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          第65回BRC セミナー その1

    場所:理研筑波研 バイオリソースセンター1階 大会議室

Professor Ami Aronheim
(Dept. of Molecular Genetics&s_comma; The B. Rappaport Faculty of Medicine&s_comma;
Technion-Israel Institute of Technology&s_comma; Israel)

「The Ras Recruitment System for the analysis of protein-protein
interaction for a wide range of proteins of interest」

The rapid advance in the genome projects provides the scientific community
with numerous proteins some of them with no assigned function. In order to
achieve a better understanding of the known and novel genes available
today&s_comma; research is focused on identification of interaction maps of
multiple protein candidates. The two-hybrid system serves as an excellent
method for this purpose&s_comma; and is commonly used to identify and isolate novel
and known protein-protein interaction partners for proteins of interest.
Although very powerful&s_comma; the two-hybrid system&s_comma; which is based on a
transcriptional readout&s_comma; exhibits several limitations and inherent problems.

To overcome the problems and limitations of the two hybrid system&s_comma; we have
developed several approaches collectively known as "Protein Recruitment
Systems". The systems are based on the absolute requirement of Ras to be
localized to the plasma membrane for functional downstream signaling. In
yeast&s_comma; a temperature sensitive mutant in the Ras guanyl exchange factor&s_comma;
CDC25 exists. At the permissive temperature&s_comma; 24oC&s_comma; cells proliferate&s_comma;
however&s_comma; when switched to 36oC cell are growth arrested. A protein of
interest (the bait) is fused to activated-cytoplasmic Ras. The bait is able
to rescue CDC25 yeast strain mutant only when is localized to the plasma
membrane. Plasma membrane translocation of the bait can be achieved via
interaction with a membrane anchored protein partner. In addition to the
yeast system&s_comma; we have developed a mammalian assay in which protein-protein
interaction is studied directly in mammalian cells. Moreover&s_comma; we have also
devised a reverse Ras Recruitment approach for the study of protein-protein
interactions with membrane proteins as baits. The potential use of the
protein recruitment systems to study protein-protein interactions for a
wide range of proteins will be demonstrated.

          第65回BRC セミナー その2

    場所:理研筑波研 バイオリソースセンター1階 大会議室

Professor Ami Aronheim
(Dept. of Molecular Genetics&s_comma; The B. Rappaport Faculty of Medicine&s_comma;
Technion-Israel Institute of Technology&s_comma; Israel)

「The c-Jun dimerization protein 2&s_comma; JDP2&s_comma; a tumor suppressor or an oncogene?」

The AP-1 transcription factor is a dimeric complex composed between members
of the basic leucine zipper (bZIP) proteins. The c-Jun dimerization protein
2&s_comma; JDP2&s_comma; is a relatively novel member of the bZIP family that was isolated
initially based on its ability to bind c-Jun. JDP2 is able to strongly
repress AP-1 dependent transcription by multiple mechanisms. JDP2 is
involved in differentiation of skeletal muscle and osteoclast cells. In
addition&s_comma; JDP2 over-expression results in inhibition of cell proliferation
and cellular transformation both in vitroand in vivo. However&s_comma; other
studies suggested that JDP2 may function as an oncogene.

We have generated transgenic mice over-expressing JDP2 specifically in the
liver under the control of the tetracycline regulated promoter. This model
can functionally inhibit AP-1 transcription in a reversible and regulated
manner and therefore has several advantages over the current available
conditional knockout mice of single components of the AP-1 transcription
Hepatocellular carcinoma is the fifth most frequent cancer in the world and
the third leading cause of cancer death. To reveal the role of JDP2 in
oncogenesis&s_comma;we are using two distinct mice models for liver cancer
resembling the etiology of heptocellular carcinoma in human. The first is a
chemically induced liver cancer model that includes a single injection of
diethylnitrosamine (DEN) that results in liver cancer within 10-12 months
of age. The second liver cancer model is: an inflammation dependent genetic
model in which the multi drug resistance 2 gene (Mdr2) is disrupted. In
this model&s_comma; liver cancer spontaneously develops at 12 months of age. By
using these two models we obtained different consequences in the
development of hepatocellular carcinoma as a result of JDP2
over-expression. The possible mechanisms involved in the differential
effects exerted by JDP2 will be discussed.


Takehide MuraTa Ph. D.
DNA Bank&s_comma; BioResource Center&s_comma; RIKEN
Tsukuba Science City&s_comma; Ibaraki 305-0074&s_comma; Japan.
e-mail: murata_t@brc.riken.jp
HP: http://www.brc.riken.jp/lab/dna/
phone: +81-29-836-3612&s_comma; fax: +81-29-836-9120

>>>> Japanese Address <<<<
305-0074 茨城県 つくば市 高野台 3-1-1
理化学研究所 筑波研究所
遺伝子材料開発室 (DNA Bank)
村田 武英,専任研究員