Rac3-Mediated Transformation Requires Multiple Effector Pathways

Research Article

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

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). The concentration of protein bound to mutagenesis as described previously (16, 27). PCR products of rac3 were glutathione-agarose beads (Sigma) was determined by colorimetric assay digested with SalI and NotI (all restriction enzymes from Invitrogen, (Bio-Rad, Hercules, CA). NIH 3T3 cells in 100-mm dishes were transfected Carlsbad, CA) at the sites introduced at the 5V and 3V ends and ligated in- with 3 Ag constructs expressing the following Rac effector proteins: pCMV6- frame with the hemagglutinin (HA) epitope tag into the vector pCGN-hyg for Myc-pak1,pcDNA3-HA-mlk3, pBABE-T7-par6 (described previously; + expression in mammalian cells. PCR products were also digested with BamHI ref. 28), pcDNA3.1 -plcb2, or pcDNA3.1-HA-mlk2 (gift of Lloyd Greene, and EcoRI and ligated in-frame with an enhanced green fluorescent protein Columbia University, New York, NY), and harvested 24 to 48 hours later. Cell (EGFP) tag into the mammalian expression vector pEGFP-C1 (BD lysates were collected as described previously (28) and precleared with Biosciences Clontech, Palo Alto, CA) that was cut previously with BglII and 50 Ag GST beads. Lysates were split and mixed with 10 to 30 Ag (depending EcoRI or in-frame with a glutathione S-transferase (GST) tag in the bacterial on effector used) of GST-only, GST-Rac1 61L, GST-Rac3 61L, or GST-Rac3 expression vector pGEX-2T (Amersham Biosciences, Piscataway, NJ). All 61L EDMs bound to beads. After incubation for 1 hour at 4jC, beads were mutations and ligation junctions were verified by forward and reverse washed and resuspended in 20 AL sample buffer. Samples were run on 12% sequencing. SDS-PAGE gels, transferred, and blocked as described above. Membranes Cell culture and transfections. NIH 3T3 mouse fibroblasts were grown were incubated for 1 hour in 1:1,000 anti-HA, anti-Myc (9E10, Sigma), anti- in DMEM (Invitrogen/Life Technologies, Gaithersburg, MD) supplemented T7 (EMD Biosciences/Novagen, San Diego, CA), or anti-PLCh2 (Santa Cruz with 10% calf serum (Invitrogen/Life Technologies) and 1% penicillin/ Biotechnology, Santa Cruz, CA) and then washed. Membranes were streptomycin (Invitrogen/Life Technologies) and maintained in 10% CO2 at incubated with secondary antibody (either anti-mouse or anti-rabbit IgG- 37jC. The day before transfection, 5 Â 105 (100-mm dish), 2.5 Â 105 (60-mm HRP) and developed as described above. dish), 1 Â 105 (per well of a six-well plate), or 3 Â 104 (per well of a 12-well Reporter gene assays. For transient luciferase assays, NIH 3T3 cells in plate) cells were plated unless otherwise indicated. Rat intestinal epithelial 6- or 12-well dishes were cotransfected with 500 ng pCGN-rac3 61L or cells (RIE-1) and COS-7 cells were grown in 5% fetal bovine serum (FBS; 500 ng pCGN-rac3 61L EDMs and 500 ng c-jun, E2F, SRF, or cyclin D1 Sigma-Aldrich, St. Louis, MO) or 10% FBS, respectively, and 1% penicillin/ reporter constructs (29). All transfections were done in at least duplicate. streptomycin and maintained as described for NIH 3T3 cells. NIH 3T3 cells Three hours after transfection by LipofectAMINE and PLUS reagent, were transfected by calcium phosphate coprecipitation as described (27) or the medium was replaced with 0.5% serum DMEM and grown for 20 to h with LipofectAMINE and PLUS reagent (Invitrogen) or FuGENE6 (Roche, 24 hours. For the PLC 2-SRF assays, cells were transfected by FuGENE6 in Indianapolis, IN) according to the manufacturer’s instructions. COS-7 cells 10% serum for 20 to 24 hours with 5 ng pCGN-rac3 61L or 25 ng pCGN-rac3 were transfected with FuGENE6 and RIE-1 cells were transfected with 61L EDMs and 150 ng of the SRF reporter construct in the presence of either + LipofectAMINE and PLUS reagent according to the manufacturer’s pcDNA3 vector or 100 ng pcDNA3.1 -plcb2. The cells were then rinsed with instructions. Stable cell lines were created in RIE-1 and NIH 3T3 cells after 1Â PBS and lysed in 1Â lysis buffer (Amersham Biosciences), and luciferase transfection with 1 Ag pCGN-hyg constructs expressing the Rac3 F28L activity was measured with enhanced chemiluminescence reagents EDMs as has been described previously (16). (Amersham Biosciences) in a Monolight 2010 luminometer (Analytical Membrane ruffling activity. NIH 3T3 cells were plated on glass Luminescence, San Diego, CA). coverslips in six-well plates. For visualization of membrane ruffles, cells Phospholipase C activity assay. COS-7 cells were seeded in 12-well were transiently transfected with 1 Ag pEGFP-C1 vector, pEGFP-rac3 61L, or culture dishes at a density of 7 Â 104 per well and transfected with 100 ng/ pEGFP-rac3 61L EDMs. After 48 hours, localization of Rac3 in live cells was well pCGN-rac3 61L or pCGN-rac3 61L EDMs and 300 ng/well pcDNA3 + visualized with a Zeiss Axioskop fluorescent microscope (Zeiss, Thornwood, vector or pcDNA3.1 -plcb2. Approximately 24 hours after transfection, NY). For each condition, at least 200 cells were counted per coverslip and culture medium was changed to inositol-free DMEM (ICN Biomedicals, scored as either ruffling or flat. Images were captured under the Â20 Aurora, OH) containing 1 ACi/well myo-[2-3H(N)]inositol (American objective with MetaMorph digital imaging software (Universal Imaging Radiolabeled Chemicals, St. Louis, MO), and metabolic labeling was allowed Corp., Downington, PA). to proceed for 12 to 16 hours. Accumulation of [3H]inositol phosphates was

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2005 American Association for Cancer Research. Rac3 Effector Pathways in Transformation initiated by the addition of 10 mmol/L LiCl to inhibit inositol mono- impairment of ruffling but instead led to enhanced ruffling, with phosphate phosphatases. The reaction was stopped after 60 minutes by >75% of the cells that were counted producing membrane ruffles. aspiration of the labeling medium and the addition of 50 mmol/L formic 3 These results suggest that Rac1 and Rac3 may use different effector acid followed by neutralization with 150 mmol/L NH4OH. [ H]Inositol pathways to promote cytoskeletal rearrangements, because the phosphates were quantified by Dowex chromatography as described F37L effector domain mutation produced the opposite effect in previously (30). Rac3 and Rac1. Mutations in the effector domain of Rac3 alter the Results morphology of transformed foci. We have shown previously All Rac3 effector domain mutants tested retain the ability to that activated GTP-bound Rac3 is capable of promoting cellular produce membrane ruffles. To better understand the role of Rac3 transformation by loss of contact inhibition, forming foci of effector pathways, we generated mutations in the core effector morphologically transformed cells in cooperation with activated domain of Rac3 analogous to mutations described previously for Raf (16). We have also noted that the foci formed by Rac3 are more Rac1 (12, 31, 32). It has been reported that a F37L mutation in Rac1 compact in morphology than those formed by Rac1.5 To determine impairs the formation of membrane ruffles and lamellipodia if mutations that impair transformation by Rac1 also affect (12, 32). To determine the effects of this mutation and others on transformation promoted by Rac3, we did a focus formation assay the ability of Rac3 to produce membrane ruffles, NIH 3T3 cells in NIH 3T3 cells transfected with Raf alone, Rac1, Rac3, or Rac3 expressing EGFP-Rac3 or Rac3 EDMs were scored as either ruffling EDMs alone or with Raf in combination with Rac1, Rac3, or Rac3 or flat (Fig. 1). For activated Rac3 without any effector domain EDMs. Unlike Rac1, where both N26D and N43D mutants were mutation, f62% of cells were characterized as ruffling (Fig. 1B). impaired in promoting loss of contact inhibition in a focus assay Whereas no single effector domain mutation caused a complete (12), all Rac3 EDMs were capable of forming foci in cooperation impairment of ruffle formation, either a N26D or a Y40C mutation with Raf with similar total numbers of foci (Fig. 2; data not shown). led to a decreased percentage of ruffling cells, 33% and 47%, However, the morphology of the foci differed depending on the respectively. A N43D mutation led to an increased percentage of mutation. Small compact foci (‘‘Rac3 like’’) were formed predom- cells that were ruffling at 70%. Unlike previous reports for Rac1, a inantly in Rac3 and Rac3 N26D-transfected cells. Larger, spread foci F37L mutation in the effector domain of Rac3 did not lead to (‘‘Rac1 like’’) were formed predominantly in Rac1 and Rac3 F37L-, Y40C-, and N43D-transfected cells. The shift of focus morphology from a Rac3-like phenotype to a Rac1-like phenotype suggests that Rac1 and Rac3 may use different subsets of effectors to promote this type of transformation and that the EDMs of Rac3 tested may shift the profile of effectors used. Only the N43D mutation retains the ability to promote anchorage-independent growth. Another characteristic of cellular transformation is the ability of cells to grow in an anchorage-independent manner. We have established previously that GTPase-defective, activated Rac3 is capable of promoting anchorage-independent growth by forming colonies in soft agar (16). Here, we establish that a fast cycling mutant of Rac3 (F28L) is also capable of promoting anchorage-independent growth (Fig. 3). We used a soft agar assay to gauge the ability NIH 3T3 cells stably expressing Rac3 EDMs to promote anchorage-independent growth. Immunoblot analysis indicated similar expression levels of activated Rac3 and Rac3 EDMs with the exception of Rac3 F37L, which consistently had decreased expression in several independent stable lines generated, as shown in Fig. 3A. Expression of the F37L mutant was confirmed transiently (Fig. 3A), suggesting that decreased expression was required for selection of stably expressing cells. In contrast to the focus formation assay, where all EDMs retained transforming ability, only the N43D mutant was capable of transformation in soft agar, producing colonies in somewhat greater number but of similar size to Rac3 lacking any effector domain mutation (Fig. 3B and C). A N26D, F37L, or Y40C mutation greatly impaired transformation in soft agar, with few colonies formed, and in those that did form, smaller colony size. For the F37L mutation, it is unclear if transformation is impaired by the consequences of the mutation on effector interaction or by the Figure 1. Rac3 N26D and Y40C have decreased, but not completely impaired, conistent down-regulation of protein expression in stable lines. membrane ruffle production. NIH 3T3 cells were transiently transfected with constructs expressing EGFP-tagged activated Rac3 or Rac3 EDMs. The pattern of expression of the EDMs and of colony formation, A, representative photos of EGFP-expressing cells as well as Rac3-expressing cells with flat and ruffled phenotypes (Â20 objective). B, columns, % total for cells that were scored as either flat or ruffling; bars, SD. Results were compiled from four independent experiments where at least 200 cells were scored for each condition. 5 Unpublished observations. www.aacrjournals.org 9885 Cancer Res 2005; 65: (21). November 1, 2005

focus formation assays combined with the opposite result seen with Rac3 N43D in soft agar assays, where, for Rac1, N43D is impaired but, for Rac3, it is not, again suggests that Rac3 uses an incompletely overlapping set of effector pathways compared with Rac1 to promote transformation. Rac3 Y40C is impaired in binding to Pak1 and Par6, whereas both Rac3 F37L and Y40C have reduced binding to phospholipase CB2. To determine how mutations in the effector domain of Rac3 affect binding to known and potential effectors, we used a GST pull-down assay, which has been described previously for determining effector interactions with RhoG (28). Lysates from NIH 3T3 cells that were expressing effector proteins (Pak1, Par6,

Figure 2. Foci formed by the N26D mutant are morphologically similar to foci formed by Rac3, whereas foci formed by the F37L, Y40C, and N43D mutants resemble Rac1. NIH 3T3 cells were transfected with activated Rac1, Rac3, or Rac3 EDMs in the presence or absence of cotransfection with activated Raf. Foci were allowed to form for 14 days, after which they were photographed under the Â4 objective, stained, and counted. Similar numbers of foci were formed for each condition (data not shown). The morphology of foci was characterized as resembling either activated Rac1 or Rac3 with no mutation in the effector domain. A, percentage of each for the various EDMs. Examples of the morphology of foci: stained plates (B) and individual foci (C). Figure 3. All EDMs, except N43D, are impaired in the ability to promote Representative of three independent experiments. anchorage-independent growth. Stable lines were created in NIH 3T3 cells expressing activated Rac3 or Rac3 with the indicated effector domain mutations. A, expression of Rac3 and Rac3 EDMs was determined by immunoblot for with only the N43D mutant retaining activity, was the same for soft the HA tag (middle). Expression of the F37L mutant was consistently lower than all others over three independently generated lines in NIH 3T3 cells. Expression agar assays done in an epithelial cell line, RIE-1 cells (data not of the F37L mutant was verified transiently (top) and total protein expression shown). Previously reported data from similar assays done with was verified by blotting for h-actin (bottom). Stable expressors were seeded into Rac1 indicated that both the N26D and the N43D mutants were 0.4% agar over a bottom layer of 0.6% agar. Colonies were allowed to form for 14 days, after which fields were photographed under the Â4 objective and impaired in focus formation and colony formation (12). The fact colony-forming activity was quantified (B and C). Representative of three that all EDMs tested for Rac3 retained transforming activity in independent experiments.

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Figure 4. Effector binding and transcriptional activation by Rac3 and Rac3 EDMs. A, NIH 3T3 cells were transfected with constructs expressing Pak1, Par6, or PLCh2. After 20 hours, lysates were harvested and split into fractions that were mixed with glutathione-agarose beads bound to GST or GST fusion proteins of activated Rac1, Rac3, or Rac3 EDMs. Effector protein that bound to Rac was identified by Western immunoblotting with the appropriate primary antibody: a-myc (Pak1), a-T7 (Par6), or a-PLCh2. B, GST-Rac effector pull-down experiments were done as in (A) with lysates expressing MLK2 or MLK3. MLK protein that bound to Rac was identified by Western immunoblotting for the HA tag. Representative of at least two independent experiments. C, NIH 3T3 cells were transiently transfected with activated Rac3 or the indicated Rac3 EDMs and luciferase reporter constructs for SRF, c-jun, cyclin D1, or E2F. After 24 hours of serum starvation (0.5%), cell lysates were analyzed for luciferase activity. Representative of at least three independent experiments. Columns, fold luciferase relative to empty vector; bars, SD. D, transient expression of Rac3 or Rac3 EDMs was determined as described in Fig. 3.

h and PLC 2) were combined with agarose beads bound with GST effectors to Rac3 (31, 35). Interaction of Rac3 with Par6 suggests a alone or GST fused to GTP-bound Rac3 or Rac3 EDMs. Beads possible role for Rac3 in modulating cell/cell junctions and bound with GST fused to GTP-bound Rac1 were used as a positive polarity, known functions of Par6, although the role that Rac1 control for effector binding. Binding to Rac3 or Rac3 EDMs was plays in Par6-mediated signaling is incompletely defined (36). determined by Western blot for the specific effector protein as Unlike Rac1, Rac3 is impaired in binding to mixed lineage shown in Fig. 4A. As expected, known Rac3 effectors Pak1 and kinases 2 and 3. GST pull-down assays were used as described h PLC 2 (21, 33) bound to wild-type Rac3. However, Rac3 Y40C above to determine if the known Rac1 effectors mixed lineage displayed impaired binding to Pak1, and both Rac3 Y40C and F37L kinases 2 and 3 (MLK2 and MLK3; refs. 37, 38) could also bind to were diminished in binding to PLCh2. Additionally, we show that activated Rac3. To our surprise, we found that MLK2 bound very Rac3 can bind to Par6, a known Rac1 effector/adaptor protein weakly to Rac3 compared with binding to Rac1 and that there was (34), and as with Pak1, only the Y40C mutant is impaired in no detectable binding of MLK3 to Rac3 despite equivalent loading binding (Fig. 4A). As Pak1 and Par6 both contain a Cdc42/Rac of the GST fusion proteins (Fig. 4B). This result suggests that Rac3 interactive and binding (CRIB) motif that is important for may differ from Rac1 in its ability to use MLK proteins to promote interaction with Rac, this suggests that, like what has been shown its downstream effects or that Rac3 may interact with MLK2/MLK3 for Rac1, Y40C mutation impairs binding of CRIB-containing through additional proteins in vivo to promote its effects. www.aacrjournals.org 9887 Cancer Res 2005; 65: (21). November 1, 2005

Mutations in the effector domain of Rac3 differentially In contrast, the N26D and N43D mutants, when transfected with impair signaling to Rac3 transcriptional pathways. We have PLCh2, promoted synergistic effects of f20- and 15-fold, respec- shown previously that Rac3 up-regulates transcription by the c-jun tively, compared with only 9- and 8-fold, respectively, for the additive h transcription factor (16). Here, we show that transient expression of contributions of Rac3 and PLC 2 alone. These results suggest that activated Rac3 also up-regulates the transcription of cyclin D1 and PLCh2 activation is a novel effector pathway used by Rac3 to the transcription of E2F- and SRF-responsive elements (Fig. 4C). To promote SRF-mediated transcription. determine the effect of specific mutations in the effector domain of Rac3 on transcriptional transactivation, we did luciferase assays to Discussion gauge the ability of Rac3 EDMs to up-regulate transcriptional We have used EDMs to evaluate relationships among Rac3 effector pathways. Activation of SRF transcriptional activity is greatly binding, transcriptional activation, membrane ruffling, and trans- reduced by Rac3 with a F37L or Y40C mutation. However, for c-jun formation (summarized in Fig. 6A). Our results indicate that the transcriptional activity, only the Y40C mutant is impaired in ability of Rac3 to promote transformation requires multiple effector signaling. Transcription of cyclin D1 is reduced by N26D, F37L, or pathways. Membrane ruffling and activation of transcriptional Y40C mutations but not by a N43D mutation. All effector domain pathways involving c-jun, SRF, and E2F are not sufficient to promote mutations tested decrease E2F transcriptional activity, with Y40C transformation. In the case of membrane ruffling, all EDMs tested and N43D showing the greatest impairment. For all transcriptional retained some level of activity, yet only the Rac3 N43D EDM pathways tested, a Y40C mutation in Rac3 led to decreased activity, promoted anchorage-independent growth (Fig. 6A). This agrees with suggesting the importance of effectors of Rac3 that are disrupted by published data for Rac1, where mutations that impaired lamellipo- this mutation, such as Pak and Par6 (Fig. 4A) or other CRIB motif– dia, such as F37L or deletion of the insert region, had no apparent containing effectors. When transient expression of Rac3 EDMs was evaluated by Western blot (Fig. 4D), all mutant proteins were expressed, indicating that impaired expression of the Y40C mutant does not account for its reduced transcriptional activity. Rac3 synergizes with phospholipase CB2 to promote transcriptional activation of serum response factor. A comparison of the effector binding and transcriptional data from Fig. 4 suggests that there is a correlation between reduced binding to the effector PLCh2 and reduced activation of SRF-mediated transcription. Although it has been shown that PLCh2 is an effector of Rac1, Rac2, and Rac3, as defined by its selective binding to Rac-GTP versus Rac-GDP, the biological functions resulting from its activation by Rac have not been determined (23, 39). To further explore this potential connection, we determined the ability of Rac3 EDMs to h activate PLC 2 by use of a PLC activity assay. As seen in Fig. 5A, the F37L mutant of Rac3 that was most impaired in binding to PLCh2 (Fig. 4A) also was the most impaired in activating PLCh2. The N43D h mutant, which retained binding to PLC 2, also retained ability to activate PLCh2 to levels similar to Rac3 without an effector domain mutation. The N26D and Y40C mutants of Rac3 displayed h intermediate ability to activate PLC 2. Although the Y40C mutant displayed reduced binding to Rac3, its binding was not completely abolished; thus, it is not surprising that it retained some ability to activate PLCh2. Although our results in Fig. 4A suggest that the N26D h mutant binds to PLC 2 with equivalent affinity to Rac3 without an effector domain mutation, we cannot rule out that the mutation has an effect structurally that may affect the efficiency of PLCh2 activation, despite robust binding, which may explain its reduced activity in the PLC activity assay. All EDMs were expressed equivalently under these assay conditions (data not shown). h To determine if Rac activation of PLC 2 contributes to SRF activity, we did luciferase reporter assays for SRF with Rac3 in the presence or absence of PLCh2. As seen in Fig. 5B, either PLCh2 alone or Rac3 alone led to roughly 3-fold activation of SRF, but when h expressed together SRF-mediated transcriptional activity was Figure 5. Rac3 uses PLC 2 to promote SRF-mediated transcription. A, activation of PLCh2 by Rac3 and Rac3 EDMs was determined by measuring boosted to f16-fold. The synergistic effect of expressing Rac3 and the generation of [3H]inositol phosphates from COS-7 cells transfected with Rac3 h PLCh2 suggests that Rac3 uses PLCh2 activity to promote SRF- or Rac3 EDMs in the presence or absence of PLC 2. Columns, counts/min (cpm); bars, SD. B, synergistic activation of SRF-mediated transcription was mediated transcriptional activation. Further bolstering this idea, determined by luciferase reporter assay with NIH 3T3 cells transfected with Rac3 h Fig. 5C shows Rac3 with reduced capability to bind to PLC 2 (F37L in the presence or absence of PLCh2. Columns, fold activation over vector control; bars, SD. C, synergistic activation of SRF-mediated transcription was and Y40C) and also exhibited reduced synergy with PLCh2 in SRF- determined by luciferase reporter assay with NIH 3T3 cells transfected with Rac3 mediated transcription, promoting only marginal increases in EDMs in the presence or absence of PLCh2. Columns, fold activation over vector activity over the additive contributions of Rac3 and PLCh2 alone. control; bars, SD. Representative of at least three independent experiments.

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Figure 6. Cyclin D1 may contribute to anchorage-independent growth promoted by Rac3. A, summary of the biological activities of Rac3 EDMs. 1, site-directed mutagenesis was used to create mutations in the effector domain of activated Rac3. Activated Rac3 protein with no effector domain mutation (À). 2, GST fusion proteins of activated Rac3 or Rac3 EDMs were used to pull down effector proteins: +, binds; +/À, decreased binding; À, does not bind. 3, transcriptional reporter activity was evaluated for the indicated pathways. Results are summarized relative to activated Rac3 without an effector domain mutation (100%): +, 0% to 25% of Rac3 parent; ++, 25% to 50%; +++, 50% to 75%; ++++, 75% to 100%. 4, EGFP fusion proteins were used to visualize Rac3 location and to determine ruffling activity. Results are summarized relative to activated Rac3 without an effector domain mutation (100%): +, 0% to 30% of Rac3 parent; ++, 30% to 60%; +++, 60% to 90%; ++++, 90% to 120%; +++++, z120%. 5, transforming activity was evaluated with a focus formation assay or with a soft agar assay: +, transforming; À, impaired in transformation. B, a comparison of data compiled in this article suggests that transcription of cyclin D1 correlates with Rac3-mediated anchorage-independent growth. We found that Rac3 binds to the Rac1 effector/adaptor Par6 but binds poorly to the Rac1 effectors MLK2 and MLK3. In addition, we have defined a new pathway from Rac3 activation of PLCh2 to SRF-mediated transcription. Characterization of Rac3 EDMs indicates that multiple signaling pathways contribute to Rac3-mediated transformation. loss of transforming ability (12, 29). For transcriptional activation of A previous study reported that the pattern of impairment was the c-jun, SRF, and E2F, the Rac3 N26D EDM retained fairly robust same for Rac1 in both transformation by loss of contact inhibition or activation of these pathways yet was clearly impaired in anchorage- gain of anchorage independence, with both Rac1 N26D and N43D independent growth (Fig. 6A), indicating that activation of c-jun-, impaired (12). This was not the case for Rac3. The variation in focus SRF-, and E2F-mediated transcriptional pathways are not sufficient morphology between Rac1 and Rac3 coupled with the different to promote transformation. Additionally, the Rac3 N26D EDM also effects of the same EDMs in Rac1- and Rac3-mediated transforma- h retained robust binding to Pak1, Par6, and PLC 2, indicating that tion suggests that there are differing pools of effectors that Rac1 these effector pathways are also not sufficient for transformation and Rac3 use to promote transformation. This may reflect differ- (Fig. 6A and B). This is in agreement with data from Rac1 EDMs and ences in innate effector binding between Rac1 and Rac3 and/or deletion mutants, showing a lack of correlation among Pak1 binding, different accessibility of effectors due to different localizations of JNK or SRF activation, and transformation (12, 31, 32). Rac1 and Rac3 within cells. Although the core effector domain is 100% identical between Rac1 Here, we describe a new connection between Rac3 activation of h and Rac3, protein sequence differences do occur in regions COOH PLC 2 and SRF-mediated transcription. Before this, it was unclear terminal to it (18, 19). Effectors and interacting proteins of Rac1, such what role PLCh2 plays downstream of Rac proteins (23, 39). Activation as Pak1, NADPH oxidase, and PI5K, are known to bind regions of PLCh isoforms leads to hydrolysis of phosphatidylinositol 4,5- outside the core effector domain (40–42). Thus, the importance of bisphosphate into the second messengers inositol 1,4,5-trisphosphate other effector binding regions in Rac proteins suggests that (IP3) and diacylglycerol (DAG; ref. 48). DAG then activates protein differential effector binding between Rac1 and Rac3 could occur. kinase C, whereas IP3 leads to an increase in cytoplasmic calcium ion This seems to be the case for in vitro binding of Rac3 to MLK2 and concentration and activation of calmodulin and calmodulin kinase MLK3. Both contain a degenerate CRIB motif that is missing one of (48, 49). Interestingly, several reports indicate the involvement of Ca2+ two conserved histidine residues, suggesting that MLK2/MLK3 may and calmodulin kinase in the regulation of SRF activity. SRF was not interact as strongly with the core effector domain where the CRIB reported to regulate increased transcription in response to elevated motif binds and may require other regions of Rac for efficient binding levels of cytoplasmic Ca2+ in a pathway involving calmodulin kinase (43). JNK activation is correlated with MLK3 activation downstream (50). Additionally, a recent report suggests that the activity of SRF of Rac1 (37, 38), and it has been shown that Rac3 can activate JNK is repressed in the nucleus by histone deacetylase 4 (HDAC4) (18, 21) as well as c-jun-mediated transcription (16). This suggests binding and that association of calmodulin kinase after Ca2+ that Rac3 may preferentially use effectors other than MLKs to stimulation with the HDAC4/SRF complex resulted in derepression activate JNK/c-jun, such as Pak, mitogen-activated/extracellular and increased SRF activity (51). SRF is a common target in many signal regulated kinase kinase kinase 1/4 (MEKK 1/4), or plenty of growth factor–induced pathways, suggesting that PLCh2 could SH3s (POSH; refs. 44–46). For Rac1-mediated activation of p38, a contribute to cellular growth activities induced by Rac activation. scaffolding molecule, JIP2, was shown to assemble a cascade from the Further studies will help to refine the pathway from PLCh2 to SRF Rac1 GEF Tiam1 to MLK3, MKK3, and p38 (47). This suggests that and to determine the potential involvement of PLCh2 in other even if Rac3 is impaired in direct binding to MLK3, scaffolding Rac-mediated signaling pathways and biological functions. molecules, such as JIP2, could still allow for signaling through MLK3 Deregulation of growth control pathways is a major means by to its downstream targets. which Rho proteins contribute to cellular transformation. Our data Our data on Rac3 EDMs and transformation also suggest that suggest a correlation between transcription of cyclin D1 and Rac3- Rac1 and Rac3 use an incompletely overlapping set of effectors. mediated anchorage-independent growth (Fig. 6A and B). Both Rac www.aacrjournals.org 9889 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 and cyclin D1 activity are required for transformation induced by Ras Acknowledgments (13, 14, 52). Furthermore, cyclin D1 transcription has been correlated with the transforming activity of several Dbl family GEFs, which Received 8/30/2004; revised 8/26/2005; accepted 9/1/2005. activate Rac1 and other Rho family proteins, and is required in Grant support: NIH grants T32-CA71341 (P.J. Keller) and CA67771 (A.D. Cox). The costs of publication of this article were defrayed in part by the payment of page mouse models for transformation by Ras (52–54). Our results suggest charges. This article must therefore be hereby marked advertisement in accordance that cyclin D1 may play an important role in transformation induced with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Krister Wennerberg for helpful discussions and advice on the GST-Rac by Rac3. Further determination of signaling pathways relevant for effector pull-down assays, Lloyd Greene for the MLK2 construct, and Laura Brenner Rac3-mediated transformation awaits future study. for technical assistance in creating the EDMs.

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Patricia J. Keller, Christyn M. Gable, Michele R. Wing, et al.

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