Regulatory mechanisms for the Ras and Rho family GTP ase network

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Regulatory mechanisms of Ras and Rho family GTP-binding proteins by guanine nucleotide exchange factors (GEFs) were explored. First, the role of Dbl in the regulation of the Rho family upon extracellular stimulation was analyzed. Dbl is tyrosine-phosphorylated by ACK-1, a target of Cdc42, and thereby its GEF activity is enhanced. In addition, Dbl forms a complex with the adaptor Grb2 and the epidermal growth factor (EGF) receptor. We showed that, upon EGF stimulation, ACK-1 and Dbl were tyrosine-phosphorylated and activated in a Cdc42 and Grb2-dependent manner, leading to actin cytoskeletal rearrangements through RhoA, Rac1, and Cdc42. Second, the function of the Sec14-like domain at the N-terminus in a set of Dbl and Dbs splice variants was analyzed. When ectopically expressed in HeLa cells, Dbl and Dbs splice variants induced different morphological alterations depending on the Sec14-like domain. The difference may be due to different subcellular localization of active Rho family members determined by the GEFs. Third, we investigated the role the CDC25 homology domain of PLCε. PLCε is a downstream target of Ras and Rap1, containing a CDC25 homology domain, which is responsible for sustained activation downstream of Rap1 in the Golgi apparatus. We demonstrated that platelet-derived growth factor-induced transient activation of PLCε is mediated by Ras, whereas sustained activation is dependent on Rap1.