Fig. 3: RNF166 mediates the polyubiquitination and proteasomal degradation of Motins.

A RNF166 mediates K48-linked polyubiquitination of AMOT. 293 T cells were cotransfected with Flag and SBP-tagged AMOT, a vector encoding HA-Ub-WT or its mutants (HA-Ub-K48 or HA-Ub-K63), and Flag-RNF166 or empty vector for 48 h. Cell lysates were incubated with streptavidin agarose for 3 h and immunoblotted with the indicated antibodies. B−E RNF166 regulates the stability of Motins. Motins protein levels were detected by immunoblots after treatment with CHX in 293 T cells transfected with Flag-RNF166 (B, C), FHC cells stably overexpressing Flag and SBP-tagged RNF166 (D), and HCT116 cells stably transfected with RNF166 shRNA (E). F−I RNF166 mediates proteasomal degradation of Motins. The effect of bortezomib treatment (0.1 μM, 12 h) on endogenous Motins were detected in 293 T cells transfected with Flag-RNF166 (F), FHC cells stably overexpressing Flag and SBP-tagged RNF166 (G), and CRC cells stably transfected with shRNF166 (H, I). J Possible ubiquitinated residues in Motins (red arrows) by combining ubiquitination sites from the PhosphoSitePlus database with conserved lysine residues. The conserved amino acids were compared with ClustalW. K Effect of RNF166 on the expression level of WT and mutant AMOT. 293 T cells were transfected with the indicated plasmids at a 1:1 ratio for 48 h and cell samples were prepared for further immunoblotting analysis. L RNF166 did not increase the ubiquitination of AMOT K464R or AMOTL2 K405R compared to wild-type Motins. 293 T cells were transfected with the corresponding plasmids and then treated as in (A). AMOT K464R was resistant to RNF166-induced degradation with or without CHX (M) and bortezomib (N) treatment. Abbreviations: CHX cycloheximide, K lysine, R arginine, SBP streptavidin-binding peptide, Ub ubiquitin, WT wild-type.