Rac3-Mediated Transformation Requires Multiple Effector Pathways
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Research Article Rac3-Mediated Transformation Requires Multiple Effector Pathways Patricia J. Keller,1 Christyn M. Gable,2 Michele R. Wing,1 and Adrienne D. Cox1,2,3,4 Departments of 1Pharmacology, 2Biology, and 3Radiation Oncology, and 4Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Abstract development and progression of cancer (5). Rac1 is essential for progression of the cell cycle and activates several pathways known to Our initial characterization of Rac3, a close relative of the small GTPase Rac1, established its ability to promote membrane be important in cellular proliferation, such as serum response factor (SRF), cyclin D1, and E2F (6, 7). Rac1 also promotes cellular survival ruffling, transformation, and activation of c-jun transcription- n al activity. The finding that Rac3 is transforming, and its by activating the nuclear factor- B pathway and by preventing similarity to Rac1, a protein that has a well-established anoikis and apoptosis (8–10). Not surprisingly, due to its role in connection to many processes important for cancer progres- promoting cell cycle progression and survival, Rac1 also promotes sion, prompted further investigation into Rac3 transformation. transformation in rodent fibroblast models and is required for We used effector domain mutants (EDMs) to explore the transformation induced by Ras (11–14). Due to its role in modulating relationship among Rac signaling, transformation, and effector the actin cytoskeleton, Rac1 also promotes motility and invasion, usage. All Rac3 EDMs tested (N26D, F37L, Y40C, and N43D) activities that are important for tumor metastasis (15). Given these retained the ability to promote membrane ruffling and focus activities, Rac1 has become a target for inhibitors that may be useful formation. In contrast, only the N43D mutant promoted in future cancer therapies (16, 17). anchorage independence. This differs from Rac1, where both Rac3, a close relative of Rac1, is less well characterized. Rac3 has 92% overall amino acid identity with Rac1, with the majority of the N26D and N43D mutants were impaired in both types of transformation. To learn more about the signaling pathways differences occurring in the COOH-terminal membrane targeting involved, we did luciferase reporter assays and glutathione region and in regions surrounding and within the Rho insert domain S-transferase pull-down assays for effector binding. We found (18, 19). Rac3 has been mapped to chromosome band 17q25.3 near a evidence for a functional link between activation of phospho- region that is commonly deleted in breast and ovarian cancers, suggesting possible transcriptional deregulation (20). It has been lipase CB2 by Rac3 and signaling to the serum response factor (SRF). Surprisingly, we also found that Rac3 binds poorly to the suggested that Rac3 but not Rac1 is hyperactivated in a subset of known Rac1 effectors mixed lineage kinases 2 and 3 (MLK2 and breast cancer cells and that inhibition of Rac3 impairs proliferation MLK3). Transcription of cyclin D1 was the only pathway that of these cells (21). In addition, a recent report suggests that Rac3 correlated with growth in soft agar. Our experiments show that plays a growth stimulatory role in mouse models of Bcr/Abl- mediated leukemias (22). We have shown previously that Rac3 can activation of membrane ruffling and transcriptional activation of c-jun, SRF, or E2F are not sufficient to promote anchorage- promote membrane ruffling, transcriptional activity of c-jun, and independent growth mediated by Rac3. Instead, multiple cellular transformation in both focus formation and soft agar assays (16). Others have shown that Rac3 can bind and activate the Rac1 effector pathways are required for Rac3 transformation, and h h these overlap partially but not completely with those used by effector phospholipase C 2 (PLC 2) and promote the activation of Rac1. (Cancer Res 2005; 65(21): 9883-90) p21-activated kinase-1 (Pak1) and c-Jun NH2-terminal kinase (JNK; refs. 18, 21, 23). Evidence that Rac1 and Rac3 share some of the same effector pathways coupled with the demonstration that Rac3 is Introduction transforming in rodent fibroblast models and plays a growth- Rac1 is one of the most heavily studied members of the Rho promoting role in mouse leukemia models suggest that Rac3 may subfamily of Ras-related small GTPases (1). Like Ras proteins, Rho have a role in human cancer. This led us to further investigate the family proteins cycle between active GTP-bound and inactive GDP- effector binding and biological properties of Rac3 that contribute to bound states. Oncogenic, activated forms of Rac proteins are either the transformation process. constitutively GTP bound (G12V or Q61L) or have greatly increased Consistent with its diverse cellular functions, a multitude of rates of GDP-GTP cycling (F28L). Activated forms of Rac proteins, effector molecules have been identified that interact with Rac1. whether through the action of guanine nucleotide exchange factors These effector molecules fall into several functional classes, such as or mutation, promote their biological effects by signaling through an serine/threonine kinases, lipid kinases, and adaptors/scaffolds, and ever-growing list of downstream effector molecules (2, 3). generally interact with the GTP-bound form of the protein (2, 3). Cytoskeletal reorganization, specifically the formation of mem- The core effector region (amino acids 26-45) of small GTPases is brane ruffles and lamellipodia, was one of the first functions conserved among Rac family members and serves as the primary site attributed to activated Rac1 (4). Since then, another major role for interaction with effectors (2, 3, 18). Mutations in this region, defined for Rac1 involves coordinating many processes related to the effector domain mutants (EDMs), were first developed for Ras proteins and have helped to uncouple certain effector pathways, such as Ras activation of Raf, from biological outcomes, such as Requests for reprints: Adrienne D. Cox, Department of Radiation Oncology, transformation (24, 25). This has led to a greater understanding of University of North Carolina at Chapel Hill, CB #7512, Chapel Hill, NC 27599-7512. how effectors of Ras contribute to its biology but has also highlighted Phone: 919-966-7713; Fax: 919-966-7681; E-mail: [email protected]. I2005 American Association for Cancer Research. the need for multiplicity and cooperation among effectors to doi:10.1158/0008-5472.CAN-04-3116 recapitulate the complex biological effects of the native protein. www.aacrjournals.org 9883 Cancer Res 2005; 65: (21). November 1, 2005 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2005 American Association for Cancer Research. Cancer Research Following the example of Ras EDMs, mutations in the core effector Transformation assays. For focus formation assays, NIH 3T3 cells were region of Rho family proteins, such as Rac1 and RhoA, have also been plated in 60-mm dishes and cotransfected in duplicate (calcium phosphate) characterized (12, 26). with either 200 ng pZIP vector or pZIP-raf 22W (NH2-terminally truncated For Rac1, EDMs were shown to selectively impair signaling and active) and 500 ng pCGN-hyg vector, pCGN-rac1/rac3 61L, or pCGN-rac3 61L EDMs. After 14 to 21 days, cells were photographed under the Â4 pathways, such as transformation (N26D or N43D mutants), objective and stained as described previously (16). Stained foci were then membrane ruffling (F37L mutant), and activation of Pak1 (Y40C counted to determine transforming activity and sorted by focus morphology. mutant; ref. 12). We made these same mutations in a Rac3 For soft agar assays, NIH 3T3 or RIE-1 cells stably expressing pCGN-hyg background to better understand how the effector binding and vector, pCGN-rac3 28L, or pCGN-rac3 28L EDMs were prepared as described signaling properties of Rac3 contribute to cellular transformation. above. Single-cell suspensions (1-5 Â 105 cells per 60-mm dish) of each stable In this study, we define a novel pathway linking the Rac effector cell line were plated in medium containing 0.4% agar on top of a bottom layer h PLC 2 and SRF-mediated transcription. We have also found that containing 0.6% agar. Colonies were allowed to form for 14 to 21 days after transcription of cyclin D1 may be important for promoting which they were photographed under the Â4 objective and counted. anchorage-independent growth but that membrane ruffling and Western immunoblotting. Stable cell lines and transiently transfected transcriptional activation of c-jun, SRF, or E2F are not sufficient. cells were harvested 2 days after plating or transfection, respectively, as These results underscore the complexity of Rac3 signaling to described previously (16). Samples were prepared in Laemmli sample buffer and 5 to 10 Ag protein from each sample were run on 12% SDS-PAGE gels. effectors necessary for transformation. Proteins were transferred at 100 V for 1 hour to polyvinylidene difluoride (Immobilon-P, Millipore, Bedford, MA). Membranes were blocked in 3% fish Materials and Methods gelatin (Sigma, St. Louis, MO), incubated for 1 hour in either 1:1,000 anti-HA Effector domain mutants of Rac3. The mutations N26D, F37L, Y40C, antibody (Covance, Philadelphia, PA) or 1:5,000 anti-h actin (Sigma), and and N43D were introduced into rac3 Q61L or rac3 F28L with the washed. Membranes were incubated for 30 minutes in 1:30,000 anti-mouse QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) IgG-horseradish peroxidase (IgG-HRP, Amersham Biosciences), washed, and according to the manufacturer’s instructions; the F28L mutation was also developed with SuperSignal West Dura Extended Duration substrate introduced in this way. The Nucleic Acids Core Facility at the University of (Pierce, Rockford, IL). North Carolina at Chapel Hill generated all primers. The rac1 and rac3 Q61L Effector pull-down assays. Activated Rac1, Rac3 (both 61L), and Rac3 constructs have been described previously (16). Restriction enzyme digestion EDMs attached to a GST tag were purified from a bacterial culture as sites were added to the 5V and 3V ends of the rac3 EDMs by PCR-based described previously (28).