Oncogene (1998) 17, 625 ± 629 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc An activated Rac mutant functions as a dominant negative for membrane ruing
Martin Alexander Schwartz, Jere E Meredith and William B Kiosses
Department of Vascular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA
Previous studies have shown that point mutations in the terminal eector domains (Westwick et al., 1997; eector domain of Rac1 block speci®c downstream Lamarche et al., 1996). Point mutants were con- pathways such as PAK, JNK/SAPK kinases and structed in V12 or Q61L backgrounds to give membrane ruing. Speci®cally, the F37A mutation, constitutively activated proteins that would show made in a constitutively activated Q61L background, dominant positive eects. These studies of Rac point activates PAK but fails to induce membrane rues. We mutations revealed that induction of membrane ruing now show that Q61L/F37A Rac not only fails to induce and cell cycle progression were independent of ruing but potently blocks membrane ruing induced by activation of PAK and other CRIB domain proteins, serum or PDGF. In the presence of serum, cells do and activation of the JNK/SAPK pathway (Westwick extend ®lopodia, suggesting that this mutant only blocks et al., 1997; Lamarche et al., 1996). a subset of the eectors that induce cytoskeletal One of these mutations, Q61L/F37A, was found to reorganization. At later times, this rac mutant induces abolish induction of membrane ruing and DNA membrane blebbing, but not apoptosis. These results synthesis but did not aect PAK or JNK/SAPK show that Q61L/F37A Rac, is constitutively activated activation (Lamarche et al., 1996). While studying with respect to PAK activation but functions as a this mutant in NIH3T3 cells, we made the observation dominant negative for another pathway, membrane that its expression caused loss of membrane rues. ruing. That an eector domain point mutant can Further analysis showed that despite the presence of a simultaneously function as a dominant negative and mutation to inhibit the GTPase and cause constitutive dominant positive for dierent pathways implies that activation, this variant not only fails to induce eects of these variants on cell functions must be membrane ruing but acts as a dominant negative interpreted with caution. for this pathway. These data indicate that cellular responses to Rac eector domain point mutations may Keywords: Rho family small GTP binding protein; be more complex than previously thought. signal transduction; actin cytoskeleton
Results Introduction Loss of membrane rues The Rho family of small GTP binding proteins A Rac1 variant containing the F37A mutation in a regulates organization of the actin cytoskeleton, gene Q61L background was expressed in NIH3T3 cells by expression, cell cycle progression and membrane injecting expression vector cDNA into cell nuclei. transport (reviewed in Ridley, 1996; Nobes and Hall, Protein expression was monitored either by staining 1994). Rac1 and Rac2 control formation of membrane ®xed cells with an antibody to the myc epitope tag or rues in ®broblasts and play important roles in cell by co-injection of a cDNA vector for green ¯uorescent growth and motility. Rac also mediates activation of protein (GFP). Protein was detectable within 1 h of the p38 kinase and jun C-terminal kinase (JNK/SAPK) injection, and eects on cell morphology were readily pathways in these cells (Coso et al., 1995; Minden et observable by 2 h. al., 1995; Bagrodia et al., 1995; Zhang et al., 1995). As previously described (Westwick et al., 1997; Rac and its close relative cdc42 appear to have a Lamarche et al., 1996), expression of Q61L/F37A number of putative eectors, including the 65 kD Rac in serum starved NIH3T3 cells had little or no protein kinase PAK (Manser et al., 1994). One group eect on morphology or cytoskeletal organization of eectors, which includes PAK, share a region of (Figure 1a). By contrast, wild type (but activated) sequence homology named the CRIB motif that Rac (Q61L Rac) induced extensive membrane ruing mediates binding to Rac and cdc42 (Burbelo et al., (Figure 1a), as did the Q61L/Y40C mutant (not 1995). These proteins bind to an N-terminal eector shown). In the presence of 10% serum, approximately domain in Rac and cdc42 spanning approximately 80% of control NIH3T3 cells showed signi®cant, residues 25 ± 45, similar to other Ras family members. sometimes extensive membrane ruing (Figure 1b). Recent reports have analysed Rac and cdc42 By 3 h after injection of DNA for Q61L/F37A Rac, variants that contain point mutations in their N- rues collapsed and cell spreading was slightly decreased. DNA for GFP alone or other control constructs including wild type Rac, Y40C/Q61L Rac and PAK did not cause similar changes (not shown). Correspondence: MA Schwartz Received 6 August 1997; revised 16 March 1998; accepted 17 March Quantitation of results showed that the percentage of 1998 cells exhibiting membrane rues decreased from Dominant negative active Rac mutant MA Schwartz et al 626
Figure 2 Time lapse analysis. An NIH3T3 cell in 10% serum was injected with Q61L/F37A Rac DNA and photographed at the times indicated. Small arrowheads indicate retraction ®bers. Large arrowheads indicate ®lopodia that grow and translocate.
These ®lopodia were observed to translocate and to disappear while other ®lopodia formed. Time lapse analysis of cells expressing Q61L/F37A Rac showed that 11 out of 11 showed formation, growth and movement of ®lopodia. Time lapse analysis of cells expressing Q61L/F37A Rac in low serum showed a drastic reduction in ®lopodia formation (not shown). For comparison, the Rac N17 mutant was exam- ined. This mutant had no discernible eect on cell Figure 1 NIH3T3 cells. (a) NIH3T3 cultured on glass coverslips morphology or cytoskeletal organization in serum for 24 h in 0.25% serum were uninjected (control) or were starved cells (Figure 1a). When expressed in cells in injected with cDNA for Q61L/F37A Rac (200 ng/ml), N17 Rac (200 ng/ml) or Q61L Rac (50 ng/ml). They were photographed 10% serum, it also induced loss of membrane rues using phase contrast optics after 4 h, or ®xed and stained as and lamellipodia (Figure 1b). In many cases, similar indicated. Arrows indicate expressing cells in cases where more actin-rich thin processes were seen in ®xed cells. than one cell is present. (b) NIH3T3 cells in 10% serum were Processes induced by N17 Rac were generally less injected (control), injected with Q61L/F37A Rac at 200 ng/ml, or dramatic, however. Furthermore, time lapse analysis with N17 Rac cDNA at the same concentration. Cells were ®xed after 3 h. (a) phase contrast microscopy. (b) cells stained with showed little or no formation or growth of ®lopodia rhodamine-phalloidin to detect F-actin. Bars=20 mm (not shown); only one out of 12 cells examined showed growth of ®lopodia. Thus, Q61L/F37A Rac causes loss of membrane rues but the cellular responses are 79+3% to 10+2% (n=3). Even in the low percentage distinct from N17 Rac. of Q61L/F37A Rac-expressing cells that continued to show some membrane ruing, the extent of ruing Blebbing was substantially decreased. Additional experiments con®rmed that expression of Rac Q61L/F37A induced At later times after expression of Q61L/F37A Rac in PAK activation in these cells (GM Bokoch, unpub- NIH3T3 cells, membrane blebbing was apparent. By lished data), as report (Lamarche et al., 1996; 7 h after injection, more than 50% of injected cells had Westwick et al., 1997). extensive blebbing (Figure 3a). This eect was observed Cells expressing Q61L/F37A Rac showed thin only at higher doses of DNA: concentrations below processes that resembled ®lopodia or retraction ®bers. 50 ng/ml were highly eective at inducing loss of Staining with rhodamine-phalloidin revealed that these membrane rues and formation of ®lopodia but did extensions contained actin (Figure 1b). To determine not trigger bleb formation, whereas 100 ng/ml or higher whether these processes were ®lopodia or retraction triggered blebbing in the majority of injected cells. ®bers, cell morphology was analysed by time lapse Rhodamine-phalloidin staining showed that these photography. Figure 2 shows that a cell expressing membrane blebs had signi®cant F-actin around their Q61L/F37A Rac contained retraction ®bers but clearly periphery (not shown). Even extensively blebbed cells, extended ®lopodia from another region of the cell. however, retained actin stress ®bers (Figure 3a), Dominant negative active Rac mutant MA Schwartz et al 627 suggesting that cells were not undergoing apoptosis. than uninjected cells but was similar to cells expressing Indeed, staining these cells for fragmented DNA using wild type Q61L Rac (Table 1). It should be noted, the TUNEL assay revealed no speci®c staining (Figure however, that higher doses of DNA that induce 3b). In addition, the nuclei remained intact, unlike blebbing do appear to inhibit replication. Cells those of apoptotic cells where chromatin condenses expressing GFP alone showed an increase in cell and the nuclei fragment. After 24 h, blebbing was number over 16 h of 51+14% (n=2), compared to noticeably diminished, but even cells that still blebbed 10+14% for cells expressing Q61L/F37A Rac. remained viable and showed no signs of nuclear Injection of control cDNA's (GFP or empty vector) condensation (Figure 3a). To con®rm that apoptotic did not inhibit cell growth (not shown). These results cells were not lost during the extended incubation, cells suggest that membrane blebbing or other eects of were injected with a mixture of Q61L/F37A Rac DNA high doses of Q61L/F37A Rac DNA are growth (200 ng/ml) plus DNA coding for GFP (20 ng/ml). The inhibitory, but these eects are most likely indirect. number of ¯uorescent cells was scored at 4 ± 6 h after injection and then again at 24 h. The total number of Swiss 3T3 cells ¯uorescent cells in two experiments increased from 49 ± 55 (10+14% increase). This increase is most likely due Because many studies of Rac and its mutants have to cell division. Taken together, these results indicate been carried out in Swiss 3T3 cells, we also examined that membrane blebs induced by Q61L/F37A are not the eects of Q61L/F37A in this system. Swiss 3T3 due to apoptosis. cells were maintained in serum-free medium for 24 h, then were injected with Rac cDNA. After 3.5 h, cells were treated with PDGF to induce membrane ruing. Cell growth Uninjected cells without PDGF show no ruing Rac has also been found to play a role in cell cycle (Figure 4). Most cells had a band of actin staining progression (Olson et al., 1995). The Q61L/F37A parallel to the edges, but some cells also had mutant did not induce DNA synthesis in starved cells perpendicular ®lament bundles that formed short (Lamarche et al., 1996; Westwick et al., 1997), but projections. Upon addition of PDGF, cells showed eects in growing cells were not examined. To extensive ruing as expected (Figure 4). Cells determine whether dominant negative eects on DNA expressing Q61L/F37A Rac in the absence of PDGF synthesis occur, cells in 10% serum were injected with tended to have more perpendicular ®lament bundles cDNA for Q61L/F37A Rac at 50 ng/ml, a concentra- leading to the edge of the cell; in higher expressors, tion that eciently blocks ruing but does not induce there also appeared to be some increase in poorly blebbing. As a control, wild type Q61L Rac was also organized actin ®laments throughout the cell, though examined. Eight hours after injections, BdUr was this eect was not dramatic. Upon addition of PDGF, added to the medium to label cells in S phase. They Q61L/F37A Rac-expressing cells showed virtually no were ®xed after 16 h of labeling and stained for both induction of rues. Quantitation of these results the myc epitope tag to detect expressing cells and with showed that ruing was reduced from 79+4% for an antibody to BdUr. The level of DNA synthesis in uninjected cells to 8+7% (n=4) for cells expressing Q61L/F37A Rac-expressing cells was slightly lower Q61L/F37A Rac; a control in which cells were injected with empty vector plus GFP DNA showed 80+4% ruing after treatment with PDGF. Thus, Q61L/F37A Rac speci®cally blocked PDGF-induced ruing in Swiss 3T3 cells. High doses of Q61L/F37A Rac DNA did not induce membrane blebbing at later times in Swiss 3T3 cells.
Discussion
The major conclusion from these studies is that an activated (Q61L) variant of Rac1 containing the F37A point mutation inhibits serum- or PDGF-induced membrane ruing in NIH3T3 and Swiss 3T3 cells. This variant therefore not only fails to induce ruing but functions as a dominant negative in this regard.
Table 1 Eect of Rac mutants on DNA synthesis Cells % Labeled Uninjected 88+1 Q6IL/F37A Rac 74+5 Figure 3 Blebbing without apoptosis. NIH3T3 cells were injected Q6IL Rac 69+2 with Q61L/F37A Rac cDNA at 200 ng/ml as above and ®xed after 7 or 24 h as indicated. DNA fragmentation was assayed by NIH3T3 cells were injected with cDNA or left uninjected as TUNEL staining. Cells treated with DNAase as a positive control indicated. After 8 h cells were labeled with BdUr for 16 h, ®xed stain brightly whereas cell expressing Q61L/F37A Rac do not and stained. The percent of control or Rac-expressing cells that had show detectable staining. Phase, bar=10 mm; ¯uorescence, labeled nuclei were scored. Values are means+range from two bar=5 mm experiments Dominant negative active Rac mutant MA Schwartz et al 628 would be hard to detect in NIH3T3 cells since in low serum these cells retain a well organized cytoskeleton. These results indicate that previous studies utilizing this mutant should be interpreted with caution. For example, the Q61L/F37A mutant may still associate with or activate some eectors of membrane ruing but downstream events would be blocked or masked by the dominant negative eects. Published work has shown that activation of PAK may mediate some eects of Rac or cdc42 on the cytoskeleton (Sells et al., 1997). Q61L/F37A Rac activates PAK, but may simultaneously block subsequent PAK-induced cytos- keletal rearrangements. Clearly the behaviour of this Rac variant is unexpected and points towards a more complex mechanism of action than previously sus- pected. Furthermore, a number of studies have used point mutants of Rac and other Ras superfamily members to correlate eects on downstream eectors and cell functions such as growth, transformation or migration. The observation that an eector domain point mutant can activate one pathway but block another introduces a signi®cant complication into the interpretation of the results from such experiments.
Figure 4 Swiss 3T3 cells. Swiss 3T3 cells were kept 24 h in serum-free DMEM (control) then injected with DNA for GFP at Materials and methods 20 ng/ml plus Q61L/F37A Rac at 50 ng/ml or N17 Rac at 100 ng/ ml. After 4 h, some cells were stimulated with 25 mg/ml PDGF for Reagents 20 min as indicated. They were then ®xed with formaldehyde and stained. Expressing cells were detected by GFP ¯uorescence and Expression vectors for Rac and mutants thereof in pCMV1 F-actin was visualized with rhodamine-phalloidin. Arrowheads were generously provided by Dr Alan Hall (University denote the expressors where more than one cell is present. College, London). The GFP vector EGFP-C1 was a gift Membrane rues were induced in uninjected cells, but not in cells from Dr Sanford Shattil (Scripps Research Institute). expressing Q61L/F37A or N17 Rac PDGF-BB and other reagents were purchased from Sigma Chemicals (St Louis, MO).
Additional eects on the cytoskeleton and/or mem- Cells brane dynamics were also observed, speci®cally NIH3T3 and Swiss 3T3 cells were grown in Dulbecco's membrane blebbing and increased formation of Modi®ed Eagles Medium (DMEM) with 10% bovine calf structures that resemble ®lopodia. serum. Swiss 3T3 cells were switched to serum-free DMEM Membrane blebbing clearly was not due to 24 h before the experiment. They were treated with 25 ng/ apoptosis, since cells showed no signs of DNA ml PDGF for 10 min to induce ruing, then ®xed as fragmentation or nuclear condensation and survived. described. In other systems, blebbing can be induced by changes in membrane-cytoskeleton connections, or in cell Injection and staining volume regulation (Cunningham, 1995; Fedier and Cells were injected into the nucleus with Rac DNAs at Keller, 1997; Myat et al., 1997). These eects were 0.2 mg/ml and GFP DNA was injected at 0.05 mg/mlunless entirely unrelated to apoptosis. Q61L/F37A Rac most otherwise noted. Cells were ®xed in 2% formaldehyde for likely perturbs one or both of these systems, but these 20 min and rinsed with phosphate-buered saline (PBS). eects are at present not understood. Cells were permeabilized with 0.4% Triton X-100 in PBS Formation of ®lopodia in response to Q61L/F37A for 2 min at room temperature. Expressed myc-tagged Rac Rac was dependent on serum and was distinct from protein was detected by staining with monoclonal anti-myc eects of N17 Rac. Membrane rues and lamellipodia ascites diluted 1 : 300 in PBS with 10% normal goat serum for 45 min, followed by anity puri®ed FITC-anti-mouse contain bundles of actin ®laments that extend outward IgG (Cappel, Durham, NC) at 1 : 200 under the same and that are connected by web-like regions, containing conditions. Actin was labeled with Rhodamine-phalloidin ®ne meshworks of micro®laments. The simplest (Molecular Probes, Eugene, OR) at 1 : 100. For conven- explanation for the observed eects is that Q61L/ tional microscopy, cells were photographed on a Nikon F37A Rac inhibits the formation of the connecting web Diaphot ¯uorescence microscope using a 406 oil immer- regions but not the rod-like projections. Thus, sion lens. For confocal microscopy, cells were examined structures similar to ®lopodia could still form and using a BioRad 1024 laser scanning microscope with a 406 translocate. This could occur if Q61L/F37A Rac forms oil immersion lens. inactive complexes with a subset of Rac eectors, thereby blocking some of the eects of endogenous Abbreviations Rac. Q61L/F37A Rac also appears to induce weak but GFP, green ¯uorescent protein; PDGF, platelet derived detectable actin polymerization or reorganization in growth factor; TUNEL, TdT-mediated UTP nick end starved Swiss 3T3 cells, indicating that it may have labeling; JNK/SAPK, jun c-terminal kinase/stress acti- some dominant eects on the cytoskeleton. Such eects vated protein kinase. Dominant negative active Rac mutant MA Schwartz et al 629 Acknowledgements London) for generously providing Rac mutants. This work We would like to thank Gary Bokoch (Scripps Research was supported by NIH grant R01 GM27214 to MAS and Institute) for helpful discussions and for communicating AHA grant 95001270 to JEM. unpublished data. We thank Alan Hall (University College,
References
Bagrodia S, Derijard B, Davis RJ and Cerione RA. (1995). J. Myat MM, Anderson S, Allen LAH and Aderem A. (1997). Biol. Chem., 270, 27995 ± 27998. Curr. Biol., 7, 611 ± 614. Burbelo PD, Drechsel D and Hall A. (1995). J. Biol. Chem., Nobes C and Hall A. (1994). Curr.Op.Gen.Dev.,4, 77 ± 81. 270, 29071 ± 29074. Olson MF, Ashworth, A and Hall, A. (1995). Science, 269, Coso OA, Chiariello M, Wu JC, Teramoto H, Crespo P, Xu 1270 ± 1272. N, Miki T and Gutkind JS. (1995). Cell, 81, 1137 ± 1146. Ridley AJ. (1996). Curr. Biol., 6, 1256 ± 1264. Cunningham CC. (1995). J. Cell Biol., 129, 1589 ± 1599. Sells MA, Knaus UG, Bagrodia S, Ambrose DM, Bokoch Fedier A and Keller HU. (1997). Cell Motil. Cytoskel., 37, GM and Chereno J. (1997). Curr. Biol., 7, 202 ± 210. 326 ± 327. Westwick JK, Lambert QT, Clark GJ, Symons M, VanAelst Lamarche N, Tapon N, Stowers L, Burbelo PD, Aspenstrom L, Pestell RG and Der CJ. (1997). Mol. Cell. Biol., 17, P, Bridges T, Chant J and Hall A. (1996). Cell, 87, 519 ± 1324 ± 1335. 529. Zhang S, Han J, Sells MA, Cherno J, Knaus UG, Ulevitch Manser E, Leung T, Salihuddin H, Zhao H and Lim L. RJ and Bokoch GM. (1995). J. Biol. Chem., 270, 23934 ± (1994). Nature, 367, 40 ± 46. 23936. Minden A, AL, Claret FX, Abo A and Karin M. (1995). Cell, 81, 1147 ± 1157.