Disordered Motifs and Domains in Cell Control - October 11-15, 2014

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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