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(Received for publication, November 21, 1994; and in revised form, December 27, 1994) From the
Although they all utilize tyrosine kinase receptors and activate
signaling pathways characterized by a similar set of phosphoproteins,
epidermal growth factor (EGF) promotes only cell division while
fibroblast growth factor (FGF) and nerve growth factor (NGF) can induce
division followed by differentiation in PC12 cells. EGF, in contrast to
NGF and FGF, cannot maintain the sustained phosphorylation and
activation of mitogen-activated protein (MAP) kinase kinase and MAP
kinases, which may account for the difference in phenotypic response.
The pretreatment of PC12 cells with staurosporine, a protein kinase
inhibitor, causes a substantial increase in both receptor and MAP
kinase phosphorylation that results in a differentiative response
(neurite proliferation). However, neurites begin to disappear after 3
days, despite the continual presence of EGF, and are largely gone after
5 days, which is not the case with NGF and FGF. Thus, the effect of
staurosporine is not permanent. Northern and Western blots indicate
that the staurosporine response mainly results from a substantial
up-regulation in EGF receptor synthesis, thus providing a much stronger
cell surface signal and supporting the view that quantitative rather
than qualitative differences distinguish the EGF versus NGF/FGF signaling pathways in these cells. Polypeptide growth factors interact with plasma membrane-bound
receptors to induce signal cascades that begin with protein tyrosine
phosphorylation. The activated kinases are either an inherent part of
the receptor or associate non-covalently with it as accessory proteins (1, 2) . The pathways that are subsequently stimulated
involve a variety of intracellular effector and adaptor proteins and
generally result overall in either a mitotic or trophic
response(3) . Rat pheochromocytoma-derived PC12 cells (4) have been extensively utilized as a paradigm to deduce the
molecular mechanisms that underlie (and distinguish) these two
responses, primarily because both division and differentiation can be
induced by several well studied
factors(5, 6, 7) . Thus, NGF ( Although the detailed mechanisms for the
induction of differentiation in PC12 cells by either NGF or FGF has not
yet been determined, recent findings have underscored the importance of
a pathway controlled by p21
Staurosporine was initially described as a specific inhibitor
of protein kinase C (26, 27) and was subsequently
shown to be a highly specific inhibitor of the NGF receptor (as well as
other neurotrophin receptors) tyrosine kinase (28, 29, 30) but not other receptor tyrosine
kinases, including those for FGF, which can also induce the
differentiative response in PC12 cells(31) . Staurosporine has
also been shown to have some neurite proliferative effects in PC12
cells at high concentrations(32, 33, 34) . As
shown in Fig. 1, at a concentration of 5
Figure 1:
Dose-dependent induction of neurites in
PC12 cells by staurosporine (
Figure 2:
The
induction of neurite outgrowth by the combined action of EGF and
staurosporine in PC12 cells. Cells were cultured in complete medium in
the presence of medium alone (CON) or with EGF (10 ng/ml) (EGF), 10 nM staurosporine (ST), or EGF (10
ng/ml) plus 10 nM staurosporine (EGF + ST). The
medium was replaced every other day. Cells were counted and scored for
the presence of neurites after 1 (D1). 3 (D3), or 5
days (D5). Bar, 100
µm.
In view of
previous reports suggesting that EGF induced a rapid decrease in its
own cell surface receptors(19) , we examined the levels of EGFR
phosphorylation as a function of time in cells stimulated with EGF in
the presence and absence of staurosporine. As shown in Fig. 3A and Fig. 4, A and C,
cells pretreated with staurosporine for 16 h clearly show more
extensive phosphorylation of the EGFR (when exposed to EGF), as
detected with anti-phosphotyrosine antibodies and
Figure 3:
Time
course of protein tyrosine phosphorylation (A) and of MAPKs
activity (B) induced by EGF in the absence and presence of
staurosporine. A, after preincubation without (leftpanel) or with (rightpanel) 10 nM staurosporine for 16 h, PC12 cells were left untreated (0) or
treated with EGF (10 ng/ml) for 2, 10, 30, 60, 120, and 240 min at 37
°C as indicated. 100 µg of protein from each whole cell lysate
was analyzed by 7.5% SDS-PAGE followed by immunoblotting with a
polyclonal anti-phosphotyrosine antibody, detection with
Figure 4:
Time course of EGF receptor tyrosine
phosphorylation (A, C, and E) and level of
EGF receptor (B, D, and F) in PC12 cells in
the absence and presence of staurosporine. After preincubation without (A, B) or with (C, D) 10 nM staurosporine for 16 h, PC12 were left untreated (lanes0) or treated with EGF (10 ng/ml) for 2, 10, 30, 60, 120,
and 240 min at 37 °C as indicated. A and C, 100
µg of protein from each whole cell lysate was analyzed by 7.5%
SDS-PAGE followed by immunoblotting with the antiphosphotyrosine
antiserum. B and D, the anti-phosphotyrosine blots
were stripped and reprobed with an anti-EGFR monoclonal antibody C13. E and F, after preincubation without or with
staurosporine for 16 h, PC12 cells were left untreated or treated with
EGF for 30 min, as indicated, lysed, and immunoprecipitated with
monoclonal anti-rat EGFR antibody. Immunoprecipitates were separated by
SDS-PAGE, transferred to Immobilon P membranes, and immunoblotted with
a polyclonal anti-phosphotyrosine antibody (E) and, after
stripping, reprobed with a monoclonal anti-EGFR antibody generated
against a peptide corresponding to the submembrane domain of the human
EGFR (F). The phosphorylation of p170, corresponding to the
EGFR, is indicated by arrowheads.
To confirm
that the increased phosphorylation of the 170-kDa species corresponds
to the EGFR, immunoprecipitation and Western blot analyses were
performed. As shown in Fig. 4, E and F, after
immunoprecipitation with anti-EGFR antibody and anti-phosphotyrosine
antibody Western blotting, cells treated with EGF (pretreated with
staurosporine) for 30 min show a more heavily phosphorylated EGFR than
cells treated with EGF alone. After stripping and reprobing the blot
with anti-EGFR antibody, it is evident that more EGFR can be
immunoprecipitated in the cells treated with either both agents or
staurosporine alone (Fig. 4F). Despite the higher level
of EGFR present in PC12 cells following their pretreatment with
staurosporine for 16 h, no tyrosine phosphorylation of this receptor
was observed until EGF was added to the culture medium. It appears that
the up-regulation of the level of EGFR in cells pretreated with
staurosporine is evident, even if in a more qualitative manner, in the
anti-EGFR Western blot of total cell extracts of PC12 cells when
comparing the first three left lanes of Fig. 4D (cells
treated with staurosporine for 16 h) to the corresponding lanes of Fig. 4B (untreated cells). As the up-regulation of
EGFR by the prolonged treatment with staurosporine cannot rule out a
possible direct effect of staurosporine on the phosphorylation of EGFR,
experiments were performed to address this issue. Upon simultaneous
addition of EGF and staurosporine, a modestly increased phosphorylation
of EGFR and MAPKs was observed when compared with the addition of EGF
alone up to 30 min (Fig. 5). This transient stimulatory effect
of staurosporine on EGF-induced protein tyrosine phosphorylation is
likely due to a direct inhibition of phosphorylation of EGFR at
Ser-1046/Ser-1047 (19) or on other
phosphorylation/dephosphorylation events that regulate EGFR activity.
Thus, they may make some contribution to the induced EGF
responsiveness.
Figure 5:
Time course of protein tyrosine
phosphorylation induced by EGF with or without the simultaneous
addition of staurosporine. PC12 cells were left untreated (0) or
treated with EGF (10 ng/ml) for 2, 10, 30, 60, 120, and 240 min at 37
°C without (leftpanel) or with the addition of
10 nM staurosporine (rightpanel). 100
µg of protein from each whole cell lysate was analyzed by 7.5%
SDS-PAGE followed by immunoblotting with a polyclonal
anti-phosphotyrosine antibody, detection with
The clear demonstration that PC12 cells treated with
staurosporine show a sustained response to EGF, including a prolonged
level of phosphorylated receptor at the plasma membrane, suggests an
altered rate of synthesis or degradation (down-regulation). To test
whether staurosporine effected the expression of EGFR, PC12 cells were
treated for 2, 4, 8, and 16 h with 10 nM drug and 20 µg of
total RNA isolated from each. Following separation on 1% agarose, blots
were probed at 65 °C with a riboprobe corresponding to the
extracellular domain of PC12 EGFR. As shown in Fig. 6,
staurosporine is a potent inducer of EGFR gene expression in these
cells. The effect appears to peak at 8 h but remains high at 16 h.
Translation of this increased level of mRNA would presumably increase
the number of plasma membrane EGFR, similar to cells transfected with
an exogenous EGFR gene (18) , and account for the increased
tyrosine phosphorylation signals induced by the added EGF ( Fig. 3and Fig. 4). It is unclear whether the mechanism by
which staurosporine effects the level of EGFR gene transcription is
related to kinase inhibition or to some other as yet undefined
activity. However, similar observations have been made with human tumor
necrosis factor receptors on myeloid and epithelial cells (38) , suggesting the phenomenon may be a more general one.
Figure 6:
Northern blot analysis of the effect of
staurosporine on the expression of EGFR mRNA level. PC12 cells were
cultured in complete medium in the absence or in the presence of 10
nM staurosporine for 0, 2, 4, 8, and 16 h as indicated. At the
end of this period, total RNA was extracted from cells, and 20 µg
of each sample was fractionated on a 1% formaldehyde-agarose gel. The
blot was first probed at 65 °C with a rat EGFR riboprobe; it was
then stripped and reprobed at 42 °C with a CHOb probe to normalize
RNA loading differences.
These findings clearly establish that the endogenous EGFR of PC12
cells is capable of inducing neurite outgrowth, effectively eliminating
the possibility that it fails to do so in wild type PC12 cells because
of some abnormality in function. These results further suggest that any
substantial increase in plasma membrane levels of EGFR will allow for
EGF-induced differentiation in these cells, in this case provided by
staurosporine-induced increases in expression or inhibition of
down-regulation. In contrast to the situation expected for stably
transfected cells(18) , the sustained expression of EGFR is
eventually overcome, i.e. the up-regulation by staurosporine
is not permanent, and neurite outgrowth begins to wane by day 5. This
provides further proof that the effect observed is related to the
number of EGFR and not to any changes in the specificity of its
receptor kinase. It also suggests that the magnitude of the signal
induced and the length of time that threshold levels of phosphorylation
can be maintained are directly related to the number of cell surface
receptors. There is greater than an order of magnitude more EGFR than
TrkA or FGF receptor on native PC12 cells ( The importance of
the duration of the MAPK signal in PC12 cells may be related to both
locational and temporal aspects of the response. Clearly, the prolonged
response induced by NGF and basic FGF causes not only the activation of
MAPK but also its translocation to the nucleus (17) . This may
be fundamental to the initiation of the neurotrophic response. It is
instructional to recall that the initial response of PC12 cells to all
of these factors is a mitotic one(41) , i.e. there is
apparently a protein(s) that must be induced before the neurotrophic
response can be manifested. This clearly requires new protein
synthesis, suggesting that the signal induced by a potential
neurotrophic factor must sustain the MAPK signal long enough to produce
an initial round of transcription (expression of the set of immediate
early response genes and their products(42) ). Cells that have
been primed, i.e. exposed previously to NGF(5) , or
variants that can respond essentially immediately to growth factor (43, 44) do not have this temporal restriction,
presumably because they have previously synthesized the required
regulatory protein(s). Although this putative entity has yet to be
described, its behavior is consistent with that expected for a mitotic
suppressor(45) . It might also be noted that the
transcriptional regulation controlled by MAPK is sufficiently extensive
that it leads to a clearly defined phenotype following 2-6 h of
stimulation by NGF or FGF that is apparently the signature of the
differentiated cell and is lacking following EGF
treatment(35) . Note Added in
Proof-After this manuscript was submitted, Isono et
al. (46) reported similar effects of K-252a, a compound
related to staurosporine, on the response of PC12 cells to EGF.
Volume 270,
Number 13,
Issue of March 31, 1995 pp. 7568-7572
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)and
FGF (basic or acidic) can promote neurite proliferation while EGF and
insulin-like growth factor-I cause only mitosis, despite the fact that
all utilize receptors containing a tyrosine kinase effector and
activate similar signaling cascades with overlapping sets of
phosphorylatable substrates. These observations have raised the
question of whether the two responses are induced by fundamentally
unique mechanisms or whether quantitative differences, i.e. the level or duration of an essential activated component(s), are
responsible(8) .
that leads to the
activation of a kinase cascade involving Raf, MAPK kinase, and
MAPK(9, 10, 11, 12, 13, 14, 15, 16) .
The adaptor proteins Shc and Grb2 (or Crk) appear to be required to
link Ras to the activated receptor (3) via a Ras
guanine-nucleotide exchange protein (SOS or C3G). Of particular
importance apparently is the sustained activation of MAPK kinase and
MAPK and the transfer of the latter to the nucleus (17, 18) . EGF, which can also activate MAPK kinase
and MAPK, cannot maintain the necessary level of phosphorylation nor
induce nuclear translocation, and it has been suggested that this may
result from the rapid internalization of occupied EGFR because of the
phosphorylation of Ser-1046/Ser-1047 (by an as yet unidentified
kinase)(19) . This would suggest that EGF, which prematurely
attenuates its own signal, induces the necessary responses to produce
differentiation, but because of the rapid loss of cell surface
receptors following ligand binding and the concomitant loss in the
transmembrane signal, it cannot do so. In this report, we show that
staurosporine, a broad inhibitor of protein kinases, can convert EGF to
a differentiative factor for PC12 cells by causing a marked
up-regulation of EGFR synthesis as well as by directly affecting
receptor phosphorylation/dephosphorylation, thus establishing that the
endogenous rat EGFR has the requisite specificity to stimulate both
mitotic and differentiative responses in PC12 cells.
Reagents
Mouse EGF was prepared by the
method of Savage and Cohen (20) or was obtained from Life
Technologies, Inc. Monoclonal anti-rat EGFR antibody (21) was a
gift from Maureen O'ConnorMcCourt (National Research Council,
Canada). A monoclonal antibody C13 corresponding to residues 996-1022
in the human EGFR (22) and an antibody made against a peptide
in the submembrane domain of human EGFR were a gift from Gordon N. Gill
(University of California, San Diego). Anti-phosphotyrosine antiserum
was obtained from Zymed Labs. Anti-ERK-1 antiserum and Protein-A/G
PLUS-agarose were from Santa Cruz Biotechnology, Inc. 
I-Labeled protein A was purchased from ICN. Staurosporine
was from Calbiochem. Molecular weight standards were from Bio-Rad and
Life Technologies, Inc. Immobilon P membranes were from Millipore.Cell Culture
PC12 cells were obtained
from E. Shooter (Stanford University) and maintained in culture in
150-cm
tissue culture flasks (Costar) in Dulbecco's
modified Eagle's medium containing 10% horse serum, 5% fetal calf
serum, and 1% Pen-strep solution (Life Technologies, Inc.) (complete
medium) in a 5% CO humidified atmosphere.Neurite Outgrowth Assay
PC12 cells were
plated in collagen-coated 6-well plates in complete medium at a density
of 0.2 
10
cells/well. After 12-16 h, the
medium was changed, and the cells were cultured in the presence of EGF
(10 ng/ml) and/or various concentrations of staurosporine. The medium
was replaced every other day. Cells were examined for the presence of
neurites at various times. Responsive cells were defined as those
bearing neurites at least 1 cell diameter in length.Immunoprecipitation and Immunoblot
Analysis
For whole cell lysates, cells were plated in
complete medium on either 100- or 150-mm collagen-coated
culture dishes and grown to 60-70% confluency. The medium was
replaced 16 h before the experiments, and 10 nM staurosporine
was added at the same time or for different lengths of time as
specified. At the end of this period, cells were treated with EGF (10
ng/ml) for the specified time, then rinsed with cold phosphate-buffered
saline containing 1 mM sodium orthovanadate and lysed in cold
lysis buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1%
Triton X-100, 5 mM EDTA, 200 µM sodium
orthovanadate, 50 mM NaF, 30 mM sodium pyrophosphate,
10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) for 15 min on ice(23) .
Insoluble material was pelleted in a microcentrifuge at 14,000 rpm for
15 min. Protein concentration was determined with the Bradford
colorimetric assay from Bio-Rad. For immunoprecipitations, lysates (2
mg) were incubated with monoclonal anti-rat EGFR antibody for 2 h at 4
°C, and immunoprecipitates were collected using protein A/G-agarose
for an additional 2 h. After being washed, the complexes were boiled
for 5 min in SDS sample buffer and resolved by 7.5% SDS-PAGE and
transferred to Immobilon P membranes by electroblotting at 63 V
overnight. The membranes were probed with antibodies as specified and
then incubated with I-labeled protein A.Northern Blot Analysis
A 
750-base
pair DNA fragment was generated by reverse transcriptase-polymerase
chain reaction using total RNA from PC12 cells and oligonucleotides
corresponding to amino acids 33-272 of the extracellular domain
of rat EGFR(24) . The amplified fragment was subcloned into
pGEM-7Zf(+) using the restriction sites BamHI and SphI. The linearized plasmid was used as a template to
generate an antisense RNA and was used for hybridization with blots at
65 °C in a hybridization solution containing 5 SSPE, 50%
formamide, 5 Denhardt, 1% SDS, 10% dextran sulfate, and 100
µg/ml salmon sperm DNA. After 20 h, blots were washed at 67 °C
in 0.2 SSPE containing 0.5% SDS. Subsequently, blots were
stripped and reprobed at 42 °C with a 0.74-kilobase BamHI
DNA fragment of CHOb (25) used to normalize RNA loading
differences.
10
M, it does show some differentiative activity in this
system. However, the presence of EGF, which alone has no neurite
proliferative capabilities, greatly enhances this response.
Interestingly, the increase in the EGF response is most pronounced
during the first 3 days of treatment and begins to decline thereafter (Fig. 2). The response to staurosporine alone is relatively
constant over this time period, suggesting that the principal effect is
an augmentation of the EGF signal rather than that induced by
staurosporine. The magnitude of the response (particularly during days
1-3) is comparable with that induced by NGF and FGF. However,
these factors do not show a diminution of response at later time
points(6) , unless the factor is removed.
) and EGF plus staurosporine
(
). PC12 cells were cultured in the presence of EGF (10 ng/ml)
and/or various concentrations of staurosporine. After 24 h of
treatment, cells were examined for the presence of neurites. Less than
1% of cells grow neurites in response to EGF. Values are averages of
quadruplicate determinations from two to three separate experiments. Verticalbars represent
S.E.
I-protein A, than cells treated with EGF alone. The
identification of the 170-kDa band as EGFR was confirmed by stripping
and reprobing the blots with anti-EGFR antibody (Fig. 4, B and D). The blots in Fig. 3also revealed an
increased level of phosphorylated proteins in the molecular weight
range expected for the two isoforms of MAPK (pp42 and pp44), which is
known to correspond to an increased activity of these
enzymes(11, 36) . This was confirmed by stripping the
blot and reprobing with antibodies specific for these entities. As
shown in the lowerpanel of Fig. 3, the levels
of phosphorylated MAPK1 and -2 (indicated by the shift in mobility in
the lanes corresponding to 2 and 10 min (leftside)
and 2, 10, 30, and 60 min (rightside)) are indeed
prolonged in the cells pretreated with staurosporine and induced by EGF
(both the phosphorylated and dephosphorylated forms are clearly visible
in the later time points of the staurosporine plus EGF samples). This
latter profile is virtually identical to that seen for NGF and basic
FGF in PC12 cells, under conditions that produce full neurite
outgrowth(37) . It is important to point out that, at the
concentration used, staurosporine by itself had no effect on the basal
level of tyrosine phosphorylation in PC12 cells and on the activity of
MAPK1 and MAPK2, as shown in Fig. 3B.
I-protein A, and autoradiography. Specific proteins are
indicated by arrowheads. The migration of molecular mass
standards (in kDa) is shown on the left and on the right for each gel. B, the anti-phosphotyrosine blots were
stripped and reprobed with anti-ERK-1 antiserum. The antiserum
recognizes both p44
and p42
as
indicated by arrowheads.
I-protein
A, and autoradiography. Specific proteins are indicated by arrowheads. The migration of molecular mass standards (in kDa)
is shown on the left and on the right for each
gel.
)(although the
relative amounts of each receptor type can vary, particularly in
different PC12 cell subspecies), which might suggest that the EGFR
tyrosine kinase has a lower specificity for substrates of the Ras
pathway. Its response is essentially identical to that of the NGF
receptor (TrkA) in the first 3 min(17, 18) , which may
represent the maximal magnitude of this response, and more EGFR may be
required to achieve this. However, in view of the rapid decay of the
signal, it is equally likely that there is no significant difference in
substrate selectivity or affinity of the EGFR for activating Ras, thus
suggesting that inherently EGF is potentially as good a neurotrophic
factor as NGF and FGF. It is interesting in this regard that it has
been reported to affect selected neurons in the central nervous
system(39, 40) , although it is not reported to act on
neuronal targets in the peripheral nervous system.
))
We thank Drs. Gordon Gill, University of California at
San Diego, and Maureen O'Connor-McCourt, National Research
Council Canada, for generously providing anti-EGFR antibodies. Also, we
thank Drs. Gill, Albert Stewart, Didier Thomas, and Yvonne Wu for
helpful discussions. The expert assistance of Gale Trudeau in the
preparation of this manuscript is appreciated.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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