 |
INTRODUCTION |
Nerve growth factor
(NGF)1 regulates the
survival, development, and differentiation of the sympathetic and
sensory neurons in the peripheral nervous system and the
differentiation of certain cholinergic neurons in the central nervous
system (1, 2). In addition, NGF promotes neuronal differentiation of
the rat pheochromocytoma cell line, PC12, which has been used
extensively to investigate ligand-receptor interactions and cellular
differentiation in response to NGF (3, 4). Upon stimulation with NGF,
these cells acquire the phenotype of sympathetic neurons as
characterized by neurite outgrowth and persistent activation of the ERK
family of mitogen-activated protein (MAP) kinase (5). Two cell surface receptors for NGF have been identified: the receptor tyrosine kinase,
TrkA, and the low-affinity neurotrophin receptor, p75NTR
(6-9). NGF exerts its growth- and survival-promoting effects on
neurons through activation of TrkA and subsequent biochemical events
that ultimately influence the expression of various genes, including
those encoding ion channels, neurotransmitter-synthesizing enzymes, and
cytoskeletal components (10). The binding of NGF to the p140 TrkA
receptor induces dimerization of TrkA receptors and stimulates rapid
tyrosine autophosphorylation of the receptor (11). The phosphotyrosines
on activated TrkA serve as docking sites for signaling substrates such
as phospholipase C-
1 (PLC-
1), phosphatidylinositol 3-kinase (PI-3
kinase), and the Shc adaptor protein (6, 12). These molecules trigger
kinase cascades resulting in the phosphorylation and activation of
transcription factors that direct gene expression. Shc triggers the
activation of Ras and the subsequent sequential phosphorylation and
activation of the kinases Raf, mitogen- and extracellular-regulated
kinase, and ERK (13, 8). The Ras-ERK pathway plays a major role in the
activation of transcriptional events by NGF and in NGF-induced neuronal
differentiation. Mutation analysis of TrkA has defined critical
tyrosines that specifically regulate the activities of PLC-
1, PI-3
kinase and Shc (14-18). PLC-
1 and Shc regulation appear to play a
major role in NGF-mediated neurite outgrowth (17, 18). In addition,
sustained PI-3 kinase activity is necessary for the neurite outgrowth
of PC12 cells induced by NGF (19).
The Csk homologous kinase (CHK) (previously referred to as
megakaryocyte-associated tyrosine kinase (MATK)) is a recently identified protein tyrosine kinase that shares high homology with Csk
(COOH-terminal SRC kinase). CHK was independently identified as MATK
(20, 21), Lsk (22), Hyl (23), Ctk (24), Ntk (25), and Batk (26). Like
Csk, CHK contains Src homology 3 (SH3), SH2, and tyrosine kinase
domains and lacks the Src family NH2-terminal myristylation
and autophosphorylation sites (20, 21). Csk is ubiquitously expressed,
whereas expression of CHK is restricted to hematopoietic cells and the
nervous system. The expression of CHK in brain increases postnatally,
whereas the expression of Csk decreases with age (27). Although the
function of CHK is still unclear, recent studies indicated that, unlike Csk, CHK interacts with receptor tyrosine kinases, such as c-Kit in
megakaryocytes (28, 29) or the ErbB-2/neu receptor in breast cancer
cells (30, 31) via its SH2 domain. Because CHK is abundantly expressed
in the nervous system, we sought to identify the signaling pathways
that involve CHK and to characterize its function in neuronal cells. In
this report, we identified and characterized the association of CHK in
TrkA signaling and investigated CHK involvement in neuronal
differentiation of PC12 cells.
 |
EXPERIMENTAL PROCEDURES |
Antibodies--
Anti-GST monoclonal antibodies were produced as
described previously (32). Affinity-purified polyclonal anti-TrkA
antibodies against the COOH-terminal peptide of the TrkA receptor were
purchased from Oncogene Science (Cambridge, MA). Anti-CHK, anti-Csk,
anti-p85/PI-3 kinase, anti-ERK1, and anti-ERK2 antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal
anti-phosphotyrosine (PY-20) was obtained from Transduction
Laboratories (Lexington, KY). The PhosphoPlus MAP kinase antibody kit
was obtained from New England BioLabs (Beverly, MA). Mouse 7S NGF was
purchased from Upstate Biotechnology (Lake Placid, NY).
Glutathione-Sepharose 4B was purchased from Amersham Pharmacia Biotech.
Reagents for electrophoresis were obtained from Bio-Rad. Horseradish
peroxidase-conjugated anti-mouse Ig antibodies, horseradish
peroxidase-conjugated anti-rabbit Ig antibodies, and enhanced
chemiluminescence (ECL) reagents were purchased from Amersham Pharmacia Biotech.
Cell Culture and Stimulation--
PC12 cells were grown as
described (3). PC12-TrkA cells were stably transfected with TrkA
cDNA (33). These cells expressed 1.3 × 105 TrkA
receptors/cell. Both PC12 and PC12-TrkA cells were grown in RPMI 1640 medium containing 10% heat-inactivated horse serum and 5% fetal
bovine serum. The cells were grown on 10-cm dishes coated with
poly-L-lysine. For stimulation, cells were stimulated with
100 ng/ml NGF for the indicated periods at 37 °C.
To establish PC12 cells that stably overexpress CHK (PC12-CHK), the
full-length CHK cDNA (1.6 kilobases) was subcloned into the
pcDNA3 vector (Invitrogen, San Diego, CA). Transfection of the CHK
cDNA into PC12 cells was performed using LipofectAMINE. The
transfected cells were selected in 750 µg/ml Geneticin. Positive transfectants were chosen based on the expression of CHK protein upon
immunoblotting with anti-CHK antibodies. PC12-CHK cells were grown in
RPMI 1640 medium containing 10% horse serum and 5% fetal bovine serum.
Immunoprecipitation and Western Blotting--
Prior to
stimulation, cells were starved for 36 h in RPMI 1640 medium
containing 0.1% horse serum and 0.1% fetal bovine serum. Cells were
then stimulated with 100 ng/ml NGF for the indicated periods at
37 °C, washed with ice-cold phosphate-buffered saline, and lysed in
lysis buffer (50 mM HEPES (pH 7.5), 10% glycerol, 150 mM NaCl, 0.5% Nonidet P-40, 1.5 mM
MgCl2, 1 mM EGTA, 25 mM NaF, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin). Lysate protein contents were normalized using the Bio-Rad
protein assay. Lysates were immunoprecipitated with affinity-purified
anti-TrkA, anti-Csk, anti-CHK polyclonal antibodies, anti-p85/PI-3
kinase, and anti-phosphotyrosine antibodies. Immunoprecipitates were
diluted with 2× SDS sample buffer 1:2 (v/v) containing
-mercaptoethanol and were separated by 7% SDS-PAGE. In some cases,
as indicated in the text, immunoprecipitates were diluted in sample
buffer without
-mercaptoethanol. Samples were separated by 7%
SDS-PAGE, and then proteins were transferred onto PVDF-Plus membranes
(Micron Separations Inc., Westboro, MA). Bound proteins were
immunoblotted with anti-phosphotyrosine antibody (PY-20),
affinity-purified polyclonal anti-TrkA, anti-CHK, or anti-Csk
antibodies. The blots were developed using the ECL system.
Precipitation with GST Fusion Proteins and Far Western
Blotting--
GST fusion proteins containing the CHK-SH3 domain, the
CHK-NH2-terminal domain plus SH3 domain
(NH2-SH3), the CHK-SH2 plus SH3 domains (CHK-SH2-SH3) or
GST alone were prepared as described previously (30). The starved
PC12-TrkA cells were stimulated with 100 ng/ml NGF and then lysed and
precipitated with various GST fusion proteins. 10 µg of GST fusion
protein bound to glutathione-Sepharose 4B were used for each binding
assay. After lysate protein content was normalized and precipitated
with GST fusion proteins, bound proteins were immunoblotted with PY-20,
affinity-purified polyclonal anti-TrkA antibody, or anti-GST antibody.
The blots were developed using the ECL system. For far Western
analysis, starved PC12-TrkA cells were stimulated with 100 ng/ml NGF
for the indicated periods, lysed, and immunoprecipitated with anti-TrkA
antibody. The immunoprecipitates were then separated on 7% SDS-PAGE
gels, transferred onto membranes, and subjected to far Western
blotting. Ten µg of GST fusion proteins were used for each binding assay.
Construction of Wild-type and Mutant TrkA cDNA Constructs and
Transfection Experiments--
Site-directed mutagenesis of full-length
TrkA cDNA in the pUC19 vector was performed using a Transformer
site-directed mutagenesis kit (CLONTECH, Palo Alto,
CA) according to the manufacturer's protocol. In order to change
selected tyrosines (Tyr) to phenylalanine (Phe), we used the following
synthetic oligonucleotides: 5'-CAC ATC ATC GAG AAC CCA CAA TTC TTC AGT
GAT GCC TGT GTT CAC-3' (Y490F), 5'-TGC CCA CCA GAG GTC TTC GCC ATC ATG
CGG GGC-3' (Y751F), and 5'-CAG GCA CCT CCT GTC TTC CTG GAT GTC CTG
GGC-3' (Y785F). The mutations were confirmed by DNA sequencing. The
mutant and wild-type TrkA inserts were excised from pUC19 vectors and
subcloned into the pcDNA3 vector. Transfection into COS-7 cells was
performed using LipofectAMINE (Life Technologies, Inc.) according to
the manufacturer's protocol.
Inhibition of the CHK-SH2 Domain/TrkA Interaction by
Phosphopeptides--
Tyrosine-phosphorylated synthetic peptides were
obtained from the Molecular Biology Core Facility at Dana Farber Cancer
Institute. Peptides were analyzed for purity by high pressure liquid
chromatography, mass spectroscopy, and amino acid analysis. Two
phosphotyrosine peptides corresponding to autophosphorylation sites of
TrkA, including the Shc association site and the PLC-
1 association
site, were synthesized. In addition, nonphosphorylated peptide
corresponding to the PLC-
1 association site was also synthesized.
Unstimulated and NGF-stimulated PC12-TrkA cell lysates were added to
the GST-CHK-SH2 fusion protein (10 µg/incubation), which was
preincubated with each phosphopeptide. The final concentration of
phosphopeptides was 100 µM for each incubation. Lysates
were incubated for 1 h at 4 °C and then precipitated by the
addition of glutathione-Sepharose 4B for 30 min at 4 °C.
Precipitates were washed and separated by 7% SDS-PAGE.
MAP Kinase Assay--
MAP kinase assay was performed as
described (34). Briefly, starved PC12 and PC12-CHK cells (three
independent clones that overexpress CHK) were stimulated with NGF (100 ng/ml). Cells were then lysed and immunoprecipitated with anti-ERK1 and
anti-ERK2 antibodies (Santa Cruz Biotechnology). MAP kinase activity
was assayed with myelin basic protein as a substrate in the presence of
[
-32P]ATP. All reactions were allowed to proceed for
10 min at 30 °C. The p44/42 MAP kinase activity was also measured in
total lysates using the p44/42 MAP kinase assay kit (New England
BioLabs). Phospho-p44/42 MAP kinase monoclonal antibody was used to
immunoprecipitate the active p44/42 MAP kinase from the cell extracts,
and then an in vitro kinase assay was performed using Elk-1
protein as a substrate.
Microinjection--
Synchronized PC12 cells were microinjected
with purified CHK-specific antibodies (at a concentration of 100 µg/ml) in microinjection buffer with GFP vector plasmid (pEGFP-C2)
(CLONTECH, Palo Alto, CA) as a marker in a final
concentration of 100 µg/ml. IgGs were purified from control
antiserum, Csk and CHK antisera using protein G columns (Mab Trap,
Amersham Pharmacia Biotech). Pooled fractions containing
electrophoretically pure IgGs were dialyzed against phosphate-buffered
saline and concentrated using Centricon concentrators (Amicon Corp.,
Danvers, MA). Following microinjection, cells were incubated for 2 h at 37 °C. Cells were refed with medium alone or medium containing
0.1% horse serum overnight. The medium was then changed to RPMI medium
containing 10% horse serum and 5% fetal bovine serum, with or without
NGF (100 ng/ml) as indicated. Cells were monitored and analyzed after
24, 36, and 48 h. Differentiated cells were defined as cells with
refractile cell bodies extending at least two processes, one of which
had to be longer than the diameter of the cell body. Subsequent
phase-contrast micrographs were recorded on Technical Pan film (2415)
(Eastman Kodak Co.).
 |
RESULTS |
CHK Is Associated with Activated TrkA Receptors upon Stimulation
with NGF--
Because CHK is a second member of the Csk family and
both kinases are expressed in brain, we investigated whether CHK and/or Csk might be involved in one of the main signaling pathways in neuronal
cells that is mediated by the neurotrophin NGF. To identify proteins
that associate with CHK and Csk in PC12 cells, we analyzed anti-CHK
immunoprecipitates from untreated or NGF-stimulated cells. The PC12
cells were starved for 36 h and stimulated with 100 ng/ml NGF for
10 min. Cells were then lysed and immunoprecipitated with either
anti-CHK antibodies, affinity-purified anti-TrkA antibodies, anti-Csk
antibodies, or control antibodies. Upon NGF stimulation, anti-CHK
antibodies, but not anti-Csk or control antibodies,
co-immunoprecipitated the tyrosine-phosphorylated protein, p140,
corresponding to the activated TrkA receptor (Fig.
1, A and B),
whereas anti-TrkA antibodies co-immunoprecipitated CHK protein (Fig.
1C) but not Csk (Fig. 1D). Therefore, these
results suggest that CHK, but not Csk, is associated with activated
TrkA receptors upon NGF stimulation.

View larger version (60K):
[in this window]
[in a new window]
|
Fig. 1.
Co-immunoprecipitation of CHK with TrkA upon
stimulation with NGF. PC12 cells were serum-starved for 36 h
and then stimulated with 100 ng/ml NGF for 10 min. The lysates were
divided into two equal portions. One-half of the samples were
immunoprecipitated with either anti-CHK antibodies, affinity-purified
anti-TrkA antibodies, anti-Csk antibodies, or control antibodies.
Immunoprecipitates were separated by 7% SDS-PAGE and immunoblotted
with anti-TrkA antibodies (A). The same blots were stripped
and reprobed with monoclonal phosphotyrosine antibodies
(PY-20) (B). The second half of the lysates were
immunoprecipitated as detailed above, and the samples were diluted 1:2
(v/v) with 2× SDS sample buffer without -mercaptoethanol. The
samples were separated by 10% SDS-PAGE and immunoblotted with anti-CHK
antibodies (C). The same blots were stripped and reprobed
with anti-Csk antibodies (D). Molecular mass markers are
indicated on the right (kDa).
|
|
Characterization of the Association of the CHK-SH2 Domain with
Activated TrkA Receptors--
In order to determine which domain of
CHK interacts with the activated TrkA receptors, we used GST fusion
proteins containing the SH2 domain of CHK (CHK-SH2). Serum-starved PC12
cells were stimulated with 100 ng/ml NGF for the indicated periods,
lysed, and then precipitated with the GST fusion protein containing the CHK-SH2 domain. The CHK-SH2 domain precipitated tyrosine-phosphorylated TrkA (Fig. 2A) upon
stimulation with NGF. The phosphorylation of TrkA upon NGF stimulation
was rapid and became prominent at 5 min. The association of CHK with
TrkA receptors was also observed after 3 days of NGF stimulation,
indicating that this association is a long-term one, based upon the
length of the stimulation (Fig. 2B). Similar results of the
association of CHK with activated TrkA receptors were obtained using
PC12-TrkA cells (PC12 cells stably transfected with TrkA receptors)
(data not shown).

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 2.
Association of the CHK-SH2 domain with the
activated TrkA receptors. A, PC12 cells were
serum-starved and stimulated with 100 ng/ml NGF for the indicated
periods. The lysates were precipitated with the GST fusion protein
containing the CHK-SH2 domain (10 µg). Precipitates were separated by
7% SDS-PAGE and immunoblotted with phosphotyrosine (PY-20)
(A, upper panel) or with anti-TrkA antibodies (A,
lower panel). B, PC12 cells were starved and then
stimulated with NGF for the indicated periods. The lysates were
precipitated with the GST fusion protein containing the CHK-SH2 domain
(10 µg). The precipitates were separated by 7% SDS-PAGE and
immunoblotted with phosphotyrosine (PY-20). Molecular mass
markers are indicated on the right (kDa).
|
|
The potential involvement of other domains of CHK in the interaction
with TrkA was examined using GST fusion proteins containing the CHK-SH3
domain, the CHK-SH3-SH2 domain, or GST protein alone. NGF-stimulated
PC12 cell lysates were incubated with the different GST fusion
proteins, analyzed by 7% SDS-PAGE, and immunoblotted with PY-20 or
anti-TrkA antibodies. Anti-phosphotyrosine and anti-TrkA Western
blotting revealed that the CHK-SH3-SH2 domain precipitated activated
TrkA receptors upon NGF stimulation (Fig.
3). However, the CHK-SH3-GST or GST alone
or a nonrelevant SH2 domain did not precipitate any proteins from the
same lysates. These results indicate that the CHK-SH2 domain
specifically precipitated activated TrkA receptors upon NGF
stimulation.

View larger version (65K):
[in this window]
[in a new window]
|
Fig. 3.
The CHK-SH2 domain specifically binds to the
activated TrkA receptors. PC12 cells were starved and then
stimulated with NGF for 10 min. The lysates were precipitated with GST
fusion proteins (10 µg) containing the CHK-SH2 domain plus SH3 domain
(SH3-SH2), the CHK-NH2-terminal domain plus SH3
domain (NH2-SH3), and the CHK SH3 domain
(SH3); GST alone as a control; or the Src-SH2 domain as a
control, and immunoblotted with phosphotyrosine (PY-20)
(A), anti-TrkA antibodies (B), or polyclonal
anti-GST antibodies (C). Molecular mass markers are
indicated on the right (kDa).
|
|
CHK Is Directly Associated with Activated TrkA Receptors--
In
order to determine whether the binding between activated TrkA receptors
and the CHK-SH2 domain is direct or indirect, we performed far Western
blotting analysis. The phosphorylation of TrkA upon NGF stimulation was
rapid, and reached maximum levels after 20 min (Fig.
4, A and B). In the
absence of NGF, the SH2 domain of CHK did not bind to TrkA, as observed
by far Western blotting (Fig. 4D). However, upon NGF
stimulation, binding of the CHK-SH2 domain to TrkA was observed at 2 min. This association was maintained up to 60 min after stimulation
with NGF (Fig. 4D). In contrast, the CHK-SH3 domain or GST
fusion protein alone did not bind to activated TrkA (Fig. 4,
C and E). These results indicate that the CHK-SH2
domain directly interacts with activated TrkA receptors upon NGF
stimulation.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 4.
CHK is directly associated with the activated
TrkA receptors upon NGF stimulation. Far Western blottings were
performed to identify the association of the CHK-SH2 domain with
activated TrkA receptors. Starved PC12 cells were stimulated with 100 ng/ml NGF for the indicated periods and lysed. TrkA receptors were
immunoprecipitated from cell lysates and then immunoblotted with
anti-TrkA antibodies (A) or phosphotyrosine
(PY-20) (B). The membrane was stripped, and far
Western blottings were performed using GST fusion proteins containing
the CHK-SH3 domain (C), the CHK-SH2 domain (D),
or GST alone as a control (E).
|
|
CHK Is Associated with Tyrosine 785 of the TrkA Receptor--
In
order to determine the binding site of the CHK-SH2 domain to TrkA
receptors, COS-7 cells were transfected with plasmids encoding
wild-type TrkA or with mutated TrkA having tyrosine-phenylalanine mutations at the Shc association site Tyr-490 (Y490F), the p85/PI-3 kinase interaction site Tyr-751 (Y751F), the PLC-
1 interaction site
Tyr-785 (Y785F), or mutations at both Tyr-490 and Tyr-751 (Y490F/Y751F), Tyr-490 and Tyr-785 (Y490F/Y785F), or Tyr-751 and Tyr-785 (Y751F/Y785F). The transfected COS-7 cells were starved and
then stimulated with 100 ng/ml NGF for 10 min. Unstimulated and
NGF-stimulated cell lysates were divided into two aliquots, and either
precipitated with the GST fusion protein containing the CHK-SH2 domain
or immunoprecipitated with anti-TrkA antibodies. Tyrosine
phosphorylation of wild-type TrkA upon stimulation with NGF was
observed in transfected cells. Furthermore, although the CHK-SH2 domain
did not precipitate the unstimulated wild-type TrkA receptor, it did
precipitate the tyrosine-phosphorylated wild-type TrkA receptor upon
NGF stimulation (Fig. 5A).
These results demonstrate that the CHK-SH2 domain interacts with
activated TrkA receptors upon NGF stimulation in TrkA-transfected COS-7 cells.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5.
Analysis of the binding site of the CHK-SH2
domain on the activated TrkA receptors. A-C, point
mutation analysis: untreated and NGF-treated COS-7 cells transfected
with wild-type (WT) TrkA cDNA were lysed and either
precipitated with the CHK-SH2 domain or immunoprecipitated with
anti-TrkA antibodies. Bound proteins were immunoblotted with anti-TrkA
antibodies (A, upper panel) or phosphotyrosine
(PY-20) (A, lower panel). COS-7 cells were
transfected with cDNA for wild-type TrkA; for mutated TrkA with
either a single mutation at Tyr-490 (Y490F), Tyr-751 (Y751F), or
Tyr-785 (Y785F) or double mutations (Y490F/Y751F, Y490F/Y785F, and
Y751F/Y785F); or for the vector control. NGF-treated cells were lysed
and either precipitated with the CHK-SH2 domain (B) or
immunoprecipitated with anti-TrkA antibodies (C) and then
immunoblotted with anti-TrkA antibodies. D, phosphotyrosine
peptide inhibition assay: Unstimulated and NGF-stimulated PC12 cell
lysates were added to the CHK SH2-GST fusion protein (10 µg)
preincubated with the indicated phosphopeptides (100 µM),
respectively, as detailed under "Experimental Procedures." Lysates
were precipitated by the addition of glutathione-Sepharose 4B.
Precipitates were immunoblotted with anti-TrkA antibodies.
|
|
Experiments in COS-7 cells transfected with TrkA wild-type or mutant
constructs revealed that the CHK-SH2 domain precipitated TrkA Y490F and
Y751F in the same manner as the wild-type TrkA receptor (Fig.
5B). The same results were observed in the binding of the
CHK-SH2 domain to TrkA Y490F/Y751F. However, the CHK-SH2 domain could
not precipitate mutant TrkA that had a Y785F mutation, such as TrkA
Y785F, TrkA Y490F/Y785F, or TrkA Y751F/Y785F. These results indicate
that the CHK-SH2 domain binds to phosphorylated Tyr-785 on the TrkA
receptor. In order to confirm the expression of wild-type or mutant
TrkA, the same amount of cell lysates was first immunoprecipitated and
then immunoblotted with anti-TrkA antibodies. Expression levels of
wild-type and mutant TrkA were similar (Fig. 5C).
Furthermore, we have studied the ability of synthetic phosphopeptides
to compete for the binding of the CHK-SH2 domain to TrkA receptors. We
synthesized two kinds of tyrosine-phosphorylated peptides derived from
the Shc binding site and the PLC-
1 association site of TrkA
receptors. A peptide containing the Shc binding site of TrkA receptors
(Y*FSDTCV, where the asterisk indicates a phosphotyrosine peptide)
could not compete the binding of the SH2 domain of CHK to the
activated TrkA receptors (Fig. 5D). The
non-tyrosine-phosphorylated control peptide (SYLDVLG) also failed
to compete the binding of the SH2 domain of CHK to the
activated TrkA receptors. On the other hand, a peptide containing
the PLC-
1 association site of TrkA receptors (SY*LDVLG) was able to
compete the binding of the SH2 domain to the activated TrkA receptors
(Fig. 5D). To further test the binding of CHK to the Tyr-785
site, we linked the tyrosine-phosphorylated SY*LDVLG peptide or the
nonphosphorylated SYLDVLG peptide to Affi-Gel 15 beads, and the
association of either the CHK-SH2 GST fusion protein or native CHK to
the beads was analyzed. GST-CHK-SH2 was associated in a phosphotyrosine
dependent manner to the phosphorylated SY*LDVLG peptide (data not
shown). Similar specificity was observed when we tested the association
of native CHK to the peptide beads. The phosphorylated SY*LDVLG was
able to associate with native CHK from extracts of PC12 or PC12-CHK
cells, whereas the nonphosphorylated peptide SYLDVLG did not associate
with native CHK (data not shown). This specificity was in agreement
with the peptide inhibition experiments. Taken together, these results,
along with the site-directed mutagenesis of TrkA and the far Western
blotting analysis, indicate that CHK binds to Tyr-785 of TrkA upon NGF stimulation.
Overexpression of CHK Results in Enhanced Activation of the MAP
Kinase Pathway upon NGF Stimulation--
Differentiation of PC12 cells
upon NGF treatment requires activation of the MAP kinase pathway (17,
18, 35). Because CHK is associated with TrkA receptors upon NGF
stimulation, we assessed the effects of overexpression of CHK on
activation of the p44 and p42 MAP kinases following stimulation with
NGF. We stably transfected PC12 cells with CHK-pcDNA3neo,
generating several PC12-CHK clones that overexpress CHK. PC12 cells and
PC12-CHK clones (three independent clones of PC12-CHK) were cultured in the absence or the presence of NGF (100 ng/ml) for the indicated times,
and MAP kinase activity using myelin basic protein as a substrate was
assayed. Although no MAP kinase activity was detected in control PC12
or PC12-CHK cells, an increase in this activity was observed in PC12
cells upon NGF stimulation, with a peak activity at 10 min, followed by
a subsequent decline in activity (Fig. 6A). In PC12 cells
overexpressing CHK, MAP kinase activity was enhanced as compared with
untreated PC12 cells, and this activity was maintained for longer
periods of up to 60 min (Fig. 6A). In addition, p44/42 MAP
kinase activity was measured using antibodies specific to the activated
p44/42 MAP kinases. The phosphokinase antibodies detect p44 and p42 MAP
kinases only when they are activated by phosphorylation at Tyr-204
(36). PC12 cells and three independent PC12-CHK clones were cultured in
the absence or presence of NGF (100 ng/ml) for the indicated times, and
MAP kinase activity was assayed. In PC12 cells overexpressing CHK,
tyrosine phosphorylation of MAP kinases was enhanced compared with
untreated PC12 cells, and this phosphorylation was maintained for
longer periods of up to 60 min (Fig. 6B). These results
indicate a role of CHK in enhancing MAP kinase activity in PC12 cells
upon NGF stimulation and thus its involvement in PC12 cell
differentiation.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of CHK overexpression on MAP kinase
activation. A, quantification of MAP kinase activity in
PC12 cells and PC12-CHK cells: PC12 and PC12-CHK cells were
serum-starved and then stimulated with NGF (100 ng/ml) for the
indicated periods. Total cell lysates were immunoprecipitated with
anti-ERK1 and anti-ERK2 antibodies and assayed for MAP kinase activity.
The phosphorylation level of the substrate, myelin basic protein, was
determined. The plot shows fold increase ± S.D. of MAP kinase
activity over the basal level. B, activation of p44/42 MAP
kinases: PC12 and PC12-CHK cells were serum-starved and then stimulated
with NGF (100 ng/ml) for the indicated periods. Total cell lysates were
prepared, and 10 µg of protein were resuspended in SDS sample buffer.
The samples were separated by 10% SDS-PAGE. The resolved proteins were
analyzed by immunoblotting with anti-phospho-MAP kinase antibody using
an antiserum specifically recognizing tyrosine-phosphorylated MAP
kinases (New England BioLabs). Columns represent the means
from densitometric scanning of three separate experiments. *,
p < 0.05, representing the difference in MAP kinase
activation of PC12-CHK clones versus PC12 cells using
Student's paired or unpaired t test.
|
|
Microinjection of CHK-specific Antibodies Inhibited Neuronal
Differentiation of PC12 Cells--
To determine the requirement for
the binding of the CHK-SH2 domain to TrkA receptors during neurite
outgrowth, we examined the effects of decreasing the intracellular
levels of CHK by microinjecting purified anti-CHK antibodies, anti-Csk
antibodies, control preimmune rabbit antiserum, or control monoclonal
antibody into living PC12 cells. Differentiation of these cells upon
NGF stimulation was inhibited, as indicated by microinjection of
purified anti-CHK antibodies, by morphological changes in neurite
outgrowth and by rapid and dramatic changes in cell shape (cell
rounding) (Fig. 7). When control
antibodies or anti-Csk kinase antibodies were microinjected into PC12
cells followed by NGF stimulation, these cells were differentiated, as
indicated by the appearance of prominent neurite outgrowth similar to
that observed for the PC12 cells treated with NGF alone (Fig. 7). These
data suggest that CHK is involved in neurite outgrowth of PC12
cells.

View larger version (59K):
[in this window]
[in a new window]
|
Fig. 7.
Microinjection of anti-CHK antibody
inhibited NGF-induced neuronal differentiation of PC12 cells.
Subconfluent PC12 cells were subjected to single cell microinjection.
A, PC12 cells were microinjected with purified anti-rabbit
IgG (a-c) and with purified anti-CHK antibody
(d-f). pEGFP-C2 vector was co-injected as a marker.
Microinjected cells were grown in the absence of NGF (a and
d) and in the presence of NGF (100 ng/ml) (b, c,
e, and f) for 48 h and then analyzed for
morphological changes under a UV microscope. Cells with
green fluorescence (c and f) are
microinjected cells shown in the same field as in b and
e. B, microinjection of anti-mouse Csk antibody
(as a negative control) and pEGFP-C2 into PC12 cells in the presence of
NGF (100 ng/ml) for 48 h. Arrows indicate microinjected
cells. C, microinjection of control nonrelevant monoclonal
antibodies (M4) and pEGFP-C2 into PC12 cells in the presence of NGF
(100 ng/ml) for 48 h. a, microinjected cells were
analyzed under a UV light microscope. b, cells in the same
field as in a were analyzed using an immunofluorescent
microscope.
|
|
 |
DISCUSSION |
CHK protein tyrosine kinase is found predominantly in neuronal and
hematopoietic cells. In the present study, we demonstrated direct
binding of CHK to the TrkA receptors, upon NGF stimulation. Furthermore, we identified the binding site of the CHK-SH2 domain to
the activated TrkA receptors as residue Tyr-785. In addition, overexpression of CHK resulted in an enhanced activation of the MAP
kinase pathway upon NGF stimulation of PC12 cells, whereas microinjection of CHK-specific antibodies inhibited the neurite outgrowth of PC12 cells upon NGF stimulation. These results indicate for the first time that CHK, but not Csk, is involved in TrkA signaling
and in neurite outgrowth of PC12 cells.
The association of the SH2 domain of CHK with activated TrkA was
demonstrated using GST fusion proteins containing various domains of
the CHK molecule. The association of CHK with TrkA occurred within the
same time period in which tyrosine phosphorylation of TrkA was observed
upon NGF stimulation. The phosphorylation of TrkA upon NGF stimulation
was rapid and declined slowly, as previously reported (11). Our results
indicated that the CHK-SH2 domain specifically interacts with the
tyrosine-phosphorylated TrkA receptors upon NGF stimulation. The
CHK-SH2 domain also precipitated other proteins, namely p112, p89, and
p77. Although we were not yet able to identify these proteins, the
possibility of their involvement in the association of CHK with TrkA
signaling remains to be investigated in future studies.
Far Western blotting revealed that the binding between activated TrkA
and the CHK-SH2 domain is direct (Fig. 4). The SH2 domain was found to
bind to activated TrkA receptors, which were tyrosine-phosphorylated upon NGF stimulation, whereas the SH3 domain or GST fusion protein failed to bind to TrkA. Therefore, the CHK-SH2 domain can bind to the
activated and phosphorylated TrkA.
Upon interaction with NGF, TrkA, a receptor tyrosine kinase, becomes
autophosphorylated on cytoplasmic tyrosine residues (11, 36), such as
Tyr-490, Tyr-670, Tyr-674, Tyr-675, or Tyr-785 (14, 18, 38). Of the
several autophosphorylated residues of TrkA, three tyrosine residues,
Tyr-490, Tyr-751, and Tyr-785, have been demonstrated to associate with
Shc, p85/PI-3 kinase, and PLC-
1, respectively (15, 16, 18, 33). All
three signaling molecules, Shc, PLC-
1, and PI-3 kinase, are
demonstrated to play a major role in NGF-mediated neurite outgrowth
(14, 17-19). In the present study, mutation analysis revealed that
residue Tyr-785 on TrkA is required for it to associate with the
CHK-SH2 domain (Fig. 5). This association was also confirmed by peptide
inhibition assay (Fig. 5D). The Tyr-785 site is known to
bind to PLC-
1. These results suggest that CHK and PLC-
1 share the
same tyrosine residue for binding with TrkA receptors. Interestingly,
platelet-derived growth factor receptor, a receptor tyrosine kinase
like TrkA, also autophosphorylates several tyrosine residues, and the
tyrosine residue sites Tyr-579 and Tyr-581 are known to bind to the SH2 domains of Shc and Src family kinases (32-39). Therefore, the signal transduction pathway through Tyr-785 on TrkA needs to be further investigated for its potential interactions with additional signaling molecules.
CHK is undetectable in early embryos and begins to be expressed around
E15 (27, 40, 41). It then increases progressively up to the time of
birth and remains high in the adult central nervous system. CHK
expression correlates with late stage development and neuronal
differentiation. In contrast, Csk is expressed throughout embryonic
development and remains high in the central nervous system until birth.
Csk is then dramatically down-regulated in the adult brain, except in
the olfactory bulb (27, 41). Such diametrically opposite temporal
expression patterns of Csk and CHK suggest distinct roles for both
these proteins, presumably mediated via different substrates. Csk is
known to inhibit Src family kinases, and this inhibition results in a
down-regulation of the MAP kinase pathway (42). However, our results
indicate that overexpression of CHK resulted in enhanced activation of the MAP kinase pathway in PC12 cells upon NGF stimulation. In addition,
we have observed that the neuronal differentiation of PC12 cells
induced by NGF was inhibited by microinjection of anti-CHK antibodies,
but not anti-Csk antibodies (Fig. 7), suggesting that CHK and Csk
regulate different targets in the signaling pathways of neuronal cells.
Thus, our findings strongly suggest that CHK plays a role in regulating
the differentiation of PC12 cells. Future studies will further
investigate the molecular mechanisms of the involvement of CHK in
neuronal differentiation.