Scholarly article on topic 'Cdc42 induces EGF receptor protein accumulation and promotes EGF receptor nuclear transport and cellular transformation'

Cdc42 induces EGF receptor protein accumulation and promotes EGF receptor nuclear transport and cellular transformation Academic research paper on "Biological sciences"

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{"Small GTP-binding protein" / "γCOP of coatomer protein complex" / "MAP kinases" / "EGF receptor" / "Cell transformation"}

Abstract of research paper on Biological sciences, author of scientific article — Xiao-Yu Wang, Ming-Xi Gan, Yong Li, Wei-Hua Zhan, Tian-Yu Han, et al.

Abstract Cdc42 is a Ras-related small GTP-binding protein. A previous study has shown that Cdc42 binding to the γ subunit of the coatomer protein complex (γCOP) is essential for Cdc42-regulated cellular transformation, but the molecular mechanism involved is not well understood. Here, we demonstrate that constitutively-active Cdc42 binding to γCOP induced the accumulation of epithelial growth factor receptor (EGFR) in the cells, sustained EGF-stimulated extracellular signal-regulated kinase (ERK), JUN amino-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K) signaling and promoted cell division. Moreover, constitutive Cdc42 activity facilitated the nuclear translocation of EGFR, and this indicates a novel mechanism through which Cdc42 might promote cellular transformation.

Academic research paper on topic "Cdc42 induces EGF receptor protein accumulation and promotes EGF receptor nuclear transport and cellular transformation"

Cdc42 induces EGF receptor protein accumulation and promotes EGF receptor nuclear transport and cellular transformation

Xiao-Yu Wanga'1, Ming-Xi Gana,1 Yong Lia, Wei-Hua Zhana, Tian-Yu Hana, Xiao-Jian Hana, Jin-Quan Cheng b, Jian-Bin Wanga'*

a Institute of Translational Medicine, Nanchang University, Jiangxi 330031, China b Department of Molecular Oncology, H. Lee Moffitt Cancer Center, Tampa, FL 33612, USA

ARTICLE INFO

ABSTRACT

Article history: Received 8 October 2014 Revised 17 November 2014 Accepted 26 November 2014 Available online 10 December 2014

Edited by Lukas Huber

Keywords:

Small GTP-binding protein

yCOP of coatomer protein complex

MAP kinases

EGF receptor

Cell transformation

Cdc42 is a Ras-related small GTP-binding protein. A previous study has shown that Cdc42 binding to the c subunit of the coatomer protein complex ("/COP) is essential for Cdc42-regulated cellular transformation, but the molecular mechanism involved is not well understood. Here, we demonstrate that constitutively-active Cdc42 binding to /COP induced the accumulation of epithelial growth factor receptor (EGFR) in the cells, sustained EGF-stimulated extracellular signal-regulated kinase (ERK), JUN amino-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K) signaling and promoted cell division. Moreover, constitutive Cdc42 activity facilitated the nuclear translocation of EGFR, and this indicates a novel mechanism through which Cdc42 might promote cellular transformation.

© 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/

licenses/by-nc-nd/3.0/).

1. Introduction

The Ras-related GTP-binding protein Cdc42 has been implicated in a variety of biological activities, including the establishment of cell polarity, regulation of cell morphology, motility, cell cycle progression, intracellular trafficking and induction of malignant transformation [1-3]. Like other members of the Rho subfamily, Cdc42 was initially implicated in the regulation of the actin cytoskeletal architecture. In particular, the microinjection of activated forms of Cdc42 were shown to cause filopodia formation whereas activated Rac1 stimulated the formation of lamellipodia and activated RhoA promoted actin stress fibers [4-6]. Subsequently, several different lines of research have implicated Cdc42, as well as other Rho-family proteins, in the control of normal cell growth or, when aberrantly regulated, in tumorigenesis and metastasis [7]. Wu et al. reported that when they searched for new Cdc42 targets in an effort to understand how Cdc42 mediates cellular transformation.

Abbreviation: EGFR epidermal growth factor receptor

* Corresponding author at: Institute of Translational Medicine, Nanchang University, No. 1299 Xuefu Road, Honggu Tan New District, Nanchang city, Jiangxi Province 330031, China. Fax: +86 791 83827160.

E-mail address: jianbinwang1@gmail.com (J.-B. Wang). 1 These authors contributed equally to this work.

They identified the y-subunit of the coatomer complex (yCOP) as a specific binding partner for activated Cdc42. This complex is important in intracellular trafficking. The binding of Cdc42 to yCOP is essential for cellular transformation [8]. However, the specific molecular mechanism remains to be defined.

Emerging evidence suggested that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, localize in the nucleus. EGF-dependent nuclear transport of EGFR is associated EGFR with yCOP [9,10]. Nuclear EGFR has been shown to be involved in transcriptional regulation, cell proliferation, DNA repair, DNA replication and chemo-resistance [11].

Cdc42 regulates actin dynamics and control Golgi-to-ER protein transport [12]. Shiga-toxin-producing Escherichia coli remain a food-borne health threat. ARHGAP 21 and Cdc42-based signaling regulates the dynein-dependent retrograde transport of Shiga toxin to the Golgi apparatus [13]. However, whether Cdc42 can regulate nuclear transport of EGFR still has not been investigated. To answer this question, we stably expressed constitutively active Cdc42 mutant Cdc42(F28L) and Cdc42(F28Lss) mutant which breaking Cdc42 and yCOP binding and blocking intracellular trafficking in NIH 3T3 cells. We found that NIH 3T3 cells stably expressing Cdc42(F28L) exhibited significantly higher levels of EGFR and a markedly extended lifetime for EGFR-coupled signaling in response to EGF and showed nuclear accumulation of EGFR.

http://dx.doi.org/10.1016/j.febslet.2014.11.049

0014-5793/® 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Conversely, the NIH 3T3 cells expressing Cdc42(F28Lss) mutant showed much less levels of EGFR in the cytoplasm and almost no accumulation of EGFR in nuclei of the cells, and thus inhibited transformed phenotype.

2. Materials and methods

2.1. Reagents and antibodies

The epidermal growth factor (EGF) was from Calbiochem. Monoclonal antibody to EGF receptor was from BD Transduction Laboratory. Phospho-Akt (Ser473), phospho-SAPK/JNK (Thr183/ Tyr185) and phospho-p44/p42 MAPK (Thr202/Tyr204), were obtained from Cell Signaling Technology. Anti-BrdU antibody was from Sigma. Oregon-Green-conjugated goat anti-mouse IgG and Rhodamine-Red-conjugated goat anti-rabbit IgG, and Tetramethylrhodamine-labeled EGF were purchased from Molecular Probes. Texas Red-conjugated philloidin and anti-vinculin antibodies were from Sigma, and Oregon Green-conjugated anti-mouse antibody was from Molecular Probes. The Oregon-Green-conjugated goat anti-mouse antibody was used to detect HA-tagged Cdc42(F28L), and Rhodamine-Red-conjugated goat anti-rabbit antibody to detect GM130(H-65), monoclonal antibody to HA was from Cov-ance and a rabbit polyclonal antibody to GM130 (a 130kDa cis-Golgi matrix protein) was from Santa Cruz.

2.2. Stable cell lines

The empty vector (pCDNA3-Neo) alone, or was cotransfected with pJ4H-Cdc42(F28L), or pJ4H-Cdc42(F28Lss) in NIH 3T3 cells. Transfected cells were maintained in Dulbecco modified Eagle medium (DMEM) (Invitrogen) supplemented with 5% calf serum and 700 ig/ml G418 (Invitrogen). After 10-14 days, G418-resistant colonies were selected.

2.3. Cell culture, immunobloting, immunofluorescence microscopy and assays for cellular transformation

Cell culture, Western blot, immunofluorescence microscopy and the detailed procedures for cellular transformation assays including growth in low serum, saturation density and soft agar were carried out as previously described [1].

2.4. Analysis of EGFR, ERK, Akt and JNK activation

NIH 3T3 cells stably expressing Cdc42(F28L), Cdc42(F28Lss) or vector control cells were seeded to 60-mm plates. Following overnight incubation in serum-free media, cells were stimulated with EGF for 5 min, 15 min, 45 min and 2 h or were left untreated, and then the cell lysates were harvested. Equivalent amounts of protein (50-80 ig) were subjected to SDS-PAGE, followed by Western blot using anti-EGFR, phospho-ERK, phospho-Akt, and phospho-JNK antibodies.

2.5. EGF receptor endocytosis assay

Vector control NIH 3T3 cells, and cells stably expressing Cdc42(F28L) or Cdc42(F28Lss) mutants were seeded on dual chamber slides and serum-starved for 12 h prior to exposure to tetramethylrhodamine-labeled EGF (0.5 ig/ml) at 4 °C for 1 h. Cells were warmed to 37 °C for the indicated times, transferred to ice, and stripped to remove surface bound EGF by stripping buffer, then washed with 1 x PBS and fixed before being observed by microscope.

2.6. Cell proliferation assay

To assay BrdU incorporation, NIH 3T3 cells stably expressing Cdc42(F28L) or Cdc42(F28Lss) were plated in 60-mm plates. Early next morning, changed the normal culture medium to 2% calf serum in DMEM without antibiotics for about 18 h. At the end of 18 h starvation, cells were trypsinized and replated in steriled chamber slides. BrdU was added to the medium, 14-16 h later, cells were processed for immunofluorescence staining with anti-BrdU monoclonal antibody, anti-HA polyclonal antibody, and Hoechst as described previously [14]. The percentage of BrdU positive cells was determined for more than 600 cells from multiple fields of each experiment.

2.7. Cellular fractionation

Nuclear fractions were isolated via NE-PER nuclear and cyto-plasmic extraction reagents (Thermo). In brief, cultured cells were harvested and resuspended in ice-cold CER I. Cells were vortexed vigorously and incubated on the ice, then were added ice-cold CER II, vortexed again and centrifuged. The resulting supernatant was the cytoplasmic extract. The pellet fraction was suspended in ice-cold CER and centrifuged. The nuclear extract fractions were then collected.

3. Results

3.1. Breaking Cdc42 and yCOP binding inhibits transformed phenotypes

There are two consecutive lysine residues within the Cdc42 C-terminal region at positions 183 and 184. Mutating these lysine residues to serine (Cdc42(F28Lss)) eliminated the binding of Cdc42 to the yCOP [12]. NIH 3T3 cells were stably expressed Cdc42(F28L), Cdc42(F28Lss) or vector control and analyzed for their ability to grow under different conditions (Western blots of the different constructs were shown in Fig. 1A, lower panel; H, M and L denote different cell lines exhibiting higher, medium and lower stable expression, respectively, of a particular construct). The upper panel in Fig. 1A shows the results of cell growth assay performed in low serum. NIH 3T3 cells expressing the constitu-tively active, fast-cycling Cdc42(F28L) mutant were extremely effective at growing in low serum whether their expressions were in higher, medium and lower levels. As compared to Cdc42(F28L), Cdc42(F28Lss) mutants in different expression levels were unable to grow under these conditions over the course of 2, 4, and 6 days. The vector control fibroblasts grew also very slow.

Similar results were obtained when examining the relative ability of the Cdc42 constructs to enable fibroblasts to increase their saturation density and to form colonies in soft agar (Fig. 1B and C). In each case, Cdc42(F28Lss) elicited a four times less transforming phenotype than Cdc42(F28L) approximately whether the expression were in higher, medium or lower levels. These results indicate that disrupting the interaction between constitutively active Cdc42 and the coatomer subunit yCOP inhibits transformed phenotypes.

3.2. Cdc42(F28Lss) and Cdc42(F28L) stimulate filopodia formation and share similar subcellular localization

In order to determine the cellular mechanism by which Cdc42(F28Lss) inhibits transformed phenotypes, we examined whether the Cdc42(F28Lss) mutant altered its ability to stimulate the actin cytoskeletal changes that have been previously associated with the activation of this GTP-binding protein. One important

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Fig. 1. Cdc42(F28Lss) mutant is unable to cause cellular transformation. (A) Growth in low serum. NIH 3T3 cells stably expressing Cdc42(F28L), Cdc42(F28Lss), or vector control cells were cultured in DMEM supplemented with 1% calf serum (low serum). At the indicated times, cells were trypsinized and counted. Data represent the average of three independent experiments. The lower images show Western blots comparing the relative expression of indicated constructs in whole cell lysates. H, M and L refer to cell lines that exhibit higher, medium and lower levels, respectively, of the stable expression of the particular protein of interest. (B) Saturation density assays. The indicated cell lines were cultured in DMEM supplemented with 10% calf serum for 6 days, trypsinized and counted. Data represent the average of three independent experiments. (C) Soft agar assays. Cells stably expressing the indicated constructs were mixed with DMEM supplemented with 03% agarose and 10% calf serum and plated on top of DMEM supplemented with 05% agarose and 10% calf serum. Colonies were scored after 14 days of growth. The data shown are the average of three independent experiments. 100% represents 500 cells counted.

characteristic for Cdc42 activation is the induction of filopodia [15]. We found that both Cdc42(F28L) and Cdc42(F28Lss) induced spike-like extensions (filopodia) into the surrounding medium in the majority of NIH 3T3 cells examined comparing with the vector control cells (Fig. 2A upper panel). This suggests that although Cdc42(F28LSS) can not induce cellular transformation, it still exhibits Cdc42 activity. Multi-molecular focal adhesion complexes associated with the plasma membrane are found in filopodia and lamellipodia induced by Cdc42 and Rac [16]. We also checked whether focal adhesion complex formation changed between the two constructs and found that they were unable to distinguish as both constructs induced this phenotype (Fig. 2A lower panel).

We next investigated whether subcelluar localization of Cdc42(F28Lss) changes comparing with Cdc42(F28L). The subcel-lular distribution of Cdc42 was determined by confocal microscope, both Cdc42(F28Lss) and Cdc42(F28L) showed a prominent perinuclear staining pattern, as detected by the anti-HA antibody (red) (Fig. 2B). The observation revealed that Cdc42(F28Lss) and Cdc42(F28L) may be present in the Golgi. To confirm this idea, the Golgi specific marker GM130 was used (green). The merged images (yellow) proved that Cdc42(F28Lss) and Cdc42(F28L) share similar subcellular localization in Golgi (Fig. 2B).

3.3. The EGF receptor is not accumulated in cells stably expressing Cdc42(F28Lss)

EGFR is a receptor tyrosine kinase involved in normal cell growth and differentiation as well as in the pathogenesis of cancer [17]. Previous studies showed that EGFR after endocytosis, are targeted for degradation via an ubiquitination reaction catalyzed by the Cbl adaptor proteins. Activated Cdc42 binds to p85Cool-1, a protein that directly associates with c-Cbl. This inhibits the binding of Cbl by the EGFR and leads to their aberrant accumulation and cellular transformation [18]. We found that Cdc42(F28Lss) mutant could still bind to p85Cool-1, then p85Cool-1 bound to Cbl to form a complex (data not show). Theoretically, EGFR should be accumulated in the cells. However, following the addition of EGF to NIH 3T3 cells expressing Cdc42(F28Lss) or Cdc42(F28L), total receptor levels in Cdc42(F28Lss) expressing cells as detected by Western blot with an anti-EGFR antibody were very low at all time points except a little bit higher at 0 min. Cells stably expressing Cdc42(F28L) showed a dramatic increase of EGFR. Additionally, the turnover of EGFR was very slow because EGFR was still detected after 2 h of incubation with EGF. The EGFR level in vector control fibroblasts was also much lower than that in NIH 3T3 cells

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Fig. 2. Cdc42(F28Lss) and Cdc42(F28L) stimulate filopodia formation and share similar subcellular localization. (A) The indicated stable cell lines were cultured on microscope chambers and fixed with 3.7% formaldehyde. Filamentous actin structures were visualized using Texas Red-conjugated Phalloidin, focal adhesion complexes were visualized with anti-vinculin antibody followed by Oregon Green conjugated anti-mouse IgG (Molecular probes). Treated slides were observed under a immunofluorescence microscope. (B) The confocal microscope was used to check subcellular localization of Cdc42 mutants. The NIH 3T3 cells stably expressing Cdc42(F28L), Cdc42(F28Lss) or vector control were fixed, permeabilized and incubated with antibodies against HA (red) and anti-GM130 (H-65) (anti-Golgi antibody) (green). Cells were treated with DAPI to visualize nuclei (blue) and composite colocalization of all three images are shown. Colocalization (yellow) of Cdc42 mutants and Golgi is detected.

expressing Cdc42(F28L) (the upper three panels of Fig. 3A). The similar results were got for lower expression cell lines (Fig. 3A middle three panels). The lower three panels of Fig. 3A shows separately that the same amount of proteins detected by anti-actin were loaded to SDS-PAGE in different samples. Therefore, disrupting the interaction between constitutively active Cdc42 and the coatomer subunit yCOP leads to down-regulation of EGFR total cellular level.

We confirmed further that EGFR was not accumulated in cells expressing Cdc42(F28Lss) by using immunofluorescence microscopy. Fig. 3B shows the rhodamine-labeled EGF bound to the receptor, as a function of time of treatment with the labeled growth factor. In cells expressing Cdc42(F28Lss), EGFR levels were significantly reduced within 15 min of treatment with labeled EGF. However, in cells expressing Cdc42(F28L), EGFR were detected and accumulated in endosomal compartments through 2 h of treatment with labeled EGF. In contrast, EGFR accumulation was not observed in vector control cells treated with EGF for more than 5 min.

3.4. Cdc42(F28Lss) mutant fails to induce ERK, JNK and PI-3K activation

For mammalian cells, MAP kinases represent a family of Ser/Thr protein kinases and are comprised of three distinct components: ERKs, JNKs and p38, they are regulated by EGFR. In different cell lines, these kinases have been shown to play important roles in

regulating cell growth [19]. Cdc42 initiates a protein kinase cascade that leads to activation of MAP kinases [20]. To determine whether the inhibitory effect of Cdc42(F28Lss) on transformed phenotypes is associated with its ability to block MAP kinases activity. We performed time-course experiments. NIH 3T3 cells stably expressing Cdc42(F28Lss), Cdc42(F28L) and vector control cells were serum starved overnight, then stimulated with EGF for different times. Fig. 4A shows that in NIH 3T3 cells stably expressing Cdc42(F28Lss) and vector control cells, the activation of ERK as examined by phosphor-p42/p44 ERK antibody was peaked within 5 min, but rapidly diminished for 15-45 min after exposure to EGF. Conversely, in NIH 3T3 cells stably expressing Cdc42(F28L), EGF-stimulated ERK activation could be detected up to 2 h of incubation with EGF. The same amount of proteins were loaded to SDS-PAGE in different samples (Fig. 4A lower panel). Similar results were obtained for JNK activation (Fig. 4B).

PI-3K is a ubiquitously expressed enzyme that plays a critical role in the regulation of many cellular processes including cellular transformation [21]. In the fibroblasts stably expressing Cdc42(F28Lss) or vector control cells, EGF-stimulated activation of Akt, a key downstream effector of PI-3K, could be detected just within 5 min and then disappeared for any longer incubation with EGF. However, in fibroblasts stably expressing Cdc42(F28L), the EGF-induced Akt activation peaked within 5 min, then reduced gradually between the span of 5 min-2 h (Fig. 4C). The same amount of proteins were loaded to SDS-PAGE in different samples (Fig. 4A lower panel).

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Fig. 3. The EGF receptor is not accumulated in cells stably expressing Cdc42(F28Lss). (A) The upper three and middle three panels show NIH 3T3 cells stably expressing Cdc42(F28L), Cdc42(F28Lss) in higher and lower levels, respectively, or vector control cells. Cells were seeded in 6-well plates and serum starved for 12 h prior to EGF (100 ng/ ml) stimulation for the indicated times at 37 "C. The levels of EGF receptor were assessed by Western blot analysis of the cell extracts using an anti-EGF receptor antibody. The expression of the different Cdc42 constructs was identical in the different cell lines (see Fig. 1A lower image). Lower three panels show that the same amount of proteins were loaded to SDS-PAGE in different samples using anti-actin antibody. (B) Vector control NIH 3T3 cells, cells stably expressing Cdc42(F28L) or Cdc42(F28Lss) were seeded on dual chamber slides and serum starved for 12 h prior to exposure to tetramethylrhodamine labeled EGF (0.5 ig/ml) at 4 "C for 1 h. Cells were warmed to 37 "C for the indicated times, transferred to ice, and stripped to remove surface bound EGF. The red punctated structures indicate tetramethylrhodamine labeled EGF receptor.

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Fig. 4. The effects of Cdc42(F28Lss) on EGF-stimulated ERK, JNK and PI-3K activities. Time course experiments. Following serum starvation overnight, NIH 3T3 cells stably expressing Cdc42(F28L), Cdc42(F28Lss) or vector control cells were stimulated with EGF (100 ng/ml) for the indicated times. Anti-phospho-p44/42 ERK antibody was used to detect activated p44/p42 ERK (A); or anti-phospho-JNK antibody to detect activated JNK (B); or anti-phospho-Akt antibody was used to detect activated PI3K (C).

3.5. Cdc42(F28Lss) mutant blocks cell cycle progression

Given the significance of Cdc42(F28L) in mediating cellular transformation, we were wondering whether Cdc42(F28Lss) mutant blocks transformed phenotypes because it inhibits cell proliferation. In BrdU assay, the levels of DNA synthesis in NIH 3T3 cells stably expressing Cdc42(F28Lss), were reduced two times comparing with those of NIH 3T3 cells stably expressing Cdc42(F28L) approximately (Fig. 5A and B). Therefore, Cdc42(F28Lss) mutant should be directly or indirectly inhibit cellular transformation by influencing cell cycle progression.

3.6. Cdc42(F28L) mutant promotes EGFR nuclear transport

Emerging evidences indicate that EGFR has been shown to localize in the nucleus of various malignant tumors [22,23]. We want to know whether Cdc42 can promote nuclear transport of EGFR in Cdc42(F28L) transformed cells. To address this issue, proteins were isolated from the cytosolic and nuclear compartments and separated by SDS-PAGE and examined by subsequent immu-noblotting to determine the effects of Cdc42 mutants on EGFR localization. As shown in Fig. 6A, ten times EGFR was appeared in the nuclei of cells expressing Cdc42(F28L) than that of the cells expressing Cdc42(F28Lss) approximately.

Next, we performed confocal microscopy to confirm the distribution of EGFR in the nuclei of different cells (Fig. 6B). NIH 3T3 cells stably expressing Cdc42(F28L), Cdc42(F28Lss) or vector control cells were immunostained with EGF receptor antibody (red) and DAPI for nuclei (blue). The nuclei of Cdc42(F28L) expressing cells were stained in red color much stronger than those of Cdc42(F28Lss) expressing cells. When merged, the nuclei of

Cdc42(F28L) cells were in a purple color; but the nuclei of Cdc42(F28Lss) were still in blue color. This suggested again that more EGFR was localized in the nuclei of Cdc42(F28L)-expressing cells than Cdc42(F28Lss)-expressing cells (Fig. 6B, left: single cell images; right: multiple cells images).

4. Discussion

EGFR is involved in numerous aspects of cell growth, survival, differentiation, migration and invasion [24,25]. EGFR regulates these diverse cell functions through interacting with and activating a number of downstream signaling proteins that organize multi-layered, distinctive, and interconnected signaling pathways [26,27]. The magnitude and duration of these signaling pathways as well as their spatial distribution all need to be delicately controlled; slight aberrations, such as EGFR overexpression or muta-tional activation or aberrant activation of downstream effectors, have been linked to the pathogenesis of cancer [28].

Cdc42 is a member of the Rho family GTPases and has been implicated in a wide range of cellular processes and signaling activities as well as in the control of normal cell growth, and when hyperactivated, in cellular transformation [29]. Cdc42 is a downstream target of EGFR that activates Cdc42 by phosphorylation. Previous evidence suggests that a positive feedback loop may exist between Cdc42 and EGFR. Treatment of cells with EGF stimulates the activation of Cdc42 [30]. Activated Cdc42, through an interaction with Cool-1, inhibits the binding of c-Cbl ubiquitin ligase to EGFR and thus leads to the aberrant accumulation of EGFR and results in malignant transformation [24].

Lin et al. reported that a novel Cdc42 mutant Cdc42(F28L), was capable of spontaneous GDP-GTP exchange and exhibited several

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Fig. 5. Cdc42(F28Lss) mutant blocks cell cycle progression. BrdU incorporation assays were performed on NIH 3T3 cells stably expressing Cdc42(F28L) or Cdc42(F28Lss). (A) The percentage of cells incorporating BrdU after 18 h is shown. Data represent the average of three independent experiments. (B) Immunofluorescence microscopy photomicrographs of the cells stained with anti-BrdU and their nuclei stained with Hoescht are shown.

Fig. 6. Activated Cdc42 promotes EGFR nuclear transport and accumulation. (A) Nuclear and cytoplasmic fractions from NIH 3T3 cells stably expressing Cdc42(F28L), Cdc42(F28Lss) or vector control cells were subjected to Western blot with antibodies against EGFR and lamin B. Lamin B was used as a marker for the nucleus. (B) NIH 3T3 cells stably expressing Cdc42(F28L), Cdc42(F28Lss) or vector control cells were immunostained with EGFR antibody (red) and the nuclei of cells were stained with DAPI (blue), and analyzed using confocal microscopy. Scale bars: 5 im. Equivalent amounts of proteins were loaded to the gel.

hallmarks of cell transformation-reduced contact inhibition, lower dependence on serum for growth and anchorage-independent growth [9]. However, when two lysine residues in position 183 and 184 were mutated to serine in Cdc42(F28L) background, this mutant Cdc42(F28Lss) did not transform the cells. To figure out the molecular mechanism, we used this mutant to eliminate Cdc42 binding to yCOP [8]. We found that Cdc42(F28Lss) fails to induce EGFR accumulation, consequently, EGF-induced activation of ERK, Akt, and JNK signals by Cdc42(F28L) were not sustained in Cdc42(F28Lss)-expressing cells. yCOP in conjunction with the GTP binding protein ARF1, forms an electron-dense coat that, when assembled onto Golgi membranes, is thought to facilitate membrane budding and fission events associated with Golgi membrane traffic [31]. EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with yCOP, one of the subunits of the COP I coatomer [32]. Taken together, we found a new molecular mechanism for Cdc42 induced cellular transformation by which Cdc42 accumulates EGFR and induces cell transformation through increased intracellular trafficking. This mechanism is different from other people's finding in which EGFR accumulation was associated with c-Cbl lost function for EGFR ubiquitination and degradation.

Multiple cell surface receptor tyrosine kinases have been reported to, localize in the nucleus. Accruing evidence points to a scenario where nuclear localization of EGFR in different tumor types has impact on not only the tumor grade but also on the resistance of tumors to therapies [33]. Endocytosis has been proven to be required for the nuclear translocation of EGFR, but after endocy-tosis the EGF-dependent nuclear transport of EGFR is regulated by a retrograde trafficking from Golgi to the ER involved an associa-

tion of EGFR with yCOP [34], yCOP can bind to Cdc42 [12]. It has not been investigated whether Cdc42 can regulate EGFR nuclear transport. In this study, we proved evidence to suggest that activated Cdc42 promotes more EGFR to enter into the nucleus. Our confocal microscopy study further confirmed that more EGFR was accumulated in nuclei of Cdc42(F28L) expressing cells than those of Cdc42(F28Lss) expressing cells.

In conclusion, our study reveals a novel mechanism by which disrupting the interaction between constitutively active Cdc42 and the coatomer subunit yCOP inhibits transformed phenotypes, and demonstrates for the first time that Cdc42 can promote EGFR nuclear transport and accumulation. These findings may provide new insight for understanding the resistance of tumors to EGFR therapies.

Acknowledgements

We thank Dr. Ceshi Chen (Kunming Institute of Zoology, Chinese Academy of Science) for discussion and reading the manuscript. This work was supported by the National Science foundation of China (81372823 and 31360282) and job starting grant (300784) from Nanchang University, China.

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