(Received for publication, April 17, 1995; and in revised form, June 5, 1995)
From the
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.
The transmembrane protein-tyrosine kinase TrkC is the high
affinity receptor for neurotrophin-3 (NT-3), ()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.
Figure 1:
NT-3-stimulated proliferation of
fibroblasts expressing TrkC variants. MG87 cells expressing full-length
TrkC () 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.''
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) () or cells
expressing full-length TrkC (
), 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.
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).
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
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.
Figure 6:
Analysis of the sites of NT-3-stimulated
TrkC phosphorylation. MG87 cells expressing full-length TrkC were
incubated with [P]P
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)).
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
, 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.
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, (
)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. (
)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.