Characterization of Alien isoforms in vertebrates : Caracterización de isoformas de Alien en vertebrados
Charakterisierung von Alien Isoformen in Vertebraten
Dokument 1.pdf (4.070 KB)
Genetisches Institut und Instituto des Investigaciones Biomedicas, Consejo Superior de Investigaciones Cientificas (CSIC), Universidad Autonoma de Madrid
Tag der mündlichen Prüfung:
Kurzfassung auf Englisch:
Alien protein isoforms have been described to be involved in a number of biological processes. Alienalpha is a corepressor of the thyroid
hormone receptor mediating transcriptional repression in a ligand-sensitive manner. Furthermore, Alienalpha is a corepressor for the
orphan receptor DAX1 and the vitamin-D3 receptor. Alienbetta/CSN2 is part of the COP9-signalosome complex that acts in protein
phosphorylation, protein degradation and cell cycle regulation.
The major goal of this work was to characterize the Alienalpha and Alienbetta isoforms. Little was known about their expression pattern and
the regulation of their expression had not been addressed.
It was determined in this work that the expression pattern of Alien is rather ubiquitous in rat tissues. Interestingly, a putative novel Alien
protein isoform and an additional alien messenger specific for adrenal gland were identified. Furthermore, it was shown in vivo and in vitro,
by in situ hybridization, Northern and Western blotting that Alien expression is regulated by thyroid hormone in the rat brain and
brain-derived cell lines. Subsequently, hints for a second T3-independent mechanism of regulation of Alien expression depending on cell
confluence or quiescence were discovered.
The comparison of Alien isoforms in functional aspects identified Rb and E2F as novel Alien-interacting proteins with similar binding
characteristics in vitro and in yeast but functional differences in vivo. Alienbetta interfered with Rb-mediated superactivation of Sp1-driven
transcription, whereas Aliena exerted strong repression on E2F transactivation. Common traits for both Alienalpha and Alienbetta are their
silencing potential, interaction with TR and activation of AP1-driven transcription.
Phosphorylation studies raised the possibility of regulation by non-hormonal signaling since Alienalpha and Alienbetta are phosphorylated
in vivo. In gel kinase assays suggested the existence of two different Alien-phosphorylating kinases. Further experiments identified MLK2
and the cell cycle kinase p34cdc2 as such kinases, suggesting a possible function of Alien in cell cycle regulation.
Taken together, the expression of Alien is regulated by thyroid hormone, and by cell density; the isoforms can be phosphorylated and can
act either as transcriptional repressors or as activators. Additional data indicate a role of Alien isoforms in cell cycle regulation through
p34cdc2 phosphorylation and isoform-specific interference with Rb and E2F.