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and Margaret M. Chou2*
Department of Pharmacology,and Department of Cell and Developmental Biology,1 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-61602
Received 23 May 2002/ Returned for modification 5 July 2002/ Accepted 18 November 2002
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ABSTRACT |
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INTRODUCTION |
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In recent years, much emphasis has been placed on understanding the molecular mechanisms by which Cdc42 and Rac exert their effects on the actin cytoskeleton. Early studies in mammalian cells revealed that expression of activated Cdc42 and Rac1 rapidly induces the formation of actin-based cell surface protrusions termed filopodia and lamellapodia (35, 45), respectively. These GTPases stimulate actin remodeling through multiple effectors (5). The best characterized are the WASP-related proteins, which include WASP and N-WASP, which are regulated directly by Cdc42, and WAVE, which is regulated by Rac via IRSp53. These proteins function by directly stimulating the actin nucleating activity of the Arp2/3 complex (30-32, 46, 49, 51). Another shared family of effectors are the p21-activated kinases (PAKs) (27). PAK1 phosphorylates and activates LIM kinase, which in turn phosphorylates and inhibits the actin depolymerizing activity of cofilin (11, 59). PAK1 has also been shown to phosphorylate myosin light chain, which stimulates actin/myosin-based contraction (43, 47). Another Cdc42-specific effector is MRCK, a kinase which also phosphorylates myosin light chain (21). Similarly, Rac uniquely targets several effectors, such as POR1, which functions through an unknown mechanism to promote actin-rich membrane protrusions (54). Another key effector of Rac is phosphatidylinositol-4-phosphate 5-kinase (52, 53), which catalyzes the production of phosphatidylinositol 4,5-bisphosphate, a potent regulator of various actin-binding proteins (50).
Studies in Saccharomyces cerevisiae have also provided fundamental insights into the functions of Cdc42. It was in yeast that Cdc42 was first shown to play a central role in actin organization and the establishment of cell polarity (15). Inactivation of Cdc42, through ablation of its guanine nucleotide exchange factor Cdc24, abolishes the polarization of actin cables that normally occurs during the budding process (4). Using a multicopy suppressor screen, Bi et al. identified proteins that could restore actin polarization in the absence of functional Cdc42. Two novel genes identified in this screen encoded the highly related proteins Msb3 and Msb4 (4). These proteins are functionally redundant, since deletion of either gene individually yielded no discernible phenotype. However, simultaneous disruption of MSB3 and MSB4 caused slowed growth and partial disorganization of the actin cytoskeleton (4). The mechanism by which Cdc42 signals to Msb3 and Msb4 remains undefined.
Sequence analysis of Msb3 and Msb4 revealed the presence of a domain that has alternatively been termed the PTM, TrH, Tbc, or Gyp domain (2, 3, 29, 44, 60). This domain is conserved throughout evolution, occurring in proteins from yeasts, Caenorhabditis elegans, Drosophila melanogaster, and mammals (34). Recent studies have revealed that these domains encode GTPase-activating proteins (GAPs) for Rab family G proteins (2, 9, 20, 42). Two PTM domain-containing proteins of particular interest were the human oncogenes, RN-tre and TRE17. RN-tre is a binding partner for the epidermal growth factor (EGF) receptor substrate, Eps8, which regulates trafficking of the receptor (20, 28). Overexpression of a truncated mutant of RN-tre causes anchorage-independent growth and enhances mitogenicity in response to EGF (28). The TRE17 gene was originally identified through a transformation-based assay using genomic DNA from a Ewing sarcoma (33). Specifically, expression of TRE17 in murine fibroblasts caused tumor formation in nude mice. However, its interaction with signaling pathways, regulation, and cellular function have remained completely unexplored.
In light of their homology to Msb3/Msb4, we explored whether TRE17 or RN-tre might function downstream of the Rho GTPases in mammalian cells. In the present study, we demonstrate that TRE17, but not RN-tre, is part of a novel effector pathway for Cdc42 and Rac1. Furthermore, we find that TRE17 is sufficient by itself to induce the accumulation of cortical actin and may therefore contribute to actin remodeling induced by Cdc42 and Rac1.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmids. The cDNAs encoding human Cdc42, Rac1, and RhoA and their mutant derivatives were subcloned into the mammalian expression vector pEBG to generate glutathione S-transferase (GST)-tagged fusion proteins (8). PBD/pEBG encodes the p21-binding domain (amino acids 70 to 117) of human PAK1.
The cDNA encoding TRE17 (clone 11-4 in pBluescript) was generously provided by Myriam Onno (33). The BamHI-SspI fragment encompassing the 5'-untranslated region and the TRE17 open reading frame was subcloned into a modified version of pcDNA3 containing a hemagglutinin (HA) epitope tag. The 5'-untranslated region was removed by digestion with BamHI-EcoRI and replaced with a PCR fragment restoring the normal TRE17 open reading frame. The sequence was confirmed by automated sequencing. HA-T17(447)/pcDNA3 encodes the first 447 amino acids of TRE17, HA-T17(
PTM)/pcDNA3 encodes a deletion of amino acids 80 to 303, and HA-T17(PTM)/pcDNA3 encompasses amino acids 80 to 327 of TRE17. In HA-T17(447/RK)/pCDNA, residue 149 was changed from arginine to lysine by PCR-mediated mutagenesis. In HA-T17(447/PA)/pCDNA, the proline-rich motifs P369RPVP373 and P389PGPP393 were mutated to ARAVA and PAGSA, respectively. Further details are available upon request.
ß-Tubulin/pEGFP was kindly provided by Paul Janmey. The cDNA encoding RN-tre was generously provided by Pier Paolo di Fiore.
Antibodies and reagents. For immunofluorescence of hemagglutinin (HA)-tagged proteins proteins, anti-HA (sc-805 [Santa Cruz] or monoclonal antibody 12CA5 [Roche]) was used; for immunoblotting, the former was used. Affinity-purified anti-GST antibody made against Escherichia coli-derived GST was used for all applications. Anti-ERK antibody was generously provided by John Blenis.
The secondary antibodies used were Cy3-conjugated donkey anti-mouse immunoglobulin G (IgG; heavy and light chain; Jackson Immunoresearch Laboratories), Alexa Fluor 633-conjugated goat anti-rabbit IgG (heavy and light chain; Molecular Probes), or FITC-conjugated donkey anti-rabbit IgG (heavy and light chain, Jackson Immunoresearch). F-actin was visualized with fluorescein isothiocyanate (FITC)-conjugated phalloidin (Molecular Probes).
Cytochalasin D (cytoD; 1 µM) and nocodazole (10 µg/ml; Sigma) were added for 1 or 2 h, respectively, where indicated.
GTPase pull-down assays. COS cells were seeded at a density of 14.0 x 105 per 100-mm dish for TRE17 or at a density of 5.0 x 105 cells per 60-mm dish for T17(447) and RN-tre(466). The following day, cells were transfected by using either 16 µg of total DNA and 48 µl of Lipofectamine per plate (TRE17) or 6 µg of total DNA and 18 µl of Lipofectamine [T17(447) and RN-tre(466)]. Cells were incubated for 4 to 5 h, washed, and then allowed to recover overnight in growth medium. Cells were washed twice in ice-cold phosphate-buffered saline (PBS) and then lysed in either 750 µl (TRE17) or 500 µl [T17(447) and RN-tre(466)] of buffer I (PBS; 5 mM MgCl2; 0.1% Triton X-100; pepstatin, leupeptin, and aprotinin at 0.7, 2, and 1 µg/ml, respectively; 1 mM dithiothreitol [DTT]; 1 mM phenylmethylsulfonyl fluoride) Lysates were incubated on ice for 10 min and then centrifuged at 16,000 x g for 10 min at 4°C. An aliquot (30 µl) of the clarified supernatant was removed for immunoblotting analysis, and the remainder was incubated with glutathione-Sepharose beads (50-µl bead volume; Amersham Pharmacia Biotech) for 4 h at 4°C with constant mixing. The beads were washed twice in buffer II (PBS; 5 mM MgCl2; 0.01% Triton X-100; pepstatin, leupeptin, and aprotinin; 1 mM DTT) and then thrice in buffer III (PBS; 5 mM MgCl2; pepstatin, leupeptin, and aprotinin; 1 mM DTT). Proteins were eluted by boiling in sample buffer (125 mM Tris [pH 6.8], 2% sodium dodecyl sulfate, 5% ß-mercaptoethanol, 7.5% glycerol, bromophenol blue). Expression of the GST-tagged GTPases and HA-tagged TRE17 or RN-tre constructs was detected by using enhanced chemiluminescence (Amersham Pharmacia Biotech).
Confocal immunofluorescence microscopy. HeLa cells were seeded on 10-mm coverslips at a density of 2 x 105 to 2.4 x 105 per 35-mm dish. The following day, cells were transfected with 2 µg of total DNA and 6 µl of FuGENE6 per plate. The next day cells were either harvested for immunofluorescence or were serum starved for 24 h, followed by various treatments as indicated. Cells were fixed in (3.7% formaldehyde, 10 mM HEPES [pH 7.4], 100 mM NaCl) for 15 min and then chased with 50 mM glycine (pH 7.4) for 10 min. Cells were washed twice with PBS, permeabilized in TBST (10 mM Tris [pH 7.5], 0.1% Triton X-100, 150 mM NaCl) for 5 min, and then blocked in blocking buffer (2% bovine serum albumin in TBST) for 15 min. Coverslips were incubated with primary antibodies diluted in blocking buffer for 2 to 3 h at room temperature and then washed three times in TBST. Samples were incubated with fluorescently labeled secondary antibodies diluted in blocking buffer for 1 h and then washed two times in TBST and once in distilled water. Coverslips were mounted with SloFade (Molecular Probes) and viewed on a Zeiss confocal microscope with LSM510 software at excitation wavelengths of 488 nm (FITC), 546 nm (Cy3), or 633 nm (Alexa Fluor 633).
For quantitation of TRE17 localization to the plasma membrane versus filaments, 100 cells expressing low to medium levels of TRE17 were scored in the absence or presence of EGF. Cells were categorized into as having predominantly filamentous staining, predominantly plasma membrane staining, discernible staining at both locations, or diffuse staining of TRE17. For quantification of TRE17-induced cortical actin accumulation, a cell was scored as positive if the cortical actin was significantly stronger than the neighboring untransfected cells, as typified by the images shown in Fig. 7. The percentages given are the fraction of cells exhibiting cortical actin accumulation relative to the total number of cells expressing the indicated construct. All quantifications were done for at least three independent experiments, and the means and standard errors are depicted in the graphs.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether TRE17 binds directly to Cdc42 and Rac1, yeast two-hybrid analysis was performed. By this assay, no interaction was observed between TRE17 and Cdc42V12 or RacV12, either with the isoprenylation motif of the GTPases intact or mutated (data not shown). Similarly, no binding was seen by using overlay assays with [32P]GTP-labeled Cdc42V12 or Rac1V12 as a probe (data not shown). Thus, it appears that the interaction between TRE17 and Cdc42/Rac1 is indirect and that TRE17 is recruited to the GTPases as part of an effector complex.
TRE17's subcellular distribution is regulated by the actin cytoskeleton and microtubules. In order to gain insight into the function of TRE17 and to explore how it might be regulated by Cdc42/Rac1, we examined its subcellular localization. Full-length HA-TRE17 was transiently transfected into HeLa cells, and its localization was analyzed by confocal microscopy. In all experiments, cells expressing low levels of TRE17 were analyzed to avoid artifacts that might arise from vast overexpression. In actively growing, unsynchronized HeLa cells, TRE17 exhibited a complex distribution, with localization at the plasma membrane, on filamentous structures, and punctate structures that were often aligned along the filaments (Fig. 2A).
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Activated Cdc42 and Rac1 induce localization of TRE17 to the plasma membrane. Since TRE17 associated with Cdc42 and Rac1, we sought to examine the effects of the GTPases on its subcellular localization. HA-TRE17 was cotransfected with the wild type (WT) or with constitutively active mutants of Rac1 or Cdc42, and their localization was analyzed by confocal microscopy. TRE17 colocalized strongly with WT Rac1 at the filamentous structures (Fig. 3A) and at the plasma membrane (data not shown). However, WT Rac1 had no effect on the subcellular distribution of TRE17. In contrast, coexpression with Rac1V12 led to a striking redistribution of TRE17, with a complete loss of filamentous staining and recruitment to the plasma membrane. TRE17 was recruited to both peripheral and dorsal ruffles, where it strongly colocalized with Rac1V12 (Fig. 3B). Treatment of cells with cytoD for 1 h reversed the effects of Rac1V12 and caused TRE17 to return to filamentous structures (Fig. 3C). Thus, Rac1V12-induced recruitment of TRE17 to the plasma membrane is dependent on integrity of the actin cytoskeleton.
Expression of Cdc42 mutants had distinct effects on TRE17 localization. WT Cdc42 induced recruitment of TRE17 to the plasma membrane, although a fraction of TRE17 remained cytosolic (Fig. 3D). Interestingly, in contrast to WT Rac1, Cdc42 did not localize to TRE17-positive filaments (Fig. 3D). More dramatic effects were seen with Cdc42V12, which induced essentially all of the TRE17 to relocalize to the plasma membrane. Furthermore, TRE17 colocalized with Cdc42V12 in filopodia (Fig. 3E). In distinct confocal sections, TRE17 did not colocalize with Cdc42 in the Golgi apparatus (data not shown). As seen with Rac1V12, Cdc42V12's ability to recruit TRE17 to the membrane was reversed by treatment with cytoD (Fig. 3F).
Together, these results indicate that activated Rac1 and Cdc42 can direct the relocalization of TRE17 to the plasma membrane in a manner that depends on integrity of the actin cytoskeleton.
TRE17 localization is regulated by growth factors. Mitogenic growth factors, such as EGF, rapidly induce changes in the actin cytoskeleton and are known to activate Cdc42 and Rac1 (7, 17, 45). We therefore sought to determine whether such factors could also regulate the localization of TRE17. TRE17-transfected HeLa cells were serum starved for 24 h and then stimulated with EGF for 10 min. In serum-starved cells, TRE17 was found predominantly on filamentous structures (Fig. 4A, left panel). However, stimulation with EGF led to a dramatic relocalization of TRE17 to the plasma membrane, accompanied by strong accumulation of F-actin at the cell cortex (Fig. 4A, middle panels). Quantification of TRE17 localization in the absence or presence of EGF is presented in Fig. 4B. Pretreatment of cells with cytoD prior to EGF stimulation completely abolished plasma membrane recruitment of TRE17 (Fig. 4A, right panel). This result demonstrates that physiological agonists such as growth factors can also regulate the localization of TRE17, again in a manner that depends on cortical actin accumulation.
EGF-induced recruitment of TRE17 to the plasma membrane requires Rac1 and Cdc42. Since Cdc42 and Rac1 are activated by EGF, we examined whether they might mediate EGF's effects on TRE17 relocalization. Coexpression of TRE17 with dominant-negative Rac1 (Rac1N17) or Cdc42 (Cdc42N17) led to an altered subcellular distribution of TRE17 under starved conditions. In Rac1N17 coexpressing cells, TRE17 was found predominantly on filamentous structures, but weak plasma membrane localization was also observed (data not shown). Stimulation of these cells with EGF resulted in no further increase in plasma membrane staining of TRE17, and a significant amount of TRE17 remained on filamentous structures (Fig. 5B). Thus, Rac1N17 partially blocked EGF-induced relocalization of TRE17. The effects of Cdc42N17 were more complicated, in part because many of the cells did not appear healthy. Under starved conditions, Cdc42N17 abolished the localization of TRE17 to filamentous structures and led to a weak recruitment to the plasma membrane (data not shown). A significant fraction of TRE17 was also found in intracellular aggregates (Fig. 5C). However, in the few healthy-looking cells coexpressing Cdc42N17 and TRE17, EGF failed to further enhance plasma membrane staining of TRE17 (Fig. 5C).
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Structural determinants of TRE17 localization.
To determine the requirements for its subcellular localization, structure-function analysis of TRE17 was performed. TRE17 contains a PTM domain near its N terminus, as well as a proline-rich domain that contains potential SH3 or WW domain binding sites (24). Three deletion constructs were generated as depicted in Fig. 6A. These mutants were transfected into HeLa cells, and their localization analyzed under serum-starved conditions. As noted above, full-length TRE17 was found predominantly on filamentous structures (Fig. 6B). However, a mutant expressing the first 447 amino acids [T17(447)] was found at the plasma membrane as well as on filamentous structures (Fig. 6B). Two additional constructs, encoding the PTM domain alone [T17(PTM)] or a deletion of the PTM domain [T17(PTM)], were diffuse in the cytoplasm; no staining of filaments or the plasma membrane was discernible (Fig. 6B).
The distinct localization of the TRE17 mutants was confirmed by using biochemical cell fractionation. Cells were hypotonically lysed and then fractionated by centrifugation at low speed. As seen in Fig. 6C, a significant fraction of full-length TRE17 and T17(447) were found in the insoluble pellet fraction, whereas T17(PTM) and T17(PTM) were largely in the soluble fraction. Together, these results indicate that the first 447 amino acids of TRE17 contain the determinants for localization at the plasma membrane and filamentous structures. Furthermore, they reveal that the PTM domain is necessary but not sufficient for normal localization of TRE17.
T17(447) stimulates cortical actin accumulation. As shown above, T17(447) localized to the plasma membrane even in the absence of growth factor stimulation. This led us to speculate that it might represent a constitutively active allele. Surprisingly, we found that expression of T17(447) in serum-starved cells induced strong accumulation of F-actin at the cell cortex, a finding similar to what was observed in some cells expressing Cdc42V12 (Fig. 7A). Quantification revealed that ca. 65% of T17(447)-expressing cells exhibited strong cortical actin staining compared to 18% of the untransfected cells. Full-length TRE17 was also able to induce cortical actin accumulation, albeit at a lower frequency than T17(447) (Fig. 7B).
In order to gain further insights into the mechanism of TRE17-induced actin remodeling, we analyzed the phenotype induced by two additional mutants. As mentioned above, TRE17 contains a Rab GAP homology domain at its N terminus. All Rab GAPs characterized to date contain a conserved arginine residue that is essential for catalytic activity; mutation of this residue to lysine completely abolishes GAP function (2, 3, 20, 42). We found that mutation of the corresponding residue in TRE17 (arginine 149) had no effect on the ability of T17(447) [mutant T17(447/RK)] to induce cortical actin accumulation (Fig. 7B). Another notable feature of T17(447) is the presence of two proline-rich motifs with potential for binding to WW or SH3 domains (24). However, we found that mutation of these motifs also left T17(447)'s actin remodeling activity intact [mutant T17(447/PA); Fig. 7B]. Both T17(447/RK) and T17(447/PA) exhibited localization patterns identical to T17(447) (data not shown). Together, these results indicate that TRE17's ability to localize to the plasma membrane and induce cortical actin accumulation is independent of its putative RabGAP activity and of interactions mediated by the proline-rich motifs.
We next tested whether T17(447)'s membrane localization or ability to induce cortical actin accumulation required Cdc42 and Rac1. Surprisingly, localization of T17(447) to the plasma membrane was not blocked by coexpression with Cdc42N17, Rac1N17, or PBD (Fig. 7C). Similarly, the accumulation of cortical actin induced by T17(447) persisted in the presence of these inhibitory molecules (Fig. 7C). These data suggest that T17(447) functions downstream of Cdc42 and Rac1 and that it may serve to mediate some of their effects on reorganization of the actin cytoskeleton. Our data further suggest a dynamic relationship between TRE17 and the actin cytoskeleton, such that cortical actin polymerization is required for stable plasma membrane recruitment of TRE17; once recruited, TRE17 may stabilize or further stimulate the accumulation of cortical actin.
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DISCUSSION |
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Taken together, our data lead to the following model for the regulation of TRE17 (Fig. 8). In quiescent cells, TRE17 is localized to intracellular filamentous and punctate structures in the cytoplasm, folded in an inactive conformation. Upon growth factor addition, Cdc42 and Rac1 become activated and recruit TRE17 to the plasma membrane. Stable membrane localization of TRE17 also requires polymerized actin. This recruitment process leads to a conformational change in TRE17, such that the N-terminal portion of the molecule further stimulates the accumulation of cortical actin. Consistent with this model, deletion of the C terminus results in constitutive activation of TRE17, such that it localizes to the plasma membrane and stimulates cortical actin accumulation in a mitogen- and Cdc42/Rac1-independent manner.
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Given that the role of Cdc42 and Rac1 in cellular transformation and actin remodeling is well established, their identification as upstream regulators of TRE17 was highly illuminating. These GTPases regulate cytoskeletal dynamics through multiple effector pathways. As discussed above, Cdc42's targets include WASP, the PAK kinases, and MRCK. Effectors of Rac1 include IRSp53/WAVE, the PAK kinases, POR1, and phosphatidylinositol-4-phosphate 5-kinase. Our results suggest that TRE17 may be an effector for both Cdc42 and Rac1 in actin remodeling. In HeLa cells, RacV12 induced peripheral and dorsal ruffles, whereas Cdc42V12 induced filopodia in ca. 75% of transfected cells and cortical actin accumulation (in the absence of pronounced filopodia) in the remaining 25%. It is interesting that the pattern of F-actin accumulation induced by T17(447) mimicked only the latter Cdc42 phenotype. How can this be reconciled with a role for TRE17 in signaling downstream of both Cdc42 and Rac1? One likely contributing factor is that T17(447) is deregulated, as demonstrated by its mitogen- and Cdc42-/Rac1-independent localization at the plasma membrane. This persistent localization may drive the accumulation of F-actin at the cell cortex in a manner distinct from the full-length protein, which is regulated more dynamically by the GTPases. Another probable factor is that both Rac1 and Cdc42 utilize multiple effectors to regulate the cytoskeleton, as discussed above. The cytoskeletal alterations induced by these GTPases represent a composite of the coordinated activities of all of these effectors. Therefore, it is not surprising that a single effector is not sufficient to recapitulate the full range of their effects. This hypothesis is supported by the literature, where overexpression of a single effector, such as WASP (49), PAK, MRCK
(21), ROCK, or mDia1 (55), is unable to phenocopy the precise actin rearrangements induced by activated Rho GTPases. Indeed, overexpression of activated PAK, which is believed to be an effector for both Cdc42 and Rac1, only induces lamellipodia and not filopodia in multiple cell types (10-12, 47, 48). Furthermore, in HeLa cells it was found that activated PAK alleles do not induce either lamellipodia or filopodia but rather inhibit stress fiber and focal adhesion formation (26).
Another possibility is that TRE17 may not function as an effector for both GTPases in actin remodeling but may instead link them to other cellular processes, such as vesicular trafficking. In this context, it is important to highlight the colocalization of TRE17 and Rac1 on filamentous structures (Fig. 3A). In HeLa cells, endogenous Rac1 has been shown to localize to similar structures which, like the TRE17-positive filaments, are enhanced in the presence of cytoD (41). These structures in fact represent a tubular endosomal compartment of a plasma membrane recycling system that is regulated by the Arf6 GTPase (6, 40, 41). Nocodazole has been shown to disrupt this compartment, leading to its collapse into a perinuclear aggregrate (40), a finding that is again similar to what was observed with TRE17. The potential localization of TRE17 to a trafficking compartment is particularly exciting since it contains a PTM/Tbc/Gyp domain. As mentioned above, the PTM/Tbc/Gyp domains of other proteins have been shown to function as GAPs for Rab family GTPases (2, 9, 20, 42), which regulate both the endocytic and secretory pathways (36).
In summary, we provide here the first insights into the cellular functions and molecular interactions of this previously uncharacterized oncogene. Future efforts will be aimed at elucidating the mechanism by which TRE17 induces actin remodeling and assessing its role in vesicular trafficking. Furthermore, its role in Cdc42- and Rac-mediated transformation will be investigated. Through these combined studies, we hope to ultimately gain insights into how TRE17 causes cellular transformation.
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ACKNOWLEDGMENTS |
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This work was supported by the NIH (grant 1 RO1 CA81415-01A1).
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FOOTNOTES |
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Present address: GlaxoSmithKline, King of Prussia, PA 19406.
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REFERENCES |
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