UC San Diego

Tankyrase (TNKS) is a poly(ADP-ribosyl) polymerase (PARP) with 20 ankyrin repeats (ANK) near the N-terminus that interact with a variety of unrelated proteins found in the nucleus and cytoplasm. Using the ANK domain, TNKS recruits substrate proteins to the C-terminal catalytic PARP domain for poly-ADP-ribosylation (PARsylation). The tankyrase ANK domain interacts specifically with proteins bearing the sequence motif RXXPDG, including Axin, TRF1 (telomere- repeat binding factor-1), and IRAP (insulin-responsive aminopeptidase). Sequence analyses suggest that the ANK domain comprises five subdomains called ARCs (ANK repeat clusters) that have evolved through exon duplication. Consequently, the ANK domain likely contains five separate binding sites for RXXPDG-containing proteins. The binding specificity of each ARC toward various RXXPDG-containing proteins remains to be systematically investigated. A better understanding of ARC binding specificity will shed light on whether adjacent ARCs may be used by TNKS to juxtapose multiple partners. It will also define subsets of TNKS partners that compete for binding to the same ARCs. Here using the bacterial vectors that I constructed, I have overexpressed and purified each of the five ARCs of TNKS. I also applied affinity precipitation and immunoblot analyses to compare a set of 18 RXXPDG-containing sequences for their affinity toward the five ARCs. I found that all 18 RXXPDG proteins can bind to at least 2 ARCs, typically ARC-IV and -V, while some versions of the RXXPDG motif can bind to four ARCs. These findings suggest possible functions for the pentavalent nature of the ANK domain of TNKS