FIGURE 4.

C terminus of RhoA interacts with AGAP1. A, peptide from RhoA C terminus binds to AGAP1. Lysates of HeLa cells expressing FLAG-AGAP1 or AGAP1-HA were mixed with biotin-tagged-RhoA C-terminal peptide (biotin-GGGLQARRGKKKSG) or the biotin-tagged CDC42 C-terminal peptide (biotin-GGGLEPPEPKKSRR). The peptides were precipitated with streptavidin-agarose. The beads were washed three times with lysis buffer, and precipitates were analyzed by SDS-PAGE followed by immunoblotting for the FLAG or HA epitope. B, effect of RhoA C terminus on GAP activity is shown. RhoA or Cdc42 C-terminal peptides were titrated into reactions containing 1.1 nm His₁₀AGAP1 and 0.5 μm myrArf·GTP. C, a lack of heat sensitivity of peptide activator is shown. The RhoA C-terminal peptide was heated for 10 min at 95 °C before being added to the GAP assay at the indicated concentration. D, the effect of peptide was observed with a nonmyristoylated Arf. RhoA or Cdc42 peptide were titrated into reactions containing 1.1 nm His₁₀AGAP1 and 0.2 μm [α³²P]GTP·[L8K]Arf1.