RhoH Downregulates Rac Through RhoGAP Family Protein

Abstract 4792

Rho GTPases have been widely studied because they are the critical regulators of cellular signaling mediated by G-protein-coupled receptors, tyrosine kinase receptors and integrins. Among more than 20 members of the Rho GTPases, Rac1, Cdc42 and RhoA have been extensively characterized. Rac plays essential roles in the control of actin cytoskeleton organization, migration, phagocytosis, metastasis, transformation, gene expression and cell-cycle progression. Moreover, it has been found that Rac plays as a key molecular switch of hematopoietic stem/progenitor cells engraftment and marrow retention. RhoH, a member of the Rho family, is specifically expressed in the hematopoietic cells, including bone marrow progenitor, differentiated myeloid and lymphoid cells. RhoH antagonizes Rac, however, the molecules that inactivate Rac downstream of RhoH have not been identified. Here we perform GTP-RhoH affinity column chromatography by using platelet lysates. Using triple quadrupole liquid chromatography tandem mass spectrometer, 83 different proteins associated with active RhoH were identified. They include Rho GTPase activating proteins (RhoGAP), serine/threonine kinases, phosphatidylinositol kinases, ERM family proteins and others. Among them, we focused on RhoGAP family. RhoGAP binds to RhoH through its GAP domain. Expression of both full length and GAP domain of RhoGAP effectively deactivates Rac, whereas GAP domain deleted RhoGAP did not. These results suggest that RhoH negatively regulates Rac through RhoGAP.