Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation
2006; Springer Nature; Volume: 25; Issue: 13 Linguagem: Inglês
10.1038/sj.emboj.7601191
ISSN1460-2075
AutoresJudit Anido, Maurizio Scaltriti, Joan Josep Bech‐Serra, Belén Santiago Josefat, Federico Rojo Todo, José Baselga, Joaquı́n Arribas,
Tópico(s)Chemical Synthesis and Analysis
ResumoArticle22 June 2006free access Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation Judit Anido Judit Anido Search for more papers by this author Maurizio Scaltriti Maurizio Scaltriti Search for more papers by this author Joan Josep Bech Serra Joan Josep Bech Serra Search for more papers by this author Belén Santiago Josefat Belén Santiago Josefat Search for more papers by this author Federico Rojo Todo Federico Rojo Todo Search for more papers by this author José Baselga José Baselga Search for more papers by this author Joaquín Arribas Corresponding Author Joaquín Arribas Medical Oncology Research Program, Vall d'Hebron University Hospital Research Institute, Barcelona, Spain Search for more papers by this author Judit Anido Judit Anido Search for more papers by this author Maurizio Scaltriti Maurizio Scaltriti Search for more papers by this author Joan Josep Bech Serra Joan Josep Bech Serra Search for more papers by this author Belén Santiago Josefat Belén Santiago Josefat Search for more papers by this author Federico Rojo Todo Federico Rojo Todo Search for more papers by this author José Baselga José Baselga Search for more papers by this author Joaquín Arribas Corresponding Author Joaquín Arribas Medical Oncology Research Program, Vall d'Hebron University Hospital Research Institute, Barcelona, Spain Search for more papers by this author Author Information Judit Anido, Maurizio Scaltriti, Joan Josep Bech Serra, Belén Santiago Josefat, Federico Rojo Todo, José Baselga and Joaquín Arribas 1 1Medical Oncology Research Program, Vall d'Hebron University Hospital Research Institute, Barcelona, Spain *Corresponding author. Medical Oncology Research Program, Vall d'Hebron University Hospital Research Institute, Psg. Vall d'Hebron 119-129, Barcelona 8035, Spain. Tel/Fax: +34 93 274 6026; E-mail: [email protected] The EMBO Journal (2006)25:3234-3244https://doi.org/10.1038/sj.emboj.7601191 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The overactivation of the HERs, a family of tyrosine kinase receptors, leads to the development of cancer. Although the canonical view contemplates HER receptors restricted to the secretory and endocytic pathways, full-length HER1, HER2 and HER3 have been detected in the nucleoplasm. Furthermore, limited proteolysis of HER4 generates nuclear C-terminal fragments (CTFs). Using cells expressing a panel of deletion and point mutants, here we show that HER2 CTFs are generated by alternative initiation of translation from methionines located near the transmembrane domain of the full-length molecule. In vitro and in vivo, HER2 CTFs are found in the cytoplasm and nucleus. Expression of HER2 CTFs to levels similar to those found in human tumors induces the growth of breast cancer xenografts in nude mice. Tumors dependent on CTFs are sensitive to inhibitors of the kinase activity but do not respond to therapeutic antibodies against HER2. Thus, the kinase domain seems necessary for the activity of HER2 CTFs and the presence of these HER2 fragments could account for the resistance to treatment with antibodies. Introduction Exposure to epidermal growth factor (EGF) leads to modifications in several aspects of the cellular behavior related to the development of cancer (Yarden and Sliwkowski, 2001). EGF binds to HER1 (also known as EGF receptor or ErbB1), which is the prototype of a family that includes three additional members: HER2 (also known as neu or ErbB2), HER3 and HER4 (also known as ErbB3 and ErbB4, respectively) (Burgess et al, 2003). The generation of HER homo- or heterooligomers induces the activation of the intrinsic tyrosine kinase activity of the receptors. The subsequent phosphorylation of intracellular tyrosine residues leads to the recruitment of factors that transfer the signal from the plasma membrane to the nucleus. This induces changes in the expression of genes that coordinately regulate proliferation, migration, adhesion, differentiation and apoptosis (Yarden and Sliwkowski, 2001). The involvement of HER receptors, and particularly HER2, in the development of a variety of cancers, including breast tumors, has led to the implementation of different therapeutic strategies (Yarden and Sliwkowski, 2001). These include monoclonal antibodies directed against the ectodomain of the receptors, such as Herceptin (also known as Trastuzumab), and small-molecule tyrosine kinase inhibitors (Baselga and Norton, 2002). Although the canonical view contemplates HER receptors restricted to the secretory and endocytic pathways, accumulating evidence indicates the presence of full-length HER receptors or fragments of them within the cell nucleoplasm. HER1, HER2 and HER3 have been detected as full-length molecules in the nucleus of a variety of cells (Lin et al, 2001; Offterdinger et al, 2002; Wang et al, 2004). A C-terminal fragment (CTF) encompassing the cytoplasmic domain of HER4, generated by the sequential action of two types of proteolytic activities, is also transported to the cell nucleus. The mechanism of solubilization and transport to the nucleus of full-length receptors is still under debate (Oksvold et al, 2002; Wells and Marti, 2002; Carpenter, 2003; Johnson et al, 2004). In contrast, the generation and transport to the nucleus of fragments consisting of the cytoplasmic domain of certain type I transmembrane molecules, including HER4, seems to be a widespread mechanism of transducing signals directly from the plasma membrane to the nucleus (Carpenter, 2003). Upon ligand binding, the extracellular domain of HER4 is cleaved by the metalloprotease disintegrin TACE (tumor necrosis factor alpha converting enzyme) in a process known as ectodomain shedding. The resulting transmembrane-cytoplasmic domain is a substrate of presenilin-like protease(s) that releases the cytoplasmic domain (Ni et al, 2001), which bears a basic amino-acid-based nuclear localization signal (NLS) (Williams et al, 2004). A series of CTFs, assumed to encompass the transmembrane and cytoplasmic domain of HER2, are frequently found in human mammary tumors (Molina et al, 2002, and references therein); in fact, these fragments are the main HER2 species in some tumors (Molina et al, 2002). Despite the functional potential and the clinical importance of these CTFs, which associate with nodal metastasis (Molina et al, 2002), the mechanism of generation is not understood. By analogy with HER4 and because ectodomain shedding is a frequent event that affects a numerous subset of cell surface proteins (reviewed in Arribas and Borroto, 2002), one possibility is that the HER2 CTFs arise through shedding of the extracellular domain of HER2. In agreement with this hypothesis, a discrete fragment of 95 kDa is generated after the shedding of HER2 by an unidentified metalloprotease (Codony-Servat et al, 1999). However, in contrast to the majority of shedding events, that of HER2 is very inefficient. It affects only a small percentage of the molecules, even when the cells are exposed to potent and nonspecific metalloprotease activators (Codony-Servat et al, 1999; Molina et al, 2001). Thus, the evidence available casts doubts on the hypothesis of ectodomain shedding as the main mechanism of generation of the HER2 CTFs in tumors. Here, we show that CTFs similar to those observed in vivo are spontaneously expressed in cells stably transfected with the cDNA encoding full-length HER2. Biochemical evidence indicate that these fragments are not generated by proteolytic processing of full-length HER2; they are largely synthesized by alternative initiation of translation from methionines 611 and 687, located right before and after the transmembrane region, respectively. In vitro and in vivo, CTFs localize to the cytoplasm and nucleus of cells. In an effort to determine their possible biological activity and relevance in tumor development, we show that overexpression of HER2 CTFs induces the growth of breast cancer xenografts in nude mice. Tumors dependent on CTFs are sensitive to inhibitors of the kinase activity but do not respond to therapeutic antibodies against HER2. Thus, the kinase domain seems necessary for the activity of HER2 CTFs and the presence of these HER2 fragments could account for the resistance to treatment with antibodies. Results Transfection of HER2 into mammalian cells leads to the spontaneous generation of CTFs As a tool to characterize the mechanism of generation of CTFs, we permanently transfected a cDNA encoding human HER2, tagged at the C-terminus with the HA and hexahistidine epitopes (Figure 1A), into CHO cells. This cell line has been frequently used as a model to study ectodomain shedding (Arribas et al, 1996). In order to select positive clones, we analyzed cell extracts by Western blot with antibodies against the cytoplasmic domain of HER2. In addition to the expected 185 kDa full-length form of the receptor, several clones expressed variable levels of ∼90–100 kDa species (data not shown), likely corresponding to fragments encompassing the intracellular domain of HER2. This conclusion was confirmed with the monoclonal antibodies L87, Herceptin, CB11and Ab3, directed against different regions of human HER2 (Figure 1A); while L87 and Herceptin recognized only full-length HER2 (data not shown), the antibodies against the cytoplasmic domain of HER2 readily detected the ∼95 kDa forms (Figure 1B). The apparent differences in the electrophoretic migration of the CTFs expressed by the three clones (Figure 1B) are attributable to the different levels of expression, because a similar analysis loading equivalent levels of CTFs showed indistinguishable electrophoretic patterns (Supplementary Figure 1A). To delimit further their length, we purified by Ni-chromatography, resolved by SDS–PAGE and trypsin digested the hexahistidine tagged CTFs. The tryptic fragments were analyzed by nano-LC-ESI ion trap mass spectrometry. We consistently identified several peptides, including those shown in Figure 1C. Thus, most of the cytoplasmic portion of HER2, likely containing the whole tyrosine kinase domain is contained in at least some of the CTFs. Figure 1.Spontaneous generation of CTFs in cells stably transfected with HER2. (A) Schematic of HER2 showing the N-terminus (N), the transmembrane domain (hatched box), the intracellular kinase domain (shaded box), the hemaglutinin antigen (HA) and hexahistidine (His) tags and the C-terminus (C). The localization of epitopes recognized by the monoclonal antibodies Herceptin® and Ab3 are shown. The monoclonal antibodies L87 and CB11 recognize undefined epitopes located in the extracellular and intracellular domain, respectively. (B) Analysis by Western blot with the indicated antibodies of parental CHO cells (C) and different clones of the same cells stably transfected with HER2 tagged with HA and His. (C) Schematic of peptides detected by mass spectrometry analysis in CTFs purified by Ni-chromatography. The sequence from lysine 676 to lysine 753 is shown. Methionines 687, 706 and 712 are shown in bold. The hatched and shaded boxes represent the transmembrane and tyrosine kinase domains, respectively. Peptides detected by mass spectrometry are indicated with thick lines. (D) Lysates from CHO cells stably transfected with untagged HER2 and from a human mammary tumor expressing CTFs were analyzed by Western blot with the antibody CB11. Download figure Download PowerPoint To compare the electrophoretic migrations of the HER2 CTFs generated in transfected cells with that of those observed in human mammary tumors, untagged HER2 was stably transfected into CHO cells. Again, Western blot analysis with CB11 antibodies showed that a number of clones expressed variable levels of HER2 CTFs (data not shown). The electrophoretic migration of CTFs present in these clones partially coincide with those of CTFs expressed in human tumors (Figure 1D). Thus, part of the CTFs generated in CHO cells seem similar, if not identical, to those generated in tumors. Ectodomain shedding is not the main mechanism of generation of HER2 CTFs The shedding of HER2 produces a soluble fragment of 110 kDa, corresponding to the extracellular domain (HER2 ECD), and a ∼95 kDa cell-associated fragment encompassing the transmembrane and intracellular domains (Codony-Servat et al, 1999). The enzyme responsible is a metalloprotease sensitive to hydroxamic acid-derived compounds such as BB94 (Codony-Servat et al, 1999). To determine whether the HER2 CTFs observed in the stably transfected CHO cell lines arise through proteolytic shedding, we analyze the effect of different concentrations of BB94. As previously shown (Molina et al, 2001), 2 and 5 μM BB94 inhibited the shedding of HER2 in BT474 cells treated with metalloprotease activators (data not shown). However, these concentrations of BB94 do not have an effect on the levels of HER2 CTFs (Figure 2A), indicating that a metalloprotease sensitive to BB-94 is not required for the generation of the HER2 CTFs in CHO cells. Figure 2.CTFs are not generated by ectodomain shedding. (A) Stably transfected CHO cells expressing different HER2 species were incubated overnight with the indicated concentrations of the metalloprotease inhibitor BB-94 and analyzed by Western blot with the monoclonal anti-HER2 antibody CB11. (B) Stably transfected CHO cells expressing CTFs (clone #1) were treated for 24 h without (NA) or with 125 nM of γ-secretase inhibitor X, 10 μM of Calpain inhibitor or 10 μM of Caspase-3 inhibitor I and analyzed by Western blot with CB11 antibodies. (C) Parental CHO cells (C) or the same cells as in (A) were metabolically labeled with 35S-Translabel for 15 min, washed, lysed and immunoprecipitated with anti-HA antibodies. Immunoprecipitates were analyzed by SDS–PAGE and fluorography. (D) Top, CHO cells were transiently transfected with a control plasmid (C) or with plasmids encoding HER2 ΔNarI or HER2 as indicated. Cell lysates from transiently transfected cells were analyzed by Western blot with CB11 antiobodies. Bottom: Schematics showing the HER2 constructs transfected. The position of the Nar I sites used to make the HER2 ΔNar I construct are shown in wild-type HER2. The HER2 ΔNar I construct contains a stop codon after glycin 312 and generates a predicted protein of 32 kDa (solid line) the rest of the sequence in this construct is out of frame and is shown in dotted lines. The N-terminus (N), the transmembrane domain (hatched box), the intracellular kinase domain (shaded box) and the C-terminus (C) are shown. The position of the first in-frame methionine is indicated with the number 1. (E) T47D cells permanently transfected with wild-type HER2 mRNA were analyzed by Western blotting with antibodies against the intracellular domain of HER2 or by Northern blotting with probes specific for the extracellular (ECD) or intracellular (ICD) domains of HER2, respectively. Download figure Download PowerPoint Certain proteolytic enzymes, such as γ-secretease, caspase-3 or calpain have been found to release the intracellular domain of transmembrane proteins (Marambaud et al, 2002; Kopan and Ilagan, 2004; Franco and Huttenlocher, 2005). To investigate the possible role of these proteases in the generation of CTFs from HER2, we treated permanently transfected cells with the corresponding inhibitors. None of the inhibitors prevented the production of CTFs (Figure 2B), arguing that the mentioned proteases do not participate in the generation of CTFs. Interestingly, the calpain inhibitor produced a slight but reproducible increase in the levels of the CTFs, indicating that a protease sensitive to this inhibitor participates in the degradation of CTFs. Since the protease inhibitors tested did not have a detectable effect on the levels of CTFs, it is possible that CTFs are not the product of full-length HER2 cleavage. To examine this possibility, we analyzed the biosynthesis of CTFs by metabolic labeling and immunoprecipitation. Full-length HER2 was undetectable after short (15 min) metabolic pulses in clones expressing predominantly CTFs (Figure 2C), indicating that CTFs are not generated through proteolysis of the full-length receptor. Despite the above results, it could be argued that the cleavage of HER2 in CHO cells is carried out by and unidentified protease that acts much faster and efficiently than expected and it is not inhibited by the compounds tested. To directly address whether CTFs are generated by proteolytic cleavage of full-length HER2, we made a HER2 cDNA deletion construct (HER2 ΔNar I). The deletion causes a frameshift after glycine 312 and a translation termination codon three amino acids downstream, preventing the formation of full-length HER2 (Figure 2D, bottom). Theoretically, this constructs generates a protein fragment with an estimated molecular weight of 32 kDa, obviously undetectable with antibodies against the cytoplasmic tail of HER2. As expected, the levels of full-length HER2 are similar in mock transfected cells and in cells transfected with the ΔNar I HER2 construct and dramatically increased in the same cells transfected with the full-length receptor (Figure 2D). However, CTFs are readily detected in cells transfected with the deletion construct (Figure 2D). This result directly shows that the generation of CTFs is independent of the presence of full-length HER2 and unveils the existence of a novel mechanism different from proteolytic processing. As shown in Figure 2D, CTFs are also generated in cells transiently transfected with the full-length cDNA, albeit with much lower efficiency. The transcription of a short transcript from a cryptic promoter could explain the generation of CTFs. However, in transiently transfected cells such as those analyzed in Figure 2D, only single transcripts, with the size expected for full-length HER2 and the HER2 ΔNar I, respectively, can be detected by Northern blotting (data not shown). Furthermore, a single transcript is also apparent in different cells stably transfected with full-length cDNA encoding HER2 that express CTFs (Figure 2E and data not shown). Therefore, although we cannot rule out the existence of an undetectable mRNA species, alternative transcripts do not seem to explain the generation of CTFs. CTFs arise from alternative initiation of translation Considering the previous results, we analyzed the possibility that the HER2 CTFs arise by alternative initiation of translation. Full-length HER2 cDNA transcribed and translated in vitro in the presence of radiolabeled methionine gives rise to the full-length protein and to three main products, similar in size to those observed in transfected cells (Figure 3, WT). The specificity of these bands was assessed by Western blot with anti-HER2 monoclonal antibodies (data not shown). Northern blotting analysis shows the existence of a single transcript (Supplementary Figure 2), further supporting that alternative transcription is not the main mechanism of generation of CTFs. As expected, the deletion construct did not produce full-length HER2, but the generation of the CTFs remained unaltered (Figure 3, ΔNar I). The deduced sequence of the cDNA encoding HER2 bears several methionines (in positions 611, 687, 706, 712, respectively) surrounding the transmembrane domain (Figure 3, schematic). To analyze the possible role of these methionines in the synthesis of the CTFs, we mutated them to alanines, and the resulting constructs were transcribed and translated in vitro. Mutation of methionine 611 prevented the synthesis of the largest protein, indicating that this HER2 species starts before the transmembrane domain. Mutation of methionine 687 prevented the generation of the following protein and induced the synthesis of a slightly smaller species, indicating that methionine 687 acts as an initiator and that in its absence the next methionine (706) is used. Finally, mutation of methionines 706 and 712 did not have any effect, indicating that these methionines are not normally used. Figure 3.Biosynthesis of HER2 species from different mutant cDNAs in vitro. cDNAs encoding wild-type HER2, a Nar I deletion construct, or the indicated point mutants, also containing the Nar I deletion, were transcribed and translated in vitro in the presence of 35S-translabel and analyzed by SDS–PAGE and fluorography. Right: schematic showing the hypothetical HER2 CTFs generated, the numbers indicate the positions of different methionines (see also, Figure 1). Download figure Download PowerPoint The smallest species is not affected by any mutation; a CTF starting in the next methionine, located at position 774, would be ∼8 kDa smaller than that initiated at methionine 706. Since the difference between the smaller CTFs corresponds to ∼1 kDa, the fastest migrating CTF likely represents the initiation at a noncanonical codon. However, we did not further investigate this possibility because this HER2 species cannot be detected in cells (see below). To determine the relevance of these methionines in vivo, the different mutant HER2 constructs were transfected into MCF7 cells. Note that these cells express higher levels of endogenous HER2, compared to CHO cells (Figure 4A and data not shown). Mutation of methionine 611 prevented the formation of the two larger and less abundant CTFs (Figure 4A, CTFs 611), indicating that this methionine works as an initiator in vivo and suggesting that one of these CTFs is a post-transductional modification of the other. Mutation of methionine 687 did not affect the species generate from methionine 611 but resulted in a slight shift in the electrophoretic migration of the shorter and predominant ones (Figure 4A, CTFs 687), likely indicating that these forms arise from methionine 687. The electrophoretic shift corresponds to ∼2 kDa, indicating that, in the absence of methionine 687, CTFs start in methionine 706. Mutation of methionine 706 did not have any effect nor did mutation in methionine 712. Figure 4.Biosynthesis of HER2 species from different mutant cDNAs in vivo. (A–C) MCF7 cells transiently transfected with a control plasmid (C) with the indicated single, double, triple or quadruple mutant constructs were lysed and cell lysates were analyzed by Western blot with CB11 antibodies. All the mutant constructs contain the ΔNar I deletion. Each panel contains the corresponding diagram showing the CTFs generated by the different constructs. Download figure Download PowerPoint Next, we transfected double, triple and quadruple mutants. In agreement with the results shown in Figure 4A, mutation of methionines 611 and 687 prevented the generation of the two larger CTFs and shifted the mobility of the two smaller ones (Figure 4B). The 611, 687 and 706 triple mutant generated a product similar to that generated by the double mutant (Figure 4B), indicating that when the first three methionines are absent CTFs can be translated from methionine 712. Supporting this conclusion, the quadruple mutant did not produce any CTF (Figure 4B). Thus, in agreement with the results of the in vitro transcription and translation, expression of mutant cDNAs in cells indicate that CTFs are translated from methionines 611 and 687; however, the predominant products arise from the latter. To confirm this conclusion we mutated methionines 611 or 687 in cDNAs containing mutations in methionines 706 and 712 (Figure 4C). In this background, mutation of methionine 611 prevented the synthesis of the largest CTFs and mutation of methionine 687 prevented that of the major HER2 species. Collectively, these results strongly support that CTFs arise from alternative initiation of translation from methionines 611 and 687. At least two products arise from methionine 611 indicating that one of them is a post-translational modification or a proteolytic product of the other. Similarly, more than one product arises from methionine 687. To analyze the possible involvement of proteolytic activities on the generation of different CTFs from the same methionine, we treated cells permanently transfected with the HER2 ΔNar I construct with different inhibitors. In agreement with the results obtained in Figure 2, inhibitors of γ-secretease, caspase-3 or calpain did not affect the generation of the different CTFs, arguing that these proteolytic activities do not participate in their generation (Figure 5). Serine protease inhibitors (leupeptin and aprotinin) or EDTA were also without effect (Figure 5). Interestingly, inhibitors of the proteasome and, to less extent, the calpain inhibitor (which also inhibits the proteasome) increased the levels of the CTFs, particularly those starting in methionine 611, suggesting that the proteasome participates in the degradation of CTFs. Furthermore, high molecular weight species of ∼120 kDa are detected in cells treated with proteasome inhibitors and EGTA (Figure 5). This result indicates that the different species observed are not proteolytic products from larger CTFs and opens the possibility that high molecular weight species of short half-life, normally degraded by the proteasome or a proteolytic activity sensitive to EGTA, are generated from methionines 611 and/or 687. Figure 5.Effect of different protease inhibitors on the expression of CTFs. T47D cells permanently transfected with the ΔNar I construct were treated for 24 h without (NA) or with 125 nM of γ-secretase inhibitor X, 5 μM of the proteasome inhibitor MG132, 10 μM of Calpain inhibitor, 10 μM of Caspase-3 inhibitor I, 10 mM of EDTA, 10 mM of EGTA, 100 μM of leupeptin or 10 μg/ml of aprotinin. Treated cells were lysed and cell lysated analyzed with CB11 antibodies. Download figure Download PowerPoint Subcellular localization of HER2 CTFs To analyze the subcellular location of HER2 CTFs, we stained cells stably expressing HER2 CTFs or full-length HER2, respectively, with antibodies against the intracellular domain of HER2. The pattern observed in cells expressing full-length HER2 is that expected for cell surface molecules and, in addition, intracellular vesicles (Figure 6A), likely representing HER2 in transit through the secretory and/or endocytic pathways. In contrast, in CTFs-expressing CHO cells diffuse cytoplasmic and a nuclear staining is apparent (Figure 6A). The same results were obtained using T47D or MCF7 cells transfected with the ΔNar I HER2 construct (data not shown), confirming that the CTFs can be transported to the nucleus of a variety of cell lines. To complement these results, we analyzed the localization of full-length HER2 and CTFs through a simple biochemical fractionation. As expected, full-length HER2 was almost exclusively detectable in the membrane fraction of CHO cells (Figure 6B). In contrast, all detectable CTFs were present in the soluble fraction (Figure 6B). Figure 6.Subcellular localization of different HER2 species. (A) Stably transfected CHO cells expressing predominantly HA-tagged full-length HER2 (#3) and HER2 CTFs (#1), respectively, were fixed, permeabilized and stained with anti-HA and FITC-labeled anti-mouse antibodies. (B) The same cells were homogenized and fractionated by ultracentrifugation. Aliquots from soluble and membrane fractions were analyzed by Western blotting and probed with CB11 antibody or, as a control, with anti-β-tubulin antibodies. (C) MDA-MB-468 cells stably expressing CTFs WT or KD versions were fixed, permeabilized and stained with anti-HER2 (CB11) and FITC-labeled anti-mouse antibodies. (D) Homogenates from tumor samples were analyzed by Western blot with the anti-HER2 monoclonal antibody CB11. (E) Immunohistochemical staining with CB11 antibodies of the same tumor samples. In the magnification, nuclear staining is marked with arrows. (F) Tumor 48 was homogenized as in B and the different fractions analyzed by Western with the monoclonal antibody CB11. Download figure Download PowerPoint Treatment of cells with tyrosine kinase inhibitors that target HER2 prevents the accumulation of the full-length receptor in the nucleus (Wang et al, 2004). Thus, we analyzed the possible role of the tyrosine kinase activity on the subcellular localization of the CTFs. To avoid interference with full-length HER2, we used MDA-MB-468 cells, which do not express detectable levels of the receptor. In agreement with the results shown in Figure 6A, analysis of MDA-MB-468 expressing CTFs cells showed nuclear staining (Figure 6C). In contrast, CTFs bearing a point mutation that disrupts the kinase activity of HER2 show a cytoplasmic distribution and accumulate in cell–cell contacts (Figure 6C). Thus, the tyrosine kinase activity seems to be required for the accumulation of CTFs in the cell nucleus. To analyze the subcellular localization of the CTFs in vivo, we analyzed by immunohistochemistry six samples from human mammary tumors: three expressing predominantly full-length HER2 and three samples expressing detectable levels of CTFs as judged by Western blot (Figure 6D and data not shown). As expected, the signal corresponding to samples expressing full-length HER2 stained with antibodies against the intracellular domain of HER2 was largely associated to membranes (Figure 6E, sample 52 and Supplementary Figure 3B). In samples expressing CTFs, in addition to the membrane pattern, a clear cytoplasmic signal and occasional nuclear staining is apparent (Figure 6E, sample 48 and Supplementary Figure 3B). In both samples, predominant surface staining was apparent with antibodies against the extracellular domain of HER2 that do not detect CTFs (supplementary Figure 3B
Referência(s)