download report - Istituto Pasteur

P a r t i c i p a n t s :
Maria Teresa Fiorenza, Arturo Bevilacqua, professors; Sonia
Canterini, researcher; Adriana Bosco, Valentina Carletti,
Valentina De Matteis, Domenico Grillo, PhD students.
C o l l a b o r a t i o n s :
Istituto Dermopatico dell’Immacolata, Roma (Dr. Giandomenico
Russo, Dr. Maria Grazia Narducci); Ohio State University,
Columbus, Ohio, USA (Prof. Carlo Croce); University of Turin (Dr.
Annalisa Buffo); University of Antwerp, Belgium (Dr. Michele
Giugliano).
Report of activity
We have exploited two different model systems, the
preimplantation mouse embryo development and the
in vitro differentiation of cerebellum granule neurons,
to investigate the control of early blastomere proliferation
and the commitment to apoptosis, respectively,
with particular reference to the functions of the oncogenic
factor T-cell Leukemia Factor 1 (TCL1) and the
putative tumor suppressor THG-1pit/Tsc22d4.
Besides T-cell leukemias, TCL1 is physiologically
expressed both in embryonic stem cells downstream
from the Oct4 gene and in early preimplantation
embryos, in which it enhances early blastomere proliferation.
TCL1 is currently believed to promote normal/tumoral
cell proliferation by binding and
transphosphorylating AKT/PKB (AKT) at the level of
plasma membrane and then mediating the phosphorylated
AKT transfer to nucleus. However, the AKT isoform(s)
that actually interacts with TCL1 and the
TCL1 requirement for AKT nuclear transfer are still
uncharacterized. We have directly approached these
questions by depleting one-cell embryos by an intracytoplasmic
microinjection of anti-AKT1, anti-AKT2 or
anti-AKT3 antibodies and then following the in vitro
development of injected embryos. Depletion of AKT2
significantly delayed/blocked embryo development to
blastocyst, as we had previously observed in Tcl1 KO
Principal investigator: Franco Mangia
Professor of General Biology
Dipartimento di Psicologia, Sezione di Neuroscienze
Tel: (+39) 06 49917784; Fax: (+39) 06 49917873
franco.mangia@uniroma1.it
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Molecular genetics of eukaryotes - AREA 3
Molecular regulation of cell proliferation and apoptosis in early
embryo blastomeres and granule neuron precursors of the mouse
embryos (Narducci et al., PNAS 2002, 99:11712-7). In
contrast, depletion of AKT1/AKT3 had no apparent
effect on embryos, pinpointing the AKT2 isoform as
the actual TCL1 interactor in preimplantation mouse
embryos. Moreover, immunofluorescence experiments
showed that AKT2, but not AKT1 nor AKT3,
migrates to nucleus in concomitance with TCL1
nuclear localization. The possibility that AKT
Ser473/Thr308 phosphorylation depended on
upstream factors/kinases, including PI3K, PDK1, and
HSP90 protein was probed by treating embryos with
specific inhibitors, showing that Ser473/Thr308-phosphorylated
AKT2 is fully inherited from oogenesis and
that, following fertilization, AKT does not undergo
significant changes in the ratio between phosphorylated
and dephosphorytlated conditions. This indirectly
indicates that TCL1 is not required for AKT phosphorylation,
whereas it represents an absolute requirement
for phosphorylated AKT transfer to nucleus. In
light of the well established finding that the AKT
Ser473 residue is phosphorylated by the mTOR-Rictor
complex (Sarbassov et al., Science 2005, 307:1098-101),
we also investigated the expression of mTOR, Raptor
and Rictor during preimplantation development by
RT-PCR and, limitedly to the blastocyst stage, by
western blot. Raptor mRNA appeared to be expressed
during entire preimplantation development. In contrast,
the mTOR message first appeared at 16-32 cell
stage (suggesting a maternal origin of the protein) and
Rictor mRNA was constantly lacking from fertilization
to blastocyst, further supporting the hypothesis that
AKT phosphorylation takes place during oogenesis,
but not during preimplantation development.
THG-1pit expression was preliminarily determined
during postnatal development by Northern/Western
blot analyses of RNA/protein extracts from cerebellum
and primary cultures of cerebellar granule neurons
(CGN) at increasing days of in vitro culture
(DIV1-6). Northern blot analysis of both cerebellum
and CGN extracts showed the presence of a single 2.7
kb transcript, corresponding to the length expected