©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Naturally Occurring Tyrosine Kinase Inserts Block High Affinity Binding of Phospholipase C and Shc to TrkC and Neurotrophin-3 Signaling (*)

(Received for publication, April 17, 1995; and in revised form, June 5, 1995)

Michelle Guiton (§) Frank J. Gunn-Moore David J. Glass (1) David R. Geis (1) George D. Yancopoulos (1) Jeremy M. Tavaré(¶)

From the Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom and Regeneron Pharmaceuticals Inc., Tarrytown, New York 10591-6707

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Neurotrophin-3 binds to the receptor tyrosine kinase, TrkC. Several naturally occurring splice variants of TrkC exist including those with 14- and 39-amino acid inserts within the tyrosine kinase homology region. When expressed in fibroblasts, full-length TrkC, but not the kinase insert variants, mediated neurotrophin-3-stimulated cell proliferation. We investigated the molecular basis of this signaling defect. The kinase inserts blocked the ability of TrkC to mediate neurotrophin-3 stimulated c-myc and c-fos transcription and activation of the AP-1 transcriptional complex. In cells expressing full-length TrkC, neurotrophin-3 promoted a sustained activation of mitogen-activated protein kinase; TrkC containing kinase inserts only mediated transient activation of mitogen-activated protein kinase. The kinase inserts specifically blocked neurotrophin-3-stimulated autophosphorylation of the phospholipase C binding site on TrkC (tyrosine 789) resulting in a severe reduction in phospholipase C association with TrkC and its tyrosine phosphorylation. Neurotrophin-3-stimulated phosphorylation of the Shc binding site (tyrosine 485) on TrkC, and tyrosine phosphorylation of Shc itself, was unaffected by the kinase inserts; however, the kinase inserts blocked high affinity Shc association with TrkC. It is proposed that the lack of high affinity binding of Shc and/or phospholipase C to the TrkC kinase insert variants may be responsible for the inability of these variants to bring about a full biological response in fibroblasts.


INTRODUCTION

The transmembrane protein-tyrosine kinase TrkC is the high affinity receptor for neurotrophin-3 (NT-3), (^1)a member of the neurotrophic factor family of related polypeptides central to the development and maintenance of the mammalian nervous system. This family also includes nerve growth factor and brain-derived neurotrophic factor, which bind to the TrkA and TrkB tyrosine kinases, respectively (for reviews see Chao(1992), Meakin and Shooter(1992), and Barbacid(1994)).

The rat TrkC locus encodes a number of differentially spliced forms. These include forms that lack the cytoplasmic tyrosine kinase domain and others that possess inserts of variable length (14, 25, and 39 amino acids) within the tyrosine kinase domain (Lamballe et al., 1993; Tsoulfas et al., 1993; Valenzuela et al., 1993). The expression of these variants and wild-type TrkC is apparently restricted to the nervous system, where they are widely expressed. When expressed in PC12 cells or fibroblasts, TrkC can mediate NT-3-stimulated neurite outgrowth and cell proliferation, respectively. However, variants of TrkC with the kinase inserts appear to be essentially incapable of mediating these biological responses (Lamballe et al., 1993; Tsoulfas et al., 1993; Valenzuela et al., 1993).

The kinase inserts of TrkC occur between subdomains VII and VIII of the tyrosine kinase homology region (as defined by Hanks et al., 1988) and immediately C-terminal to tyrosines 674, 678, and 679. In TrkB the equivalent tyrosines (residues 670, 674, and 675) are major ligand-stimulated autophosphorylation sites (Guiton et al., 1994; Middlemas et al., 1994). How such kinase inserts interfere in normal TrkC signaling is not clear. The kinase inserts do not appear to affect tyrosine autophosphorylation of TrkC, as assessed by Western blotting with anti-phosphotyrosine antibodies but block tyrosine phosphorylation of phospholipase C and phosphatidylinositol 3-OH kinase (PtdIns 3-kinase) activation through an undefined mechanism (Lamballe et al., 1993; Valenzuela et al., 1993). Phospholipase C (PLC) has been proposed to directly bind to phosphorylated Trk family members through its Src homology 2 (SH2) domain (Lamballe et al., 1993; Obermeier et al., 1993a). The mechanism by which the Trk family activates PtdIns 3-kinase is more controversial. It has been proposed that PtdIns 3-kinase binds directly to the receptor (Soltoff et al., 1992; Obermeier et al., 1993a), whereas others have failed to detect such an association (Ohmichi et al., 1992).

The identification of SH2 domains has resulted in considerable advances in our understanding of the early events initiated after the binding of neurotrophic factors to the Trk family. In addition to the autophosphorylation of tyrosines 670, 674, and 675 (numbering based on the amino acid sequence of TrkB) (Guiton et al., 1994; Middlemas et al., 1994), it appears that two other tyrosines are involved in direct binding of effectors to Trk family members via SH2 domains. The most C-terminal tyrosine (residue 785 in TrkB) has been indirectly demonstrated to be autophosphorylated and forms a binding site for the SH2 domain of phospholipase C (Middlemas et al., 1994). Shc binding, via its PTB (phosphotyrosine binding) domain, has been reported to be blocked by a tyrosine 490 for phenylalanine substitution within a chimeric receptor comprising the Trk tyrosine kinase domain (from the gag-Trk oncogene) fused to the EGF receptor ligand binding domain (EGFR-Trk) (Obermeier et al., 1993b; Dikic et al., 1995). However, this tyrosine has yet to be directly shown to constitute a bona fide autophosphorylation site. Mutagenesis of tyrosine 751, which lies within a putative PtdIns 3-kinase binding consensus sequence (YXXM), was reported to block PtdIns 3-kinase association with the EGFR-Trk tyrosine kinase domain (Obermeier et al., 1993b). By phosphopeptide mapping, however, we have been unable to detect the phosphorylation of tyrosine 751 in TrkB, suggesting that PtdIns 3-kinase activation occurs via a route that does not involve its direct association with Trk (Guiton et al., 1994).

In the current study we directly demonstrate that the putative phospholipase C and Shc binding sites on TrkB (tyrosines 484 and 785, respectively) are indeed autophosphorylated in TrkB. We further demonstrate that these sites are also autophosphorylated in TrkC (tyrosines 485 and 789, respectively), in addition to the putative regulatory tyrosines 674, 678, and 679. We also demonstrate that the kinase inserts specifically block the autophosphorylation of tyrosine 785 and that this prevents phospholipase C association with TrkC and its tyrosine phosphorylation. The phosphorylation of other sites on TrkC is unaffected, although high affinity Shc binding, but not tyrosine phosphorylation, is also blocked by the kinase inserts. These data help provide a molecular explanation for the defect in TrkC signaling induced by kinase inserts and define a novel role for such sequences in modulating receptor tyrosine kinase signaling.


EXPERIMENTAL PROCEDURES

Materials

[P]Orthophosphate and ECL reagents were from Amersham International (Amersham, United Kingdom). Polyclonal anti-Trk C-terminal peptide antibody (immunoreactive toward TrkA, TrkB, and TrkC) was as described in Guiton et al.(1994). Antisera toward phosphotyrosine (4G10), Shc, and PLC were from Upstate Biotechnology Inc. (UBI, Lake Placid, NY). Nitrocellulose and Immobilon-P were from Schleicher & Schuell and Millipore Corp., respectively. Sequencing grade trypsin was from Boehringer Mannheim (Lewes, UK). Unless otherwise stated all biochemicals were supplied by Sigma, and general purpose laboratory reagents were of analytical grade and from BDH (Poole, UK).

Growth Assays

Stable transfections of a laboratory isolate of NIH 3T3 cells, which we term MG87, were performed as described previously (Glass et al., 1991). Growth assays were performed as described in Ip et al.(1993), with the number of viable cells being determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide binding assay.

Northern Analysis and AP-1 Complex Assays

Northern analysis for the induction of c-myc and c-fos transcripts was performed as described in Squinto et al.(1991) and Ip et al.(1993). AP-1 complex activity was measured in fibroblasts transiently transfected with the plasmid pCol.Luc (collagenase promoter upstream of luciferase cDNA) as described in Guiton et al.(1994) after incubation of cells for 24 h with 50 ng/ml NT-3.

Mitogen-activated Protein (MAP) Kinase Assays

Transfected fibroblasts were grown to approximately 80% confluence in 60-mm dishes. Cells were serum-starved for 2 h prior to incubation with NT-3 for the times and at the concentrations indicated in the figure legends. Cells were washed and extracted, and MAP kinase activity in the cell lysate was assayed essentially as described by Young et al.(1994) but with 200 µM T669 peptide (KRELVEPLTPSGEAPNQALLR) as substrate.

Tyrosine Phosphorylation Assays

MG87 fibroblasts stably transfected with the various TrkC-expressing plasmids (Valenzuela et al., 1993) in the pCMX vector (Davis et al., 1991) were grown to confluence and serum-starved for 3 h in Dulbecco's modified Eagle's medium prior to the indicated treatments for 5 min at 37 °C. The cells were lysed in phosphate-buffered saline containing 1% Nonidet P-40 and 1 mM phenylmethylsulfonyl fluoride, 0.14 units/ml aprotinin, 1 mM EDTA and 1 mM Na(3)VO(4). The lysates were immunoprecipitated as indicated in the text and Western blotted with anti-phosphotyrosine antiserum, 4G10. Following incubation of the blots with horseradish peroxidase-conjugated goat anti-rabbit antibodies (Caltag), bands were visualized by ECL (Amersham).

Site-directed Mutagenesis

In vitro mutagenesis was performed on plasmid pBSK-TrkB (rat TrkB sequence cloned into Bluescript SK (Strategene)) as outlined in ``Muta-Gene Phagemid In Vitro Mutagenesis Version 2'' (Bio-Rad) with the following primers: 5`-AACCCCCAGTTCTTCGGTATC-3` for modifying tyrosine 484 to phenylalanine (Y484F) and 5`-TCGCCCGTCTTCCTGGACATC-3` for modifying tyrosine 785 for phenylalanine (Y785F). Mutant and wild-type receptors were subcloned into the mammalian expression vector pECE (Ellis et al., 1986), and the entire subcloned region containing the mutated residues was resequenced to confirm the specificity of the mutation.

Transient Transfection of COS Cells and Analysis of TrkB Receptor Phosphorylation Sites

COS7 cells were seeded at 4 10^5 cells/60-mm dish and transfected 24 h later with 5 µg of DNA using DEAE-dextran as described by Gluzman(1981). Experiments were performed 48 h after transfection. Transiently transfected COS cells, or MG87 cells expressing the TrkC variants, were metabolically labeled with [P]orthophosphate and treated with or without NT-3 (100 ng/ml) for 5 min prior to extraction in 0.5 ml of ice-cold lysis buffer exactly as described in Guiton et al. (1994). P-Labeled TrkB or TrkC was immunopurified using the anti-Trk antibody coupled to protein A-Sepharose, and phosphopeptide mapping or phosphoamino acid analysis was performed using a two-dimensional thin-layer chromatography system described by Guiton et al.(1994).


RESULTS

Kinase Inserts Block the Ability of TrkC to Mediate NT-3-stimulated Cell Proliferation in MG87 Cells

TrkC, TrkC.ki14, and TrkC.ki39 were stably expressed at approximately equal receptor numbers in a fibroblast cell line, MG87, which lacks endogenous Trk family members. NT-3 promoted a pronounced increase in the proliferation of MG87 cells expressing full-length TrkC (EC 5-10 ng/ml NT-3; Fig. 1). In contrast, NT-3 had little or no effect on the proliferation of MG87 cells expressing either TrkC.ki14 or TrkC.ki39 (Fig. 1). Since previous studies had shown the kinase inserts to have little apparent effect on NT-3-stimulated TrkC tyrosine autophosphorylation (as judged by Western blotting with anti-phosphotyrosine antibodies), we further investigated the molecular basis of the effect of the kinase inserts on TrkC signaling.


Figure 1: NT-3-stimulated proliferation of fibroblasts expressing TrkC variants. MG87 cells expressing full-length TrkC (black square) or the TrkC(ki14) (⊡) or TrkC(ki39) () variants were incubated in the presence of the indicated concentrations of NT-3, and the increase in cell number was measured by monitoring 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide binding as described under ``Experimental Procedures.''



Effects of the Kinase Inserts on TrkC Signaling to the MAP Kinase Pathway and Regulation of Gene Transcription

We first assessed the effect of the kinase inserts on NT-3-stimulated transcription of the early response genes c-myc and c-fos, as well as the stimulation of AP-1 complex activity (a heterodimer between the c-fos and c-jun transcription factors). All these events may be critical to the proliferative response of fibroblasts.

Northern analysis of c-myc and c-fos transcripts demonstrates that NT-3 has a rapid and sustained stimulatory effect on their transcription in cells expressing full-length TrkC (Fig. 2). Cells expressing TrkC.ki14 or TrkC.ki39 mediated only a small transient burst of c-myc induction and very little induction of c-fos transcription in response to NT-3 (Fig. 2). NT-3 also induced a pronounced activation of the AP-1 complex in cells expressing TrkC, which was totally blocked upon introduction of the kinase inserts (Table 1).


Figure 2: Induction of c-myc and c-fos transcripts by NT-3 in MG87 cells. MG87 cells expressing full-length TrkC or the TrkC(ki14) or TrkC(ki39) variants were incubated in the absence or presence of NT-3 (50 ng/ml) for the times indicated. The expression of the c-fos and c-myc transcripts was determined by Northern analysis using probes specific for these early response genes, as described under ``Experimental Procedures.''





Both p62, which is involved in the induction of c-fos mRNA, and the AP-1 complex have been proposed to be directly regulated by phosphorylation by the MAP kinases Erk1 and Erk2 (Marais et al., 1993; Pulverer et al., 1991). These kinases are activated as the result of a Raf-1 MAP kinase kinase MAP kinase cascade (Davis, 1993). Thus we assessed the ability of NT-3 to activate MAP kinase as a measure of the ability of the TrkC variants to utilize this pathway. MAP kinase was assayed in crude cell lysates using a peptide based on the MAP kinase phosphorylation site in the EGF receptor (EGFR669 peptide).

As shown in Fig. 3, full-length TrkC promoted a rapid activation of MAP kinase, which peaked at 10 min and was sustained at an elevated level for at least 60 min. In contrast, TrkC.ki14 and TrkC.ki39 mediated only a transient NT3-stimulated MAP kinase activation, although activity was significantly above basal level at 60 min (Fig. 3). MAP kinase was not activated by NT-3 in the parental fibroblasts (Fig. 3).


Figure 3: Activation of MAP kinase by NT-3 in cells expressing TrkC variants. Parental MG87 cells (MG87) (black square) or cells expressing full-length TrkC (diamond, filled), TrkC(ki14) (), or TrkC(ki39) () variants were incubated with NT-3 (50 ng/ml) for the indicated times. The cells were lysed, and the activity of MAP kinase was assayed in the resulting extract using the EGFR669 peptide. The results are representative of three separate experiments.



Engagement with, and Phosphorylation of, Shc and Phospholipase C by TrkC

Trk activation leads to the association of a number of cellular effector molecules with the receptor through SH2 domain interactions. This includes the association and phosphorylation of Shc and PLC. Shc, when tyrosine-phosphorylated, forms a tight complex with Grb2 and Sos. Sos then catalyzes nucleotide exchange and thus activation of Ras. Active Ras then initiates the MAP kinase cascade (Davis, 1993). Tyrosine-phosphorylated PLC, on the other hand, should theoretically promote a rise in cytosolic Ca and diacylglycerol through enhancement of phosphatidylinositol 4,5-bisphosphate turnover. The increase in diacylglycerol could lead to the activation of protein kinase C and consequent activation of c-Raf through its phosphorylation (Sozeri et al., 1992). Thus we next investigated the ability of the various forms of TrkC to bind and phosphorylate Shc and PLC.

Cells were incubated in the presence or absence of NT-3 and rapidly extracted, and TrkC, PLC, or Shc was immunoisolated with specific antisera. In agreement with previous studies, antiphosphotyrosine antibody blotting of anti-Trk precipitates revealed that all three forms of TrkC were tyrosine-phosphorylated to equivalent extents in response to NT-3 (Fig. 4A). By contrast, PLC phosphorylation was only stimulated in the presence of NT-3 in the cells expressing full-length TrkC (Fig. 4B). Furthermore, the co-precipitation of TrkC with PLC was dramatically inhibited upon introduction of the kinase inserts. This presumably reflects the high affinity association of full-length TrkC with PLC via its SH2 domain, and the decrease in association of the TrkC variants with PLC is probably responsible for the lack of PLC tyrosine phosphorylation.


Figure 4: Association of TrkC with PLC and Shc. Parental MG87 cells (MG) or cells expressing full-length TrkC, TrkC(ki14), or TrkC(ki39) were incubated in the absence or presence of NT-3 (50 ng/ml) and extracted, and TrkC (panelA), PLC (panelB), or Shc (panelC) was immunoprecipitated with specific antisera. The precipitates were separated by SDS-polyacrylamide gel electrophoresis and Western blotted with the antisera indicated to the left of each panel followed by ECL detection. The relative migrations of TrkC, PLC, and Shc are indicated.



Anti-Shc precipitates showed that all three forms of TrkC were capable of mediating a pronounced NT-3-dependent tyrosine phosphorylation of Shc (Fig. 4C). Interestingly, only full-length TrkC (and not TrkC.ki14 or TrkC.ki39) co-precipitated with Shc (Fig. 4C).

Are the Shc and PLC Binding Sites Phosphorylated in Trk?

The inability of the variant forms of TrkC to bind Shc and PLC could be due to the lack of phosphorylation of their binding sites on TrkC. A small reduction in phosphorylation of TrkC might not be detectable by anti-phosphotyrosine antibody blotting (Fig. 4A), so we analyzed the individual tyrosine autophosphorylation sites on TrkC.

In a previous study we directly identified three of the five major brain-derived neurotrophic factor-stimulated tyrosine autophosphorylation sites in TrkB as tyrosines 670, 674, and 675 (Guiton et al., 1994). Our previous phosphopeptide analysis suggested the presence of only two further unidentified phosphotyrosine-containing peptides, and other work has implied that tyrosines 484 and 785 constituted binding sites for the SH2 domains of Shc and PLC, respectively (see Introduction). Therefore, we began by substituting these residues for phenylalanines.

The mutant TrkB constructs were transiently expressed in COS cells for in vivoP labeling. This cell system allows high levels of receptor expression, consequently easing analysis by phosphopeptide mapping. It should be noted that TrkB exhibits ligand-independent and constitutive phosphorylation when overexpressed in COS cells, probably as a result of ligand-independent receptor dimerization and hence kinase activation (see Guiton et al., 1994).

Consistent with our previous studies, the maps of P-labeled wild-type TrkB (Fig. 5B) revealed five phosphotyrosine-containing peptides. Peptides T4, T5, and T6 are mono-, bis-, and trisphosphorylated derivatives of the tryptic peptide DVYSTDYYR containing tyrosines 670, 674, and 675 (see Fig. 5A for a key and Guiton et al.(1994) for identification and further discussion). Substitution of tyrosine 785 for phenylalanine resulted in the disappearance of peptide T1 (Fig. 5C). In COS cells, phosphopeptide T3 was phosphorylated rather poorly and, in some experiments, was found close to another unidentified minor phosphopeptide. However, peptide T3 (the upper peptide of the doublet) (i) was absent from a mutant TrkB where tyrosine 484 was substituted with phenylalanine (Fig. 5D) and (ii) co-migrated with a synthetic tryptic phosphopeptide containing the sequence surrounding tyrosine 484 (Guiton et al., 1994). It should be noted that the migration of these phosphopeptides is consistent with their approximate predicted charges at pH 3.5 of -1.0 (for peptide T1; sequence ASPVY(P)LDILG) and 0 (for peptide T3; sequence IPVIENPQY(P)FGITNSQLK) (Boyle et al., 1991). Note that due to electroendosmosis, the peptides have a tendency to migrate with a slightly greater net positive charge than predicted (see Dickens and Tavaré, 1992).


Figure 5: Analysis of TrkB phosphorylation sites by two-dimensional phosphopeptide mapping. COS cells were transfected with wild-type TrkB (panelB) or TrkB mutants with Y785F (panelC) or Y484F (panelD) substitutions. The cells were labeled with [P]P(i) and radiolabeled TrkB was immunoisolated and digested with trypsin as described under ``Experimental Procedures.'' The tryptic phosphopeptides were separated by two-dimensional thin layer chromatography, and labeled peptides were detected using a PhosphorImager. PanelA shows a key to the identification of the phosphopeptides.



Analysis of the Sites of NT-3-stimulated Tyrosine Phosphorylation in TrkC

We next examined the phosphopeptide maps obtained from full-length TrkC in MG87 cells. The pattern of phosphopeptides generated from NT-3-stimulated P-labeled TrkC (see Fig. 6B) was almost identical to that found in TrkB expressed in COS cells (Fig. 5B) and MG86 cells (see Guiton et al., 1994). NT-3 stimulated the phosphorylation of all phosphopeptides detected (Fig. 6).


Figure 6: Analysis of the sites of NT-3-stimulated TrkC phosphorylation. MG87 cells expressing full-length TrkC were incubated with [P]P(i) and then in the absence (panelA) or presence (panelB) of NT-3 (100 ng/ml) for 5 min. TrkC was immunoisolated, and tryptic phosphopeptide mapping was performed as described in the legend to Fig. 5.



Tryptic digestion of TrkC generated three peptides (T4-T6), which exactly co-migrated with the equivalent peptides of TrkB (Fig. 5B). This suggests that T4, T5, and T6 correspond to the mono-, bis-, and trisphosphorylated forms of the peptide DVYSTDYYR derived from the putative ``regulatory'' domain of the kinase, which is completely conserved between TrkB and TrkC. In TrkC this peptide contains the tyrosine phosphorylation sites 674, 678, and 679.

Peptide T1 of TrkC co-migrated with peptide T1 of TrkB. The sequence of the predicted tryptic peptide (ATPIY(P)LDILG) from TrkC has the same charge and hydrophobicity as peptide T1 of TrkB (ASPVY(P)LDILG). This peptide, therefore, corresponds to the PLC binding site of TrkC (tyrosine 789; by analogy with tyrosine 785 of TrkB) (Obermeier et al., 1993a; Middlemas et al., 1994).

Peptide T3 of TrkC co-migrates with peptide T3 of TrkB, suggesting that they too are related. In TrkB this peptide has the sequence IPVIENPQY(P)FGITNSQLK. In TrkC the peptide would be predicted to be shorter (IPVIENPQY(P)FR) but carry the same charge at pH 3.5 and the same hydrophobicity as its homologue in TrkB. Thus peptide T3 of TrkC is the presumptive Shc binding site containing tyrosine 485.

The only major difference between the pattern of TrkB and TrkC phosphorylation in fibroblasts is that peptide T3 of TrkC is consistently and extensively labeled, while its phosphorylation in TrkB is highly variable. Indeed under most circumstances we see very little P incorporation into peptide T3 in TrkB (see Fig. 5B and further discussion in Guiton et al. (1994)).

Phosphopeptide Mapping of TrkC Variants

We next investigated whether the lack of PLC tyrosine phosphorylation, as well as Shc and PLC association with the TrkC variants, was due to a reduction in phosphorylation of their binding sites on TrkC (tyrosines 485 and 789, respectively). The three forms of NT-3-stimulated TrkC were subjected to phosphopeptide mapping. Peptide T1, the PLC binding site, was extensively phosphorylated in full-length TrkC (Fig. 7A), but this phosphorylation was dramatically reduced upon introduction of the kinase inserts (see upperarrow in Fig. 7, B and C). Peptide T3, the Shc binding site, was phosphorylated to an equivalent extent in all three forms of TrkC. The only other significant difference was a reduction in trisphosphorylation of tyrosines 674, 678, and 679 in TrkC.ki39 (i.e. peptide T6) and the appearance, in the same variant, of a new phosphopeptide migrating close to the origin (Fig. 7C).


Figure 7: Effects of kinase inserts on the pattern of TrkC tryptic phosphopeptides. MG87 cells expressing full-length TrkC (panelA), TrkC(ki14) (panelB) or TrkC(ki39) (panelC) were labeled with [P]P(i), and phosphopeptide mapping was performed on the immunoisolated TrkC as described in the legend to Fig. 6. The downwardarrow highlights the phosphopeptide containing tyrosine 789, which is the PLC binding site. The leftwardarrow highlights a novel phosphopeptide only observed with the TrkC(ki39) variant.




DISCUSSION

In this study we show that the inability of the kinase insert variants of TrkC to mediate NT-3-stimulated cell proliferation in fibroblasts (Fig. 1) is accompanied by a reduced ability of the variants to mediate NT-3-stimulated c-fos and c-myc induction and activation of the AP-1 transcriptional complex ( Fig. 2and Table 1). Such changes in early gene responses are believed to be central to the proliferative response of fibroblasts (Angel and Karin, 1991).

The induction of c-fos and the activation of the AP-1 complex by growth factors and neurotrophins are thought be mediated by MAP kinase (see Hill and Treisman(1995) for review). The fact that only full-length TrkC promotes sustained MAP kinase activation, while TrkC.ki14 and TrkC.ki39 promote a short transient burst of MAP kinase activity (Fig. 3), is consistent with this hypothesis. Interestingly, the induction of c-myc by full-length TrkC is sustained over 45 min, whereas that induced by TrkC.ki14 and TrkC.ki39 is transient. The mechanism by which neurotrophins and growth factors activate the c-myc promoter is not well understood, but our data could also be interpreted as suggesting an involvement of MAP kinase. Importantly, however, the fact that the kinase insert variants of TrkC can mediate a quite significant initial phase of c-myc induction and MAP kinase activation suggests that these receptor variants are capable of a degree of acute signaling.

It has been proposed that the neurite outgrowth response of PC12 cells to ligand stimulation requires sustained MAP kinase activation (see Marshall(1995) for review). The fact that only the full-length TrkC mediates sustained MAP kinase activation in fibroblasts (Fig. 3) is entirely consistent with this hypothesis, since only this form of TrkC can mediate NT-3-stimulated neurite outgrowth of PC12 cells (Lamballe et al., 1993; Tsoulfas et al., 1993; Valenzuela et al., 1993). However, this can only be rigorously tested once we have examined MAP kinase activation profiles in PC12 cells stably transfected with the various TrkC isoforms.

We further investigated the molecular basis for the reduced ability of the TrkC variants to promote sustained MAP kinase activation in MG87 cells. Full-length TrkC is phosphorylated in response to NT-3 on five tyrosine residues (tyrosines 485, 674, 678, 679, and 789). Tyrosines 674, 678, and 679 of TrkC are homologous to tyrosines 670, 674, and 675 of TrkB, and tyrosines 1158, 1162, and 1163 of the insulin receptor, all of which are phosphorylated in response to ligand binding (Guiton et al., 1994; Middlemas et al., 1994; Tavaréet al., 1988; Tornqvist et al., 1988; White et al., 1988). Indeed in both TrkB and the insulin receptor, these tyrosines play a critical role in intracellular signaling (Ellis et al., 1986; Guiton et al., 1994).

The TrkC kinase inserts are spliced into the kinase domain just two amino acids C-terminal to the tyrosine 679 autophosphorylation site of TrkC. Since this region would lie close to the peptide binding pocket of the tyrosine kinase domain of TrkC (as predicted from the crystal structure of the highly homologous insulin receptor tyrosine kinase domain) (Hubbard et al., 1994) it might be expected that the inserts would have profound effects on tyrosine autophosphorylation of TrkC. However, this appears not to be the case. The 14-amino acid kinase insert has no apparent effect on the autophosphorylation of tyrosine 674, 678, or 679. The 39-amino acid insert causes a small reduction in the trisphosphorylation of tyrosines 674, 678, and 679 (peptide T6) such that peptides containing these tyrosines are predominantly bisphosphorylated (peptide T5; see Fig. 7).

Interestingly, the introduction of the 39-amino acid insert into TrkC results in the appearance of a new phosphopeptide (in Fig. 7C, arrow). While we have not confirmed that this peptide contains phosphotyrosine (due to lack of sufficient P incorporation), the 39-amino acid insert contains a single tyrosine residue at position 4, which could serve as a phosphate acceptor. While this tyrosine does not appear to lie in a putative SH2 domain-binding consensus sequence, the possibility that it could engage a downstream effector molecule cannot be discounted.

Tyrosine 485 of TrkC (tyrosine 484 in TrkB) plays an important role in the binding of the PTB domain of Shc to Trk family members, as first shown for an EGF receptor-Trk chimera (Obermeier et al., 1993b; Dikic et al., 1995). However, the introduction of a kinase insert into TrkC has no apparent effect on the autophosphorylation of this residue (Fig. 7). In contrast, the ability of Shc to bind to this phosphotyrosine is disrupted, as assessed by co-precipitation of Shc with TrkC (Fig. 4C). Despite this, however, Shc still serves as an efficient substrate for the TrkC tyrosine kinase in the presence of kinase inserts (Fig. 4C).

Interestingly, the insulin receptor possesses a tyrosine phosphorylation site (tyrosine 972), which is in a topologically equivalent position to tyrosine 485 of TrkC and is related in the surrounding sequence (i.e. NPEY in the insulin receptor and NPQY in TrkC). The insulin receptor does not form a high affinity complex with Shc, but it is perfectly capable of promoting Shc phosphorylation in intact cells stimulated with insulin (Pronk et al., 1993). Insulin generally only promotes transient MAP kinase activation in a number of cell types (e.g. see Dickens et al.(1992), Young et al.(1994), and Haystead et al.(1994)). Since only full-length TrkC is capable of forming a high affinity Shc complex, sustained MAP kinase activation may only occur if such a complex can form. A TrkC-Shc complex would engage the Grb2-Sos complex in the vicinity of the membrane and thus juxtapose with its substrate, Ras. On the other hand, insulin receptors and TrkC.ki14 or TrkC.ki39, which do not form a high affinity complex with Shc, only promote transient MAP kinase activation perhaps because the Grb2-Sos is not localized at the plasma membrane close to Ras. This hypothesis will require identification of the subcellular localization of Shc-Grb2-Sos complexes after ligand stimulation.

The reasons for the inability of Shc to form a high affinity complex with tyrosine 485 in the kinase insert variants is not known but may simply be explained by steric hindrance due to the presence of the bulky polypeptide insert close to the kinase active site.

Neither TrkC.ki14 or TrkC.ki39 can mediate significant NT-3-stimulated PLC tyrosine phosphorylation unlike full-length TrkC (Fig. 4B), confirming previous observations (Lamballe et al., 1993). Here, however, we demonstrate a possible molecular explanation for this defect. The kinase inserts very specifically block the phosphorylation of the C-terminal PLC binding site (tyrosine 789; peptide T1 in Fig. 7). This is likely to prevent the formation of a high affinity TrkC-PLC complex and thus decrease the affinity of PLC as a substrate for phosphorylation. Consistent with this hypothesis, very small amounts of TrkC.ki14 and TrkC.ki39 still co-precipitated with PLC (Fig. 4B), and peptide mapping of these variants did consistently reveal a very low level of phosphorylation of the PLC binding site on TrkC (tyrosine 789, peptide T1; Fig. 7, B and C). However, the additional possibility that the kinase inserts sterically hinder PLC binding cannot be excluded.

The role of PLC in neurotrophic factor signaling is rather obscure. Mutagenesis of this tyrosine to phenylalanine has no affect on the ability of Trk family members to promote ligand-dependent neurite outgrowth in PC12 cells (Loeb et al., 1994) or induction of AP-1 complex activity in Chinese hamster ovary cells, (^2)although it does prevent the induction of peripherin intermediate filament mRNA (Loeb et al., 1994). We have been unable to demonstrate any changes in cytosolic Ca in Fura-loaded MG87 cells expressing full-length TrkC and incubated with NT-3. (^3)Furthermore, there are only a few reports that nerve growth factor activates protein kinase C (e.g. see Heasley and Johnson (1989)). Despite this, however, we cannot rule out the possibility that the lack of PLC association and phosphorylation leads in part to the failure of TrkC.ki14 and TrkC.ki39 to promote sustained MAP kinase activation in response to NT-3. It is well established that phorbol esters, acting through protein kinase C (and presumably c-raf phosphorylation), can activate the MAP kinase cascade (Davis, 1993).

In conclusion, the kinase inserts block the phosphorylation of the PLC binding site on TrkC but not that of the Shc binding site. Despite this, the high affinity association of both Shc and PLC with TrkC is prevented by kinase inserts, and this leads, by some as yet unidentified mechanism, to the inability of TrkC.ki14 and TrkC.ki39 to mediate sustained MAP kinase activation. This, in turn, may prevent NT-3-stimulated induction of early genes and the AP-1 complex and thus to the ultimate inability of the kinase insert variants to stimulate cell proliferation in fibroblasts. Similar events may also be responsible for the inability of TrkC.ki14 and TrkC.ki39 to promote NT-3-stimulated neurite outgrowth in PC12 cells.

Kinase inserts are characteristic of several receptor tyrosine kinases (e.g. platelet-derived growth factor receptors) and usually possess tyrosine autophosphorylation sites responsible for engaging the SH2 domains of effectors such as PtdIns 3-kinase, Grb2, Nck, and GAP (for a review see Panayotou and Waterfield(1993)). The kinase inserts of TrkC define a novel set of inserts that disrupt rather than mediate biological signaling. In vivo TrkC.ki14 and TrkC.ki39 may still be capable of a more subtle level of signaling than full-length TrkC. For example, it is possible that the splice variants could mediate NT-3-dependent neurite outgrowth and neuronal survival in concert with other ligands that activate PLC-dependent pathways or ligands that further potentiate Shc-dependent pathways. This possibility requires further investigation.


FOOTNOTES

*
This work was supported in part by the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Funded by a Medical Research Council studentship.

A British Diabetic Association Senior Research Fellow. To whom correspondence should be addressed. Tel.: 44-117-928-8273; Fax: 44-117-928-8274; tavare{at}bsa.bristol.ac.uk.

(^1)
The abbreviations used are: NT-3, neurotrophin-3; EGF, epidermal growth factor; EGFR, EGF receptor; PtdIns 3-kinase, phosphatidylinositol 3-OH kinase; PLC, phospholipase C; MAP kinase, mitogen-activated protein kinase; SH2, Src homology 2.

(^2)
F. J. Gunn-Moore and J. M. Tavaré, unpublished observations.

(^3)
M. Guiton, M. Conner, and J. M. Tavaré, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to Professor P. Cohen for advice on MAP kinase assays.


REFERENCES

  1. Angel, P., and Karin, M. (1991) Biochim. Biophys. Acta 1072,129-157 [CrossRef][Medline] [Order article via Infotrieve]
  2. Barbacid, M. (1994) J. Neurobiol. 25,1386-1403 [Medline] [Order article via Infotrieve]
  3. Boyle, W. J., van der Geer, P., and Hunter, T. (1991) Methods Enzymol. 201,110-149 [Medline] [Order article via Infotrieve]
  4. Chao, M. V. (1992) Neuron 9,583-593 [Medline] [Order article via Infotrieve]
  5. Davis, R. J. (1993) J. Biol. Chem. 268,14553-14556 [Free Full Text]
  6. Davis, S., Aldrich, T. H., Valenzuela, D. M., Wong, V., Furth, M. E., Squinto, S. P., and Yancopoulos, G. D. (1991) Science 253,59-63 [Medline] [Order article via Infotrieve]
  7. Dickens, M., and Tavaré, J. M. (1992) Biochem. Biophys. Res. Commun. 186,244-250 [Medline] [Order article via Infotrieve]
  8. Dickens, M., Chin J. E., Roth R. A., Ellis, L., Denton, R. M., and Tavaré, J. M. (1992) Biochem. J. 287,201-209 [Medline] [Order article via Infotrieve]
  9. Dikic, I., Batzer, A. G., Blaikie, P., Obermeier, A., Ullr, A., Schlessinger, J., and Margolis, B. (1995) J. Biol. Chem. 270,15125-15129 [Abstract/Free Full Text]
  10. Ellis, L., Clauser, E., Morgan, D. O., Edery, M., Roth, R. A., and Rutter, W. J. (1986) Cell 45,721-732 [Medline] [Order article via Infotrieve]
  11. Glass, D. J., Nye, S. H., Hantzopoulos, P., Macchi, M. J., Squinto, S. P., Goldfarb, M., and Yancopoulos, G. D. (1991) Cell 66,405-413 [Medline] [Order article via Infotrieve]
  12. Gluzman, Y. (1981) Cell 23,175-182 [Medline] [Order article via Infotrieve]
  13. Guiton, M., Gunn-Moore, F. J., Stitt, T. N., Yancopoulos, G. D., and Tavaré, J. M. (1994) J. Biol. Chem. 269,30370-30377 [Abstract/Free Full Text]
  14. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241,42-52 [Medline] [Order article via Infotrieve]
  15. Haystead, C. M. M., Gregory, P., Shirazi, A., Fadden, P., Mosse, C., Dent, P., and Haystead, T. A. J. (1994) J. Biol. Chem. 269,12804-12808 [Abstract/Free Full Text]
  16. Heasley, L. E., and Johnson, G. L. (1989) J. Biol. Chem 264,8646-8652 [Abstract/Free Full Text]
  17. Hill, C. S., and Treisman, R. (1995) Cell 80,199-211 [Medline] [Order article via Infotrieve]
  18. Hubbard, S. R., Wei, L., Ellis, L., and Hendrickson, W. A. (1994) Nature 372,746-754 [CrossRef][Medline] [Order article via Infotrieve]
  19. Ip, N. Y., Stitt, T. N., Tapley, P., Klein, R., Glass, D. J., Fandl, J., Greene, L. A., Barbacid, M., and Yancopoulos, G. D. (1993) Neuron 10,137-149 [Medline] [Order article via Infotrieve]
  20. Lamballe, F., Tapley, P., and Barbacid, M. (1993) EMBO J. 12,3083-3094 [Abstract]
  21. Loeb, D. M., Stephens, R. M., Copeland, T., Kaplan, D. R., Greene, L. A. (1994) J. Biol. Chem. 269,8901-8910 [Abstract/Free Full Text]
  22. Marais, R., Wynne, J., and Treisman, R. (1993) Cell 73,381-393 [Medline] [Order article via Infotrieve]
  23. Marshall, C. J. (1995) Cell 80,179-185 [Medline] [Order article via Infotrieve]
  24. Meakin, S. O., and Shooter, E. M. (1992) Trends Neurosci. 15,323-331 [CrossRef][Medline] [Order article via Infotrieve]
  25. Middlemas, D. S., Meisenhelder, J., and Hunter, T. (1994) J. Biol. Chem. 269,5458-5466 [Abstract/Free Full Text]
  26. Obermeier, A., Halfter, H., Wiesmuller, K. H., Jung, G., Schlessinger, J., and Ullrich, A. (1993a) EMBO J. 12,933-941 [Abstract]
  27. Obermeier, A., Lammers, R., Wiesmuller, K. H., Jung, G., Schlessinger, J., and Ullrich, A. (1993b) J. Biol. Chem. 268,22963-22966 [Abstract/Free Full Text]
  28. Ohmichi, M., Decker, S. J., and Saltiel, A. R. (1992) Neuron 9,769-777 [Medline] [Order article via Infotrieve]
  29. Panayotou, G., and Waterfield, M. D. (1993) BioEssays 15,171-177 [Medline] [Order article via Infotrieve]
  30. Pronk, G. J., McGlade, J., Pelicci, G., Pawson, T., and Bos, J. L. (1993) J. Biol. Chem. 268,5748-5753 [Abstract/Free Full Text]
  31. Pulverer, B. J., Kyriakis, J. M., Avruch, J., Nikolakaki, E., and Woodgett, J. R. (1991) Nature 353,670-674 [CrossRef][Medline] [Order article via Infotrieve]
  32. Soltoff, S. P., Rabin, S. L., Cantley, L. C., and Kaplan, D. R. (1992) J. Biol. Chem. 267,17472-17477 [Abstract/Free Full Text]
  33. Sozeri, O., Vollmer, K., Liyanage, M., Frith, D., Kour, G., Mark, G. E., and Stabel, S. (1992) Oncogene 7,2259-2262 [Medline] [Order article via Infotrieve]
  34. Squinto, S. P., Stitt, T. N., Aldrich, T. H., Davis, S., Bianco, S. M., Radziejewski, C., Glass, D. J., Masiakowski, P., Furth, M. E., Valenzuela, D. M., Distefano, P. S., and Yancopoulos, G. D. (1991) Cell 65,885-893 [Medline] [Order article via Infotrieve]
  35. Tavaré, J. M., O'Brien, R. M., Siddle, K., and Denton, R. M. (1988) Biochem. J. 253,783-788 [Medline] [Order article via Infotrieve]
  36. Tornqvist, H. E., Gunsalus, J. R., Nemenoff, R. A., Frackelton, A. R., Pierce, M., and Avruch, J. (1988) J. Biol. Chem. 263,350-359 [Abstract/Free Full Text]
  37. Tsoulfas, P., Soppet, D., Escandon, E., Tessarollo, L., Mendoza-Ramirez, J. L., Rosenthal, A., Nikolics, K., and Parada, L. F. (1993) Neuron 10,975-990 [Medline] [Order article via Infotrieve]
  38. Valenzuela, D. M., Maisonpierre, P. C., Glass, D. J., Rojas, E., Nunez, L., Kong, Y., Geis, D. R., Stitt, T. N., Ip, N. Y., and Yancopoulos, G. D. (1993) Neuron 10,963-974 [Medline] [Order article via Infotrieve]
  39. White, M. F., Shoelson, S. E., Keutmann, H., and Kahn, C. R. (1988) J. Biol. Chem. 263,2969-2980 [Abstract/Free Full Text]
  40. Young, S. W., Dickens, M., and Tavaré, J. M. (1994) FEBS Lett. 338,212-216 [CrossRef][Medline] [Order article via Infotrieve]

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