HER2 Carboxyl-terminal Fragments Regulate Cell Migration and Cortactin Phosphorylation
2009; Elsevier BV; Volume: 284; Issue: 37 Linguagem: Inglês
10.1074/jbc.m109.001982
ISSN1083-351X
AutoresJesús García‐Castillo, Kim Pedersen, Pierdavide Angelini, Joan Josep Bech‐Serra, Núria Colomé, Matthew Cunningham, Josep Lluís Parra-Palau, Françesc Canals, José Baselga, Joaquı́n Arribas,
Tópico(s)Advanced Biosensing Techniques and Applications
ResumoA group of breast cancer patients with a higher probability of developing metastasis expresses a series of carboxyl-terminal fragments (CTFs) of the tyrosine kinase receptor HER2. One of these fragments, 611-CTF, is a hyperactive form of HER2 that constitutively establishes homodimers maintained by disulfide bonds, making it an excellent model to study overactivation of HER2 during tumor progression and metastasis. Here we show that expression of 611-CTF increases cell motility in a variety of assays. Since cell motility is frequently regulated by phosphorylation/dephosphorylation, we looked for phosphoproteins mediating the effect of 611-CTF using two alternative proteomic approaches, stable isotope labeling with amino acids in cell culture and difference gel electrophoresis, and found that the latter is particularly well suited to detect changes in multiphosphorylated proteins. The difference gel electrophoresis screening identified cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, as a phosphoprotein probably regulated by 611-CTF. This result was validated by characterizing cortactin in cells expressing this HER2 fragment. Finally, we showed that the knockdown of cortactin impairs 611-CTF-induced cell migration. These results suggest that cortactin is a target of 611-CTF involved in the regulation of cell migration and, thus, in the metastatic behavior of breast tumors expressing this CTF. A group of breast cancer patients with a higher probability of developing metastasis expresses a series of carboxyl-terminal fragments (CTFs) of the tyrosine kinase receptor HER2. One of these fragments, 611-CTF, is a hyperactive form of HER2 that constitutively establishes homodimers maintained by disulfide bonds, making it an excellent model to study overactivation of HER2 during tumor progression and metastasis. Here we show that expression of 611-CTF increases cell motility in a variety of assays. Since cell motility is frequently regulated by phosphorylation/dephosphorylation, we looked for phosphoproteins mediating the effect of 611-CTF using two alternative proteomic approaches, stable isotope labeling with amino acids in cell culture and difference gel electrophoresis, and found that the latter is particularly well suited to detect changes in multiphosphorylated proteins. The difference gel electrophoresis screening identified cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, as a phosphoprotein probably regulated by 611-CTF. This result was validated by characterizing cortactin in cells expressing this HER2 fragment. Finally, we showed that the knockdown of cortactin impairs 611-CTF-induced cell migration. These results suggest that cortactin is a target of 611-CTF involved in the regulation of cell migration and, thus, in the metastatic behavior of breast tumors expressing this CTF. Deregulation of the epidermal growth factor receptor signaling network contributes to initiate and/or maintain malignant growth (1Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Cell Biol. 2001; 2: 127-137Crossref PubMed Scopus (5628) Google Scholar). One of these alterations, aberrant cellular motility, is necessary for invasive growth, which eventually culminates with the establishment of distant metastases, the leading cause of death in patients with cancer. The epidermal growth factor receptor is the prototype of a family that also includes HER2 (ErbB2, Neu), HER3, and HER4 (ErbB3 and ErbB4). The analysis of cells expressing various HER receptors indicated that HER2 plays a critical role in the regulation of motility (2Spencer K.S. Graus-Porta D. Leng J. Hynes N.E. Klemke R.L. J. Cell Biol. 2000; 148: 385-397Crossref PubMed Scopus (157) Google Scholar, 3Brandt B.H. Roetger A. Dittmar T. Nikolai G. Seeling M. Merschjann A. Nofer J.R. Dehmer-Möller G. Junker R. Assmann G. Zaenker K.S. FASEB J. 1999; 13: 1939-1949Crossref PubMed Scopus (90) Google Scholar). Upon activation through homo- or heterodimerization with other HER receptors, several tyrosines in the cytoplasmic tail of HER2 are phosphorylated and initiate intracellular signaling pathways, including the phospholipase C-γ1 and phosphatidylinositol 3-kinase pathways (4Dittmar T. Husemann A. Schewe Y. Nofer J.R. Niggemann B. Zänker K.S. Brandt B.H. FASEB J. 2002; 16: 1823-1825Crossref PubMed Google Scholar), which, in turn, promote cell migration through partially understood cascades. These cascades are largely regulated by phosphorylation/dephosphorylation of cellular components. A subgroup of HER2-positive patients expresses a series of carboxyl-terminal fragments (CTFs) 5The abbreviations used are:CTFcarboxyl-terminal fragmentDIGEdifference gel electrophoresisSILACstable isotope labeling with amino acids in cell cultureDAPI4′,6-diamidino-2-phenylindoleMSmass spectrometryCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidDoxdoxycycline. of HER2. HER2 CTFs can be generated by two independent mechanisms: proteolytic processing and alternative initiation of translation. Metalloproteases with the so-called α-secretase activity shed the extracellular domain of HER2, leaving behind a fragment, known as P95, that starts around alanine 648 (5Yuan C.X. Lasut A.L. Wynn R. Neff N.T. Hollis G.F. Ramaker M.L. Rupar M.J. Liu P. Meade R. Protein Expr. Purif. 2003; 29: 217-222Crossref PubMed Scopus (36) Google Scholar) (see also Fig. 1A). Alternative initiation of translation of the mRNA encoding HER2 from the methionine codons 611 and 687 generates two fragments: 611- and 687-CTF. These differ by a stretch of 76 amino acids, which includes the transmembrane domain and a cysteine-rich short extracellular domain (6Anido J. Scaltriti M. Bech Serra J.J. Josefat B.S. Todo F.R. Baselga J. Arribas J. EMBO J. 2006; 25: 3234-3244Crossref PubMed Scopus (180) Google Scholar) (see also Fig. 1A). carboxyl-terminal fragment difference gel electrophoresis stable isotope labeling with amino acids in cell culture 4′,6-diamidino-2-phenylindole mass spectrometry 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid doxycycline. We have recently shown that 687-CTF seems to be inactive (7Pedersen K. Angelini P.D. Laos S. Bach-Faig A. Cunningham M.P. Ferrer-Ramón C. Luque-García A. García-Castillo J. Parra-Palau J.L. Scaltriti M. Ramón y Cajal S. Baselga J. Arribas J. Mol. Cell. Biol. 2009; 29: 3319-3331Crossref PubMed Scopus (135) Google Scholar). In contrast, the two CTFs containing the transmembrane domain, 648- and 611-CTFs, expressed at levels similar to those found in human breast tumors, can activate different intracellular signal transduction pathways (7Pedersen K. Angelini P.D. Laos S. Bach-Faig A. Cunningham M.P. Ferrer-Ramón C. Luque-García A. García-Castillo J. Parra-Palau J.L. Scaltriti M. Ramón y Cajal S. Baselga J. Arribas J. Mol. Cell. Biol. 2009; 29: 3319-3331Crossref PubMed Scopus (135) Google Scholar). The level of activation of these pathways by HER2 CTFs is quite different. 611-CTF activates the mitogen-activated protein kinase and the Akt pathways to a greater extent because it constitutively forms homodimers maintained through disulfide bonds (7Pedersen K. Angelini P.D. Laos S. Bach-Faig A. Cunningham M.P. Ferrer-Ramón C. Luque-García A. García-Castillo J. Parra-Palau J.L. Scaltriti M. Ramón y Cajal S. Baselga J. Arribas J. Mol. Cell. Biol. 2009; 29: 3319-3331Crossref PubMed Scopus (135) Google Scholar). In contrast, 648-CTF does not seem to form homodimers, and its activity is comparable with that of full-length HER2 (7Pedersen K. Angelini P.D. Laos S. Bach-Faig A. Cunningham M.P. Ferrer-Ramón C. Luque-García A. García-Castillo J. Parra-Palau J.L. Scaltriti M. Ramón y Cajal S. Baselga J. Arribas J. Mol. Cell. Biol. 2009; 29: 3319-3331Crossref PubMed Scopus (135) Google Scholar). Therefore, cells expressing transmembrane CTFs, particularly 611-CTF, constitute a relevant model to study the consequences of the overactivation of HER2 signaling in tumors. Supporting this conclusion, it has been shown that breast cancer patients expressing CTFs have worse prognosis and are more likely to develop nodal metastasis compared with patients expressing predominantly full-length HER2 (8Molina M.A. Sáez R. Ramsey E.E. Garcia-Barchino M.J. Rojo F. Evans A.J. Albanell J. Keenan E.J. Lluch A. García-Conde J. Baselga J. Clinton G.M. Clin. Cancer Res. 2002; 8: 347-353PubMed Google Scholar). Here we show that expression of 611-CTF enhances the migration of breast cancer cells as judged by monitoring single-cell migration, transwell migration, and wound healing assays. Since cell migration is frequently regulated by phosphorylation/dephosphorylation, we searched for phosphoproteins regulated by 611-CTF and probably contributing to cell migration using two independent proteomic approaches. The results of these analyses showed that difference gel electrophoresis (DIGE) is a particularly convenient methodology to analyze the regulation of multiphosphorylated proteins. Cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, was identified by DIGE as a phosphoprotein likely to be regulated by 611-CTF. Several assays showed that expression of 611-CTF leads to an increase in the phosphorylation of cortactin and to the generation of cell protrusions resembling lamellipodia or invadopodia. Confirming a role of cortactin on the increased cell migration induced by 611-CTF, down-modulation of the former with short hairpin RNAs leads to an impairment of the cell migration induced by the HER2 fragment. These results unveil a role of cortactin in the increased cell migration induced by hyperactive HER2 and strongly suggest that cortactin-dependent increased cell migration contributes to the tendency of breast tumors expressing CTFs to metastasize. MCF7 Tet-Off (BD Biosciences) transfected with full-length HER2, 611-CTF, 648-CTF, and 687-CTF have been recently characterized (7Pedersen K. Angelini P.D. Laos S. Bach-Faig A. Cunningham M.P. Ferrer-Ramón C. Luque-García A. García-Castillo J. Parra-Palau J.L. Scaltriti M. Ramón y Cajal S. Baselga J. Arribas J. Mol. Cell. Biol. 2009; 29: 3319-3331Crossref PubMed Scopus (135) Google Scholar). To generate cortactin knockdowns, MCF7 Tet-Off/611-CTF cells were transfected with empty pRetroSuper vector or with the same vector expressing a hairpin targeting cortactin (5′-GAT CCC CCC ACA GAA TTT GCT AAT ATT TCA AGA GAA TAT TAG CAA ATT CTG TGG TTT TTA-3′). Stable transfectants were selected with 1 μg/ml puromycin, and the levels of cortactin were analyzed by Western blot. Two independent clones, 611 sh1 and 611 sh2, with ∼3.5- and ∼7-fold lower levels of cortactin compared with cells transfected with vector, respectively, were chosen for further analysis. Antibodies were from Cell Signaling (P-Erk1/2 (catalog number 9101), Erk1/2 (catalog number 9102), P-Akt (catalog numbers 9275 and 9271), and Akt (catalog number 9272)), BioGenex (anti-HER2 (CB11)), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) (anti-cortactin (clone H-191)), Abcam (anti-cortactin Y-421 (catalog number ab47768)) Sigma (anti-FLAG epitope), Upstate Biotechnology (anti-phosphotyrosine (clone 4G10) and anti-cortactin (clone 4F11)), Neomarkers (anit-p21WAF1), BD Pharmigen (anti-Bid), Amersham Biosciences (anti-mouse and anti-rabbit horseradish peroxidase-conjugated), and Invitrogen (anti-rabbit and anti-mouse IgGs linked to Alexa Fluor 488 or Alexa 568). Anti-EF2 antibody was a kind gift from Dr. C. G. Proud (University of British Columbia, Vancouver, Canada). Extracts for immunoblots were prepared in modified radioimmune precipitation buffer (20 mm NaH2PO4/NaOH, pH 7.4, 150 mm NaCl, 1% Triton X-100, 5 mm EDTA, 100 mm phenylmethylsulfonyl fluoride, 25 mm NaF, 16 μg/ml aprotinin, 10 μg/ml leupeptin, and 1.3 mm Na3VO4), and protein concentrations were determined with DC protein assay reagents (Bio-Rad). Samples were mixed with loading buffer (final concentrations: 62 mm Tris, pH 6.8, 12% glycerol, 2.5% SDS) with 5% β-mercaptoethanol and incubated at 99 °C for 5 min before fractionation of 15 μg of protein by SDS-PAGE. Where appropriate, signals in Western blots were quantified with the software ImageJ 1.38 (National Institutes of Health, Bethesda, MD). For two-dimensional blots, protein extracts were isoelectrofocused on immobilized pH gradient strips (7 cm; pH 4–7) using an Ettan IPGphor system. After focusing, the strips were equilibrated for 15 min in a reducing solution (6 m urea, 100 mm Tris-HCl, pH 8, 30% (v/v) glycerol, 2% (w/v) SDS, 5 mg/ml dithiothreitol), and the focused proteins were resolved by SDS-PAGE. Finally, the proteins were transferred to a nitrocellulose membrane (Bio-Rad) and immunoblotted with anti-cortactin antibodies. PP2 (Src inhibitor) and PD98056 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor) were both from Calbiochem. Lapatinib was kindly provided by GlaxoSmithKline (Research Triangle Park, NJ). Cell motility data were generated using a single-cell time lapse video microscopy system consisting of an inverted microscope with ×10 magnification differential interference contrast objective (Olympus IX-81, Olympus, Japan); motorized xy-stage, z-focus drive, and shutter (Olympus, Japan); charge-coupled device camera (Olympus, Japan); and automated data acquisition software (CELL-R, Olympus, Japan). Individual cell tracks were manually drawn, from digital images taken every 30 min for 24 h, by plotting the position of the cell nucleus. 2.5 × 104 cells were plated onto serum-coated transwells (8-μm pore membranes; Corning Glass). After 48 h, cells of the upper side of the membrane were removed with a cotton swab, and the migrated cells in the lower side were fixed with 2.5% glutaraldehyde for 20 min at room temperature. Finally, membranes were excised and mounted on glass slides, using mounting medium with DAPI (VectaShield; Vector Laboratories). Cells in the lower side of the membranes were counted in an epifluorescence microscope Nikkon Eclipse TE2000-S, assessing five random fields at ×20 magnification. Confluent cell monolayers were scratch-wounded with a yellow Gilson pipette tip and further cultured in the presence or absence of doxycycline. The average migration distance was calculated by subtracting the wound width after 24 h of migration from that of the reference wound. MCF7 Tet-Off/611-CTF cells were seeded in SILAC DMEM medium (Invitrogen) supplemented with dialyzed serum (amino acid-free) and the appropriate normal or isotopically labeled amino acids: 12C14N ("light") arginine and 12C ("light") lysine or 13C15N ("heavy") arginine and 13C ("heavy") lysine. Cells were incubated for a total of 7 days, corresponding to ∼5 cell doublings. Cells labeled with light or heavy isotopes were cultured with or without doxycycline, respectively, for 18 h in the presence of serum and an additional 6 h in serum-free media and lysed. Proteins in cell lysates were quantified and mixed 1:1. Phosphoproteins were purified with the Qiagen phosphoprotein purification kit according to the manufacturer's instructions and in the presence of phosphatase inhibitors. Purified phosphoproteins were resuspended in SDS-PAGE loading buffer and subjected to one-dimensional electrophoresis on a 10% polyacrylamide-SDS gel. The one-dimensional gel lanes were cut into 20 horizontal slices, and each slice was subjected to in-gel tryptic digestion using modified porcine trypsin (Promega). The digests were then analyzed on an Esquire HCT IT mass spectrometer (Bruker, Bremen), coupled to a nano-high pressure liquid chromatography system (Ultimate; LC Packings). Peptide mixtures were first concentrated on a 300-mm inner diameter, 1-mm PepMap nanotrapping column and then loaded onto a 75-mm inner diameter, 15-cm PepMap nanoseparation column (LC Packings). Peptides were then eluted by an acetonitrile gradient (0–60% B in 150 min, where B is 80% ACN, 0.1% formic acid in water; flow rate ∼300 nl/min) through a PicoTip emitter nanospray needle (NewObjective, Woburn, MA) onto the nanospray ionization source of the IT mass spectrometer. MS/MS fragmentation (1.9 s, 100–2800 m/z) was performed on two of the most intense ions, as determined from a 1.2-s MS survey scan (310–1500 m/z), using a dynamic exclusion time of 1.2 min for precursor selection and excluding single-charged ions. An automated optimization of MS/MS fragmentation amplitude, starting from 0.60 V, was used. Data processing for protein identification and quantitation was performed using WARP-LC 1.1 (Bruker, Bremen), a software platform integrating liquid chromatography-MS run data processing, protein identification through data base search of MS/MS spectra, and protein quantitation based on the integration of the chromatographic peaks of MS-extracted ion chromatograms for each precursor. Proteins were identified using Mascot (Matrix Science, London, UK) to search the International Protein Index-Human 3.26 data base (67,665 sequences, 28,462,007 residues) (9Kersey P.J. Duarte J. Williams A. Karavidopoulou Y. Birney E. Apweiler R. Proteomics. 2004; 4: 1985-1988Crossref PubMed Scopus (640) Google Scholar). MS/MS spectra were searched with a precursor mass tolerance of 1.5 Da, fragment tolerance of 0.5 Da, trypsin specificity with a maximum of 1 missed cleavage, cysteine carbamidomethylation set as fixed modification, and methionine oxidation and the corresponding Lys and Arg SILAC labels as variable modifications. The positive identification criterion was set as an individual Mascot score for each peptide MS/MS spectrum higher than the corresponding homology threshold score. The false positive rate for Mascot protein identification was measured by searching a randomized decoy data base, as described by Elias and Gygi (10Elias J.E. Gygi S.P. Nat. Methods. 2007; 4: 207-214Crossref PubMed Scopus (2839) Google Scholar), and estimated to be under 5%. A second round search restricted to the proteins positively identified in the first Mascot search was performed setting Ser, Thr, and Tyr phosphorylation as additional variable modifications. For protein quantitation, heavy/light (H/L) ratios were calculated averaging the measured H/L ratios for the observed peptides, after discarding outliers. For selected proteins of interest, quantitation data obtained from the automated WARP-LC analysis was manually reviewed. 107 MCF7 Tet-Off/611-CTF cells were seeded and cultured with or without doxycycline for 18 h in the presence of serum and an additional 6 h in serum-free media and lysed. Phosphoproteins were purified from cell lysates with the Qiagen phosphoprotein purification kit according to the manufacturer's instructions and in the presence of phosphatase inhibitors. Purified phosphoproteins were adjusted to a concentration of 2 mg/ml by the addition of DIGE labeling buffer. 50 mg of each sample were labeled by the addition of 400 pmol of either Cy3 or Cy5 cyanine dyes (GE Healthcare) in 1 ml of anhydrous dimethylformamide. After 30 min of incubation on ice in the dark, the reaction was quenched by the addition of 10 mm lysine followed by a further 10 min of incubation. After labeling, the samples corresponding to cells not expressing (Cy3) and expressing 611-CTF (Cy5) were mixed and diluted 2-fold with isoelectric focusing sample buffer (8 m urea, 4% (w/v) CHAPS, 2% dithiothreitol, 2% pharmalytes, pH 3–10). Two-dimensional electrophoresis was performed using GE Healthcare reagents and equipment. First dimension isoelectric focusing was performed on immobilized pH gradient strips (24 cm; pH 3–10) using an Ettan IPGphor system. Samples were applied via cup loading near the basic end of the strips, previously rehydrated overnight in 450 ml of rehydration buffer (8 m urea, 4% (w/v) CHAPS, 1% pharmalytes, pH 3–10, 100 mm DeStreak). After focusing for a total of 67 kV-h, the strips were equilibrated first for 15 min in 6 ml of reducing solution (6 m urea, 100 mm Tris-HCl, pH 8, 30% (v/v) glycerol, 2% (w/v) SDS, 5 mg/ml dithiothreitol) and then in 6 ml of alkylating solution (6 m urea, 100 mm Tris-HCl, pH 8, 30% (v/v) glycerol, 2% (w/v) SDS, 22.5 mg/ml iodoacetamide) for a further 15 min on a rocking platform. Second dimension SDS-PAGE was run by overlaying the strips on 12.5% isocratic Laemmli gels (24 × 20 cm), cast in low fluorescence glass plates, on an Ettan DALT VI system. Gels were run at 20 °C, at 2.5 watts/gel constant power during 30 min followed by 17 watts/gel until the bromphenol blue tracking front had run off the bottom of the gels (about 5 h). Fluorescence images of the gels were acquired on a Typhoon 9400 scanner (GE Healthcare). Cy3 and Cy5 images were scanned at 532-nm excitation/580-nm emission and 633-nm excitation/670-nm emission, respectively, at a 100 mm resolution. Image analysis was performed using DeCyder version 5.0 software (GE Healthcare). Selected proteins of interest were excised from the gels, subjected to trypsin digestion, and identified by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry as described (11Bech-Serra J.J. Santiago-Josefat B. Esselens C. Saftig P. Baselga J. Arribas J. Canals F. Mol. Cell. Biol. 2006; 26: 5086-5095Crossref PubMed Scopus (99) Google Scholar). FLAG-tagged constructs were purified by immunoprecipitation with M2-anti-FLAG-agarose beads (Sigma) from transiently transfected HEK293 cells. Cells were lysed in immunoprecipitation buffer, containing 150 mm NaCl, 50 mm Tris, pH 7.4, 1% Nonidet P-40, 2 mm EDTA, 5 mm NaF, 10 μg/ml aprotinin, 1 mm Na3VO4, 10 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mm phenylmethylsulfonyl fluoride. The kinase activity was determined by incubating the immunoprecipitated material for 4 h with lysates of MCF7 Tet-Off cells stably transfected with 611-CTF treated or not with doxycycline, in 150 mm NaCl, 20 mm HEPES, pH 7.9, 0.1% Nonidet P-40, 5 mm MgCl2, 5 mm MnCl2, 1 mm dithiothreitol, 1 μm cold ATP, and 0.3 μm [γ-32P]ATP (3000 Ci/mmol). To analyze in vitro kinase activity of 611-CTF, it was purified by immunoprecipitation with CB11 antibody from MCF7 Tet-Off cells treated or not with doxycycline and incubated for 24 h with cortactin-FLAG purified as above in the presence of [γ-32P]ATP. Cells for immunofluorescence microscopy seeded on glass coverslips were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.2% Triton X-100 for 10 min. For blocking and antibody binding, we used phosphate-buffered saline with 1% bovine serum albumin, 0.1% saponin, and 0.02% NaN3, and for mounting, we used Vectashield with DAPI (Vector Laboratories). To analyze the effect of individual CTFs on cell migration, we used MCF7 Tet-Off cells stably transfected with plasmids encoding HER2 or 611-, 648-, or 687-CTFs (Fig. 1A; see also Ref. 7Pedersen K. Angelini P.D. Laos S. Bach-Faig A. Cunningham M.P. Ferrer-Ramón C. Luque-García A. García-Castillo J. Parra-Palau J.L. Scaltriti M. Ramón y Cajal S. Baselga J. Arribas J. Mol. Cell. Biol. 2009; 29: 3319-3331Crossref PubMed Scopus (135) Google Scholar) and, as control, the same cells transfected with the empty vector. Analysis by Western blot showed that, except for cells transfected with 611-CTF, each cell line predominantly expressed HER2 species of the expected molecular weight (Fig. 1B). In cells transfected with 611-CTF, we detected two isoforms (Fig. 1B). Previous characterization showed that the faster migrating one is an intracellular precursor, and the slower migrating form is N-glycosidated and located at the cell surface (7Pedersen K. Angelini P.D. Laos S. Bach-Faig A. Cunningham M.P. Ferrer-Ramón C. Luque-García A. García-Castillo J. Parra-Palau J.L. Scaltriti M. Ramón y Cajal S. Baselga J. Arribas J. Mol. Cell. Biol. 2009; 29: 3319-3331Crossref PubMed Scopus (135) Google Scholar). Analysis of cell migration tracks by time lapse video microscopy showed that, while the expression of 687-CTF had no effect on cell migration, expression of full-length HER2 and 648-CTF induced a slight (∼1.5-fold) increase in cell migration speed (Fig. 1E). In contrast, the expression of 611-CTF led to a higher (∼3-fold) increase in the same assay and to the formation of very dynamic cell protrusions typical of migrating cells (Fig. 1, C–E, and supplemental videos). Equivalent results were observed when assaying cell migration in serum-coated transwells (Fig. 1, F and G). Although 687-CTF had no effect and HER2 and 648-CTF induced a modest increase in the number of cells that migrated through the transwell membrane (Fig. 1G), 611-CTF induced a ∼2.5-fold increase (Fig. 1, F and G). Finally, and in agreement with the previous assays, cells expressing 611-CTF closed the wounds made in a confluent cell monolayer faster than control cells or cells expressing HER2 or 648- or 687-CTF (Fig. 1, H and I). Similar results were obtained with an independent 611-CTF clone (data not shown) that expresses one-fifth the levels of the clone shown in Fig. 1. The levels of 611-CTF in this low expressing clone are similar to those found in human tumors (7Pedersen K. Angelini P.D. Laos S. Bach-Faig A. Cunningham M.P. Ferrer-Ramón C. Luque-García A. García-Castillo J. Parra-Palau J.L. Scaltriti M. Ramón y Cajal S. Baselga J. Arribas J. Mol. Cell. Biol. 2009; 29: 3319-3331Crossref PubMed Scopus (135) Google Scholar). In summary, the results of the different assays consistently show that the expression of 611-CTF increases the ability of cells to migrate. Cell motility is frequently regulated by phosphorylation/dephosphorylation of cellular components. Consistently, treatment with lapatinib, a potent tyrosine kinase inhibitor that targets both HER2 and the epidermal growth factor receptor (12Rusnak D.W. Affleck K. Cockerill S.G. Stubberfield C. Harris R. Page M. Smith K.J. Guntrip S.B. Carter M.C. Shaw R.J. Jowett A. Stables J. Topley P. Wood E.R. Brignola P.S. Kadwell S.H. Reep B.R. Mullin R.J. Alligood K.J. Keith B.R. Crosby R.M. Murray D.M. Knight W.B. Gilmer T.M. Lackey K. Cancer Res. 2001; 61: 7196-7203PubMed Google Scholar) and is commonly used to analyze HER2-dependent processes (e.g. see Ref. 13Scaltriti M. Verma C. Guzman M. Jimenez J. Parra J.L. Pedersen K. Smith D.J. Landolfi S. Ramón y Cajal S. Arribas J. Baselga J. Oncogene. 2009; 28: 803-814Crossref PubMed Scopus (329) Google Scholar), prevented the increase in cell migration observed in the transwell migration assay (supplemental Fig. S1). To identify phosphoproteins regulated by 611-CTF, we used two different proteomic techniques, SILAC (stable isotope labeling by amino acids in cell culture) (14Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4581) Google Scholar) and DIGE (15Van den Bergh G. Arckens L. Curr. Opin. Biotechnol. 2004; 15: 38-43Crossref PubMed Scopus (167) Google Scholar). SILAC is considered the most accurate way of quantifying differential protein expression and is based on the labeling of proteins with isotopic variants of the same amino acids (16Everley P.A. Zetter B.R. Ann. N.Y. Acad. Sci. 2005; 1059: 1-10Crossref PubMed Scopus (14) Google Scholar). DIGE is an alternative quantitative proteomic technique, which is based on the labeling of proteins with different fluorochromes. Labeled proteins are loaded onto a single two-dimensional gel, thereby overcoming the lack of reproducibility inherent to two-dimensional electrophoresis (15Van den Bergh G. Arckens L. Curr. Opin. Biotechnol. 2004; 15: 38-43Crossref PubMed Scopus (167) Google Scholar). To perform the SILAC analysis, we cultured MCF7 Tet-Off/611-CTF cells with arginine and lysine labeled with light and heavy isotopes (Fig. 2A) (see "Experimental Procedures"). Then the expression of 611-CTF was induced by removal of doxycycline from the culture medium of cells labeled with heavy isotopes. Next, cells expressing 611-CTF as well as non-expressing control cells (i.e. cells labeled with light arginine and lysine and kept in the presence of doxycycline) were lysed (Fig. 2A). Cell lysates were mixed one to one, and phosphoproteins, purified from this mixture using metal affinity chromatography, were fractionated by SDS-PAGE (Fig. 2A). After separation, the gel was cut horizontally in 20 slices, and these gel segments were separately processed (see "Experimental Procedures") to identify peptides by mass spectrometry and compare their relative abundance in lysates from control cells and cells expressing 611-CTF (Fig. 2A). 3260 peptides from 658 individual proteins were identified. The distribution of the H/L ratios indicated that the majority of phosphoproteins were not affected by the expression of 611-CTF (Fig. 2B). Proteins exhibiting average H/L ratios outside the interval 0.8–1.3 are shown in supplemental Table S1. To illustrate these results, peptide doublets from BID, a proapoptotic factor that belongs to the Bcl-2 protein family, the cyclin-dependent kinase inhibitor 1A (p21), and EF2 (translation elongation factor 2) are shown in Fig. 2C (upper panels). Direct determination of the levels of BID, p21, and EF2 in phosphoprotein fractions of cells treated without or with doxycycline (Fig. 2C, bottom panels) validated the results of the SILAC analysis. However, none of the proteins identified appeared to be firm candidates to regulate cell migration (supplemental Table S1). To identify additional phosphoproteins regulated by 611-CTF, we performed a DIGE screening (Fig. 3A). Phosphoproteins from cells expressing 611-CTF or control cells were purified by metal affinity chromatography. Purified phosphoproteins were labeled with the cyanine dyes Cy3 or Cy5 and analyzed by two-dimensional gel electrophoresis (Fig. 3A). To visualize the protein spots, the gel was scanned with fluorophore-specific excitation and emission wavelengths, and two independent images
Referência(s)