From the Department of Biological Sciences, Program in Cellular and Molecular Biosciences, Auburn University, Auburn, Alabama 36849
Received for publication, August 19, 2002, and in revised form, December 1, 2002
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ABSTRACT |
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Previous work demonstrated an essential role for
the atypical protein kinase C interacting protein, p62, in neurotrophin
survival and differentiation signaling. Here we show that p62 interacts not only with TrkA but also with TrkB and TrkC, which are the primary
receptors for brain-derived neurotrophic factor and neurotrophin-3. The
interaction of p62 with TrkA requires the kinase activity of TrkA.
Mapping analysis indicates that p62 does not compete with Shc for
binding to TrkA, and p62 association was confined to the juxtamembrane
region of TrkA, amino acids 472-493. By immunofluorescence the
colocalization of p62 and TrkA was observed 30 min post-nerve growth
factor treatment within overlapping vesicular structures. Upon
subcellular fractionation, activated TrkA colocalized to an endosomal
compartment and p62 was coassociated with the receptor post-nerve
growth factor stimulation. Moreover, an absence of p62 blocked
internalization of TrkA without an effect on phosphorylation of either
TrkA or MAPK; however, Erk5 signaling was selectively abrogated. We
propose that p62 plays a novel role in connecting receptor signals with
the endosomal signaling network required for mediating TrkA-induced differentiation.
Nerve growth factor
(NGF)1 regulates survival,
differentiation, and maintenance of neurons. NGF belongs to a family of
structurally related neurotrophins such as brain-derived neurotrophic
factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5)
(1). The biological effects of the neurotrophins are mediated by two classes of cell surface receptors, namely a high affinity tyrosine kinase Trk receptor and a low affinity p75NTR receptor.
TrkA is the prototype of the Trk family and specifically binds NGF,
whereas TrkB and TrkC are the primary receptors for BDNF and NT-3,
respectively (2). NGF binding to TrkA stimulates dimerization and
autophosphorylation of tyrosine residues Tyr499,
Tyr679, Tyr683, Tyr684, and
Tyr794 in the intracellular domain of the receptor, thereby
creating sites for binding and activation of signaling intermediates
(3) such as phospholipase C (PLC)- Previous work in our laboratory has shown that the atypical protein
kinase C-interacting protein, p62, interacts with TrkA and binds tumor
necrosis factor receptor-associated factor 6 (TRAF6), which in turn
interacts with p75. TRAF6-p62 forms a complex that serves as a bridge
to link the common neurotrophin receptor, p75, with TrkA (12). p62 then
recruits atypical protein kinase C (aPKC) to phosphorylate I In this present study, we observed that the interaction of p62 with
TrkA requires the tyrosine phosphorylation of TrkA. Mapping analysis
indicates that p62 does not compete with Shc for binding to the TrkA,
and p62 association was confined to the juxtamembrane region of TrkA.
Both p62 and TrkA were colocalized within similar vesicular structures.
We also demonstrate the intracellular localization of activated TrkA
along with p62 in an endosomal compartment upon NGF stimulation. In
addition, antisense p62 was found to block the internalization of TrkA
receptor with a specific effect on the activation of Erk5 pathway. Our
observations, viewed in the context of existing knowledge, are
compatible with a model where p62 may play a role in trafficking of the
TrkA receptor to the endocytic pathway.
Materials--
Anti-TrkA serum, which recognizes the
extracellular domain of TrkA, was obtained from Louis Reichardt,
University of California, San Francisco. The
anti-pantothenate Trk polyclonal antibody (C-14), anti-Trk
monoclonal antibody (B-3), raised against the C-terminal region of
TrkA, and monoclonal Trk antibody (E-6) that recognizes Tyr496 phosphorylated TrkA (19) were purchased from Santa
Cruz Biotechnology. Anti-phosphotyrosine (PY20) anti-p62 was purchased
from BD Transduction Laboratories, and anti-Myc, anti-HA, and anti-GST
monoclonal antibody and rabbit anti-HA were from Santa Cruz
Biotechnology. Erk1/2 and Erk5 antibodies were obtained from Upstate
Biotechnology, inc. 2.5 S NGF was obtained from Bioproducts for
Science, and 125I (5 mCi) was from PerkinElmer Life
Sciences. Reagents for SDS-PAGE and protein molecular weight standards
were bought from Bio-Rad. Enhanced chemiluminescence (ECL) reagents, a
horseradish peroxidase-conjugated secondary antibody, and hyperfilm
were purchased from Amersham Biosciences.
Cell Culture--
Human embryonic kidney 293 (HEK 293) cells
were cultured in high glucose Dulbecco's modified Eagle's medium
(DMEM) containing 10% heat-inactivated fetal calf serum. PC12 cells
were grown on culture and were coated with rat tail collagen in DMEM
containing 10% heat-inactivated horse serum, 5% heat-inactivated
fetal calf serum, 50 µg/ml streptomycin, and 50 units/ml penicillin.
For all experiments, 24 h prior to stimulation, the medium was
replaced with medium containing reduced serum at a ratio of 1 part
complete medium/5 parts serum-free medium and then treated with 50 ng/ml NGF at 37 °C. For inhibition of tyrosine phosphorylation of
TrkA, PC12 cells were treated with different concentrations of K252a (Biomol Research Laboratories Inc.) for 60 min before adding NGF. PC12
cells were gently washed with ice-cold phosphate-buffered saline (PBS)
and harvested by centrifugation. The cell pellets were incubated on ice
for 30 min and then disrupted by sonication for 5 s in 300 µl of
lysis buffer (20 mM Tris-Cl, pH 7.4, 150 mM
NaCl, 1% Triton X-100, 20 mM NaF, 2 mM
p-nitrophenyl phosphate, 1 µg/ml leupeptin, and 2 mM sodium orthovanadate). The larger cellular debris was
removed by centrifugation at 12,000 × g in a
microcentrifuge at 4 °C for 3 min. Protein concentration of the
supernatants was determined using the Bio-Rad reagent based on Bradford
procedure with bovine serum albumin (BSA) as a standard.
Preparation of GST-Shc Fusion Protein and Subcellular
Fractionation--
GST-Shc in glutathione-agarose beads was prepared
from a single colony of Escherichia coli cells containing
the recombinant GST-Shc plasmid as described previously (17). The
protein was released from the beads by adding buffer containing 10 mM glutathione in 50 mM Tris, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol and rotated for
2 h at 4 °C. The supernatant containing the GST-Shc protein was
collected by centrifugation at 3000 × g at 4 °C.
Protein concentration was determined using the Bio-Rad reagent.
Subcellular fractions of cytosol, endosomes, Golgi, lysosomes, plasma
membrane, small membrane, and nucleus were isolated from PC12 cells as
characterized and described previously (17). The protein at the 32/45%
sucrose interface has been characterized as endosomes (17) and was used for immunoprecipitation.
DNA Transfections--
Both PC12 and HEK 293 cells were
transfected using LipofectAMINE 2000; alternatively, in some
experiments HEK 293 cells were transfected by the calcium phosphate
procedure (12). After 36 h of transfection, cells were stimulated
or not with 50 ng/ml NGF. Cells were harvested and lysed in 1 ml of PD
buffer (40 mM Tris-HCl, pH 8.0, 500 mM NaCl,
0.1% Nonidet P-40, 6 mM EDTA, 6 mM EGTA, 10 mM Immunoprecipitation and Western Blot Analysis--
Equal volume
of whole cell lysates (750 µg) were incubated with 3 µg of
appropriate primary antibody for 3 h at 4 °C, followed by 40 µl of agarose-coupled secondary antibody for an additional 2 h
at 4 °C. The immunoprecipitates were then washed five times with PD
buffer. The proteins were released by boiling for 2 min in SDS-PAGE
sample buffer electrophoresed on 10% SDS-PAGE, followed by transfer to
nitrocellulose membranes, and subjected to Western blot analysis with
the corresponding antibodies (20). In some experiments, whole cell
lysates were blotted with antibody to either phosphorylated Erk1/2 or
non-phosphorylated Erk5 (21). Erk5 activity was measured by immune
complex kinase assay with myelin basic protein as substrate, employing
buffers and conditions as described previously (22).
GST Pull-down Assay--
The HEK 293 cell lysates were set up to
equal protein concentration (750 µg). GST-ZIP protein in beads was
blocked for 1 h at 4 °C with 0.5% BSA in PBS, and the beads
were washed 3 times with binding buffer (20 mM Tris, 100 mM NaCl, 2 mM EDTA, 0.1% Triton X-100, 2 mM dithiothreitol, 0.05% BSA, and 5% glycerol). 5 µg of
GST-ZIP was added to the cell lysates and rotated for 90 min at
4 °C. The samples were washed 5 times with GST wash buffer (2.5 mM Tris, 2.5 mM EDTA, 250 mM NaCl,
0.1% Triton X-100, and 10% glycerol), and SDS-PAGE sample buffer was
added, and the samples were boiled for analysis.
Immunofluorescence and Confocal Microscopy--
PC12 cells were
grown on coverslips in 24-well plates in DMEM. Cells were serum-starved
24 h before treating with 50 ng/ml NGF at 4 °C for 1 h.
Fresh warm serum-free DMEM was added and chased NGF by incubating at
37 °C for different times. Cells were gently washed two times with
phosphate-buffered saline (PBS), fixed with methanol for 5 min at
4 °C, and washed three times with PBS. The cells were blocked by
adding 3% BSA in PBS for 1 h at room temperature, followed by
addition of primary antibody in 0.2% BSA in PBS and incubated
overnight at 4 °C. Cells were washed three times in PBS
and followed by addition of secondary antibody in 0.2% BSA in PBS and
allowed to incubate for 1 h in the dark at room temperature. The
cells were finally washed four times in PBS containing 0.05% Tween 20, rinsed in PBS, distilled H2O, blotted dry, mounted, and
sealed onto slides for observation by confocal microscopy. The images
were captured with an MRC-1024 laser scanning confocal microscope
(Bio-Rad) employing five passes with a Kalman filter. The images were
processed by Confocal Assistant 4.02 (Bio-Rad) and Adobe Photoshop 5.0 (Adobe Systems, Mountain View, CA).
FACS Analysis--
PC12 cells overexpressing TrkA in 60-mm
plates were allowed to bind NGF (50 ng/ml) for 1 h at 4 °C. The
cells were harvested in PBS and incubated at 37 °C for different
times, followed by addition of 500 µl of 1:5000 dilution of anti-TrkA
serum, which recognizes the extracellular domain of TrkA (23) in 0.2%
BSA in PBS at 4 °C for 1 h. The cells were washed three times
with PBS followed by incubation with 500 µl of 1:1000 dilution of
fluorescein isothiocyanate in 0.2% BSA in PBS at 4 °C for 30 min.
Cells were washed five times with PBS and fixed in 1 ml of 2%
paraformaldehyde in PBS at room temperature for 30 min, and 10,000 cells per sample were analyzed in FACScan (Elite model,
Beckman-Coulter) equipped with CellQuest software (24, 25).
Iodination of NGF--
Iodination of NGF was
performed as described previously (26). The reaction was carried at
room temperature with a mixture of 4 mCi of Na125I, 75 µl
of 0.1 M phosphate buffer, pH 7.4, 15 µl of
lactoperoxidase (30 µg/ml) in the phosphate buffer, 50 µg Ligand-Receptor Complex Internalization Assay--
The acid-wash
technique (27) was used to determine the kinetics of NGF-induced
internalization of TrkA. PC12 cells expressing TrkA were allowed to
bind 0.4 µCi/ml 125I-NGF alone, or with excess NGF (200 ng/ml), and 0.1 mg/ml BSA at 4 °C for 1 h. The medium was
subsequently removed and fresh warm serum-free DMEM was added and
incubated at 37 °C for the indicated time. The cells were washed
with 1 ml of ice-cold 0.2 M acetic acid and 0.5 M NaCl for 6 min to remove the surface
125I-NGF. The cells were then washed once with 1 ml of
ice-cold PBS and lysed with 1 ml of 1% SDS with 0.1 N
NaOH. The radioactive cell lysate was then counted on a p62 Directly Binds the Neurotrophin Trk Receptors in a
Phosphotyrosine-dependent Manner--
As a preliminary step,
we analyzed the ability of p62 to interact with various Trk receptors.
HEK cells were cotransfected with cDNAs encoding HA-tagged TrkA,
TrkB, or TrkC along with Myc-tagged p62 followed by stimulation with or
without NGF, BDNF, or NT-3 respectively. The lysates were
immunoprecipitated with anti-HA or anti-Myc antibody and probed with
anti-HA. In control immunoprecipitates (anti-HA), all three
neurotrophin receptors were equally expressed; p62
coprecipitated with all three Trk receptors. Moreover, the association
of p62 with Trk was dependent upon the stimulation with neurotrophin
(Fig. 1). However, BNDF treatment
consistently resulted in enhanced association of p62 with TrkB. By
comparison, TrkA or -C both possessed a large degree of p62 associated
with the receptor in the basal unstimulated state. Collectively, these results reveal that p62 interacts not only with the NGF receptor TrkA,
but also with TrkB and TrkC, which are the primary receptors for
brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3).
To determine whether the association of p62 with TrkA receptor required
the activation of TrkA, HEK 293 cells were cotransfected with wild-type
or kinase-inactive form of TrkA (K538A) and Myc-tagged p62, followed by
NGF treatment. TrkA was immunoprecipitated from lysates and Western
blotted with anti-phosphotyrosine and Myc antibody. We observed that
wild-type TrkA was autophosphorylated and interacted with p62 on NGF
stimulation (Fig. 2A,
3rd lane), whereas the kinase inactive form was not
tyrosine-phosphorylated and poorly interacted with p62 (Fig.
2A, 4th lane).
We next conducted a parallel experiment in PC12 cells treated with the
kinase inhibitor K252a, which prevents the tyrosine phosphorylation of
TrkA upon NGF stimulation (28, 29). When immunoprecipitation was
carried out with anti-TrkA followed by Western blotting for
anti-phosphotyrosine or anti-p62 antibody, we observed that K252a
treatment prevented the interaction between p62 and TrkA (Fig.
2B, 2nd to 4th lanes), when compared
with cells stimulated with NGF, which were not treated with K252a (Fig.
2B, 2nd lane). Thus, p62 interacts with TrkA in a
phosphotyrosine-dependent manner.
Mapping the Interaction of p62 with TrkA--
To map the binding
site within TrkA that mediates p62 interaction, HA-tagged TrkA receptor
mutants: S17 (
p62 interacts with TrkA and recruits aPKC, which leads to
phosphorylation of I
To explore further the p62-interacting region within TrkA, a series of
deletion mutants were employed; TrkA-(1-452), TrkA-(1-472), TrkA-(1-493), TrkA-(1-501), and TrkA-(1-522) (Fig.
3A) were transfected in HEK
293 cells. After NGF stimulation the cell lysates were immunoprecipitated with anti-p62 (Fig. 3B). Alternatively
the lysates were also subjected to in vitro GST-p62
pull-down assay (Fig. 3C), followed by Western blotting for
TrkA employing a TrkA antibody that recognizes the extracellular domain
of TrkA (23) to detect TrkA complexed with p62. Full-length TrkA and
TrkA-(1-522) coprecipitated endogenous p62 (lanes 2 and
3), whereas TrkA-(1-501) and TrkA-(1-493) showed slight
interaction with p62 (4th and 5th lanes);
however, no interaction was observed with TrkA-(1-472) or
TrkA-(1-452) (lanes 6 and 7) by either
coimmunoprecipitation or GST pull-down assay (Fig. 3, B and
C). These findings reveal that amino acids 472-493 in the
juxtamembrane region play a role in the interaction of p62 with
TrkA.
Colocalization of p62 with TrkA in Endocytic Vesicles--
Because
p62 interacts with the TrkA receptor, which is subsequently
internalized to an endosomal compartment (18), we next analyzed whether
p62 colocalizes with TrkA within vesicular structures by double
immunofluorescence and confocal microscopy (Fig.
4). To examine the intracellular
colocalization of p62 with TrkA, PC12 cells overexpressing TrkA were
treated with NGF for 1 h at 4 °C to allow binding at that
temperature which inhibits membrane trafficking (7). The unbound NGF
was extensively washed, and the cells were warmed for different times
at 37 °C to permit endocytosis. Both p62 and TrkA showed a similar
punctate staining pattern within endocytic vesicles (Fig. 4) throughout
the course of NGF treatment. By 30 min, there was increased
colocalization of p62 with TrkA in a majority of the vesicles. All of
the p62 containing vesicular structures colocalized with internalized
TrkA; however, there were numerous TrkA containing vesicles that did
not colocalize with p62. It thus appears that p62 only associates with
a fraction of the intracellular receptor pool of activated TrkA
receptors. In addition, as a fraction of the TrkA pool was observed to
recycle to the cell membrane (60 min), p62 containing vesicles remained coassociated with intracellular vesicles. Taken together, these results
provide evidence for the presence of p62 in the same intracellular sites of trafficking as TrkA receptor.
Interaction of Activated TrkA with p62 in Endosomes--
Previous
studies have shown that p62 colocalizes with aPKC in endosomes (16),
and also p62 acts as a shuttling protein involved in routing activated
aPKC to an endosomal compartment (16, 17). In PC12 cells, p62 has been
purified as a component of the late endosomal compartment and
colocalizes with specific marker proteins of the endosomal-lysosomal
network (17). Upon NGF treatment most of the activated TrkA has been
shown to localize to the endosomal network (18, 24). Because p62
colocalizes with internalized TrkA in endocytic vesicles (Fig. 4), it
is possible that upon NGF stimulation p62 may be routed along with
activated TrkA to the endosomes and may play a role in degradation of
TrkA. In order to test this possibility, we employed a previously
characterized subcellular fractionation procedure that had been adapted
for the subcellular fractionation of membranes employing a sucrose step
gradient (17). PC12 cells overexpressing TrkA were allowed to bind NGF
at 4 °C for 1 h and chased at 37 °C for 0 and 15 min, followed by subcellular fractionation. When the equivalent protein of
various fractions (small membrane, plasma membrane, cytoplasm, nucleus,
lysosomes, endosomes, and Golgi) were Western-blotted with an anti-TrkA
antibody (E-6) that recognizes only phosphorylated and activated TrkA
(19), an increased level of phosphorylated TrkA was observed in both
endosomes and nucleus at 15 min of NGF stimulation (Fig.
5A, lanes N and
E). It is likely that the increase in the nucleus reflects
contamination of the nuclear pellet with the membrane fraction because
the fractionation procedure produces a crude nuclear pellet (17). The
presence of activated TrkA within the endosomal compartment in resting
cells is consistent with other studies that have observed a significant
amount of activated TrkA within the endosomes of resting PC12 cells
(24). As a measure of purity for the separation of the vesicular
network, an aliquot of the separated fractions were Western-blotted
with affinity-purified Rab7 antibody, a protein that is enriched in the
late endosomes (16, 17). The 18/32% interface collected as the
endosomal fraction was enriched in Rab7 (Fig. 5B). To
determine whether p62 and TrkA are cotrafficked and remain coassociated within the endosome, equivalent protein from the endosomal fraction was
immunoprecipitated with anti-TrkA and Western-blotted with anti-phosphotyrosine (PY20) or p62 antibody. We observed that TrkA in
the endosome was tyrosine-phosphorylated and hence confirmed the
sequestration of activated TrkA receptor in the endosome (Fig. 5B, lane P). Moreover, TrkA complexes contained
p62, and the interaction of p62 with TrkA was dependent upon treatment
with NGF (Fig. 5C, 2nd lane). Collectively, these
findings reveal that p62 associates with activated TrkA and is
cotrafficked to an endosomal compartment along with the receptor.
p62 Influences TrkA Trafficking--
Because p62 localized with
TrkA to the endosomal network, and remained associated with a fraction
of activated TrkA along the endocytic route (Fig. 4), we sought to
investigate whether p62 may affect trafficking of TrkA receptor. PC12
cells were transfected with Myc-tagged p62 (OEp62) or a full-length
antisense construct of p62 (ASp62), which has been shown to deplete the
levels of p62 upon transfection (12, 15). As control, the expression levels of TrkA and p62 were examined employing equal protein
concentration of whole cell lysate (Fig.
6A). The level of
TrkA was the same in all the lysates, but p62 expression increased in
cells transfected with Myc-tagged p62 (2nd lane) and was
significantly decreased in cells transfected with ASp62 (3rd
lane).
To examine the effect of p62 upon trafficking of TrkA, the cells were
transfected with a full-length antisense p62 and treated with NGF for
1 h at 4 °C and chased at 37 °C for 30 min. Transfection of
antisense (AS) p62 blocked trafficking of TrkA into the vesicular punctate structures. The majority of TrkA staining was at the cell
surface, and little or no p62 staining was observed when compared with
the control non-transfected cells (Fig. 6B), thus confirming
decrease in p62 expression induced by ASp62 construct.
As a separate independent measure, the effect of p62 upon NGF receptor
internalization was examined by employing fluorescence-activated cell
sorting (FACS) to detect cell surface TrkA employing an antibody, which
recognizes the extracellular domain of TrkA (23) (Fig. 6C).
The cell surface expression of TrkA was measured in control, cells
transfected with antisense p62, and in cells overexpressing p62. PC12
cells overexpressing TrkA were treated with NGF for 1 h at 4 °C
to allow binding and were chased for different times at 37 °C to
permit internalization. As shown in Fig. 6C, 30 min post-warming, NGF reduced the expression of TrkA at the cell surface in
both control and in cells overexpressing p62, representing the
internalization of TrkA in these cells. On incubation at 37 °C for
60 min, there was a significant increase in the expression of TrkA at
the cell surface. However, no change in the expression of cell surface
TrkA receptor was observed in cells transfected with antisense p62.
In addition, the extent of TrkA internalized was also assessed by
125I-NGF binding assay in control, PC12 cells transfected
with antisense p62 and in cells overexpressing p62. Cells were
incubated with 125I-NGF alone or with excess unlabeled NGF
for 1 h at 4 °C, and then warmed at 37 °C for different
times. The cell surface 125I-NGF was eliminated by acid
washing; the cells were lysed, and internalized 125I-NGF
was estimated in a Effects of p62 on TrkA Receptor Signaling--
NGF binding to the
TrkA receptor plays a role in the activation of two distinct signaling
cascades, MAPK/Erk kinase 1/2 (11) and the Erk5 pathways (21). Both
pathways are dependent upon TrkA kinase activity, as K252a pretreatment
abolishes NGF induced activation of these pathways. However, Erk5
activation is also dependent upon internalization of TrkA (21), and
likewise TrkA delivery to the signaling endosome is required for
NGF-mediated differentiation (11). Because p62 modulated
internalization of TrkA (Fig. 6, A-D), as well as
NGF-mediated differentiation (17), we set out to examine whether p62
expression affected TrkA signaling of either MAPK or Erk5 (Fig.
7). PC12 cells were transfected with
either ASp62 or Myc-tagged p62 (OEp62). The cells were stimulated with
NGF for different times followed by immunoprecipitation of TrkA. NGF
induced transient phosphorylation of TrkA, which coincided with both
MAPK and Erk5 activation (Fig. 7A). Overexpression of p62
was without any effect on either the phosphorylation of TrkA or
downstream signaling targets. However, the removal of p62 with the
antisense construct slightly enhanced MAPK signaling when compared with
either the control cells or those overexpressing p62. We next set out
to examine the effect of p62 removal on the activation of Erk5.
By employing two separate methods, the examination of Erk5
phosphorylation by Western blot analysis (Fig. 7A) and immune complex kinase assay (Fig. 7B) demonstrated that
depletion of p62 severely abrogated NGF-induced activation of Erk5,
whereas overexpression of p62 enhanced Erk5 activity compared with
control. Altogether these findings reveal that p62 connects the
trafficking/internalization of TrkA to the Erk5 pathway.
Intracellular Colocalization of TrkA and p62 in Differentiated
Cells--
Because transport of NGF is required for mediating the
longer term effects of NGF on differentiation of PC12 cells (11), we
sought to examine whether p62 and TrkA were colocalized in NGF-differentiated PC12 cells. Hence PC12 cells were allowed to differentiate and form neurites by treatment with NGF. Both p62 and
TrkA showed punctate staining pattern in the cell body (×63) and along
the neurites (×100) by double labeling as shown in Fig. 8. These findings suggest that
cotransport of p62 with TrkA may play a role in prolonged signaling
required for NGF differentiation via moving signals from axon terminals
to neuronal cell bodies.
Our findings support a model where p62 interaction with TrkA
regulates the internalization and localization of the receptor (Fig.
9). Results obtained by
coexpression/immunoprecipitation, native coassociation, and
immunofluorescence confirm the interaction and colocalization of p62
with TrkA. Moreover, the coassociation between the two proteins is
retained upon trafficking of the receptor to the endosomal compartment.
Interestingly, an absence of p62 blocks receptor internalization as
measured by three independent means: immunofluorescence staining, FACS
analysis of cell surface TrkA expression, or association of
125I-NGF within the cell. In this study we observed a
significant amount of TrkA or 125I-NGF recirculation to the
cell surface. The kinetics and reappearance of TrkA at the cell surface
correlate well with earlier more extensive studies on receptor
internalization/recycling (32, 33). We have shown previously (12) that
the interaction between p62 and TrkA peaks at 15 min post-NGF
treatment, whereas internalization of NGF peaks at 30 min. The kinetics
are thus consistent with a model whereby p62 binding precedes the
internalization of TrkA. On the other hand, p62 can also bind the
common neurotropin receptor p75 through the signaling adapter TRAF6
(12). The kinetics of binding between p62 and p75 is an early rapid
event, taking place between 1 and 5 min (12). Recent studies (24)
suggest that p75 is capable of modulating TrkA internalization. In the
context of p62, we speculate that p75-TRAF6-p62 may prime the complex causing a conformational change thereby allowing for optimal
interaction between p62/TrkA. Further studies will be needed to explore
this possibility.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (4), adapter proteins Shc and
FRS2 (5), and phosphatidylinositol 3'-kinase (6) that initiates the
intracellular signaling cascades. It has been observed that NGF rapidly
induces internalization of TrkA receptor to endocytic vesicles (7-9).
The receptors within signaling vesicles remain catalytically active and
phosphorylated because they are less accessible to membrane-associated
phosphatases (10). Internalized TrkA receptors induce a higher peak
level of mitogen-activated protein kinase (ERK/MAPK) activation thereby
regulating cell differentiation (11).
B kinase
and leads to the activation of the transcription factor nuclear
factor-
B (NF-
B). Expression of antisense p62 in PC12 cells
inhibits NGF-induced NF-
B activation (12). In contrast, it has been
shown that a dominant-negative mutant of the Shc adaptor protein
effectively blocks TrkA-mediated activation of NF-
B (13). p62 also
serves as a scaffold for the NF-
B pathway through tumor necrosis
factor-
and interleukin-1 receptor signaling cascades (14, 15).
Endogenous and ectopically expressed p62 has been shown to colocalize
with
/
PKC and
PKC in lysosome-targeted endosomes (16, 17). p62
binds the NGF receptor, TrkA (12), which likewise localizes to late
endosomal vesicles. Recent studies have shown that internalized TrkA
receptors continue to signal within the endosomal compartment (18) and are required to mediate NGF-induced differentiation (10). Moreover, inhibition of p62 expression has been shown to block NGF-induced neurite outgrowth (17). Hence, it is possible that p62 may be critical
for the transport of TrkA from the plasma membrane to the endosome.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, 10 mM NaF, 10 mM phenyl phosphate, 300 µM
Na3VO4, 1 mM benzamide, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM dithiothreitol) for 30 min on ice, followed by centrifugation at 1400 rpm for 15 min at 4 °C to remove the insoluble fraction. The protein
concentration of the supernatant was determined by the Bradford method.
NGF,
and 15 µl of 1:104 dilution of
H2O2 (30%) in phosphate buffer. After 30 min
another 15 µl of H2O2 was added, and the
reaction was allowed for an additional 1 h. Iodinated NGF was
separated from free 125I through a Sephadex G-50 column.
Fractions of 500 µl were collected, and aliquots of 5 µl were
counted on a
-counter (Topcount NXT microplate scintillation and
luminescence counter, Packard Instruments), and a molecular weight of
125I-NGF was estimated by SDS-PAGE. The peak fractions
recovered from the column were used in subsequent receptor binding assays.
-counter,
and specific internalization (internalized 125I-NGF minus
internalized 125I-NGF with 200 ng/ml NGF) was assessed for
each time point.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Interaction of p62 with TrkA, -B, and
-C. HEK 293 cells were cotransfected with HA-tagged TrkA, -B, or
-C along with Myc-tagged p62 followed by stimulation with or without
NGF, BDNF, and NT-3 (50 ng/ml) for 15 min as shown. The interaction was
determined by immunoprecipitation (IP) of the cell lysates
(750 µg) with anti-HA and anti-Myc and Western blotting
(WB) with anti-HA as shown. As control, a fraction of
the lysate (40 µg) was blotted with anti-HA or Myc to
check for the expression of Trks and p62. This experiment is
representative of three separate experiments.
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Fig. 2.
Kinase activity of TrkA is required for
interaction with p62. A, subconfluent cultures of HEK 293 cells were cotransfected with wild-type TrkA or kinase-inactive K538A
TrkA and Myc-tagged p62 followed by treatment with NGF (50 ng/ml) for
15 min. The lysates (750 µg) were immunoprecipitated (IP)
with anti-TrkA (B-3) and followed by Western blot (WB)
analysis with anti-phosphotyrosine antibody, PY20 or Myc. The blot was
reprobed with anti-TrkA (C-14) antibody. The lysates (40 µg) were
Western-blotted with anti-TrkA (C-14) or Myc to verify the protein
expression levels. B, PC12 cells were pretreated with K252a
(0-300 nM) or not ( ) for 1 h prior to stimulation
with 50 ng/ml NGF for 15 min. Tyrosine phosphorylation of TrkA was
determined by immunoprecipitation with anti-TrkA (E-6) followed by
Western blotting with anti-phosphotyrosine antibody, PY20, or p62. The
blot was reprobed with anti-TrkA (C-14) antibody. Cell lysate was
analyzed by blotting with anti-TrkA (B-3) or p62. This experiment is
representative of three separate experiments.
450KFG452), S3
(
493IMENP497), S8 (Y499F), and S9 (Y794F)
that affect specific signaling pathways were used. Shc binds
Tyr499; PLC
-1 binds Tyr794; both
493IMENP497 and
450KFG452 stimulate NGF-dependent
cell cycle arrest or neuronal differentiation, and
450KFG452 is also involved in phosphorylation
of FRS2 (30, 31). TrkA mutants were coexpressed with Myc-tagged p62 in
HEK 293 cells, followed by immunoprecipitation with anti-HA and Western
blotting for HA-TrkA and Myc-p62. Wild-type TrkA and all the TrkA point mutants (S17, S3, S8, and S9) coprecipitated with Myc-p62 (data not
shown). These results suggest that these amino acids of TrkA were not
required for its interaction with p62.
B kinase leading to the activation of NF-
B (12). The adapter protein Shc has also been shown to bind TrkA on NGF
stimulation (31). Moreover, a dominant-negative mutant of Shc inhibits
NF-
B activation (13). As both p62 and Shc are involved in NF-
B
pathway, we also examined whether p62 might compete for Shc binding to
TrkA. We transfected HEK 293 cells with HA-tagged TrkA and Myc-tagged
p62 cDNAs, followed by immunoprecipitation with anti-HA in the
presence of increasing concentrations of GST-Shc, and we tested for the
interaction of p62 or Shc with TrkA. We observed that as the
concentration of GST-Shc increases, the interaction of p62 with TrkA
was not affected, thus confirming that p62 does not compete with the
Shc-binding site for interaction with TrkA (data not shown).
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Fig. 3.
Mapping the p62 interacting region in
TrkA. A, schematic representation of the TrkA deletion
mutants TrkA-(1-452), TrkA-(1-472), TrkA-(1-493), TrkA-(1-501), and
TrkA-(1-522). B, TrkA, wild-type and mutants were expressed
in HEK 293 cells followed by NGF (50 ng/ml) stimulation for 15 min. The
interaction was determined by immunoprecipitation (IP) of
the cell lysates (750 µg) with anti-p62 and Western blotting
(WB) with anti-TrkA, which recognizes the extracellular
domain of TrkA or p62. C, HEK 293 cell lysates (500 µg)
expressing various TrkA mutants were interacted with an equivalent
amount of GST-p62 in a pull-down assay. The association of TrkA was
determined by Western blotting with TrkA antibody. These experiments
are representative of three separate experiments.
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Fig. 4.
Confocal microscopic analysis of the
codistribution of p62 and TrkA in PC12 cells overexpressing TrkA.
Cells were treated with NGF (50 ng/ml) for 1 h at 4 °C and
chased at 37 °C for 0, 10, 30, and 60 min, followed by fixation and
staining with both anti-p62 and anti-TrkA (B-3) antibody. The extent of
colocalization was assessed by superimposing red (p62, Texas Red) and
green (TrkA, Oregon Green) signals. Areas of colocalization are noted
by the presence of arrows. These experiments are
representative of three separate experiments.
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Fig. 5.
Colocalization of activated TrkA with p62 in
endosomes. A, PC12 cells were treated with NGF (50 ng/ml) for 1 h at 4 °C and chased at 37 °C for 0 and 15 min.
Cells were fractionated into small membrane (S), plasma
membrane (P), cytoplasm (C), nuclei
(N), lysosomes (L), endosomes (E), and
Golgi (G). Equivalent protein from each fraction (30 µg)
was Western-blotted (WB) with anti-TrkA (E-6), which
recognizes the activated TrkA receptor. B, the separated
fractions were also Western-blotted with affinity-purified Rab7
antibody. C, the endosomal fraction (350 µg) was
immunoprecipitated (IP) with anti-TrkA (C-14) followed by
Western blotting with anti-phosphotyrosine antibody, PY20 or p62. The
TrkA blot was stripped and reprobed with anti-TrKA (C-14)
antibody.
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Fig. 6.
p62 influences TrkA trafficking.
A, PC12 cells overexpressing TrkA were transfected with
Myc-tagged p62 (OE) or ASp62. 60 µg of protein from the
cell lysates were Western-blotted (WB) with anti-TrkA (B-3)
and anti-p62 antibody to check the expression level of each protein.
B, PC12 cells expressing TrkA were transfected with or
without antisense (AS) p62. Cells were treated with NGF (50 ng/ml) for 1 h at 4 °C and chased at 37 °C for 30 min
followed by staining for anti-p62 and anti-TrkA (B-3) antibody and
examined by confocal microscopy. C, PC12 cells expressing
TrkA were transiently transfected with ASp62 or Myc-tagged p62. The
cells were allowed to bind NGF (50 ng/ml) for 1 h at 4 °C and
chased at 37 °C for 0, 10, 30, and 60 min. Thereafter, the cells were incubated with rabbit anti-TrkA serum
which recognizes the extracellular domain of TrkA at 4 °C for 1 h, washed, and incubated with fluorescein isothiocyanate-labeled goat
anti-rabbit at 4 °C for 30 min. The cells were washed, fixed, and
subjected to FACS analysis. The graph represents the percentage of cell
surface TrkA receptor at different time points. D, the cells
were transfected as above and were incubated with 0.4 µCi/ml
125I-NGF alone or with excess unlabeled NGF (200 ng/ml) for
1 h at 4 °C and chased at 37 °C for 0, 10, 30, and 60 min.
The cell surface 125I-NGF was removed by acid wash,
followed by lysis with 1% SDS in 0.1 N NaOH. Specific
internalized counts (125I-NGF internalized minus
125I-NGF internalized in presence of excess NGF) were
determined by -counting. The data shown are mean ± S.D. of the
internalized NGF receptor for three different experiments. The data
were analyzed using Student's t test (#, p < 0.005; *, p < 0.001).
-counter. Cells transfected with antisense p62
did not internalize 125I-NGF during any period of NGF
treatment. In cells overexpressing p62, there was a significant
(p < 0.005) increase in internalization of
125I-NGF at 30 min of NGF incubation at 37 °C when
compared with control (Fig. 6D). When cells were incubated
for 60 min at 37 °C, a significant proportion of the
125I-NGF appeared on the cell surface in both control and
cells overexpressing p62. These findings reveal that a significant
amount of 125I-NGF can bypass a lysosomal/degradative
pathway in the cell and recirculates to the cell surface after
internalization, which is consistent with previous study on
internalization/trafficking of TrkA (32, 33). Altogether these findings
reveal that p62 may play an important role in trafficking of the cell
surface TrkA receptor to the signaling vesicle.
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Fig. 7.
Effects of p62 on TrkA receptor
signaling. A, PC12 cells overexpressing TrkA were
transfected with either ASp62 or Myc-tagged p62 (OEp62). The
transfected cells were treated with 50 ng/ml NGF for 0, 10, 30, and 60 min at 37 °C. Equivalent cell lysate (750 µg) was
immunoprecipitated (IP) with either anti-TrkA (C-14) or
anti-Erk5 and Western-blotted (WB) with anti-phosphotyrosine
antibody, PY20. The blot was stripped and reprobed with anti-TrkA (B-3)
or anti-Erk5 antibody. Equivalent protein from cell lysates (60 µg)
was Western-blotted with anti-p-MAPK antibody. The p- MAPK
blot were stripped and reprobed with non-phospho-MAPK antibody as
shown. B, lysates from control and transfected cells were
Western-blotted with anti-Erk5. Alternatively, lysates (750 µg) were
immunoprecipitated with anti-Erk5 antibody and subjected to immune
complex kinase assay. Shown is the activity of Erk5 as measured
employing myelin basic protein (MBP) as substrate. The
autoradiogram was scanned, and relative activity of Erk5 was plotted.
These findings are representative of two independent experiments with
similar results.
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Fig. 8.
Immunofluorescence analysis of TrkA and p62
in NGF-differentiated cells. PC12 cells stably overexpressing TrkA
were treated with 50 ng/ml NGF for 4 days to stimulate differentiation
and outgrowth of neurites. The cells were fixed with methanol and
double-labeled with anti-p62 and anti-TrkA (B-3) antibody, and the
extent of colocalization was examined by confocal microscopy.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Model depicting the role of p62 in
trafficking. In the presence of p62 the NGF-TrkA receptor complex
is endocytosed to the signaling vesicle, whereas in the absence of p62
blocks NGF-TrkA receptor internalization and downstream Erk5
activation.
Antisense p62 has been shown to block NGF-mediated differentiation of PC12 cells (17). These data shed light on the underlying mechanism whereby p62 can affect NGF-mediated differentiation. Previous studies (11) have shown that internalized TrkA receptors within the signaling endosome are the site of the differentiation signal for NGF/TrkA. The region where p62 binds TrkA lies within amino acids 472-493, and the TrkA juxtamembrane domain has been implicated previously as critical for TrkA-mediated differentiation (30). Interestingly, another signaling adapter GIPC binds to TrkA employing the same amino acids (19). We have examined whether p62 binds to GIPC and have failed to observe a direct interaction.2 Thus, we propose that p62 interacts directly with TrkA. A conserved domain search employing amino acids 472-493 of TrkA reveals no domain or motif specific to these amino acids. Because TrkA phosphorylation is required for optimal interaction with p62, we favor a model whereby the interaction domain for p62 within TrkA is unmasked by conformational changes that follow receptor activation and autophosphorylation.
Binding of neurotrophins to Trk receptors is known to stimulate activation of both MAP kinase (Erk1/2) and Erk5, which have been shown to be critical to both the differentiation and survival of PC12 cells (11, 21). Moreover, TrkA internalization and retrograde transport has been shown to activate specifically the Erk5 pathway (21). Depletion of p62 by use of the antisense p62 construct was without any effect upon TrkA phosphorylation or the MAP kinases (Erk1/2); however, NGF-induced Erk5 activity was specifically affected. Our findings suggest that it may not be activation of TrkA which drives Erk5 phosphorylation/activation, but rather, it is the coupling of TrkA to p62 that may control Erk5 activation. Upstream MEK5 has been shown to be a direct and specific activator of Erk5 (22). Interestingly, MEK5 contains an aPKC interaction domain, and deletion of this region impairs formation of a complex between aPKC and MEK5 (35). Moreover, it has been shown that aPKC recruitment into a complex triggers the activation of ErK5 in a manner dependent on MEK5 (35); thus, activation of aPKC is capable of triggering the ErK5 cascade. We propose a model whereby TrkA activation results in binding of p62 scaffold to the receptor and recruitment of aPKC (20), followed by activation of MEK5 (35), leading to activation of Erk5 (Fig. 9). It is interesting to note that a newly isolated protein, Pincher (36), has been shown to participate in clathrin-independent internalization. A dominant-negative mutant form of Pincher inhibited NGF-induced endocytosis of TrkA and selectively blocked TrkA-mediated signaling of Erk5 but not Erk1/2 kinases. Given the similarities between p62 and Pincher in their ability to modulate Erk5 and TrkA internalization, it is possible that p62 may connect with the Pincher-mediated internalization pathway. Altogether, these findings underscore the role of p62 as a critical regulator of not only NGF survival signaling (12, 37) but also in intracellular signaling of activated TrkA. We propose that p62 is a component of a targeting pathway leading to delivery of TrkA and subsequently degradation of TrkA in the lysosomes.
TrkA as well as p75 independently activate the NF-B pathway (13).
With respect to TrkA, it has been shown that Shc is required for this
pathway (13, 34, 38). The failure of Shc to compete for with p62 for
binding to TrkA reveals that p62 activates the
B pathway
independently of this site. In addition, the p62-binding site within
TrkA-(472-493) lies outside the Shc-binding site (493-497). When both
receptors, p75 and TrkA, are coexpressed, NGF-activated
B signal is
attenuated (37); inhibition of TrkA by K252a enhances survival
signaling, impairs TrkA internalization, and promotes activation of
NF-
B. Alternatively, overexpression of p62 can enhance survival
signaling and activation of NF-
B (37, 12). Thus, our data support a
model whereby survival signaling occurs predominantly from NGF bound at
the cell surface (11). Collectively, these findings underscore the
critical nature p62 plays in mediating the balance in survival
signaling versus differentiation signaling, by providing a
scaffold for selective activation of NF-
B (12, 37) as well as
modulating internalization and signaling of activated TrkA. Given the
colocalization of p62 with TrkA along the neurite of differentiating
cells, we propose that p62 may be a possible target for mediating
retrograde NF-
B activation in neurons.
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ACKNOWLEDGEMENTS |
---|
We are indebted to Susan Meakin and Moses Chao for TrkA mutants, Maria T. Diaz-Meco and Jorge Moscat for providing us with p62 constructs, and Louis Reichardt for anti-TrkA serum. We thank Michael Miller for helping us carry out the confocal microscopy analysis, Lamar Seibenhener for assistance in graphics, and Randy White for assistance with flow cytometry.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant NS 33661.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence and reprint requests should be addressed:
Dept. of Biological Sciences, 331 Funchess Hall, Auburn University, AL
36849. Tel.: 334-844-9245; Fax: 334-844-9234; E-mail:
mwwooten@acesag.auburn.edu.
Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M208468200
2 T. Geetha and M. W. Wooten, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
NGF, nerve growth
factor;
FACS, fluorescence-activated cell sorter;
GST, glutathione
S-transferase;
MAPK, mitogen-activated protein kinase;
Erk, extracellular signal-related protein kinase;
PKC, protein kinase C;
ASp62, antisense p62;
OEp62, overexpressed p62;
p-TrkA, phosphorylated
TrkA;
NF-B, nuclear factor-
B;
aPKC, atypical protein kinase C;
BDNF, brain-derived neurotrophic factor;
NT, neurotrophin;
BSA, bovine
serum albumin;
DMEM, Dulbecco's modified Eagle's medium;
PBS, phosphate-buffered saline;
TRAF6, tumor necrosis factor
receptor-associated factor 6.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Chao, M. V. (2000) J. Neurosci. Res. 59, 353-355[CrossRef][Medline] [Order article via Infotrieve] |
2. | Patapoutian, A., and Reichardt, L. F. (2001) Curr. Opin. Neurobiol. 11, 272-280[CrossRef][Medline] [Order article via Infotrieve] |
3. | Stephens, R. M., Loeb, D. M., Copeland, T. D., Pawson, T., Greene, L. A., and Kaplan, D. R. (1994) Neuron 12, 691-705[Medline] [Order article via Infotrieve] |
4. | Kaplan, D. R., and Miller, F. D. (1997) Curr. Opin. Cell Biol. 9, 213-221[CrossRef][Medline] [Order article via Infotrieve] |
5. | Kouhara, H., Hadari, Y. R., Spivak-Kroizman, T., Schilling, J., Bar-Sagi, D., Lax, I., and Schlessinger, J. (1997) Cell 89, 693-702[Medline] [Order article via Infotrieve] |
6. |
Holgado-Madruga, M.,
Moscatello, D. K.,
Emlet, D. R.,
Dieterich, R.,
and Wong, A. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12419-12424 |
7. |
Grimes, M. L.,
Zhou, J.,
Beattie, E.,
Yuen, E.,
Hall, D.,
Valletta, J.,
Topp, K.,
LaVail, J.,
Bunnett, N.,
and Mobley, W. C.
(1996)
J. Neurosci.
16,
7950-7964 |
8. |
Grimes, M. L.,
Beattie, E.,
and Mobley, W. C.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
9909-9914 |
9. |
Ricco, A.,
Pierchala, B. A.,
Ciarallo, C. L.,
and Ginty, D. D.
(1997)
Science
277,
1097-1100 |
10. | Tisi, M., Xie, Y., Yeo, T., and Longo, F. (2000) J. Neurobiol. 42, 477-486[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Zhang, Y.,
Moheban, D. B.,
Conway, B. R.,
Bhattacharyya, A.,
and Segal, R. A.
(2000)
J. Neurosci.
20,
5671-5678 |
12. |
Wooten, M. W.,
Seibenhener, M. L.,
Mamidipudi, V.,
Diaz-Meco, M. T.,
Barker, P. A.,
and Moscat, J.
(2001)
J. Biol. Chem.
276,
7709-7712 |
13. |
Foehr, E. D.,
Lin, X.,
O'Mahony, A.,
Gelezinunas, R.,
Bradshaw, R. A.,
and Greene, W. C.
(2000)
J. Neurosci.
20,
7556-7563 |
14. |
Sanz, L.,
Sanchez, P.,
Lallena, M. J.,
Diaz-Meco, M. T.,
and Moscat, J.
(1999)
EMBO J.
18,
3044-3053 |
15. |
Sanz, L.,
Diaz-Meco, M. T.,
Nakano, H.,
and Moscat, J.
(2000)
EMBO J.
19,
1576-1586 |
16. |
Sanchez, P., De,
Carcer, G.,
Sandoval, I. V.,
Moscat, J.,
and Diaz-Meco, M. T.
(1998)
Mol. Cell. Biol.
18,
3069-3080 |
17. | Samuels, I. S., Seibenhener, M. L., Neidigh, K. B. W., and Wooten, M. W. (2001) J. Cell. Biochem. 82, 452-466[CrossRef][Medline] [Order article via Infotrieve] |
18. | Howe, C. L., Valletta, J. S., Rusnak, A. S., and Mobley, W. C. (2001) Neuron 32, 801-814[Medline] [Order article via Infotrieve] |
19. |
Lou, X.,
Yano, H.,
Lee, F.,
Chao, M. V.,
and Farquhar, M. G.
(2001)
Mol. Biol. Cell
12,
615-627 |
20. |
Wooten, M. W.,
Vandenplas, M. L.,
Seibenhener, M. L.,
Geetha, T.,
and Diaz-Meco, M. T.
(2001)
Mol. Cell. Biol.
21,
8414-8427 |
21. | Watson, F. L., Heerssen, H. M., Bhattacharya, A., Klesse, L., Lin, M. Z., and Segal, R. A. (2001) Nat. Neurosci. 4, 981-988[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Kamakura, S.,
Moriguchi, T.,
and Nishida, E.
(1999)
J. Biol. Chem.
274,
26563-26571 |
23. | Clary, D. O., Weskamp, G., Austin, L. R., and Reichardt, L. F. (1994) Mol. Biol. Cell 5, 549-563[Abstract] |
24. |
Jullien, J.,
Guili, V.,
Reichardt, L. F.,
and Rudkin, B. B.
(2002)
J. Biol. Chem.
277,
38700-38708 |
25. |
Fan, G. H.,
Yang, W.,
Sai, J.,
and Richmond, A.
(2002)
J. Biol. Chem.
277,
6590-6597 |
26. | Sutter, A., Riopelle, R. J., Harris-Warrick, R. M., and Shooter, E. M. (1979) J. Cell Biol. 254, 5972-5982 |
27. | Eveleth, D. D., and Bradshaw, R. A. (1992) J. Cell Biol. 117, 291-299[Abstract] |
28. | Barbacid, M., Lamballe, F., Pulido, D., and Klein, R. (1991) Biochim. Biophys. Acta 1072, 115-127[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Berg, M. M.,
Sternberg, D. W.,
Parada, L. F.,
and Chao, M. V.
(1992)
J. Biol. Chem.
267,
13-16 |
30. | Meakin, S. O., and MacDonald, J. I. S. (1998) J. Neurochem. 71, 1875-1888[Medline] [Order article via Infotrieve] |
31. |
Meakin, S. O.,
MacDonald, J. I. S.,
Gryz, E. A.,
Kubu, C. J.,
and Verdi, J. M.
(1999)
J. Biol. Chem.
274,
9861-9870 |
32. |
Buxser, S.,
Decker, D.,
and Ruppel, P.
(1990)
J. Biol. Chem.
265,
12701-12710 |
33. |
Zapf-Colby, A.,
and Olefsky, J. M.
(1998)
Endocrinology
139,
3232-3240 |
34. |
Raffioni, S.,
Thomas, D.,
Foehr, E. D.,
Thompson, L. M.,
and Bradshaw, R. A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
7178-7183 |
35. |
Diaz-Meco, M. T.,
and Moscat, J.
(2001)
Mol. Cell. Biol.
21,
1218-1227 |
36. |
Shao, Y.,
Akmentin, W.,
Toledo-Aral, J.,
Rosenbaum, J.,
Valdez, G.,
Cabot, J.,
Hilbush, B.,
and Halegoua, S.
(2002)
J. Cell Biol.
157,
679-691 |
37. |
Mamidipudi, V., Li, X.,
and Wooten, M. W.
(2002)
J. Biol. Chem.
277,
28010-28018 |
38. |
Thomas, D.,
and Bradshaw, R. A.
(1997)
J. Biol. Chem.
272,
22293-22299 |