**2. Categorization**

Rho GTPases belong to the Ras superfamily of low molecular mass (~21 kDa) proteins that are widely expressed in mammalian cells (DerMardirossian and Bokoch 2001). In mammals, the Rho family of GTPases contains 22 members which can be classified into six groups: Rho (RhoA, RhoB, RhoC), Rac (Rac1, Rac2, Rac3, RhoG), Cdc42 (Cdc42, TC10, TCL, Chp, Wrch-1), Rnd (Rnd1, Rnd2, Rnd3/RhoE), RhoBTB (RhoBTB1, RhoBTB2) and Miro (Miro-1, Miro-2) (Wennerberg and Der 2004). RhoD, Rif and RhoH/TTF have not been grouped yet. RhoA, Rac1 and Cdc42 are the best-characterized family members of Rho family GTPases. Each controls the formation of a distinct cytoskeletal element in mammalian cells. Activation of Rac induces Actin polymerization to form lamellipodia (Ridley, Paterson et al. 1992), whereas activation of CDC42 stimulates the polymerization of actin to filopodia or microspikes (Nobes and Hall 1995). In contrast, Rho regulates bundling of actin filaments into stress fibers and the formation of focal adhesion complexes (Keely, Westwick et al. 1997).

Rho GTPases and Breast Cancer 561

formation and tumorigesis when expressed in NIH-3T3 cells (Eva and Aaronson 1985). It has 29% sequence identity with the *Saccharomyces cerevisiae* cell division protein Cdc24, which is found upstream of the yeast small GTP-binding protein Cdc42 in the bud assembly pathway (Ron, Zannini et al. 1991). This was the first clue that DB1 functions as a GEF. Biochemical study has confirmed that Db1 is able to release GDP from the human homolog of Cdc42 *in vitro*. Further study suggested that the DH domain is essential and sufficient for the catalytic activity and that this domain was also necessary to induce oncogenicity (Zheng,

After the discovery of Dbl, a number of mammalian proteins containing DH and PH domain have been studied (Cerione and Zheng 1996). Many of these have been identified as oncogenes in transfection assays. Tiam, however, was first identified as an invasioninducing gene using proviral tagging and *in vitro* selection for invasiveness (Habets, Scholtes et al. 1994). Two other members of the DH/PH-containing protein family, Fgd1 and Vav, have been shown to be essential for normal embryonic development (Pasteris, Cadle et al. 1994; Tarakhovsky, Turner et al. 1995). Moreover, some members of the DH protein family (such as Dbl) have been shown to exhibit exchange activity *in vitro* for a broad range of Rho-like GTPases, whereas others appear to be more specific. For example, Lbc and oncoproteins Lfc and Lsc, are specific for Rho, whereas Fgd1 is specific for Cdc42 (Glaven, Whitehead et al. 1996). Although Vav was originally identified as an activator of Ras (Gulbins, Coggeshall et al. 1993), it has been demonstrated more recently to function as a GEF for members of the Rho family (Crespo, Schuebel et al. 1997; Han, Das et al. 1997).

The first GAP protein specific for the Rho family GTPases was purified from cell extracts using recombinant Rho. This protein, designated p50Rho-GAP, was shown to have GAP activity toward Rho, Cdc42 and Rac *in vitro* (Hall 1990; Lancaster, Taylor-Harris et al. 1994). Since then, a growing number of proteins that present GAP activity for Rho GTPases have been identified in mammalian cells, all of which share a related GAP domain that spans 140 amino acids without significant resemblance to Ras GAP. In addition to accelerating the hydrolysis of GTP, Rho GAPs also mediate other downstream functions of Rho proteins in mammalian systems. For example, it has been reported that the p190GAP plays a role in

The ubiquitously expressed protein Rho GDI was the first GDI identified for the members of the Rho family. It was isolated as a cytosolic protein that preferentially associated with the GDP-bound form of RhoA and RhoB and thereby inhibited the dissociation of GDP (Fukumoto, Kaibuchi et al. 1990; Ueda, Kikuchi et al. 1990). Rho GDI was found to be active on Cdc42 and Rac as well (Abo, Pick et al. 1991; Leonard, Hart et al. 1992). Further studies demonstrated that Rho GDI also associated weakly with the GTP-bound form of Rho, Rac, and Cdc42 (Hart, Maru et al. 1992; Chuang, Xu et al. 1993), leading to an inhibition of the intrinsic and GAP-stimulated GTPase activity of the Rho GTPases. Therefore, Rho GDI appears to be a molecule capable of blocking both the GDP/GTP exchange step and the GTP hydrolytic step. It was also reported that the Rho GDIs play a crucial role in the translocation of the Rho GTPases between membranes and the cytoplasm. In resting cells, the Rho proteins are found in the cytosol as a complex with Rho GDIs, which inhibit their

Zangrilli et al. 1996).

**3.1.2 GAPs** 

**3.1.3 GDIs** 

cytoskeletal rearrangement (Chang, Gill et al. 1995).
