Figure 5. The Rho-GTPase CDC42 is a substrate of UBA6-dependent ubiquitination and is involved in EMT induced by UBA6 deficiency and growth factor deprivation.

(A) UBA6 depletion downregulates polyubiquitinated forms of CDC42. HEK293 cells were transfected with plasmids for expression of CDC42 and poly-histidine-HA-tagged ubiquitin (Ub) and treated with 10 μM MG132 for 90 min. Cells were then harvested and analyzed by immunoprecipitation (IP) with anti-CDC42 antibody, followed by immunoblotting (IB) for the HA tag. IgG, control immunoprecipitation with normal rabbit IgG. The right panels indicate immunoblotting with total cell lysates for the indicated proteins. (B) Uba6 targets CDC42 to degradation. MCF10A control cells and shUBA6^Top cells were treated with 100 μg/mL cycloheximide for the indicated hours, and then examined by immunoblotting for CDC42 to determine its stability. (C) Formation of island-like cell clusters on monolayer of MCF10A-shUBA6^Mix cells after 7-day incubation in minimal medium supplemented with the CDC42 inhibitor ML141 at the indicated doses. (D) Box-and-whisker plots showing significant differences in sizes of island-like clusters after ML141 treatment (n1 = 107, n2 = 63, n3=48, n4= 61, n5=26). ^*p<0.05; ^**p<0.01; N.S., not significant. (E) ML141-mediatd suppression of the EMT process in shUBA6^Mix cells induced by incubation in minimal medium. Cells were harvested after 11-day incubation with ML141 at indicated doses in minimal medium, and analyzed by immunoblotting.