- Email: [email protected]
Signal transduction by the neutrophin receptors
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Signal transduction by the neurotrophin receptors David R Kaplan* and Freda D Millert The neurotrophins signal cell survival, differentiation, growth cessation, and apoptosis through two cell surface receptors, the Trks and p75NTR (p75 neurotrophin receptor). Recent advances indicate that the particular events that are mediated by neurotrophins are dependent upon the cell type and the expression pattern of each neurotrophin receptor. For example, TrkA activation induces cell death of neural tumor cells, and survival and differentiation of neurons. Likewise, p75NTR, when activated in the absence of a strong Trk signal, induces apoptosis of neurons, while in the presence of "Irk it enhances responses to neurotrophin. These differing responses point to a complex interplay between neurotrophin-stimulated survival, differentiation, and apoptosis pathways.
lose the neurons of the sympathetic ganglia soon after birth and show extensive loss of the thermosensitive and pain-sensitive sensory neurons of the dorsal root ganglia [2]. In addition to their influence on neuronal development, N G F and the other neurotrophins play important roles in the maintenance and repair of the adult nervous system, and have been implicated as both oncogenic agents and tumor suppressors in various human tumors [3-5]. T h e mechanisms by which the Trk receptors signal differentiation events have been the subject of recent reviews [6,7°,8°,9]. We will therefore focus this review on recent concepts pertaining to Trk signaling in neural tumor cells and neurons, and on the striking data demonstrating the cell death inducing effects of the p75NTR.
Addresses *Brain Tumor Research Centre, and #Centre for Neuronal Survival, Montreal Neurological Institute, 3801 University Street, Montreal, PQ, Canada, H3A 2B4 *e-mail: [email protected] re-mail: [email protected]
Trk signal transduction pathways
Current Opinion in Cell Biology 1997, 9:213-221
Electronic identifier: 0955-06?4-009-00213 © Current Biology Ltd ISSN 0955-0674 Abbreviations brain-derived neurotrophic factor BDNF extracellular signal regulated kinase ERK insulin-like growth factor IGF JNK Jun amino-terminal kinase MAPK mitogen-activated protein kinase MAPK and ERK kinase MEK nerve growth factor NGF neurotrophin NT PI3-kinase phosphatidylinositol 3-kinase PLC phospholipase C receptor R Src homology SH SHP SH2-containing tyrosine phosphatase Sucl-associated neurotrophic factor target SNT TNFRI tumor necrosis factor receptor I
Introduction
T h e neurotrophins have traditionally been studied in the context of their survival- and differentiation-inducing effects on neurons. T h e neurotrophin family, consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, NT-4, and NT-6, together with their receptors, TrkA, TrkB, TrkC, and p75NTR (where R is receptor), play important roles in the development of the nervous system. Mice in which either the neurotrophins or their receptors have been selectively deleted by homologous recombination display severe defects in peripheral and motor neurons (reviewed in [1]). For example, the NGF-knockout mice
via multiple signaling
Studies by many laboratories, emphasizing the use of the PC12 cell system, have elucidated the signal transduction pathways used by Trk to promote the differentiation of neural cells. NGF addition to PC12 cells stimulates their differentiation into cells with many properties of sympathetic neurons [10]. The NGF-treated cells cease proliferating, acquire neurites and a dependence upon N G F as a survival factor in serum-free medium, and exhibit somatic hypertrophy. Previous work using Trk mutants defective in associating with, or stimulating the activities of, intracellular signaling proteins has indicated the existence of several signal transduction pathways used by the neurotrophins to mediate cell differentiation events (reviewed in [7",8°1). At least three proteins, namely, the adaptor proteins She and its neural-specific isoform N-Shc [11], the phospholipase PLC-y1 (phospholipase C-y1), and the phosphotyrosine phosphatase SHP (SH2-containing tyrosine phosphatase)-l, directly associate with NGFactivated Trk (reviewed in [8°]). These proteins couple Trk to several intracellular signaling pathways. For example, She association with, and tyrosine phosphorylation by, Trk results in the rapid activation of phosphatidylinositol 3-kinase (PI3-kinase), Ras, and the serine/threonine kinases B-Raf and mitogen-activated protein kinase (MAPK) ([8*,12,13]; B Hallberg, R Stephens, D Kaplan, L Greene, J Downward, unpublished data). Although the absence of Shc activity and subsequent downstream events does not appreciably a h e r N G F induced differentiation of PC12 cells, mutagenesis studies with the TrkA receptor indicate that the Shc pathway acts in concert with PLC-y1 to regulate the elongation and maintenance of neurites and cell survival ([14,15]; L Greene, D Kaplan, unpublished data). However, in similar studies, elimination of the Shc/Ras and PLC-y1 signaling pathways using Trk mutants did not prevent
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the NGF-induced initiation of neurite outgrowth and somatic hypertrophy [14,16]. These events were instead suppressed by deletion of a site on Trk that regulates the tyrosine phosphorylation of the nuclear protein S N T (Sucl-associated neurotrophic factor target) but that does not affect the tyrosine phosphorylation of Shc and PLC-y1 [16]. Mutation of all three sites regulating SNT, PLC-y1, and Shc tyrosine phosphorylation suppresses most of the NGF-induced differentiation of PC12 cells (M Ashcroft, R Stephens, L Greene, D Kaplan, unpublished data). Therefore, at least two separable pathways, one defined by the Shc- and PLC-yl-binding sites on Trk (tyrosines 490 and 785 of human TrkA, respectively), and another defined by the site stimulating S N T tyrosine phosphorylation (amino acids 441-443 of human TrkA) (Fig. 1), regulate a number of the observable phenotypic properties of PC12 cells treated with NGF. T h e R a s p a t h w a y is i n s u f f i c i e n t differentiation responses
for many
T h e Ras pathway plays a prominent role in many N G F mediated differentiation events (reviewed in [7",8°,13]). In PC12 cells and rat sympathetic neurons, two individual components of the Shc pathway, Ras and MEK (MAPK and ERK [extracellular signal regulated kinase} kinase), have been shown to be necessary for NGF-induced neuritogenesis or survival, using neutralizing Ras antibodies [13,17], dominant-inhibitory approaches [13,18], or the selective MEK inhibitor PD90859 [19]. T h e ability of Ras to induce differentiative events correlates with sustained MEK and MAPK activities, leading to the hypothesis that the sustained activity of neurotrophic factor receptors, rather than of specific receptor substrates, determines
whether a differentiative response will occur (reviewed in [8°,20]). In support of this hypothesis, mitogens, such as epidermal growth factor (EGF), for PCI2 cells induce transient intracellular activations of signaling pathways such as Ras that last minutes (<30minutes), while differentiation factors such as N G F and fibroblast growth factor (FGF) induce sustained signaling (>2 hours) (reviewed in [20]). Furthermore, sustained activation of M E K in PC12 cells suppresses serum- and NGF-induced apoptosis mediated by MKK (MAPK kinase)-JNK (Jun amino-terminal kinase) signaling pathways [21°°]. Recent evidence, however, has not substantiated the hypothesis of the sufficiency of sustained Ras, MEK, or MAPK activity for many aspects of neuronal differentiation. T h e first study indicating that Ras was not involved in certain neurotrophin-mediated neuronal differentiation events was that of Borasio et al. [22], who showed that the activity of Ras was neither necessary nor sufficient for the NGF-mediated survival or neuritogenesis of chick sympathetic neurons. More recent studies have indicated that sustained Ras, MEK, or MAPK activities are insufficient for many of the responses of PC12 cells, immortalized hippocampal neurons, and primary neurons to neurotrophic factors. These studies showed that: first, neither sustained MAPK activity nor expression of activated M E K was sufficient to induce neuritogenesis in immortalized rat hippocampal cells [23*]; second, persistent stimulation of the Ras/MAPK pathway was insufficient for platelet-derived growth factor (PDGF)-induced neuritogenesis in PC12 cells expressing mutant P D G F receptors [24]; third, sustained activation of Ras and MAPK was insufficient for survival responses of
Figure 1
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Y Y Y
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I
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i Y674 Y751
© 199'7 Current Opinion in Cell Biology
Regulatory sequences of TrkA. The single-letter amino acid code is used. Sequences in human TrkA that are important for NGF-mediated interactions with, and activations of, substrates include amino acids NPQY, which constitute an Shc-association site when Y490 is phosphorylated, and YLDV, a PLC-yl-association site when Y785 is phosphorylated. The Shc-association site is also important for PI3-kinase (PI3-K) activation. Amino acids KFG (441-443) are necessary for the tyrosine phosphorylation of SNT. Tyrosines Y751, Y670, Y674, and Y6"75 are involved in the regulation of Trk kinase activity and transphosphorylation [79]. K538 is an ATP-binding site.
Signal transductionby the neurotrophinreceptorsKaplanand Miller
hippocampal pyramidal neurons [25]; fourth, expression of activated Ras in PC12 cells was not sufficient (although it was necessary) for increases in calcium channel currents [26]; and fifth, inhibition of MEK activity with the MEK inhibitor PD98059 failed to block NGF-dependent survival of rat sympathetic neurons [27,28]. This same inhibitor also failed to block NGF-mediated neurite outgrowth from chick sensory and sympathetic neurons, indicating that MEK and MAPK might be unnecessary for both neuritogenesis and survival responses in peripheral neurons [29]. Even the hypothesis that sustained Trk activity determines the timing and extent of neuronal differentiation is in question, particularly with regard to primary neurons. In post-mitotic rat sympathetic neurons, N G F is much more efficient than NT-3 at inducing survival and neuritogenesis even though both of these factors induce a temporally similar sustained activation of TrkA [30°]. These reports, like those using Trk mutants described above, point to the necessity of other Trk signaling pathways that act in concert with the Ras pathway to induce the multitude of neuronal differentiation responses. T h e y also suggest that specific intracellular targets of Trk, rather than sustained Trk/Ras/MEK/MAPK activities, regulate different aspects of the differentiation process, particularly in primary neurons. What is the nature of these other signaling pathways? They include the PLC-y1 pathway, which mediates NGFinduced peripherin expression in PC12 cells (reviewed in [8°]) and may be required for BDNF-stimulated survival of cerebellar neurons [31], and as yet uncharacterized pathways that regulate the activity of Src, S N T (reviewed in [8°]), and SHP-1 [32]. The adaptor protein Crk may also influence neuritogenesis events via a signaling pathway that functions independently of Shc; expression of a dominant-inhibitory Crk mutant in PC12 cells delays neurite elongation and actin filament organization at the growth cone without affecting She tyrosine phosphorylation [33°]. The PI3-kinase pathway, as described below, is another promising candidate for a novel pathway that controls differentiation responses.
T h e P I 3 - k i n a s e / A k t s i g n a l i n g p a t h w a y as a m o d u l a t o r of n e u r i t o g e n e s i s and cell survival A number of recent studies have implicated PI3-kinase and its downstream substrate, the serine/threonine kinase Akt, in neuronal differentiation and survival. Two initial studies, which largely utilized selective though not specific PI3-kinase inhibitors, suggested a role for PI3-kinase in mediating NGF-induced neuritogenesis [34] or survival [35] in PCI2 cells. In spite of these observations, however, the necessity of PI3-kinase for neuritogenesis and cell survival is still unclear. For example, in PC12 cells, PI3-kinase activity is controlled through the Shc-binding site on Trk [12], which is dispensable for NGF-mediated cell survival and initiation of neurites [8°,14,15]. Thus, PI3-kinase can be considered as a member of a pathway
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that may collaborate with other signaling pathways to regulate survival and neuritogenesis events. Recent data have indicated that Akt, a serine/threonine kinase that can be activated in vivo and in vitro by phosphatidylinositol 3,4-bisphosphate [36], a phospholipid second messenger generated by PI3-kinase activity, is necessary and sufficient for survival of cultured rat cerebellar neurons [37°°]. In these experiments, overexpression of Akt was sufficient to allow neurons to survive in the absence of survival factors, and dominant-inhibitory Akt blocked much of KCI- and insulin-mediated survival. The requirement for Akt activity in this system suggests that this protein is a key component of the cell survival machinery of cerebellar neurons. In support of this idea, PI3-kinase was recently shown to mediate IGF-1 (insulin-like growth factor-1)-induced survival of primary cerebellar neurons [37°°,38]. It is not yet known whether PI3-kinase or Akt participates in neurotrophin-dependent cell survival of neurons, or how these proteins interact with the cell signaling machinery to inhibit apoptosis.
TrkA as an a p o p t o s i s - i n d u c i n g receptor for neural t u m o r cells Although the "Irks mediate neuritogenesis and survival of neurons, they induce two very different responses in neural tumor cells. In PC12 pheochromocytoma and neuroblastoma cells, TrkA activity inhibits cell growth and neuritogenesis [10,39]. The antimitotic responses of NGF-activated TrkA may be due to an increase in the levels of the cyclin dependent kinase inhibitor p21, at least in PC12 cells [40]. Surprisingly, NGF-stimulated TrkA activity does not induce neuritogenesis or growth cessation of TrkA-transfected medulloblastoma cells, but instead induces cellular apoptosis within 24 hours [41°°]. Similarly, rats injected with C6 glioma cells expressing TrkA survived much longer than rats injected with C6 cells expressing kinase-inactive TrkA [42°°], probably because the cells expressing wild-type TrkA were less invasive and had a greater rate of apoptosis. As TrkA has never been reported to be apoptotic in postmitotic neurons, this finding may reflect a fatal consequence of a conflict between antiproliferative "Irk signaling and the proliferative state of tumor cells. These data are of considerable interest in the neural tumor field, particularly because expression of TrkA and TrkC correlates with tumor prognosis; high expression of TrkA correlates with favorable prognosis in neuroblastoma [4,43-46], while high expression of TrkC correlates with favorable prognosis in medulloblastoma [5,47]. On the basis of these findings, we can propose a potential therapy for neural tumors involving the induction or overexpression of TrkA/C in neural tumors, combined with NGF or NT-3 administration, to induce the death of tumor cells. Interestingly, not all Trk receptors show antimitotic or apoptosis-inducing activity in neural tumor cells. TrkB expression in neuroblastoma stimulates not only neurito-
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genesis but also cell invasiveness, growth, disaggregation, survival, and resistance to toxicity by the chemotherapeutic drug vinblastine [48°,49,50]. These later responses are all characteristics of metastatic cells. In contrast, TrkA expression in the same neuroblastoma cells (cell line SH-SY5Y) mediates neuritogenesis and the cessation of cell growth, with none of the metastatic cell type effects. As many neuroblastoma cell lines and tumors express the TrkB ligand B D N F [50,51], perhaps a T r k B - B D N F autocrine loop may promote cell growth and survival of neuroblastomas in vivo. These results reflect fundamental signaling differences among Trk receptors expressed in neural tumor cells.
p 7 5 N T R as an a p o p t o s i s - i n d u c i n g receptor for neurons Although the p75 neurotrophin receptor (p75NTR) was the first-isolated neurotrophin receptor, as well as the first-reported member of the p 7 5 N T R / F a s / T N F R I (tumor necrosis factor receptor I) family (reviewed in [6]), definition of its functional role and/or signaling capacity has remained elusive. However, the past year has seen significant advances in our understanding of how this receptor signals, and the biological significance of such signaling. T h e first insights into the biochemical function of the p 7 5 N T R derived from two distinct lines of investigation (reviewed in [6,8°]). In one group of studies, p 7 5 N T R was shown to modify the function of the TrkA receptor. Ligand-binding by p 7 5 N T R led to an increase in high-affinity TrkA binding sites [52], enhanced TrkA autophosphorylation in response to N G F [53-55], and increased selectivity of TrkA for its neurotrophin ligands [56,57]. These findings led to the hypothesis that the function of p75NTR was to enhance TrkA responsiveness and selectivity for N G F [6]. This was thought to occur either by p75NTR 'concentrating' N G F within the TrkA local environment [54] or by p 7 5 N T R forming a molecular complex with TrkA that modified ligand binding [52]. T h e first report of the p 7 5 N T R - / - mouse [58] apparently supported this hypothesis, as this mouse appeared to be an attenuated phenocopy of the TrkA-/- mouse, at least with regard to sensory neurons. A second concurrent line of investigation focused on the possibility that p 7 5 N T R might signal independently of TrkA. T h e first report that it could do so was by Dobrowsky et al. [59], who demonstrated that, in glioma cells that did not express TrkA, binding of N G F to p 7 5 N T R led to an increase in intracellular ceramide levels. Interestingly, in PC12 cells that express TrkA, N G F did not induce a similar increase, although BDNF, which binds p75NTR but not TrkA, did [60°°]. This finding was the first biochemical hint that there was antagonistic cross-talk between these two receptors, with
Trk being able to silence at least some p 7 5 N T R signaling pathways. Biological data on sympathetic neurons also led to speculation that p 7 5 N T R could downregulate some TrkA functions [61].
p75NTR signal transducUon T h e past year has seen confirmation that p 7 5 N T R signals in a similar way to other members of the T N F R I / F a s receptor family. In one recent report, Carter et al. [62 °°] demonstrated that activation of p 7 5 N T R on cells that did not express TrkA led to the activation of the transcription factor NFtcB. In a second report, Casaccia-Bonnefil et al. [63 °°] demonstrated that NGF-mediated activation of p 7 5 N T R in cultured oligodendrocytes, which also do not express TrkA, led to an increase in ceramide levels and an activation of JNK. In neither of these systems were B D N F or NT-3 able to mediate these signaling events. In contrast, in cultured sympathetic neurons which express TrkA but not the B D N F receptor TrkB, BDNF-mediated activation of p 7 5 N T R led to increased phosphorylation of c-jun (R Aloyz, S Bamji, FD Miller, unpublished data), an event mediated by the JNK family. These data support two major conclusions. First, p 7 5 N T R signals via ceramide [59], the JNK family [63°'], and NF~:B [62°°], as do other members of this receptor family [59]. Second, p 7 5 N T R signaling is inhibited by coactivation of a Trk receptor expressed on the same cell: in PC12 cells and sympathetic neurons, only ligands that do not efficiently bind coexpressed TrkA are able to activate p 7 5 N T R signalling [60°°], while in Schwann cells and oligodendrocytes, which express Trk receptors other than TrkA, only N G F (the TrkA ligand) activates p75NTR. These data also raise a number of questions. First, what is the mechanism whereby TrkA activation 'silences' p75NTR-mediated signaling? Although this question has not yet been addressed, we know that it must occur at a level proximal to the receptor itself, as all downstream signaling events that have been measured are abrogated by Trk coactivation. It is possible, at least for TrkA, that the mechanism involves a p 7 5 N T R - T r k A complex, as the evidence that such a molecular association exists is now compelling, as measured in biophysical experiments. For example, p 7 5 N T R colocalizes on cell surfaces with TrkA (but not with TrkB or other receptor tyrosine kinases) [64°], and TrkA will limit the diffusion of p75NTR in the cytoplasmic membrane [65]. Second, can p 7 5 N T R signaling similarly antagonize TrkA signalling? Although there is now good cellular evidence that p 7 5 N T R signaling can 'override' TrkA signaling (see below; Fig. 2), it is, as yet, unclear how this occurs. However, recent data suggest that it may occur in part at the level of the TrkA receptor itself (P Barker, personal communication); data show that BDNF-mediated activation of p 7 5 N T R in PC12 cells reduced TrkA autophosphorylation in experiments utilizing a mutant form of N G F that recognizes TrkA and not p75NTR.
Signal transduction by the neurotrophin receptors Kaplan and Miller
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Figure 2
(a)
(b)
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p75NTR
Ceramide
Ceramide
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~>,
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Apoptosis
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High Trk and high p75NTR activity O 1997
Current Opinionin Cell Biology
Signaling mechanisms of p75NTR, p75NTR can induce apoptosis, cell migration, and aspects of cell differentiation on its own when highly activated (a), suppress Trk signaling and induce apoptosis when highly activated in the presence of low levels of Trk activity (b), and enhance Trk signaling and Trk-mediated cell differentiation when co-activated with Trk (¢). Ceramide, NF~B and JNK are signaling proteins that are activated by p75NTR in certain cellular environments, while Ras, PI3-kinase (PI3-K) and SNT are signaling proteins that are activated by TrkA.
These findings make it clear that the cellular ratio of p 7 5 N T R to Trk will be a fundamental determinant of the signaling mediated by exposure to a given neurotrophin. This is particularly important for ceils in which the levels of these two receptor classes are independently regulated. For example, on sensory, sympathetic, and basal forebrain cholinergic neurons, p 7 5 N T R levels are dramatically upregulated by N G F exposure [66,67], while, at least in sympathetic neurons, TrkA levels remain constant [30"]. Moreover, on sympathetic neurons, there is partial compartmentalization of these two receptors; p 7 5 N T R is equally distributed over the entirety of the neuron, while TrkA is present at approximately threefold higher density on cell bodies (J Kohn, FD Miller, unpublished data). Thus, the neuronal signaling events mediated in response to N G F may differ in axons versus cell bodies, and as a function of long-term exposure to N G F itself.
p75NTR,a mediatorof neuralcell death What is the cellular outcome of p 7 5 N T R signaling? Recent evidence indicates that, like other members of this family, p 7 5 N T R can signal to mediate apoptosis. T h e first report implicating p 7 5 N T R in apoptosis was by Rabizadeh et al. [68] who studied an immortalized neural cell line. However, in this case, p 7 5 N T R activation was thought to be ligand-independent, and it was unclear what the involvement of Trk receptors was in this system. Three subsequent reports implicated p 7 5 N T R in neuronal apoptosis. In one report selective activation of p 7 5 N T R by
N G F led to increased death of neurons in the developing chick isthmo-optic nucleus [69], while in a second report inhibition of N G F in the developing chick eye using function-blocking antibodies led to decreased cell death in the retina [70"']. In a third report, antisense inhibition of p 7 5 N T R in developing sensory neurons increased cell death at one stage, and decreased death at another [71]. However, in none of these systems was it possible to define the putative role of Trk receptors and/or to gain insights into potential p 7 5 N T R signalling mechanisms. Two more recent studies have addressed these issues. Casaccia-Bonnefil et al. [63"'] used cultured oligodendrocytes that expressed p 7 5 N T R but not TrkA, and demonstrated that N G F exposure led to rapid apoptosis accompanied by increases in ceramide levels and JNK activity. In a second set of experiments, BDNF-mediated activation of p 7 5 N T R in cultured sympathetic neurons was shown to override suboptimal survival signals generated either via TrkA or via KCI to lead to apoptosis (DJ Belliveau, S Bamji, FD Miller, unpublished data). p 7 5 N T R was, however, unable to mediate apoptosis when TrkA was activated to higher levels, supporting the concept that these two receptors both functionally and biochemically antagonize each other in some situations. As in oligodendrocytes, the p75NTR-mediated apoptosis of sympathetic neurons was accompanied by hyperphosphorylation of c-jun. Interestingly, previous studies demonstrated that overexpression of c-jun in sympathetic
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neurons is sufficient to cause apoptosis [72°°], and that functional inhibition of c-jun is sufficient to inhibit sympathetic neuron [72•°,73] or PC12 cell [21"] apoptosis induced by neurotrophin withdrawal. It is as yet unclear, however, whether JNK family activation is necessary or sufficient for the induction of neuronal apoptosis by p75NTR. Recent studies of transgenic mice support the conclusion that p75NTR plays a physiological role in regulating neuronal apoptosis. A re-examination of the p75NTR--/mouse demonstrated that there are increased numbers of basal forebrain cholinergic neurons [74"] and less injuryinduced death of sensory and motor neurons (K-F Lee, personal communication). Expression of the intracellular domain of the p75NTR in neurons of transgenic mice demonstrated that this truncated receptor could function as an 'apoptoprotein', or constitutive apoptosis-inducing protein, leading to increased death of developing sensory and sympathetic neurons, and inducible death of mature, injured motor neurons (M Majdan, FD Miller, P Barker, unpublished data). Finally, studies of BDNF-/mice demonstrated an increased number of sympathetic neurons (C Pozniak, C Causing, FD Miller, unpublished data), a result consistent with the in vitro data showing that BDNF-mediated activation of p75NTR led to apoptosis of these neurons. Together with the recent studies from Barde's laboratory [70"] that demonstrated that the functional inhibition of neurotrophin actions decreases cellular apoptosis in the developing retina, these data indicate that neurotrophin-mediated activation of p75NTR plays an essential role in naturally occurring cell death, and is likely to be responsible for some types of neuronal apoptosis that follow injury of the mature nervous system.
with several additional but important functions. Most prominent of these functions is an apparently essential role as an apoptosis mediator in the nervous system, both during naturally occurring cell death and following injury. Important questions remain to be answered about the mechanisms used by p75NTR to induce cell death, and about how p75NTR can function as both a positive effector of Trk-mediated cell differentiation and an apoptosis:inducing protein. It is also clear that, depending upon cellular context and relative expression or activation levels, p75NTR can antagonize Trk-mediated cell survival responses, and, conversely, that Trk can inhibit p75NTR-induced apoptosis. The mechanisms whereby these two receptors influence each other's function is an important area of research for the future. Progress has also been made in understanding the mechanisms whereby the Trk receptors mediate neuronal survival. These survival pathways are only now being identified, with recent data implicating the Ras pathway and that of PI3-kinase and its downstream target, Akt. In this regard, the major questions to be asked are whether all neurons use these same survival pathways, and which signaling proteins are used by Trk to suppress p75NTR-induced apoptosis. One suggestion is that components of the Ras pathway, particularly those regulating the MAPKs, suppress cell death pathways mediated by c-jun kinases, which are activated during both p75NTR- and NGF-withdrawal-induced apoptosis of sympathetic neurons. The next year should yield much information about the signaling mechanisms used by the apoptosis- and survival-inducing neurotrophin receptors.
Acknowledgements We thank P Barker, A Ross, K-I: Lee, and L Greene for sharing data prior to publication.
References and recommended reading It is equally clear, however, that apoptosis is only one cellular outcome of p75NTR-mediated signaling, p75NTR activation in the absence of Trk activation has been shown to cause enhanced cellular migration [75], induction of the gene for neural adhesion molecule NILE/L1 [76], release of dopamine [77], and enhancement of extracellular matrix penetration of human melanoma cells [78]. Moreover, coactivation of Trk and p75NTR in the same cell does not lead to apoptosis (except in cases where Trk activation is suboptimal; Fig. 2), and the ultimate outcome in this situation is likely to be determined by the amount of cross-talk between a given Trk receptor and p75NTR, as well as by the cellular context.
3.
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Conclusions
4.
Nakagawara K, Arima-Nakagawara M, Scavarda NJ, Azar CG, Cantor AB, Brodeur GM: Association between high levels of
The significant breakthrough in the past year in the study of neurotrophin signal transduction has been the recognition of the role of the p75NTR as an apoptosisinducing receptor for neural cells and neurons, p75NTR has evolved in our view from a receptor with a limited role as a positive modifier of Trk activity to a protein
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15.
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16.
Peng X, Greene LA, Kaplan DR, Stephens RM: Deletion of a conserved juxtamembrane sequence in "Irk abolishes NGFpromoted neuritogenesis. Neuron 1995, 15:395-406.
17.
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18.
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219
30. •
Belliveau DJ, Krivko I, Kohn J, Lachance C, Pozniak C, Rusakov D, Kaplan D, Miller FD: NGF and NT-3 both activate TrkA on sympathetic neurons but differentially regulate survival and neuritogenesis. J Cell Biol 1997, 136:375-388. First report that, in neurons, both NGF and NT-3 mediate several different biological responses through the same receptor, TrkA. Each factor induced prolonged TrkA activity, suggesting that specific receptor substrates, rather than sustained activation of a particular substrate, are responsible for differentiation responses. 31.
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32.
Vambutas V, Kaplan DR, Sells MA, Chernoff J: Nerve growth factor stimulates tyrosine phosphorylation and activation of Src homology-containing protein tyrosine phosphatase 1 in PC12 cells. J Biol Chem 1995, 270:25629-25633.
33.
21. •.
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Borasio GD, Markus A, Wittinghofer A, Barde Y-A, Heumann R: Involvement of ras p21 in neurotrophin-inducad response of sensory but not sympathetic neurons. J Cell Biol 1993, 121:665-672.
23. •
Kuo W-L, Abe M, Rhee J, Eves EM, McCarthy SA, Yan M, Teml~letonDJ, McMahon M, Rosner MR: Raf, but not MEK or
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34.
Kimura K, Hattori S, Kabuyama Y, Shizawa Y, Takayanagi J, Nakamura S, Toki S, Matsuda Y, Onodera K, Fukui Y: Neurite outgrowth of PC12 cells is suppressed by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. J Bio/Chem 1994, 269:18961-1896?.
35.
Yao RI, Cooper GM: Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 1995, 267:5206-5208.
36.
Franke TF, Kaplan DK, Cantley LC, Toker A: Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4bisphosohate. Science 1997. 275:665-668.
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37. =o
Dudek H, Datta SD, Franke TF, Birnbaum MJ, Yao R, Cooper GM, Segal RA, Kaplan DK, Greenberg ME: Regulation of neuronal survival by the serine-threonine kinase Akt. Science 1997, 275:661-665. Reports that expression of the serine/threonine kinase Akt in primary cerebellar neurons is both necessary and sufficient for much of the survival response of these neurons to neurotrophic factors. 38.
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D'Mello SR, Borodezt K, Soltoff S: Insulin-like growth factor and potassium depolarization maintain neuronal survival by distinct signaling pathways: possible involvement of PI3-kinase in IGF-I signaling. J Neurosci 199'7, 17:1548-1560. Matsushima H, Bogenmann E: Expression of trkA cDNA in neuroblastoma mediates differentiation in vitro and in vivo. Mo/ Cell Bio11993, 13:7447-7456. Yan GZ, Ziff EB: NGF regulates the PC12 cell cycle machinery through specific inhibition of the cdk kinases and induction of cyclin D1. J Neurosci 1995, 15:6200-6212.
Muragaki Y, Chou TT, Kaplan DR, Trojanowski JG, Lee VMY: Nerve growth factor induces apoptosis in human medulloblastoma cell lines that express trkA receptors. J Neurosci 1997, 17:530-542. First report that expression of TrkA in a neural tumor cell induces NGF o mediated apoptosis.
52.
1991,350:6?8-683.
53.
Verdi JM, Birren SJ, Ibanez CF, Persson H, Kaplan DR, Benedetti M, Chao MV, Anderson DJ: p75 LNGFRregulates Trk signal transduction and NGF-induced neuronal differentiation in MAH cells. Neuron 1994, 12:733-745.
54.
Barker PA, Shooter EM: Disruption of NGF binding to the low affinity neurotrophin receptor p75LNTR reduces NGF binding to TrkA on PC12 cells. Neuron 1994, 13:203-215.
55.
Hantzopoulos PA, Suri C, Glass MP, Goldfarb MP, Yancopolous GD: The low affinity NGF receptor, p75, can collaborate with each of the Trks to potentiate functional responses to the neurotrophins. Neuron 1994, 13:187-201.
56.
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57.
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58.
Lee KF, Li E, Huber I_J, Landis SC, Sharpe AH, Chao MY, Jaenisch R: Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system. Ceil 1992, 69:737-749.
59.
Dobrowsky RT, Werner MH, Castellino AM, Chao MV, Hannun Y: Activation of the sphingomyelin cycle through the low-affinity neurotrophin receptor. Science 1994, 265:1569-1599.
41. o,
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LachyankarMB, Ross AH, Litofsky NS, Condon PJ, Guesenberry PJ, Recht LD: TrkA expression decreases the in vivo aggressiveness of C6 glioma cells. Cancer Res 1997, in press. Demonstration that TrkA expression in a glioblastoma cell line decreases the aggressiveness, invasiveness, and perhaps survival of these cells in vivo. 43.
Kogner P, Barbany G, Dominici C, Castello MA, Rashella G, Persson H: Coexpression of messenger RNA for Trk protooncogene and low affinity nerve growth factor receptor in neuroblastoma with favorable prognosis. Cancer Res 1993, 53:2044-2050.
44.
Hoehner JC, Olsen L, Sandstedt B, Kaplan DR, Pahlman S: Association of neurotrophin receptor expression and differentiation in human neuroblastoma. Am J Path t995, 147:102-113.
45.
Kramsr K, Gerald W, Lesauteur L, Saragovi HU, Dheung NKV: Prognostic value of TrkA protein detection by monoclonal antibody 5c3 in neuroblastoma. C/in Cancer Res 1996, 8:1361-1367.
46.
Suzuki T, Bogenmann E, Shimada H, Stram D, Seeger RC: Lack of high affinity nerve growth factor receptors in aggressive neuroblastomas. J Nat/Cancer Inst 1993, 85:377-384.
47.
Ryden M, Sehgal R, Dominici C, Schilling FH, Ibanez CF, Kogner P: Expression of mRNA for the neurotrophin receptor trkC in neuroblastomas with favourable tumour stage and good prognosis. Brit J Cancer 1996, 74:773-779.
48.
Matsumoto K, Wada R, Yamashiro J, Kaplan DR, Thiele CJ: Expression of brain-derived neurotrophic factor (BDNF) and p145Trke affects survival, differentiation, and invasiveness of human neuroblastoma cells. Cancer Res 1995, 55:1798-1806. Indicates that TrkB, in contrast to TrkA, is a potent survival- and invasivenessinducing receptor for neuroblastoma cells. •
49.
50.
60. =•
Dobrowsky RT, Jenkins GM, Hannun YA: Neurotrophins induce sphingomyelin hydrolysis: modulation by co-expression of p75 with Trk receptors. J Biol Chem 1995, 270:22135-22142. First evidence that the activity of Trk antagonizes p75NTR-mediated signal transduction. Coactivation of TrkA and p75NTR suppressed p75NTRinduced sphingomyelin hydrolysis, while activation of p?5NTR in the absence of TrkA activation resulted in sphingomyelin hydrolysis. 61.
Miller FD: NGF and neuronal gene expression. Prog Brain Res 1994, 103:23-33.
62. ••
Carter BD, Kaltschmidt C, Kaltschmidt B, Offenhauser N, BohmMatthaei R, Baeuerle PA, Bards YA: Selective activation of NFKB by nerve growth factor through the neurotrophin receptor p75. Science 1996, 272:542-545. The first report of a Trk-independent signaling pathway induced by p75NTR in primary neural cells. NGF, but not BDNF and NT-3, stimulated the activity of the NFKB transcription factor in rat Schwann cells. 63. *,
Casaccia-Bonnefil P, Carter BD, Dobrowsky RT, Chao MV: Death of oligodendrocytes mediated by the interaction of nerve growth factor with its receptor p75. Nature 1996, 383:716-719. First evidence that p75NTR activity, in the absence of Trk receptor signaling, induces apoptosis of neural cells, p?5NTR also stimulated the generation of ceramide and the activity of JNK. 64. •
Wosikowski K, Giannakakou P, Vails P, Biedler JL, Spengler BA, Lucarelli E, Bates SE, Thiele CJ: Brain-derived neurotrophic factor protects neuroblastoma cells from vinblastine toxicity. Cancer Res 1996, 56:3737-3742.
Ross AH, Daou M-C, McKinnon CA, Condon PJ, Lachyankar MB, Stephens RM, Kaplan DR, Wolf DE: The neurotrophin receptor, gp75, forms a complex with the receptor tyrosine kinase TrkA. J Cell Biol 1996, 132:945-953. Reports the specific ligand-independent association of TrkA and p75NTR on cell surfaces. Both extracellular and intracellular domains of TrkA and p75NTR were required for the interactions between these receptors.
Nakagawara A, Azar CG, Scavarda NJ, Brodeur GM: Expression
65.
Wolf DE, McKinnon CA, Daou M-C, Stephens RM, Kaplan DR, Ross AH: Interaction with TrkA immobilizes gp75 in the high affinity growth factor receptor complex. J Biol Chem 1995, 270:2133-2138.
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and function of trk-B and BDNF in human neuroblastomas. Mol Cell Biol 1994, 14:759-767.
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Kaplan DR, Matsumoto K, Lucarelli E, Thiele C J: Induction of TrkB by retinoic acid mediates biological responsiveness to neurotrophins and differentiation of neuroblastema cells. Neuron 1993, 11:321-331.
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Frade JM, Rodriguez-Tebar A, Barde Y-A: Induction of cell death by endogenous nerve growth factor through its p75 receptor. Nature 1996, 383:166-168. Shows that, during development, endogenous NGF causes the death of rat retinal neurons that express p75NTR but not TrkA. 71.
Barrett GL, Bartlett PF: The p75 nerve growth factor receptor mediates survival or death depending on the stage of sensory neuron development. Proc Nat/Acad Sci USA 1994, 91:6501-6505.
Ham J, Babij C, Whitfield J, Pfarr CM, Lallemand D, Yaniv M, Rubin LL: A c-jun dominant negative mutant protect sympathetic neurons against programmed cell death. Neuron 1995, 14:927-939. Describes an essential role for the transcription factor c-jun in the induction of apoptosis of sympathetic neurons following NGF deprivation. Expression of c-jun was sufficient to induce cell death, while expression of dominantinhibitory c-jun prevented cell death.
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Van der Zee CEM, Ross GM, Riopelle PJ, Hagg T: Survival of cholinergic forebrain neurons in developing p75NGFR-deficient mice. Science 1996, 274:1729-1732. Reports that mice lacking p75NTR have an increased number of cholinergic forebrain neurons, suggesting that p75NTR is an apoptosis-inducing receptor during neuronal development. 75.
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Herrmann JL, Mentr DG, Hamda J-I, Marchetti D, Nakajima M, Nicolson GL: Mediation of NGF-stimulated extracellular matrix invasion by the human melanoma low-affinity p75 neurotrophin receptor: melanoma p75 functions independently of trkA. Mo/Bio/Cell 1993, 4:1205-1216.
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Cunningham ME, Stephens RM, Kaplan DR, Greene LA: Autophosphorylation of activation loop tyrosines regulates signaling by the Trk NGF receptor. J Biol Chem 1997, in press.
72. •o
Signal transduction by the neurotrophin receptors David R Kaplan* and Freda D Millert The neurotrophins signal cell survival, differentiation, growth cessation, and apoptosis through two cell surface receptors, the Trks and p75NTR (p75 neurotrophin receptor). Recent advances indicate that the particular events that are mediated by neurotrophins are dependent upon the cell type and the expression pattern of each neurotrophin receptor. For example, TrkA activation induces cell death of neural tumor cells, and survival and differentiation of neurons. Likewise, p75NTR, when activated in the absence of a strong Trk signal, induces apoptosis of neurons, while in the presence of "Irk it enhances responses to neurotrophin. These differing responses point to a complex interplay between neurotrophin-stimulated survival, differentiation, and apoptosis pathways.
lose the neurons of the sympathetic ganglia soon after birth and show extensive loss of the thermosensitive and pain-sensitive sensory neurons of the dorsal root ganglia [2]. In addition to their influence on neuronal development, N G F and the other neurotrophins play important roles in the maintenance and repair of the adult nervous system, and have been implicated as both oncogenic agents and tumor suppressors in various human tumors [3-5]. T h e mechanisms by which the Trk receptors signal differentiation events have been the subject of recent reviews [6,7°,8°,9]. We will therefore focus this review on recent concepts pertaining to Trk signaling in neural tumor cells and neurons, and on the striking data demonstrating the cell death inducing effects of the p75NTR.
Addresses *Brain Tumor Research Centre, and #Centre for Neuronal Survival, Montreal Neurological Institute, 3801 University Street, Montreal, PQ, Canada, H3A 2B4 *e-mail: [email protected] re-mail: [email protected]
Trk signal transduction pathways
Current Opinion in Cell Biology 1997, 9:213-221
Electronic identifier: 0955-06?4-009-00213 © Current Biology Ltd ISSN 0955-0674 Abbreviations brain-derived neurotrophic factor BDNF extracellular signal regulated kinase ERK insulin-like growth factor IGF JNK Jun amino-terminal kinase MAPK mitogen-activated protein kinase MAPK and ERK kinase MEK nerve growth factor NGF neurotrophin NT PI3-kinase phosphatidylinositol 3-kinase PLC phospholipase C receptor R Src homology SH SHP SH2-containing tyrosine phosphatase Sucl-associated neurotrophic factor target SNT TNFRI tumor necrosis factor receptor I
Introduction
T h e neurotrophins have traditionally been studied in the context of their survival- and differentiation-inducing effects on neurons. T h e neurotrophin family, consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, NT-4, and NT-6, together with their receptors, TrkA, TrkB, TrkC, and p75NTR (where R is receptor), play important roles in the development of the nervous system. Mice in which either the neurotrophins or their receptors have been selectively deleted by homologous recombination display severe defects in peripheral and motor neurons (reviewed in [1]). For example, the NGF-knockout mice
via multiple signaling
Studies by many laboratories, emphasizing the use of the PC12 cell system, have elucidated the signal transduction pathways used by Trk to promote the differentiation of neural cells. NGF addition to PC12 cells stimulates their differentiation into cells with many properties of sympathetic neurons [10]. The NGF-treated cells cease proliferating, acquire neurites and a dependence upon N G F as a survival factor in serum-free medium, and exhibit somatic hypertrophy. Previous work using Trk mutants defective in associating with, or stimulating the activities of, intracellular signaling proteins has indicated the existence of several signal transduction pathways used by the neurotrophins to mediate cell differentiation events (reviewed in [7",8°1). At least three proteins, namely, the adaptor proteins She and its neural-specific isoform N-Shc [11], the phospholipase PLC-y1 (phospholipase C-y1), and the phosphotyrosine phosphatase SHP (SH2-containing tyrosine phosphatase)-l, directly associate with NGFactivated Trk (reviewed in [8°]). These proteins couple Trk to several intracellular signaling pathways. For example, She association with, and tyrosine phosphorylation by, Trk results in the rapid activation of phosphatidylinositol 3-kinase (PI3-kinase), Ras, and the serine/threonine kinases B-Raf and mitogen-activated protein kinase (MAPK) ([8*,12,13]; B Hallberg, R Stephens, D Kaplan, L Greene, J Downward, unpublished data). Although the absence of Shc activity and subsequent downstream events does not appreciably a h e r N G F induced differentiation of PC12 cells, mutagenesis studies with the TrkA receptor indicate that the Shc pathway acts in concert with PLC-y1 to regulate the elongation and maintenance of neurites and cell survival ([14,15]; L Greene, D Kaplan, unpublished data). However, in similar studies, elimination of the Shc/Ras and PLC-y1 signaling pathways using Trk mutants did not prevent
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the NGF-induced initiation of neurite outgrowth and somatic hypertrophy [14,16]. These events were instead suppressed by deletion of a site on Trk that regulates the tyrosine phosphorylation of the nuclear protein S N T (Sucl-associated neurotrophic factor target) but that does not affect the tyrosine phosphorylation of Shc and PLC-y1 [16]. Mutation of all three sites regulating SNT, PLC-y1, and Shc tyrosine phosphorylation suppresses most of the NGF-induced differentiation of PC12 cells (M Ashcroft, R Stephens, L Greene, D Kaplan, unpublished data). Therefore, at least two separable pathways, one defined by the Shc- and PLC-yl-binding sites on Trk (tyrosines 490 and 785 of human TrkA, respectively), and another defined by the site stimulating S N T tyrosine phosphorylation (amino acids 441-443 of human TrkA) (Fig. 1), regulate a number of the observable phenotypic properties of PC12 cells treated with NGF. T h e R a s p a t h w a y is i n s u f f i c i e n t differentiation responses
for many
T h e Ras pathway plays a prominent role in many N G F mediated differentiation events (reviewed in [7",8°,13]). In PC12 cells and rat sympathetic neurons, two individual components of the Shc pathway, Ras and MEK (MAPK and ERK [extracellular signal regulated kinase} kinase), have been shown to be necessary for NGF-induced neuritogenesis or survival, using neutralizing Ras antibodies [13,17], dominant-inhibitory approaches [13,18], or the selective MEK inhibitor PD90859 [19]. T h e ability of Ras to induce differentiative events correlates with sustained MEK and MAPK activities, leading to the hypothesis that the sustained activity of neurotrophic factor receptors, rather than of specific receptor substrates, determines
whether a differentiative response will occur (reviewed in [8°,20]). In support of this hypothesis, mitogens, such as epidermal growth factor (EGF), for PCI2 cells induce transient intracellular activations of signaling pathways such as Ras that last minutes (<30minutes), while differentiation factors such as N G F and fibroblast growth factor (FGF) induce sustained signaling (>2 hours) (reviewed in [20]). Furthermore, sustained activation of M E K in PC12 cells suppresses serum- and NGF-induced apoptosis mediated by MKK (MAPK kinase)-JNK (Jun amino-terminal kinase) signaling pathways [21°°]. Recent evidence, however, has not substantiated the hypothesis of the sufficiency of sustained Ras, MEK, or MAPK activity for many aspects of neuronal differentiation. T h e first study indicating that Ras was not involved in certain neurotrophin-mediated neuronal differentiation events was that of Borasio et al. [22], who showed that the activity of Ras was neither necessary nor sufficient for the NGF-mediated survival or neuritogenesis of chick sympathetic neurons. More recent studies have indicated that sustained Ras, MEK, or MAPK activities are insufficient for many of the responses of PC12 cells, immortalized hippocampal neurons, and primary neurons to neurotrophic factors. These studies showed that: first, neither sustained MAPK activity nor expression of activated M E K was sufficient to induce neuritogenesis in immortalized rat hippocampal cells [23*]; second, persistent stimulation of the Ras/MAPK pathway was insufficient for platelet-derived growth factor (PDGF)-induced neuritogenesis in PC12 cells expressing mutant P D G F receptors [24]; third, sustained activation of Ras and MAPK was insufficient for survival responses of
Figure 1
!~ Ii!s
KFG
NPQY i
Y Y Y
rranl- I ( ~ Phosphorylation
membrane I domain
I
441~-443
Y49
i Y674 Y751
© 199'7 Current Opinion in Cell Biology
Regulatory sequences of TrkA. The single-letter amino acid code is used. Sequences in human TrkA that are important for NGF-mediated interactions with, and activations of, substrates include amino acids NPQY, which constitute an Shc-association site when Y490 is phosphorylated, and YLDV, a PLC-yl-association site when Y785 is phosphorylated. The Shc-association site is also important for PI3-kinase (PI3-K) activation. Amino acids KFG (441-443) are necessary for the tyrosine phosphorylation of SNT. Tyrosines Y751, Y670, Y674, and Y6"75 are involved in the regulation of Trk kinase activity and transphosphorylation [79]. K538 is an ATP-binding site.
Signal transductionby the neurotrophinreceptorsKaplanand Miller
hippocampal pyramidal neurons [25]; fourth, expression of activated Ras in PC12 cells was not sufficient (although it was necessary) for increases in calcium channel currents [26]; and fifth, inhibition of MEK activity with the MEK inhibitor PD98059 failed to block NGF-dependent survival of rat sympathetic neurons [27,28]. This same inhibitor also failed to block NGF-mediated neurite outgrowth from chick sensory and sympathetic neurons, indicating that MEK and MAPK might be unnecessary for both neuritogenesis and survival responses in peripheral neurons [29]. Even the hypothesis that sustained Trk activity determines the timing and extent of neuronal differentiation is in question, particularly with regard to primary neurons. In post-mitotic rat sympathetic neurons, N G F is much more efficient than NT-3 at inducing survival and neuritogenesis even though both of these factors induce a temporally similar sustained activation of TrkA [30°]. These reports, like those using Trk mutants described above, point to the necessity of other Trk signaling pathways that act in concert with the Ras pathway to induce the multitude of neuronal differentiation responses. T h e y also suggest that specific intracellular targets of Trk, rather than sustained Trk/Ras/MEK/MAPK activities, regulate different aspects of the differentiation process, particularly in primary neurons. What is the nature of these other signaling pathways? They include the PLC-y1 pathway, which mediates NGFinduced peripherin expression in PC12 cells (reviewed in [8°]) and may be required for BDNF-stimulated survival of cerebellar neurons [31], and as yet uncharacterized pathways that regulate the activity of Src, S N T (reviewed in [8°]), and SHP-1 [32]. The adaptor protein Crk may also influence neuritogenesis events via a signaling pathway that functions independently of Shc; expression of a dominant-inhibitory Crk mutant in PC12 cells delays neurite elongation and actin filament organization at the growth cone without affecting She tyrosine phosphorylation [33°]. The PI3-kinase pathway, as described below, is another promising candidate for a novel pathway that controls differentiation responses.
T h e P I 3 - k i n a s e / A k t s i g n a l i n g p a t h w a y as a m o d u l a t o r of n e u r i t o g e n e s i s and cell survival A number of recent studies have implicated PI3-kinase and its downstream substrate, the serine/threonine kinase Akt, in neuronal differentiation and survival. Two initial studies, which largely utilized selective though not specific PI3-kinase inhibitors, suggested a role for PI3-kinase in mediating NGF-induced neuritogenesis [34] or survival [35] in PCI2 cells. In spite of these observations, however, the necessity of PI3-kinase for neuritogenesis and cell survival is still unclear. For example, in PC12 cells, PI3-kinase activity is controlled through the Shc-binding site on Trk [12], which is dispensable for NGF-mediated cell survival and initiation of neurites [8°,14,15]. Thus, PI3-kinase can be considered as a member of a pathway
215
that may collaborate with other signaling pathways to regulate survival and neuritogenesis events. Recent data have indicated that Akt, a serine/threonine kinase that can be activated in vivo and in vitro by phosphatidylinositol 3,4-bisphosphate [36], a phospholipid second messenger generated by PI3-kinase activity, is necessary and sufficient for survival of cultured rat cerebellar neurons [37°°]. In these experiments, overexpression of Akt was sufficient to allow neurons to survive in the absence of survival factors, and dominant-inhibitory Akt blocked much of KCI- and insulin-mediated survival. The requirement for Akt activity in this system suggests that this protein is a key component of the cell survival machinery of cerebellar neurons. In support of this idea, PI3-kinase was recently shown to mediate IGF-1 (insulin-like growth factor-1)-induced survival of primary cerebellar neurons [37°°,38]. It is not yet known whether PI3-kinase or Akt participates in neurotrophin-dependent cell survival of neurons, or how these proteins interact with the cell signaling machinery to inhibit apoptosis.
TrkA as an a p o p t o s i s - i n d u c i n g receptor for neural t u m o r cells Although the "Irks mediate neuritogenesis and survival of neurons, they induce two very different responses in neural tumor cells. In PC12 pheochromocytoma and neuroblastoma cells, TrkA activity inhibits cell growth and neuritogenesis [10,39]. The antimitotic responses of NGF-activated TrkA may be due to an increase in the levels of the cyclin dependent kinase inhibitor p21, at least in PC12 cells [40]. Surprisingly, NGF-stimulated TrkA activity does not induce neuritogenesis or growth cessation of TrkA-transfected medulloblastoma cells, but instead induces cellular apoptosis within 24 hours [41°°]. Similarly, rats injected with C6 glioma cells expressing TrkA survived much longer than rats injected with C6 cells expressing kinase-inactive TrkA [42°°], probably because the cells expressing wild-type TrkA were less invasive and had a greater rate of apoptosis. As TrkA has never been reported to be apoptotic in postmitotic neurons, this finding may reflect a fatal consequence of a conflict between antiproliferative "Irk signaling and the proliferative state of tumor cells. These data are of considerable interest in the neural tumor field, particularly because expression of TrkA and TrkC correlates with tumor prognosis; high expression of TrkA correlates with favorable prognosis in neuroblastoma [4,43-46], while high expression of TrkC correlates with favorable prognosis in medulloblastoma [5,47]. On the basis of these findings, we can propose a potential therapy for neural tumors involving the induction or overexpression of TrkA/C in neural tumors, combined with NGF or NT-3 administration, to induce the death of tumor cells. Interestingly, not all Trk receptors show antimitotic or apoptosis-inducing activity in neural tumor cells. TrkB expression in neuroblastoma stimulates not only neurito-
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Cellregulation
genesis but also cell invasiveness, growth, disaggregation, survival, and resistance to toxicity by the chemotherapeutic drug vinblastine [48°,49,50]. These later responses are all characteristics of metastatic cells. In contrast, TrkA expression in the same neuroblastoma cells (cell line SH-SY5Y) mediates neuritogenesis and the cessation of cell growth, with none of the metastatic cell type effects. As many neuroblastoma cell lines and tumors express the TrkB ligand B D N F [50,51], perhaps a T r k B - B D N F autocrine loop may promote cell growth and survival of neuroblastomas in vivo. These results reflect fundamental signaling differences among Trk receptors expressed in neural tumor cells.
p 7 5 N T R as an a p o p t o s i s - i n d u c i n g receptor for neurons Although the p75 neurotrophin receptor (p75NTR) was the first-isolated neurotrophin receptor, as well as the first-reported member of the p 7 5 N T R / F a s / T N F R I (tumor necrosis factor receptor I) family (reviewed in [6]), definition of its functional role and/or signaling capacity has remained elusive. However, the past year has seen significant advances in our understanding of how this receptor signals, and the biological significance of such signaling. T h e first insights into the biochemical function of the p 7 5 N T R derived from two distinct lines of investigation (reviewed in [6,8°]). In one group of studies, p 7 5 N T R was shown to modify the function of the TrkA receptor. Ligand-binding by p 7 5 N T R led to an increase in high-affinity TrkA binding sites [52], enhanced TrkA autophosphorylation in response to N G F [53-55], and increased selectivity of TrkA for its neurotrophin ligands [56,57]. These findings led to the hypothesis that the function of p75NTR was to enhance TrkA responsiveness and selectivity for N G F [6]. This was thought to occur either by p75NTR 'concentrating' N G F within the TrkA local environment [54] or by p 7 5 N T R forming a molecular complex with TrkA that modified ligand binding [52]. T h e first report of the p 7 5 N T R - / - mouse [58] apparently supported this hypothesis, as this mouse appeared to be an attenuated phenocopy of the TrkA-/- mouse, at least with regard to sensory neurons. A second concurrent line of investigation focused on the possibility that p 7 5 N T R might signal independently of TrkA. T h e first report that it could do so was by Dobrowsky et al. [59], who demonstrated that, in glioma cells that did not express TrkA, binding of N G F to p 7 5 N T R led to an increase in intracellular ceramide levels. Interestingly, in PC12 cells that express TrkA, N G F did not induce a similar increase, although BDNF, which binds p75NTR but not TrkA, did [60°°]. This finding was the first biochemical hint that there was antagonistic cross-talk between these two receptors, with
Trk being able to silence at least some p 7 5 N T R signaling pathways. Biological data on sympathetic neurons also led to speculation that p 7 5 N T R could downregulate some TrkA functions [61].
p75NTR signal transducUon T h e past year has seen confirmation that p 7 5 N T R signals in a similar way to other members of the T N F R I / F a s receptor family. In one recent report, Carter et al. [62 °°] demonstrated that activation of p 7 5 N T R on cells that did not express TrkA led to the activation of the transcription factor NFtcB. In a second report, Casaccia-Bonnefil et al. [63 °°] demonstrated that NGF-mediated activation of p 7 5 N T R in cultured oligodendrocytes, which also do not express TrkA, led to an increase in ceramide levels and an activation of JNK. In neither of these systems were B D N F or NT-3 able to mediate these signaling events. In contrast, in cultured sympathetic neurons which express TrkA but not the B D N F receptor TrkB, BDNF-mediated activation of p 7 5 N T R led to increased phosphorylation of c-jun (R Aloyz, S Bamji, FD Miller, unpublished data), an event mediated by the JNK family. These data support two major conclusions. First, p 7 5 N T R signals via ceramide [59], the JNK family [63°'], and NF~:B [62°°], as do other members of this receptor family [59]. Second, p 7 5 N T R signaling is inhibited by coactivation of a Trk receptor expressed on the same cell: in PC12 cells and sympathetic neurons, only ligands that do not efficiently bind coexpressed TrkA are able to activate p 7 5 N T R signalling [60°°], while in Schwann cells and oligodendrocytes, which express Trk receptors other than TrkA, only N G F (the TrkA ligand) activates p75NTR. These data also raise a number of questions. First, what is the mechanism whereby TrkA activation 'silences' p75NTR-mediated signaling? Although this question has not yet been addressed, we know that it must occur at a level proximal to the receptor itself, as all downstream signaling events that have been measured are abrogated by Trk coactivation. It is possible, at least for TrkA, that the mechanism involves a p 7 5 N T R - T r k A complex, as the evidence that such a molecular association exists is now compelling, as measured in biophysical experiments. For example, p 7 5 N T R colocalizes on cell surfaces with TrkA (but not with TrkB or other receptor tyrosine kinases) [64°], and TrkA will limit the diffusion of p75NTR in the cytoplasmic membrane [65]. Second, can p 7 5 N T R signaling similarly antagonize TrkA signalling? Although there is now good cellular evidence that p 7 5 N T R signaling can 'override' TrkA signaling (see below; Fig. 2), it is, as yet, unclear how this occurs. However, recent data suggest that it may occur in part at the level of the TrkA receptor itself (P Barker, personal communication); data show that BDNF-mediated activation of p 7 5 N T R in PC12 cells reduced TrkA autophosphorylation in experiments utilizing a mutant form of N G F that recognizes TrkA and not p75NTR.
Signal transduction by the neurotrophin receptors Kaplan and Miller
217
Figure 2
(a)
(b)
p75NTR
p75NTR
Ceramide
Ceramide
(c)
1 TrkA
p75NTR
i
~>,
TrkA
Ras
PI3-K SNT
NFK:B~JNK
NF~:B~~JNK
Differentiation Migration Apoptosis
Apoptosis
High p75NTR activity
Survival suppressed
Low Trk activity High p75NTR activity
Apoptosis suppressed
Differentiation
Survival
High Trk and high p75NTR activity O 1997
Current Opinionin Cell Biology
Signaling mechanisms of p75NTR, p75NTR can induce apoptosis, cell migration, and aspects of cell differentiation on its own when highly activated (a), suppress Trk signaling and induce apoptosis when highly activated in the presence of low levels of Trk activity (b), and enhance Trk signaling and Trk-mediated cell differentiation when co-activated with Trk (¢). Ceramide, NF~B and JNK are signaling proteins that are activated by p75NTR in certain cellular environments, while Ras, PI3-kinase (PI3-K) and SNT are signaling proteins that are activated by TrkA.
These findings make it clear that the cellular ratio of p 7 5 N T R to Trk will be a fundamental determinant of the signaling mediated by exposure to a given neurotrophin. This is particularly important for ceils in which the levels of these two receptor classes are independently regulated. For example, on sensory, sympathetic, and basal forebrain cholinergic neurons, p 7 5 N T R levels are dramatically upregulated by N G F exposure [66,67], while, at least in sympathetic neurons, TrkA levels remain constant [30"]. Moreover, on sympathetic neurons, there is partial compartmentalization of these two receptors; p 7 5 N T R is equally distributed over the entirety of the neuron, while TrkA is present at approximately threefold higher density on cell bodies (J Kohn, FD Miller, unpublished data). Thus, the neuronal signaling events mediated in response to N G F may differ in axons versus cell bodies, and as a function of long-term exposure to N G F itself.
p75NTR,a mediatorof neuralcell death What is the cellular outcome of p 7 5 N T R signaling? Recent evidence indicates that, like other members of this family, p 7 5 N T R can signal to mediate apoptosis. T h e first report implicating p 7 5 N T R in apoptosis was by Rabizadeh et al. [68] who studied an immortalized neural cell line. However, in this case, p 7 5 N T R activation was thought to be ligand-independent, and it was unclear what the involvement of Trk receptors was in this system. Three subsequent reports implicated p 7 5 N T R in neuronal apoptosis. In one report selective activation of p 7 5 N T R by
N G F led to increased death of neurons in the developing chick isthmo-optic nucleus [69], while in a second report inhibition of N G F in the developing chick eye using function-blocking antibodies led to decreased cell death in the retina [70"']. In a third report, antisense inhibition of p 7 5 N T R in developing sensory neurons increased cell death at one stage, and decreased death at another [71]. However, in none of these systems was it possible to define the putative role of Trk receptors and/or to gain insights into potential p 7 5 N T R signalling mechanisms. Two more recent studies have addressed these issues. Casaccia-Bonnefil et al. [63"'] used cultured oligodendrocytes that expressed p 7 5 N T R but not TrkA, and demonstrated that N G F exposure led to rapid apoptosis accompanied by increases in ceramide levels and JNK activity. In a second set of experiments, BDNF-mediated activation of p 7 5 N T R in cultured sympathetic neurons was shown to override suboptimal survival signals generated either via TrkA or via KCI to lead to apoptosis (DJ Belliveau, S Bamji, FD Miller, unpublished data). p 7 5 N T R was, however, unable to mediate apoptosis when TrkA was activated to higher levels, supporting the concept that these two receptors both functionally and biochemically antagonize each other in some situations. As in oligodendrocytes, the p75NTR-mediated apoptosis of sympathetic neurons was accompanied by hyperphosphorylation of c-jun. Interestingly, previous studies demonstrated that overexpression of c-jun in sympathetic
218
Cell regulation
neurons is sufficient to cause apoptosis [72°°], and that functional inhibition of c-jun is sufficient to inhibit sympathetic neuron [72•°,73] or PC12 cell [21"] apoptosis induced by neurotrophin withdrawal. It is as yet unclear, however, whether JNK family activation is necessary or sufficient for the induction of neuronal apoptosis by p75NTR. Recent studies of transgenic mice support the conclusion that p75NTR plays a physiological role in regulating neuronal apoptosis. A re-examination of the p75NTR--/mouse demonstrated that there are increased numbers of basal forebrain cholinergic neurons [74"] and less injuryinduced death of sensory and motor neurons (K-F Lee, personal communication). Expression of the intracellular domain of the p75NTR in neurons of transgenic mice demonstrated that this truncated receptor could function as an 'apoptoprotein', or constitutive apoptosis-inducing protein, leading to increased death of developing sensory and sympathetic neurons, and inducible death of mature, injured motor neurons (M Majdan, FD Miller, P Barker, unpublished data). Finally, studies of BDNF-/mice demonstrated an increased number of sympathetic neurons (C Pozniak, C Causing, FD Miller, unpublished data), a result consistent with the in vitro data showing that BDNF-mediated activation of p75NTR led to apoptosis of these neurons. Together with the recent studies from Barde's laboratory [70"] that demonstrated that the functional inhibition of neurotrophin actions decreases cellular apoptosis in the developing retina, these data indicate that neurotrophin-mediated activation of p75NTR plays an essential role in naturally occurring cell death, and is likely to be responsible for some types of neuronal apoptosis that follow injury of the mature nervous system.
with several additional but important functions. Most prominent of these functions is an apparently essential role as an apoptosis mediator in the nervous system, both during naturally occurring cell death and following injury. Important questions remain to be answered about the mechanisms used by p75NTR to induce cell death, and about how p75NTR can function as both a positive effector of Trk-mediated cell differentiation and an apoptosis:inducing protein. It is also clear that, depending upon cellular context and relative expression or activation levels, p75NTR can antagonize Trk-mediated cell survival responses, and, conversely, that Trk can inhibit p75NTR-induced apoptosis. The mechanisms whereby these two receptors influence each other's function is an important area of research for the future. Progress has also been made in understanding the mechanisms whereby the Trk receptors mediate neuronal survival. These survival pathways are only now being identified, with recent data implicating the Ras pathway and that of PI3-kinase and its downstream target, Akt. In this regard, the major questions to be asked are whether all neurons use these same survival pathways, and which signaling proteins are used by Trk to suppress p75NTR-induced apoptosis. One suggestion is that components of the Ras pathway, particularly those regulating the MAPKs, suppress cell death pathways mediated by c-jun kinases, which are activated during both p75NTR- and NGF-withdrawal-induced apoptosis of sympathetic neurons. The next year should yield much information about the signaling mechanisms used by the apoptosis- and survival-inducing neurotrophin receptors.
Acknowledgements We thank P Barker, A Ross, K-I: Lee, and L Greene for sharing data prior to publication.
References and recommended reading It is equally clear, however, that apoptosis is only one cellular outcome of p75NTR-mediated signaling, p75NTR activation in the absence of Trk activation has been shown to cause enhanced cellular migration [75], induction of the gene for neural adhesion molecule NILE/L1 [76], release of dopamine [77], and enhancement of extracellular matrix penetration of human melanoma cells [78]. Moreover, coactivation of Trk and p75NTR in the same cell does not lead to apoptosis (except in cases where Trk activation is suboptimal; Fig. 2), and the ultimate outcome in this situation is likely to be determined by the amount of cross-talk between a given Trk receptor and p75NTR, as well as by the cellular context.
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Nakagawara K, Arima-Nakagawara M, Scavarda NJ, Azar CG, Cantor AB, Brodeur GM: Association between high levels of
The significant breakthrough in the past year in the study of neurotrophin signal transduction has been the recognition of the role of the p75NTR as an apoptosisinducing receptor for neural cells and neurons, p75NTR has evolved in our view from a receptor with a limited role as a positive modifier of Trk activity to a protein
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