FIGURE 6:

Y940 of the DH core of LARG is critical for binding to RhoA, and mutation leads to apoptosis. (A) HeLa cells were transfected with indicated plasmid. At 48 h posttransfection, cell lysates were harvested and subject to GST-G17A-RhoA pull down. Relative binding to RhoA was determined by immunoblot for LARG and pull down and input fractions compared for each respective LARG plasmid. I, 5% total input; P, 50% total pull down. Top bands, exogenous mCherry-tagged LARG proteins; bottom bands, endogenous LARG. (B) HeLa cells were transfected with indicated plasmid for 48 h, cell lysates harvested, and relative apoptosis determined by immunoblot for PARP cleavage. (C) Crystal structure of DH/PH domain of LARG in complex with RhoA from PDB database (1X86; Kristelly et al., 2003) depicting residues (pink) of the DH domain (gray) targeted here in mutation studies and relative locations to RhoA (brown). (D) Multiple RhoGEFs exhibit temporally distinct functions for the proper completion of mitosis. See the text for discussion.