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1 State Key Laboratory of Biotherapy, West China Hospital, College of Life Science, Sichuan University, Chengdu, Sichuan, China
2 Laboratory of Signal Transduction, Department of Obstetrics and Gynecology, St. Joseph's Hospital and Medical Center, Phoenix, Arizona, USA
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Introduction |
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Over the past several decades, massive efforts in this direction have resulted in the identification of a number of important endothelial cell-specific molecules (ECSMs), for example, platelet endothelial cell adhesion molecule (PECAM-1 or CD31) (Newman et al. 1990) and vascular endothelial growth factor (VEGF) (Ferrara et al. 1991), etc. Bioinformatics strategies, such as database mining and virtual screening of public gene libraries, led to the discovery of additional novel endothelial-specific genes, including many Uni-genes/expressed sequence tags (Huminiecki & Bicknell 2000; Ho et al. 2003). One of them was named ECSM2 (UniGene ID Hs.30089) (Huminiecki & Bicknell 2000) and its sequence was identical to a cDNA from the patent ‘cDNA encoding novel polypeptide from human umbilical vein endothelial cells’ (EMBL acc. E10591 [GenBank] ) (Shibayama et al. 1997). It was predicted to encode a 205-amino acid protein with a suggested role in cell adhesion because of its abundant serine and proline residues (Huminiecki & Bicknell 2000). However, the exact function of ECSM2 has not been defined.
The cytoskeleton, consisting of actin, microtubule, and intermediate filaments, is a highly organized, dynamic biological architecture that maintains cell shape and size, enables cellular motion, and regulates diverse functions depending on cell type (Hall 1998; Small & Resch 2005). Multiple signal transduction pathways converge to induce the rearrangement of actin cytoskeleton to mediate cell motility, shape change, and cell–substratum (extracellular matrix) interactions, which is a prerequisite for many physiological and pathological conditions including angiogenesis, cancer progression and metastasis (Affolter & Weijer 2005; Lamalice et al. 2007; Vignjevic & Montagnac 2008). Classically, it is believed that dynamic remodeling of the actin cytoskeleton into filopodia, lamellipodia, and stress fibers and precise regulation of the formation of these structures are essential to drive the multistep actin-based cell motility. These included sensing of motile stimuli by filopodia-like projections, forward extension by protrusions of lamellipodia, attachment of the protrusions to substratum, forward progress by stress fiber-mediated contraction, detachment of adhesions at the rear, and recycling of adhesion and signaling molecules (Webb et al. 2002; Ridley et al. 2003). Numerous signaling molecules have been implicated in coordinating these events, including the Rho family of small GTPases, focal adhesion proteins, and receptor tyrosine kinases (RTKs) (Hall 1998; Parsons 2003; Affolter & Weijer 2005; Bryan & D’Amore 2007).
Epidermal growth factor receptor (EGFR) signaling represents a paradigm for RTK signal transduction and has been extensively studied partially due to its therapeutic significance. Upon ligand engagement, EGFR intrinsic tyrosine kinase is activated via autophosphorylation, which in turn activates several downstream signaling pathways including the Shc-Ras-Raf-MEK-ERK (p42/p44 MAP Kinases) pathway (Grant et al. 2002). The EGFR-mediated signaling pathways play important roles in the dynamic changes of actin cytoskeletal networks, and their actual biological effects may vary depending on the cellular context (Xie et al. 1998; Marcoux & Vuori 2003, 2005; Toral et al. 2003; Faisal et al. 2004; Cáceres et al. 2005). However, how other effectors influence the EGFR signaling and thereby regulate the remodeling of actin cytoskeleton and cell motility, remains to be explored.
The current work was carried out to investigate the cellular function of ECSM2. We showed that ECSM2 is an evolutionarily conserved gene with a highly restricted expression pattern in endothelial cells and blood vessels. More importantly, we demonstrated, in several reconstitution mammalian expression systems, that human ECSM2 is a plasma membrane protein and is critically involved in the reorganization of actin cytoskeleton and modulation of EGFR signaling to regulate cell shaping and migration.
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Results |
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We first performed structure prediction of the deduced amino acid sequence of human ECSM2 using the ExPASy proteomics tools. Based on a Dense Alignment Surface method (Cserzö et al. 1997), human ECSM2 is likely a membrane protein with a putative cleavable approximately 18-amino acid signal peptide at its N-terminus and approximately 28-amino acid transmembrane (TM) region closer to the C-terminus (Fig. 1A). In addition, an N-glycosylation site and an O-glycosylation site located at N-terminal extracellular domain were predicted (Fig. 1A, indicated by arrows). Furthermore, with BLAST searches (http://www.ncbi.nlm.nih.gov/blast), we obtained several homologous hypothetical proteins across species, including human, chimpanzee, rat, mouse, cattle, horse, chicken and zebrafish (Supporting Information Fig. S1). Alignment of these sequences and homology analysis resulted in a dendrogram indicating their evolutionary relationship (Fig. 1B). The sequence identities and similarities are shown in Fig. 1C. All these data suggest that ECSM2 is an evolutionarily conserved protein. Notably, the multiple sequence alignment results (Supporting Information Fig. S1) indicated that the C-terminal segments containing the TM region and cytoplasmic domain are highly conserved across species, suggesting that the cytoplasmic domain may be functionally important (e.g. interacting with intracellular proteins to initiate signaling and regulate endothelial functions). However, using currently available molecular biology software combined with high stringent screening, we were not able to identify any apparent functional motifs, such as kinase domains, phosphorylation sites or protein binding sites, in the putative cytoplasmic domains of all ECSM2 proteins.
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ECSM2 is involved in cell shape change and actin cytoskeleton rearrangement
Our data suggested that the ECSM2 is highly expressed in endothelial cells and blood vessels, and is evolutionarily conserved. Here we sought to explore its potential function at the cellular level using a reconstitution expression system. Human embryonic kidney (HEK) 293 cells that do not express endogenous ECSM2 (Fig. 2A), were transfected with the pECSM2-GFP plasmid, which encodes the human ECSM2 with its C-terminus fused to the green fluorescent protein (GFP). Stable transfectants expressing ECSM2-GFP were selected as pools to avoid clonal variations. The expression of ECSM2-GFP protein was confirmed by immunoblotting with an anti-GFP antibody (Fig. 3A, lane 2). We then examined the subcellular localization of ECSM2-GFP by fluorescence microscopy (Fig. 3B). In contrast to a diffused intracellular expression pattern of GFP alone, we observed a prominent cell surface expression of ECSM2-GFP (Fig. 3B, arrows), which is consistent with the existence of a predicted transmembrane domain (TM) in human ECSM2 (Fig. 1A and Supporting Information Fig. S1). Interestingly, unlike the GFP control cells, the morphology of ECSM2-GFP-expressing HEK293 cells was remarkably irregular; there were numerous filopodia-like microspike structures, that is, long hair-like or finger-like cellular extensions or projections from the cell periphery (Fig. 3C, b, arrows). These extensions often had a thick root and were branched at their distal parts as thin microspikes or filopodia (Faix & Rottner 2006). We also observed these filopodia-like structures in other cell types, including mouse C2C12 myoblasts (Fig. 3C, d), human endometrial cancer Ishikawa cells, breast cancer MCF-7 cells, and hepatoma HepG2 cells, upon transient expression of ECSM2-GFP (data not shown). In particular, we transiently over-expressed ECSM2-GFP in a mouse endothelial cell line, MS1, and examined its subcellular localization and cell morphology (Fig. 3C, f). Our results in MS1 cells supported the notion that ECSM2-GFP resides at cell membrane and is involved in cell shape changes (e.g., promoting the formation of filopodia).
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Cell signaling is known to be essential for a variety of cellular processes. Therefore, we further probed which signaling aspects might be altered by ectopic expression of ECSM2-GFP. We first compared the overall tyrosine phosphorylation levels between GFP control and ECSM2-GFP-expressing HEK293 cells by immunoblotting with an anti-phosphotyrosine antibody (pTyr). Interestingly, we observed diminished overall tyrosine phosphorylation signaling in ECSM2-GFP-expressing cells when compared to the GFP control cells (Fig. 5A). The Shc family of adaptor/docking proteins is an important component of signaling pathways induced by various extracellular signals, such as growth factors, cytokines, integrins, and extracellular matrix. Previous studies suggested that Shc plays a role in cytoskeletal reorganization-induced signaling (Gu et al. 1999; Faisal et al. 2004). Here we also assessed the Shc activation or phosphorylation by immunoblotting with an anti-phospho-Shc (pShc) antibody. Consistent with the inhibition of general tyrosine phosphorylation (Fig. 5A), ectopic expression of ECSM2-GFP suppressed the Shc activation (Fig. 5B, upper panel). Immunoblotting with an anti-Shc antibody recognizing all Shc isoforms revealed the equal loading of proteins and predominant forms of p52 and p46 Shc present in the HEK293 cells (Fig. 5B, lower panel). Densitometric analysis of the anti-pShc blots from three independent experiments indicated that ECSM2 inhibited the Shc activation by approximately 37% (Fig. 5C).
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Our findings that expression of ECSM2 suppresses tyrosine phosphorylation signaling and Shc activation in HEK293 cells strongly prompted us to investigate the relationship between ECSM2 and EGFR, a prototype of RTK superfamily. Since HEK293 cells do not express EGFR (Kloth et al. 2002; Arao et al. 2004), we first generated HEK293 cells stably expressing EGFR alone or co-expressing EGFR and ECSM2-GFP. The expression of EGFR alone or co-expression of EGFR/ECSM2-GFP was confirmed by fluorescent microscopy and immunoblotting with anti-EGFR and anti-GFP antibodies, respectively (Fig. 6A). As expected, co-expression of EGFR did not alter the predominant membrane localization of ECSM2-GFP (Fig. 6A, right). Likewise, the EGFR/ECSM2-GFP-co-expressing cells exhibited substantial filopodia-like structures (Fig. 6A, arrows). Comparison of F-actin staining patterns showed long stress fibers in the EGFR-expressing cells versus destructed stress fibers in the cells co-expressing EGFR/ECSM2-GFP (data not shown), which was similar to the difference between GFP- and ECSM2-GFP-expressing cells (Fig. 4). These results again supported the notion that ECSM2 is involved in cell morphological changes and actin cytoskeletal rearrangement.
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ECSM2 impacts the EGF-induced cell migration partially through regulation of p44/42 MAPK (ERK1/2) pathway
It is known that EGF exerts its biological effects via binding to the EGFR to elicit downstream signaling pathways. Here we evaluated whether ECSM2 could attenuate the EGF-induced EGFR tyrosine phosphorylation (or kinase activity) and activation of downstream signaling, which may underlie its effects on cell motility. First, we assessed EGFR phosphorylation/activation by immunoblotting with a panel of state-specific antibodies correlated with EGFR kinase activation and/or tyrosine phosphorylation, Tyr-845, Tyr-1045, and Tyr-1068. We observed the basal EGFR tyrosine phosphorylation in the EGFR-expressing cells in serum-free medium (Fig. 7A, lane 1), possibly due to the constitutive expression (over-expression) of EGFR in HEK293 cells. As expected, acute EGF stimulation (15 min) of the EGFR-expressing cells resulted in elevated EGFR tyrosine phosphorylation signals (Fig. 7A, lane 3 vs. 1), confirming the robust EGF/EGFR signaling in these cells. Noticeably, co-expression of ECSM2-GFP in the EGFR-expressing cells diminished both basal and EGF-induced EGFR phosphorylation (Fig. 7A, lane 2 vs. 1; lane 4 vs. 3), suggesting that ECSM2-GFP had a dampening effect on EGFR kinase activation. The difference in the SDS-PAGE migration of EGFR seen between the two cell lines (Fig. 7A, bottom panel) could partially reflect the changes of EGFR phosphorylation and/or other post-translational modifications mediated by the presence of ECSM2-GFP.
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In summary, in the present study, we identified, by database mining, several hypothetical proteins that shared substantial homology to the human ECSM2. We also characterized their structures and expression patterns. Our results suggested that ECSM2 is a unique molecule with considerable evolutionary conservation and a highly restricted pattern of cellular and developmental expression. Furthermore, in a reconstitution mammalian cell system, we demonstrated that ECSM2-GFP is a plasma membrane protein, critically involved in cell shaping, actin cytoskeletal rearrangement, and suppresses tyrosine phosphorylation signaling. Finally, we suggested that ECSM2-GFP may have the ability to cross-talk with EGFR, attenuating EGF-induced cell migration and inhibiting EGF/EGFR downstream signaling such as the Shc-ERK MAPK pathway.
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Discussion |
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To this end, we ectopically expressed the GFP-tagged human ECSM2 in the HEK293 cells that lack endogenous ECSM2. We were particularly intrigued by the dramatic differences in cell morphology between the ECSM2-GFP-expressing and GFP control cells. Cell adhesion to the substratum elicits a series of intracellular signaling cascades that render the cell ability to shape and migrate. At least three actin-based infrastructures including filopodia (for sensing), lamellipodia (for forward extension) and stress fibers (for cell body contraction and rear retraction) are crucial for cell migration. The formation of these structures involves the small GTPases of the Rho family, Rho, Cdc42, and Rac. In constitutive cell culture systems, Rho promotes the assembly of contractile stress fibers, whereas Cdc42 and Rac promote the actin polymerization to form peripheral filopodial and lamellipodial protrusions, respectively (Hall 1998). Activation of Cdc42 leads to the sequential activation of Rac and Rho. In addition, all three GTPases promote the assembly of integrin-based cell–substratum tight adhesions (focal adhesions). The ability of these GTPases to cycle between active and inactivate states allows the cell to respond to extracellular signals (Nobes & Hall 1995; Hall 1998). Any effectors that break this tightly regulated dynamic balance could lead to the abnormality in cell shaping and motility. Indeed, the F-actin staining patterns in the ECSM2-GFP-expressing and GFP control cells were remarkably distinct. The stress fibers were largely destructed in the ECSM2-GFP-expressing cells. Although further experiments are needed in the future to evaluate the impact of ECSM2 on activation/inactivation of GTPases, our current findings implied that ECSM2 may be an effector promoting Cdc42 signaling to form filopodia-like structures but inhibiting Rho signaling required for actin stress fiber formation that allows cell contraction. On the other hand, we propose that the roles of ECSM2 in regulating the Rho GTPase switch and coordinating the assembly and disassembly of actin-based structures could be indirect, since no functional motifs (e.g., kinase domains, phosphorylation sites or protein binding sites) were identified (this study and Huminiecki & Bicknell 2000). Thus, future studies should also attempt to identify partner(s) potentially interacted with ECSM2 in parallel with investigating its effects on activation of the small GTPases of the Rho family.
In the search for signaling mechanisms underlying the ECSM2's effects on cell morphological changes and cytoskeletal dynamics, we uncovered that ECSM2-GFP down-regulated overall tyrosine phosphorylation and Shc activation. This could provide a potential connection between ECSM2 and actin cytoskeletal rearrangement. We then addressed whether ECSM2-GFP could cross-talk with EGFR signaling (a paradigm for RTK signaling). Interestingly, we found that co-expression of ECSM2-GFP with EGFR in HEK293 cells markedly suppressed the EGF-induced cell migration when compared to expression of EGFR alone. Since the stress fibers were largely destructed in the cells co-expressing EGFR/ECSM2-GFP, we concluded that the inhibitory effect of ECSM2-GFP on the EGF-induced cell motility is most likely a result of the redistribution of actin cytoskeleton in the EGFR/ECSM2-GFP-coepxressing cells. The cell motile process requires the activation of various signaling pathways that converge on actin cytoskeletal reorganization. Growth factor and RTK signaling networks play a pivotal role in chemotactic movement (chemotaxis) usually directed by soluble chemoattractants and their cognate cell–surface receptors (e.g. EGF and EGFR) (Affolter & Weijer 2005). In this study, we demonstrated that ECSM2-GFP has the ability to cross-talk with the EGF/EGFR signaling system to attenuate the EGFR, Shc and ERK activation.
Although the detailed mechanism(s) is currently unknown, here we propose a model of how ECSM2 modulates EGF/EGFR signaling, possibly extending to other growth factors/RTKs such as VEGF, platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and their respective receptor signaling in endothelial cells, to impact actin cytoskeletal remodeling and subsequent cell movement (Fig. 8). One potential link is though Shc-ERK (p44/p42 MAPK) pathway. The growth factor-activated ERK pathway has been well-documented to regulate the cell morphogenetic and motile responses (Huang et al. 2004; Katz et al. 2007b; Pullikuth & Catling 2007). The ERK pathway can directly promote cell motility (Klemke et al. 1997; Zeigler et al. 1999; Krueger et al. 2001), and/or regulate Rho and Rac activity to impact cell motility and invasion (Sahai et al. 2001; Vial et al. 2003; Vial & Pouysségur 2004). The adaptor/docking protein, Shc, mainly includes three isoforms (p42, p52 and p66) and is an important signaling molecule that can be activated by multiple RTKs including EGFR and utilized as an upstream effector of the Ras–ERK pathway (Ravichandran 2001). All three Shc isoforms were involved in the regulation of actin cytoskeleton via ERKs (Faisal et al. 2004; Natalicchio et al. 2004; Kleiner et al. 2007). In this study, we showed that ECSM2-GFP suppresses EGF-induced EGFR tyrosine phosphorylation and activation of p46/p52 Shc proteins and p42/p44 ERKs, and retards EGF-mediated cell migration. These novel findings support the idea that ECSM2 could exert its effects through the EGFR-Shc-Ras-ERK pathway to regulate actin cytoskeletal rearrangement and thereby cell motility (Fig. 8). Interestingly, CD31 (PECAM-1), an adhesion molecule highly expressed in the blood and endothelial cells, has emerged as an important receptor that has diverse functions in vascular biology including modulation of integrin-mediated cell adhesion, transendothelial migration, angiogenesis, immune responses, platelet function, and thrombosis (Woodfin et al. 2007). Notably, an early study reported that transfection of CD31 into NIH 3T3 fibroblast cells diminished cell migration (Schimmenti et al. 1992). Accumulating evidence has also implicated CD31 as an inhibitor of cellular activation via protein tyrosine kinase-dependent signaling pathways (Newman & Newman 2003). In this context, although demonstrated in a reconstitution system, our findings that ECSM2-GFP cross-talks with EGF/EGFR signaling system to suppress cell migration may imply important physiological and pathological roles of ECSM2 (e.g., endothelial cell migration during angiogenesis). It also raises the possibility that ECSM2 may interact with the VEGF/VEGFR signaling pathways in endothelial cells. This will become an avenue of future investigation with the availability of an effective anti-ECSM2 antibody.
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In this report, we provide the experimental evidence that ECSM2-GFP is a plasma membrane protein when over-expressed in multiple cell types including a mouse endothelial cell line. This is consistent with our structural prediction results. Although GFP-fusion proteins are widely used in functional studies, we cannot rule out the possibility that a small tag (GFP) could interfere with the actual properties of ECSM2, such as cellular localization and function. Again, these important questions cannot be addressed in endothelial cells until an anti-ECSM2 antibody is available. How ECSM2 interacts with the cell–surface EGFR (directly or indirectly) to exert its biological effects is also a puzzle to be solved. Our initial co-immunoprecitation experiments suggested that ECSM2-GFP is likely associated with EGFR when ectopically expressed in HEK293 cells (F. Ma and Y. Huang, unpublished observations). However, other approaches are needed to support this finding. Taken together, our results suggest that ECSM2 is not only an evolutionarily conserved endothelial cell-specific membrane protein present in a range of organisms from zebrafish to human, but also profoundly involved in the actin cytoskeleton remodeling and modulation of EGFR signaling. In particular, the inhibitory effects of ECSM2 on the EGF-induced activation of EGFR and downstream Shc-ERK (MAPK) pathway as well as the EGF-mediated cell migration highlight its potential therapeutic importance. Thus, future studies will be directed toward delineating the precise roles of ECSM2 in cell–substratum interaction, focal adhesion turnover, actin dynamics as well as determining whether ECSM2 plays a role in VEGF/VEGFR-mediated signaling and cellular processes, which may contribute to vascular functions.
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Experimental procedures |
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Recombinant human EGF was from Invitrogen (Carlsbad, CA). All routine chemicals and reagents were from Sigma (St. Louis, MO) and Fisher (Pittsburgh, PA) unless otherwise noted. Anti-EGFR and anti-Shc antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine antibody 4G10, anti-MAPK antibody (anti-ERK1/2) and anti-phospho-Shc (Tyr-239) were from Upstate Biotechnology (Lake Placid, NY). Anti-active MAPK antibody (recognizing the dually phosphorylated Thr183 and Tyr185 corresponding to the active forms of ERK1 and ERK2) was from Promega (Madison, WI). Anti-phospho-EGFR antibodies (Tyr-845, Tyr-1045, and Tyr-1068) were from Cell Signaling (Beverly, MA). Anti-GFP antibody and phalloidin-tetramethylrhodamine B isothiocyanate (TRITC) were from Sigma.
Cell culture
HEK293 (human kidney epithelial cells), C2C12 (mouse myoblast cells), and MS1 (mouse endothelial cells) were purchased from ATCC (Manassas, VA), and grown in Dulbecco's modified Eagle's medium (DMEM) (Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) (Sigma), 100 units/mL penicillin, and 100 µg/mL streptomycin (Mediatech).
Whole-mount in situ hybridization (WISH)
Zebrafish (wild-type AB line) embryos were fixed with 4% formaldehyde in phosphate-buffered saline (PBS, pH 7.4). A cDNA encoding a zebrafish ECSM2 segment was obtained by RT-PCR. Digoxigenin-labeled RNA probe was transcribed from a linear cDNA construct according to the manufacturer's instructions (Roche, Indianapolis, IN). Whole-mount in situ hybridization assays were performed at 67 °C as described elsewhere (Bennett et al. 2001; Thisse et al. 2004). The probe was developed with 4-Nitro blue tetrazolium (NBT)/5-Bromo-4-chloro-3-indolyl-phosphate (BCIP) (Roche) to produce purple color.
RNA extraction and reverse transcription-PCR (RT-PCR)
Total RNAs from young Wistar rat (15 days old) tissues, cultured cell lines, and zebrafish embryos were isolated using TRIzol Reagent (Invitrogen). RT-PCR and semi-quantitative PCR were carried out as described elsewhere (Wu et al. 2007). All PCR primers were designed using Primer3 internet version. The PCR products were resolved on agarose gels and photographed using a gel documentation system.
Plasmid construction, cell transfection and generation of stable cell lines
A cDNA encoding full-length human ECSM2 was obtained by RT-PCR using total RNAs of human umbilical vein endothelial cells (HUVEC) and cloned into the pEGFP-N1 vector (Clontech Laboratories, Palo Alto, CA), referred to as pECSM2-GFP. Full-length human EGFR in the plasmid pX-EGFR (kindly provided by Dr R. Davis, University of Massachusetts Medical School, Worcester, MA) was subcloned into the pcDNA3.1 vector (Invitrogen), named pcDNA-EGFR. Plasmid DNAs were transfected into HEK293, C2C12 or MS1 cells using Lipofectamine 2000 (Invitrogen). To generate HEK293 cells stably expressing GFP or ECSM2-GFP, transfectants were selected in 1 mg/mL of G418 (Mediatech) and stable pools were maintained in culture medium containing 200 µg/mL of G418. To generate HEK293 cells stably expressing EGFR alone or co-expressing EGFR and ECSM2-GFP, a clone of HEK293 cells stably expressing EGFR was first obtained by selection in 1 mg/mL of G418 after transfection with pcDNA-EGFR as described previously (Huang et al. 2003). This clone was then employed as the recipient for pECSM2-GFP plasmid to ensure the same EGFR expression level before and after the secondary transfection. The EGFR/ECSM2-GFP-coexpressing cells were further obtained by combining green fluorescent colonies.
Cell starvation, stimulation protein extraction and immunoblotting
Cell starvation, stimulation, protein extraction and immunoblotting were performed as previously described (Huang et al. 2003, 2006). Briefly, cells were starved in serum-free medium containing 0.5% bovine serum albumin (BSA) (Roche) for 16 h, then stimulated with 1 nM EGF or 10% FBS for 15 min at 37 °C, harvested, and lysed in the lysis buffer containing 1% Triton X-100 and protease and phosphatase inhibitors (Huang et al. 2003, 2006). Proteins were quantitated using bicinchoninic acid (BCA) reagents (Pierce, Rockford, IL), resolved by SDS-PAGE and transferred onto Hybond ECL nitrocellulose membranes (Amersham Biosciences) followed by immunoblotting with antibodies as specified in each experiment. The bound antibodies were detected with SuperSignal chemiluminescent substrate (Pierce) and images were captured using a Kodak 4000 MM molecular imager.
Fluorescence staining and microscopy
Cells expressing GFP, ECSM2-GFP, EGFR, or co-expressing EGFR and ECSM2-GFP were grown on glass coverslips precoated with gelatin in 24-well plates for 24 h in the culture medium until reaching approximately 30% confluence. The cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 in PBS containing 1% BSA for 15 min, and incubated with phalloidin-TRITC (0.25 µM) for 1 h at room temperature. The coverslips were mounted onto microscope slides in Vectorshield mounting medium for fluorescence (Vector Laboratories, Burlingame, CA) and fluorescent images were visualized and captured using a Zeiss Axio Imager upright fluorescent microscope, as described previously (Tu et al. 2001).
Cell migration
Wound closure assays were performed as previously described (Zhang et al. 2002). Briefly, HEK293 cells were grown in 6-well plates until reaching confluence and then starved for 16 h. A scratch wound was introduced into the monolayer using a plastic pipette tip. All wounds were verified by microscopy to ensure that they initially had the same width. Cells were further cultured in the serum-free medium supplemented with 0.5% BSA with or without the addition of EGF (1 nM) to allow the cells to migrate into a scraped, acellular area. Wound closure was monitored by microscopy, and images of at least 10 different segments of the cell-free area were captured using a Zeiss Axiovert 200 M inverted microscope at 0, 24, 40, and 48 h after wounding. The wound width was measured using the Zeiss AxioVision software.
Densitometry and statistical analysis
Densitometric quantification of digital immunoblotting images was performed using Kodak Molecular Imaging Software (Version 4.0). All statistical data were from multiple experiments or measurements and presented as mean ± SE. The significance (P value) of differences was estimated using unpaired t-test and P < 0.05 was considered significant.
Multiple sequence alignment, homology comparison and bioinformatics analysis
Multiple sequence alignment and phylogenetic tree were made using the MegAlign program of DNAStar package. The homology was calculated using the AlignX software of the Vector NT1 Suite 9 package (Invitrogen). All other bioinformatics analyses were performed via the ExPASy (Expert Protein Analysis System) Server (http://us.expasy.org/tools) as indicated in the text and figure legends.
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Acknowledgements |
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Footnotes |
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* Correspondence: yhuang{at}chw.edu or xiao-zhao{at}126.com
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References |
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Received: 24 July 2008
Accepted: 20 November 2008
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