Disordered Motifs and Domains in Cell Control
Poster Session II
47-POS
Board 23
The Human hCdc4/Fbw7 has a Multisite Phosphorylation-dependent Dynamic Interaction
with its Binding Partner c-Jun Facilitating Tight Cell-cycle Control
Veronika Csizmok
1
, Hong Lin
1
, Ranjith Muhandiram
2
, Julie D. Forman-Kay
1,3
.
1
The Hospital for Sick Children, Toronto, ON, Canada,
2
University of Toronto, Toronto, ON,
Canada,
3
University of Toronto, Toronto, ON, Canada.
Cancer-associated proteins, including the oncoprotein c-Jun, are highly enriched in disordered
protein regions that mediate complex regulation of cell cycle and other signalling pathways. The
complexity of regulation is often facilitated by dynamic interactions of disordered proteins, not
leading to a static ordering upon binding. The transcription factor c-Jun is targeted for
degradation by the human orthologue of Cdc4 (hCdc4). We hypothesize that the hCdc4 has a
multisite phosphorylation-dependent dynamic interaction with its binding target c-Jun as it
contains many possible weak CPD (Cdc4 phosphodegron) sites. We assigned the backbone
resonances in non-phosphorylated and phosphorylated states of two overlapping c-Jun constructs
containing 4 (Jun123) and 6 (Jun276) CPD sites and performed structural and binding studies.
Our data show that both Jun123 and Jun276 are disordered, and interestingly Jun276 has 10 fold
greater binding affinity to the isolated WD40 domain of Cdc4 than Jun123. The NMR analysis
shows broadening on many phosphorylated residues in both Jun123 and Jun276, without
significant ordering of Jun upon binding. Together, our data demonstrate that a multisite,
dynamic interaction occurs between the Cdc4 and c-Jun. To determine how multiple CPD sites
affect Jun stability in cells, we transfected HeLA cell with WT and different CPD site mutants
Jun and followed the degradation of Jun. The preliminary data show that degradation of
individual N-terminal mutants is similar to WT, degradation of the protein mutated on all four N-
terminal sites is slower, and degradation of C-terminal mutants and the all sites mutant is even
slower, consistent with our hypothesis of the importance of multiple CPD sites. These results
provide a detailed picture of interactions of the cancer-associated hCdc4 and facilitate
understanding of the oncogenic process mediated by Jun.
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