FIGURE 6.
Mutation of the UBS prevents RhoA targeting in vitro and interferes with binding of a monoubiquitylated form of RhoA. a, Smurf2 Y459A mutant monoubiquitylates, but does not polyubiquitylate RhoA. Constitutively active Smurf2 F-29/30-A with either wild type or the Y459A mutant UBS were used in an in vitro ubiquitylation assay with RhoA substrate. RhoA was immunoprecipitated (IP) from ubiquitylation reactions and analyzed by immunoblotting (top panel), whereas polyubiquitylated RhoA was analyzed by immunoblotting ubiquitylation reactions as indicated (as indicated, the top and bottom panels are an analysis of the lower molecular weight and higher molecular weight regions, respectively). RhoA and Smurf2 input were detected by immunoblotting (IP) as indicated. b, ubiquitylation of RhoA using UbNOK. RhoA was subject to an in vitro ubiquitylation reaction as in a, except that UbNOK was employed in the reaction. Ubiquitylated RhoA was detected by subjecting total reactions to immunoblotting with an anti-RhoA antibody as indicated. c, the UBS promotes Smurf2 interaction with a Ub-RhoA fusion protein. Bacterially expressed GST or GST-Smurf2 WW-HECT domain were bound to GST beads and incubated with purified RhoA or Ub-RhoA. Smurf2-bound RhoA or Ub-RhoA (top panel) or totals (bottom panel) were analyzed by immunoblotting with α-RhoA antibodies.