Fig. 1.

Identification of ARHGEF11 as a ZO-1–binding protein. (A) Domain structure of ZO-1 and the fragments used to construct bait plasmids. Plasmids encoding the underlined fragments, aa 181 to 503, 423 to 862, and 1520 to 1745, were used to screen a mouse embryo cDNA prey library. The positive clone, prey202, contained a fragment encoding the C-terminal region of ARHGEF11, which possesses PDZ, RGS, DH, and PH domains. (B) In vitro binding assay between ARHGEF11 and ZO-1. The GST protein (control) or the GST fusion proteins encoding ZO-1 fragments aa 181 to 503, 423 to 862, or 1520 to 1745 (asterisks) bound to glutathione-Sepharose beads, was incubated with or without the lysate of Escherichia coli-expressing prey202 fused to the MBP (arrowhead). The eluates from beads incubated with a glutathione-containing buffer were separated by SDS/PAGE and analyzed by Coomassie brilliant blue staining. Two asterisks mark the degradation products of the native proteins. (C and D) Lysates of HeLa cells transfected with Myc-ARHGEF11 or Myc-ARHGEF1 were incubated with GST or GST-ZO-1(1520–1745) coupled with glutathione-Sepharose beads (C); lysates from Myc-ZO-1 or Myc-ZO-2 transfectants were incubated with GST or GST-ARG11 (C-term), which is the C-terminal region of ARHGEF11 corresponding to prey202 (D). Coprecipitated proteins were detected with anti-Myc antibodies. (E) Endogenous ARHGEF11 was immunoprecipitated from MCF7 cells, and the immunoprecipitants were analyzed by immunoblotting for ARHGEF11 and ZO-1. Mouse IgG was used as a control.