Abstract
Rho guanine exchange factors (RhoGEFs) control many aspects of the cellular cytoskeleton, and thereby influence cell processes such as cell migration, cell adhesion and proliferation. Misregulation of RhoGEF activity is implicated in various aspects of cancer. TGAT is a splice variant of the RhoGEF Trio, with high oncogenic potential. The last 15 amino acids of the C-terminus of TGAT are critical for its oncogenic potential, but the precise molecular origins remain elusive. We find that TGAT is partially located at the Golgi apparatus and that this localization depends on two cysteine residues in the C-terminal tail. Accordingly, TGAT lacking the last 15 amino acids (TGATΔ15) is localized to the cytoplasm. To examine potential plasma membrane association we developed a novel, highly sensitive image analysis method. The analysis revealed that a fraction of wild-type TGAT and its cysteine mutants co-localized with the plasma membrane. The functional importance of membrane localization was determined by measuring TGAT activity, using single cell RhoA GTP loading and F-actin levels as a read-out. Strikingly, mutants of TGAT devoid of one or more palmitoylation sites in the C-tail retain similar activation characteristics as wild-type TGAT, whereas the activity was clearly abrogated for TGATΔ15. Synthetic recruitment of TGATΔ15 to membranes using a rapamycin based heterodimerization system confirmed that TGAT effectively activates RhoA at the plasma membrane. Together, our results show that membrane association of TGAT is critical for its activity, but that palmitoylation is dispensable.