(Received for publication, October 19, 1995; and in revised form, November 27, 1995)
From the
The genetic locus for the TrkC/neurotrophin 3 (NT-3) receptor
tyrosine kinase encodes multiple isoforms including receptors with
inserts in the catalytic domain. This study examines the signaling
capabilities of TrkC and related kinase insert isoforms TrkC14 and
TrkC25. We show that in PC12 cells expressing both TrkC and TrkA/nerve
growth factor (NGF) receptors, different morphological changes occur
upon addition of NGF or NT-3. NT-3-treated cells exhibit longer
neurites and larger cell bodies as compared to NGF-treated cells. Both
TrkC and TrkA mediate qualitatively similar increases in the tyrosine
phosphorylation of phospholipase C (PLC)-1, Shc, SNT, and MAPK and
the transcription of the c-fos, c-jun, NGFI-A, and NGFI-B immediate early genes. However,
the TrkC kinase insert forms fail to stimulate these events.
Furthermore, TrkC14 and TrkC25 have only a low intrinsic tyrosine
kinase activity, and insertion of the TrkC14 kinase insert into TrkA at
an equivalent position results in a dramatic reduction of the kinase
activity and signaling capabilities of TrkA. The TrkC14 and -25
isoforms may fail to transmit signals due to their low intrinsic kinase
activity and failure to activate and/or tyrosine phosphorylate targets
shown to be involved in neurotrophin signal transduction pathways.
Many developmental processes are controlled by growth factors that activate receptor tyrosine kinases(1) . For example, during the development of the nervous system, the trk gene family that includes the TrkA, TrkB, and TrkC receptor tyrosine kinases, play prominent regulatory roles(2) . In vivo analysis of Trk and their ligands, the neurotrophins, has verified their importance for normal development and survival of the majority of sensory and sympathetic neurons(2) . For example, mice lacking expression of TrkC or its ligand, NT-3, exhibit loss of specific subsets of sensory neurons(3, 4, 5, 6) .
Another
useful system for studying Trk function is the PC12 pheochromocytoma
tumor cell line(7) . Nerve growth factor (NGF), ()the major ligand for
TrkA(8, 9, 10) , promotes the differentiation
of the PC12 cell line into cells resembling sympathetic
neurons(7) . NGF exerts its effects by binding to the second
immunoglobulin-like domain of TrkA(11, 12) . The
kinase domain is rapidly transphosphorylated on specific tyrosine
residues in the catalytic and noncatalytic intracellular domains of the
receptor (13) . For example, the phosphorylated residue Tyr-490
has been shown to bind and mediate the tyrosine phosphorylation of the
Shc proteins, that in turn associate with the small adaptor protein,
Grb2(13, 14) . The coupling of TrkA to Shc stimulates
the activity of Ras and the serine/threonine kinases of the Ras pathway
that include B-Raf, MEK, MAPK, and p90
(15) . The
other major autophosphorylation site in the noncatalytic domain,
Tyr-785, interacts with PLC-
1(16, 55) . This site
has been shown to participate in a variety of effects on NGF-treated
PC12 cells, including the stimulation of peripherin transcription and
the activation of MAPK(16) . Site-directed mutagenesis of
Tyr-490 and Tyr-785 has established these residues as primary effectors
of NGF-dependent neurite outgrowth and survival responses in PC12
cells(13, 16, 17) . Expression of dominant
inhibitory or activated alleles or overexpression of Ras, Raf, Shc,
Mek, and MAPK in PC12 cells can mimic some but not all of NGF-induced
differentiation
events(18, 19, 20, 21) , indicating
the importance of these proteins in the differentiation process while
suggesting that alternative Ras-independent signaling pathways mediate
some of the effects of NGF(15) . One such pathway, defined by a
deletion in TrkA at amino acids 441-443 (
KFG), may involve
the nuclear protein SNT (24) . TrkA encoding this deletion is
defective in mediating the NGF-induced initiation of neurite outgrowth,
somatic hypertrophy, and cessation of cell proliferation(24) .
A third pathway involving phosphatidylinositol 1,4,5-trisphosphate
kinase may modulate NGF-induced neurite outgrowth and survival
responses(22, 23) . Taken together, these results
suggest that several signaling pathways cooperate to regulate the
establishment and maintenance of the neuronal phenotype of PC12 cells.
The PC12 cell system has also been useful for studying the function of TrkB and TrkC. Brain-derived neurotrophic factor, a member of the NGF family of growth factors, can activate TrkB (receptor tyrosine kinase) and induce neurite outgrowth in PC12 cells transfected with this receptor(25) . Similarly, NT-3 will stimulate neurite outgrowth in PC12 cells transfected with TrkC(26) . The TrkC locus encodes at least eight isoforms including forms without the kinase domain or with kinase insertions adjacent to the major autophosphorylation site(26, 27) . These forms arise by alternative splicing events and are expressed in different tissues and cell types(26, 27, 28, 29) . We and others have shown that the mammalian kinase insert forms of TrkC, TrkC14, and TrkC25 do not have the capability to induce PC12 cells transiently expressing these receptors to differentiate in response to NT-3(26, 27, 28) . The basis of the lack of signaling potential of the TrkC kinase insert isoforms in neuronal cells has not been investigated. Here, we analyze the signal transduction capabilities of TrkC and its related kinase insert isoforms relative to TrkA in the same cellular background, PC12 cells. We show that TrkC uses the same known signal transduction pathways as TrkA, although the morphological changes in PC12 cells induced by these two receptors are distinct. We show that the TrkC kinase insert isoforms fail to transduce signals to known substrates, induce immediate early gene transcription, or efficiently autophosphorylate in vitro. Placement of the TrkC14 insert at a similar position in the kinase domain of the TrkA receptor results in a dramatic reduction of the tyrosine kinase activity of TrkA and its signaling capabilities.
Human TrkA
containing the TrkC14 kinase insert (TrkA14) was generated by
conventional mutagenesis (Bio-Rad) using human trkA as
template. The residues corresponding to the trkC14 insert were
inserted into position 606 and confirmed by sequencing. The PLC-1
baculovirus was a gift of V. Cleghon and D. Morrison (NCI-FCRDC).
TrkA14, TrkA(16) , and PLC-
1 baculoviruses were used as
described(16) . For recombinant protein production, 2
10
Sf9 cells were co-infected with PLC-
1 and either
TrkA or TrkA14 at a multiplicity of infection of 5 and lysed at 26 h
postinfection.
Figure 1: NGF and NT-3 mediate specific tyrosine autophosphorylation of their respective receptors. Parental PC12 cells (PC12) or PC12 cells expressing TrkC receptors (PC12 TRKC) were treated with 100 ng/ml NGF or NT-3 for 5 min and lysed, and equal amounts of lysate (normalized for total protein) were subjected to immunoprecipitation with anti-pan Trk443 or anti-TrkC656. The immunoprecipitates were electrophoresed on 7.5% SDS-polyacrylamide gels, proteins were transferred to nitrocellulose filters, and the filters were probed with anti-Tyr(P). Shown is an autoradiograph of a Western blot developed using enhanced chemiluminescence.
Figure 2: The TrkC kinase insert isoforms display reduced tyrosine autophosphorylation in response to NT-3. Dose-response of TrkC isoforms to NT-3. TrkC, TrkC14 and TrkC25 expressing PC12 cells were treated with 0, 1, 10, 25, or 100 ng/ml NT-3 for 5 min at 37 °C and lysed, and equal amounts of lysate (normalized for total protein) were subjected to immunoprecipitation with anti-TrkC656. Proteins were probed with anti-Tyr(P) (left panel). To confirm that the amounts of immunoprecipitated TrkC were similar in each lane, the blots in the left panel were stripped of antibody and reprobed with anti-TrkC656 (right panel). The apparent molecular weights of protein standards are indicated by the arrows on the right, and the position of the TrkC-encoded proteins is indicated by the triangle.
Figure 3: Neurite outgrowth assay. PC12 cell lines expressing TrkC, TrkC14, and TrkC25 were plated on collagen-coated dishes. The cells were treated with medium alone (top panel), medium supplemented with 100 ng/ml NT-3 (middle panel), or medium with 100 ng/ml NGF (bottom panel). Representative fields were photographed at 3, 10, and 14 days after treatment.
Figure 4:
Tyrosine phosphorylation of PLC-1,
Shc, and SNT in PC12 cells expressing TrkC and TrkC kinase insert
isoforms. Lysates were prepared from mock-treated (0), NGF-treated (100
ng/ml for 5 min), or NT-3-treated (100 ng/ml for 5 min) cells
expressing the TrkC, TrkC14, and TrkC25 receptors and equalized for
cell protein. A, tyrosine phosphorylation of PLC-
1.
Lysates were immunoprecipitated with anti-PLC-
1 and probed with
anti-Tyr(P) (top panel). To verify that the amounts of
immunoprecipitated PLC-
1 were similar in each lane, the blot in
the top panel was stripped of antibody and reprobed with
anti-PLC-
1. The positions of PLC-
1 and Trk proteins are
indicated with arrows. B, tyrosine phosphorylation of
Shc. Lysates were immunoprecipitated with anti-Shc and probed with
anti-Tyr(P) (top panel) followed by reprobing with anti-Shc (bottom panel). The positions of Shc are indicated by arrows. C, tyrosine phosphorylation of SNT. SNT
proteins were precipitated with p13
-agarose and
probed with anti-Tyr(P).
Figure 5: Tyrosine phosphorylation and time course of MAP kinases. PC12 cell lines expressing TrkC (PC12 TRKC), TrkC14 (PC12 TRKC14), and TrkC25 (PC12 TRKC25) were mock-treated (0), NGF-treated (100 ng/ml), or NT-3 treated (100 ng/ml) for 5 min at 37 °C. Lysates were normalized for cell protein. A, lysates from all cell lines were immunoprecipitated with anti-Erk and probed with anti-Tyr(P) (top panel) followed by reprobing with anti-Erk (bottom panel). B, time course of tyrosine phosphorylation of MAP kinases in PC12TRKC cell line. Lysates prepared from cells treated for the indicated times with 100 ng/ml NGF or NT-3 were immunoprecipitated with anti-Erk. The immunoprecipitates were probed with anti-Tyr(P) (top panel). The gel in the top panel was stripped of antibody and reprobed with anti-Erk (bottom panel). The positions of Erk1 and Erk2 are indicated by arrows.
Figure 6: Differential expression of NGF- or NT-3-inducible early genes in PC12 cells expressing TrkC and TrkC14. Expression of early response genes c-fos (A), c-jun (B), NGFI-B/TIS1 (C), and NGFI-A/TIS8 (D) was determined by RNA transfer analysis of total RNA isolated from cells treated with NGF or NT-3 for the indicated times (0, 30, 60, and 180 min). Filters were probed with radiolabeled cDNA probes, followed by reprobing with a cyclophilin cDNA probe to control for RNA loading (E).
Figure 7:
Impaired in vitro kinase activity
of TrkC14 and TrkC25. PC12 cells expressing TrkC, TrkC14, or TrkC25
were treated with 100 ng/ml NGF or NT-3, and the cell lysates were
immunoprecipitated with anti-pan Trk203(31) . The
immunoprecipitates were washed, and Trk proteins were assessed for in vitro kinase activity using 5 µg of a peptide encoding
amino acids 664-681 of human TrkA (peptide 1251) in the presence
of 30 mM Hepes, pH 7.4, 10 mM MnCl, 5
µM ATP, and 1 µCi of
[
-
P]ATP for 3 min at 25 °C. Shown is an
autoradiograph of phosphorylated
I-peptide
electrophoresed on a 15% Tricine gel. The three bands correspond to the
peptide phosphorylated once, twice, or three
times.
Figure 8:
TrkA
encoding the kinase insert from TrkC14 (TrkA14) is defective
in NT-3-induced tyrosine autophosphorylation and PLC-1 tyrosine
phosphorylation in vivo. Sf9 cells co-expressing TrkA and
bovine PLC-
1 or TrkA14 and PLC-
1 were treated for 5 min with
100 ng/ml NGF, Trk, and PLC-
1 immunoprecipitated with specific
antibody, and Trk (A) and PLC-
1 (C) tyrosine
phosphorylation was assessed in Western blots using anti-Tyr(P). The
blots were stripped and reprobed with anti-pan Trk203 (B) or
anti-PLC-
1 (D).
In response to NGF, PC12 cells undergo dramatic alterations in phenotype, including changes in cell shape and hypertrophy, extension of long neurites, and the acquisition of electrical excitability(44) . Recent studies have identified correlations between biological responses and the activation of selective intracellular signaling pathways mediated by NGF-activated TrkA in PC12 cells(13, 15, 16, 17, 24) . We likewise used the PC12 cell system to explore and compare the cellular modifications and signaling pathways utilized by TrkC and related kinase insert isoforms to those used by TrkA. Our results indicate that TrkA and TrkC show quantitative and qualitative differences in their abilities to induce neuronal differentiation, and that the consequence of inserts in TrkC is to impair biological and biochemical signaling capacities.
To compare the signaling
potentials of TrkA and TrkC, these receptors were expressed such that
they exhibited similar levels of ligand-induced tyrosine
phosphorylation in PC12 cells. When bound by ligand, these receptors
did not appear to form heterodimeric complexes, since
immunoprecipitates of NT-3-activated TrkC did not contain TrkA. Neurite
outgrowth and cell hypertrophy were more pronounced in cells exposed to
NT-3 than those exposed to NGF. Thus, TrkC and TrkA, when
autophosphorylated to similar extents, have distinct morphological
effects in PC12 cells. One explanation for more rapid responses
mediated by TrkC is that this receptor causes a prolonged activation of
the proteins of the Ras signaling pathway. Sustained signaling through
Ras has been suggested to be responsible for the ability of normally
mitogenic receptors such as the insulin and epidermal growth factor
receptors to elicit neurites when overexpressed in PC12
cells(32, 33, 45, 46) . However, the
tyrosine phosphorylation of Shc and PLC-1 was reduced in
NT-3-treated cells as compared to NGF, and the strength and duration of
Erk tyrosine phosphorylation and the induction of the c-fos,
c-jun, NGFI-A, and NGFI-B genes were
similar. The differences in biological responses elicited by NGF and
NT-3, therefore, cannot be correlated with the duration and strength of
signaling through targets of Ras such as Erk. Quantitative differences
in the tyrosine phosphorylation of proteins such as SNT could account
for differential biological response. However, the precise molecular
nature of SNT is not yet known, and other cellular proteins that are
activated or phosphorylated by neurotrophin treatment such as
phosphoinositide 3-kinase(14, 47, 48) ,
SH-PTP1(49) , and Nck (50) may be responsible for the
differences in TrkA and TrkC signaling.
We also show that the
inability of TrkC14 and TrkC25 to induce phenotypic changes correlates
with their inability to stimulate the tyrosine phosphorylation of Shc,
PLC-1, Erk, and SNT or to mediate immediate early gene induction
events. This is in accordance with other studies showing that TrkC14
and TrkC25 fail to activate PLC-
1 and phosphoinositide 3-kinase
and induce biological responses in NIH-3T3
cells(28, 56) . We suggest that the inability of
TrkC14 and TrkC25 to stimulate morphological and signaling responses is
most likely due to their inefficiency at ligand-induced
autophosphorylation and kinase activity ( Fig. 7and Fig. 8).
It is not known what structural constraints the kinase inserts confer to the kinase domain that prevent proper activation of the receptor and stimulation of primary effectors. The crystal structure of the catalytic domain for the human insulin receptor has been determined, and a general mechanism for the activation of receptor tyrosine kinases has been proposed, known as the cis-inhibition and trans-activation model(51) . The activation loop in the insulin receptor contains three tyrosines at positions 1157, 1162, and 1163 (YETDYY) that become autophosphorylated upon receptor activation. However, in the unphosphorylated state, one of these residues, Tyr-1162, is engaged in the active site with the catalytic base of Asp-1132. This engagement prevents the accessibility of exogenous substrate and ATP binding, thus repressing kinase activity. When insulin binds the receptor, Tyr-1162 is trans-autophosphorylated and disengaged from the active site, and ATP is bound. Consequently, the activation loop is stabilized in a new conformation. These intermolecular changes result in increased ATP and substrate accessibility and greatly enhanced kinase activity. As in the insulin receptor, the Trk receptors contain three tyrosines, YXXDYY, located in a similar position in a potential activation loop. In TrkA, these three tyrosines have been found to be major targets of autophosphorylation(13) . The 14- and 25-amino acid inserts in the kinase domain of TrkC are found within two amino acids from the carboxyl-terminal of the tyrosine residues. The presence of additional sequences directly following the activation loop and their proximity to the tyrosines that become autophosphorylated during receptor activation may adversely affect the disengagement of the tyrosine from the active site or access to substrate. Such a mechanism could reduce the efficiency of the trans-autophosphorylation that alters the final conformation of the intracellular domains of TrkC that would result in a lack of interaction or access of this receptor to its substrates. Our data are consistent with such a model. The autophosphorylation of TrkC insert isoforms was reduced substantially in PC12 cells, and the intrinsic kinase activity of these receptors in vitro was reduced greatly. Furthermore, when the TrkC14 insert form was inserted in a similar position in TrkA, tyrosine kinase activity was greatly diminished. We conclude that the TrkC kinase inserts confer functional constrains to other Trk family members, indicating that the impaired tyrosine kinase activity of TrkC14 is not specific to TrkC.
In summary, our observations support the concept
that the TrkC insert isoforms have a low signaling capability in
neuronal cells. While TrkC kinase inserts are expressed in the same
cell populations as TrkC, it remains unclear whether both classes of
receptors are co-expressed in the same cell. In neurons, TrkC insert
isoforms may modulate signal transduction, either by the formation of
heterodimers with TrkC or by competitive binding of available ligand.
We have observed that TrkC insert isoforms are expressed in the central
nervous system during postnatal and adult life, ()which
could explain the low level of NT-3-induced tyrosine phosphorylation of
TrkC in isolated adult brain tissues(52) . However, since the
TrkC kinase insert isoforms have a limited signaling capability when
overexpressed in a non-neuronal background(28, 56) ,
it is possible that these receptors might have some functional
capability in vivo.