Fig. 4: RNF166 regulates Motins through TNKS mediated PARsylation.

A Schematic diagrams of WT and different truncated constructs of the AMOT. The key domains (rectangles) and motifs (vertical lines) are labeled above. B The N-terminal region (aa 1-411) of AMOT was required for AMOT binding to RNF166. The relevant plasmids were transfected into 293 T cells for 48 h. Interactions were detected by streptavidin pulldown, and different truncations of AMOT are labeled with asterisks. C AMOT aa 501–700 was essential for interaction with RNF166. D Effect of TNKS inhibitors on disrupting RNF166 binding to AMOT. After transiently transfecting 293 T cells with the indicated plasmids for 30 h, cells were then treated with DMSO or XAV939 (2 μM) for 18 h. These samples were further analyzed by streptavidin pulldown and the pulled proteins were immunoblotted. (E), XAV939 treatment affected the interaction between RNF166 and AMOTL2 in xenograft mode. F AMOT aa 1-542 mediated the interaction with RNF166. G Detection of PARsylation on AMOT WT and mutations at possible PARsylated sites. The experiment was performed as the ubiquitination assay but required the addition of PDD00017273 (5 μM) to the cell lysates. H The interaction between RNF166 and AMOT WT or PARsylated site mutations. D/E4A mutations abolished AMOT PARsylation (I) and prevented their binding to RNF166 (J). Effects of RNF166 and XAV939 on the stabilization of Motins in 293 T and FHC cells. 293 T cells transiently expressed Flag-AMOT and Flag-RNF166 (K), whereas FHC cells stably overexpressed Flag and SBP-tagged RNF166 (L). M Assay of ubiquitination of WT and D/E4A mutations of AMOT with DMSO or XAV939. Cells were transfected with the indicated plasmids, treated with DMSO or XAV939 (2 μM, 18 h) and prepared for the ubiquitination assay. N Effects of RNF166 overexpression, bortezomib, and XAV939 treatment on the stabilization of WT and D/E4A mutant AMOT. O Effect of RNF166 overexpression and XAV939 treatment on protein stabilization of WT and K464R AMOT. Abbreviations: A, alanine; aa, amino acids; ABI angiostatin binding domain, CC coiled coil; D/E4A, simultaneously mutated 4 sites (D506, E513, E516 and D528) to alanine (A) of AMOT; DMSO, dimethyl sulfoxide; PDD00017273, the inhibitor against poly (ADP ribose) glycohydrolase; WT, wild type.