The armadillo protein SmgGDS promotes guanine nucleotide exchange by small GTPases containing a C-terminal polybasic region (PBR), such as Rac1 and RhoA. Because the PBR resembles a nuclear localization signal (NLS) sequence, we investigated the nuclear transport of SmgGDS with Rac1 or RhoA. We show that the Rac1 PBR has significant NLS activity when it is fused to green fluorescent protein (GFP) or in the context of full-length Rac1. In contrast, the RhoA PBR has very poor NLS activity when it is fused to GFP or in the context of full-length RhoA. The nuclear accumulation of both Rac1 and SmgGDS is enhanced by Rac1 activation and diminished by mutation of the Rac1 PBR. Conversely, SmgGDS nuclear accumulation is diminished by interactions with RhoA. An SmgGDS nuclear export signal sequence that we identified promotes SmgGDS nuclear export. These results suggest that SmgGDS⅐ Rac1 complexes accumulate in the nucleus because the Rac1 PBR has NLS activity and because Rac1 supplies the appropriate GTP-dependent signal. In contrast, SmgGDS⅐RhoA complexes accumulate in the cytoplasm because the RhoA PBR does not have NLS activity. This model may be applicable to other armadillo proteins in addition to SmgGDS, because we demonstrate that activated Rac1 and RhoA also provide stimulatory and inhibitory signals, respectively, for the nuclear accumulation of p120 catenin. These results indicate that small GTPases with a PBR can regulate the nuclear transport of armadillo proteins.Armadillo (ARM) 1 family proteins that contain multiple copies of the ϳ42-amino acid (aa) ARM motif include SmgGDS, p120 catenin (p120ctn), ␤-catenin, plakoglobin, APC, karyopherin ␣ (also known as importin ␣), and several other proteins (reviewed in Refs. 1-3). Nucleocytoplasmic shuttling by many ARM proteins allows them to regulate events in different cellular compartments, including gene transcription and cell adhesion (reviewed in Refs. 2-7). ARM proteins enter the nucleus by different mechanisms (reviewed in Refs. 2-7). Karyopherin ␣ enters the nucleus when it associates with proteins containing a nuclear localization signal (NLS) sequence consisting of a series of adjacent lysines or arginines (reviewed in Ref. 3). The NLS sequence is believed to anchor within the long surface groove formed by the multiple ARM repeats of karyopherin ␣, promoting the nuclear import of both the NLS-containing protein and karyopherin ␣ (3, 5). APC possesses two NLS sequences and may enter the nucleus by associating with karyopherin ␣ or related proteins (4, 7). The mechanisms by which other ARM proteins enter the nucleus are less clear, because some of these proteins neither possess classic NLS sequences, nor have they been reported to associate with NLS-containing proteins.Several ARM proteins interact with the Rho family of small GTPases (8 -15) or with guanine nucleotide exchange factors (GEFs) for these GTPases (16,17). SmgGDS promotes guanine nucleotide exchange by small GTPases containing a C-terminal polybasic region (PBR), which is a series of adjacent l...

We observed evolutionary conservation of canonical nuclear localization signal sequences (K(K/R)X(K/R)) in the C-terminal polybasic regions (PBRs) of some Rac and Rho isoforms. Canonical D-box sequences (RXXL), which target proteins for proteasome-mediated degradation, are also evolutionarily conserved near the PBRs of these small GTPases. We show that the Rac1 PBR (PVKKRKRK) promotes Rac1 nuclear accumulation, whereas the RhoA PBR (RRGKKKSG) keeps RhoA in the cytoplasm. A mutant Rac1 protein named Rac1 (pbrRhoA), in which the RhoA PBR replaces the Rac1 PBR, has greater cytoplasmic localization, enhanced resistance to proteasome-mediated degradation, and higher protein levels than Rac1. Mutating the D-box by substituting alanines at amino acids 174 and 177 significantly increases the protein levels of Rac1 but not Rac1(pbrRhoA). These results suggest that Rac1 (pbrRhoA) is more resistant than Rac1 to proteasomemediated degradative pathways involving the D-box. The cytoplasmic localization of Rac1(pbrRhoA) provides the most obvious reason for its resistance to proteasome-mediated degradation, because we show that Rac1(pbrRhoA) does not greatly differ from Rac1 in its ability to stimulate membrane ruffling or to interact with SmgGDS and IQGAP1-calmodulin complexes. These findings support the model that nuclear localization signal sequences in the PBR direct Rac1 to the nucleus, where Rac1 participates in signaling pathways that ultimately target it for degradation.

Val-12 after prolonged mAChR activation. We also demonstrate that Rac1 participates in mAChR-induced cell-cell compaction and c-Jun phosphorylation. These results indicate that M 3 mAChR activation converts Rac1 to the GTP-bound form, alters interactions between Rac1, IQGAP1, and actin, and causes the junctional accumulation of Rac1 and IQGAP1.The small GTPase Rac1 is emerging as an important participant in a variety of signaling pathways. Activation of Rac1 contributes to many responses in smooth muscle cells including c-Jun NH 2 -terminal kinase (JNK) 1 activation (1), reactive oxygen species generation (2), and contraction (3). The involvement of Rac1 in neuronal growth cone remodeling and neurite outgrowth implicates Rac1 as an intriguing regulator of axonal pathfinding and synaptogenesis (4 -6). The participation of Rac1 in the cadherin-mediated adhesion of epithelial and endothelial cells indicates that Rac1 signaling cascades may also regulate wound healing and vascular permeability (reviewed in Refs. 7-9). In many instances, activation of heterotrimeric G proteincoupled receptors (GPCR) initiates these Rac1-dependent cellular responses (1, 2, 5, 9). Activation of GPCR stimulates the conversion of Rac from the GDP-bound form to the GTP-bound form (1, 10). In addition to this event, GPCR activation undoubtedly induces additional uncharacterized changes in Rac1, such as altered interactions with protein partners or translocation to unique intracellular sites that promote the participation of Rac1 in these different signaling pathways. However, little is known regarding the concurrent changes in GTP binding activity, protein interactions, and subcellular localization of Rac1, which are induced by GPCR activation.The M 3 muscarinic acetylcholine receptor (mAChR) is a likely candidate to regulate Rac1 activity in a variety of cell types. The M 3 mAChR and the closely related M 1 mAChR are GPCR that are expressed in a wide variety of cells including smooth muscle cells, neurons, and epithelial and endothelial cells (reviewed in Ref. 11). Activation of M 3 mAChR induces several cellular responses that may involve Rac1 including JNK activation (12), reactive oxygen species generation (13), smooth muscle contraction (reviewed in Ref. 14), and cadherinmediated adhesion (11,15). Many of these responses have important physiological effects. For example, smooth muscle contraction induced by M 3 mAChR activation significantly alters pulmonary and cardiovascular function (reviewed in Refs. 16 and 17). The induction of E-cadherin-mediated adhesion by M 3 mAChR activation in lung carcinoma cells may diminish metastatic potential (reviewed in Refs. 11 and 15). The M 3 mAChR-mediated activation of JNK (12) may play an important role in AP-1-mediated transcription in a variety of cell types (reviewed in Ref. 18). The probability that these M 3 mAChR-dependent functions involve Rac1 provides a strong rationale for investigating how M 3 mAChR activation alters Rac1. * This work was supported by Grant R01 HL63921 from the NHLBI...