The Rho GTPase Rac1 controls cell adhesion and motility. The effector loop of Rac1 mediates interactions with downstream effectors, whereas its C-terminus binds the exchange factor b-Pix, which mediates Rac1 targeting and activation. Here, we report that Rac1, through its C-terminus, also binds the nuclear oncogene SET/I2PP2A, an inhibitor of the serine/threonine phosphatase PP2A. We found that SET translocates to the plasma membrane in cells that express active Rac1 as well as in migrating cells. Membrane targeting of SET stimulates cell migration in a Rac1-dependent manner. Conversely, reduction of SET expression inhibits Rac1-induced migration, indicating that efficient Rac1 signalling requires membrane recruitment of SET. The recruitment of the SET oncogene to the plasma membrane represents a new feature of Rac1 signalling. Our results suggest a model in which Rac1-stimulated cell motility requires both effector loop-based downstream signalling and recruitment of a signalling amplifier, that is, SET, through the hypervariable C-terminus.

Rho-like GTPases control a wide range of cellular functions such as integrin-and cadherin-mediated adhesion, cell motility, and gene expression. The hypervariable C-terminal domain of these GTPases has been implicated in membrane association and effector binding. We found that cell-permeable peptides, encoding the C termini of Rac1, Rac2, RhoA, and Cdc42, interfere with GTPase signaling in a specific fashion in a variety of cellular models. Pull-down assays showed that the C terminus of Rac1 does not associate to either RhoGDI or to Pak. In contrast, the C terminus of Rac1 (but not Rac2 or Cdc42) binds to phosphatidylinositol 4,5-phosphate kinase (PIP5K) via amino acids 185-187 (RKR). Moreover, Rac1 associates to the adapter protein Crk via the N-terminal Src homology 3 (SH3) domain of Crk and the proline-rich stretch in the Rac1 C terminus. These differential interactions mediate Rac1 localization, as well as Rac1 signaling, toward membrane ruffling, cell-cell adhesion, and migration. These data show that the Cterminal, hypervariable domain of Rac1 encodes two distinct binding motifs for signaling proteins and regulates intracellular targeting and differential signaling in a unique and non-redundant fashion.Rho-like GTPases drive temporally and spatially coordinated actin polymerization to control cell motility, cadherin-based cell-cell adhesion, and cytokinesis (1-4). To relay their signals, Rho-like GTPases interact with a wide range of effector proteins. These interactions are generally thought to occur at the plasma membrane to which GTPases are translocated prior to activation by guanine nucleotide exchange factors. This membrane association is dependent on lipid modifications of specific cysteine residues in the extreme C terminus (i.e. prenylation), as well as on the C-terminal polybasic region (5).Rho-like GTPases share a high level of homology, despite the fact that their effects on cellular morphology or function can be very distinct. This has been attributed to sequence diversity in the effector domain (residues 26 -45) and the so-called insert region (amino acids 124 -135). However, the most divergent region between otherwise very homologous GTPases (e.g. Rac 1066 CX, and Rac2) is the C-terminal polybasic region. This domain was shown to be required for activation of the neutrophil NADPH oxidase (6) and was claimed for Rac1 to drive activation of PAK 1 by some (7) but not by others (6). Most of the studies that have addressed the role of this domain relied on deletion or mutation strategies, testing the role of this region in the context of, for instance, a constitutively active variant of the GTPase.Tao et al. (5) showed recently that deleting the C-terminal polybasic region affects the efficiency of prenylation of Rac2. Moreover, del Pozo et al. (8) have shown that mutation of the crucial cysteine residue to prevent Rac prenylation blocked activation of PAK. This means that mutating or deleting the C-terminal domain may affect downstream signaling in an indirect manner, complicating the interpret...