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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 55:B125-B134 (2000)
© 2000 The Gerontological Society of America

Age-Related Decline in Ras/ERK Mitogen-Activated Protein Kinase Cascade Is Linked to a Reduced Association Between Shc and EGF Receptor

Dorothy Huttera, Yoshikage Yob, Wei Chena, Pinghu Liua, Nikki J. Holbrooka, George S. Rothb and Yusen Liua

a Laboratory of Biological Chemistry, National Institute on Aging, Gerontology Research Center,
b Laboratory of Cellular and Molecular Biology, National Institute on Aging, Gerontology Research Center,

Yusen Liu, Laboratory of Biological Chemistry, National Institute on Aging, Gerontology Research Center, 5600 Nathan Shock Drive, Baltimore, MD 21224 E-mail: yusenliu{at}nih.gov.

Decision Editor: Jay Roberts, PhD


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
Numerous studies have demonstrated that the proliferative capacity of cells declines with age. Using rat primary hepatocytes as a model system, we recently demonstrated that this age-related decline in the proliferative response to mitogenic stimulation is associated with decreased activities of both extracellular signal-regulated kinase (ERK) and p70 S6 kinase (p70S6k). To unravel the molecular basis for age-related defects in the ERK pathway, we have now characterized the upstream signaling events that occur after epidermal growth factor (EGF) stimulation in young and aged hepatocytes. As previously noted for ERK, the activities of both MEK (the kinase immediately upstream of ERK) and Ras following EGF stimulation were significantly lower in aged hepatocytes. An examination of the EGF receptor (EGFR) revealed a similar amount of EGFR in the two age groups. Likewise, EGFR and Shc, an adaptor protein that plays a crucial role in linking EGFR to Ras activation, underwent tyrosine phosphorylation to a similar degree in both young and aged hepatocytes. However, in aged cells Shc was unable to form stable complexes with EGFR after EGF stimulation. Our results suggest that a decrease in the association between Shc and EGFR in aged cells underlies the age-related declines in the ERK signaling cascade and in proliferative capacity.

GROWTH factor stimulation triggers a series of complex signaling events that lead to an enhanced transcription of a group of proliferation-associated genes and an increased translation rate of proteins necessary for cell proliferation (1)(2). The mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK) cascade plays a crucial role in regulating cell growth and differentiation (3). The initiation of this signaling pathway by means of growth factor receptors has been studied extensively. Ligand-mediated dimerization of growth factor receptors stimulates their intrinsic tyrosine kinase activities, resulting in the autophosphorylation of tyrosine residues (4). These residues then serve as docking sites for the recruitment of downstream signaling mediators necessary for the activation of membrane-localized Ras. The adaptor protein Grb2 can form complexes through its Src homology 3 domain with the Ras guanine nucleotide-exchange factor, Son of Sevenless (Sos). Grb2 can directly bind to the receptor phosphotyrosine residues through its Src homology 2 (SH2) domains to translocate Sos from the cytoplasm to the vicinity of Ras. In addition, the Grb2·Sos complexes can be recruited to the receptors through another adaptor protein, Shc (5). Shc binds to certain receptor phosphotyrosine sites through its phosphotyrosine-binding (PTB) domain, becomes tyrosine phosphorylated itself, and thereby provides additional docking sites for Grb2 (4)(5). The recruitment of Grb2·Sos complexes to the phosphotyrosine sites on membrane-associated proteins enables Sos to catalyze the GTP/GDP exchange reaction on Ras, resulting in Ras activation. GTP-bound Ras interacts with the oncoprotein Raf, facilitating the translocation of Raf to the plasma membrane for further activation. The activation of Raf marks the start of the sequential phosphorylation cascade. Raf phosphorylates MEK, which in turn phosphorylates ERK on the threonine and tyrosine residues in the kinase subdomain VIII, resulting in ERK activation (3). ERK is responsible for the phosphorylation of a variety of cellular proteins, including the p90RSK protein kinase, transcription factors, and components of the protein synthesis machinery (2)(3)(6).

Evidence from several different model systems has indicated that cellular aging is associated with a loss in proliferative capacity (7)(8). For in vitro aged senescent human diploid fibroblasts, the decline in proliferative capacity is correlated with a reduced activation of both serum response factor and AP-1 transcription factor complexes, and attenuated c-fos induction (9)(10). Liver regeneration following partial hepatectomy has been used as a model for studying the effect of aging on the proliferative response in vivo. Following partial hepatectomy, DNA synthesis is delayed and reduced in magnitude in aged rats compared with young adult rats (11)(12). This age-related proliferative decline is, at least partially, due to changes at the cellular level, because epidermal growth factor (EGF)-stimulated DNA synthesis in primary hepatocytes from aged rats is significantly lower than that seen in hepatocytes from young animals (13). We have used primary cultured rat hepatocytes to study the molecular mechanisms responsible for the age-associated loss of proliferative response. Previously, we demonstrated that hepatocytes from aged animals exhibit a significant attenuation in EGF-induced activation of ERK MAP kinase and expression of ERK-regulated genes relative to hepatocytes from young rats (14). In addition, the activation of p70S6k in response to EGF is also attenuated in aged hepatocytes (13). As the p70S6k pathway is mediated by a distinct set of molecules, it seems likely that a defect in aged cells could originate at an early signaling event that is common to the activation of both the ERK MAP kinase and p70S6k signaling pathways.

In the studies described here, we have characterized the early signaling events that occur immediately following EGF stimulation in young and aged hepatocytes. In a comparison with young hepatocytes, the aged hepatocytes exhibited a significant decline in EGF-stimulated MEK activity. Although Ras was activated following EGF stimulation in both young and aged hepatocytes, the maximal Ras activity reached was higher and relatively sustained in the young cells, whereas it was lower and more transient in the aged cells. Nevertheless, there were no significant differences observed between the two age groups in either the levels of EGF receptor (EGFR) or Shc and Grb2 proteins, or the overall tyrosine phosphorylation profiles. Surprisingly, we found that in aged cells there is a dramatic reduction in the amount of Shc·EGFR complexes formed in response to EGF stimulation, although both EGFR and Shc were similarly tyrosine phosphorylated. These results suggest that a defect in either the recruitment of Shc to EGFR or its retention in the complexes contributes to the age-associated decline in proliferative capacity.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Isolation, Culture, and Treatment of Rat Hepatocytes
Hepatocytes were isolated from young (6-month old) and aged (24-month old) male Wistar rats by the collagenase perfusion method of Seglen (15). Isolated cells were plated in Williams E medium containing 5% fetal bovine serum and cultured for 2 hours at 37°C and 5% CO2 to allow attachment. The medium was then replaced with serum-free Williams E medium, and the cells were cultured for an additional 16 hours prior to treatment. This procedure results in less than 5% contamination with nonhepatocytes. EGF was added to the medium to a final concentration of 100 ng/ml.

Antibodies, Immunoprecipitation, and Western Blot Analysis
Polyclonal anti-Shc, anti-Sos, monoclonal anti-Grb2, anti-MEK1, anti-MEK2, and anti-p90RSK were purchased from Transduction Laboratories (Lexington, KY). Polyclonal antibodies against EGFR and ERK2 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal anti-phospho-MEK was purchased from New England Biolabs, Inc. (Beverly, MA). Anti-phosphotyrosine monoclonal antibody 4G10 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Horseradish peroxidase (HRP) conjugated antimouse and antirabbit secondary antibodies were purchased from Amersham Co. (Arlington Heights, IL).

Western blot analysis was carried out as described previously (16). Briefly, hepatocytes were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in 1 ml of lysis buffer (20 mM of N-2-hydroxyethylpiperazine-N1-2-ethane sulfonic acid (HEPES), pH 7.4, 50 mM of ß-glycerophosphate, 1% Triton X-100, 10% glycerol, 2 mM of Ethylene bis(oxyethylenenitrilo) tetraacetic acid (EGTA), 1 mM of dithiothreitol, 10 mM of sodium fluoride, 1 mM of sodium orthovanadate, 2 µM of leupeptin, 2 µM of aprotinin, 1 mM of phenylmethylsulfonyl fluoride, and 0.5 µM of okadaic acid). The cell lysates were clarified by centrifugation at 14,000 rpm for 10 minutes. Samples normalized for total protein content were resolved by electrophoresis through 10% or 4–12% gradient NuPAGE Bis-Tris gels (Novex) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). Enhanced chemiluminescence (ECL) reagent (Amersham Co.) was used for the detection of the immunoreactive bands. For reprobing, blots were stripped with a buffer containing 50 mM of Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), and 0.1 M of ß-mercaptoethanol.

For immunoprecipitation, soluble cell lysates normalized for total protein content were precleaned by incubation with 20 µl of protein A-Sepharose beads (Pharmacia Biotech Inc., Piscataway, NJ) at 4°C for 30 minutes. After incubation, the supernatants were collected by brief centrifugation and incubated with the indicated rabbit polyclonal antibody and 20 µl of protein A-Sepharose beads for 16 hours at 4°C while they gently rotated. The immunoprecipitates were washed four times with 1 ml of lysis buffer and separated through the NuPAGE gel system (Novex). A Western blot analysis of the immunoprecipitates was carried out as described above.

Immune Complex Kinase Assay
The kinase activity of MEK1 was assessed by an immune complex kinase assay as previously described, except for the use of recombinant ERK2 K-R protein as a substrate (17). Briefly, soluble cell lysates containing 800 µg of protein were incubated with 1 µg of rabbit polyclonal anti-MEK1 antibody (Babco, Berkeley, CA) and 20 µl of protein A-Sepharose beads at 4°C for 16 hours with gentle rotation. The immunoprecipitates were then washed three times with lysis buffer, three times with wash buffer (500 mM of LiCl, 100 mM of Tris-HCl, pH 7.6, 0.1% Triton X-100, and 1 mM of dithiothreitol) and three times with kinase assay buffer (20 mM of 3-(4-Morpholino) propane sulfonic acid (MOPS), pH 7.2, 2 mM of EGTA, 10 mM of MgCl2, 1 mM of dithiothreitol, and 0.1% Triton X-100). The kinase reactions were carried out at 30°C for 20 minutes in a 55-µl kinase assay buffer containing 10 µM of adenosine triphosphate (ATP), 10 µCi of {gamma}-32P-ATP, 20 mM of MgCl2, and 3 µg of recombinant, kinase-dead ERK2 (ERK2 K-R) tagged with a polyhistidine peptide. The polyhistidine-tagged recombinant ERK2 K-R was produced in Escherichia coli and purified through Ni-affinity chromatography as previously described (18). After completion of the reaction, the proteins were resolved by SDS-polyacrylamide gel electrophoresis. Incorporation of 32P into the substrates was quantitated by using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The kinase activity of ERK was assessed by an immune complex kinase assay as previously described, using myelin basic protein (MBP) as a substrate (17). Similarly, p90RSK was immunoprecipitated by using a rabbit polyclonal antibody against the carboxyl-terminal 20 amino acid sequence of avian p90RSK (Babco, Berkeley, CA). Its kinase activity was assessed in a reaction containing 10 µM of ATP (10 µCi of {gamma}-32P-ATP), 65 nM of protein kinase A inhibitor (Santa Cruz Biotechnology Inc., Santa Cruz, CA), 30 mM of p-nitrophenol phosphate, and 5 µg of a peptide corresponding to the phosphorylation domain of ribosomal protein S6 (Santa Cruz Biotechnology Inc., Santa Cruz, CA). After termination of the kinase reaction, the peptide substrate in a 10-µl reaction solution was loaded onto a SpinZyme Phosphocellulose unit (Pierce, Rockford, IL), and washed four times with 150 mM of phosphoric acid to remove free ATP. Incorporation of 32P into the S6 peptide was determined by scintillation counting.

Ras Activity Assay
Ras activity assays were carried out according to a procedure developed by Taylor and Shalloway (19). Briefly, the Ras interacting domain (RID) of Raf was expressed in E. coli as a glutathione S-transferase (GST) fusion protein and purified by glutathione-Sepharose (Pharmacia Biotech Inc., Piscataway, NJ) affinity chromatography. Twenty micrograms of the GST-Raf RID fusion protein on the glutathione-Sepharose beads (Pharmacia Biotech Inc., Piscataway, NJ) were incubated with soluble lysates containing 1 mg of protein at 4°C for 30 minutes. The Sepharose beads were recovered through gentle centrifugation and rapidly washed three times with 1 ml of ice-cold lysis buffer. Proteins recovered with the GST-Raf RID fusion protein were separated through a 10% NuPAGE Bis-Tris gel in 4-Morpholine ethane sulfonic acid (MES) buffer (Novex, San Diego, CA), and they were subjected to a Western blot analysis with a monoclonal antibody against pan-Ras (Calbiochem, La Jolla, CA).

Statistical Analysis
Data are presented as means ±SEM. Effects of age, EGF treatment, and their interaction were determined by repeated measures analysis of variance. The interaction was statistically significant (p = .0001). This means that the effect of age is different at the various time points. To identify the time at which young hepatocytes differed from the old hepatocytes, we compared the means at these points by using a Bonferroni correction. A p value of <.0125 was considered significant.


   Results
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 Abstract
 Methods
 Results
Discussion
 References
 
The Activities of ERK MAP Kinase and ERK-Regulated p90RSK Decline with Age
The ERK MAP kinase plays a crucial role in regulating cell growth. In hepatocytes, a pharmacological inhibitor specific for MEK has been shown to potently inhibit EGF-stimulated hepatocytes' DNA synthesis, supporting the critical importance of this pathway in hepatocyte proliferation (20). We have previously reported that aged hepatocytes show reduced DNA synthesis and decreased cell cycle progression in response to EGF stimulation (13)(14). This age-associated decline in proliferative capacity is correlated with a reduction in ERK MAP kinase activity, a decreased expression of ERK-regulated genes (i.e., c-fos and c-jun), and lower levels of AP-1 DNA-binding activity (13)(14). In the current study, ERK was rapidly and transiently activated by EGF stimulation in both young and aged hepatocytes (Fig. 1). EGF stimulation of young cells resulted in a more than 60-fold increase in ERK activity within 5 minutes, declining thereafter. However, even 30 minutes following the EGF treatment, ERK activity remained about 20-fold elevated relative to untreated cells. Although the overall kinetics of ERK activation in aged cells were similar to those seen in the young cells, the maximal activity achieved in aged cells was less than 50% of that observed in young cells.



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  		Figure 1. EGF-triggered activation of ERK and p90RSK in hepatocytes from young and old rats. A, Kinetics of ERK2 activation in young and old hepatocytes. Data are expressed as the percentage relative to the maximal kinase activity (young hepatocytes, 5 minutes after EGF treatment). Data shown are from a representative experiment. B, Kinetics of p90RSK activation in young and old hepatocytes. Values are the means ±SEM of three independent experiments. The asterisks denote a significant difference from young animals at the indicated time point (p < .0125). C, Effect of EGF stimulation (100 ng/ml) on p90RSK protein levels in young and old hepatocytes; p90RSK was detected by a Western blot analysis, using a monoclonal antibody.

 
p90RSK represents a group of highly homologous serine/threonine protein kinases whose activities are directly regulated through phosphorylation by ERK MAP kinases. It has been shown that inactive p90RSK forms complexes with ERK MAP kinases in the cytoplasm. After activation, p90RSK disassociates from ERK and translocates into the nuclear compartment (21). Because p90RSK can phosphorylate transcription factors including c-fos, CREB, and I{kappa}B (11)(22)(23)(24), it has been suggested that this group of kinases may contribute to the transcriptional regulation of genes important for mitogenesis (24)(25). Therefore, given the difference in ERK MAP kinase activity between young and aged cells, we examined p90RSK activity in the different aged hepatocytes. p90RSK activity was assessed by an immune complex kinase assay, using a peptide substrate corresponding to the ribosomal protein S6. Consistent with the notion that p90RSK activity is controlled by ERK MAP kinases, we observed a 50% decline in EGF-stimulated p90RSK activity in aged hepatocytes compared with that seen in young cells (Fig. 1). Unlike basal ERK activity, which did not differ between the two age groups, the basal level of p90RSK activity in aged cells was consistently lower than that in young cells. The age-related differences in p90RSK activities were not due to an alteration in p90RSK content, as a Western blot analysis of total cell lysates revealed no difference in protein levels between young and aged cells (Fig. 1).

EGF-Induced MEK Activation in Young and Aged Hepatocytes
Because MEK protein kinases are the immediate upstream regulators of ERK MAP kinases, we investigated whether EGF-stimulated MEK activities differed between the two age groups. MEK is activated by its phosphorylation on serine-218 and serine-222 by the oncoprotein Raf. Hence, MEK activity can be assessed by a Western blot analysis, using an antibody specific to the phosphorylated form of MEK. As shown in Fig. 2 (upper panel), EGF treatment of young cells resulted in a transient increase in the amount of phosphorylated MEK. The dramatic increase observed at 5 minutes is consistent with that seen for rapid activation of the downstream target ERK. In contrast to that seen in young cells, no detectable increase in phosphorylated MEK was observed in aged cells, even immediately after stimulation with EGF. Importantly, there were no detectable differences in the levels of either MEK1 or MEK2 proteins in young versus aged cells (Fig. 2, middle and lower panels). MEK1 activity was also assessed by an immune complex kinase assay, using a kinase-dead recombinant ERK2 as a substrate (Fig. 2). This assay, which directly measures kinase activity, is more sensitive than the Western blot analysis used above and allows for quantitative measurement of the MEK activity. EGF stimulation resulted in a time-dependent increase in MEK1 activity in young hepatocytes, an effect that was significantly reduced in aged cells. Overall, the kinetics of MEK1 activation were very similar to those of ERK. A nearly 20-fold increase in MEK1 activity was detected in young cells within 5 minutes after EGF stimulation, whereas in the aged cells there was only an eightfold elevation at the same time point. Although MEK1 activity dropped rapidly after 5 minutes, the activity in young cells was consistently higher than that in aged cells at all time points examined. The results of the statistical analysis comparing MEK activation in three sets of young versus aged cells are indicated in Fig. 2.



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  		Figure 2. EGF-triggered MEK activation in hepatocytes from young and old rats. A, MEK phosphorylation in young and old hepatocytes in response to EGF stimulation. Phospho-MEK (upper panel) was detected by a Western blot analysis, using a polyclonal antibody specific to active/phosphorylated MEK. MEK1 and MEK2 protein levels in the cell lysates were detected by using monoclonal antibodies specific for MEK1 (middle panel) and MEK2 (lower panel). B, MEK1 activity in young and old hepatocytes following EGF stimulation. C, Statistical analysis of MEK1 activation in hepatocytes from multiple young and aged rats. MEK1 activity was expressed as the percentage relative to the maximal kinase activity (young hepatocytes, 5 minutes after EGF treatment). Values are the means ±SEM of three independent experiments. The asterisks denote a significant difference from young animals at the indicated time point (p < .0125).

 
Ras Activation in Response to EGF Stimulation Is Attenuated in Aged Hepatocytes
Activation of Ras has been shown to be a crucial event in initiating the phosphorylation cascade leading to ERK activation. Therefore, we investigated whether Ras activity induced by EGF was attenuated with age. Activated Ras was detected by the procedures developed by Taylor and Shalloway (19) based on the fact that GTP-bound (activated) Ras, but not GDP-bound (unactivated) Ras, interacts with Raf through an amino terminal RID. Briefly, a recombinant GST-Raf RID fusion protein containing the amino acids 1-149 of c-Raf-1 was generated and incubated with lysates from control and EGF-stimulated cells to purify GTP-bound Ras. Then, the purified Ras was detected by a Western blot analysis, using an anti-pan-Ras monoclonal antibody. As shown in Fig. 3, a protein of 21 kDa was detected in both young and aged cells after EGF stimulation, but not in the untreated controls. Most importantly, this protein comigrated with p21Ras detected from the total cell lysates, verifying that it did indeed represent the GTP-bound form of p21Ras (data not shown). Ras was activated to maximum levels of about 12-fold within 5 minutes of EGF treatment in young hepatocytes. Following this peak level of activation, active Ras levels in young hepatocytes decreased gradually. By 30 minutes after EGF stimulation, Ras activity decreased to 50% of the maximal level. Although in aged cells EGF treatment also resulted in rapid Ras activation, the maximal Ras activity was significantly lower than that in their young counterparts. In contrast to the gradual decrease in the level of GTP-bound Ras seen in young cells, the levels of GTP-bound Ras in aged hepatocytes rapidly declined to close to basal levels within 10 minutes of EGF treatment. A statistical analysis of the data further indicated that Ras activity declined at a faster pace in the aged hepatocytes than in the young cells, suggesting that aged hepatocytes may have alterations in their Ras regulatory machinery (Fig. 3). A Western blot analysis indicated that there was no change in the abundance of total Ras proteins observed during the course of the experiment (Fig. 3, lower panel), nor were there differences in total Ras content found between cells from the two age groups (Fig. 3).



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  		Figure 3. EGF-stimulated Ras activation in hepatocytes from young and old rats. A, Ras activation in young and old hepatocytes in response to EGF stimulation. Upper panel: GTP-bound Ras. Lower panel: Ras protein levels in young and old hepatocytes. B, Statistical analysis of Ras activation in hepatocytes from multiple young and old rats. Ras activity was expressed as the percentage relative to the maximal arbitrary density (young hepatocytes, 5 minutes after EGF treatment). Values are the means ±SEM of five independent experiments. The asterisks denote a significant difference from young animals at the indicated time point (p < .0125). C, Comparison of total Ras levels in hepatocytes from multiple young and aged animals. Results shown in A and C are from representative experiments.

 
EGF Induces a Comparable Tyrosine Phosphorylation of EGFR in Both Young and Aged Hepatocytes
Activation of Ras-mediated ERK cascade by EGF is mediated through GDP/GTP exchange catalyzed by guanine nucleotide-exchange factor Sos proteins, which are translocated to the plasma membrane through the adaptor proteins Grb2 and Shc (4). Because an attenuated ERK signaling cascade in EGF-treated aged hepatocytes might be a reflection of an age-associated reduction in the protein levels of the Ras modulators, we investigated whether there were age-related changes in the protein levels of Sos, Grb2, and Shc. A Western blot analysis with total cell lysates from multiple animals revealed no significant difference in Sos protein levels between young and aged hepatocytes (data not shown). Similarly, no age-related differences were detected in the levels of either Grb2 or Shc (data not shown).

Protein tyrosine phosphorylation is among the earliest signaling events that occur following EGF stimulation (4). It mediates the initiation of all downstream signaling cascades, including the Ras/ERK MAP kinase pathway. Therefore, we investigated whether age-related differences in the overall protein tyrosine phosphorylation profiles could be observed. Protein tyrosine phosphorylation was detected by Western blot analysis, using the 4G10 monoclonal antiphosphotyrosine antibody. As shown in Fig. 4, both young and aged cells responded to EGF treatment with the rapid tyrosine phosphorylation of a group of proteins with molecular weights of approximately 170 K, 120 K, 54 K, 50 K, 44 K, and 42 K. A Western blot analysis on the same membrane indicated that the 170-kDa protein comigrated with the EGFR, while the 54- and 50-kDa proteins comigrated with the p52Shc and p47Shc isoforms (Fig. 4, right two lanes). A comparison of the overall levels of tyrosine-phosphorylated proteins at 5 minutes revealed no obvious differences between responses of the two age groups (Fig. 4).



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  		Figure 4. Protein tyrosine phosphorylation profile of young and aged hepatocytes. A, Time course (left panel) of protein tyrosine phosphorylation in response to EGF stimulation. The major tyrosine-phosphorylated bands induced by EGF are indicated. The identical blot was then stripped and probed with EGFR (middle panel) and Shc (right panel). B, Comparison of protein tyrosine phosphorylation profiles in young and aged hepatocytes. C, Western blot analysis of EGFR levels in untreated hepatocytes from four young and four aged rats. D, Time course of EGFR tyrosine phosphorylation in response to EGF stimulation. EGFR was immunoprecipitated from hepatocyte cell lysates and then subjected to a Western blot analysis for protein tyrosine phosphorylation. E, Direct comparison of EGFR tyrosine phosphorylation in multiple young and aged hepatocytes. Young and aged hepatocytes were treated with 100 ng/ml of EGF for 5 minutes or left untreated. The positions where IgG heavy chain migrated are marked; results shown are from representative experiments.

 
In earlier studies utilizing ligand-binding assays, it was reported that young and aged hepatocytes possessed comparable EGF-binding capacities (26)(27), both with respect to the number of receptors and their affinity for EGF. However, EGFR protein levels were not directly measured, nor were other functional properties of the receptor examined in these early studies. Hence, we undertook experiments to address these issues. We first examined the levels of EGFR protein in young and aged hepatocytes by means of a Western blot analysis. Consistent with the comparable EGF-binding capacity seen in earlier studies, we observed no difference in the amount of EGFR protein in young and aged hepatocytes (Fig. 4).

In order to directly assess the tyrosine phosphorylation of EGFR in response to EGF stimulation and to examine the functional ability of EGFR, EGFR was immunoprecipitated with a rabbit polyclonal antibody raised against the carboxyl terminal of human EGFR. The immune complexes were then separated by SDS-PAGE, and tyrosine phosphorylation of EGFR was detected by a Western blot analysis, using the 4G10 antiphosphotyrosine antibody. Consistent with the findings presented above (Fig. 4 and Fig. 4), EGF stimulation triggered the rapid tyrosine phosphorylation of EGFR in both young and aged hepatocytes with very similar kinetics (Fig. 4). A comparison of the levels of EGF-induced EGFR tyrosine phosphorylation in multiple samples revealed no significant difference in its ability to undergo autophosphorylation (Fig. 4).

Age-Related Decline in the EGF-Induced Association Between Shc and EGFR
Shc is another adaptor protein that has been shown to play an important role in mediating the EGFR-initiated Ras/ERK signaling cascade (5)(28)(29). Through its PTB domain, Shc is recruited to certain phosphotyrosine residues of growth factor receptors. It then undergoes phosphorylation, and thereby provides higher affinity docking sites for Grb2·Sos complexes (29)(30). The fact that aged cells exhibited similar levels of EGFR tyrosine phosphorylation compared with young cells, but an attenuated ERK signaling cascade, prompted us to investigate the involvement of Shc in the age-related decline of the Ras/ERK signaling cascade. Shc was immunoprecipitated from lysates prepared from either control or EGF-treated young and aged cells, and tyrosine-phosphorylated proteins in these immune complexes were then detected by using the 4G10 antiphosphotyrosine antibody (Fig. 5, upper panel). Consistent with levels of Shc tyrosine phosphorylation detected in total cell lysates in Fig. 4, the levels of tyrosine-phosphorylated Shc itself in the anti-Shc immunoprecipitates were comparable at various time points between the two age groups. However, we saw a striking difference in the amounts of two tyrosine-phosphorylated proteins with molecular masses around 170 kDa and 120 kDa that coimmunoprecipitated with Shc in young versus old cells. The 120-kDa and 170-kDa proteins were much more abundant in young cells relative to those in aged hepatocytes. In contrast, a slightly higher level of a 70-kDa tyrosine-phosphorylated protein was present in the immunoprecipitates from aged cells. Reprobing the identical blot with anti-Grb2 antibody detected a single 25-kDa band in EGF-stimulated Shc immune complexes but not the controls. No significant age-related differences were detected in the amounts of Grb2 present in the Shc immune complexes (Fig. 5, lower panel). A Western blot analysis of the same Shc immunoprecipitates with anti-EGFR antibody confirmed that the 170-kDa band detected by the antiphosphotyrosine antibody contained EGFR (Fig. 5, middle panel). These findings, demonstrating a reduced association of EGFR and Shc in aged hepatocytes, have been consistently reproduced in four separate experiments. The identities of the 120- and 70-kDa tyrosine-phosphorylated proteins in the Shc immune complexes are currently unclear, but they may represent other signaling molecules that interact with either Shc or EGFR. Taken together, our results indicated that aged rat primary hepatocytes exhibit a defect in the recruitment or retention of Shc to EGFR. This age-related defect in organizing signaling complexes may lead to a decline in the EGF-stimulated Ras/ERK MAP kinase signaling cascade, and it may contribute to an age-associated decrease in proliferative capacity.



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  		Figure 5. Age-related decline in association between Shc and EGFR in EGF-treated rat hepatocytes. Young and aged hepatocytes were stimulated with 100 ng/ml of EGF for the indicated time. Shc was immunoprecipitated from cell lysates and then subjected to Western blot analysis, using either 4G10 antiphosphotyrosine (upper panel) or anti-EGFR polyclonal antibody (middle panel). The blot probed with anti-Shc antibody was stripped and reprobed with monoclonal anti-Grb2 antibody (lower panel). Similar results were obtained in four independent experiments. Results shown are from a representative experiment.

 

   Discussion
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 Abstract
 Methods
 Results
 Discussion
References
 
Previously, we reported that primary rat hepatocytes exhibit an age-associated decline in their proliferative response to EGF as indicated by a reduction in DNA synthesis (13)(26). This decline is correlated with reductions in ERK MAP kinase activation, the expression of ERK-regulated genes, and AP-1 DNA binding activity (14). Recently, we also reported an age-related decline in the p70S6k pathway (13), which represents another important mitogenic signaling pathway. As the two pathways, for the most part, are mediated through a distinct set of molecules, it is likely that aged hepatocytes exhibit a defect(s) in the early steps of the signaling events common to both pathways. In order to understand the mechanisms contributing to these phenomena, we have examined upstream signaling events known to be involved in the EGF signaling pathway. Therefore, we concentrated on the functional properties of the EGFR and other signaling events intermediate to ERK activation. These included the following: (a) activation of MEK, the kinase positioned directly upstream of ERK; (b) activation of Ras, the molecular switch lying upstream of the ERK MAP kinase pathway; (c) the tyrosine phosphorylation of EGFR and the Shc adaptor protein; and (d) the ability of EGFR to form complexes with Shc. A direct comparison of MEK1 activity between the young and aged hepatocytes indicated that EGF-stimulated MEK1 activity also exhibited a significant age-related decline (Fig. 2). Likewise, the age-related discrepancy was also detected at the level of Ras activation. Although there was no marked difference in the basal levels of Ras activities between the two age groups, the maximal activity stimulated by EGF treatment was significantly lower in aged hepatocytes than in their young counterparts. Additionally, even though maximal Ras activity was reached within 5 minutes after addition of EGF to the medium, it appeared that Ras activity decreased more rapidly in aged hepatocytes than in the young hepatocytes (Fig. 3). The difference in Ras activation indicated that the defect(s) in aged cells lies above Ras in the early steps of the signaling cascade.

Earlier studies utilizing classical ligand-binding assays had demonstrated that neither the number of EGFR nor the affinity of EGFR for its ligand was altered with aging (26)(27). In keeping with the earlier studies, we saw no evidence for alterations in the amount of EGFR protein between old and young cells. A further examination of the tyrosine phosphorylation of EGFR and Shc in response to EGF stimulation revealed no significant difference between the young and aged hepatocytes. However, aged cells showed a strikingly lower amount of Shc·EGFR complexes compared with that seen in young cells. While this article was under its initial review, a report was published by Palmer and colleagues that showed that this same aging effect occurs in hepatocytes from Fischer 344/Brown Norway rats (31). Thus, the phenomenon is not restricted to the Wistar rat strain. Therefore, it is likely to represent a fundamental alteration with aging, which contributes to the reduced proliferative capacity of aged hepatocytes.

The Shc adapter protein plays an important role in the transmission of proximal receptor tyrosine kinase signals to the distal effector Ras. Following the tyrosine phosphorylation of the Shc protein, Shc can then engage the SH2 domain of Grb2 and form ternary complexes with Grb2 and Sos (32)(33). In addition to binding the Grb2·Sos complex through its phosphotyrosine sites, Shc can also interact with the activated EGFR through its amino-proximal PTB domain and carboxyl-terminal SH2 domain (5)(33). The Shc mediated association of the Grb2·Sos complex with the tyrosine-phosphorylated EGFR brings Sos in close proximity to the plasma membrane location of Ras, thereby effecting guanylnucleotide exchange of GTP for the bound GDP (4)(5). Although a small fraction of the Grb2·Sos complexes can be targeted to activated EGFR through direct Grb2-EGFR interaction (34), in most systems recruitment of Grb2·Sos complexes to the EGFR is mainly mediated through Shc (28). Thus, association of Shc with activated EGFR represents a critical step in the signaling cascade linking EGFR activation to the Ras/ERK MAP kinase pathway. Therefore, an age-related decrease in the association between Shc and activated EGFR would be expected to lead to a decline in the activity of Ras, and the Ras-mediated ERK MAP kinase cascade. This hypothesis is consistent with our observation that the activities of Ras, MEK, ERK, and p90RSK are all significantly attenuated in aged hepatocytes, although we have not directly demonstrated such a reduction in the activity of Raf. In addition to the ERK MAP kinase signaling pathway, Ras also participates in the regulation of a number of signaling cascades, including activation of phosphatidylinositol-3 kinase that leads to the p70S6k (35). It is possible that a decrease in the activity of Ras, as a result of an attenuated Shc-EGFR interaction, may also contribute to the age-associated decline in p70S6k activity as we previously reported.

The molecular basis for the decline in the association between activated EGFR and Shc in aged hepatocytes is unclear. Several possibilities exist to explain this age-related defect. Although we have not observed a significant difference in gross EGFR tyrosine phosphorylation, we cannot exclude the possibility that a specific phosphotyrosine residue such as tyr1173, which serves as a docking site for the Shc PTB domain, undergoes preferential dephosphorylation. Because EGFR undergoes tyrosine phosphorylation at multiple sites (33), a subtle difference in the phosphorylation state of a specific residue may not be detected by our assay. Alternatively, the interaction between the EGFR and Shc molecules could be downregulated by an inhibitory mechanism specific for aged hepatocytes. In fact, a tyrosine phosphatase SHP-1 has recently been shown to bind to the same site on EGFR as Shc (36). The picture is further complicated by the fact that other EGFR-related tyrosine kinases such as erbB2 and erbB3 can dimerize with EGFR and participate in mediating these signaling events (37)(38). A further characterization of the proteins capable of interacting with EGFR and Shc may enable us to better understand the cause of the age-related decline in the association between these two regulatory molecules. Nevertheless, our results demonstrating that the association between Shc and EGFR in hepatocytes is reduced with age offers a molecular mechanism for the age-related attenuation in the ERK signaling cascade and provides an explanation for the age-associated decline in the proliferative capacity of hepatocytes. Future studies using different model systems will be explored to determine whether alterations in the association between Shc and growth factor receptors occur as a general feature of aging.


   Acknowledgments
 
We thank Drs. A. Thoms-Chesley and C. Morrell for the statistical analysis and G. Kokkonen for assistance in preparing hepatocytes. We are greatly indebted to Drs. M. Weber, M. Cobb, and S. Taylor for providing us with valuable reagents. We are grateful to Drs. R. Wange, J. Staros, and S. Guyer for stimulating discussions and valuable suggestions.


   Footnotes
 
The first three authors contributed equally to the work.

Received February 23, 1999

Accepted August 10, 1999


   References
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 References
 

  1. Curran T, Bravo R, Muller R, 1985. Transient induction of c-fos and c-myc in an immediate consequence of growth factor stimulation. Cancer Surv. 4:655-681. [Medline]
  2. Brown EJ, Schreiber SL, 1996. A signaling pathway to translational control. Cell. 86:517-520. [Medline]
  3. Davis RJ, 1993. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 268:14553-14556. [Free Full Text]
  4. Schlessinger J, 1993. How receptor tyrosine kinases activate Ras. Trends Biochem Sci. 18:273-275. [Medline]
  5. Bonfini L, Migliaccio E, Pelicci G, Lanfrancone L, Pelicci PG, 1996. Not all Shc's roads lead to Ras. Trends Biochem Sci. 21:257-261. [Medline]
  6. Wood KW, Sarnecki C, Roberts TM, Blenis J, 1992. Ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell. 68:1041-1050. [Medline]
  7. Hayflick L, 1965. The limited in vitro lifetime of human diploid cell strains. Exp Cell Res. 37:614-636. [Medline]
  8. Rubin H, 1997. Cell aging in vivo and in vitro. Mech Ageing Dev. 98:1-35. [Medline]
  9. Riabowol K, Schiff J, Gilman MZ, 1992. Transcription factor AP-1 activity is required for initiation of DNA synthesis and is lost during cellular aging. Proc Natl Acad Sci USA. 89:157-161. [Abstract/Free Full Text]
  10. Atadja PW, Stringer KF, Riabowol KT, 1994. Loss of serum response element-binding activity and hyperphosphorylation of serum response factor during cellular aging. Mol Cell Biol. 14:4991-4999. [Abstract/Free Full Text]
  11. Schouten GJ, Vertegaal AC, Whiteside ST, et al. 1997. I{kappa}B {alpha} is a target for the mitogen-activated 90 kDa ribosomal S6 kinase. EMBO J. 16:3133-3144. [Medline]
  12. Timchenko NA, Wilde M, Kosai KI, et al. 1998. Regenerating livers of old rats contain high levels of C/EBP {alpha} that correlate with altered expression of cell cycle associated proteins. Nucleic Acids Res. 26:3293-3299. [Abstract/Free Full Text]
  13. Liu Y, Gorospe M, Kokkonen GC, et al. 1998. Impairments in both p70 S6 kinase and extracellular signal-regulated kinase signaling pathways contribute to the decline in proliferative capacity of aged hepatocytes. Exp Cell Res. 240:40-48. [Medline]
  14. Liu Y, Guyton KZ, Gorospe M, et al. 1996. Age-related decline in mitogen-activated protein kinase activity in epidermal growth factor-stimulated rat hepatocytes. J Biol Chem. 271:3604-3607. [Abstract/Free Full Text]
  15. Seglen PO, 1976. Preparation of isolated rat liver cells. Methods Cell Biol. 13:29-83. [Medline]
  16. Chen W, Martindale JL, Holbrook NJ, Liu Y, 1998. Tumor promoter arse-nite activates extracellular signal-regulated kinase through a signaling pathway mediated by epidermal growth factor receptor and Shc. Mol Cell Biol. 18:5178-5188. [Abstract/Free Full Text]
  17. Liu Y, Gorospe M, Yang C, Holbrook NJ, 1995. Role of mitogen-activated protein kinase phosphatase during the cellular response to genotoxic stress. Inhibition of c-Jun N-terminal kinase activity and AP-1-dependent gene activation. J Biol Chem. 270:8377-8380. [Abstract/Free Full Text]
  18. Canagarajah BJ, Khokhlatchev A, Cobb MH, Goldsmith EJ, 1997. Activation mechanism of the MAP kinase ERK2 by dual phosphorylation. Cell. 90:859-869. [Medline]
  19. Taylor SJ, Shalloway D, 1996. Cell cycle-dependent activation of Ras. Curr Biol. 6:1621-1627. [Medline]
  20. Kitano S, Fawcett TW, Yo Y, Roth GS, 1998. Molecular mechanisms of impaired stimulation of DNA synthesis in cultured hepatocytes of aged rats. Am J Physiol. 275:C146-C154. [Abstract/Free Full Text]
  21. Chen RH, Sarnecki C, Blenis J, 1992. Nuclear localization and regulation of Erk- and Rsk-encoded protein kinases. Mol Cell Biol. 12:915-927. [Abstract/Free Full Text]
  22. Chen RH, Abate C, Blenis J, 1993. Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase. Proc Natl Acad Sci USA. 90:10952-10956. [Abstract/Free Full Text]
  23. Ghoda L, Lin X, Greene WC, 1997. The 90-kDa ribosomal S6 kinase (pp90RSK) phosphorylates the N-terminal regulatory domain of I{kappa}B{alpha} and stimulates its degradation in vitro. J Biol Chem. 272:21281-21288. [Abstract/Free Full Text]
  24. Xing J, Ginty DD, Greenberg ME, 1996. Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase. Science. 273:959-963. [Abstract]
  25. De CD, Jacquot S, Hanauer A, Sassone-Corsi P, 1998. Rsk-2 activity is necessary for epidermal growth factor-induced phosphorylation of CREB protein and transcription of c-fos gene. Proc Natl Acad Sci USA. 95:12202-12207. [Abstract/Free Full Text]
  26. Ishigami A, Reed TD, Roth GS, 1993. Effect of aging on EGF stimulated DNA synthesis and EGF receptor levels in primary cultured rat hepatocytes. Biochem Biophys Res Commun. 196:181-186. [Medline]
  27. Sawada N, Ishikawa T, 1988. Reduction of potential for replicative but not unscheduled DNA synthesis in hepatocytes isolated from aged as compared to young rats. Cancer Res. 48:1618-1622. [Abstract/Free Full Text]
  28. Sakaguchi K, Okabayashi Y, Kido Y, et al. 1998. Shc phosphotyrosine-binding domain dominantly interacts with epidermal growth factor receptors and mediates Ras activation in intact cells. Mol Endocrinol. 12:536-543. [Abstract/Free Full Text]
  29. Basu T, Warne PH, Downward J, 1994. Role of Shc in the activation of Ras in response to epidermal growth factor and nerve growth factor. Oncogene. 9:3483-3491. [Medline]
  30. Chook YM, Gish GD, Kay CM, Pai EF, Pawson T, 1996. The Grb2-mSos1 complex binds phosphopeptides with higher affinity than Grb2. J Biol Chem. 271:30472-30478. [Abstract/Free Full Text]
  31. Palmer HJ, Tuzon CT, Paulson KE, 1999. Age-dependent decline in mitogenic stimulation of hepatocytes. Reduced association between Shc and the epidermal growth factor receptor is coupled to decreased activation of Raf and extracellular signal-regulated kinases. J Biol Chem. 274:11424-11430. [Abstract/Free Full Text]
  32. Li N, Batzer A, Daly R, et al. 1993. Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature. 363:85-88. [Medline]
  33. Batzer AG, Rotin D, Urena JM, Skolnik EY, Schlessinger J, 1994. Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor. Mol Cell Biol. 14:5192-5201. [Abstract/Free Full Text]
  34. Rozakis-Adcock M, Fernley R, Wade J, Pawson T, Bowtell D, 1993. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature. 363:83-85. [Medline]
  35. Rodriguez-Viciana P, Warne PH, Dhand R, et al. 1994. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature. 370:527-532. [Medline]
  36. Keilhack H, Tenev T, Nyakatura E, et al. 1998. Phosphotyrosine 1173 mediates binding of the protein-tyrosine phosphatase SHP-1 to the epidermal growth factor receptor and attenuation of receptor signaling. J Biol Chem. 273:24839-24846. [Abstract/Free Full Text]
  37. Sasaoka T, Langlois WJ, Bai F, et al. 1996. Involvement of ErbB2 in the signaling pathway leading to cell cycle progression from a truncated epidermal growth factor receptor lacking the C-terminal autophosphorylation sites. J Biol Chem. 271:8338-8344. [Abstract/Free Full Text]
  38. Karunagaran D, Tzahar E, Beerli RR, et al. 1996. ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. EMBO J. 15:254-264. [Medline]



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