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

Disordered Motifs and Domains in Cell Control

Tuesday Speaker Abstracts

The Role of Multivalent Interactions of Tumor Suppressor SPOP with Gli3 in Regulating Ubiquitination Melissa Marzah, Wendy Pierce, Jihun Lee, Amanda Nourse, Suresh Marada, Stacey Ogden, Tanja Mittag . 1 St. Jude Children's Research Hospital, Memphis, TN, USA Multivalent protein interactions can give rise to avidity effects, ultrasensitivity, phase separation and spatial organization. The tumor suppressor SPOP, the substrate adaptor of a ubiquitin ligase, self-associates into large oligomers. We predict that its predominantly intrinsically disordered substrate Gli3 contains many weak SPOP binding motifs. Multivalency of SPOP and Gli3 for each other, i.e. the ability of SPOP oligomers to bind many SPOP binding motifs in Gli3 and of Gli3 molecules to interact with many SPOP monomeric units, suggests a highly concentration- dependent interaction. We used a peptide microarray, NMR spectroscopy, fluorescence methods and analytical ultracentrifugation (AUC) to identify SPOP binding motifs in Gli3. Importantly, even very weak motifs contributed substantially to ubiquitination demonstrating their functional relevance. Using AUC, size exclusion chromatography, and static light scattering, we demonstrated that two distinct dimerization domains in SPOP mediate the formation of concentration-dependent higher-order SPOP homo-oligomers. Our thermodynamic characterization supports an isodesmic self-association model, indicating that the size of the oligomers is limited only by protein availability. Importantly, our full description of SPOP’s valency as a function of concentration now permits the quantitative characterization of its interactions with multivalent binding partners. Does the multivalent Gli3/SPOP interaction give rise to avidity effects in small complexes or to large cross-linked complexes? We show that Gli3 and SPOP co-localize into nuclear “bodies” in cells suggesting that these “bodies” may be mediated by (1) specific oligomerization through defined interfaces in SPOP; and (2) the weak multivalent interactions between Gli3 and SPOP. It has been suggested that concentration-dependent assembly/disassembly of large, multivalent complexes provides a mechanism for fine-tuning signaling cascades. Multivalent interactions of SPOP with its substrates may be an avenue to regulate levels of proteins critical in development and proliferation in a concentration-dependent manner.

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