Differential binding of ZAK to RhoGDIβ and phosphorylation by ZAK. 293T cells were transiently transfected with the indicated GFP-tagged wild-type or mutant ZAK. After 48 h, cell lysates were harvested and 300 μg of total lysate was used in a pull-down assay with glutathione-Sepharose bead-bound GST-RhoGDIβ. (A) Wild-type ZAK (ZAK), dominant-negative (dn) ZAK, or constitutively active (E/E) ZAK. Bottom panel shows the binding affinity of RhoGDIβ to different forms of ZAK. The relative binding affinity was calculated by the amount of bound form divided by the total loading of RhoGDIβ. (B) Binding of RhoGDIβ to ZAK may maintain ZAK in a hypophosphorylated, inactive state. Unbound (U) ZAK in cell lysates consisted of both hyperphosphorylated (ppZAK) and hypophosphorylated (pZAK) forms. The hypophosphorylated form of ZAK was pulled down by GST-RhoGDIβ. (C) ZAK induces autophosphorylation and phosphorylation of GST-RhoGDIβ. Cell lysates from 293T cells transfected with pEGFPC1 empty vector (C), wild-type ZAK (ZAK), or dominant-negative ZAK (dn) were immunoprecipitated with anti-GFP. The immunopurified ZAK or ZAKdn was incubated with GST-RhoGDIβ in the presence of [γ-32P]ATP. Data shown are representative of three independent experiments.