Figure 4.

Superoxide induces RhoGDI phosphorylation by PKCζ, leading to RhoGTPase liberation from RhoGDI. (a) After SASH1 cells were treated with calphostin C for 15 min, they were stimulated with (open bars) or without (closed bars) superoxide for 2 h. Cell motility was measured and shown in the bar graph. *, P < 0 .01, compared with the value without any treatment. Error bars indicate SEM. (b) After 15 min of pretreatment of calphostin C, SASH1 cells were treated with or without superoxide for 2 min, and RhoGTPase activity was measured by pull-down assay. (c, e, and f) SASH1 cells were treated with or without superoxide, lysed, and subjected to IP using anti-RhoGDI antibody. The samples were then electrophoresed and immunoblotted by the antibodies indicated in the figures. Lane 1, positive controls; lane 2, without superoxide treatment; lane 3, 2 min of superoxide treatment. (c) SASH1 cells; (e) SASH1 cells pretreated with 40 mM NAC for 20 min; (f) Cu-Zn SOD overexpressed SASH1 cells (clone 1). (d) Translocation assay of PKCζ in SASH1 cells treated with superoxide. 4 × 10⁷ cells were treated with superoxide for the indicated times and subjected to subcellular fractionation. Plasma membrane fractions were electrophoresed and immunoblotted by the antibody indicated in the figure. (g) Activity of PKCζ was measured by its phosphorylation state as described in Materials and methods. SASH1 cells were pretreated by calphostin C of the concentrations indicated in the figure. (top) Phosphorylated PKCζ confirmed by autoradiography; (bottom) immunoblotting of PKCζ to confirm that an equal amount of the protein was loaded. (h) Superoxide phosphorylates threonine residue of RhoGDI. SASH1 cells with or without 2 min of superoxide stimulation were lysed and immunoprecipitated with anti-RhoGDI antibody. Immunoprecipitates were electrophoresed and immunoblotted by antibodies indicated in the figure. (i) Recombinant active PKCζ phosphorylated RhoGDI-1 immunoprecipitated from SASH1 cell lysate. In vitro kinase assay was performed as described in Materials and methods. (top) [³²P]RhoGDI-1; (bottom) RhoGDI-1 immunoblotting. (j) PKCζ associated with and RhoGTPases dissociated from RhoGDI after superoxide stimulation. SASH1 cells with or without superoxide treatment were subjected to IP by anti-RhoGDI antibody, and the samples were immunoblotted by the antibodies indicated in the figure.