- Email: [email protected]
A novel Cdc42Hs mutant induces cellular transformation
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Brief Communication
A novel Cdc42Hs mutant induces cellular transformation R. Lin, S. Bagrodia, R. Cerione and D. Manor Cdc42Hs is a small GTPase of the Rho-subfamily, which regulates signaling pathways that influence cell morphology and polarity, cell-cycle progression and transcription [1–7]. An essential role for Cdc42Hs in cell growth regulation has been suggested by the finding that the Dbl oncoprotein is an upstream activator — a guanine nucleotide exchange factor (GEF) — for Cdc42Hs [8,9], and that activated mutants of the closely related GTPases Rac and Rho are transforming [10–13]. As we were unable to obtain significant over-expression of GTPase-defective Cdc42Hs mutants, we have generated a mutant, Cdc42Hs(F28L), which can undergo spontaneous GTP–GDP exchange while maintaining full GTPase activity, and thus should exhibit functional activities normally imparted by Dbl. In cultured fibroblasts, Cdc42Hs(F28L) activated the c-Jun kinase (JNK1) and stimulated filopodia formation. Cells stably expressing Cdc42Hs(F28L) also exhibited several hallmarks of transformation — reduced contact inhibition, lower dependence on serum for growth, and anchorageindependent growth. Our findings indicate that Cdc42Hs plays a role in cell proliferation, and is a likely physiological mediator of Dbl-induced transformation. Address: Department of Pharmacology, Veterinary Medical Center, Cornell University, Ithaca, New York 14853-6401, USA. Correspondence: D. Manor Email: [email protected] Received: 22 May 1997 Revised: 27 June 1997 Accepted: 30 July 1997 Current Biology 1997, 7:794–797 http://biomednet.com/elecref/0960982200700794 © Current Biology Ltd ISSN 0960-9822
Results and discussion Cdc42Hs(F28L) rapidly cycles between the GTP- and GDP-bound states
In Ras-related proteins, highly conserved residue Phe28 is located in the nucleotide-binding pocket, where it has been proposed to stabilize the guanine ring moiety through pi-orbital interactions [14–16]. Mutation of Phe28 in Ras was shown to increase the GDP dissociation constant ~150-fold and activate the protein in vivo [17]. We replaced Phe28 in Cdc42Hs with a leucine and purified the mutant protein from over-expressing bacteria [18]. We first examined the ability of this mutant to undergo [35S]GTPγS—GDP exchange (activation) using a filterbinding assay (Figure 1a). As is typically the case for GTPbinding proteins, wild-type Cdc42Hs shows minimal [35S] GTPγ-S binding in the presence of physiological Mg2+
concentrations [19]. In vivo, GEFs are needed to stimulate GDP dissociation and accelerate GTP–GDP exchange; this can be mimicked in vitro by the addition of EDTA, which chelates the tightly bound Mg2+, thereby weakening GDP binding and facilitating nucleotide exchange (Figure 1a). Cdc42Hs(F28L), however, exhibits near maximal [35S]-GTPγS binding even in the presence of 10 mM MgCl2 (Figure 1a). These data suggest that Cdc42Hs(F28L) has a greater nucleotide exchange activity than the wild-type protein. In Figure 1b, the nucleotide exchange kinetics are directly monitored by the enhancement in fluorescence of the GDP analog Mant-GDP on binding to Cdc42Hs [20]. Nucleotide exchange occurs very slowly in wild-type Cdc42Hs, but the reaction can then be markedly stimulated (27-fold) by addition of Dbl. In the case of Cdc42Hs(F28L), however, exchange is rapid, even in the absence of added Dbl and despite the presence of 5 mM MgCl2 in the assay. This is further demonstrated by kinetic analyses of nucleotide exchange (Figure 1c). These data clearly indicate that Cdc42Hs(F28L) is capable of spontaneously undergoing guanine nucleotide exchange. To determine the GTPase activity of Cdc42Hs(F28L), we used a spectrofluorimetric assay that monitors the nucleotide-sensitive emission of Trp97 [20]. Both the intrinsic and GAP-stimulated GTPase activities of the Cdc42Hs(F28L) were essentially identical to wild type (Figure 1d). Cdc42Hs(F28L) activates JNK in vivo
Cdc42Hs initiates a protein kinase cascade that leads to activation of the MAP kinases, c-Jun kinase (JNK1) and p38 [4–7]. To see whether Cdc42Hs(F28L) undergoes spontaneous activation in cells and stimulates JNK1 activity, COS-7 cells were transiently co-transfected with Cdc42Hs mutants and Flag-tagged JNK1. JNK1 was then immunoprecipitated and assayed for kinase activity in vitro using GST-c-Jun as a substrate [7]. As previously reported [7], activated Cdc42Hs, Cdc42Hs(Q61L), stimulated JNK activity > 2-fold more than wild-type (Figure 2a). Cdc42Hs(F28L) also clearly stimulated JNK1 activity, often yielding a stronger stimulation than observed with the GTPase-defective Cdc42Hs mutant. We established NIH3T3 cell lines stably expressing HAtagged Cdc42Hs(F28L). Two clones showing significantly different Cdc42Hs(F28L) expression levels were studied further. Using agarose-immobilized GST-c-Jun as an affinity reagent, endogenous JNK was precipitated from lysates of the stably transfected cell lines and assayed in vitro. As shown in Figure 2b, we found a correlation between the expression levels of Cdc42Hs(F28L) and endogenous JNK
Brief Communication
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Cdc42Hs(F28L) is fast-cycling in vitro. (a) Cdc42Hs(F28L) spontaneously binds GTP-γ-35S. Wild-type or F28L Cdc42Hs proteins (6 µM) were incubated with 50 µM GTP-γ-35S (357 Ci/mmole) in the absence (solid bars) or presence (hatched bars) of 20 mM EDTA, and protein-bound radioactivity was measured as described [24]. Shown are the means of two duplicate measurements (S.E.M.< 10%). (b) Nucleotide exchange on Cdc42Hs(F28L) is not dependent on GEFs. The kinetics of nucleotide exchange on wild-type and F28L Cdc42Hs proteins were assayed using a fluorescence assay described earlier [16]. Cdc42Hs (0.5 µM) was added to a cuvette
containing 0.25 µM Mant-GDP at the indicated arrows. Dbl (100 nM) and unlabeled GDP (100 µM) were added where indicated. The rising portions of the fluorescence change were fit to a single exponential process, and the extracted rate constant is shown in (c). (d) Cdc42Hs(F28L) exhibits normal GTP hydrolysis. The GTPase reaction was measured using the fluorescence assay described previously [16]. To 1 µM Cdc42Hs and 2 mM GTP, EDTA was added to induce nucleotide exchange, and hydrolysis was initiated by addition of 15 mM MgCl2. Initial rates in the presence (hatched bars) or absence (solid bars) of 100 nM Cdc42-GAP are shown.
activity, further demonstrating that Cdc42Hs(F28L) is spontaneously activated in cells.
activated, GTPase-defective forms of Cdc42Hs into Swiss3T3 cells [3]. It has also been reported that Dbl can activate Rac in vivo, and that the resultant production of lamellipodia can obscure the detection of filopodia. That expression of Cdc42Hs(F28L) does not cause significant lamellipodia formation then argues against the notion that the Dbl-mediated activation of Rac is a downstream event from the activation of Cdc42Hs in NIH3T3 cells [3, 23].
Morphological changes associated with Cdc42Hs(F28L) expression
NIH3T3 cells stably expressing Cdc42Hs(F28L) exhibit some morphological characteristics very similar to those of cells expressing the oncogenic Dbl. Approximately 9% of the total cell population is composed of giant, multi-nucleated cells (see Figures 3a,b), similar to what was observed in the original studies of Dbl [21,22]. However, while expression of oncogenic Dbl gives rise to ‘membrane-ruffles’ (lamellipodia), cells in sub-confluent cultures expressing Cdc42Hs(F28L) show prominent ‘spike-like’ extensions (filopodia) into the surrounding medium (Figure 3c). This is similar to what was observed on micro-injection of
Growth characteristics of Cdc42Hs(F28L)-expressing cells
Figure 4a compares the growth rates of different Cdc42Hs(F28L)-expressing cell lines with those of Dblexpressing lines and control NIH3T3 cells. While there is no significant difference between all cell lines examined in the presence of high serum (10%), a strikingly different growth profile is observed in reduced-serum medium
Phosphorylation of GST-c-Jun α-Flag immunoblot
anti-Cdc42 antibody to assay the Cdc42 expression level in the different clones. The migrational positions of the endogenous and recombinant (HA-tagged) Cdc42Hs proteins
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Cdc42Hs(F28L) is activated in vivo. (a) Transient transfection of cells with Cdc42Hs(F28L) results in JNK activation. COS-7 cells were transiently transfected with pCDNA3 constructs containing the indicated Cdc42Hs variants, together with pCDNA3Flag-JNK. JNK was immunoprecipitated from lysates and assayed in vitro using recombinant GST-c-Jun as a substrate [7]. (b) Endogenous JNK activity is elevated in stably transfected Cdc42Hs(F28L) cell lines. Lysates from different clones of NIH3T3 cells stably transfected with HA-Cdc42Hs(F28L) or HA-Dbl were assayed for endogenous JNK activity as described earlier [7]. Lower panels: lysates were immunoblotted with anti-HA or
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Figure 3 3T3
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Morphological characteristics of Cdc42Hs (F28L)-expressing fibroblasts. NIH3T3 cells stably transfected with pCDNA3-Neo, pJ4HCdc42Hs(F28L), and pJ4H-Dbl were fixed, stained with Trypan Blue and visualized by light microscopy: (a) magnification 100X; (b) highmagnification (200X) view of giant multi-nucleated cells.
(c) Cdc42Hs(F28L)- and Dbl-expressing cell lines were fixed and stained specifically for F-actin using Texas-red-conjugated phalloidin stain (magnification 400X). Lamellipodia (l) and actin micro-spikes (filopodia; m) are indicated by arrows.
(0.5–1.0%). Control (neomycin-selected) fibroblasts showed virtually no detectable growth, whereas Cdc42Hs(F28L) expressing cells displayed significant proliferation, which correlated with the levels of Cdc42Hs(F28L) expression (compare Figure 4a with Figure 2b). The highest expressing Cdc42Hs(F28L) cell line approached the growth rate of cells transformed with the Dbl oncogene, and showed a significantly higher saturation density than control cells. Fibroblasts stably expressing Cdc42Hs(F28L) also showed anchorage-independent growth when cultured in semi-solid agar (Figures 5a,5b), which correlated well with the level of Cdc42Hs(F28L) expression. Taken together, these data show that cells expressing Cdc42Hs(F28L) exhibit three of the hall-marks of transformation: diminished contact inhibition, reduced dependence on serum for growth and anchorage-independent growth.
of effects of oncogenic Dbl, including the generation of giant, multi-nucleate cells and growth in low serum or in soft-agar. These results suggest that Cdc42Hs is a true cellular mediator of Dbl transformation and is capable of stimulating cell growth. While completing this work, a study was reported that reached a similar conclusion [24]. In that study, relatively low-level expression of Cdc42Hs(G12V) in Rat1 fibroblasts was shown to result in anchorage-independent growth and tumorigenicity, although, interestingly, not in serum-independent growth. We have tried to generate NIH3T3 cell lines that stably express GTPase-defective Cdc42Hs mutants and have failed; in fact, all of our attempts to overexpress GTPase-defective Cdc42Hs (at levels that exceed endogenous Cdc42Hs levels) have yielded significant growth inhibition. This may mean that over-expression of GTPase-defective Cdc42Hs, perhaps by sequestering or persistently stimulating target molecules, has deleterious effects on cell growth. Nonetheless, given the relatively straightforward capability for generating stable cell lines expressing Cdc42Hs(F28L), we now are in position to examine in detail how activated Cdc42Hs gives rise to a diversity of biological responses.
Conclusions In these studies, we have generated and biochemically characterized a mutant Cdc42Hs, Cdc42Hs(F28L), which undergoes accelerated GTP–GDP exchange. Expression of Cdc42Hs(F28L) in cells mimics a number Figure 4 (a)
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Fibroblasts stably expressing Cdc42Hs(F28L) exhibit diminished serum-dependence and contact inhibition. (a) Cell lines were cultured in the presence of 1% serum and at the indicated times trypsinized and counted. Data are representative of two experiments. Clone #21 has similar Cdc42Hs(F28L) expression levels as clone #20 (see Figure 2). (b) Saturation density of Cdc42Hs(F28L)expressing NIH3T3 cells. Cells were counted after 5 days of culture in DMEM (5% calf serum). Data are the average of four plates .
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Brief Communication
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Figure 5
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Materials and methods [35S] GTP-γ-S binding activities were measured as described earlier [8]. Kinetics of nucleotide exchange reactions and GTP hydrolysis were measured by the fluorescence assays described earlier [20]. Conditions for cell culture, transfections, immunoprecipitations and JNK assays were described in [7].
Supplementary material Detailed experimental protocols are published with this paper on the internet.
Acknowledgements This work was supported by Grant GM47458 from the National Institutes of Health, USA.
References 1. Olson ME, Ashworth A, Hall A: An essential role for Rho, Rac, and CDC42 GTPases in cell cycle progression through G1. Science 1995, 269:53-62. 2. Kozma R, Ahmed S, Best A, Lim L: The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts Molec Cell Biol 1995, 15:1942-1952. 3. Nobes CD, Hall A: Rho, Rac and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 1995, 81:53-62. 4. Coso OA, Chiariell M, Yu JC, Teramoto H, Crespo P, Xu N, Miki T, Gutkind JS: The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell 1995, 81:1137-1146. 5. Minden A, Lin A, Claret FX, Abo A, Karin M: Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42. Cell 1995, 81:1147-1157. 6. Zhang S, Han J, Sells NA, Chernoff J, Knaus UG, Ulevitch RJ, Bokoch GM: Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1. J Biol Chem 1995, 270:23934-23936. 7. Bagrodia S, Derigard B, Davis RJ, Cerione RA: Cdc42 and PAKmediated signaling leads to Jun kinase and p38 mitogenactivated protein kinase activation. J Biol Chem 1995, 270:27995-27998. 8. Hart MJ, Eva A, Evans T, Aaronson SA, Cerione RA: Catalysis of guanine nucleotide exchange on the Cdc42Hs protein by the dbl oncogene product. Nature 1991, 354:311-314. 9. Hart MJ, Eva A, Zangrilli D, Aaronson SA, Evans T, Cerione RA, Zheng Y: Cellular transformation and guanine nucleotide exchange activity are catalyzed by a common domain on the dbl oncogene product. J Biol Chem 1994, 269:62-65. 10. Avraham H, Weinberg R: Characterization and expression of the human rhoH12 gene product. Molec Cell Biol 1989, 9:2058-2066. 11. Khosravi-Far R, Solski PA, Clarck GJ, Kinch MS, Der CJ: Activation of Rac1, RhoA, and mitogen-activated protein kinase is required for Ras transformation. Molec Cell Biol 1995, 15:6443-6453.
F28L
Dbl
(b) Growth in soft agar (%)
Fibroblasts stably expressing Cdc42Hs(F28L) exhibit anchorageindependent growth. (a) NIH3T3 cells that stably express Cdc42Hs(F28L), Dbl or the pCDNA3 vector were mixed with media supplemented with 0.3% agar and 10% fetal calf serum, and plated on top of a 0.5% agarose layer (magnification 40X). (b) Growing colonies were scored after 14 days for the various cell-lines shown in Figure 4. Values shown are the average of two to three independent experiments. 100% represents 90-140 cells counted.
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12. Qiu RG, Chen J, McCormick F, Symons M: A role for rho in ras transformation. Proc Natl Acad Sci USA 1995, 92:11781-11785. 13. Qiu RG, Chen J, Kirn D, McCormick F, Symons M: An essential role for Rac in Ras transformation. Nature 1995, 374:457-459. 14. Pai EF, Kabsch W, Krengel U, Holmes KC, John J., Wittinghofer A: Structure of the guanine nucleotide binding domain of the HaRas oncogene product p21 in the triphosphate conformation. Nature 1989, 341:209-214. 15. Hirshberg M, Stockley RW, Dodson G, Webb MR: The crystal structure of human Rac1, a member of the Rho-family, complexed with a GTP analogue. Nature Struct Biol 1997, 4:147-151. 16. Feltham J, Dotch V, Raza S, Manor D, Cerione R, Sutcliffe M, Wagner G, Oswald R: Definition of the switch surface in the solution structure of Cdc42Hs. Biochemistry 1997, 36:8755-8766. 17. Reinstein J, Schlichting I, Frech M, Goody R, Wittinghofer A: p21 with a phenylalanin 28 leucine mutation reacts normally with the GTPase activating protein GAP but nevertheless has transforming properties. J Biol Chem 1991, 266:17700-17706. 18. Leonard DA, Sartoskar R, Wu JW, Cerione RA, Manor D: Use of a fluorescence spectroscopic read-out to characterize the interactions of Cdc42Hs with its target/effector, mPAK-3. Biochemistry 1997, 36:1173-1180. 19. Hall A, Self AJ: The Effect of Mg2+ on the guanine nucleotide exchange rate of p21 n-ras. J Biol Chem 1986, 261:10963-10965. 20. Leonard DA, Evans T, Hart M, Cerione RA, Manor D: Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy. Biochemistry 1994, 33:12323-12328. 21. Ron D, Zannini M, Lewis M, Wickner RB, Hunt LT, Graziani G, Tronick SR, Aaronson SA, Eva A: A region of proto-dbl essential for its transforming activity shows sequence similarity to a yeast cell cycle gene, CDC24, at the human breakpoint cluster gene, BCR. The New Biologist 1991, 3:372-379. 22. Zangrilli A, Eva A: Cell transformation by the dbl oncogene. Methods Enzymol 1995, 256:347-358. 23. Olson MF, Pasteris NG, Gorski JL, Hall A: Faciogenital dysplasia protein (FGD1) and Vav, two related proteins required for normal embryonic development, are upstream regulators of Rho GTPases. Curr Biol 1996, 6:1628-1633. 24. Qiu R, Abo A, McCormick F, Symons M: Cdc42 regulates anchorage-independent growth and is necessary for ras transformation. Molec Cell Biol 1997, 17:3449-3458.
®
Brief Communication
A novel Cdc42Hs mutant induces cellular transformation R. Lin, S. Bagrodia, R. Cerione and D. Manor Cdc42Hs is a small GTPase of the Rho-subfamily, which regulates signaling pathways that influence cell morphology and polarity, cell-cycle progression and transcription [1–7]. An essential role for Cdc42Hs in cell growth regulation has been suggested by the finding that the Dbl oncoprotein is an upstream activator — a guanine nucleotide exchange factor (GEF) — for Cdc42Hs [8,9], and that activated mutants of the closely related GTPases Rac and Rho are transforming [10–13]. As we were unable to obtain significant over-expression of GTPase-defective Cdc42Hs mutants, we have generated a mutant, Cdc42Hs(F28L), which can undergo spontaneous GTP–GDP exchange while maintaining full GTPase activity, and thus should exhibit functional activities normally imparted by Dbl. In cultured fibroblasts, Cdc42Hs(F28L) activated the c-Jun kinase (JNK1) and stimulated filopodia formation. Cells stably expressing Cdc42Hs(F28L) also exhibited several hallmarks of transformation — reduced contact inhibition, lower dependence on serum for growth, and anchorageindependent growth. Our findings indicate that Cdc42Hs plays a role in cell proliferation, and is a likely physiological mediator of Dbl-induced transformation. Address: Department of Pharmacology, Veterinary Medical Center, Cornell University, Ithaca, New York 14853-6401, USA. Correspondence: D. Manor Email: [email protected] Received: 22 May 1997 Revised: 27 June 1997 Accepted: 30 July 1997 Current Biology 1997, 7:794–797 http://biomednet.com/elecref/0960982200700794 © Current Biology Ltd ISSN 0960-9822
Results and discussion Cdc42Hs(F28L) rapidly cycles between the GTP- and GDP-bound states
In Ras-related proteins, highly conserved residue Phe28 is located in the nucleotide-binding pocket, where it has been proposed to stabilize the guanine ring moiety through pi-orbital interactions [14–16]. Mutation of Phe28 in Ras was shown to increase the GDP dissociation constant ~150-fold and activate the protein in vivo [17]. We replaced Phe28 in Cdc42Hs with a leucine and purified the mutant protein from over-expressing bacteria [18]. We first examined the ability of this mutant to undergo [35S]GTPγS—GDP exchange (activation) using a filterbinding assay (Figure 1a). As is typically the case for GTPbinding proteins, wild-type Cdc42Hs shows minimal [35S] GTPγ-S binding in the presence of physiological Mg2+
concentrations [19]. In vivo, GEFs are needed to stimulate GDP dissociation and accelerate GTP–GDP exchange; this can be mimicked in vitro by the addition of EDTA, which chelates the tightly bound Mg2+, thereby weakening GDP binding and facilitating nucleotide exchange (Figure 1a). Cdc42Hs(F28L), however, exhibits near maximal [35S]-GTPγS binding even in the presence of 10 mM MgCl2 (Figure 1a). These data suggest that Cdc42Hs(F28L) has a greater nucleotide exchange activity than the wild-type protein. In Figure 1b, the nucleotide exchange kinetics are directly monitored by the enhancement in fluorescence of the GDP analog Mant-GDP on binding to Cdc42Hs [20]. Nucleotide exchange occurs very slowly in wild-type Cdc42Hs, but the reaction can then be markedly stimulated (27-fold) by addition of Dbl. In the case of Cdc42Hs(F28L), however, exchange is rapid, even in the absence of added Dbl and despite the presence of 5 mM MgCl2 in the assay. This is further demonstrated by kinetic analyses of nucleotide exchange (Figure 1c). These data clearly indicate that Cdc42Hs(F28L) is capable of spontaneously undergoing guanine nucleotide exchange. To determine the GTPase activity of Cdc42Hs(F28L), we used a spectrofluorimetric assay that monitors the nucleotide-sensitive emission of Trp97 [20]. Both the intrinsic and GAP-stimulated GTPase activities of the Cdc42Hs(F28L) were essentially identical to wild type (Figure 1d). Cdc42Hs(F28L) activates JNK in vivo
Cdc42Hs initiates a protein kinase cascade that leads to activation of the MAP kinases, c-Jun kinase (JNK1) and p38 [4–7]. To see whether Cdc42Hs(F28L) undergoes spontaneous activation in cells and stimulates JNK1 activity, COS-7 cells were transiently co-transfected with Cdc42Hs mutants and Flag-tagged JNK1. JNK1 was then immunoprecipitated and assayed for kinase activity in vitro using GST-c-Jun as a substrate [7]. As previously reported [7], activated Cdc42Hs, Cdc42Hs(Q61L), stimulated JNK activity > 2-fold more than wild-type (Figure 2a). Cdc42Hs(F28L) also clearly stimulated JNK1 activity, often yielding a stronger stimulation than observed with the GTPase-defective Cdc42Hs mutant. We established NIH3T3 cell lines stably expressing HAtagged Cdc42Hs(F28L). Two clones showing significantly different Cdc42Hs(F28L) expression levels were studied further. Using agarose-immobilized GST-c-Jun as an affinity reagent, endogenous JNK was precipitated from lysates of the stably transfected cell lines and assayed in vitro. As shown in Figure 2b, we found a correlation between the expression levels of Cdc42Hs(F28L) and endogenous JNK
Brief Communication
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Cdc42Hs(F28L) is fast-cycling in vitro. (a) Cdc42Hs(F28L) spontaneously binds GTP-γ-35S. Wild-type or F28L Cdc42Hs proteins (6 µM) were incubated with 50 µM GTP-γ-35S (357 Ci/mmole) in the absence (solid bars) or presence (hatched bars) of 20 mM EDTA, and protein-bound radioactivity was measured as described [24]. Shown are the means of two duplicate measurements (S.E.M.< 10%). (b) Nucleotide exchange on Cdc42Hs(F28L) is not dependent on GEFs. The kinetics of nucleotide exchange on wild-type and F28L Cdc42Hs proteins were assayed using a fluorescence assay described earlier [16]. Cdc42Hs (0.5 µM) was added to a cuvette
containing 0.25 µM Mant-GDP at the indicated arrows. Dbl (100 nM) and unlabeled GDP (100 µM) were added where indicated. The rising portions of the fluorescence change were fit to a single exponential process, and the extracted rate constant is shown in (c). (d) Cdc42Hs(F28L) exhibits normal GTP hydrolysis. The GTPase reaction was measured using the fluorescence assay described previously [16]. To 1 µM Cdc42Hs and 2 mM GTP, EDTA was added to induce nucleotide exchange, and hydrolysis was initiated by addition of 15 mM MgCl2. Initial rates in the presence (hatched bars) or absence (solid bars) of 100 nM Cdc42-GAP are shown.
activity, further demonstrating that Cdc42Hs(F28L) is spontaneously activated in cells.
activated, GTPase-defective forms of Cdc42Hs into Swiss3T3 cells [3]. It has also been reported that Dbl can activate Rac in vivo, and that the resultant production of lamellipodia can obscure the detection of filopodia. That expression of Cdc42Hs(F28L) does not cause significant lamellipodia formation then argues against the notion that the Dbl-mediated activation of Rac is a downstream event from the activation of Cdc42Hs in NIH3T3 cells [3, 23].
Morphological changes associated with Cdc42Hs(F28L) expression
NIH3T3 cells stably expressing Cdc42Hs(F28L) exhibit some morphological characteristics very similar to those of cells expressing the oncogenic Dbl. Approximately 9% of the total cell population is composed of giant, multi-nucleated cells (see Figures 3a,b), similar to what was observed in the original studies of Dbl [21,22]. However, while expression of oncogenic Dbl gives rise to ‘membrane-ruffles’ (lamellipodia), cells in sub-confluent cultures expressing Cdc42Hs(F28L) show prominent ‘spike-like’ extensions (filopodia) into the surrounding medium (Figure 3c). This is similar to what was observed on micro-injection of
Growth characteristics of Cdc42Hs(F28L)-expressing cells
Figure 4a compares the growth rates of different Cdc42Hs(F28L)-expressing cell lines with those of Dblexpressing lines and control NIH3T3 cells. While there is no significant difference between all cell lines examined in the presence of high serum (10%), a strikingly different growth profile is observed in reduced-serum medium
Phosphorylation of GST-c-Jun α-Flag immunoblot
anti-Cdc42 antibody to assay the Cdc42 expression level in the different clones. The migrational positions of the endogenous and recombinant (HA-tagged) Cdc42Hs proteins
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Cdc42Hs(F28L) is activated in vivo. (a) Transient transfection of cells with Cdc42Hs(F28L) results in JNK activation. COS-7 cells were transiently transfected with pCDNA3 constructs containing the indicated Cdc42Hs variants, together with pCDNA3Flag-JNK. JNK was immunoprecipitated from lysates and assayed in vitro using recombinant GST-c-Jun as a substrate [7]. (b) Endogenous JNK activity is elevated in stably transfected Cdc42Hs(F28L) cell lines. Lysates from different clones of NIH3T3 cells stably transfected with HA-Cdc42Hs(F28L) or HA-Dbl were assayed for endogenous JNK activity as described earlier [7]. Lower panels: lysates were immunoblotted with anti-HA or
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Figure 3 3T3
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Morphological characteristics of Cdc42Hs (F28L)-expressing fibroblasts. NIH3T3 cells stably transfected with pCDNA3-Neo, pJ4HCdc42Hs(F28L), and pJ4H-Dbl were fixed, stained with Trypan Blue and visualized by light microscopy: (a) magnification 100X; (b) highmagnification (200X) view of giant multi-nucleated cells.
(c) Cdc42Hs(F28L)- and Dbl-expressing cell lines were fixed and stained specifically for F-actin using Texas-red-conjugated phalloidin stain (magnification 400X). Lamellipodia (l) and actin micro-spikes (filopodia; m) are indicated by arrows.
(0.5–1.0%). Control (neomycin-selected) fibroblasts showed virtually no detectable growth, whereas Cdc42Hs(F28L) expressing cells displayed significant proliferation, which correlated with the levels of Cdc42Hs(F28L) expression (compare Figure 4a with Figure 2b). The highest expressing Cdc42Hs(F28L) cell line approached the growth rate of cells transformed with the Dbl oncogene, and showed a significantly higher saturation density than control cells. Fibroblasts stably expressing Cdc42Hs(F28L) also showed anchorage-independent growth when cultured in semi-solid agar (Figures 5a,5b), which correlated well with the level of Cdc42Hs(F28L) expression. Taken together, these data show that cells expressing Cdc42Hs(F28L) exhibit three of the hall-marks of transformation: diminished contact inhibition, reduced dependence on serum for growth and anchorage-independent growth.
of effects of oncogenic Dbl, including the generation of giant, multi-nucleate cells and growth in low serum or in soft-agar. These results suggest that Cdc42Hs is a true cellular mediator of Dbl transformation and is capable of stimulating cell growth. While completing this work, a study was reported that reached a similar conclusion [24]. In that study, relatively low-level expression of Cdc42Hs(G12V) in Rat1 fibroblasts was shown to result in anchorage-independent growth and tumorigenicity, although, interestingly, not in serum-independent growth. We have tried to generate NIH3T3 cell lines that stably express GTPase-defective Cdc42Hs mutants and have failed; in fact, all of our attempts to overexpress GTPase-defective Cdc42Hs (at levels that exceed endogenous Cdc42Hs levels) have yielded significant growth inhibition. This may mean that over-expression of GTPase-defective Cdc42Hs, perhaps by sequestering or persistently stimulating target molecules, has deleterious effects on cell growth. Nonetheless, given the relatively straightforward capability for generating stable cell lines expressing Cdc42Hs(F28L), we now are in position to examine in detail how activated Cdc42Hs gives rise to a diversity of biological responses.
Conclusions In these studies, we have generated and biochemically characterized a mutant Cdc42Hs, Cdc42Hs(F28L), which undergoes accelerated GTP–GDP exchange. Expression of Cdc42Hs(F28L) in cells mimics a number Figure 4 (a)
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Fibroblasts stably expressing Cdc42Hs(F28L) exhibit diminished serum-dependence and contact inhibition. (a) Cell lines were cultured in the presence of 1% serum and at the indicated times trypsinized and counted. Data are representative of two experiments. Clone #21 has similar Cdc42Hs(F28L) expression levels as clone #20 (see Figure 2). (b) Saturation density of Cdc42Hs(F28L)expressing NIH3T3 cells. Cells were counted after 5 days of culture in DMEM (5% calf serum). Data are the average of four plates .
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Materials and methods [35S] GTP-γ-S binding activities were measured as described earlier [8]. Kinetics of nucleotide exchange reactions and GTP hydrolysis were measured by the fluorescence assays described earlier [20]. Conditions for cell culture, transfections, immunoprecipitations and JNK assays were described in [7].
Supplementary material Detailed experimental protocols are published with this paper on the internet.
Acknowledgements This work was supported by Grant GM47458 from the National Institutes of Health, USA.
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Fibroblasts stably expressing Cdc42Hs(F28L) exhibit anchorageindependent growth. (a) NIH3T3 cells that stably express Cdc42Hs(F28L), Dbl or the pCDNA3 vector were mixed with media supplemented with 0.3% agar and 10% fetal calf serum, and plated on top of a 0.5% agarose layer (magnification 40X). (b) Growing colonies were scored after 14 days for the various cell-lines shown in Figure 4. Values shown are the average of two to three independent experiments. 100% represents 90-140 cells counted.
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