Fig. 2.

USP34 confers ubiquitin protease activity to the axin protein complex. (A) Cleavage of K48-linked ubiquitin by recombinant USP2 and USP34 core domains and by purified AXIN1 protein complexes but not by AXIN1 protein complexes isolated from cells where USP34 expression was knocked down using shRNA. Cleavage efficiency is monitored with the appearance of monoubiquitin from the polyubiquitin chains. (B) Quantification of ubiquitin protease activity using the ubiquitin-PLA2 assay. Purified AXIN1 complexes from SBP-HA-CBP-AXIN1 cells but not from cells expressing a USP34 shRNA exhibited USP activity. A similar amount of the unrelated RADIL protein complex showed no activity. Recombinant USP2 and USP34 were used as positive controls in this assay. (C) Western blot verification of endogenous USP34 knockdown in HEK293T SBP-HA-CBP-AXIN1 cells stably expressing USP34 shRNA. (D) Cleavage specificity of the USP34 core domains. UB-, SUMO3-, ISG15-, and NEDD8-PLA₂ assays were used to demonstrate that the USP34 core domain preferentially cleaves ubiquitin. (E) Ubiquitinated axin is sensitive to USP activity. Cells stably expressing SBP-HA-CBP-AXIN1 were transfected with FLAG-ubiquitin. Input lysates prepared for streptavidin affinity purification were left untreated (lane 1) or treated with the nonspecific cysteine protease inhibitor NEM (lane 2). Ubiquitin-linked axin conjugates were resolved by SDS-PAGE and detected by using anti-FLAG antibodies (top panel). Equivalent pulldown of axin was monitored using anti-HA antibodies (bottom panel). The inhibition of USP activity robustly increased the amount of ubiquitinated axin (compare lane 2 to lane 1). (F) The USP34 core domain deubiquitinates axin in vitro. FLAG-ubiquitin-axin conjugates, wild-type (WT), and catalytically inactive (C1903S) core domains of USP34 were separately purified from transfected cells using affinity purification. FLAG-UB-axin proteins were then incubated alone (lane 1) or with WT (lane 2) or catalytically inactive USP34 core domain (lane 3) proteins for 1 h. The reaction was stopped by addition of 2× sample buffer, and samples were run on an 8% SDS-PAGE. UB-axin conjugates were detected by using FLAG antibodies and AXIN1 and the core domains of USP34 with HA antibodies.