- Email: [email protected]
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Nuclear localization and possible functions of receptor tyrosine kinases Graham Carpenter Recent data have renewed interest in the possible nuclear localization of receptor tyrosine kinases, as well as their ligands. In one case, that of ErbB-4, the receptor is processed by two membrane-localized proteases to produce a soluble cytoplasmic domain fragment that includes the tyrosine kinase domain. This fragment, generated by a metalloproteasedependent ectodomain cleavage followed by g-secretase cleavage within the transmembrane domain, is also found in the nucleus. Three other receptor tyrosine kinases have been detected in the nucleus in the absence of proteolytic processing. In some instances, nuclear localization of receptor tyrosine kinases is growth-factor-dependent and tentative evidence suggests a role in transcription. Addresses Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA e-mail: [email protected]
Current Opinion in Cell Biology 2003, 15:143–148 This review comes from a themed issue on Cell regulation Edited by Pier Paolo Di Fiore and Pier Giuseppe Pelicci 0955-0674/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0955-0674(03)00015-2
Abbreviations APP Alzheimer’s precursor protein EGF epidermal growth factor ER endoplasmic reticulum FGF fibroblast growth factor FGFR FGF receptor RTK receptor tyrosine kinase STAT signal transducers and activators of transcription TACE tumor necrosis factor a converting enzyme TPA 12-O-tetradecanoylphorbol-13-acetate
Introduction Receptor tyrosine kinases (RTKs) are type I transmembrane molecules positioned at the cell surface to detect the presence of cognate growth factors produced in the extracellular milieu by neighboring cells. This recognition event activates the receptors’ intrinsic tyrosine kinase activity and initiates a network of signaling pathways that relay cell surface information to the nucleus and other points in the cell [1,2]. Sequentially acting components, such as those of the Ras/MAPK (mitogen-activated protein kinase) pathway, or single component systems, such as the STAT pathway, constitute the mechanism by www.current-opinion.com
which this intracellular transfer of biochemical information is mediated. Current thinking is that the combinatorial information provided by these signal transduction pathways can explain the biological responses of cells to growth factors. Growth factor–RTK complexes formed at the plasma membrane are not stagnant or restricted to the cell surface. That the complexes are rapidly internalized through clathrin-coated pits into an endocytic pathway has been recognized for several years. Subsequent to internalization, receptor complexes remain active, but eventually are sorted either to the lysosome and degraded or recycled back to the cell surface. More recently, evidence has begun to accumulate that the endocytic pathway may also be a site for the generation of signal transduction events [3,4]. In this review, I describe recent data that combine RTK signaling and intracellular trafficking in a novel way. Recent reports indicate that receptors or fragments of receptors travel from the plasma membrane to the nucleus by different mechanisms and may, in the process, constitute biochemical signals that regulate cell function.
Routes to the nucleus RTKs are found in the nucleus in two forms — either the intact molecule or its cytoplasmic domain fragment. While the means by which an intact receptor is translocated from the plasma membrane to the nucleus is not understood, the mechanism for fragment formation and translocation is, in general, known and supported by precedents of other cell-surface transmembrane molecules (Figure 1).
The protease-dependent route The Notch receptor (a non-RTK) is cleaved following ligand binding such that two large fragments are produced by the sequential action of two distinct membrane-localized proteases, and one fragment is translocated to the nucleus [5,6]. A similar scenario has been described for proteolytic processing of the Alzheimer’s precursor protein (APP). This mechanism has now been extended to ErbB-4 [7], an RTK that binds the growth factors heregulin/ neuregulin, betacellulin, epiregulin and heparin-binding epidermal growth factor [8,9]. Interestingly, the latter three growth factors also are recognized by ErbB-1, whereas heregulin/neuregulin is also recognized by ErbB-3. The binding of ligand to ErbB-4 [10] or the activation of protein kinase C by TPA (12-O-tetradecanoylphorbol-13acetate) [11] provokes an ectodomain cleavage that Current Opinion in Cell Biology 2003, 15:143–148
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Figure 1
Growth factor
TPA TACE
PS-1 + X
Internalization
TACE
PM
PS-1
X
Intracellular vesicle
Degradative path to lysosome
Recycling path to cell surface
Golgi Ligand-binding ectodomain ER Transmembrane domain Tyrosine kinase domain Carboxy-terminal domain
Nucleus X
Current Opinion in Cell Biology
Pathways for the translocation of RTKs from the plasma membrane (PM) to the nucleoplasm. Evidence exists for a dual protease system (TACE, PS-1) that acts on ErbB-4 following TPA or heregulin addition. The TPA-dependent mechanism does not involve internalization of the receptor, but the growth-factor-initiated pathway does. In either case, sequential cleavage by TACE, removing the ectodomain, and g-secretase activity (PS-1), cleaving within the transmembrane domain, produces a soluble cytoplasmic tyrosine kinase domain that is found in the cytoplasm and nucleus. The pathway by which intact RTKs together with their cognate ligands arrive in the nucleus from the plasma membrane is entirely speculative. Since a known ER system (ERAD) exists to remove full-length transmembrane proteins from this organelle into the cytoplasm, the hypothetical mechanism is centered on delivery of RTK from the cell surface to the ER, extraction into the cytoplasm, and translocation into the nucleoplasm. Also, it has been suggested by others (reviewed in [34]) that an unknown mechanism may remove intact RTKs from the cell surface with the assistance of unidentified factors, designated X. Dashed lines represent hypothetical pathways.
releases a 120 kDa ectodomain fragment into the extracellular milieu and generates a membrane-associated 80 kDa fragment that includes the receptor’s tyrosine kinase function. This cleavage requires metalloprotease activity and is the initial regulated event in agonistdependent ErbB-4 processing and eventual nuclear translocation. Also, there is a low but detectable basal level of ErbB-4 ectodomain cleavage in various cells [12], but how it is controlled, if at all, is not known. This constitutive Current Opinion in Cell Biology 2003, 15:143–148
cleavage of ErbB-4 is analogous to the generation of amyloid peptides from the cleavage of APP, which has no known ligand. TPA fails to stimulate ErbB-4 ectodomain cleavage in tumor necrosis factor a converting enzyme (TACE)-null cells [13] and TACE (ADAM 17) or closely related ADAMs play a general role of in mediating cell surface shedding of numerous membrane molecules [14]; hence, is clear that www.current-opinion.com
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TACE participates in ErbB-4 ectodomain cleavage. However, that TACE directly executes this cleavage event has not been demonstrated. TACE and other ADAMs are single-transmembrane molecules that have a metalloprotease active site within their ectodomain [14]. While TPA or heregulin stimulate this initial cleavage of ErbB-4, the mechanisms seem to be distinct [10]. For example, a general protein kinase C inhibitor blocks the TPA-induced cleavage, but not that induced by heregulin. Additionally, the evidence suggests that heregulininduced cleavage is associated with ErbB-4 endocytosis, while TPA is not. Ligand-induced proteolytic cleavage of Notch is also associated with endocytosis [6]. TPA induces cleavage of ErbB-4 in all cell backgrounds; however, heregulin-induced cleavage is only detectable in a subset of cell lines [10], but the meaning of this is unclear. That the ectodomain cleavage of ErbB-4 has biological significance is suggested by the fact that a non-cleavable isoform of ErbB-4 has been characterized in mouse tissues [15] and human tumor specimens [16,17]. This isoform is apparently generated by the use of alternative exons to alter the ErbB-4 coding sequence in the stalk region of the receptor’s ectodomain, the likely site of TACE-induced cleavage. ErbB-4 isoforms are reviewed elsewhere [18]. There is evidence that at least one of the products derived from ErbB-4 ectodomain cleavage is functional. The cellassociated 80 kDa fragment remains, at least in vitro, an active tyrosine kinase. [12]. There is evidence for several RTKs that loss of the ectodomain activates tyrosine kinase activity. This membrane-associated 80 kDa ErbB-4 fragment is a substrate for two protease systems. With time, the fragment is ubiquitinated and degraded by proteosome activity [12]. However, this fragment also serves as a substrate for g-secretase (PS-1) activity [7,19], which typically cleaves a membrane protein within its transmembrane domain and thereby provokes release of the cytoplasmic domain into the cytosol [6] (Figure 1). g-Secretase activity converts the membraneassociated 80 kDa ErbB-4 fragment to a soluble or cytosolic fragment termed ‘s80’. The action of g-secretase on transmembrane proteins, including ErbB-4, requires preceding ectodomain cleavage for reasons that are not known [6] (see also Update). Treatment of ErbB-4-expressing cells with heregulin or TPA provokes release of the s80 cytoplasmic domain fragment into the cytoplasm and the fragment is then rapidly detected in the nucleus [7]. Also, expression of a green fluorescent protein (GFP)-tagged ErbB-4 cytoplasmic domain fragment in recipient cells showed significant accumulation in the nucleus, particularly if leptomycin B, an inhibitor of nuclear export, was present. Putative nuclear import and export sequences in this ErbB-4 www.current-opinion.com
fragment have been identified [7,19], but have not been mutated. Interestingly, immunohistochemical studies of human tissues have noted the presence of nuclear ErbB-4 [20–22]. There are two well-established precedents for this cleavage mechanism: the Notch receptor and APP, neither of which are RTKs. In both instances, the released cytoplasmic domain fragments are translocated to the nucleus and function to regulate transcription [5,6]. That this pathway may be more widespread is indicated by analogous cleavage events reported for the adhesion molecules CD44 [23,24] and E-cadherin [25], plus the low-density lipoprotein receptor-related protein [26]. TPA-induced ectodomain shedding has been reported for several other RTKs (e.g. CSF-1, c-Kit, MGF, Axl, TrkA, Met and Tie-1), and ligand-induced ectodomain cleavage has been reported for TrkA [27] and the discoidin domain1 collagen receptor [28]. Whether any of these or other RTKs are also processed by g-secretase activity remains to be seen.
The holoreceptor route Three recent papers have added substantially to previously published data reporting the presence of fulllength RTKs in the nucleus. In the case of ErbB-1, the epidermal growth factor (EGF) receptor, addition of the cognate ligand is required for nuclear localization, and, in fact, the complete ligand–receptor complex was reported to be present in the nucleus [29]. That this ligand– receptor complex is trafficked to the nucleus needs to be evaluated in light of older studies that defined the endocytic pathway using EGF labeled with fluorescein, ferritin or 125 I and failed to report the appearance of labeled ligand in the nucleus. A second RTK ErbB-3, which binds the growth factor heregulin, was reported to be constitutively present in an uncleaved form in the nucleus and the addition of its ligand influenced distribution of the receptor between the nucleus and cytosol [30]. When ErbB-3-expressing cells were grown on filters to induce epithelial polarity, the ErbB-3 signal was concentrated in nucleoli. The third RTK is fibroblast growth factor receptor I (FGFR-I). Nuclear localization of the full-length receptor is promoted by the addition of FGF to cells [31,32] and appears to involve interaction of this receptor with importin b [33]. Depletion of cellular ATP not only augmented the FGF-dependent nuclear accumulation of FGFR-I, but was also sufficient to promote nuclear accumulation in the absence of FGF. My conclusion is that ATP depletion results in an increased level of cytoplasmic importin b, which facilitates nuclear localization of this RTK. The available reports suggest that these nuclear RTKs are not found in the nuclear envelope, but rather are Current Opinion in Cell Biology 2003, 15:143–148
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present in the nucleoplasm in a non-membranous environment. In the cases of ErbB-1 and FGFR-I, nuclear localization is ligand-dependent and could involve endocytosis together with an unknown mechanism to traffic these RTKs to the nucleus (Figure 1). Hence, the main complication would seem to be a mechanism to remove the intact receptor from a membrane bilayer, either at the cell surface or within an intracellular compartment, such as the endosome (see [34] for a review). While no such mechanism is known for this mode of intracellular receptor trafficking, a mechanism does exist in the endoplasmic reticulum (ER) for the extraction of intact transmembrane proteins into the cytoplasm. This is the ER-associated degradation (ERAD) system, which functions to detect malfolded proteins in the ER and to deliver such molecules intact to the cytoplasm, where they are ubiquitinated and degraded [35]. One could envision a model in which receptor-bearing endosomes fuse with the Golgi, are retrograde-transported to the ER, extracted by the ERAD system, and not degraded but transported to the nucleus (Figure 1). Several toxins added to the extracellular environment gain entrance to the cytoplasm by this route [36]. Recently it has been demonstrated that caveolar uptake of virus leads to the ER without the intervention of endosomes [37]. It is possible, therefore, that some RTK internalization, especially in receptor-overexpressing cells, occurs through non-coated pit mechanisms such as caveolae and ultimately results in delivery to the ER.
Nuclear functions for receptor tyrosine kinases Obviously, the physiologic importance of RTK nuclear localization has to be established by identifying their nuclear targets and demonstrating that these targets are required for a growth factor cellular response. In the case of ErbB-4 proteolytic processing, g-secretase inhibition blocks heregulin-dependent growth inhibition of T47 cells [7]. Although the inhibitor could have other unknown effects, it did not influence EGF-dependent growth stimulation of the same cells. This remains the only demonstration of the physiological significance of RTK nuclear localization in terms of growth factor action. Several experiments hint at a role for RTKs in the control of gene expression. The most extensive data are for ErbB-1 [29]. Those data include a large stimulation of a GAL4 fusion protein reporter assay in several cell lines, indicative of a transactivation function for the ErbB-1 carboxy-terminal domain. In the same assay, the ErbB-4 carboxy-terminal domain provoked a much more modest transcription increase [7]. Two points need to be made. First, this assay is known to be quite sensitive and false positives occur frequently. Second, while the ErbB-1 and ErbB-4 carboxy-terminal domains (about 200 residues) scored positive in this assay, the Current Opinion in Cell Biology 2003, 15:143–148
entire cytoplasmic domains (about 600 residues), which include the carboxy-terminal domains, were without effect and there is no evidence that free carboxy-terminal domains are found in the nucleus. Additional evidence for a role of ErbB-1 in transcription includes an in vitro demonstration of this RTK binding to a specific DNA sequence (designated ATRS [adenine/ thymidine-rich sequence]) [29]. Subsequent experiments in vivo show the EGF-dependent stimulation of a reporter construct containing the ATRS sequence, a chromatin immunoprecipitation assay demonstrating ErbB-1 bound to the cyclin D1 promoter, which contains an ARTS sequence, and the EGF-dependent stimulation of cyclin D1 mRNA levels [29]. The capacity of FGF-2 to induce expression of c-Jun or cyclin D1 mRNA is sensitive to the presence/absence of an artificial nuclear localization sequence incorporated into a transfected FGFR-I construct [33]. Another recent study has provided evidence that FGFR-I acts as a transcription factor at the FGF-2 promoter [38]. However, since the liganddependent nuclear localization of ErbB-1 or FGFR-I cannot be blocked, it remains to be shown whether these growth-factor-dependent transcription responses are dependent on nuclear localization of the receptor. Since RTKs contains an enzymatic function, it is plausible that the modification of nuclear proteins is another consequence for nuclear-localized receptors or their cytoplasmic domain fragments. Nuclear non-receptor tyrosine kinases, such as c-Abl, are known, but the relevant nuclear substrates are not clear. Finally, it is possible that RTKs carry other molecules into the nucleus and that these receptor-associated molecules are functional in the nucleus. It has been proposed that ErbB-1 may transport STAT-1, a tyrosine-phosphorylated transcription factor, from the cytosol into the nucleus [39]. In the case of ErbB-1 and perhaps FGF-I, it would seem that the cognate growth factor is carried by the receptor into the nucleus. The possible importance of growth factors in the nucleus has been reviewed elsewhere [40–42]. The nuclear localization of ErbB-1 recalls data published some years ago that showed DNA topoisomerase activity in highly purified preparations of this RTK [43]. A subsequent report confirmed this, but showed that the topoisomerase activity could be separated from the receptor by an additional purification step [44]. Nevertheless, these two reports do suggest that topoisomerase activity is physically associated with ErbB-1 at least in vitro.
Conclusions The new observations discussed above raise provocative ideas about the trafficking and signaling mechanisms of RTKs. The nuclear localization of other cell surface molecules or their cytoplasmic domain fragments are www.current-opinion.com
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parallel examples of direct communication between these two cellular compartments. It seems logical to expect that additional examples, including other RTKs, will be added in the near future. The most important questions that remain to solidify the importance of these observations are twofold. First, how do intact receptors move from the cell surface to the nucleus; and second, is the nuclear localization of an RTK a necessary part of the cellular response to growth factors?
Update Previous reports have identified PSD-95 as a molecule that associates with a PDZ-domain recognition motif at the carboxyl terminus of ErbB-4 [45–47]. Those reports suggest that ErbB-4 association with PSD-95 facilitates ligand-dependent activation of ErbB-4, ErbB-4 oligomerization, or both. A new report indicates that when the carboxy-terminal three residues of ErbB-4 are deleted, a mutation that prevents association with PDZ-domaincontaining proteins, ErbB-4 proteolytic processing by g-secretase is significantly impaired [48]. However, ectodomain proteolytic processing is not impaired in this mutant. These data suggest a mechanism to facilitate ErbB-4 processing by g-secretase.
Acknowledgements Space limitations preclude highlighting all relevant work, particularly that reported before 2000. The author appreciates the contributions of Sue Carpenter and Lori Bennett in manuscript and figure preparation, respectively. I am also grateful to two colleagues, Bruce Carter and Scott Hiebert, for reading the manuscript and offering suggestions. The support of the National Cancer Institute, USA, (grant number CA97456) is acknowledged.
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36. Sandvig K, van Deurs B: Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives. EMBO J 2000, 19:5943-5950. 37. Pelkmans L, Kartenbeck J, Helenius A: Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat Cell Biol 2001, 3:473-483. 38. Peng H, Moffett J, Myers J, Fang X, Stachowiak EK, Maher P, Kratz E, Hines J, Fluharty SJ, Mizukoshi E et al.: Novel nuclear signaling pathway mediates activation of fibroblast growth factor-2 gene by type 1 and type 2 angiotensin II receptors. Mol Biol Cell 2001, 12:449-462. 39. Bild AH, Turkson J, Jove R: Cytoplasmic transport of Stat3 by receptor-mediated endocytosis. EMBO J 2002, 21:3255-3263. In this paper, the authors describe a possible carrier function for internalized ErbB-1. STAT-1 is tyrosine-phosphorylation by ErbB-1 and then translocates to the nucleus to act as a transcription factor. The authors suggest the endosomal ErbB-1 carries STAT-1 and may be involved in its nuclear translocation. 40. Pederson T: Growth factors in the nucleolus? J Cell Biol 1998, 143:279-281.
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Nuclear localization and possible functions of receptor tyrosine kinases Graham Carpenter Recent data have renewed interest in the possible nuclear localization of receptor tyrosine kinases, as well as their ligands. In one case, that of ErbB-4, the receptor is processed by two membrane-localized proteases to produce a soluble cytoplasmic domain fragment that includes the tyrosine kinase domain. This fragment, generated by a metalloproteasedependent ectodomain cleavage followed by g-secretase cleavage within the transmembrane domain, is also found in the nucleus. Three other receptor tyrosine kinases have been detected in the nucleus in the absence of proteolytic processing. In some instances, nuclear localization of receptor tyrosine kinases is growth-factor-dependent and tentative evidence suggests a role in transcription. Addresses Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA e-mail: [email protected]
Current Opinion in Cell Biology 2003, 15:143–148 This review comes from a themed issue on Cell regulation Edited by Pier Paolo Di Fiore and Pier Giuseppe Pelicci 0955-0674/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0955-0674(03)00015-2
Abbreviations APP Alzheimer’s precursor protein EGF epidermal growth factor ER endoplasmic reticulum FGF fibroblast growth factor FGFR FGF receptor RTK receptor tyrosine kinase STAT signal transducers and activators of transcription TACE tumor necrosis factor a converting enzyme TPA 12-O-tetradecanoylphorbol-13-acetate
Introduction Receptor tyrosine kinases (RTKs) are type I transmembrane molecules positioned at the cell surface to detect the presence of cognate growth factors produced in the extracellular milieu by neighboring cells. This recognition event activates the receptors’ intrinsic tyrosine kinase activity and initiates a network of signaling pathways that relay cell surface information to the nucleus and other points in the cell [1,2]. Sequentially acting components, such as those of the Ras/MAPK (mitogen-activated protein kinase) pathway, or single component systems, such as the STAT pathway, constitute the mechanism by www.current-opinion.com
which this intracellular transfer of biochemical information is mediated. Current thinking is that the combinatorial information provided by these signal transduction pathways can explain the biological responses of cells to growth factors. Growth factor–RTK complexes formed at the plasma membrane are not stagnant or restricted to the cell surface. That the complexes are rapidly internalized through clathrin-coated pits into an endocytic pathway has been recognized for several years. Subsequent to internalization, receptor complexes remain active, but eventually are sorted either to the lysosome and degraded or recycled back to the cell surface. More recently, evidence has begun to accumulate that the endocytic pathway may also be a site for the generation of signal transduction events [3,4]. In this review, I describe recent data that combine RTK signaling and intracellular trafficking in a novel way. Recent reports indicate that receptors or fragments of receptors travel from the plasma membrane to the nucleus by different mechanisms and may, in the process, constitute biochemical signals that regulate cell function.
Routes to the nucleus RTKs are found in the nucleus in two forms — either the intact molecule or its cytoplasmic domain fragment. While the means by which an intact receptor is translocated from the plasma membrane to the nucleus is not understood, the mechanism for fragment formation and translocation is, in general, known and supported by precedents of other cell-surface transmembrane molecules (Figure 1).
The protease-dependent route The Notch receptor (a non-RTK) is cleaved following ligand binding such that two large fragments are produced by the sequential action of two distinct membrane-localized proteases, and one fragment is translocated to the nucleus [5,6]. A similar scenario has been described for proteolytic processing of the Alzheimer’s precursor protein (APP). This mechanism has now been extended to ErbB-4 [7], an RTK that binds the growth factors heregulin/ neuregulin, betacellulin, epiregulin and heparin-binding epidermal growth factor [8,9]. Interestingly, the latter three growth factors also are recognized by ErbB-1, whereas heregulin/neuregulin is also recognized by ErbB-3. The binding of ligand to ErbB-4 [10] or the activation of protein kinase C by TPA (12-O-tetradecanoylphorbol-13acetate) [11] provokes an ectodomain cleavage that Current Opinion in Cell Biology 2003, 15:143–148
144 Cell regulation
Figure 1
Growth factor
TPA TACE
PS-1 + X
Internalization
TACE
PM
PS-1
X
Intracellular vesicle
Degradative path to lysosome
Recycling path to cell surface
Golgi Ligand-binding ectodomain ER Transmembrane domain Tyrosine kinase domain Carboxy-terminal domain
Nucleus X
Current Opinion in Cell Biology
Pathways for the translocation of RTKs from the plasma membrane (PM) to the nucleoplasm. Evidence exists for a dual protease system (TACE, PS-1) that acts on ErbB-4 following TPA or heregulin addition. The TPA-dependent mechanism does not involve internalization of the receptor, but the growth-factor-initiated pathway does. In either case, sequential cleavage by TACE, removing the ectodomain, and g-secretase activity (PS-1), cleaving within the transmembrane domain, produces a soluble cytoplasmic tyrosine kinase domain that is found in the cytoplasm and nucleus. The pathway by which intact RTKs together with their cognate ligands arrive in the nucleus from the plasma membrane is entirely speculative. Since a known ER system (ERAD) exists to remove full-length transmembrane proteins from this organelle into the cytoplasm, the hypothetical mechanism is centered on delivery of RTK from the cell surface to the ER, extraction into the cytoplasm, and translocation into the nucleoplasm. Also, it has been suggested by others (reviewed in [34]) that an unknown mechanism may remove intact RTKs from the cell surface with the assistance of unidentified factors, designated X. Dashed lines represent hypothetical pathways.
releases a 120 kDa ectodomain fragment into the extracellular milieu and generates a membrane-associated 80 kDa fragment that includes the receptor’s tyrosine kinase function. This cleavage requires metalloprotease activity and is the initial regulated event in agonistdependent ErbB-4 processing and eventual nuclear translocation. Also, there is a low but detectable basal level of ErbB-4 ectodomain cleavage in various cells [12], but how it is controlled, if at all, is not known. This constitutive Current Opinion in Cell Biology 2003, 15:143–148
cleavage of ErbB-4 is analogous to the generation of amyloid peptides from the cleavage of APP, which has no known ligand. TPA fails to stimulate ErbB-4 ectodomain cleavage in tumor necrosis factor a converting enzyme (TACE)-null cells [13] and TACE (ADAM 17) or closely related ADAMs play a general role of in mediating cell surface shedding of numerous membrane molecules [14]; hence, is clear that www.current-opinion.com
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TACE participates in ErbB-4 ectodomain cleavage. However, that TACE directly executes this cleavage event has not been demonstrated. TACE and other ADAMs are single-transmembrane molecules that have a metalloprotease active site within their ectodomain [14]. While TPA or heregulin stimulate this initial cleavage of ErbB-4, the mechanisms seem to be distinct [10]. For example, a general protein kinase C inhibitor blocks the TPA-induced cleavage, but not that induced by heregulin. Additionally, the evidence suggests that heregulininduced cleavage is associated with ErbB-4 endocytosis, while TPA is not. Ligand-induced proteolytic cleavage of Notch is also associated with endocytosis [6]. TPA induces cleavage of ErbB-4 in all cell backgrounds; however, heregulin-induced cleavage is only detectable in a subset of cell lines [10], but the meaning of this is unclear. That the ectodomain cleavage of ErbB-4 has biological significance is suggested by the fact that a non-cleavable isoform of ErbB-4 has been characterized in mouse tissues [15] and human tumor specimens [16,17]. This isoform is apparently generated by the use of alternative exons to alter the ErbB-4 coding sequence in the stalk region of the receptor’s ectodomain, the likely site of TACE-induced cleavage. ErbB-4 isoforms are reviewed elsewhere [18]. There is evidence that at least one of the products derived from ErbB-4 ectodomain cleavage is functional. The cellassociated 80 kDa fragment remains, at least in vitro, an active tyrosine kinase. [12]. There is evidence for several RTKs that loss of the ectodomain activates tyrosine kinase activity. This membrane-associated 80 kDa ErbB-4 fragment is a substrate for two protease systems. With time, the fragment is ubiquitinated and degraded by proteosome activity [12]. However, this fragment also serves as a substrate for g-secretase (PS-1) activity [7,19], which typically cleaves a membrane protein within its transmembrane domain and thereby provokes release of the cytoplasmic domain into the cytosol [6] (Figure 1). g-Secretase activity converts the membraneassociated 80 kDa ErbB-4 fragment to a soluble or cytosolic fragment termed ‘s80’. The action of g-secretase on transmembrane proteins, including ErbB-4, requires preceding ectodomain cleavage for reasons that are not known [6] (see also Update). Treatment of ErbB-4-expressing cells with heregulin or TPA provokes release of the s80 cytoplasmic domain fragment into the cytoplasm and the fragment is then rapidly detected in the nucleus [7]. Also, expression of a green fluorescent protein (GFP)-tagged ErbB-4 cytoplasmic domain fragment in recipient cells showed significant accumulation in the nucleus, particularly if leptomycin B, an inhibitor of nuclear export, was present. Putative nuclear import and export sequences in this ErbB-4 www.current-opinion.com
fragment have been identified [7,19], but have not been mutated. Interestingly, immunohistochemical studies of human tissues have noted the presence of nuclear ErbB-4 [20–22]. There are two well-established precedents for this cleavage mechanism: the Notch receptor and APP, neither of which are RTKs. In both instances, the released cytoplasmic domain fragments are translocated to the nucleus and function to regulate transcription [5,6]. That this pathway may be more widespread is indicated by analogous cleavage events reported for the adhesion molecules CD44 [23,24] and E-cadherin [25], plus the low-density lipoprotein receptor-related protein [26]. TPA-induced ectodomain shedding has been reported for several other RTKs (e.g. CSF-1, c-Kit, MGF, Axl, TrkA, Met and Tie-1), and ligand-induced ectodomain cleavage has been reported for TrkA [27] and the discoidin domain1 collagen receptor [28]. Whether any of these or other RTKs are also processed by g-secretase activity remains to be seen.
The holoreceptor route Three recent papers have added substantially to previously published data reporting the presence of fulllength RTKs in the nucleus. In the case of ErbB-1, the epidermal growth factor (EGF) receptor, addition of the cognate ligand is required for nuclear localization, and, in fact, the complete ligand–receptor complex was reported to be present in the nucleus [29]. That this ligand– receptor complex is trafficked to the nucleus needs to be evaluated in light of older studies that defined the endocytic pathway using EGF labeled with fluorescein, ferritin or 125 I and failed to report the appearance of labeled ligand in the nucleus. A second RTK ErbB-3, which binds the growth factor heregulin, was reported to be constitutively present in an uncleaved form in the nucleus and the addition of its ligand influenced distribution of the receptor between the nucleus and cytosol [30]. When ErbB-3-expressing cells were grown on filters to induce epithelial polarity, the ErbB-3 signal was concentrated in nucleoli. The third RTK is fibroblast growth factor receptor I (FGFR-I). Nuclear localization of the full-length receptor is promoted by the addition of FGF to cells [31,32] and appears to involve interaction of this receptor with importin b [33]. Depletion of cellular ATP not only augmented the FGF-dependent nuclear accumulation of FGFR-I, but was also sufficient to promote nuclear accumulation in the absence of FGF. My conclusion is that ATP depletion results in an increased level of cytoplasmic importin b, which facilitates nuclear localization of this RTK. The available reports suggest that these nuclear RTKs are not found in the nuclear envelope, but rather are Current Opinion in Cell Biology 2003, 15:143–148
146 Cell regulation
present in the nucleoplasm in a non-membranous environment. In the cases of ErbB-1 and FGFR-I, nuclear localization is ligand-dependent and could involve endocytosis together with an unknown mechanism to traffic these RTKs to the nucleus (Figure 1). Hence, the main complication would seem to be a mechanism to remove the intact receptor from a membrane bilayer, either at the cell surface or within an intracellular compartment, such as the endosome (see [34] for a review). While no such mechanism is known for this mode of intracellular receptor trafficking, a mechanism does exist in the endoplasmic reticulum (ER) for the extraction of intact transmembrane proteins into the cytoplasm. This is the ER-associated degradation (ERAD) system, which functions to detect malfolded proteins in the ER and to deliver such molecules intact to the cytoplasm, where they are ubiquitinated and degraded [35]. One could envision a model in which receptor-bearing endosomes fuse with the Golgi, are retrograde-transported to the ER, extracted by the ERAD system, and not degraded but transported to the nucleus (Figure 1). Several toxins added to the extracellular environment gain entrance to the cytoplasm by this route [36]. Recently it has been demonstrated that caveolar uptake of virus leads to the ER without the intervention of endosomes [37]. It is possible, therefore, that some RTK internalization, especially in receptor-overexpressing cells, occurs through non-coated pit mechanisms such as caveolae and ultimately results in delivery to the ER.
Nuclear functions for receptor tyrosine kinases Obviously, the physiologic importance of RTK nuclear localization has to be established by identifying their nuclear targets and demonstrating that these targets are required for a growth factor cellular response. In the case of ErbB-4 proteolytic processing, g-secretase inhibition blocks heregulin-dependent growth inhibition of T47 cells [7]. Although the inhibitor could have other unknown effects, it did not influence EGF-dependent growth stimulation of the same cells. This remains the only demonstration of the physiological significance of RTK nuclear localization in terms of growth factor action. Several experiments hint at a role for RTKs in the control of gene expression. The most extensive data are for ErbB-1 [29]. Those data include a large stimulation of a GAL4 fusion protein reporter assay in several cell lines, indicative of a transactivation function for the ErbB-1 carboxy-terminal domain. In the same assay, the ErbB-4 carboxy-terminal domain provoked a much more modest transcription increase [7]. Two points need to be made. First, this assay is known to be quite sensitive and false positives occur frequently. Second, while the ErbB-1 and ErbB-4 carboxy-terminal domains (about 200 residues) scored positive in this assay, the Current Opinion in Cell Biology 2003, 15:143–148
entire cytoplasmic domains (about 600 residues), which include the carboxy-terminal domains, were without effect and there is no evidence that free carboxy-terminal domains are found in the nucleus. Additional evidence for a role of ErbB-1 in transcription includes an in vitro demonstration of this RTK binding to a specific DNA sequence (designated ATRS [adenine/ thymidine-rich sequence]) [29]. Subsequent experiments in vivo show the EGF-dependent stimulation of a reporter construct containing the ATRS sequence, a chromatin immunoprecipitation assay demonstrating ErbB-1 bound to the cyclin D1 promoter, which contains an ARTS sequence, and the EGF-dependent stimulation of cyclin D1 mRNA levels [29]. The capacity of FGF-2 to induce expression of c-Jun or cyclin D1 mRNA is sensitive to the presence/absence of an artificial nuclear localization sequence incorporated into a transfected FGFR-I construct [33]. Another recent study has provided evidence that FGFR-I acts as a transcription factor at the FGF-2 promoter [38]. However, since the liganddependent nuclear localization of ErbB-1 or FGFR-I cannot be blocked, it remains to be shown whether these growth-factor-dependent transcription responses are dependent on nuclear localization of the receptor. Since RTKs contains an enzymatic function, it is plausible that the modification of nuclear proteins is another consequence for nuclear-localized receptors or their cytoplasmic domain fragments. Nuclear non-receptor tyrosine kinases, such as c-Abl, are known, but the relevant nuclear substrates are not clear. Finally, it is possible that RTKs carry other molecules into the nucleus and that these receptor-associated molecules are functional in the nucleus. It has been proposed that ErbB-1 may transport STAT-1, a tyrosine-phosphorylated transcription factor, from the cytosol into the nucleus [39]. In the case of ErbB-1 and perhaps FGF-I, it would seem that the cognate growth factor is carried by the receptor into the nucleus. The possible importance of growth factors in the nucleus has been reviewed elsewhere [40–42]. The nuclear localization of ErbB-1 recalls data published some years ago that showed DNA topoisomerase activity in highly purified preparations of this RTK [43]. A subsequent report confirmed this, but showed that the topoisomerase activity could be separated from the receptor by an additional purification step [44]. Nevertheless, these two reports do suggest that topoisomerase activity is physically associated with ErbB-1 at least in vitro.
Conclusions The new observations discussed above raise provocative ideas about the trafficking and signaling mechanisms of RTKs. The nuclear localization of other cell surface molecules or their cytoplasmic domain fragments are www.current-opinion.com
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parallel examples of direct communication between these two cellular compartments. It seems logical to expect that additional examples, including other RTKs, will be added in the near future. The most important questions that remain to solidify the importance of these observations are twofold. First, how do intact receptors move from the cell surface to the nucleus; and second, is the nuclear localization of an RTK a necessary part of the cellular response to growth factors?
Update Previous reports have identified PSD-95 as a molecule that associates with a PDZ-domain recognition motif at the carboxyl terminus of ErbB-4 [45–47]. Those reports suggest that ErbB-4 association with PSD-95 facilitates ligand-dependent activation of ErbB-4, ErbB-4 oligomerization, or both. A new report indicates that when the carboxy-terminal three residues of ErbB-4 are deleted, a mutation that prevents association with PDZ-domaincontaining proteins, ErbB-4 proteolytic processing by g-secretase is significantly impaired [48]. However, ectodomain proteolytic processing is not impaired in this mutant. These data suggest a mechanism to facilitate ErbB-4 processing by g-secretase.
Acknowledgements Space limitations preclude highlighting all relevant work, particularly that reported before 2000. The author appreciates the contributions of Sue Carpenter and Lori Bennett in manuscript and figure preparation, respectively. I am also grateful to two colleagues, Bruce Carter and Scott Hiebert, for reading the manuscript and offering suggestions. The support of the National Cancer Institute, USA, (grant number CA97456) is acknowledged.
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