Pollock, K., Ranes, M., Collins, I. & Guettler, S.
(2017). Identifying and Validating Tankyrase Binders and Substrates: A Candidate Approach. Methods mol biol,
The poly(ADP-ribose)polymerase (PARP) enzyme tankyrase (TNKS/ARTD5, TNKS2/ARTD6) uses its ankyrin repeat clusters (ARCs) to recognize degenerate peptide motifs in a wide range of proteins, thereby recruiting such proteins and their complexes for scaffolding and/or poly(ADP-ribosyl)ation. Here, we provide guidance for predicting putative tankyrase-binding motifs, based on the previously delineated peptide sequence rules and existing structural information. We present a general method for the expression and purification of tankyrase ARCs from Escherichia coli and outline a fluorescence polarization assay to quantitatively assess direct ARC-TBM peptide interactions. We provide a basic protocol for evaluating binding and poly(ADP-ribosyl)ation of full-length candidate interacting proteins by full-length tankyrase in mammalian cells..
Mariotti, L., Templeton, C.M., Ranes, M., Paracuellos, P., Cronin, N., Beuron, F., Morris, E. & Guettler, S.
(2016). Tankyrase Requires SAM Domain-Dependent Polymerization to Support Wnt-β-Catenin Signaling. Molecular cell,
(2016). AXIN Shapes Tankyrase ARChitecture. Structure,
Guettler, S., LaRose, J., Petsalaki, E., Gish, G., Scotter, A., Pawson, T., Rottapel, R. & Sicheri, F.
(2011). Structural basis and sequence rules for substrate recognition by Tankyrase explain the basis for cherubism disease. Cell,
The poly(ADP-ribose)polymerases Tankyrase 1/2 (TNKS/TNKS2) catalyze the covalent linkage of ADP-ribose polymer chains onto target proteins, regulating their ubiquitylation, stability, and function. Dysregulation of substrate recognition by Tankyrases underlies the human disease cherubism. Tankyrases recruit specific motifs (often called RxxPDG "hexapeptides") in their substrates via an N-terminal region of ankyrin repeats. These ankyrin repeats form five domains termed ankyrin repeat clusters (ARCs), each predicted to bind substrate. Here we report crystal structures of a representative ARC of TNKS2 bound to targeting peptides from six substrates. Using a solution-based peptide library screen, we derive a rule-based consensus for Tankyrase substrates common to four functionally conserved ARCs. This 8-residue consensus allows us to rationalize all known Tankyrase substrates and explains the basis for cherubism-causing mutations in the Tankyrase substrate 3BP2. Structural and sequence information allows us to also predict and validate other Tankyrase targets, including Disc1, Striatin, Fat4, RAD54, BCR, and MERIT40..
Guettler, S., Mariotti, L. & Pollock, K.
Regulation of Wnt/ß-catenin signalling by tankyrase-dependent poly(ADP-ribosyl)ation and scaffolding. British journal of pharmacology,