Regulatory mechanisms for the Ras and Rho family GTP ase network

探讨鸟嘌呤核苷酸交换因子（GEFs）对Ras和Rho家族GTP结合蛋白的调控机制。首先，分析Dbl在细胞外刺激后Rho家族的调节中的作用。Dbl被Cdc 42的靶标ACK-1酪氨酸磷酸化，从而增强其GEF活性。此外，Dbl与衔接子Grb 2和表皮生长因子（EGF）受体形成复合物。我们发现，在EGF刺激，ACK-1和Dbl酪氨酸磷酸化和激活的Cdc 42和Grb 2依赖的方式，导致肌动蛋白细胞骨架重排通过RhoA，Rac 1，和Cdc 42。其次，在一组Dbl和Dbs剪接变体中，分析N-末端的Sec 14样结构域的功能。当在HeLa细胞中异位表达时，Dbl和Dbs剪接变体根据Sec 14样结构域诱导不同的形态学改变。这种差异可能是由于不同的亚细胞定位的活性Rho家族成员所确定的GEF。第三，我们研究了PLCε的CDC 25同源结构域的作用。PLCε是Ras和Rap 1的下游靶点，含有CDC 25同源结构域，其负责高尔基体中Rap 1下游的持续激活。我们证明，血小板衍生生长因子诱导的PLCε的瞬时激活是由Ras介导的，而持续激活依赖于Rap 1。

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.