A large body of literature documents in both mice and Drosophila the
involvement of Insulin pathway in growth regulation, probably due to its role
in glucose and lipid import, nutrient storage, and translation of RNAs
implicated in ribosome biogenesis (Vanhaesebroeck et al. 2001). Moreover
several lines of evidence implicate this pathway as a causal factor in cancer
(Sale, 2008; Zeng and Yee 2007; Hursting et al., 2007; Chan et al., 2008).
With regards to Myc, studies in cell culture have implied this family of
transcription factors as regulators of the cell cycle that are rapidly induced in
response to growth factors. Myc is a potent oncogene, rearranged and
overexpressed in a wide range of human tumors and necessary during
development. Its conditional knock-out in mice results in reduction of body
weight due to defect in cell proliferation (Trumpp et al. 2001).
Evidence from in vivo studies in Drosophila and mammals suggests a
critical function for myc in cell growth regulation (Iritani and Eisenman 1999;
Johnston et al. 1999; Kim et al. 2000; de Alboran et al. 2001; Douglas et al.
2001). This role is supported by our analysis of Myc target genes in
Drosophila, which include genes involved in RNA binding, processing,
ribosome biogenesis and nucleolar function (Orain et al 2003, Bellosta et al.,
2005, Hulf et al, 2005).
The fact that Insulin signaling and Myc have both been associated
with growth control suggests that they may interact with each other.
However, genetic evidence suggesting that Insulin signaling regulates Myc
in vivo is lacking.
In this work we were able to show, for the first time, a direct
modulation of dMyc in response to Insulin stimulation/silencing both in vitro
and in vivo. Our results suggest that dMyc up-regulation in response to
DILPs signaling occurs both at the mRNA and potein level. We believe
dMyc protein accumulation after Insulin signaling activation is conditioned to
AKT-dependent GSK3β/sgg inactivation. In fact, we were able to
demonstate that dMyc protein stabilization through phosphorylation is a
conserved feature between Drosophila and vertebrates and requires
multiple events. The final phosphorylation step, that results in a non-stable
form of dMyc protein, ready to be degraded by the proteasome, is
performed by GSK3β/sgg kinase (Sears, 2004). At the same time we
demonstrated that CKI family of protein kinase are required to prime dMyc
phosphorylation.
DILPs and TOR/Nutrient signalings are known to communicate at
several levels (Neufeld, 2003). For this reason we further investigated TOR
contribution to dMyc-dependent growth regulation. dMyc protein
accumulates in S2 cells after aminoacid stimulation, while its mRNA does
not seem to be affected upon TORC1 inhibition, suggesting that the Nutrient
pathway regulates dMyc mostly post-transcriptionally.
In support to this hypothesis, we observed a TORC1-dependent
GSK3β/sgg inactivation, further confirming a synergic effect of DILPs and
Nutrients on dMyc protein stability.
On the other hand, our data show that Rheb but not S6K, both
downstream of the TOR kinase, contributes to the dMyc-induced growth of
the eye tissue, suggesting that Rheb controls growth independently of S6K..
Moreover, Rheb seems to be able to regulate organ size during
development inducing cell death, a mechanism no longer occurring in
absence of dmyc. These observations suggest that Rheb might control
growth through a new pathway independent of TOR/S6K but still dependent
on dMyc.
In order to dissect the mechanism of dMyc regulation in response to
these events, we analyzed the relative contribution of Rheb, TOR and S6K
to dMyc expression, biochemically in S2 cells and in vivo in morphogenetic
clones and we further confirmed an interplay between Rheb and Myc that
seems to be indipendent from TOR.
In this work we clarified the mechanisms that stabilize dMyc protein in
vitro and in vivo and we observed for the first time dMyc responsiveness to
DILPs and TOR. At the same time, we discovered a new branch of the
Nutrient pathway that appears to drive growth through dMyc but
indipendently from TOR.
We believe our work shed light on the mechanisms cells use to grow
or restrain growth in presence/absence of growth promoting cues and for
this reason it contributes to understand the physiology of growth control.
| Identifer | oai:union.ndltd.org:unibo.it/oai:amsdottorato.cib.unibo.it:1567 |
| Date | 21 April 2009 |
| Creators | Parisi, Federica <1981> |
| Contributors | Pession, Annalisa |
| Publisher | Alma Mater Studiorum - Università di Bologna |
| Source Sets | Università di Bologna |
| Language | English |
| Detected Language | English |
| Type | Doctoral Thesis, PeerReviewed |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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