Abstract
Abstract
The evolutionary conserved Wnt/b-catenin signalling
pathway regulates a variety of biological processes. Abberant activation of
the pathway for instance leads to tumourigenesis. Therefore, the abundance of
the central effector molecule b-catenin is tightly
controlled by proteasomal degradation. The N-terminal phosphorylation of b-catenin
that initiates its turnover is achieved in a cytoplasmic multiprotein complex.
The scaffold of this so-called degradation complex is provided by conductin
or its homologue axin, both of which are essential for efficient b-catenin
degradation. Using a yeast-2-hybrid approach, this study aims to identify novel
conductin interacting proteins and to characterize their regulatory impact on
the Wnt signalling pathway.
Initial experiments focused on the impact of conductin on the signalling activity
of b-catenin. Conductin was identified in our group,
and the first part of this study is part of the functional characterization
by Behrens et al. (1998). A synthetic reportergene-assay was established and
provides evidence for conductins role as a negative regulator of the Wnt/b-catenin
signalling pathway. Using this assay, the functional aspects of the domain structure
of conductin was clarified.
Using the yeast-2-hybrid system, I-mf was identified as a novel binding partner
of conductin. In addition, I-mf a is capable of interacting with the GSK3b-inhibitor
FRAT-1. I-mf a modulates the competitive binding of conductin and FRAT-1 to
GSK3b. I-mf a shields the conductin-bound GSK3b
from the competitive attack of FRAT-1. Dissociation of GSK3b
fom the degradation complex by is considered a key mechanism how molecules like
FRAT-1, but also Dishevelled, activate b-catenin
signalling. Thus, the results establish a novel mechanism to regulate such a
dissociation behavior.
Diversin was identified in our group by virtue of its binding to conductin,
too. Diversin is able to potentiate FRAT-1 mediated activation of the b-catenin
signal in a conductin-dependent manner. This synergy is not achieved through
increased dissociation of GSK3b fom the dgradation
complex. Rather, it can be demonstrated that ternary complexes consisting of
conductin, diversin, and GSK3b are essential to the
synergistic activation of b-catenin. Ternary complex
formation is dependent on conductin dimerization, since dimerization-deficient
conductin mutants fail to facilitate triple complex assembly and therefore block
the synergy of diversin and FRAT-1. In addition to dimerization, the DIX domain
of conductin is important for efficient ternary complex formation, and hence,
synergy. Based on these data, diversin is thought to provide an optimal configuration
of the conductin-based degradation complex to allow for enhanced FRAT-1 action
on the conductin-bound GSK3b. In the canonical Wnt
pathway, diversin may regulate lateral inputs coming from FRAT-1 and thus adds
a new layer of regulatory complexity.
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