From the Jean Mayer U.S. Department of Agriculture
Human Nutrition Research Center on Aging at Tufts University and the
¶ Department of Biochemistry, Tufts University School of Medicine,
Boston, Massachusetts 02111
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ABSTRACT |
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The proliferative potential of the
liver has been well documented to decline with age. However, the
molecular mechanism of this phenomenon is not well understood. Cellular
proliferation is the result of growth factor-receptor binding and
activation of cellular signaling pathways to regulate specific gene
transcription. To determine the mechanism of the age-related difference
in proliferation, we evaluated extracellular signal-regulated
kinase-mitogen-activated protein kinase activation and events upstream
in the signaling pathway in epidermal growth factor (EGF)-stimulated
hepatocytes isolated from young and old rats. We confirm the
age-associated decrease in extracellular signal-regulated
kinase-mitogen-activated protein kinase activation in response to EGF
that has been previously reported. We also find that the activity of
the upstream kinase, Raf kinase, is decreased in hepatocytes from old
compared with young rats. An early age-related difference in the
EGF-stimulated pathway is shown to be the decreased ability of the
adapter protein, Shc, to associate with the EGF receptor through the
Shc phosphotyrosine binding domain. To address the mechanism of
decreased Shc/EGF receptor interaction, we examined the phosphorylation
of the EGF receptor at tyrosine 1173, a site recognized by the Shc
phosphotyrosine binding domain. Tyrosine 1173 of the EGF receptor is
underphosphorylated in the hepatocytes from old animals compared
with young in a Western blot analysis using a phosphospecific antibody
that recognizes phosphotyrosine 1173 of the EGF receptor. These data
suggest that a molecular mechanism underlying the age-associated
decrease in hepatocyte proliferation involves an
age-dependent regulation of site-specific tyrosine residue
phosphorylation on the EGF receptor.
The proliferative potential of several tissues, including the
liver, declines with age (1, 2). The ability to proliferate or
regenerate hepatocytes is particularly important in the elderly, who,
due to drug metabolism and other environmental exposures, need to
replace cells that are destroyed due to toxic reactions. Thus, an
age-related decline in the ability to proliferate may limit the ability
of the elderly to recover from toxic exposures. The molecular mechanism
underlying the age-related decrease in proliferative response is not
well understood. Cellular proliferation is activated by a cascade of
signals initiated at the cell membrane in response to growth factor
binding to its receptor. In response to the mitogen, epidermal growth
factor (EGF),1 hepatocytes
from old rats compared with young rats have diminished induction of DNA
synthesis (2). However, hepatocyte EGF receptor number and binding to
the receptor are similar with age (3). Such data suggest that
age-related differences in proliferation may involve differences in the
signal transduction pathways stimulated by growth factors.
The binding of EGF to its receptor initiates a series of signaling
events to induce DNA synthesis and cell division. In response to ligand
binding, the EGF receptor is phosphorylated, which leads to membrane
recruitment of adapter and exchange proteins for activation of Ras and
subsequent Raf kinase activation (4-6). Downstream of Raf kinase,
activation of a series of kinases proceeds to activate primarily
ERK-MAP kinase in response to EGF (7, 8). The MAP kinase family of
proteins are activated by dual phosphorylation on Thr-X-Tyr
residues (reviewed in Refs. 9 and 10). Other members of the MAP kinase
family include JNK and p38 MAP kinases, which are primarily activated
by proinflammatory cytokines and cellular stress (reviewed in Ref. 11).
Upstream activators of these kinases can function both specifically and
with cross-specificity, resulting in activation of more than one MAP
kinase. For example, in response to EGF, ERK-MAP kinase is activated as
much as 20-fold, while JNK-MAP kinase is activated up to 6-fold
(12-14). Activation of ERK-MAP kinase results in phosphorylation of a
number of target proteins including Elk-1 of the ternary complex
factor. Elk-1 mediates transcriptional activation of many genes,
including c-fos and, consequently, AP-1 target genes
(15-19).
We have used a model of primary hepatocytes obtained from young
(4-6-month) or old (>32-month) rats to investigate the mechanism for
the age-related differences in signal transduction pathways that are
activated in response to EGF. ERK-MAP kinase activity was previously
reported to be substantially reduced in hepatocytes from old compared
with young rats (20). In addition to decreased ERK-MAP kinase
activation by EGF, we found that activity of Raf kinase, an upstream
signal transducer, was also substantially decreased without a
change in Raf protein level in hepatocytes from old rats. The
tyrosine phosphorylation of the EGF receptor was similar in hepatocytes
from young and old after EGF stimulation, suggesting that the
age-related difference is after receptor activation and before Raf
kinase activation. Therefore, we examined the ability of the adapter
protein, Shc, to associate with the EGF receptor. Using both direct
immunoprecipitation and GST fusion protein capture assays, we observed
a decrease in the association of the activated EGF receptor with Shc in
hepatocytes from old animals compared with young. Furthermore, we show
by Western blot analysis that site-specific phosphorylation at tyrosine
1173 of the EGF receptor, a Shc interaction site, is reduced in
hepatocytes from old compared with the young. These results indicate
that the reduced ability of the EGF receptor to associate with Shc is
an early step in the EGF signal transduction pathway that is altered
with age.
Hepatocyte Isolation--
Hepatocytes were isolated from young
(4-6-month) or old (>30-month) male F1 F344 X BN rats by perfusion of
the liver with collagenase, type 2 (Worthington) according to the
procedure of Seglen (21) with the modification of using a HEPES (10 mM)-based buffer (22). Cell viability (>85%) was
determined by trypan blue exclusion. The cells (5.5 × 106/100-mm dish) were plated in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum, penicillin
(100 µg/ml), and streptomycin (100 µg/ml) on tissue culture dishes
precoated with Matrigel (1.2 mg/100-mm dish; Collaborative Research,
Lexington, MA). Matrigel supports the differentiated state of
hepatocytes similar to that in the native liver (22). Two hours after
plating, the medium was removed, cells were washed gently two times
with Dulbecco's modified Eagle's medium alone, and treatment medium was added (Dulbecco's modified Eagle's medium supplemented with 0.5%
fetal bovine serum plus glucose, 5 mM; insulin, 0.14 µM; hydrocortisone, 5 µM; sodium selenite,
0.2 µg/ml; and transferrin, 1 µg/ml).
EGF Treatment and Cell Harvest--
Following plating of the
cells (24 h), human EGF (50 ng/ml; Life Technologies, Inc.) was added
to the medium. Cells were harvested by washing with ice-cold
phosphate-buffered saline (twice), scraped into phosphate-buffered
saline, and pelleted by centrifugation, and protein extracts were
prepared by extraction into a whole cell extract buffer (25 mM HEPES, pH 7.5, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.05%
Triton X-100, 20 mM Immune Complex Kinase Assay--
ERK-MAP kinase, JNK-MAP kinase,
and Raf kinase activities were measured using an immune complex kinase
assay (23). Briefly, whole cell extract (50 µg of protein) was
immunoprecipitated using specific antibodies (Santa Cruz Biotechnology,
Inc., Santa Cruz, CA) bound to protein A-Sepharose beads (Repligen
Corp., Needham, MA). Myelin basic protein (Sigma) was used as the
substrate for the p44 ERK kinase assay and the N-terminal c-Jun
obtained from a GST fusion protein purified on glutathione-agarose
beads (Amersham Pharmacia Biotech, Uppsala, Sweden) was used as
substrate for p46 JNK kinase. Purified MEK protein (Santa Cruz
Biotechnology) was used as substrate for Raf kinase. Activity was
visualized by autoradiography of the dried 10% SDS-polyacrylamide gel,
and quantitation was assessed by PhosphorImager analysis using
Molecular Dynamics phosphor imaging equipment.
Immunoprecipitation and Western Blot Analysis--
Specific
antibody was bound to Protein A-Sepharose beads (Repligen Corp.,
Needham, MA). Human Shc antibody and phosphotyrosine antibody, PY20,
were obtained from Transduction Laboratories (Lexington, KY). The
phosphospecific antibody, pY 1173, of the EGF receptor was obtained
from Upstate Biotechnology, Inc. (Lake Placid, NY). The specificity of
the antibody was confirmed by Drs. H. Hoschuetzky and P. Schuessler,
nanoTools Antikorpertechnik, Denzlingen,
Germany.2 All other
antibodies were purchased from Santa Cruz Biotechnology. Whole cell
extracts (100-400 µg of protein) were immunoprecipitated with the
bead-antibody complex. The beads were washed with Triton lysis buffer
(0.1% Triton X-100, 20 mM Tris, pH 7.4, 137 mM
NaCl, 2 mM EDTA, 25 mM Shc Domain GST "Capture"--
Bacterially expressed GST-Shc
domain fusion proteins were generated from pGEX-Shc-PTB and pGEX-Shc
SH2 domain constructs obtained from K. Ravichandran (University of
Virginia, Charlottesville, VA). The expressed proteins were bound to
glutathione Sepharose beads. After washing three times with buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM
EDTA, 0.5% Nonidet P-40, 0.4 mM phenylmethylsulfonyl fluoride, leupeptin (1 µg/ml), and pepstatin (1 µg/ml)), whole cell
extracts (120 µg of protein) were mixed with the beads/fusion protein
and rocked for 2-4 h at 4 °C. After washing, the "captured" proteins were eluted into 4× Laemmli's buffer, boiled, and
electrophoresed by SDS-polyacrylamide gel electrophoresis. The
"captured" EGF receptor was identified by Western blot analysis
using the EGF receptor antibody and detected with chemiluminescent
reagents (NEN Life Science Products).
Age-related Differences in MAP Kinase Activation--
In the
signal transduction cascade to activate transcription of genes involved
in proliferation, ERK-MAP kinase is the final regulatory step that
directly interacts with transcriptional regulators. Therefore, we first
measured activation of ERK-MAP kinase in response to EGF in hepatocytes
isolated from young and old rats. In hepatocytes isolated from young
rats, ERK-MAP kinase was maximally activated 15-fold (15 min, Fig.
1). Activation of ERK-MAP kinase was only 5-fold in hepatocytes from old animals (Fig. 1). The activity returned
to near base-line levels by 2 h. after the addition of EGF to the
medium. (Note that the Fig. 1, top panels, show a higher than base line
response of one pair of animals at 2 h. The average of all animals
at this time was near base line.) JNK-MAP kinase was modestly activated
(5-fold) by 60 min after the addition of EGF in cells from young
animals (Fig. 2). Cells from old animals showed little EGF-stimulated JNK-MAP kinase activity compared with
cells from young animals. The amount of protein determined by Western
blot analysis for either JNK or ERK-MAP kinases stimulated with EGF was
not different in the hepatocytes from young compared with old (Figs. 1
and 2). These results are similar to the data reported for ERK- and
JNK-MAP kinases previously (20) and confirm that there is a significant
decline in EGF-dependent signaling in hepatocytes from old
animals.
MKP-1 Protein Levels in Young and Old Animals--
MKP-1 has been
identified as a dual specificity phosphatase that inhibits ERK-MAP
kinase activity (24, 25). An age-dependent increase in
basal MKP-1 RNA expression was previously suggested to be involved in
the mechanism for the decreased ERK-MAP kinase activation in response
to EGF (13). We measured the protein level of MKP-1 in whole cell
extracts from hepatocytes of young and old rats as well as the level in
the native liver. Fig. 3 shows that there
is actually a decline in the amount of MKP-1 protein in both the liver
(4.5-fold) and cultured hepatocytes (3-fold) with age. This suggests
that the mechanism for the age-related difference in ERK-MAP kinase
activation does not involve MKP-1.
EGF-stimulated Raf Kinase Activity--
To determine if the
age-dependent decline in ERK-MAP kinase activity was due to
differences in upstream activation of the kinase pathway, we measured
the activity of Raf kinase. Raf kinase is activated by Ras GTPase (6).
Ras is induced into the active GTP-bound form following the coupling of
Ras to the EGF receptor via the SOS-GRB2-SHC complex (5). Subsequent to
Raf kinase activation, a series of kinases including MEK1 and -2 are
activated by phosphorylation leading to activation of ERK-MAP kinase.
Raf kinase activity was measured using an immune complex kinase assay with purified MEK as substrate. Raf kinase activity was significantly reduced at 15 min in EGF-stimulated hepatocytes from old animals compared with those from young animals (Fig.
4). Importantly, Raf kinase activity was
greater in the young than the old without a change in the protein
level, indicating that specific activity of Raf kinase declined with
age. These data suggest that age-related changes in the activation of
the ERK-MAP kinase pathway may be upstream of Raf kinase.
Total Tyrosine Phosphorylation of the EGF Receptor--
Upon
activation of the EGF receptor, autophosphorylation of specific
receptor tyrosines initiate the signaling cascade by creating
phosphotyrosine sites for binding of SH2 domain-containing proteins
that are critical for the recruitment of SHC, GRB2, and SOS proteins to
the membrane. To establish if age-related differences in Raf kinase
activity were due to differences in signaling initiated at the
membrane, we determined the amount of tyrosine-phosphorylated EGF
receptor by Western blot analysis. As shown in Fig.
5, no age-related differences in the
amount of EGF receptor protein nor in the phosphorylation of the
receptor upon EGF stimulation were found. Although no age-related
changes in total phosphorylation of the EGF receptor were detected, we
could not conclude that membrane signaling events are not different
with age, since phosphorylation levels at specific sites were not
determined by these methods. Therefore, as a functional measure of
membrane membrane-coupled signaling, we examined the adapter protein
Shc, in response to EGF.
Tyrosine Phosphorylation of Shc and Association with the EGF
Receptor--
The adapter protein Shc associates with phosphotyrosine
residues at positions 1148 and 1173 of the EGF receptor (26, 27) and,
itself, is phosphorylated on tyrosine residues upon activation of the
EGF receptor (28, 29). These Shc phosphotyrosine residues create
binding sites for the SH2 domain of the adapter protein, Grb2 (29, 30).
To determine differences in the age-associated ability to phosphorylate
Shc, extracts from hepatocytes isolated from young and old rats were
immunoprecipitated with Shc antibody and immunoblotted with a
phosphotyrosine antibody. The age-dependent ability to
phosphorylate Shc in response to EGF was not different (Fig.
6A), nor was there any
age-associated difference in the amount of Shc protein (data not
shown). However, the association of a 170-kDa protein with Shc was
markedly reduced in extracts from hepatocytes of old animals compared
with those from young animals (Fig. 6A). In order to
determine if this 170-kDa protein was the EGF receptor, Shc
immunoprecipitates were analyzed by Western blot analysis using an EGF
receptor antibody. As shown in Fig. 6B, the Shc-associated
EGF receptor protein was decreased in hepatocytes from old animals as
compared with the young.
To confirm the age-related decrease in the association between the EGF
receptor and Shc, a GST capture experiment using bacterial fusion
proteins of GST-Shc domains was performed. Three domains (shown in Fig.
7A) have been identified in
the Shc protein. The N-terminal phosphotyrosine binding (PTB) domain
and the C-terminal SH2 domain recognize phosphotyrosine residues of the
activated EGF receptor (27, 28). The internal collagen homology domain contains tyrosine residues that are phosphorylated upon activation and
create the binding sites for the SH2 domain of Grb2 (29, 30). As shown
in Fig. 7B, the amount of EGF receptor "captured" from
the extracts of the hepatocytes from old animals is decreased in
comparison with the young when using the bacterially expressed PTB
domain of the Shc protein. The amount of decreased association between
the proteins ranged from 2- to 3-fold in three independent experiments.
Tyrosine 1148 has been identified to be a recognition site for binding
of the Shc PTB domain (27, 32), and tyrosine 1173 has been reported to
be recognized by the Shc-SH2 domain (26, 32) as well as the PTB domain
(32). Therefore, it is possible that the EGF receptor may be less
phosphorylated specifically at tyrosine 1148 and/or 1173 with age. A
difference in the phosphorylation state at these positions could exist
and would not have been detectable by Western blot analysis for total
phosphotyrosine in the EGF receptor-immunoprecipitated extracts (Fig.
5) due to the abundance of tyrosine phosphorylation on multiple
tyrosines on the EGF receptor. Furthermore, due to the low levels of
EGF receptor in primary hepatocytes, we could not directly map the
amount of phosphotyrosine residues at these specific sites.
Phosphorylation Level of Tyrosine 1173 on the EGF Receptor--
To
determine whether the age-related difference in the ability of Shc to
associate with the EGF receptor is due to a difference in level of
phosphorylation at a specific site on the EGF receptor, we determined
the amount of EGF receptor phosphorylation at tyrosine 1173 by Western
blot analysis. Whole cell extracts were immunoprecipitated with the EGF
receptor antibody and immunoblotted with an antibody specific for the
epitope containing phosphotyrosine 1173 of the EGF receptor. We found
decreased levels of EGF receptor phosphotyrosine 1173 in response to
EGF in hepatocytes isolated from old rats compared with young (Fig.
8).
The results with both direct Shc immunoprecipitation and the GST-Shc
capture assay clearly suggest that a major cause of the age-related
decrease in ERK-MAP kinase signaling pathway is due to an alteration in
the EGF receptor coupling to the membrane signaling machinery.
Furthermore, the results of the GST capture experiment (Fig. 7), using
the PTB domain of Shc, indicate that the age-related difference is in
the activated EGF receptor protein. Western blotting for
phosphotyrosine 1173 (Fig. 8) confirms that the age-related difference
in the ability of the EGF receptor to associate with Shc is due to a
site-specific difference in tyrosine phosphorylation of the EGF receptor.
The molecular basis of the age-related decrease in the capacity of
some tissues to proliferate is not well understood. Cellular proliferation is initiated by growth factor binding to specific receptors to initiate signal transduction. Two models of age-related responses to proliferative stimuli have shown differences in either receptor number and/or receptor phosphorylation (33-35). However, such
differences are not found in the hepatocyte model for the liver (3). In
a human skin fibroblast model of aging, the EGF receptor number and
phosphorylation kinetics were reduced in old compared with young (33,
34). However, the comparison was done in cells derived from different
locations i.e. foreskin (young) and forearm (old). This may
reflect two very different sources of cells that are influenced by
differences in environmental exposures as well as aging. Age-related
differences were also reported in tyrosine phosphorylation of the
CD3 MAP kinases are key regulators of transcription, since these are the
catalysts for phosphorylation of transcription factors regulating
proliferation. Blocking the ERK-MAP kinase pathway has been shown to
down-regulate the mitogenic response (36). Reduced activity of ERK-MAP
kinase in response to EGF was previously identified in hepatocytes
isolated from old rats compared with young. The mechanism of this
decreased activation was suggested to be due to an increase in the
basal RNA expression of the dual specificity phosphatase, MKP-1 (13).
MKP-1 functions as a dual site phosphatase to inactivate the dually
phosphorylated ERK-MAP kinase (24, 25) and inhibits cell division (37).
Our data also show ERK-MAP kinase activity to be diminished in
hepatocytes from old animals in response to EGF (Fig. 1). However,
rather than measuring MKP-1 RNA expression, we determined the amount of
MKP-1 protein and found the levels to be decreased in hepatocytes as
well as in the native livers from old rats (Fig. 3), suggesting that
MKP-1 is not regulating the age-related decrease in ERK-MAP kinase
activity. Further support for the lack of a role for MKP-1 in
regulating the decrease in ERK-MAP kinase comes from the work of
Shapiro and Ahn (38). They reported that MKP-1 has a positive effect on
Raf kinase activity independent of the basal inhibition of ERK-MAP
kinase activity. These results are consistent with our own, where we
observed a decrease in Raf kinase activation in hepatocytes from old
compared with young (Fig. 4), not an increase as would be expected if
MKP-1 were increased.
Our results indicate that the age-related difference in the
proliferative response to EGF is upstream from ERK-MAP kinase activation. Since Raf kinase activation also declined with age (Fig.
4), we examined the coupling of the EGF receptor to the ERK-MAP kinase
signaling pathway. Age-related differences in the membrane complexes
involved in signaling have previously been reported in T-lymphocytes
(35, 39, 40) and B-cells (41). We have identified an early step in the
EGF receptor signal transduction pathway to be a decrease in the
association of the adapter protein, Shc, with the activated EGF
receptor in hepatocytes from old compared with young (Fig. 6).
Furthermore, the results of the GST capture experiment (Fig.
7B), which employed bacterially expressed GST fusion protein
containing the PTB domain of Shc, indicate that the age-related
difference in hepatocytes is in the activated EGF receptor protein. The
PTB domain recognizes the tyrosine phosphorylation at position 1148 of
the EGF receptor (27, 32, 57) and position 1173 (32, 57). We also
observed a similar age-related decrease in the association of the EGF
receptor with the SH2 domain of the Shc protein, which recognizes
tyrosine 1173, although the magnitude of the association was not as
great as with the Shc-PTB domain (data not shown). The idea that
reduced association of Shc with the EGF receptor due to defects in
autophosphorylation sites with the effect of reducing mitogenic
activity is supported by the data of Gotoh et al. (42). A
cell line containing a deletion mutant of the EGF receptor that lacked
the autophosphorylation sites downstream of residue 1011 was used to
show that EGF treatment could still result in stimulation of Shc
phosphorylation but retained only approximately 20% of its mitogenic
activity when measured as cell growth. Others have shown that
EGF-stimulated mutant EGF receptors containing a deleted C-terminal
portion of the EGF receptor or site-specific mutations of C-terminal
autophosphorylation sites also stimulate Shc phosphorylation in NIH 3T3
cells (43, 44). Shc phosphorylation in the absence of binding to the
EGF receptor (Figs. 6 and 7; Refs. 42-44) may be in response to other
non-autophosphorylation-dependent pathways initiated by EGF
transphosphorylations, such as through the ErbB3 receptor (45, 46). The
transforming ability of the C-terminal deletion mutant of the EGF
receptor (42) is not inconsistent with our data, since it can be
mediated in some cell types by Eps8, a substrate for EGF receptor
tyrosine kinase activity (47, 48). Clearly, the complexities of the
pathways that regulate the mitogenic response initiated by EGF ligand
binding are not completely understood.
The mechanism of the age-related decline in the ability of the liver to
proliferate is identified from our data to probably involve factors
that regulate the phosphorylation of the EGF receptor at the specific
tyrosine residues that function as sites for binding of the adapter
protein Shc. Our data identify tyrosine 1173 of the EGF receptor as one
of the residues that is differentially phosphorylated with age in
response to EGF (Fig. 8). Candidates of regulatory enzymes for the
tyrosine phosphorylation include both kinases and phosphatases.
Phosphatases may function as negative or positive regulators of
signaling by directly acting on a phosphorylated tyrosine. For example,
the phosphatase SHP-2 has been suggested to have a positive effect on
EGF signaling (49-51). We found no age-related difference in the level
of SHP-2 protein expression or in the association of SHP-2 in the
complex that was immunoprecipitated by Shc antibody following EGF
stimulation (data not shown). Another phosphatase, SHP-1, has recently
been reported to bind the activated EGF receptor at tyrosine 1173 (52).
Binding at this site resulted in decreased ERK-MAP kinase activity in
response to EGF. Clearly, the balance between kinase and phosphatase
activity is important in regulating the EGF signaling pathway. An
alternative mechanism for regulation of signal transduction is
suggested by the ability of proteins to mask specific SH2 domains of
EGF receptor-coupled proteins (53). However, our data, particularly the
GST capture experiments, do not suggest such a mechanism in the
age-dependent decline in EGF signal transduction.
Understanding the molecular mechanisms for the age-related differences
in the proliferative response to EGF will require more information
about the subtle mechanisms that modulate the EGF receptor-ERK
signaling pathway. There are certain to be newly discovered regulators
that will play a role. For example, it has only recently been shown
through genetic and biochemical studies that the kinase suppressor of
Ras is an important regulator of Raf activity (54, 55); however, this
regulation has also been reported to occur in a kinase-independent
manner (56). Thus, the complexity of the molecular mechanisms of
age-related differences in response to proliferative stimuli in
different tissues will require a more in depth knowledge of the EGF
signaling pathway.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, 0.1 mM orthovanadate, 0.5 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, leupeptin (1 µg/ml),
and pepstatin (1 µg/ml)) for 30 min at 4 °C. Extracted cells were
centrifuged at 12,000 × g for 15 min. Supernatants
were frozen in aliquots at
70 °C.
-glycerophosphate, 1 mM sodium orthovanadate, 2 mM sodium
pyrophosphate, 10% glycerol, 0.4 mM phenylmethylsulfonyl fluoride, leupeptin (1 µg/ml), and pepstatin (1 µg/ml)), and the protein was eluted into 4× Laemmli's buffer, boiled, and
electrophoresed by SDS-polyacrylamide gel electrophoresis. Western blot
analysis was done by standard procedures after protein transfer to
nitrocellulose membrane. Proteins were detected by a chemiluminescent
procedure (NEN Life Science Products).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ERK-MAP kinase activation in response to EGF
in hepatocytes from young and old rats. Top
panels, representative activity (ACT.) of ERK-MAP
kinase after 15, 60, or 120 min of EGF treatment (50 ng/ml) of
hepatocytes isolated from young and old rats. The activity was
determined by an immune complex kinase assay using myelin basic protein
as a substrate. Middle panels, Effect of EGF on
ERK-MAP kinase protein levels in primary hepatocytes as detected by
Western blot analysis using an ERK-1 antibody. Bottom
panels, graphic results of ERK-MAP kinase activity in
response to EGF treatment of hepatocytes from three pairs of animals.
Results are expressed as the mean ± S.E. and normalized to the
average activity of the young controls.
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Fig. 2.
JNK-MAP kinase activation in response to EGF
in hepatocytes from young and old rats. Top
panel, representative activity (ACT.) of JNK-MAP
kinase after 15, 60, or 120 min of EGF treatment (50 ng/ml) of
hepatocytes isolated from young and old rats. The activity was
determined by an immune complex kinase assay using the GST fusion
protein of N-terminal c-Jun. Middle panel, effect
of EGF on JNK-MAP kinase protein levels in primary hepatocytes as
detected by Western blot analysis using a JNK-1 antibody.
Bottom panel, graphic results of JNK-MAP kinase
activity in response to EGF treatment of hepatocytes from three pairs
of animals. Results are expressed as the mean ± S.E. and
normalized to the average activity of the young controls.
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Fig. 3.
MKP-1 protein levels in hepatocytes and liver
from young and old rats. MKP-1 protein was assayed by Western blot
analysis of whole cell extracts from hepatocytes or native liver of
young or old rats.
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Fig. 4.
Raf kinase activity in response to EGF
treatment in hepatocytes from young and old rats. Top
panel, representative activity (ACT.) of Raf
kinase after 15 or 60 min of EGF treatment (50 ng/ml) of hepatocytes
isolated from young and old rats. The activity was determined by an
immune complex kinase assay using MEK-1 as a substrate.
Middle panel, effect of EGF on Raf kinase protein
levels in primary hepatocytes as detected by Western blot analysis
using a Raf-1 antibody. Bottom panel, graphic
results of Raf kinase activity in response to EGF treatment of
hepatocytes from four pairs of animals. Results are expressed as the
mean ± S.E. and normalized to the young control.
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Fig. 5.
Total amount and tyrosine phosphorylation of
the EGF receptor in response to EGF in hepatocytes from young and old
rats. A, total tyrosine phosphorylation of the EGF
receptor was determined by immunoprecipitating the whole cell extracts
with the EGF receptor antibody followed by immunoblotting with an
antibody for phosphotyrosine. The results shown here are representative
of analysis done on three pairs of animals. B, the amount of
EGF receptor was analyzed by Western blot using the human EGF receptor
antibody. The results shown here are representative of analysis done on
three pairs of animals.
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Fig. 6.
Tyrosine phosphorylation of Shc and proteins
associated with Shc in response to EGF treatment of hepatocytes from
young and old rats. A, whole cell extracts were
immunoprecipitated (IP) with the Shc antibody and
immunoblotted (IB) with an antibody for phosphotyrosine. The
results shown are from two pairs of animals. B, whole cell
extracts were immunoprecipitated with the Shc antibody and
immunoblotted with an antibody for the EGF receptor in order to
identify the 170-kDa tyrosine phosphorylated protein that was found to
be immunoprecipitated by the Shc antibody in A. The results
shown are from two pairs of animals. Lysate from human fibroblast
(HF) cells was used as a positive control.
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Fig. 7.
The "capture" of the EGF receptor by the
PTB domain of the Shc protein. A, schematic showing the
three domains of the Shc protein. B, whole cell extracts
from control or EGF-treated (15 min) hepatocytes were incubated with
glutathione-Sepharose bound to either GST protein or the GST-Shc-PTB
domain. The "captured" protein was electrophoresed and
immunoblotted with an antibody for the EGF receptor. The results shown
here are representative of three independent experiments.
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Fig. 8.
Western blot analysis of the level of
phosphotyrosine 1173 on the EGF receptor. A, whole cell
extracts were immunoprecipitated (IP) with the EGF receptor
antibody and immunoblotted (IB) with an antibody that
specifically recognizes the epitope for phosphotyrosine at 1173 on the
EGF receptor. The results are representative of experiments performed
with three pairs of animals. B, the membrane of the Western
blot shown in A was "stripped" and reblotted with the
EGF receptor antibody to show equal loading of the protein.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain of the T-cell receptor (35). Additionally, these
age-related differences suggest cell type differences in the mechanism
of responses to proliferative stimuli with age. Our results agree with
those of Ishigami (3) and show that EGF receptor protein levels are
similar, and in addition that total tyrosine phosphorylation of the
receptor is similar in hepatocytes from young or old rats. Thus, the
age-related differences in EGF-stimulated hepatocyte proliferation do
not seem likely to be caused by gross overall differences in receptor number or phosphorylation. These data suggested that the mechanistic age-related differences in the response to proliferative stimuli in
hepatocytes may be downstream in the signal transduction pathway.
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ACKNOWLEDGEMENTS |
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We thank Drs. Trudy Kokkonen, Doug Jefferson, and David Johnston for information about hepatocyte isolation and culture conditions; Dr. Ben Neel for helpful discussion of the data, K. Ravichandron for GST-Shc domain constructs; and Drs. Brent Cochran and Larry Feig for reviewing the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1 DK50442 (to K. E. P.) and by the Center for Gastroenterology Research on Absorptive and Secretory Processes, NEMCH (NIDDK, NIH, Grant P30 DK34928). This project was also funded in part with federal funds from the U. S. Department of Agriculture Agricultural Research Service under contract 53-3K06-01 (to K. E. P.). The contents of this publication do not necessarily reflect the views or policies of the USDA nor does mention of trade names, commercial products, or organizations imply endorsement by the U. S. government.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.
§ Present address: University of Colorado Health Science Center, Dept. of Biochemistry and Molecular Genetics, 4200 E. Ninth Ave., Denver, CO 80220.
To whom correspondence should be addressed: Jean Mayer USDA
Human Nutrition Research Center on Aging at Tufts University, 711 Washington St., Boston, MA 02111. Tel.: 617-556-3112; Fax: 617-556-3344; E-mail: kpaulson{at}hnrc.tufts.edu.
2 H. Hoschuetzky and P. Schuessler, personal communication; see also, on the World Wide Web, http://www.nanotools.de.
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ABBREVIATIONS |
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The abbreviations used are: EGF, epidermal growth factor; MAP, mitogen-activated protein; GST, glutathione S-transferase; MKP, mitogen-activated protein kinase phosphatase; PTB, phosphotyrosine binding; SH2, Src homology 2; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.
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REFERENCES |
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