Strain-dependent viral dynamics and virus-cell interactions in a novel in vitro system supporting the life cycle of blood-borne hepatitis C virus. Hepatology. 50(3): 689-696, 2009. 4. Arimoto K, Takahashi H, Hishiki T. Konishi H, Fujita T and Shimotohno K., Negative regulation of the RIG-I signaling by the novel ubiquitin ligase RNF125. Proc. Natl. Acd. Sci. USA 104: 7500-7505, 2007 5. Miyanari Y, Atsuzawa K, Usuda N, Watashi K, Hishiki T, Zayas M, Bartenschlager R, Wakita T, Hijikata M, Shimotohno K. The lipid droplet is an important organelle for hepatitis C virus production. Nat Cell Biol. 9(9): 1089-1097, 2007 6. Watashi K, Inoue D, Hijikata M, Goto K, Aly HH, Shimotohno K. Anti-hepatitis C virus activity of tamoxifen reveals the functional association of estrogen receptor with viral RNA polymerase NS5B. J Biol Chem. 282(45): 32765-32772, 2007 7. Ishii N, Watashi K, Hishiki T, Goto K, Inoue D, Hijikata M, Wakita T, Kato N, Shimotohno K. Diverse effects of cyclosporine on hepatitis C virus strain replication. J Virol. 80: 4510-4520, 2006. 8. Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, Shimotohno K. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell. 19: 111-122, 2005.
36 Day-2| 11:45-12:20 Session 2-2
Immunological Dysregulation and Oncogenesis by Human T-Cell Leukemia Virus Type 1
Masao Matsuoka Institute for Virus Research, Kyoto University 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
Human T-cell leukemia virus type 1 (HTLV-1) is a causal agent of adult T-cell leukemia (ATL) and inflammatory diseases including HTLV-1 associated myelopathy/tropical spastic paraparesis [1]. After infection of HTLV-1, some carriers will develop ATL after a long latent period. Among these viral genes, previous study focused on the tax gene since Tax has pleiotpropic functions. However, analyses of leukemic cells in ATL patients revealed that Tax expression was frequently inactivated in leukemic cells [1]. The HTLV-1 bZIP factor (HBZ) gene is encoded by the minus strand of provirus, and transcribed as anti-sense transcript from 3’-LTR. Analyses of proviral sequences in ATL cells showed that nonsense mutations of viral genes except HBZ gene were generated by APOBEC3G before the integration, indicating that only HBZ gene is essential for oncogenesis by HTLV-1 [2]. We found that HBZ gene was expressed in all ATL cases. Functional analyses of the HBZ gene on proliferation of T-lymphocytes reveal that HBZ RNA promotes proliferation of T-lymphocytes whereas HBZ protein suppresses Tax mediated transactivation through 5’LTR [3]. In the transgenic mice that expressing HBZ gene by CD4 specific promoter, we found development of T-cell lymphomas and systemic inflammations. In the tissues, CD4+ T-cells infiltrate into skin and lung, indicating that HBZ gene is associated with infiltrative phenotype of HTLV-I infected cells. We found that effector/memory T cells and regulatory T-cells increased in HBZ-Tg mice. As a mechanism to increase regulatory T-cells, HBZ directly promoted the transcription of Foxp3 gene [4]. Further, HBZ interacted with Foxp3 and impaired its functions in regulatory T-cells. Thus, HBZ plays critical roles in pathogenesis by HTLV-1.
References 1. Matsuoka M and Jeang KT. Nat Rev Cancer 7, 270-80, 2007. 2. Fan J. et al. J Virol, 84, 7278-7287, 2010. 3. Satou Y. et al. Proc Natl Acad Sci USA 103, 720-725, 2006. 4. Satou Y. et al. PloS Pathog, in press.
37 Masao Matsuoka Laboratory of Virus Control, Institute for Virus research, Kyoto University E-mail: [email protected]
EDUCATIONS/TRAINING Kumamoto University MD Medicine 1982 Kumamoto University PhD Medicine 1988 UC Berkeley Postdoc Molecular biology 1988-1992
POSITIONS AND HONORS 1986-1991 Assistant Professor, Dept of Internal Medicine, Kumamoto University Medical School 1995-1999 Associate Professor, 1999-present Professor, Institute for Virus Research, Kyoto University 2010-presrnt Director of Institute for Virus Research, Kyoto University
1989: Leukemia Research Foundation Fellowship 1995: Award for young researcher from Japanese society of Hematology
2005-present: Editorial Board, Retrovirology 2009-present: Associate Editor, Cancer Science
SELECTED RECENT PUBLICATIONS
1. Satou Y, Yasunaga J, Zhao T, Yoshida M, Miyazato P, Takai K, Shimizu K, Ohshima K, Green PL, Ohkura N, Yamaguchi T, Ono M, Sakaguchi S, Matsuoka M. HTLV-1 bZIP factor induces T-cell lymphoma and systemic inflammation in vivo. PloS Pathog (in press).
2. Fan J, Ma G, Nosaka K, Tanabe J, Satou Y, Koito A, Wain-Hobson S, Vartanian JP, Matsuoka M. APOBEC3G generates nonsense mutations in HTLV-1 proviral genomes in vivo. J Virol 84: 7278-7287, 2010.
3. Matsuoka M and Jeang KT. Human T cell leukemia virus type 1 (HTLV-1) and leukemic transformation: viral infectivity, Tax, HBZ, and therapy. Oncogene (in press).
4. Zhao T, Yasunaga J-I, Satou Y, Nakao M, Takahashi M, Fujii M, Matsuoka M. Human T-cell leukemia virus type 1 bZIP factor selectively suppresses the classical pathway of NF-!B. Blood 113: 2755-2764, 2009.
5. Matsuoka M and Jeang KT. Human T-cell leukemia virus type 1 (HTLV-1) infectivity and cellular transformation. Nat Rev Cancer 7: 270-80, 2007.
6. Satou Y, Yasunaga J-I, Yoshida M, Matsuoka M. The HTLV- I bZIP factor gene mRNA supports proliferation of adult T-cell leukemia cells. Proc Natl Acad Sci USA 103: 720-725, 2006.
38 Day-2| 14:00-14:45 Special Lecture
Regulation of Immune Responses and Oncogenesis by the IRF Family of Transcription Factors and Their Partners
Tadatsugu Taniguchi Department of Immunology, Graduate School of Medicine, University of Tokyo Hongo 7-3-1, Bunkyo-city, Tokyo 113-0033, Japan
My research career in immunology began with the initial isolation, identification and characterization of a cytokine gene, type I interferon (IFN) beta. It was during the course of our studies of the regulation of the IFN system that my laboratory made its next major achievement, the discovery and characterization of a family of transcription factors termed interferon regulatory factors (IRFs). The IRF family consists of nine members in man and mouse, and its role in the regulation of immunity and oncogenesis has since been extensively studied. In this talk, I will first summarize the versatile functions of IRFs in host defense and then present our recent data on the role of select IRFs in tumor immunosurveillance and pathogen recognition receptor signaling. We first show a critical role for IRF5 in the suppression of tumor growth by cells other than T cells and natural killer cells, which are currently understood to be the critical immune effector cells in this context. Next, we address a critical aspect of an immune response, the polarization of the response towards a given pathogen. Two classes of pathogen recognition receptors (PRRs), membrane-bound Toll-like receptors (TLRs) and cytosolic receptors such as RIG-I-like receptors (RLRs), typically trigger the innate immune system. Depending on the pathogen, activation of each class of PRRs may evoke distinct immune responses appropriate to the pathogenic challenge. This issue has not been rigorously addressed. We show that in antigen presenting cells the most notable feature of RLR signaling induced by viruses or mimetic agonists is the activation of type I IFN and silencing of interleukin (IL)-12p40 gene expression, whereas the opposite observation is made by TLR signaling stimulated by bacteria or mimetic agonists. As a result, TLR signaling strongly favors Th1-type responses and RLR signaling Th2 responses in vivo. We present evidence that activation of IRF3 upon RLR, but not by TLR, signaling underlies these distinct responses. Thus, this ingenious mechanism of transcriptional regulation may underlie the PRR-mediated orientation of anti-viral vs. anti-bacterial immune responses, which may have clinical implications.
39 Tadatsugu Taniguchi Department of Immunology, Graduate School of Medicine, University of Tokyo E-mail: [email protected]
EDUCATIONAL BACKGROUND 1971: B. Sci. in Biology, Tokyo University of Education Tokyo, Japan 1978: Ph.D. in Molecular Biology, University of Zurich Zurich, Switzerland (Thesis advisor; Prof. Dr. Charles Weissmann)
PROFESSIONAL EXPERIENCE 1978 - 1980: Associate, Department of Biochemistry, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan 1980 - 1983: Associate Member, Department of Biochemistry, Cancer Institute, Japanese Foundation for Cancer Research 1980 - 1982: Visiting Associate Professor, New York University Medical Center, New York, U.S.A. 1983 - 1984: Member and Chief, Department of Biochemistry, Cancer Institute, Japanese Foundation for Cancer Research 1984 - 1994: Professor, Division of Molecular Biology, Institute for Molecular and Cellular Biology, Osaka University 1995 - 1997: Professor, Department of Immunology, Faculty of Medicine, University of Tokyo 1997-present: Professor, Department of Immunology, Graduate School of Medicine and Faculty of Medicine, University of Tokyo 1998-2000: Member of the Science Council, Ministry of Education, Culture, Sports, and Science of Japan. 2002-2008: Co-chair person, International Affairs Committee, American Association for Cancer Research 2003-present Foreign Associate Member, National Academy of Sciences, U.S.A. 2005-present Member of the Science Council of Japan 2006-present Visiting Professor, Wakayama Medical University 2006-present Adjunct Professor, New York University Medical School, U.S.A.
HONORS 1978: Prix Jacques de Bedriaga (University of Zurich) 1981: Japanese Biochemical Society Shoreisho Award 1983: The Scientific Award of Japanese Foundation for Cancer Research 1985: The Noguchi Memorial Medical Award 1986: Hammer Prize (U.S.A.) 1988: Behring-Kitasato Prize (Japan/Germany) 1988: The Milstein Award (International Society of Interferon Research) 1989: The Asahi Prize 1989: Osaka Science Prize 1989: Wakayama Prefecture Culture Award 3rd Prize 1991: Robert-Koch Prize (Germany) 1993: Honorary Member, The American Association of Immunologists 1994: The Uehara Prize 1995: Academic Award of the Mochida Memorial Foundation 1995: The Fulbright 50th Anniversary Distinguished Fellow (U.S.A.) 1996: The Prize of Princess Takamatsunomiya Cancer Research Foundation 1996: Fujihara Award 1997: Wakayama Prefecture Culture Award Grand Prize 1997: Keio Medical Science Prize
40 1999: Lifetime Honorary Membership Award (International Cytokine Society) 2000: Japan Academy Prize 2000: ISI Citation Laureate Award 2003: Foreign Associate Member, National Academy of Sciences, U.S.A. 2006: "Laurea Honoris Causa" from University of Verona (Italy) 2006: The Harvey Lecture (Harvey Society, New York) 2006: Pezcoller Foundation-AACR International Award for Cancer Research (Italy/ U.S.A) 2007: The Feodor Lynen Lecture Award (U.S.A) 2007: Honorable Doctorate Degree, University of Zurich (Switzerland) 2008: The Tomizo Yoshida Prize (Japanese Cancer Association) 2009: Person of Cultural Merit (Government of Japan) 2010: The Naito Foundation Research Prize 2010: Honorary Citizenship, Aridagawa-cho, Wakayama Prefecture, Japan
SELECTED RECENT PUBLICATIONS 1. Takaoka, A., Wang, Z., Choi, MK., Yanai, H., Negishi, H., Ban, T., Yan, L., Miyagishi, M., Kodama, T., Honda, K., Ohba, Y. and Taniguchi, T.; DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. (2007). Nature. 448(26), 501-506. 2. Kano, S., Sato, K., Morishita, Y., Vollstedt, S., Kim, S., Bishop, K., Honda, K., Kubo, M., and Taniguchi, T.; The contribution of transcription factor IRF1 to the + interferon-#-interleukin 12 signaling axis and TH-17 differentiation of CD4 T cells. (2008). Nat Immunol, 9(1), 34-41. 3. Couzinet. A., Tamura. K., Chen. H., Nishimura. K., Wang. Z., Morishita. Y., Takeda. K., Yagita. H., Yanai. H., Taniguchi. T., and Tamura. T.; A cell type-specific requirement for IRF5 in Fas-induced apoptosis. (2008). Proc. Natl. Acad. Sci. USA., 105, 2556-2561. 4. Tamura. T., Yanai. H., Savitsky. D., and Taniguchi. T.; The IRF Family Transcription Factors in Immunity and Oncogenesis. (2008) Annu. Rev. Immunol., 26, 535-584. 5. Taniguchi, T.; Aimez-vous Brahms? A story capriccioso from the discovery of a cytokine family and its regulators. (2009) Nat Immunol. 10(5), 447-449. 6. Choi,.MK, Wang, Z., Ban. T., Yanai, H., Lu, Y., Koshiba, R., Nakaima, Y., Hangai, S., Savitsky, D., Nakasato, M., Negishi, H., Takeuchi, O., Honda, K., Akira, S., Tamura, T., and Taniguchi, T.; A selective contribution of the RIG-I-like receptor pathway to type I interferon responses activated by cytosolic DNA. (2009) Proc. Natl. Acad. Sci. U S A. 106, 19870-17875. 7. Yanai, H., Ban, T., Wang, Z., Choi, MK, Kawamura, T., Negishi, H., Nakasato, M., Lu, Y., Hangai, S., Koshiba, R., Savitsky, D., Ronfani, R., Akira, S., Marco E. Bianchi, ME, Honda, K., Tamura, T., Kodama, T., and Taniguchi, T.; HMGB proteins function as universal sentinels for nucleic acid-mediated innate immune responses. (2009) Nature, 462, 99-103.
41 Day-2| 14:45-15:20 Session 2-3
Functional Divergence of RNA-Sensing Systems in Dendritic Cells for Induction of Antitumor Effectors
Tsukasa Seya, Hiroaki Shime, Masahiro Azuma, Jun Kasamatsu, Hroyuki Oshiumi, Misako Matsumoto
Department of Microbiology and Immunology, Hokkaido University Graduate School of Medicine, kita-15, Nishi-7, kita-ku, Sapporo 060-8638, Japan
Myeloid dendritic cells (mDC) sense virus-derived RNA (a representative analog polyI:C) by TLR3 or RIG-I/MDA5 and induce production of type I IFN. These pathways further derive antiviral natural killer (NK) [1] and CD8+ T cells (CTL) [2] independent of the function of type I IFN. TLR3 links the adaptor TICAM-1 (TRIF) while RIG-I takes the adaptor IPS-1. The TICAM-1 pathway and IPS-1 pathway are converged to activate IRF-3, which in turn induces type I interferon (IFN) and mDC maturation [3]. Here we additionally employ newly synthetic derivatives for specifically stimulating either one of the IFN-inducing pathways. What we have known is that participation of each pathway in mDC-derived effectors is not the same, depending on the reflection of their differential role in provoking antitumor immunity. Here we focus on how immune responses differ in these two RNA-sensing pathways by using gene-disrupted mice and syngenic implant tumors. A characteristic feature for activation of the TICAM-1 pathway in murine CD8+ DCs is to drive NK activation and CD8+ CTL induction. The IPS-1 pathway in CD8+ DC has a minimal role in CTL induction, while it induces potent NK activation. MHC low and high tumors loaded are regressed by i.p. administration of the RNA adjuvants through inducing NK and CTL. It is notable that myeloid-derived suppressor cells (MDSC) sense polyI:C by TLR3 leading to tumor regression in a manner different from that of mDC. The effector-inducing profiles are rooted in interferon-regulatory factors (IRF) 3/7. In NK cell activation, a mDC surface molecule is inducible by IRF-3 to facilitate cell-cell contact with effector cells [4], while in CTL proliferation cytoplasmic IRF-3/7-inducing molecules play a key role for mDC cross-priming. The in vivo roles in tumor-suppression of the molecules expressed on mDC by RNA pattern recognition are currently analyzed. Immunotherapy is an attractive strategy for cancer treatment since it confers high QOL on patients. Although a number of tumor-associated antigens have been discovered, lack of appropriate adjuvants for immunotherapy could be a serious barrier for generalizing this therapy [5]. The purpose of this study is to develop a RNA-based adjuvant for driving effective antitumor effectors. Clinical merits of the synthetic RNA adjuvants will be further discussed.
1. Akazawa T., et al. Proc. Natl. Acad. Sci. USA. 104: 252-257, 2007. 2. Ebihara, T., et al. Hepatology. 48: 48-58, 2008. 3. Sasai, M., et al. J. Immunol. 177: 8676-8683, 2006. 4. Ebihara, T., et al. J. Exp. Med. 207: 2675-2687, 2010. 5. Matsumoto, M., and T. Seya. Adv. Drug Deliv. Rev. 60: 805-812, 2008.
42 Tsukasa Seya Department of Microbiology and Immunology, Hokkaido University Graduate School of Medicine E-mail: [email protected]
EDUCATIONS/TRAINING Hokkaido University PhD Pharmaceutical Sciences 1984 Hokkaido University MD Medicine 1987 Washington Univ. Postdoc Innate Immunity 1984-1987
POSITIONS AND HONORS 1987-1998 Associate Director, Department of Immunology, Osaka Medical Cancer for Cancer and Cardiovascular Diseases. 1994-1997 A chief investigator of PRESTO (directed by K. Toyoshima) 1998-2004 Professor (concurrently), Nara Institute of Science and Technology. 1998-2001 Director, Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases. 2001-2004 Director-in-Chief, Research Institute of Osaka Medical Center for Cancer. 2002-2007 A chief investigator of CREST (directed by T. Kishimoto) 2002-2004 Professor (concurrently), Osaka University School of Medicine. 2004- Professor, Hokkaido University Graduate School of Medicine (Department of Microbiology and Immunology)
SELECTED RECENT PUBLICATIONS 1. Oshiumi, H., M. Matsumoto, K. Funami, T. Akazawa, and T. Seya. 2003. TICAM-1, an adapter molecule that participates in Toll-like receptor 3-mediated interferon-beta induction. Nature Immunol. 4: 161-167. 2. Matsumoto, M., K. Funami, M. Tanabe, H. Oshiumi, M. Shingai, Y. Seto, A. Yamamoto, and T. Seya. 2003. Subcellular localization of Toll-like receptor 3 in human dendritic cells. J. Immunol. 171: 3154-3162.
3. Akazawa T., H. Masuda, Y. Saeki, M. Matsumoto, K. Takeda, S. Akira, K. Tsujimura, K. Kuzushima, T. Takahashi, I. Azuma, S. Akira, K. Toyoshima, and T. Seya. 2004. Adjuvant-mediated tumor regression and tumor-specific cytotoxic response are impaired in MyD88-deficient mice. Cancer Res. 64: 757-764.
4. Ebihara, T., M. Azuma, H. Oshiumi, J. Kasamatsu, K. Iwabuchi, K. Matsumoto, H. Saito, T. Taniguchi, M. Matsumoto, and T. Seya. 2010. Identification of a polyI:C-inducible membrane protein, that participates in dendritic cell-mediated natural killer cell activation. J. Exp. Med. 207: 2675-2687.
5. Oshiumi, H., M. Miyashita, N. Inoue, M. Okabe, M. Matsumoto, and T. Seya. 2010. Essential role of Riplet in RIG-I-dependent antiviral innate immune responses. Cell host microbe. (in press)
43 Day-2| 15:20-15:55 Session 2-3
Polysaccharide Nanogel DDS for Cancer Immunotherapy
Kazunari Akiyoshi Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University Katsura, Nishikyo-ku, Kyoto, 615-8510, Japan
Application of biomaterials, such as polymer nano-particles, liposomes and nanogels, has a great potential in vaccine development and immunotherapy. We have developed a new method of creating a series of functional nanogels via self-assembly of associating polymer such as cholesteryl group-bearing pullulan (CHP). The CHP nanogels trap various proteins by mainly hydrophobic interactions and acquire chaperon-like activity because the proteins are trapped inside of a hydrated nanogel polymer network (nano-matrix) without aggregating and are gradually released in the native form [1]. We recently reported cationic CHP nanogel as a novel adjuvant-free antigen-delivery vehicle for intranasal vaccines [2]. In this talk, polysaccharide nanogels for cancer immunotherapy were described. We developed a novel polysaccharide nanogel/oncoprotein complex vaccine. Phase I trials have clearly shown that subcutaneous injection of CHP nanogel carrying the cancer antigen HER2 (CHP-HER2) or NY-ESO-1 (CHP-NY-ESO-1) effectively induces antigen-specific CD8+ cytotoxic T lymphocyte responses and antibody production [3]. We also reported CHP nanogel delivery system for cytokine such as recombinant murine IL-12 (rmIL-12). Repetitive administrations of the CHP/rmIL-12, but not rmIL-12 alone, induced drastic growth retardation of preestablished subcutaneous fibrosarcoma without causing any serious toxic event [4]. The results propose a novel therapeutic technology that is taking advantage of slow and sustained release of bioactive cytokines from the nanogels.
References 1. Y. Sasaki, K. Akiyoshi, The Chemical Record, 10, 366-376 (2010) 2. T. Nochi, et al., Nature Materials, 9, 572-578 (2010). 3. A. Uenaka, et al., Cancer Immunity, 7, 9-20 (2007) 4. T. Shimizu, et al., BBRC., 367, 330-335 (2008)
44 Kazunari Akiyoshi Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University E-mail: [email protected]
EDUCATIONS/TRAINING Kyushu University M.Sc. Chemistry 1982 Kyushu University PhD Chemistry 1985 Purdue University, USA Postdoc Chemistry 1985-1987
POSITIONS AND HONORS 1987-1988 Lecturer, Faculty of Engineering, Nagasaki University, Japan 1989-1993 Assistant Professor, Department of Polymer Chemistry, Kyoto University, Japan 1993-2002 Associate Professor, Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Japan 1997 Visiting Associate Professor, Department of Chemistry, Luis Pasteur University, France 1999-2002 Research member, Precursory Research for Embryonic Science and Technology, 21(PRESTO), JST, Japan 2002-2010 Professor, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Japan 2005-2007 Visiting professor, Biotic Integration Engineering (Guest Chair) Division, Precision and Intelligence Laboratory, Tokyo Institute of Technology, Japan 2010-present Professor, Department of Polymer Chemistry, Kyoto University 1993: Young Investigate Lecture Awards of the Chemical Society of Japan 1998: The Award of the Society of Polymer Science, Japan, The Society of Polymer Science, Japan 2001: Barre Lecturer Awards (2001), the University of Montreal, Canada 2007: Best paper awards IEEE 2007, International Symposium on Micro-Nanomechatronics and Human Science (MHS2007), Japan 2009-present: Regional Editors, Journal of Bioactive and Compatible Polymers
SELECTED RECENT PUBLICATIONS 1. T. Nochi, Y. Yuki, H. Takahashi, S. Sawada, M. Mejima, T. Kohda, N. Harada, G. Kong, A. Sato, N. Kataoka, D. Tokuhara, S. Kurokawa, Y. Takahashi, H. Tsukada, S. Kozaki, K. Akiyoshi, H. Kiyono, Nanogel antigenic protein delivery system for adjuvant-free intranasal vaccines, Nature Materials, 9, 572-578 (2010) 2. S. Toita, N. Morimoto, K. Akiyoshi, Functional cycloamylose-based biomaterial: application in a gene delivery system, Biomacromolecules, 11, 397- 401(2010). 3. U. Hasegawa, S. Sawada, T. Shimizu, T. Kishida, E. Otsuji, O. Mazda, K. Akiyoshi, Raspberry-Like Assembly of Cross-Linked Nanogels for Protein Delivery, J. Controlled Release, 140, 312-317(2009) 4. M. Kaneda, S. M. Nomura, S. Ichinose, S. Kondo, K. Nakahama, K. Akiyoshi, I. Morita, Direct formation of proteo-liposomes by in vitro synthesis and cellular cytosolic delivery with connexin-expressing liposomes, Biomaterials, 30, 3971-3977 (2009). 5. C. Hayashi, U. Hasegawa, Y. Saita, H. Hemmi, T. Hayata, K. Nakashima, Y. Ezura, T. Amagasa, K. Akiyoshi, M. Noda, Osteoblastic bone formation is induced by using nanogel-crosslinking hydrogel as novel scaffold for bone growth factor, J. Cell. Phys., 220(1), 1-7 (2009). 6. H. Ayame, N. Morimoto, K. Akiyoshi, Self-assembled cationic nanogels for intracellular protein delivery system, Bioconjugate Chem., 19, 882-890 (2008) 7. S. Kageyama, S. Kitano, M. Hirayama, Y. Nagata, H. Imai, T. Shiraishi, K. Akiyoshi, A. Scott, R. Murphy, E. Hoffman, L. Old, N. Katayama, and H. Shiku, Humoral immune responses in patients vaccinated with 1-146 HER2 protein complexed with cholesteryl pullulan nanogel (CHP-HER2), Cancer Sci., 99, 601-607 (2008).
45 Day-2| 16:10-16:45 Session 2-4
Tyrosine Phosphatases in Health and Disease
Benjamin G. Neel Department of Stem Cell and Developmental Biology, Ontario Cancer Institute and Campbell Family Research Institute, Princess Margaret Hospital; Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
Tyrosyl phosphorylation, which is regulated by the combined actions of protein-tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), plays a critical role in the control of cellular pathways regulating proliferation, differentiation and survival. Not surprisingly, disorders of these pathways can result in diseases such as cancer. Although PTK abnormalities have been identified in many different human cancers, the role of specific PTPs is less clear. One exception is the SH2 domain-containing protein-tyrosine phosphatase SHP2, encoded by PTPN11 in humans (Ptpn11 in mice). Shp2 plays a key role in RAS/ERK activation in response to most, if not all, receptor tyrosine kinase (RTK), cytokine receptor and integrin signaling pathways, and may also have cell-type and signal-dependent functions in other downstream signaling cascades. Somatic PTPN11 mutations cause juvenile myelomonocytic leukemia (JMML) and contribute to other hematological malignancies. Previous studies in our lab have implicated the Shp2 binding protein Gab2 in the pathogenesis of Bcr/Abl-associated malignancies and in HER2-Neu induced breast cancer. We have now found that Shp2 binding is required for Bcr/Abl-induced myeloid and lymphoid leukemogenesis in mice. Furthermore, studies using an inducible allele of Shp2 is required for HER2/Neu-induced mouse mammary carcinogenesis. These data suggest that Shp2 might be a good target for anti-neoplastic drug development. However, Shp2 deletion in hematopoietic stem cells results in bone marrow aplasia, due to stem cell and progenitor cell death. Finally, we have developed a global, quantitative methodology to assay PTP expression and redox regulation. Using this assay, we have found distinct patterns of PTP oxidation in response to different types of oncogenes and in different tumor types. These data suggest that PTP oxidation/inactivation may be an important mechanism of oncogenesis by distinct types of transforming events.
46 Benjamin G. Neel, M.D., PhD. Ontario Cancer Institute, Princess Margaret Hospital, Canada [email protected]
EDUCATIONS 1977 A.B. Cornell University College of Arts and Sciences, Ithaca, NY 1982 Ph.D. Rockefeller University, New York, NY 1983 M.D. Cornell University Medical College, New York, NY
ACADEMIC POSITIONS AND APPOINTMENTS 1988-1993 Assistant Professor of Medicine, Harvard Medical School, Boston, MA 1993-1999 Associate Professor of Medicine, Harvard Medical School, Boston, MA 1994- Director, Cancer Biology Prgram, Beth Israel Deaconess Medical Center 1999- Professor of Medicine, Harvard Medical School, Boston, MA 2006 - William B. Castle Professor of Medicine, Harvard Medical School, Boston, MA 2007 - Professor of Medical Biophysics, University of Toronto 2007 - Senior Scientist, Stem Cell and Developmental biology, University Health Network 2007 - Director, Ontario Cancer Institute, University Health Network 2007 - Canada Research Chair, Tier 1
SELECTED RECENT PUBLICATIONS (Bold indicates Neel Lab members) 1. Wu, X., Simpson, J., Hong, JH., Kim, KH., Thavarajah NK., Backx,PB., Neel,BG. Araki, T., Modulating MEK- ERK pathway ameliorates disease phenotypes in a mouse model of Noonan syndrome-associated Raf1 mutation , J Clin Invest, In Press 2. Marin,TM., Keith, K, Davies, B., Conner,DA., Guha,P., Kalaitzidis,D., Wu,X., Lauriol, J., Wang, B., Bauer, M., Bronson8, R., Franchini, KG., Neel,BG., Kontaridis, MI., Rapamycin normalizes hypertrophic cardiomyopathy in a mouse model of LEOPARD Syndrome-associated PTPN11 mutation ,J Clin Invest , In Press 3. Matsuo K, Delibegovic M, Matsuo I, Nagata N, Liu S, Bettaieb A, Xi Y, Araki K, Yang W, Kahn BB, Neel BG, Haj FG., Altered glucose homeostasis in mice with liver-specific deletion of Src homology phosphatase 2, J Biol Chem. 2010 Sep 14. [Epub ahead of print] 4. Stewart R.A., Sanda T., Widlund H.R., Zhu S., Swanson K.D., Hurley A.D., Bentires-Alj M., Fisher D.E., Kontaridis A., Look T., Neel B.G. Phosphatase-Dependent and –Independent Functions of Shp2 in Neural Crest Cells Underlie LEOPARD Syndrome Pathogenesis. Developmental Cell, May 18, 2010 18(5)pp.750-762 5. Araki T, Chan G, Newbigging S, Morikawa L, Bronson RT, Neel BG. Noonan Syndrome cardiac defects are due to PTPN11 acting in endocardium to enhance endocardial-mesenchymal transformation. Proc Natl Acad Sci, Mar 24 2009; 106(12): 4736-41Epub Feb 27 2009 6. Chan G, Kalaitzidis D, Usenko T, Kutok JL, Yang W, Mohi MG, Neel BG. Leukemogenic Ptpn 11 causes fatal myeloproliferative disorder via cell-autonomous effects on multiple stages of hematopoiesis. Blood. Apr 30 2009; 113(18): 4414-4424 Epub Jan 29 2009 7. Kontaridis MI, Yang W, Bence KK, Cullen D, Wang B, Bodyak N, Ke Q, Hinek A, Kang PM, Liao R, Neel BG. Deletion of Ptpn11 (Shp2) in cardiomyocytes causes dilated Cardiomyopathy via effects on the extracellular signal-regulated kinase/mitogen-activated Protein kinase and RhoA signaling pathways. Circulation. 2008 Mar 18;117(11):1423-35. Epub 2008 Mar 3. Erratum in: Circulation.2008 Apr 15;117(15) e314.
47 Day-2| 16:45-17:20 Session 2-4
Role of SHP2 Tyrosine Phosphatase in Gastric Carcinogenesis
Masanori Hatakeyama Division of Microbiology, Graduate School of Medicine University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Most if not all human gastric carcinomas arise from stomach mucosa persistently infected with Helicobacter pylori cagA-positive strains. The cagA gene product CagA is delivered into host gastric epithelial cells via a type IV secretion-mediated interaction of CagA with host membrane phosphatidylserine (1). Delivered CagA undergoes tyrosine phosphorylation by Src family kinases and thereby acquires the ability to specifically interact with SHP2 tyrosine phosphatase, a bona-fide human oncoprotein. CagA-bound SHP2 exhibits elevated phosphatase activity and aberrantly activates pro-mitogenic Erk MAP kinase signaling (2,3), which paradoxically triggers oncogenic stress that gives rise to aberrant p21 accumulation and premature cell senescence. In polarized epithelial cells, however, CagA not only activates the Erk signal but also provokes junctional and polarity defects by binding and inhibiting PAR1 polarity-regulating kinase. As a result, expression of CagA in polarized epithelial cells stimulates RhoA-specific GEF-H1, which in turn activates the RhoA/ROCK/c-Myc/microRNA pathway that prevents Erk-dependent accumulation of p21 and thereby overrides oncogenic stress-induced cell senescence (4,5). These observations indicate that CagA preferentially promotes transformation of polarized epithelial cells, legitimate cells during in vivo H. pylori infection. Consistent with the oncogenic potential of CagA, transgenic expression of CagA in mice induces gastrointestinal malignancies in a manner that is dependent on CagA tyrosine phosphorylation, a CagA modification that is essential for the CagA-SHP2 interaction (6).
References 1. Murata-Kamiya N. et al. Cell Host Microbe 7, 399-411, 2010. 2. Higashi H. et al. Science 295, 683-686, 2002. 3. Hatakeyama M. et al. Nat Rev Cancer 4, 688-694, 2004. 4. Saadat I. et al. Nature 447, 330-333, 2007. 5. Saito Y. et al. J Exp Med 207, 2157-2174, 2010. 6. Ohnishi et al. Proc Natl Acad Sci USA 105, 1003-1008, 2008.
48 Masanori Hatakeyama Division of Microbiology, Graduate School of Medicine, University of Tokyo E-mail: [email protected]
EDUCATIONS/TRAINING Hokkaido University MD Medicine 1981 Hokkaido University PhD Medicine 1986 Whitehead Institute, MIT Postdoc Cancer Biology 1991-1994
POSITIONS AND HONORS 1986-1992 Assistant Professor, Institute for Molecular and Cellular Biology, Osaka University 1995-2000 Member and Chief, Department of Viral Oncology, The Cancer Institute, Japanese Foundation for Cancer Research (JFCR) 1999-2009 Professor, Institute for Genetic Medicine, Hokkaido University 2009- present Professor, Graduate School of Medicine, University of Tokyo
1991: Incitement Award of the Japanese Cancer Association 1991: Human Frontier Science Program (HFSP) Long-term Fellowship 1994: Leukemia Research Foundation Scholarship 2006: JCA-Mauvernay Award
2005-present: Editorial Board, International Journal of Cancer 2006-present: Associate Editor, Cancer Science
SELECTED RECENT PUBLICATIONS 1. Saito Y, Murata-Kamiya N, Hirayama T, Ohba Y and Hatakeyama M. Conversion of Helicobacter pylori CagA from senescence inducer to oncogenic driver through polarity-dependent regulation of p21. J Exp Med 207, 2157-2174, 2010
2. Murata-Kamiya N, Kikuchi K, Hayashi T, Higashi H and Hatakeyama M. Helicobacter pylori exploits host membrane phosphatidylserine for delivery, localization and pathophysiological action of the CagA oncoprotein. Cell Host Microbe, 7, 399-411, 2010
3. Hatakeyama M. Linking epithelial polarity and carcinogenesis by multitasking Helicobacter pylori virulence factor CagA. Oncogene 27, 7047-7054, 2008
4. Ohnishi N, Yuasa H, Tanaka S, Sawa H, Miura M, Matsui A, Higashi H, Musashi M, Iwabuchi K, Suzuki M, Yamada G, Azuma T and Hatakeyama M. Transgenic expression of Helicobacter pylori CagA induces gastrointestinal and hematopoietic neoplasms in mouse. Proc Natl Acad Sci USA 105, 1003-1008, 2008
5. Saadat I, Higashi H, Obuse C, Umeda M, Murata-Kamiya N, Saito Y, Lu H, Ohnishi N, Azuma T, Suzuki A, Ohno S and Hatakeyama M. Helicobacter pylori CagA targets PAR-1/MARK kinase to disrupt epithelial cell polarity. Nature 447, 330-333, 2007
49 Day-2| 17:20-17:55 Session 2-4
Identification of PTPN11/Shp2 as a Tumor Suppressor in Liver Cancer
Gen-Sheng Feng Department of Pathology, University of California San Diego, La Jolla, California 92093-0864, USA
Shp2, an intracellular tyrosine phosphatase with two SH2 domains, has been found to promote activation of the Ras-Erk pathway by growth factors, cytokines and hormones. Furthermore, dominantly activating mutations in the human gene PTPN11, coding for Shp2, have been detected in nearly 50% of Noonan syndrome patients who have higher risk of suffering juvenile myelomonocytic leukemia (JMML), and somatic mutations constitutively activating Shp2 have also been found in several types of leukemia. Therefore, PTPN11 has been proposed as the first proto-oncogene that encodes a tyrosine phosphatase. In previous work, we generated a liver-specific Shp2 knockout (LSKO) mouse model and found that Shp2 deletion suppressed Erk signal and hepatocyte proliferation following partial hepatectomy, which revealed a positive role of Shp2 in upregulation of proliferative signaling in hepatocytes. However, we present data here that suggests a tumor-inhibiting function of Shp2 in liver malignancy. LSKO mice exhibited hepatic necrosis and inflammation, and upregulation of hepatic inflammatory signals, particularly the IL-6/Stat3 pathway. Liver damage and inflammation triggered compensatory proliferation, resulting in spontaneous development of hepatocellular adenoma in aged LSKO mice. Furthermore, Shp2 ablation dramatically enhanced diethyl nitrosamine (DEN)-induced hepatocellular carcinoma (HCC) development. Decreased Shp2 expression was detected in a sub-fraction of human hepatocellular carcinoma specimens. Thus, in contrast to the leukemogenic effect of dominantly activating mutants, Shp2 can inhibit neoplastic proliferation in the liver through suppression of inflammatory signaling. PTPN11/Shp2 may function as a tumor promoter or suppressor depending on cellular or pathological context. References 1. Tartaglia, M., and Gelb, B.D. (2005). Germ-line and somatic PTPN11 mutations in human disease. Eur J Med Genet 48, 81-96. 2. Chan, R.J., and Feng, G.S. (2007). PTPN11 is the first identified proto-oncogene that encodes a tyrosine phosphatase. Blood 109, 862-867. 3. Bard-Chapeau, E.A., Yuan, J., Droin, N., Long, S., Zhang, E.E., Nguyen, T.V., and Feng, G.S. (2006). Concerted functions of Gab1 and Shp2 in liver regeneration and hepatoprotection. Mol Cell Biol 26, 4664-4674.
50 Gen-Sheng Feng Department of Pathology, School of Medicine, and Section of Molecular Biology, Division of Biological Sciences, University of California San Diego, USA Email: [email protected]
EDUCATIONS/TRAINING Hangzhou University, Hangzhou, China BS in Biology 1981 2nd Medical School of Army, Shanghai, China, MS in Immunology 1984 Indiana University Bloomington, USA PhD in Molecular Biology 1990 Hospital for Sick Children, Toronto, Canada Postdoc 1990-91 Mount Sinai Hospital, Toronto, Canada Postdoc 1991-94
POSITIONS AND HONORS 1994–99. Assistant Professor of Biochemistry, Indiana University School of Medicine 1999–99 Associate Professor, Indiana University School of Medicine, Indianapolis 2000–05 Associate Professor, Sanford/Burnham Medical Research Institute, California 2005–09 Professor, Sanford/Burnham Medical Research Institute, California 2009- Professor, University of California, San Diego (UCSD), La Jolla, California 1995-97 Career Development Award of American Diabetes Association 1990 Outstanding Associate Instructor Award, Howard Hughes Medical Inst. 2001 Distinguished Lecture, Cleveland Clinic Foundation, Cleveland, Ohio 2006- Editorial Board: Molecular and Cellular Biology (MCB) 2006- Editorial Board: Journal of Biological Chemistry (JBC)
SELECTED RECENT PUBLICATIONS 1. Chapeau EA. L Hevener, S Long, EE Zhang, JM Olefsky and GS Feng. Deletion of Gab1 in the liver leads to enhanced glucose tolerance and improved hepatic insulin action. Nature Medicine 11, 567-571, 2005. 2. Bard-Chapeau EA, J Yuan, N Droin, S Long, EE Zhang, TV Nguyen, and GS Feng. Concerted functions of Gab1 and Shp2 in liver regeneration and hepatoprotection. MCB 26, 4664-4674, 2006. 3. Chan, R.J. and G.S. Feng. PTPN11 is the first identified proto-oncogene that encodes a tyrosine phosphatase (Invited review). Blood 109, 862-867, 2007. 4. Ke, Y., D. Wu, F. Princen, T. Nguyen, Y. Pang, J. Lesperance, W. J. Muller, R.G. Oshima, and G.S. Feng. Role of Gab2 in mammary tumorigenesis and metastasis. Oncogene 26, 4951-4960, 2007. 5. Ke, K., E.E. Zhang, K. Hagihara, D. Wu, Y.H. Pang, R. Klein, T. Curran, B. Ranscht, and G.S. Feng. Deletion of Shp2 in the brain leads to defective proliferation and differentiation in neural stem cells and early postnatal lethality. MCB 27, 6706-6717, 2007. 6. Princen, F, E Bard, F Sheikh, SS Zhang, J Wang, W Zago, D Wu, RD Trelles, B Bailly-Maitre, JC Reed, M Mercola, G Tong, J Chen, and GS Feng. Selective deletion of tyrosine phosphatase Shp2 in muscle leads to cardiomyopathy, insulin resistance and premature death. MCB 29, 378-388, 2009. 7. Zhang, SS, E Hao, J Yu, W Liu, J Wang, F Levine, and GS Feng. Coordinated regulation by Shp2 tyrosine phosphatase of signaling events controlling insulin biosynthesis in pancreatic beta-cells. PNAS 106, 7531-7536, 2009.
51 Day-2| 17:55-18:30 Session 2-4
Helicobacter pylori Infection and Gastric Cancer: Relationship between Gastric Cancer and Diversity of Helicobacter pylori
Takeshi Azuma Department of Gastroenterology, Graduate School of Medicine Kobe University 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
Helicobacter pylori (H. pylori) is a human pathogen responsible for chronic active gastritis, and infection with this organism is an important risk factor for gastric cancer. H. pylori CagA protein is considered a major virulence factor associated with gastric cancer. CagA is directly injected from the H. pylori into the gastric epithelial cells via the bacterial type IV secretion system and undergoes tyrosine phosphorylation in the host cells. The translocated CagA forms a physical complex with SHP-2, and stimulates phosphatase activity. Deregulation of SHP-2 by CagA may induce abnormal proliferation and movement of gastric epithelial cells. In addition, the CagA protein has the molecular diversity at the phosphorylation site, and there are two major types, the Western and East Asian type. East Asian-type CagA confers stronger SHP-2 binding and transforming activities to Western-type CagA. The diversity of the CagA phosphorylation site, which collectively determines binding affinity of CagA to SHP-2, is an important variable in determining the clinical outcome of infection by different H. pylori strains. The prevalence of East Asian CagA-positive H. pylori was significantly higher in patients with gastric cancer than in patients with chronic gastritis. Therefore, patients harboring East Asian CagA-positive H. pylori are at a higher risk for developing gastric cancer than those infected with Western CagA-positive strains.
References 1. Azuma T, Yamazaki S, Yamakawa A, Ohtani M, Muramatsu A, Suto H, Ito Y, Dojo M, Yamazaki Y, Kuriyama M, Keida H, Higashi H, Hatakeyama M. Association between diversity in the Src homology 2 domain-containing tyrosine phosphatase binding site of Helicobacter pylori CagA protein and gastric atrophy and cancer. J Infect Dis 189:820-827,2004. 2. Higashi H, Tsutsumi R, Muto S, Sugiyama T, Azuma T, Asaka M, Hatakeyama M. SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein. Science 295: 683-686, 2002.
52 Takeshi Azuma Department of Gastroenterology, Graduate School of Medicine, Kobe University E-mail: [email protected]
EDUCATIONS/TRAINING Kyoto Prefectural University of Medicine MD Medicine 1981 Kyoto Prefectural University of Medicine PhD Medicine 1987 Wayne State University Postdoc Molecular Biology and Genetics 1987-1989
POSITIONS AND HONORS 1989-1992 Assistant Professor, Department of Preventive Medicine, Kyoto Prefectural University of Medicine 1993 Assistant Professor, Second Department of Internal Medicine, Fukui Medical University 1995 Lecturer, Second Department of Internal Medicine, Fukui Medical University 2001 Associate Professor, Second Department of Internal Medicine, Fukui Medical University 2005 Professor, Frontier Medical Science in Gastroenterology, International Center for Medical Research and Treatment, Graduate School of Medicine, Kobe University 2007-present Professor, Department of Gastroenterology, Graduate School of Medicine, Kobe University
1994: Young Investigator Award of the Japanese Society of Gastroenterology.
SELECTED RECENT PUBLICATIONS
1. Tanaka H, Yoshida M, Nishiumi S, Ohnishi N, Kobayashi K, Yamamoto K, Fujita T, Hatakeyama M, Azuma T. The CagA protein of Helicobacter pylori suppresses the function of dendritic cell in mice. Arch Biochem Biophys 498:35-42, 2010.
2. Truong BX, Chi Mai VT, Tanaka H, Ly LT, Thong TM, Hai HH, Long DV, Furumatsu K, Yoshida M, Kutsumi H and Azuma T. Diverse Characteristics of the cagA Gene of Helicobacter pylori strains in gastric cancer and peptic ulcer patients from Southern Vietnam. J Clin Microbiol 47:4021-4028,2009.
3. Fukase K, Kato M, Kikuchi S, Inoue K, Uemura N, Okamoto S, Terao S, Amagai K, Hayashi S, Asaka M: Japan Gast Study Group. Effect of eradication of Helicobacter pylori on incidence of metachronous gastric carcinoma after endoscopic resection of early gastric cancer: an open-label, randomized controlled trial. Lancet 372:392-397, 2008.
4. Saadat I, Higashi H, Obuse C, Umeda M, Murata-Kamiya N, Saito Y, Lu H, Ohnishi N, Azuma T, Suzuki A, Ohno S and Hatakeyama M. Helicobacter pylori CagA targets PAR-1/MARK kinase to disrupt epithelial cell polarity. Nature 447, 330-333, 2007
5. Matsumoto Y, Marusawa H, Kinoshita K, Endo Y, Kou T, Morisawa T, Azuma T, Okazaki I, Honjo T, Chiba T. Helicobacter pylori infection triggers aberrant expression of activation-induced cytidine deaminase in gastric epithelium. Nat Med 13:470-176, 2007.
53 Day-3| 9:00-9:35 Session 3-1
Regulation of Intestinal Immunity by SAP-1, a Microvillus-Specific Receptor-Type Protein Tyrosine Phosphatase
Takashi Matozaki Division of Molecular and Cellular Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
The intestinal epithelial cells are thought to be important for regulation of intestinal immunity, the molecular mechanism for such regulations remains largely unknown, however. SAP-1/PTPRH is a receptor-type protein tyrosine phosphatase that specifically localizes to the microvilli (MV) of the brush border in gastrointestinal epithelial cells [1-3]. We here show that SAP-1 ablation markedly increased the severity of colitis, with elevation of mRNA expression of various cytokines and chemokines, in interleukin-10-deficient mice. Biochemical analysis of MV membranes showed that the tyrosine phosphorylation of ~100 kDa protein (p100) was markedly increased in the colonic epithelium of the SAP-1-deficient mice, suggesting that p100 is a substrate for SAP-1. p100 is also an intestinal MV-specific immunoglobulin-superfamily transmembrane protein, of which tyrosine phosphorylation is triggered by Src family kinases. Such tyrosine phosphorylation of p100 promotes the binding of Syk to p100. These results thus indicate a novel regulatory mechanism for the intestinal immunity by the counterbalance between SAP-1 and p100.
References 1. Matozaki T. et al. J Biol Chem 269, 2075-2081, 1994 2. Sadakata H. et al. Genes Cells 14, 295-308, 2009 3. Matozaki T. et al. Cell signal 22, 1811-1817, 2010
54 Takashi Matozaki Division of Molecular and Cellular Signaling, Kobe University E-mail: [email protected]
EDUCATIONS/TRAINING Kobe University MD Medicine 1981 Kobe University PhD Medicine 1988 University of Michigan Postdoc Physiology 1988-1990
POSITIONS AND HONORS 1993-1998 Assistant Professor, Second Department of Internal Medicine, Kobe University School of Medicine 1998-2000 Assistant Professor, Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine 2000-2000 Associate Professor, Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine 2000-2004 Professor, Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University 2004-2010 Professor, Laboratory of Biosignal Sciences, Institute for Molecular and Cellular Regulation, Gunma University 2010-present Professor, Division of Molecular and Cellular Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine Visiting Professor, Laboratory of Biosignal Sciences, Institute for Molecular and Cellular Regulation, Gunma University
1998: Encouragement Award, Japanese Society of Gastroenterology
SELECTED RECENT PUBLICATIONS 1. Matozaki T, Murata Y, Mori M, Kotani T, Okazawa H and Ohnishi H. Expression, localization and biological function of the R3 subtype of receptor-type protein tyrosine phosphatases in mammals. Cell Signal 22, 1811-1817, 2010 2. Kotani T, Murata Y, Ohnishi H, Mori M, Kusakari S, Saito Y, Okazawa H, Bixby JL and Matozaki T. Expression of PTPRO in the interneurons of adult mouse olfactory bulb. J Comp Neurol, 518, 119-136, 2010 3. Mori M, Murata Y, Kotani T, Kusakari S, Ohnishi H, Saito Y, Okazawa H, Ishizuka T, Mori M and Matozaki T. Promotion of cell spreading and migration by vascular endothelial-protein tyrosine phosphatase (VE-PTP) in cooperation with integrins. J Cell Physiol 224, 195-204, 2010 4. Murata, Y, Mori M, Kotani T, Supriatna Y, Okazawa H, Kusakari S, Saito Y, Ohnishi H and Matozaki T. Tyrosine phosphorylation of R3 subtype receptor-type protein tyrosine phosphatases and their complex formations with Grb2 or Fyn. Genes Cells 15, 513-524, 2010 5. Matozaki T, Murata Y, Okazawa H and Ohnishi H. Functions and molecular mechanisms of the CD47-SIRP! signalling pathway. Trends Cell Biol 19, 72-80, 2009
55 Day-3| 9:35-10:10 Session 3-1
Beyond Insulin Secretion: Substrate Identification of a Mitochondrial Phosphatase
Ji Zhang and Jack E. Dixon Department of Pharmacology, School of Medicine University of California San Diego 9500 Gilman Drive, La Jolla, CA 92093-0721, USA
The Protein Tyrosine Phosphatase family, all of which contain a highly conserved active site motif, Cys-x5-Arg (Cx5R), are key mediators of a wide variety of cellular processes, including growth, metabolism, differentiation, and motility 1,2. Our laboratory previously discovered a protein tyrosine phosphatase (PTP) that localized 3 exclusively to the inner mitochondrial membrane . This is the first CX5R phosphatase found in mitochondria, and as a result of its unique localization we named it PTPMT1 (PTP localized to the Mitochondrion 1). Notably, knockdown of PTPMT1 has a profound effect on ATP production and potentiates glucose-stimulated insulin secretion 3. Although initially characterized as a lipid phosphatase with activity towards phosphatidylinositol 5-phosphate (PI(5)P) in vitro 4, our efforts failed to demonstrate that PI(5)P was a biologically important substrate. To study the function of PTPMT1 in a physiologically relevant context, we genetically engineered mice in which the Ptpmt1 gene has been ablated through homologous recombination. Interestingly, whole body deletion of Ptpmt1 leads to death of the early embryo, suggesting an essential role for PTPMT1 during mouse development. Contrary to our prediction, PTPMT1-deficiency in cells severely compromised mitochondrial respiration and resulted in abnormal mitochondrial morphology. Furthermore, lipid analysis of Ptpmt1-deficient cells revealed an accumulation of PGP along with a concomitant decrease in PG content. PGP is an essential intermediate in the biosynthetic pathway that leads to the production of cardiolipin, a mitochondrial-specific phospholipid that plays pleiotropic roles in regulating the membrane integrity and activities of the organelle 5. We further demonstrated that PTPMT1 specifically dephosphorylated PGP in vitro. Loss of PTPMT1 led to dramatic diminution of cardiolipin. Together, our study identifies PTPMT1 as the mammalian PGP phosphatase and points to its role as a novel regulator of mitochondrial activities. This work was supported by grants from the National Institutes of Health. (DK18024 and NIDDK DK18849 to J.E.D., DK054441 to A.N.M), and the Larry L. Hillblom Foundation (2007-D-016-FEL) to J.Z.
56 References 1 Alonso, A. et al., Protein tyrosine phosphatases in the human genome. Cell 117 (6), 699 (2004). 2 Tonks, N. K., Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol 7 (11), 833 (2006). 3 Pagliarini, D. J. et al., Involvement of a mitochondrial phosphatase in the regulation of ATP production and insulin secretion in pancreatic beta cells. Mol Cell 19 (2), 197 (2005). 4 Pagliarini, D. J., Worby, C. A., and Dixon, J. E., A PTEN-like phosphatase with a novel substrate specificity. J Biol Chem 279 (37), 38590 (2004). 5 Schlame, M., Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes. J Lipid Res 49 (8), 1607 (2008).
Ji Zhang Department of Pharmacology, University of California San Diego, USA E-mail: [email protected]
EDUCATIONS/TRAINING Beijing Medical University Bachelor of Medicine Medicine 1996 Duke University PhD Pharmacology 2004 University of California San Diego Postdoc Pharmacology 2005-present
SELECTED RECENT PUBLICATIONS
1. Zhang, J., Guan, Z., Murphy, A.N., Wiley, S.E., Perkins, G.A., Worby, C.A., Engel, J.L., Heacock, P., Nguyen, O.K., Wang, J., Raetz, C.R.H., Dowhan, W., Dixon, J.E. Substrate Identification of A Mitochondrial “Protein” Phosphatase: An unexpected role in cardiolipin biosynthesis. (manuscript in preparation)
2. Doughty-Shenton, D., Joseph, J.D., Zhang, J., Pagliarini, D.J., Kim, Y., Lu, D., Dixon, J.E., Casey, P.J. Pharmacological targeting of the mitochondrial phosphatase PTPMT1. J Pharmacol Exp Ther. 2010 May; 333(2): 584-92.
3. Zhang, J., Bao, S., Furumei, R., Kucera, K., Ali, A., Dean, N.M., Wang, X.-F. Protein phsophatase 5 is required for ATR-mediated checkpoint activation. Molecular and Cellular Biology 2005, 25(22): 9910-9
4. Ali, A.*, Zhang, J.*, Bao, S., Liu, I., Otterness, D., Dean, N.M., Abraham, R.T., Wang, X.-F. (2004) Requirement of protein phosphatase 5 in DNA damage-induced ATM activation. Gene & Development 18:249-254 *Contribute equally to this work
57 Day-3| 10:10-10:25 Session 3-1
Shear Stress Regulates Cellular Localization of Vascular Endothelial–Protein Tyrosine Phosphatase (VE-PTP)
Munemasa Mori1, Yoji Murata2, Kemala Isnainiasih Mantilidewi1, Hiroshi Ohnishi1 and Takashi Matozaki1,2 1Laboratory of Biosignal Sciences, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan 2Division of Molecular and Cellular Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan
Shear stress (SS) caused by blood flow plays important roles in the regulation of endothelial cell (EC) functions, such as cell proliferation, cell migration, nitric oxide production and cytokine production. Vascular endothelial–protein tyrosine phosphatase (VE-PTP) is a receptor-type protein tyrosine phosphatase that is expressed specifically in ECs, and it is implicated in the regulation of angiogenesis. We have shown here that in static condition VE-PTP was diffusely localized in mouse endothelioma bEnd.3 cells or human umbilical vein endothelial cells (HUVECs). By contrast, SS induced the accumulation of VE-PTP protein at the downstream edges of these cells. The SS-induced redistribution of VE-PTP in b.End3 cells was inhibited by the treatment with cytochalasin D, an inhibitor for actin polymerization, or by the inhibition of Cdc42. The forced expression of a dominant-negative mutant of Rab5 in bEnd.3 cells also prevented the SS-induced redistribution of VE-PTP. In addition, SS increased the immunoreactivity for tyrosine-phosphorylated proteins at the downstream edges of HUVECs compared with that at the upstream edges. Such increase was attenuated by either the forced expression of a phosphatase-inactive mutant of VE-PTP or the knockdown of endogenous VE-PTP in HUVECs. These results suggest that the SS-induced redistribution of VE-PTP requires actin cytoskeletal reorganization and endocytosis. VE-PTP may participate in the SS-mediated EC functions through regulation of protein tyrosine phosphorylation.
58 Day-3| 10:25-10:45 Session 3-1
Phospho-Ppaxillin Is an Oncogenic in Vivo Target of the Tumor Suppressor Receptor Protein Tyrosine Phosphatase T
Toshio Watanabe Division of Molecular Oncology, Institute for Genetic Medicine Nara Women’s University Kituoya-nishi-machi, Nara 630-8506, Japan
Receptor protein tyrosine phosphatase T (PTPRT) is originally isolated as a new member of meprin-A5 antigen-PTP (MAM) PTPsaes family, and later is revealed to be the most frequently mutated protein tyrosine phosphatase in human cancers. However, the cell signaling pathways regulated by PTPRT largely remain to be elucidated. We previously identified five novel putative substrates for PTPRT and demonstrated here that paxillin is a direct substrate of PTPRT. PTPRT specifically regulates paxillin phosphorylation at the tyrosine 88 (Y88) residue in colorectal cancer (CRC) cells. A recent mass spectrometric analysis indicates that tyrosine phosphorylation occurs at multiple sites on paxillin. Although paxillin Y31 and Y118 phosphorylation has been implicated in cell adhesion and migration, the physiological relevance of tyrosine phosphorylation at other residues remained to be determined. Paxillin Y88F mutant knock-in CRC cells greatly reduce their abilities in cell migration and anchorage-independent growth, fail to form xenograft tumors in nude nice, and affect p130CAS, SHP2 and AKT phosphorylation. The pY88 paxillin is up-regulated in PTPRT knockout mouse colon and a majority of human colon carcinomas. Moreover, PTPRT knockout mice are highly susceptible to carcinogen azoxymethane-induced colon tumor, providing critical in vivo evidence that PTPRT normally functions as a tumor suppressor. These studies reveal a novel signaling pathway that plays an important role in colorectal tumorigenesis.
References 1. MacAndrew PE et al. Brain Res. Mol. Brain Res., 59, 9-21, 1998. 2. Wang Z et al. Science 304, 1164-1166, 2005. 3. Zhao Y et al. Proc. Natl. Acad. Sci., USA, 107, 2592-2597, 2010.
59 Toshio Watanabe Department of Biological Sciences, Graduate School of Humanities and Sciences, Nara Women’s University E-mail: [email protected]
EDUCATIONS/TRAINING University of Tokyo PhD Biochemistry 1987 Max-Planck Institute Postdoc Developmental Biology 1992-1994
POSITIONS AND HONORS 1987-1996 Assistant Professor, Institute for Molecular and Cellular Biology, Osaka University 1992-1994 Visiting Scientist, Max-Planck Institute of Immunobiology, Freiburg, Germany 1996-2006 Associate Professor, Institute of Development, Aging, and Cancer, Tohoku University 2006-present Professor, Department of Biological Sciences, Graduate School of Humanities and Sciences, Nara Wmen’s University
1989: Kato Memorial Award of the Molecular Biology Society of Japan 1992: Human Frontier Science Program (HFSP) Long-term Fellowship
SELECTED RECENT PUBLICATIONS 1. Zhao Y, Zhang X, Guda K, Lawrence E, Sun Q, Watanabe T, Iwakura Y, Asano M,Wei L, Yang Z, Zheng W, Dawson D, Willis J, Markowitz SD, Satake M, Wang Z. Identification and functional characterization of paxillin as a target of protein tyrosine phosphatase receptor T. Proc. Natl. Acad. Sci., USA, 107, 2592-2597, 2010. 2. Wong WF, Nakazato M, Watanabe T, Kohu K, Ogata T, Yoshida N, Sotomaru Y, Ito M, Araki K, Telfer J, Fukumoto M, Suzuki D, Sato T, Hozumi K, Habu S, Satake M. Over-expression of Runx1 transcription factor impairs the development of thymocytes from the double negative to double positive stages. Immunology 130, 243-253, 2010. 3. Nakagawa K, Sugahara M, Yamasaki T, Kajiho H, Takahashi S, Hirayama J, Minami Y, Ohta Y, Watanabe T, Hata Y, Katada T, Nishina H. Hematopoiesis-dependent expression of CD44 in murine hepatic progenitor cells. Biochem. Biophys. Res. Commun. 379, 817-823, 2010. 4. Nakamichi S, Yamanaka K, Suzuki M, Watanabe T, Kagiwada S. Human VAPA and the yeast VAP Scs2p with an altered proline distribution can phenocopy amyotrophic lateral sclerosis-associated VAPB(P56S). Biochem. Biophys. Res. Commun. in press. 5. Sakakura I, Tanabe K, Nouki N, Suzuki M, Satake M, Watanabe T. The carboxyl-terminal region of SMAP2 is important for its specific subcellular localizations as well as its target specificity for Arf proteins. Biochem. Biophys. Res. Commun. in press. 6. Ohata S, Nawa M, Kasama T, Yamasaki T, Sawanobori K, Hata S, Nakamura T, Asaoka Y, Watanabe T, Okamoto H, Hara T, Terai S, Sakaida I, Katada T, Nishina H. Hematopoiesis-dependent expression of CD44 in murine hepatic progenitor cells. Biochem. Biophys.
60 Res. Commun. 379, 817-823, 2009. 7. Kon, S., Tanabe, K., Watanabe, T., Sabe H., and Satake, M. Clathrin-dependent endocytosis of E-cadherin is regulated by the ArfGAP isoform SMAP1. Experimental Cell Research, 314, 1415-1428, 2008. 8. Tanabe, K., Kon, S., Ichijo, Funaki, T., Watanabe, T., Satrake, M. Methods in Enzymology, 438, 155-170, 2008. 9. Natsume, W., Tanabe, K., Kon S., Yoshida N., Watanabe, T. Torii, T., and Satake M. SMAP2, a novel Arf GTPase-activating protein, interacts with clathrin and clathrin assembly protein, and functions on the AP-1-positive early endosome/trans-Golgi-network. Mol. Biol. Cell 17; 2592-2603.
61 Day-3| 11:00-11:20 Session 3-2
Regulation of Cellular Stress Response by Mitochondrial Protein Phosphatase PGAM5
Kohsuke Takeda Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences The University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Exposure of cells to cytotoxic stressors causes mitochondrial dysfunctions, such as mitochondrial DNA damage, decreased ATP synthesis and accumulation of misfolded mitochondrial proteins, which profoundly affect cell fate by inducing apoptosis and necrosis. Accumulating evidence has suggested that these mitochondrial dysfunctions lead to various human disorders, such as neurodegenerative diseases, metabolic diseases, cancers and accelerated senescence. Thus, the sensing system of mitochondrial damage is crucial for cellular stress response. Here we show that phosphoglycerate mutase 5 (PGAM5) functions as a component of such system. We identified PGAM5 as an interacting protein and activator of the stress-activated kinase ASK1 and have shown that PGAM5 lacks phosphoglycerate mutase activity but instead acts as a novel type of protein Ser/Thr phosphatase. The mutant flies lacking the Drosophila ortholog of PGAM5 (dPGAM5) were vulnerable to heat shock, suggesting that PGAM5 is involved in stress response. PGAM5 was localized to the mitochondria through its N-terminal transmembrane domain and was cleaved upon loss of mitochondrial membrane potential. Moreover, the cleavage site was within the transmembrane domain, suggesting that PGAM5 is a novel substrate of intramembrane-cleaving proteases (I-CliPs). In this symposium, we will discuss the possible role of PGAM5 as a mitochondrial damage sensor that plays a role as an intermediate between mitochondrial dysfunctions and cellular stress response.
References Takeda K. et al. Proc Natl Acad Sci USA 106, 12301-12305, 2009 Imai Y. et al. PLoS Genet 6, e1001229, 2010
62 Kohsuke Takeda Graduate School of Pharmaceutical Sciences, The University of Tokyo E-mail: [email protected]
EDUCATIONS/TRAINING Tokyo Medical & Dental University DDS Dentistry 1989 Tokyo Medical & Dental University PhD Dentistry 1995 The Cancer Institute of the Japanese Foundation for Cancer Research (JFCR) Fellow in Cancer Research of the Japan Society for the Promotion of Science 1995-1999
POSITIONS AND HONORS 1999-2003 Research Assistant, Faculty of Dentistry, Tokyo Medical & Dental University 2003-2005 Lecturer, Graduate School of Pharmaceutical Sciences, The University of Tokyo 2005-present Associate Professor, Graduate School of Pharmaceutical Sciences, The University of Tokyo
2000: Rising Members Award of Japanese Association for Oral Biology 2004: Incitement Award of the Japanese Cancer Association 2010: Lion Dental Research Award of Japanese Association for Oral Biology
2009-present: Editorial Board, Journal of Signal Transduction 2010-present: Editorial Board, Journal of Oral Biosciences
SELECTED RECENT PUBLICATIONS
1. Takeda, K., Komuro, Y., Hayakawa, T., Oguchi, H., Ishida, Y., Murakami, S., Noguchi, T., Kinoshita, H., Sekine, Y., Iemura, S., Natsume, T. and Ichijo, H. Mitochondrial phosphoglycerate mutase 5 uses alternate catalytic activity as a protein serine/threonine phosphatase to activate ASK1. Proc. Natl. Acad. Sci. USA. 106, 12301-12305, 2009
2. Iriyama, T., Takeda, K., Nakamura, H., Morimoto, Y., Kuroiwa, T., Mizukami, J., Umeda, T., Noguchi, T., Naguro, I., Nishitoh, H., Saegusa, K., Tobiume, K., Homma, T., Shimada, Y., Tsuda, H., Aiko, S., Imoto, I., Inazawa, J., Chida, K., Kamei, Y., Kozuma, S., Taketani, Y., Matsuzawa, A. and Ichijo, H. ASK1 and ASK2 differentially regulate the counteracting roles of apoptosis and inflammation in tumorigenesis. EMBO J 28, 843-853, 2009
3. Nishitoh, H., Kadowaki, H., Nagai, A., Maruyama, T., Yokota, T., Fukutomi, H., Noguchi, T., Matsuzawa, A., Takeda, K. and Ichijo, H. ALS-linked mutant SOD1 induces ER stress- and ASK1-dependent motor neuron death by targeting Derlin-1. Genes Dev 22, 1451-1464, 2008
4. Takeda, K., Noguchi, T., Naguro, I. and Ichijo, H. Apoptosis signal-regulating kinase 1 in stress and immune response. Annu Rev Pharmacol Toxicol 48, 199-225, 2008
5. Takeda, K., Shimozono, R., Noguchi, T., Umeda, T., Morimoto, Y., Naguro, I., Tobiume, K., Saitoh, M., Matsuzawa, A. and Ichijo, H. Apoptosis signal-regulating kinase 2 (ASK2) functions as a MAP3K in a heteromeric complex with ASK1. J Biol Chem 282, 7522-7531, 2007
63 Day-3| 11:20-11:55 Session 3-2
Translation Repression in the Management of Protein Misfolding Disorders
Shirish Shenolikar Professor and Associate Dean, Office of Research Duke-NUS Graduate Medical School Singapore 8 College Road, Singapore 169857
Wei Zhou1, Matthew H. Brush3, Meng Shyan Choy1 and Shirish Shenolikar1,2,3. 1Signature Programs in Cardiovascular and Metabolic Disorders & Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School Singapore; Departments of 2Psychiatry and Behavioral Sciences & 3Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
A universal response of eukaryotic cells to many environmental stresses is to temporarily halt general protein translation. This allows cells to redirect energy towards expressing stress proteins that help to resolve the stress and promote cell survival. However, prolonged inhibition of protein synthesis is clearly incompatible with normal cell function and viability. Thus, a stress-induced protein, GADD34, assembles a cellular serine/threonine phosphatase complex that functions in a feedback loop to dephosphorylate the eukaryotic initiation factor, eIF2! and reinitiate protein translation. As excessive protein synthesis also creates stress by overloading the cell’s protein quality control machinery in the endoplasmic reticulum, cells cannot tolerate the prolonged expression of GADD34 without triggering cell death. Thus, all cells have developed complex mechanisms to degrade the GADD34 protein to permit a successful recovery of cells from stress. In this presentation, we will describe our studies of cellular mechanisms that regulate cellular GADD34 levels and provide an update on ongoing efforts directed at attenuating GADD34 function using small molecule inhibitors, which provide a promising new cytoprotective strategy for the treatment of diabetes, cancer and neurodegenerative diseases.
64 Shirish Shenolikar, Ph.D.
Shirish Shenolikar is Professor and Associate Dean, Office of Research, at Duke-NUS Graduate Medical School Singapore (Duke-NUS) and member of Signature Research Programs in Cardiovascular and Metabolic Disorders and Neuroscience and Behavioral Disorders. He also holds appointments as Professor of Psychiatry and Behavioral Sciences and Professor of Pharmacology and Cancer Biology at Duke University (USA).
Dr. Shenolikar received B.Sc. Hons in Biochemistry from University College, London and Ph.D. in Biochemistry from University of Leeds.
Prior to arriving in Singapore, he was Research Fellow in Mechanistic and Target Biology (Head of Enzymology - Molecular Pharmacology) and Senior Director for Cardiovascular Molecular Sciences at Pfizer Global Research and Development. Dr. Shenolikar has held faculty positions at University of Leeds (UK), University of South Alabama School of Medicine, University of Texas Health Science Center in Houston and Duke University, where he was Vice-Chair of Pharmacology and Cancer Biology.
He has won awards for his teaching and research. He co-chaired the FASEB Conference of Protein Phosphatases, AAAS Symposium on Protein Kinases and Phosphatases and has been a member of the organizing committees of numerous International Conferences on protein phosphorylation.
65 Day-3| 11:55-12:15 Session 3-2
What Is “Information” in Signal Transduction?
Hidetaka Yakura University of Paris Diderot, Paris, France
In the field of signal transduction, metaphorical words such as signal, message, transduction, communication and information are used almost on a daily basis probably without examining what they really mean. What is “information” that is processed, transferred or transmitted inside the cell upon binding of a ligand to its receptor? Although biology (or physics, for that matter) is flourishing without the exact knowledge of life (or energy/mass/time), I think the efforts to define life should give us a broader and deeper understanding of what life is or what distinguishes living from non-living. Given the claim that the information transfer is one of the key factors to differentiate between the biotic and the non-biotic worlds, rigorous examination of the meaning of information may give us a profound, if not a new, perspective not only on biochemical events in the cell but also on the nature of life. With this in mind, I would like to review the evolution of the concept of information in biology and give some thoughts on the information transfer.
66
Poster Presentation (13:30-15:30 on February 1, 2011)
67 P-1 !"#$#%&"'(")"*+#!!=45$4*(54;/3*<-7-59 “Stress-Activated Protein Kinase Signaling Regulates Ca2+-Calcineurin Signaling”
P-2 ,-$#%,"."'/!!=45$4*(54;/3*<-7-59! “MADS-Box Transcription Factor Mbx1 Is Involved in the Regulation of Calcineurin Signaling through Ca2+-Homeostasis Modulation”
P-3 0$#+#1/%2"$34"!!=45$4*(54;/3*<-7-59! “The Actin Binding Protein Cis3/PSTPIP Is Involved in the Regulation of the Pmk1 MAPK Signaling Pathway and Cytokinesis”
P-4 0$#$/%5"$"6/.#*!@8$$-4.8*(54;/3*<-7-59 “Calcineurin-Binding Proteins, RanBP3 and CaNBP75 Regulate Subcellular Localization and Phosphatase Activity of Calcineurin”
P-5 5/1#)-$#%7-3)/*+#!!=-6-K-*(54;/3*<-7-59! “Functional Processing of Nuclear Ca2+/Calmodulin-Dependent Protein Kinase Phosphatase (CaMKP-N): Evidence for a Critical Role of Proteolytic Processing in the Regulation of Its Catalytic Activity, Subcellular Localization and Substrate Targeting in Vivo”
P-6 5/1#8-.#%7+#/4"!!:828$#*(54;/3*<-7-59! “Imbalance of CaMKII and PP1 Activities Is Associated with Abnormal Dendritic Spine Formation in the ATRX Mutant Mouse Brain”
P-7 2"$3*+#%9+"."%!T->-6#124*(54;/3*<-7-59 “Endotoxin Conditioning Induces VCP/p97-Mediated and iNOS-Dependent Tyr284 Nitration in Protein Phosphatase 2A”
P-8 2"$")"*-%&/(")"*+#!!:828$#*(54;/3*<-7-59! “AMP Activated Protein Kinase (AMPK) Is Regulated by Multiple Protein Phosphatases”
P-9 ,--$#%&/:"$"#!!@8$$-4.8*(54;/3*<-7-59! “Increase in Number of Nucleoli by Protein Phosphatase PPM1D Overexpression”
P-10 ;#1/"$#%,"<#*!@8$$-4.8*(54;/3*<-7-59! “Novel Small Molecule Inhibitors Specific for p53-Inducible Protein Phosphatase PPM1D”
P-11 ,-*-$3%=."(3>>-!!HE-$-*(54;/3*<-7-59 “Control of Phosphorylation and Intracellular Localization of Gln3 by Protein Phosphatase Siw14 in Saccharomyces cerevisiae”
68 P-12 5/(-+#1/%2"'-."!!04F-64*A-51%,*A%5?%,*G%E%-,12*D5E?/3*<-7-59* “Phospho-Regulation of Spliceosomal Proten, Sap155/Sf3b1”
P-13 7+#/1#%?-1"$".#!!(54;/*:8$F83*<-7-59* “Mitochondrial Membrane Potential Loss Induces Intramembrane Proteolysis of the Mitochondria-Resident Protein Phosphatase PGAM5”
P-14 ,/*-$3%=*+#4"!!(54;/*:8$F83*<-7-59* “Roles of a Protein Phosphatase PGAM5 in Heat Shock Stress Response”
P-15 0$#+#1/%@-A#$"B"*!'-?485-C*D5E?4?#?%*N8,*J-E41*J48C86F3*<-7-59* “Consensus Substrate Sequence for Ptprz”
P-16 2/*+#%?"1-)"."%!+#5>-*(54;/3*<-7-59 “Hypothermia-Induced Tyrosine Phosphorylation of SIRP! in the Brain”
P-17 ;3C3'%D"C.31%!G4$$F8*(54;/3*<-7-59* “PTP-PEST Ser-39 Phosphorylation and Its Regulation in Jurkat Cells Costimulated with CD3 and CD28”
P-18 ,/$/%0/$#!!:828$#*(54;/3*<-7-59* “Molecular and Clinical Analysis of RAF1 in Noonan Syndrome and Related Disorders: Dephosphorylation of Serine 259 as the Essential Mechanism for Mutant Activation”
P-19 06*-*+#%2"$"+"*+#!!(54;/*:8$F83*<-7-59 “Identification of a Tumor Suppressor Parafibromin/Cdc73 as a Nuclear Substrate for the SHP2 Tyrosine Phosphatase”
P-20 E)/-+3#%2*-6*-.#%!(54;/*:8$F83*<-7-59* “Regulation of Intracellular Distribution of SHP2 Phosphatase”
P-21 ;#1/$/%9*+#."%!=-5-M-K-*(54;/3*<-7-59* “Downregulation of Tumor Suppressor MicroRNA in Inflammatory Microenvironment”
P-22 73#A#%2/1##%!+#5>-*(54;/3*<-7-59* “Basal Autophagy Regulates Oxidative Stress-Associated Cell Death in Neural Cells”
P-23 ?3'<%7+)"'%F+/) (Duke-NUS Graduate Medical School, Singapore) “Tracking the Mobility of GADD34 and eIF2! Phosphatase Assembly using Fluorescence Live Cell Imaging”
69 [P-1] Stress-Activated Protein Kinase Signaling Regulates Ca2+-Calcineurin Signaling
Daiki Kanbayashi, Aiko Nishida, Ayako Kita and Reiko Sugiura Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Higashi-Osaka 577-8502, Japan
Calcineurin (CN) is a highly conserved Ca2+/calmodulin-dependent Serine/Threonine protein phosphatase that plays a critical role in various physiological functions, including T-cell activation, cardiac development, and hypertrophy, through the activation of the transcription factor NF-AT. In order to elucidate the regulatory mechanisms of the Ca2+-CN signaling, we have been studying the calcineurin and MAPK signaling pathways using fission yeast Schizosaccharomyces pombe as a model organism. Stress-activated protein kinase (SAPK) cascade is also highly conserved through evolution. The Atf1 transcription factor functions downstream of the SAPK in fission yeast and controls gene expression that is crucial for combating cytotoxic stress. Here, we demonstrated that SAPK signaling regulates Ca2+-CN signaling based on the following findings. Atf1 deletion cells are hypersensitive to Ca2+. Moreover, Atf1 deletion exhibited a dramatic decrease in the intracellular Ca2+ levels compared with that in wild-type cells, suggesting that Atf1 is involved in the maintenance of Ca2+ homeostasis. Surprisingly, the in vivo real-time monitoring of CN signaling demonstrated that Atf1 deletion displayed a marked decrease in the CN activation. Consistently, Atf1 regulates gene expression implicated in Ca2+-CN signaling. Thus, SAPK signaling plays an important role in the regulation of Ca2+ homeostasis and CN activity. The possible mechanism of the cross-talk between SAPK and CN will be discussed.
70 [P-2] MADS-Box Transcription Factor Mbx1 Is Involved in the Regulation of Calcineurin Signaling through Ca2+-Homeostasis Modulation
Yuki Yamano, Atsushi Uchida, Sayako Moriuchi, Yuta Asayama, Naoyuki Yamagishi, Masaki Ashida, Ayako Kita, Syunji Ishiwata, Reiko Sugiura Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University
The MADS-box transcription factor MEF2 (myocyte enhancer factor) plays a critical role in regulating gene expression of cardiac muscle–related genes. It has been reported that the transcriptional activity of MEF2 depends on intracellular Ca2+ and calcineurin (CN) signaling. However, the detailed control mechanism and physiology of MEF2 in CN signaling is poorly understood. We have been studying the CN-mediated signal transduction pathway using fission yeast Schizosaccharomyces pombe as a model system, because this organism is amenable to genetics and has many advantages in terms of its relevance to higher system. Here, we identified the MADS-box transcription factor Mbx1, a homologue of MEF2 in mammals, and characterized its role in the Ca2+/CN signaling pathway. We found that the mRNA levels of Mbx1 were dependent on Ca2+/CN signaling. We further identified the dom1+ gene (Downstream of mbx1) that encodes a novel membrane transporter as a transcriptional target of Mbx1. Interestingly, the mbx1-null and dom1-null cells displayed a dramatic decrease in the intracellular Ca2+ concentration, suggesting that Mbx1 and Dom1 are involved in the regulation of the Ca2+-homeostasis in fission yeast. Notably, the in vivo real-time assay of CN activity showed that CN was down-regulated in these mutant cells. Thus, Mbx1/Dom1 signaling may be involved in the positive feedback regulation of CN signaling through Ca2+-homeostasis control.
71 [P-3] The Actin Binding Protein Cis3/PSTPIP Is Involved in the Regulation of the Pmk1 MAPK Signaling Pathway and Cytokinesis
Akihiro Takeda, Satoru Takamura, Tomoyuki Takabe, Keiko Sugimoto, Yuka Shimamura, Ayako Kita, Shunji Ishiwata, and Reiko Sugiura Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Osaka, Japan
Mitogen-activated protein kinases (MAPKs), found in all eukaryotes, play a central role in various biological processes including cell proliferation, cell differentiation, and cell cycle. Calcineurin (CN) is a conserved Ca2+/calmodulin-dependent Ser/Thr protein phosphatase that plays a critical role in Ca2+ signaling. We have been studying signaling pathway regulated by these enzymes using fission yeast Schizosaccharomyces pombe as a model organism. We have previously demonstrated that CN and Pmk1 MAPK signaling play antagonistic roles in the Cl- homeostasis (Sugiura et al., Nature 1999, 2003). Our genetic screen utilizing the functional interaction between CN and Pmk1 identified various phosphatases including the pmp1+ encoding a dual-specificity phosphatase for Pmk1 MAPK, as well as ptc1+ and ptc3+, both encoding a type 2C phosphatase (Sugiura et al., Nature 2003, Takada et al. Mol. Biol. Cell 2007). Here, we identified cis3+ gene that encodes an actin binding protein highly homologous to the murine PSTPIP. The cis3 mutants displayed defective cytokinesis, and the GFP-Cis3 localized to the contractile actin ring consistent with its role in actin cytoskeleton. Interestingly we found that the phosphorylation state of Pmk1 MAPK was markedly increased upon Cis3 overproduction, and the activated Pmk1 MAPK phosphorylated Cis3. We discuss the possible role of Cis3 in the regulation of the Pmk1 MAPK signaling and cytokinesis.
72 [P-4] Calcineurin-Binding Proteins, RanBP3 and CaNBP75 Regulate Subcellular Localization and Phosphatase Activity of Calcineurin
Akiko Nakatomi1, Kei Sasaki2, Kenta Ohata2, Michio Yazawa1, and Yota Murakami1 1Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, Japan 2Department of Chemistry, Graduate School of Science, Hokkaido University, Sapporo, Japan
Calcineurin (CaN), serine/threonine-specific protein phosphatase 2B, is a Ca2+/calmodulin (CaM)-dependent phosphatase that plays an important role in a variety of cell types. Recently, a growing numbers of CaN-binding proteins have been discovered from various tissues, most of which function as tissue specific regulator for CaN. We found novel gonad specific CaN-binding protein CaNBP75 from scallop testis, and also found that RanBP3, mammalian homolog of CaNBP75, also interacts with CaN. In order to identify the role of CaNBP75 and RanBP3 (CaNBPs) as CaN regulator, we tried to investigate the effects of CaNBPs on localization and phosphatase activity of CaN. Fluorescence microscopic analysis revealed that GFP-CaNBPs were predominantly localized in the nucleus, and acted as shuttling protein between cytoplasm and nucleus. Co-expression of CaN and CaNBPs influenced their subcellular localization, and CaNBPs facilitated the nuclear entry of CaN. In vitro experiments revealed that CaNBPs interacted with CaN in a Ca2+/CaM-dependent manner, and increased the CaM affinity of CaN without changing the maximum activity. This character of CaNBPs is different from those of previously reported CaN-binding proteins that inhibit phosphatase activity of CaN. These observations may indicate that CaNBPs activate CaN at lower Ca2+ concentration in nucleus, which lead effective activation of the CaN target proteins such as a transcription factor NFAT.
73 [P-5] Functional Processing of Nuclear Ca2+/Calmodulin-Dependent Protein Kinase Phosphatase (CaMKP-N): Evidence for a Critical Role of Proteolytic Processing in the Regulation of Its Catalytic Activity, Subcellular Localization and Substrate Targeting in Vivo
Noriyuki Sueyoshi,1 Takaki Nimura,1 Takashi Onouchi,1 Hiromi Baba,1 Shinobu Takenaka1, Atsuhiko Ishida2, Isamu Kameshita1 1Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan 2Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan
Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear homolog CaMKP-N are Ser/Thr protein phosphatases that belong to the PPM family. These phosphatases are highly specific for multifunctional CaM kinases and negatively regulate their activities. CaMKP-N is only expressed in the brain and specifically localized in the nucleus. In this study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing in both the zebrafish brain and Neuro2a cells. In Neuro2a cells, zCaMKP-N was ubiquitinated for proteolysis at the C-terminal region containing the nuclear localization signal. The proteolytic processing was effectively inhibited by the proteasome inhibitors MG-132, Epoxomicin, and Lactacystin, suggesting that the ubiquitin-proteasome pathway was involved in this processing. Using MG-132, we found that the proteolytic processing changed the subcellular localization of zCaMKP-N from the nucleus to the cytosol. Accompanying this change, the cellular targets of zCaMKP-N in Neuro2a cells were significantly altered. Furthermore, we obtained evidence that the zCaMKP-N activity was markedly activated when the C-terminal domain was removed by the processing. Thus, the proteolytic processing of zCaMKP-N at the C-terminal region regulates its catalytic activity, subcellular localization and substrate targeting in vivo.
74 [P-6] Imbalance of CaMKII and PP1 Activities Is Associated with Abnormal Dendritic Spine Formation in the ATRX Mutant Mouse Brain
Norifumi Shioda1, Hideyuki Beppu2, Isao Kitajima2, Kohji Fukunaga1 1Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan 2Department of Clinical and Molecular Pathology, Faculty of Medicine, University of Toyama, Toyama, Japan.
In humans, mutations in the gene encoding ATRX, a chromatin remodeling protein of the SNF-2 family, cause several mental retardation disorders, including alpha-thalassemia X-linked (ATR-X) syndrome. We generated ATRX mutant mice lacking exon 2 (ATRX!E2 mice), a mutation that mimics exon 2 mutations seen in human patients who represent a mild mental retardation symptom. ATRX!E2 mice exhibited abnormal dendritic spine formation in the medial prefrontal cortex (mPFC). ATRX!E2 mice exhibited longer and thinner dendritic spines compared to wild-type mice. Interestingly, an aberrant increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity was observed in the mPFC of ATRX!E2 mice. The increased CaMKII autophosphorylation and activity were associated with increased phosphorylation of the Rac1-GEFs, Tiam1 and Kalirin-7. We also confirmed increased phosphorylation of p21-activated kinases (PAKs) in mPFC extracts. Furthermore, reduced protein expression and activity of protein phosphatase 1 (PP1) was evident in the mPFC of ATRX!E2 mice. Taken together, our data strongly suggest that aberrant CaMKII activation likely mediates abnormal spine formation in the mPFC. Such morphological changes with elevated Rac1-GEF/PAK signaling seen in ATRX!E2 mouse brain may contribute to define the mechanisms underlying mental retardation syndromes in human patients.
75 [P-7] Endotoxin Conditioning Induces VCP/p97-Mediated and iNOS-Dependent Tyr284 Nitration in Protein Phosphatase 2A
Takashi Ohama1,2 and David L. Brautigan2 1Department of Veterinary Pharmacology, Yamaguchi University, Japan 2Center for Cell Signaling, University of Virginia, USA
Exposure to stresses, such as endotoxin, cytokines, ischemia, hypoxia, etc., reprograms cells to be refractory to a secondary stress exposure. This adaptive phenomenon termed tolerance or preconditioning protects immune, cardiovascular and neurological systems, and limits tissue damage from recurrent insults. It is known that one type of primary stress can attenuate different types of secondary stress-induced injury, e.g. preconditioning with endotoxin diminishes ischemia/reperfusion injury, called cross-tolerance, suggesting the existence of some common mechanisms. Here we show this involves accumulation of a complex between endogenous protein phosphatase 2A (PP2Ac) and VCP/p97, a conserved AAA ATPase involved in ubiquitination and deubiquitination of proteins. PP2A activates VCP/p97, reversing the effects of Akt and VCP/p97 recruits inducible NO synthetase (iNOS), known to be a key regulator for tolerance, to mediate tyrosine nitration of PP2Ac. Peroxynitrite reaction with purified PP2Ac primarily nitrates on Tyr284 and results in dissociation from its scaffolding A subunit, which restricts PP2Ac activity. VCP/p97 brings together two phosphatases, PP2Ac and DUSP1/MKP1. Endotoxin preconditioning is mimicked by transient over-expression of either PP2Ac or VCP/p97 that elevates levels of DUSP1, reduces p38, ERK and JNK activation and suppresses release of TNF-alpha. Thus, VCP/p97 recruits PP2Ac, promotes its Tyr nitration and targets DUSP1 to produce stress-induced tolerance. We propose Tyr nitration of PP2Ac as a marker for pre-conditioning and for stress-induced damage.
76 [P-8] AMP Activated Protein Kinase (AMPK) Is Regulated by Multiple Protein Phosphatases
Takayasu Kobayashi, Toko Chida, Masakatsu Ando, Tasuku Matsuki, Yutaro Masu, Yusuke Kanto, Yuko Nagaura, Shinri Tamura Department of Biochemistry, Institution of Development, Aging and Cancer, Tohoku University, Japan
AMPK is a major sensor of cellular and whole body energy. The elevation of cellular AMP/ATP ratio activates AMPK, which then switches on catabolic processes including glucose uptake and " oxidation and turn off anabolic systems such as gluconeogenesis and fatty acid synthesis. AMPK is a heterotrimer that comprises a catalytic ! subunit (!1 or !2) and regulatory " and # subunits and is only active after phosphorylation at Thr172 of ! subunit by upstream kinases. Protein phosphatase 2C (PP2C) family is a group of monomeric metal ion-dependent and okadaic acid resistant phosphatases including 14 members. Although PP2C-like phosphatase activity was reported to be involved in inactivation of AMPK in hepatocyte, specific isoform(s) that are responsible for dephosphorylation of AMPK are unknown. In this study, we examined the effect of overexpression of PP2C members on phosphorylation at Thr172 of AMPK!1 and !2. We found that overexpression of PP2C! (PPM1A) or PP2C" (PPM1B) dramatically decreased phosphorylation at Thr172 of AMPK!1 and AMPK!2, while CaMKP-N (PPM1E) specifically dephospholyated AMPK!2. In contrast, overexpression of NERPP (PPM1H) increased phosphorylation of both AMPK!1 and AMPK!2. We also found that G2A mutation of PP2C! (PPM1A) and PP2C" (PPM1B) reduced their ability to dephosphorylate AMPK without affecting enzymatic activity. These results suggested that multiple phosphatases are involved in regulation of AMPK activation and that myristoylation may play important role in function of PP2C! (PPM1A) and PP2C" (PPM1B).
77 [P-9] Increase in Number of Nucleoli by Protein Phosphatase PPM1D Overexpression
Yuuki Kozakai, Yoshiro Chuman, Hiroaki Yagi, Youhei Teduka, Toshiaki Imagawa, and Kazuyasu Sakaguchi Laboratory of Biological Chemistry, Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan
Nucleolus is a highly dynamic nuclear compartment that plays a key role in ribosome biogenesis. The changes in number and morphology of nucleolus were observed in various cancer tissues. Nucleophosmin (NPM) is a nucleolar protein and is involved in various cellular activities such as ribosome biogenesis and centrosome duplication. It was reported that these cellular activities require phosphorylation at several sites of NPM. PPM1D (Wip1, PP2C$) is a p53-inducible Ser/Thr phosphatase. One of PPM1D functions in normal cells is a negative feedback regulation by dephosphorylating cell-cycle checkpoint kinases and tumor suppressor p53. On the other hand, gene amplification and protein overexpression of PPM1D were reported in many human tumors. In this study, we analyzed the effect of PPM1D on nucleolus formation in cells. In normal cells, PPM1D was localized in nucleolus. However, PPM1D was mainly observed in nucleolus localized with Nucleophosmin, and also in nucleoplasm when overexpressed. In MCF7 cells that overexpress PPM1D, the knockdown of the PPM1D expression decreased the number of nucleoli. The number of nucleoli in MCF7 was also significantly reduced by the incubation with the PPM1D inhibitor, SPI-001. Moreover, in p53-null H1299 cells, the overexpression of PPM1D leads to increase the number of nucleoli. We showed that PPM1D associated with NPM by co-immunoprecipitation assay and that PPM1D dephosphorylated the NPM-S(P)4 peptide in vitro. These results suggested that PPM1D affected the number of nucleolus through the regulation of phosphorylation on NPM in a p53-independent manner.
78 [P-10] Novel Small Molecule Inhibitors Specific for p53-Inducible Protein Phosphatase PPM1D
Hiroaki Yagi1, Yoshiro Chuman1,Yuuki Kozakai1, Toshiaki Imagawa1, Fumihiko Yoshimura2, Keiji Tanino2 and Kazuyasu Sakaguchi1 1Laboratiry of Biological Chemistry, Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan 2Laboratory of Organic Chemistry II, Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan p53-inducible Ser/Thr protein phosphatase PPM1D (Wip1, PP2C !) is a member of PPM1 (PP2C) family. The PPM1D gene amplification and overexpression have been reported in many human tumors, including breast cancer and neuroblastoma. Because the phosphoatase activity of PPM1D is essential for its oncogenic role, PPM1D inhibitor should be a viable anti-cancer agent. In this study, we identified highly potent and specific PPM1D inhibitors, SPI-001 and SPI-002, by screening of compounds in our own chemical library. The compounds strongly inhibited PPM1D activity in noncompetitive matter. The Ki value of SPI-001 is determined to be 0.59 µM. Both of the compounds inhibited PPM1D phosphatase activity in PPM1D-overexpressed human cells. Structure activity relationship analysis suggested that the special configuration of two hydrophobic moieties in the compounds was important for its inhibitory activity. Circular dichroism analysis showed that SPI-001 induced conformational change of PPM1D upon binding, suggesting that SPI-001 interacted with PPM1D at the allostric site. Furthermore, we showed the P-loop, the PPM1D-specific proline rich region in the catalytic domain, was important for PPM1D inhibition by SPI-001. Proline rich seaquence frequently plays an important role in protein-protein interaction. The results suggested that the P-loop is involved in regulation of PPM1D activity through the interaction with other proteins.
79 [P-11] Control of Phosphorylation and Intracellular Localization of Gln3 by Protein Phosphatase Siw14 in Saccharomyces cerevisiae
Yusuke Imabeppu, Minori Numamoto, Yusuke Ueda, Masataka Hirasaki, Minetaka Sugiyama, Yoshinobu Kaneko and Satoshi Harashima Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan
Disruption of S.cerevisiae SIW14, which encodes a protein tyrosine phosphatase, causes caffeine sensitivity. We found that caffeine sensitivity of the Dsiw14 disruptant was suppressed by disruption of GLN3. Gln3 is a transcriptional activator for nitorogen catabolite repression genes in response to nutrient availability and its activity is regulated by subcellular localization. In the absence of caffeine, Gln3 is hyper-phosphorylated and localizes in the cytoplasm. Treatment with caffeine causes Gln3 dephosphorylation and translocation into nucleus. In the Dsiw14 disruptant, Gln3 accumulates in nucleus and the phosphorylation level of Gln3 decreases despite the presence or absence of caffeine. Therefore, we suggested in a previous study that Siw14 controls Gln3 localization by regulating phosphorylation level of Gln3. However, phosphorylation sites which control Gln3 intracellular localization are unknown. In this study, to identify the phosphorylation sites which affect intracellular localization, we focused on nuclear localization site of Gln3. We found that the Gln3 with the mutation of Ser-355 residue to phosphorylation-incompetent Ala accumulates in nucleus in the presence and absence of caffeine, and that like wild type Gln3, Gln3 with the mutation of Ser-355 to phosphorylation-mimic Asp localizes in cytoplasm in the absence of caffeine and in nucleus in the presence of caffeine. These results suggest that Siw14 controls Gln3 localization through phosphorylation of Ser-355. However, Siw14 cannot phosphorylate Ser-355 of Gln3 directly since Siw14 is a protein phosphatase but not protein kinase. Therefore, we suggest that unknown protein kinase phosphorylates Ser-355 of Gln3 and Siw14 controls that protein kinase.
80 [P-12] Phospho-Regulation of Spliceosomal Proten, Sap155/Sf3b1
Tanuma N., Nomura M., Sato M., Yamashita Y., Shiiba K., Katakura R. and Shima H. Div. Cancer Chemother., Miyagi Cancer Center Research Inst., Japan
Pre-mRNA splicing entails reversible phosphorylation of spliceosomal proteins. Our recent work has revealed PP1-NIPP1 holoenzyme is one of phosphatase responsible for dephosphorylation of Sap155/Sf3b1, an essential component of U2 snRNP (1). Sap155 becomes hyper-phosphorylated concomitant with or just after the first catalytic step of splicing in vitro. In this study, we identified in vivo phosphorylation sites of Sap155 purified from HeLa cells, and developed phospho-specifc antibodies against those sites. One of antibodies worked well in several immuno-applications. Analysis using the phospho-Sap155 antibody will be reported. 1) Tanuma N. et al. J. Biol. Chem., 283(51): 35805-14, 2008
81 [P-13] Mitochondrial Membrane Potential Loss Induces Intramembrane Proteolysis of the Mitochondria-Resident Protein Phosphatase PGAM5
Shiori Murakami, Ayako Nishihara, Hidenori Ichijo and Kohsuke Takeda Cell Signaling, Grad. Sch. Pharmaceut. Sci., Univ. of Tokyo, Japan
PGAM5, a member of the phosphoglycerate mutase (PGAM) family, lacks mutase activity, but instead acts as a protein Ser/Thr phosphatase. To further elucidate the function of PGAM5, we focused on an N-terminal transmembrane domain that is a unique structure for PGAM5 among PGAM family members. Immunocytochemistry and sucrose density gradient centrifugation experiments revealed that PGAM5 was an innermembrane-resident protein. To examine the topology of PGAM5, we treated the isolated mitochondria with trypsin. Reactivity of mitochondrial PGAM5 to the antibody that recognizes the C-terminal end of PGAM5 was abolished upon the treatment with trypsin, suggesting that the most region of this molecule including the catalytic domain faces the mitochondrial intermembrane space. Moreover, we found that PGAM5 was cleaved in its transmembrane domain, when cells were treated with the mitochondrial uncoupler CCCP. Recently, it has been reported that cleavage of mitochondrial proteins, such as OPA1 and PINK1, is regulated depending on the mitochondrial membrane potential (%&m). Our results therefore suggest that PGAM5 is another hot example that is proteolytically regulated by %&m. These mitochondrial proteolysis regulations by %&m may be one of the general stress responses of mitochondria, and we consider that PGAM5 is a unique protein phosphatase that may sense and respond to stress-induced changes in states of the mitochondria.
82 [P-14] Roles of a Protein Phosphatase PGAM5 in Heat Shock Stress Response
Yosuke Ishida, Yusuke Sekine, Hidenori Ichijo and Kohsuke Takeda Cell Signaling, Grad. Sch. Pharmaceut. Sci., Univ. of Tokyo, Japan
PGAM5 is a novel type of Ser/Thr-specific protein phosphatase that we identified as an interacting protein and activator of ASK1, which is a stress-responsive MAP3 kinase of the JNK and p38 MAPK pathways. We have shown that the Ser/Thr phosphatase activity as well as the primary structure of PGAM5 is highly conserved among species including Drosophila, suggesting the evolutionarily conserved roles of PGAM5. Here we show that dPGAM5, the Drosophila ortholog of mammalian PGAM5, is involved in heat shock stress response. We first generated and analyzed dPGAM5 null mutant flies and found that they were vulnerable to heat shock stress. The flies in which dPGAM5 was knocked down specifically in the mushroom body (MB) in the brain also exhibited the vulnerability to heat shock, suggesting that dPGAM5 in the MB plays a role in the whole body response to heat shock. As we previously found that overexpression of dPGAM5 induced rough eye phenotype through the Drosophila p38 (Dp38) pathway, we further investigated the involvement of the dPGAM5-Dp38 pathway in heat shock response. Knockdown of Dp38 in the MB increased the vulnerability to heat shock, and heat shock-induced activation of Dp38 was reduced in the head, but not the body (thorax and abdomen), of dPGAM5 mutant flies. Moreover, expression of p35, a caspase inhibitory protein, in the MB rescued the vulnerability of dPGAM5 mutants to heat shock. These results suggest that regulation of the Dp38 pathway and caspase activity by dPGAM5 in the MB may play a crucial role in protecting organisms from heat shock stress.
83 [P-15] Consensus Substrate Sequence for Ptprz
Akihiro Fujikawa1, Masahide Fukada1, Yoshikazu Makioka2, Ryoko Suzuki1, Jeremy P. H. Chow1 and Masaharu Noda1 1Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki, Japan 2Synthetic Organic Division, Tokyo Institute of Technology, Yokohama, Japan
Protein-tyrosine phosphatase receptor type Z (Ptprz/PTPz/RPTPb) is predominantly expressed in the CNS. The physiological importance of this molecule has been demonstrated through studies of Ptprz-deficient mice. The major defect we found is a maturation-dependent impairment in the hippocampal function. Ptprz is expressed also in the stomach, where it functions as a receptor of VacA, a cytotoxin secreted by Helicobacter pylori. Ptprz-deficient mice are resistant to gastric ulcer induction by VacA. Although our understanding about physiological functions of Ptprz is thus getting on, our knowledge about its biochemical properties such as substrate specificity are still limited. We previously developed a genetic method named ”yeast substrate-trapping system” to screen for PTP substrates. Using this method, we identified several substrates for Ptprz, such as G protein-coupled receptor kinase-interactor 1 (Git1), membrane associated guanylate kinase, WW and PDZ domain containing 1 (Magi1), and GTPase-activating protein for Rho GTPase (p190 RhoGAP). Here, we show that Ptprz selectively dephosphorylates specific phospho-tyrosine residues on these proteins in in vitro and cell-based assays. Alignment of the primary sequences surrounding the dephosphorylation sites revealed considerable similarity. We next performed a kinetic analysis using fluorescent substrate peptides with replacement at various positions to elucidate the motif structure. The consensus sequence thus obtained predicted paxillin as a novel substrate candidate. Indeed, the site in paxillin was efficiently dephosphorylated by Ptprz in our cell-based assay.
84 [P-16] Hypothermia-Induced Tyrosine Phosphorylation of SIRP" in the Brain
Toshi Maruyama1,2, Hiroshi Ohnishi1, Shinya Kusakari1, Yuriko Hayashi1, Yoji Murata3, Yasuyuki Saito1, Per-Arne Oldenborg4, Shoji Kishi2 and Takashi Matozaki1,3 1Laboratory of Biosignal Sciences, Institute for Molecular and Cellular Regulation, Gunma University, Gunma, Japan 2Department of Ophthalmology, Gunma University Graduate School of Medicine, Gunma, Japan 3Division of Molecular and Cellular Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan 4Department of Integrative Medical Biology, Section for Histology and Cell Biology, Umeå University, Umeå, Sweden
SIRP" is a membrane protein that undergoes tyrosine phosphorylation in the brain in response to forced swim (FS) stress. We now show that the increase in tyrosine phosphorylation of SIRP" by FS stress primarily depends on the water temperature. FS in cold water (23 °C), but not in warm water (37 °C), induced a significant increase in the level of tyrosine phosphorylation of SIRP" in the mouse brain. The core body temperature (CbT) of the mice was markedly decreased by 10 min-FS in cold water from 37-38 °C to 27-30 °C, while the same parameter was not affected by FS in warm water. Immersion of the restrained mice in cold water also induced tyrosine phosphorylation of SIRP" in the brain in parallel with a decrease in CbT. An increase in the tyrosine phosphorylation of SIRP" in the brain was also induced by administration of ethanol or picrotoxin, severe starvation, or cooling of anesthetized mice; these treatments indeed induced hypothermia. Furthermore, exposure to low temperature of cultured hippocampal neurons promoted tyrosine phosphorylation of SIRP". By contrast, hypothermia-induced tyrosine phosphorylation of SIRP" in the brain was markedly decreased in mice deficient of CD47, a ligand for SIRP". Our data suggest that tyrosine phosphorylation of SIRP" is a novel cellular response to cold stress in neurons. It might participate in the regulation of behavioral immobility during FS.
85 [P-17] PTP-PEST Ser-39 Phosphorylation and Its Regulation in Jurkat Cells Costimulated with CD3 and CD28
Helen E. F. Palmer and Keisuke Mashima Department of Life Sciences, Faculty of Science, Rikkyo University, Japan
Protein tyrosine phosphatase-PEST (PTP-PEST), encoded by the PTPN12 gene, is expressed in a wide variety of cell types is an efficient regulator of cell adhesion, spreading and migration in adherent cells, and antigen receptor-mediated signaling in lymphocytes. Previous studies clarified that the PTP activity of PTP-PEST was regulated by Ser-39 phosphorylation, which is known to be catalyzed by PKA and/or PKC. On the other hand, the cellular function of Ser-39 phosphorylation remains unknown. Here, we showed that the phosphorylation of Ser-39 was regulated by the costimulation of CD3/CD28 antibodies in PTP-PEST expressed Jurkat cells. Phosphorylation of Ser-39 was induced after 5 minutes of CD3/C28 costimulation, however, these levels decreased after 10 minutes. The CD3/CD28 costimulation-induced Ser-39 phosphorylation levels were suppressed in cells pre-treated with PKC # - specific inhibitor, indicating that CD3/CD28 costimulation mediated Ser-39 phosphorylation through PKC # activation. Now we seek to further elucidate the regulation of CD3/CD28 mediated Ser-39 phosphorylation and dephosphorylation in Jurkat cells.
86 [P-18] Molecular and Clinical Analysis of RAF1 in Noonan Syndrome and Related Disorders: Dephosphorylation of Serine 259 as the Essential Mechanism for Mutant Activation
Yoko Aoki, Tomoko Kobayashi, Tetsuya Niihori and Yoichi Matsubara Department of Medical Genetics, Tohoku University School of Medicine, Sendai, Japan
Noonan syndrome (NS) and related disorders are autosomal dominant disorders characterized by heart defects, facial dysmorphism, ectodermal abnormalities and mental retardation. The dysregulation of the RAS/MAPK pathway appears to be a common molecular pathogenesis of these disorders: mutations in PTPN11, KRAS and SOS1 have been identified in patients with NS, those in KRAS, BRAF and MAP2K1/2 in patients with CFC syndrome and those in HRAS mutations in Costello syndrome patients. Recently, mutations in RAF1 have been also identified in patients with NS and two patients with LEOPARD (multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth and sensorineural deafness) syndrome. In the current study, we identified eight RAF1 mutations in 18 of 119 patients with NS and related conditions without mutations in known genes. We summarized clinical manifestations in patients with RAF1 mutations as well as those in NS patients with PTPN11, SOS1 or KRAS mutations previously reported. Hypertrophic cardiomyopathy and short stature were found to be more frequently observed in patients with RAF1 mutations. Mutations in RAF1 were clustered in the conserved region 2 (CR2) domain, which carries an inhibitory phosphorylation site (serine at position 259; S259). Functional studies revealed that the RAF1 mutants located in the CR2 domain resulted in the decreased phosphorylation of S259, and that mutant RAF1 then dissociated from 14-3-3, leading to a partial ERK activation. Our results suggest that the dephosphorylation of S259 is the primary pathogenic mechanism in the activation of RAF1 mutants located in the CR2 domain as well as of downstream ERK.
87 [P-19] Identification of a Tumor Suppressor Parafibromin/Cdc73 as a Nuclear Substrate for the SHP2 Tyrosine Phosphatase
Atsushi Takahashi1, Ryouhei Tsutsumi1, Ippei Kikuchi1, Azadeh Seidi1, Chikashi Obuse2 and Masanori Hatakeyama1 1Div. Microbiol., Grad. Sch. Med., Univ. Tokyo, Japan 2Div. Mol. Life Sci., Grad. Sch. Life Sci., Hokkaido Univ., Japan
SHP2 is a ubiquitously expressed protein tyrosine phosphatase that is encoded by the PTPN11 gene. Deregulation of SHP2, which is caused by the gain-of-function mutations in the PTPN11 or bacterial infection with cagA+ Helicobacter pylori, is associated with the development of human malignancies such as childhood leukemia and gastric cancer, indicating that SHP2 is a bona-fide oncoprotein. While SHP2 has been primarily reported to promote the RAS-ERK pathway in the cytoplasm, the roles of SHP2 present in the nucleus are poorly understood. To elucidate the whole picture of SHP2-mediated cellular events, we screened SHP2 substrates by combining “substrate-trapping” immunoprecipitation and LC-MS/MS, and identified the RNA Polymerase II-Associated Factor (PAF) complex, a nuclear multiprotein complex that regulates various processes of transcription, as a potential SHP2 substrate. We then found that parafibromin/Cdc73, a member of the PAF complex that is also recognized as a tumor suppressor, undergoes tyrosine phosphorylation, whose level was elevated upon SHP2-specific knockdown. We also identified the tyrosine phosphorylation sites of parafibromin, which are dephosphorylated by SHP2. These results indicate that parafibromin is a novel substrate of SHP2 and suggest that SHP2 not only acts as a mitogenic signal transducer in the cytoplasm but also functions as a transcriptional regulator in the nucleus, deregulation of which may also contribute to carcinogenesis.
88 [P-20] Regulation of Intracellular Distribution of SHP2 Phosphatase
Ryouhei Tsutsumi, Atsushi Takahashi, Ippei Kikuchi and Masanori Hatakeyama Division of Microbiology, Graduate School of Medicine, University of Tokyo, Japan
SHP2, encoded by PTPN11, is a ubiquitously expressed tyrosine phosphatase which possesses two tandem SH2 domain followed by PTP domain and C-terminal tail. Germline- and somatic gain-of-function mutations of PTPN11 are associated with Noonan syndrome, juvenile myelomonocytic leukemias (JMML), adult leukemia and several solid tumors. Although SHP2 had been recognized as a growth signal mediator under several growth factor receptors and scaffold proteins at plasma membrane, it also localizes in cellular nucleus. To understand whole picture of SHP2 signaling, it is necessary to reveal both cytoplasmic and nuclear function of SHP2. Recently, we identified a nuclear PAF complex component, parafibromin as a substrate of SHP2, indicating the evidence for a nuclear function of the phosphatase. In this study, we show the translocalization of SHP2 between cytoplasm and nuclear in a manner dependent on growth signal. We observed SHP2 abundantly presents in the nucleus of proliferating cells. However, in cells that ceased proliferation at an elevated density, SHP2 was excluded from nucleus and mostly distributed to the cytoplasm. In addition, expression of K-RASV12 rescued the density dependent exclusion of SHP2 from cellular nucleus. Furthermore, observed dynamic translocalization altered nuclear SHP2 activity. SHP2 functions not only at plasma membrane but also in cellular nucleus. Present study indicates a molecular mechanism for the regulation of nuclear SHP2 activity, which may underlie the important roles of SHP2 in human development and diseases.
89 [P-21] Downregulation of Tumor Suppressor MicroRNA in Inflammatory Microenvironment
Hiroko Oshima, Dan Kong, Tomo-o Ishikawa, Masanobu Oshima Division of Genetics, Cancer Research Institute, Kanazawa Univiersity, Japan
Inflammatory responses play an important role in cancer development by induction of growth factors as well as suppression of apoptosis and immune surveillance. On the other hand, accumulating evidence has indicated that expression changes of microRNAs contribute to cancer development through downregulation of tumor suppressor genes or upregulation of oncogenes. Here, we have investigated expression changes of tumor-related microRNAs and its role in tumorigenesis using gastric cancer mouse models and human gastric cancer cell lines, respectively. We previously constructed mouse models for gastritis (K19-C2mE mice) and gastric cancer (Gan mice) by transgenic expression of prostaglandin E2 (PGE2) and combination of Wnt1 and PGE2, respectively. Microarray analyses showed that expression of several microRNAs (miR-7, miR-10b, miR-143, and miR-145) was significant downregulated in mouse gastric cancer tissues. Notably, these microRNAs were also downregulated in the non-tumor gastritis tissues. These expression changes were confirmed by real-time RT-PCR. Among these microRNAs, we further examined miR-7 because miR-7 has shown to be a possible tumor suppressor in brain tumors, lung and breast cancers. By luciferase reporter assays, we found that conditioned medium from activated macrophages but not PGE2 itself suppresses expression of miR-7 in human gastric cancer cells. Moreover, we confirmed that transfection of miR-7 precursor suppressed tumor cell proliferation and soft-agar colony formation. These results suggest that macrophage-derived factor(s) suppresses expression of tumor suppressor miR-7 in gastric epithelial cells, which contribute to promotion of tumorigenesis.
90 [P-22] Basal Autophagy Regulates Oxidative Stress-Associated Cell Death in Neural Cells
Seiji Torii, Chisato Kubota, Hideaki Imai, and Toshiyuki Takeuchi Institute for Molecular and Cellular Regulation, Gunma University, Japan Reactive oxygen species (ROS) are involved in several cell death processes through the oxidation of cellular components including protein phosphatases. Now we suggest a new idea on the relationship among ROS, autophagy, and necrosis that is one of the topics in the cell death field. We found that glutamate-induced ROS accumulation and the associated neural cell death were prevented by inhibitors to autophagy or lysosomal activity. Glutamate, however, did not stimulate autophagy, and neither changes in organization of mitochondria nor lysosomal membrane permeabilization were observed. Fluorescent analyses by a redox probe revealed that autophagosomes are the major sites for basal ROS generation in addition to mitochondria. Treatments with autophagic/lysosomal inhibitors decreased their basal ROS production and caused a burst of mitochondrial ROS to be delayed. On the other hand, attenuation of mitochondrial activity by serum depletion or by high cell-density culture resulted in the loss of both constitutive ROS production and a ROS burst in mitochondria. Thus, constitutive ROS production within mitochondria and autophagosomes/lysosomes enables cells to be susceptible to glutamate-induced oxidative cytotoxicity. Corroboratively, the inhibitors reduced neural cell death in an ischemia model in rats. We suggest that cell death during periods of ischemia is regulated by the interplay between autophagosomes/lysosomes and mitochondria on the regulation of intracellular ROS.
91 [P-23] Tracking the Mobility of GADD34 and eIF2! Phosphatase Assembly using Fluorescence Live Cell Imaging
Meng Shyan Choy and Shirish Shenolikar Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857
GADD34 (Growth Arrest and DNA Damage-Inducible Protein) is a PP1 regulator that is expressed in cells recovering from cellular stress. GADD34 binds to PP1 at the C-terminus (KVRF motif) and assembles the eIF2! phosphatase. This binding confers the specificity of the complex to dephosphorylate phospho-eIF2!, allows the protein synthesis in the cells to resume upon the removal of cellular stress. Although GADD34 localizes in the endoplasmic reticulum (ER) membrane, some fraction of GADD34 can be found in the cytosol. Why GADD34 is found both in the ER and cytosol is not clear but the localization of GADD34 might be important for its stability and complex assembly. Using Fluorescence Recovery after Photobleaching (FRAP) and Bimolecular Fluorescence Complementation (BiFC) techniques, we set to track the mobility of GADD34 in live cells and to pinpoint the location of GADD34/PP1/eIF2! complex assembly. Our results show that GADD34 is more dynamic compared to the exclusively membrane bound homolog CReP (Constitutively expressed repressor of eIF2! phosphorylation), a characteristic due probably to its presence in both cytosol and membrane fractions. Using a novel trimeric BiFC assay, we are able to demonstrate that the assembly of GADD34, PP1! and eIF2! complex occurs primarily in the site of GADD34 localization i.e. ER and cytosol. No assembly of GADD34, PP1! and eIF2! was observed in the nucleus even though a nucleus targeted mutant 513-674 GADD34 (C-terminus of GADD34) was used.
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