Activation of ErbB2 during Wallerian Degeneration of Sciatic Nerve
Yunhee Kim Kwon,1,2,5Anita Bhattacharyya,2,5John A. Alberta,2William V. Giannobile,2,4Kangwoo Cheon,1
Charles D. Stiles,2and Scott L. Pomeroy,3
1Department of Biology, College of Arts and Sciences, KyungHee University, Seoul, 130-701 Korea,2Department of
Microbiology and Molecular Genetics, Harvard Medical School and the Dana-Farber Cancer Institute, Boston,
Massachusetts 02115,3Department of Neurology, Harvard Medical School and Children’s Hospital, Boston,
Massachusetts 02115, and4Department of Periodontology, Harvard School of Dental Medicine,
Boston, Massachusetts 02115
We used anti-phosphopeptide-immunodetecting antibodies as
immunohistochemical reagents to define the location and ac-
tivity state of p185erbB2during Wallerian degeneration. Nerve
damage induces a phosphorylation event at Y1248, a site that
couples p185erbB2to the Ras–Raf–MAP kinase signal trans-
duction pathway. Phosphorylation of p185erbB2occurs within
Schwann cells and coincides in time and space with Schwann
cell mitotic activity, as measured by bromodeoxyuridine uptake.
These visual images of receptor autophosphorylation link acti-
vation of p185erbB2to the Schwann cell proliferation that ac-
companies nerve regeneration.
Key words: neuregulin; erbB2; receptor tyrosine kinase;
Schwann cell; Wallerian degeneration; phosphotyrosine
Unlike elements of the CNS, peripheral nerves can regenerate
when damaged. Understanding the regulation of this process has
practical implications for treatment of peripheral neuropathies
such as those secondary to diabetes, cancer chemotherapeutic
agents, and other toxins. Moreover, insights into peripheral nerve
regeneration may be transferable to treatment of spinal cord
injuries. After peripheral nerves are damaged, they initially un-
dergo Wallerian degeneration. Axons distal to the site of injury
degenerate, and their myelin sheaths break down. Schwann cells
then proliferate, providing a context for axonal regrowth and
nerve regeneration (Waller, 1851; Fawcett and Keynes, 1990).
Although Schwann cell proliferation is a prominent feature of
nerve regeneration, the molecular signals driving the mitotic
response have not been characterized.
One viable candidate for regulating the Schwann cell prolifer-
ation that accompanies regeneration of peripheral nerves is the
transmembrane tyrosine protein kinase p185erbB2. Schwann cells
express p185erbB2both in culture and in vivo (Jin et al., 1993;
Marchionni et al., 1993). The tyrosine kinase activity of p185erbB2
is activated by a family of ligands known collectively as the
neuregulins (glial growth factor, acetylcholine receptor-inducing
activity, Neu differentiation factor, and heregulin) that are en-
coded as splice variant transcripts of a common gene. Neuregulins
are expressed by neurons in the peripheral nervous system (Mar-
chionni et al., 1993; Dong et al., 1995), and they promote the
proliferation of Schwann cells in vitro (Marchionni et al., 1993;
Marchionni, 1995; Morrissey et al., 1995).
Activation of the p185erbB2tyrosine kinase results in the auto-
phosphorylation of specific tyrosines on the intracellular domain
of the receptor. This autophosphorylation can be monitored with
anti-phosphotyrosine antibodies. However, reactivity with ge-
neric antibodies to phosphotyrosine provides no specific insight
into the catalytic or signaling activities of a growth factor recep-
tor. Moreover, anti-phosphotyrosine antibodies cannot be used as
receptor-specific histochemical reagents. To determine the cellu-
lar location and activity state of p185erbB2during Wallerian
degeneration, we exploited the fact that synthetic tyrosine phos-
phopeptides, corresponding to major autophosphorylation motifs,
can be used to raise anti-phosphopeptide-immunodetecting
(APHID) antibodies (Bangalore et al., 1992; Epstein et al., 1992).
These APHID antibodies report the phosphorylation state of
specific tyrosine residues within a targeted growth factor receptor
or phosphoprotein. In the present study, we use an APHID
antibody to monitor the activation state and signaling functions of
p185erbB2in an injured sciatic nerve. We show that p185erbB2
becomes phosphorylated in proliferating Schwann cells during
Wallerian degeneration. Moreover, phosphorylation occurs at a
position that couples p185erbB2to the Ras–Raf–MAP kinase
signal transduction pathway.
MATERIALS AND METHODS
Cell culture. The G8/DHFR cell line was a generous gift from the
laboratory of Robert Weinberg (Massachusetts Institute of Technolo-
gy). These murine fibroblasts, which overexpress the rat c-erbB2 gene
product, were cultured, and cell lysates containing either unactivated
or activated p185erbB2were prepared as described previously (Epstein
et al., 1992).
Surgical procedures. To obtain nerve samples, male Sprague Dawley
rats (?250 gm) were anesthetized with sodium pentobarbital (50 mg/kg).
Using aseptic technique, the right sciatic nerve was exposed 1.0 cm distal
to the sciatic notch, doubly ligated, and transected. The rats were allowed
to recover from surgery. Later, the animals were anesthetized to harvest
the sciatic nerves.
Immunoprecipitation and immunoblotting. Immunoprecipitates of
Received June 19, 1997; revised Aug. 5, 1997; accepted Aug. 11, 1997.
The work was supported by National Institutes of Health Grants NS27773 and
HD18655 (S.L.P.) and HD24926 (C.D.S.), and from Korean Science and Engineer-
ing Foundation Grant 95-0403-03 for cell differentiation through BIOTECH 2000,
(S.R.C.), and KyungHee University (Y.K.K.). We thank Pieter Dikkes for technical
In compliance with Harvard Medical School guidelines on possible conflict of
interest, we disclose that C.D.S. has consulting relationships with Upstate Biotech-
nology and Sandoz Pharmaceuticals Inc.
Y.K.K. and A.B. contributed equally to this work.
Correspondence should be addressed to Scott L. Pomeroy, Department of Neu-
rology, Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115.
Copyright © 1997 Society for Neuroscience 0270-6474/97/178293-07$05.00/0
The Journal of Neuroscience, November 1, 1997, 17(21):8293–8299
p185erbB2were prepared from uncut sciatic nerve or from the distal
stump of the nerve at 5–28 d into Wallerian degeneration. To obtain
samples for immunoprecipitation and immunoblotting, the proximal and
distal stumps of the cut nerve and the opposite uncut nerve were excised
and snap frozen in liquid nitrogen. To prepare lysates, frozen nerve
samples were minced with a razor blade on top of dry ice. The samples
were then homogenized with eight strokes in a Dounce homogenizer in
lysis buffer (1% NP-40, 20 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol,
1 mM sodium orthovanadate, 4 gm/l NaF, 8.8 gm/l sodium pyrophos-
phate decahydrate, 1 mM PMSF, 10 ?g/ml aprotinin, and 20 ?M leupep-
tin) and clarified by centrifuging for 10 min in a microcentrifuge at 4°C.
One milligram aliquots of each sample were immunoprecipitated with an
antibody to p185erbB2[polyclonal antibody 1 (pAb-1) rabbit polyclonal
from Zymed Laboratories, Inc., South San Francisco, CA] using estab-
lished protocols (Harlow and Lane, 1988). The immunoprecipitates were
size-fractionated on 7.0% SDS polyacrylamide gels and immunoblotted
with a monoclonal antibody to phosphotyrosine (4G10; a generous gift
from Tom Roberts, Harvard Medical School).
Antibody specificity was determined by competition experiments in
immunoblots of G8/DHFR cells. Competing peptides (14 mer at 100 nM)
were preincubated with antibody at 4°C for 2 hr before immunoblotting.
The negative and positive specificity controls were, respectively, the
peptide and phosphopeptide counterparts of the sequence used to raise
the p185erbB2APHID antibody (tyrosine 1248 in the sequence AEN-
PEpYLGLDVPV). Other specificity controls were tyrosine phosphopep-
tides containing the closely related NPXY motifs in the C-terminal
domain of epidermal growth factor (EGF) receptor (phosphotyrosine
1197 in the sequence AENAEpYLRVAPQS) and the erbB4 gene prod-
uct (phosphotyrosine 1284 in the sequence AENPEpYLSEFSLK). The
C-terminal portion of p180erbB3has no positional equivalent of the
Immunohistochemistry. For immunohistochemistry, anesthetized ani-
mals were killed by intracardiac perfusion with 4% paraformaldehyde in
PBS with 1 mM sodium orthovanadate added to inhibit endogenous
phosphatase activity. Cryostat sections (7.5–10 ?m) from proximal and
distal stumps of sciatic nerve were permeabilized with 0.5% NP-40 in
Tris-buffered saline (in mM: 20 Tris, pH 7.4, and 150 NaCl) and blocked
with 5% normal goat serum. Aliquots of primary APHID antibody (100
?l) were mixed with BSA (final concentration, 1 mg/ml), solvent control,
or competing peptide (100 nM) as indicated in a final volume of 150 ?l.
Antibody and competing peptides were mixed with gentle rocking at 4°C
for 2 hr before incubating with nerve sections (150 ?l over each sample
on a glass slide) for 24–36 hours at 4°C, followed by incubation in
biotinylated secondary antibody (Vector Laboratories, Burlingame, CA).
Staining was visualized with True Blue (KPL, Gaithersburg, MD) or
with DAB per instructions of the Vectastain ABC kit (Vector Labora-
tories). Identical procedures were used for immunohistochemical stain-
ing with commercial antisera to antibody p185erbB2(Triton Biosciences,
Alameda, CA) and with antisera to S100 protein (Dako, Carpinteria,
CA). For double-labeling experiments, sections were permeabilized,
blocked, and incubated with primary APHID antibody overnight at 4°C,
followed by biotinylated goat anti-rabbit IgG (Vector Laboratories) and
avidin-Cy3 (Jackson Immunochemicals, West Grove, PA). The sections
in sciatic nerve during Wallerian degeneration. p185erbB2immunopre-
cipitates were prepared from uncut sciatic nerve (cnt) or from the
distal stump of nerves from 5 to 28 d into Wallerian degeneration as
described in Materials and Methods. The immunoprecipitates were
size-fractionated on 7.0% SDS-polyacrylamide gels and immunoblot-
ted with a monoclonal antibody to phosphotyrosine. The arrow on the
left indicates position of p185erbB2.
Tyrosine phosphorylation indicative of p185erbB2activation
of p185erbB2on tyrosine. BrdU uptake was monitored in parallel with
immunoprecipitation studies using separate animals. BrdU-labeled nuclei
(mean ? SD) were counted in five high-powered fields or nerve segments
(2 animals per time point; W, weeks after lesion). The highest number of
labeled nuclei was found in the distal nerve stump (D) 2 weeks after
transection, coinciding with the peak of p185erbB2activation shown in
Figure 1. Nuclear labeling increased to a lesser extent in the proximal
stump (P). By immunoblotting, lesser amounts of activated p185erbB2also
were detected in the proximal stump of the lesioned nerve (inset). C,
Schwann cell proliferation is associated with phosphorylation
tyrosine 1248 within activated p185erbB2. A, G8/
DHFR cells were treated with phorbol or with sol-
vent control to generated inactive (?) or activated
(?) p185erbB2as described in Materials and Meth-
ods. Cell lysates were size-fractionated by SDS-
PAGE (40 ?g/lane) and immunoblotted with the
APHID antibody to p185erbB2(APHID) or with a
commercial antibody that recognizes the receptor
irrespective of activation state ( pAb-1). B, Lysates
fractionated and immunoblotted with the APHID
antibody to p185erbB2as A, except that the antibody
was preincubated with synthetic peptides or phos-
phopeptides corresponding to NPXY motifs in
p185erbB2, EGF receptor, or p180erbB4as described
in Materials and Methods. The double-ended arrow
indicates position of 200 kDa molecular weight
APHID antibody is specific for phospho-
8294 J. Neurosci., November 1, 1997, 17(21):8293–8299Kwon et al. • erbB2 Activation during Wallerian Degeneration
were then incubated overnight at 4°C with an S100-? monoclonal anti-
body (Sigma, St. Louis, MO) followed by Cy2-conjugated goat anti-
mouse IgG (Jackson Immunochemicals).
Bromodeoxyuridine labeling. For mitotic labeling, animals were injected
with bromodeoxyuridine (BrdU; 50 mg/kg, i.p.). After 1 hr, the animals
were killed by intracardiac perfusion with 4% paraformaldehyde in PBS.
Cryostat sections (7.5 ?m) of the nerves were prepared and stained with a
monoclonal anti-BrdU primary antibody and fluorescein-conjugated sec-
ondary antibody according to the manufacturer’s specifications (Boehringer
Mannheim, Indianapolis, IN). Labeled nuclei were counted in five high-
powered fields per nerve segment (two animals per time point) viewed by
fluorescence optics (Leitz, Wetzlar, Germany; 40? objective).
Y1248 visualized in sciatic nerve Schwann
cells during Wallerian degeneration. Top
panels, Cryostat sections of control and dis-
tal stump of rat sciatic nerve at 2 weeks
into Wallerian degeneration were immu-
nostained with the APHID antibody to
p185erbB2as described in Materials and
Methods. Visualization with True Blue re-
sults in blue-green color over areas of im-
munoreactivity. Bottom panels, Controls
for specificity of APHID antibody immu-
nohistochemical stain. Sections of the dis-
tal stump of rat sciatic nerve at 2 weeks
into Wallerian degeneration were stained
with APHID antibody preincubated with
peptides or phosphopeptides correspond-
ing to NPXY motifs in p185erbB2, EGF
receptor, or p180erbB4as described in Ma-
terials and Methods. Scale bars, 15 ?M.
Phosphorylation of p185erbB2at
receptors in cells expressing p185erbB2. G8/DHFR cells were treated
with phorbol or solvent control to generate inactive (?) or activated
(?) p185erbB2as described in Materials and Methods. The cells were
immunostained with a commercial antibody that recognizes p185erbB2
irrespective of activation state (top panels) or with the APHID antibody
to activated p185erbB2(bottom panels) and visualized with DAB. For
scale reference, G8/DHFR cell nuclei are ?10 ?M in diameter.
APHID antibody immunostaining is specific for activated
Kwon et al. • erbB2 Activation during Wallerian DegenerationJ. Neurosci., November 1, 1997, 17(21):8293–8299 8295
of proximal and distal stumps of rat sciatic nerve at 2 weeks into Wallerian degeneration were stained for BrdU uptake (top row) or with the APHID
antibody (bottom row). Schematic diagram of sciatic nerve (cell bodies to left, nerve termini to right) illustrates approximate position of the nerve
transection together with relative positions of the edge, center, and end segments. A control, uncut nerve section (top) stained for BrdU uptake is shown
for comparison. Scale bars, 15 ?M.
Top. Tight positional relationship between p185erbB2activation and BrdU labeling during Wallerian degeneration in sciatic nerve. Sections
8296 J. Neurosci., November 1, 1997, 17(21):8293–8299Kwon et al. • erbB2 Activation during Wallerian Degeneration
Schwann cell proliferation is temporally associated
with activation of p185erbB2
As an initial probe into the activation state of p185erbB2during
Wallerian degeneration, we surgically transected rat sciatic nerve
and ligated both the proximal and distal stumps. We monitored
tyrosine phosphorylation of p185erbB2within the nerve segments
5–28 d after nerve transection, the period of Wallerian degener-
ation (Bradley and Asbury, 1970; Clemence et al., 1989). Sciatic
nerve extracts were immunoprecipitated with a pan-p185erbB2
antibody, and the immunoprecipitates were immunoblotted with
antiphosphotyrosine. Relative to control nerve sections, in-
creased p185erbB2tyrosine phosphorylation was found in the
distal nerve stump between 5 and 18 d after transection (Fig. 1).
To explore the temporal relationship between p185erbB2tyrosine
phosphorylation and Schwann cell mitotic activity we monitored
BrdU uptake over the same time frame. The BrdU uptake results
(Fig. 2) show, as noted by others (Cohen et al., 1992), that mitotic
activity is enhanced within 1 week after surgical transection of the
nerve. Mitotic activity remains above baseline levels for at least
14 d but returns to control level by 28 d after injury. Thus
p185erbB2tyrosine phosphorylation and Schwann cell mitotic
activity occur over the same broad time frame after nerve injury.
To explore positional relationships between p185erbB2tyrosine
phosphorylation and Schwann cell mitotic activity, we turned to
an activation state-specific antibody targeted to p185erbB2.
Receptor specificity and activation state specificity of
an APHID antibody to p185erbB2
In previous studies, we raised an APHID antibody directed to
Y1248 in human p185erbB2(Epstein et al., 1992). This tyrosine
lies within an NPXY motif which is a canonical target for the Shc
adapter protein (Campbell et al., 1994; Dilworth et al., 1994;
Stephens et al., 1994). Amino acid substitution experiments indi-
cate that Y1248 is necessary for transforming activity of activated
p185erbB2(Segatto et al., 1990; Akiyama et al., 1991; Mikami et
al., 1992). Moreover, reconstitution experiments indicate that
phosphorylation of Y1248 couples p185erbB2to the Ras–Raf–
MAP kinase signal transduction pathway and is sufficient for
transforming activity (Ben-Levy et al., 1994).
The erbB2 gene is a member of the subgroup I receptor
tyrosine kinase family (Ullrich and Schlessinger, 1990) that in-
cludes the EGF receptor, p185erbB2(Peles et al., 1992), erbB3
(Kraus et al., 1989), and erbB4 (Plowman et al., 1993). To
document receptor specificity and activation state specificity of
the APHID antibody targeted to Y1248, we used an indicator cell
line, G8/DHFR. This is a murine fibroblast line that overex-
presses the rat c-erbB2 gene product. Semiconfluent G8/DHFR
cells express an intermediate level of c-erbB2 receptor tyrosine
kinase activity. This intermediate level of activity is suppressed
transmodulate the receptor (Epstein et al., 1990). As shown by
immunoblot analysis of G8/DHFR cells (Fig. 3), our APHID
antibody recognizes p185erbB2only in the activated state, whereas
a conventional antibody reacts with the protein in the activated or
inactivated state. Peptide competition experiments (Fig. 3) doc-
ument receptor selectivity of the APHID antibody directed to
Y1248 in p185erbB2. The synthetic tyrosine phosphopeptide used
to raise the antibody competes for recognition of activated
p185erbB2. An identical peptide without a phosphate at Y1248
does not compete. Phosphopeptides corresponding to positionally
equivalent NPXY motifs in the C-terminal domain, the EGF
receptor, and p180erbB4do not compete with the APHID anti-
body for recognition of activated p185erbB2. The C-terminal por-
tion of p180erbB3has no positional equivalent of the p185erbB2
NPXY motif and was therefore not tested.
Phosphorylation of p185erbB2at Y1248 visualized in
sciatic nerve Schwann cells
To determine whether the APHID antibody can be used as an
immunocytochemical reagent to visualize p185erbB2signaling in
vivo, we stained cryostat sections from control nerve and from the
distal stump of sciatic nerve at 2 weeks into Wallerian degener-
ation, a time when receptor activation is at its zenith, as shown by
immunoblot analysis with conventional reagents (Fig. 1). As
shown in Figure 4, top panel, sections from transected sciatic
nerve show much more immunoreactivity with the APHID anti-
body than control nerve sections. Selectivity of the APHID
antibody as an immunocytochemical reagent is established by
competition with the subgroup I receptor peptides and phos-
phopeptides. As shown in Figure 4, bottom panel, histochemical
staining was completely ablated by competitions with the erbB2
phosphopeptide. Competitions with erbB2 peptide or other sub-
group I receptor phosphopeptides had little or no effect on
staining. Double labeling with an antibody to S100 protein con-
firms that cells that react with APHID antibody are Schwann cells
(see Fig. 7). In both proximal and distal stump sections, the
APHID antibody immunostaining is localized diffusely over the
surface of reactive cells, as predicted for a cell surface protein in
7.5 ?m cryostat sections. We obtained a similar pattern of immu-
nostaining using a different APHID antibody directed to ty-
rosines 1221 and 1222 that also are autophosphorylated in acti-
vated p185erbB2(data not shown). As a positive control for
immunohistochemistry in sciatic nerve sections, we used the
panels, Sections of distal stumps of rat sciatic nerve at 2 weeks into Wallerian degeneration were costained with a monoclonal antibody to S100 protein
and the APHID antibody to p185erbB2followed by secondary antibodies conjugated to Cy2 (S100) or Cy3 (p185erbB2). Colocalization of S100 ( green)
and activated p185erbB2(red) is indicated in the color overlay ( yellow). Bottom panels, Sections were immunostained with the APHID antibody to
p185erbB2and visualized with Cy3. BrdU uptake was visualized in the same sections with an antibody to BrdU followed by a Cy2-conjugated secondary
antibody. Colocalization of BrdU uptake ( green) and activated p185erbB2(red) is indicated in the color overlay. The somewhat inferior quality of
activated p185erbB2immunostaining reflects the fact that the sections must be exposed to partially denaturing conditions for BrdU immunostaining before
staining for activated p185erbB2.
Middle. Colocalization of activated p185erbB2, Schwann cell marker protein, and BrdU incorporation by double immunofluorescence. Top
(distal stump) at 2 weeks into Wallerian degeneration were stained with the APHID antibody to p185erbB2(top row) or a conventional antibody to
p185erbB2(pAb-1, bottom row). To aid in visualization of stained cells, photomicrographs were taken at identical exposure settings, scanned, and digitized
into the Adobe Photoshop program. Background color was deleted electronically. Weakly stained cells in the processed images from uncut nerve and
distal stump (arrows) can be matched to positional equivalents in the unprocessed photomicrographs.
Bottom. Nerve damage increases the amount of activated p185erbB2 per cell. Cryostat sections of uncut sciatic nerve or transected nerve
Kwon et al. • erbB2 Activation during Wallerian DegenerationJ. Neurosci., November 1, 1997, 17(21):8293–8299 8297
G8/DHFR cell line. As shown in Figure 5, the APHID antibody
recognizes p185erbB2on G8/DHFR cells only when p185erbB2is
active. By contrast, a conventional antibody to p185erbB2(pAb-1)
recognizes p185erbB2irrespective of its activation state. Collec-
tively, these data indicate that the staining differential between
control and transected nerve sections shown in Figure 4, top
panel, most likely reflects the activation of p185erbB2in Schwann
Tight linkage between p185erbB2activation and BrdU
labeling during Wallerian degeneration in sciatic nerve
Using the APHID antibody as an immunohistochemical reagent,
we established a tight positional relationship between Schwann
cell mitotic activity and p185erbB2signaling functions during
Wallerian degeneration (Fig. 6). In the distal stump of transected
nerve, cells that stain with the APHID antibody are distributed
uniformly from the edge of the lesion to the end of the nerve
segment. Double-labeling experiments indicate that BrdU incor-
poration occurs predominantly within Schwann cells (Fig. 7).
Thus, the distribution of APHID antibody immunostained cells
tracks well with Schwann cell mitotic activity, as visualized by
BrdU labeling. The proximal stump of the nerves reveals a
different immunostaining pattern for both APHID antibody and
BrdU label but with the same tight positional coincidence. Here,
APHID antibody staining and mitotic activity are seen at the
edge of the nerve lesion. However, beyond the edge of the lesion
proximal toward the nerve cell bodies, APHID antibody staining
and mitotic activity are attenuated to the level seen in uncut
Enhanced Schwann cell staining with APHID antibody
reflects activation state, rather than abundance,
Both erbB2 mRNA and p185erbB2accumulate in the distal stump
of sciatic nerve during Wallerian degeneration (Cohen et al.,
1992; Carroll et al., 1997). The number of Schwann cells in the
distal stump also increases as a consequence of mitotic activity.
Accordingly, we wondered whether the increased staining with
APHID antibody reflects a gain in the number of cells that
display activated p185erbB2or an increase in the amount of
activated p185erbB2per cell or both. To address this question, we
compared immunostaining patterns with our APHID antibody
with those obtained with a commercial antibody that recognizes
p185erbB2irrespective of activation state. To visualize the stain-
ing differential more readily, we used image-processing software
to subtract background color in the stained sections.
Relative to uncut nerve, distal stump sections stained with
APHID antibody show a gain in both number of stained cells and
staining intensity per cell (Fig. 8, top row). By contrast (Fig. 8,
bottom row), identical sections stained with conventional antibody
show a gain in the number of stained cells (in confirmation of
Cohen et al., 1992), but the staining intensity per cell is compa-
rable in uncut nerve and distal stump. This comparison is some-
what subjective, because immunohistochemical staining reactions
are not inherently linear. However, the differential intensity of
cell staining with the APHID antibody was apparent to the eye in
Targeted disruption studies show that the neuregulin and erbB2
gene products play vital roles in development of the embryonic
heart, Schwann cell precursors, and cranial nerve ganglia (Gassman
et al., 1995; Lee et al., 1995; Marchionni, 1995; Meyer and Birch-
meier, 1995). However, because null mutants of either gene die in
utero at day 10.5, the functions of neuregulins and their receptors
in adult animals cannot be discerned by gene disruption. The
APHID antibody images shown here indicate that these proteins
play an active role in the regeneration of injured nerves in adult
Activation of p185erbB2after nerve damage could, in principle,
reflect suppression of a negative regulator. In tissue culture model
systems, the activity state of p185erbB2is negatively regulated by
protein kinase C agonists (Dougall et al., 1994). For several
reasons, however, we favor the view that activation of p185erbB2
reflects increased availability of p185erbB2-activating ligands, the
neuregulins. Neuregulins are produced by neurons (Marchionni
et al., 1993; Dong et al., 1995) in a variety of splice variant
transcripts, some of which encode a membrane-bound ligand
(Marchionni et al., 1993). Immunohistochemical studies demon-
strate neuregulins within axons (Sandrock et al., 1995), and neu-
ronal membrane preparations stimulate the growth of cultured
Schwann cells (Salzer et al., 1980) through the activation of
p185erbB2(Morrissey et al., 1995).
Neuregulins are thought to interact with p185erbB2by inducing
the formation of heterodimers between p185erbB2and either
p180erbB3or p180erbB4. Neuregulin-induced heterodimer forma-
tion between p185erbB2and p180erbB3constitutes an interesting
example of a symbiotic relationship in receptor signaling. By itself,
p185erbB2cannot interact with neuregulins. Although p180erbB3is
a competent neuregulin-binding protein, it appears to have no
intrinsic tyrosine kinase activity (Carraway and Cantley, 1994).
Formation of a heterodimeric p185erbB2–p180erbB3complex gen-
erates a fully competent growth factor receptor (Carraway and
Cantley, 1994; Marchionni, 1995; Wallasch et al., 1995). In prelim-
inary studies, we have noted that p180erbB3is present in rat sciatic
nerve lysates (data not shown) (Carroll et al., 1997).
The anatomy of sciatic nerve raises an interesting question. If
neuregulin synthesis is confined to neurons, what serves as a
source of neuregulin for Schwann cells in the distal stump of a
regenerating nerve? Neuronal protein synthesis is mainly con-
fined to the nerve cell bodies. Accordingly, only neuregulins
synthesized before transecting the nerve would have access to
Schwann cells in the distal stump. Fischbach and associates have
described a slow release mechanism for neuregulins at the sciatic
nerve ending. Here within the neuromuscular junction, neuregu-
lin is bound to extracellular matrix and inhibited from activating
p185erbB2on striated muscle until it is released by proteolysis
(Goodearl et al., 1995; Loeb and Fischbach, 1995; Sandrock et al.,
1995). Conceivably, stored reservoirs of inactive neuregulin are
distributed along the length of the axon to be activated and slowly
released as axons degenerate after transection of the nerve.
Alternatively, neuregulins may be synthesized by non-neuronal
cells during Wallerian degeneration, perhaps by Schwann cells
themselves in an autocrine growth mode (Carroll et al., 1997).
Finally, the phosphorylation of p185erbB2may be triggered by a
novel and presently uncharacterized ligand that is released after
Future studies with APHID antibodies targeted to other mem-
bers of the subgroup I receptor family may identify heterodimeric
partners and the source of ligand for p185erbB2during nerve
regeneration. In the meantime, it is worth noting that the ap-
proach taken here to provide images of p185erbB2activation in
vivo has general utility. Synthetic phosphopeptides can, in prin-
ciple, be used to raise APHID antibodies targeted to any growth
factor receptor or signal-generating protein that is regulated by
8298 J. Neurosci., November 1, 1997, 17(21):8293–8299 Kwon et al. • erbB2 Activation during Wallerian Degeneration
tyrosine phosphorylation events. As immunochemical probes, Download full-text
these APHID antibodies are more selective than conventional
reagents, and they can be used as immunohistochemical reagents
to display the activation state of specific receptors or signal
generators in situ.
Akiyama T, Matsuda S, Namba Y, Saito T, Toyoshima K, Yamaoto T
(1991) The transforming potential of the c-erbB-2 protein is regulated
by its autophosphorylation at the carboxyl-terminal domain. Mol Cell
Bangalore L, Tanner AJ, Laudano AP, Stern DF (1992) Antiserum
raised against a synthetic phosphotyrosine-containing peptide selec-
tively recognizes p185neu/erbB-2and the epidermal growth factor recep-
tor. Proc Natl Acad Sci USA 89:11637–11641.
Ben-Levy RB, Paterson HF, Marshall CJ, Yarden Y (1994) A single
autophosphorylation site confers oncogenicity to the Neu/ErbB-2 re-
ceptor and enables coupling to the MAP kinase pathway. EMBO J
Bradley WG, Asbury AK (1970) Duration of synthesis phase in neuri-
lemma cells in mouse sciatic nerve during regeneration. Exp Neurol
Campbell KS, Ogris O, Burke B, Su W, Auger KR, Druker BJ, Schaff-
hausen BS, Roberts TM, Pallas DC (1994) Polyoma middle tumor
antigen interacts with SHC protein via the NPTY (Asn-Pro-Thr-Tyr)
motif in middle tumor antigen. Proc Natl Acad Sci USA 91:6344–6348.
Carraway KI, Cantley L (1994) A neu acquaintance for ErbB3 and
ErbB4: a role for receptor heterodimerization in growth signaling. Cell
Carroll SL, Miller ML, Frohnert PW, Kim SS, Corbett JA (1997) Ex-
pression of neuregulins and their putative receptors, erbB2 and erbB3,
is induced during Wallerian degeneration. J Neurosci 17:1642–1659.
ClemenceA, Mirsky R, Jessen
Schwann cells proliferate rapidly during Wallerian degeneration in the
rat sciatic nerve. J Neurocytol 18:185–192.
Cohen J, Yachnis A, Arai M, Davis J, Scherer S (1992) Expression of
the neu proto-oncogene by Schwann cells during peripheral nerve
development and Wallerian degeneration. J Neurosci Res 31:622–634.
Dilworth SM, Brewster CEP, Jonew MD, Lanfrancone L, Pelicci G,
Pelicci PG (1994) Transformation by polyoma virus middle T-antigen
involves the binding and tyrosine phosphorylation of Shc. Nature
Dong Z, Brennan A, Liu N, Yarden Y, Lefkowitz G, Mirsky R, Jessen K
(1995) Neu differentiation factor is a neuron-glia signal and regulates
survival, proliferation and maturation of rat Schwann cell precursors.
Dougall W, Qian X, Peterson N, Miller M, Samanta A, Greene M (1994)
The neu-oncogene: signal transduction pathways, transformation mech-
anisms and evolving therapies. Oncogene 9:2109–2132.
Epstein RJ, Druker BJ, Roberts TM, Stiles CD (1990) Modulation of a
Mr 175,000 c-neu receptor isoform in G8/DHFR cells by serum star-
vation. J Biol Chem 265:10746–10751.
Epstein R, Druker B, Roberts T, Stiles CD (1992) Synthetic phos-
phopeptide immunogens yield activation-specific antibodies to the c-
erbB-2 receptor. Proc Natl Acad Sci USA 89:10435–10439.
Fawcett JW, Keynes RJ (1990) Peripheral nerve regeneration. Annu
Rev Neurosci 13:43–60.
Gassman M, Casagranda F, Orioli D, Simon H, Lai C, Klein R, Lemke
G (1995) Aberrant neural and cardiac development in mice lacking
the erbB4 neuregulin receptor. Nature 378:390–394.
Goodearl A, Yee A, Sandrock AJ, Corfas G, Fischbach G (1995) ARIA
is concentrated in the synaptic basal lamina of the developing chick
neuromuscular junction. J Cell Biol 130:1423–1434.
KR (1989) Non-myelin-forming
Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring
Harbor, NY: Cold Spring Harbor Laboratory.
Jin J, Nikitin A, Rajewsky M (1993) Schwann cell lineage-specific
neu(erbB-2) gene expression in the developing rat nervous system. Cell
Growth Differ 4:227–237.
Kraus M, Issing W, Miki T, Popescu N, Aaronson S (1989) Isolation and
characterization of erbB3, a third member of the erbB/epidermal
growth factor receptor family: evidence for overexpression in a subset
of human mammary tumors. Proc Natl Acad Sci USA 86:9193–9197.
Lee K-F, Simon H, Chen H, Bates B, Hung M-C, Hauser C (1995)
Requirement for neuregulin receptor erbB2 in neural and cardiac
development. Nature 378:394–398.
Loeb J, Fischbach G (1995) ARIA can be released from extracellular
matrix through cleavage of a heparin-binding domain. J Cell Biol
Marchionni M (1995) Neu tack on neuregulin. Nature 378:334–335.
Marchionni M, Goodearl A, Chen M, McDonogh O, Kirk C, Hendricks
M, Danehy F, Misumi D, Sudhalter J, Kobayashi K, Wroblewki D,
Lynch C, Baldassare M, Hiles I, Davis J, Hsuan J, Totty N, Otsu M,
McBurney R, Waterfield M, Stroobant P, Gwynne D (1993) Glial
growth factors are alternatively spliced erbB2 ligands expressed in the
nervous system. Nature 362:312–318.
Meyer D, Birchmeier C (1995) Multiple essential functions of neuregu-
lin in development. Nature 378:386–389.
Mikami Y, Davis JG, Dobashi K, Dougall WC, Myers JN, Brown V,
Greene MI (1992) Carboxyl-terminal deletion and point mutations
decrease the transforming potential of the activated rat neu oncogene
product. Proc Natl Acad Sci USA 89:7335–7339.
Morrissey T, Levi A, Nuijens A, Sliwkowski M, Bunge R (1995) Axon-
induced mitogenesis of human Schwann cells involves heregulin and
p185erbB2. Proc Natl Acad Sci USA 92:1431–1435.
Peles E, Bacus EE, Koski RA, Lu HS, Wen D, Ogden SG, Levy RB,
Yarden Y (1992) Isolation of the Neu/HER-2 stimulatory ligand: a 44
kDa glycoprotein that induces differentiation of mammary tumor cells.
Plowman G, Culouscou J-M, Whitney G, Green J, Carlton G, Foy L,
Newbauer M, Shoyab M (1993) Ligand-specific activation of HER4/
p180erbB4, a fourth member of the epidermal growth factor receptor
family. Proc Natl Acad Sci USA 90:1746–1750.
Salzer J, Williams A, Glaser L, Bunge R (1980) Studies of Schwann cell
proliferation. II. Characterization of the stimulation and specificity of
the response to a neurite fraction. J Cell Biol 84:753–766.
Sandrock AJ, Goodearl A, Yin Q-W, Chang D, Fischbach G (1995)
ARIA is concentrated in nerve terminals at neuromuscular junctions
and at other synapses. J Neurosci 15:6124–6136.
Segatto O, Lonardo F, Pierce JH, Bottaro DP, DiFiore PP (1990) The
role of autophosphorylation in modulation of erbB-2 transforming
function. New Biol 2:187–195.
Stephens RM, Loeb DM, Copeland TD, Pawson T, Greene LA, Kaplan
DR (1994) Trk receptors use redundant signal transduction pathways
involving SHC and PLC-?1 to mediate NGF responses. Neuron
Ullrich A, Schlessinger J (1990) Signal transduction by receptors with
tyrosine kinase activity. Cell 61:203–212.
Wallasch C, Weiss FU, Niederfellner G, Jallal B, Issing W, Ullrich A
(1995) Heregulin-dependent regulation of HER2/neu oncogenic sig-
naling by heterodimerization with HER3. EMBO J 14:4267–4275.
Waller A (1851) Experiments on the section of the glosso-pharyngeal
and hypoglossal nerves of the frog, and observations of the alterations
produced thereby in the structures of their primitive fibers. Edinburgh
Med Surg J 76:369–376.
Kwon et al. • erbB2 Activation during Wallerian DegenerationJ. Neurosci., November 1, 1997, 17(21):8293–8299 8299