INTRODUCTION

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A major signaling pathway through which virtually all known mitogens exert intracellular responses is the mitogen-activated protein (MAP)¹ kinase cascade (1, 2). A primary mechanism by which growth factor receptor tyrosine kinases activate MAP kinases involves tyrosine phosphorylation of an adaptor protein, Shc. Shc binds to a second adaptor protein, Grb2, by SH2/3 interactions. This complex incorporates a guanyl nucleotide exchange factor, Sos, which interacts with and activates Ras. The GTP-bound Ras recruits Raf kinase to the cell membrane and initiates a protein kinase cascade that phosphorylates and activates a MAP kinase kinase termed MEK (MAP kinase or extracellular signal-regulated kinase kinase). MEK in turn phosphorylates MAP kinases on both tyrosyl and threonyl sites. Active MAP kinases translocate to the nucleus where they phosphorylate other serine-threonine kinases and transcription factors which can mediate immediate early gene induction.

The growth and development of the fetus during late gestation is an intricate process dependent upon multiple regulatory mechanisms. Changes in hepatic growth tend to parallel effects on fetal somatic growth (3, 4), making liver an appropriate organ for studying fetal growth control. When we embarked on the present studies we expected that the proliferation of fetal rat hepatocytes, both in vivo and in vitro, would require the action of fetal growth factors. However, late gestation (19 day; E19) rat fetal hepatocytes cultured without serum, growth factors, or insulin were found to be highly proliferative (5, 6). By using a variety of approaches, we were able to find no direct evidence for the production of autocrine growth factors. Thus, we went on to employ MAP kinase activity as an indirect indicator of mitogenic signaling. Studies showed that the MAP kinases present in cultured fetal hepatocytes and in fetal liver were minimally active, although MAP kinase could be activated in cultures of primary fetal hepatocytes by at least two mitogens, transforming growth factor- (TGF-) and hepatocyte growth factor (7).

Given the abundance of these growth factors in the late gestation fetal rat, we hypothesized that the relatively low activity of hepatic MAP kinases in vivo might be the result of negative feedback inhibition. In our initial studies (8), we demonstrated that intraperitoneal injection of epidermal growth factor (EGF)² into E19 fetuses or adult rats resulted in the activation of the proximal portion of the EGF signaling pathway in the liver. This was based on the EGF-induced tyrosine phosphorylation of Shc and Shc-Grb2 complex formation. When the downstream activation of MAP kinases was examined, EGF was shown to result in the marked (~50-fold) activation of MAP kinase in the adult liver. However, minimal MAP kinase activation occurred in the fetus (8).

The present studies were performed following our initial observation of uncoupling of the MAP kinase signaling cascade in E19 fetal liver. We have determined when in development uncoupling of the hepatic MAP kinase cascade occurs as well as the site in this cascade where the actual uncoupling of MAP kinase activation takes place. We proceeded to characterize the constituents of the MAP kinase pathway to better understand this physiologic mechanism for down-regulation of mitogenic signaling.

	EXPERIMENTAL PROCEDURES

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Animals-- Pregnant Sprague-Dawley rats (Charles River Breeding Laboratory, Wilmington, MA), of known gestation (term specified as 21 days), were delivered by cesarean section under pentobarbital anesthesia (50 mg/kg, intraperitoneally). Male Sprague-Dawley rats (150-175 g) were used for adult liver preparation. Rats were injected intraperitoneally with EGF (0.5 µg/g body weight) or phosphate-buffered saline and sacrificed 15 min later.

Hepatocyte Isolation and Primary Culture-- Fetal hepatocytes were isolated on day 19 of gestation by collagenase digestion as described previously (6). Cell suspensions were diluted to 3 × 10⁶ cells per 100-mm Primaria tissue culture plate with supplemented minimum essential medium (6) containing 5% fetal bovine serum. After a 2-h attachment period, the medium was removed and replaced with supplemented minimum essential medium without fetal bovine serum. Media contained 0.1 mg/ml bovine serum albumin to avoid nonspecific binding of growth factors.

Immune Complex Kinase Assay-- Rat liver homogenates were prepared as described previously for MAP kinase activity (7). Homogenates corrected for protein (4 mg) were precleared with Protein A-Sepharose CL-4B (Pharmacia Biotech Inc.). MAP kinase immunoprecipitation was accomplished with a peptide antibody against the rat ERK-1 sequence (anti-MAP kinase R2, Upstate Biotechnology, Lake Placid, NY) that had been covalently coupled to Protein A-Sepharose CL-4B using dimethyl pimelimidate (20 mM in 0.1 M sodium borate, pH 9.0). The immunoprecipitates were washed 4 × in 10 mM Tris, pH 7.6, 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 100 µM Na₃VO₄, 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 25 µg/ml phenylmethylsulfonyl fluoride, and then 1 × with 25 mM -glycerophosphate, 50 µM Na₃VO₄, 10 mM MgCl₂, 0.5 mM EGTA, 10 mM NaF, 0.2% Triton X-100, plus the above protease inhibitors. The immunoprecipitated kinase was resuspended in 25 µl of this buffer. MAP kinase was assayed by adding an equal volume of 2 × reaction mixture containing 50 mM -glycerophosphate, pH 7.2, 100 µM Na₃VO₄, 20 mM MgCl₂, 1 mM EGTA, 200 µM [-³²P]ATP (0.05 µCi/nmol), and 0.667 mg/ml MBP and incubating for 20 min at 30 °C. The assays were terminated by adding 10 µl of 250 mM EDTA. MBP was separated on a 12% polyacrylamide gel under reducing conditions. The dried gel was exposed to Kodak XAR-5 film at 70 °C in the presence of intensifying screens, and phosphate incorporation into MBP was quantified with a Hoefer model 300S scanning densitometer connected to a Hewlett-Packard model 3390A integrator.

Mono Q Fractionation and Kinase Assays-- Rat liver homogenates and hepatocyte lysates were prepared for MAP kinase activity determinations as described previously (7). Where indicated, samples were fractionated using Mono Q fast protein liquid chromatography. MAP kinase activity was determined using myelin basic protein (MBP) as substrate (7).

In Vitro Activation of Rat Liver MAP Kinase-- Livers from fetal and adult rats injected with EGF were homogenized in 50 mM Hepes, pH 7.2, 50 mM NaCl, 1 mM Na₃VO₄, 20 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 25 µg/ml phenylmethylsulfonyl fluoride. After centrifugation at 100,000 × g for 1 h, 250 µl was loaded onto a Superose 6 gel filtration column (Pharmacia), run at 0.5 ml/min with the above buffer. Fifty 0.5-ml fractions were collected to separate active MEK. The reaction was started by mixing 10 µl of each fraction with 5 µl of either control fetal rat liver MAP kinase partially purified by Mono Q chromatography (7), recombinant human ERK-1 (0.4 µg), or MAP kinase Mono Q buffer A as control (7). After incubation for 3 min at 30 °C, 5 µl of 20 mM magnesium acetate plus 800 µM ATP was added. This mixture was incubated for 20 min at 30 °C, after which was added 80 µl of 50 mM Tris, pH 7.4, 0.125 mM EGTA, 0.125 mM Na₃VO₄, 12.5 mM magnesium acetate, 250 µM [-³²P]ATP (0.05 µCi/nmol), and 0.4 mg/ml MBP. After an additional 10 min incubation, the reaction was stopped with 10 µl of 250 mM EDTA. Proteins were separated on a 10% polyacrylamide gel and analyzed as above.

Partial Purification of Activated MEK-- Liver from an adult rat injected with EGF was fractionated on a Mono Q column. For these experiments, Triton X-100 was not added to Mono Q buffers. Fractions containing MEK activity were pooled and then precipitated by adding a volume of 3.6 M ammonium sulfate (2 M final concentration). The pellet was resuspended in 25 mM Tris, pH 7.4, 1 mM EDTA, 5% glycerol (v/v), 0.02% Brij 35 (w/v), 25 mM NaF, 0.1% -mercaptoethanol, and 1 mM benzamidine. The solution was dialyzed overnight using the same buffer plus 50% glycerol.

Western Immunoblotting-- For detection of immunoreactive kinases, samples were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting with the MAP kinase R2 antibody or antibodies to either MEK-1 or MEK-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Western immunoblots were also performed using an antibody to the active dually phosphorylated (Thr/Tyr) MAP kinase (Promega, Madison, WI), as well as an antibody to active dually phosphorylated (Ser-217/221) MEK-1/2 (New England Biolabs, Beverly, MA). In all cases, detection employed chemiluminescence (ECL, Amersham Corp.).

	RESULTS

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MAP Kinase Activation in Developing Rat Liver-- To confirm our previous observations that MAP kinase activation in fetal liver is uncoupled (8), we immunoprecipitated MAP kinase in fetal liver homogenates from E19 fetal and adult rats given an intraperitoneal injection of phosphate-buffered saline (control) or EGF. MAP kinase activity was then measured directly on immunoprecipitated MAP kinase. Results showed that control fetal and adult rats showed minimal MAP kinase activity (Fig. 1). Upon EGF administration, there was a slight increase in fetal MAP kinase activity, whereas adult MAP kinase was seen to increase approximately 15-fold (Fig. 1). Western immunoblotting of parallel immunoprecipitates (Fig. 1, bottom) showed similar MAP kinase content in fetal and adult samples, indicating that the diminished ability of EGF to stimulate MAP kinase activity in the fetal rat was not due to a decrease in MAP kinase content.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   In vivo activation of fetal and adult rat liver MAP kinase by EGF. Livers from 19-day fetal and adult rats were collected 15 min after intraperitoneal injection of 0.5 µg of EGF/g of body weight (+) or from animals injected with phosphate-buffered saline (). Homogenates were prepared, and equal amounts of protein (4 mg) were immunoprecipitated with immobilized MAP kinase R2 antibody. MAP kinase (MAPK) activity was measured as described under "Experimental Procedures" using MBP as substrate. The graph depicts densitometric analysis of the resulting autoradiogram shown below the figure as phosphorylation of MBP. Each lane and its corresponding bar represents a separate animal. Samples immunoprecipitated in parallel were immunoblotted with the MAP kinase R2 antibody to assess total MAP kinase content (bottom). The positions of the 44- and 42-kDa MAP kinases are shown to the right of the immunoblot.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Ontogeny of MAP kinase activation in developing rat liver. Livers from 21-day fetal rats, and 1-, 4-, 7-, and 28-day postnatal (P) rats were collected 15 min after intraperitoneal injection of 0.5 µg of EGF/g of body weight (solid circles) or from uninjected animals (control, open circles). Homogenates were prepared and fractionated by Mono Q chromatography (7). Individual fractions were assayed for MAP kinase activity (7). Previous studies have established the elution of 42- and 44-kDa MAP kinases in fractions 32-46.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Levels of total versus active MAP kinase in developing rat liver. Fractions 32-46 from each chromatogram in Fig. 2 (uninjected () and EGF-injected (+) animals) were pooled, subjected to SDS-PAGE, transferred to nitrocellulose, and then immunoblotted with anti-MAP kinase R2. These samples were also immunoblotted with an antibody to the dually phosphorylated (Thr/Tyr) MAP kinase. Numbers to the right of the autoradiograms indicate apparent molecular mass in kilodaltons.

View larger version (51K): [in this window] [in a new window]   Fig. 4.   Translocation of fetal versus adult liver MAP kinase from the cytosol to the nucleus in control () and EGF-injected (+) rats. E19 fetal (Fet) and adult (Ad) rats were injected with phosphate-buffered saline (control) or 0.5 µg of EGF/g of body weight. Fifteen minutes later livers were removed and homogenized, and nuclei were separated from cytosol as described previously (11). Cytosol (2.9 mg of protein) and nuclear extracts (0.46 mg of protein) were immunoprecipitated using anti-MAP kinase R2, separated by SDS-PAGE, and Western immunoblotted with the MAP kinase R2 antibody. Nuclear fraction autoradiograms were exposed to film for 30 s, whereas cytosolic fraction autoradiograms were exposed for 2 s. Arrows indicate the positions of the 44- and 42-kDa MAP kinases.

In Vivo Activation of Raf and MEK-- We hypothesized that the attenuation of growth factor-mediated phosphorylation and activation of MAP kinase were due to the failure to activate the kinases upstream of this enzyme. The activity of Raf (MEK kinase), the proximal kinase in the signaling pathway, was examined to determine indirectly if Ras activation was intact. Liver homogenates were fractionated by Mono Q ion exchange chromatography. Individual fractions were assayed for MEK kinase activity using recombinant, kinase-deficient MEK-1 as substrate. Fetuses injected with EGF showed marked activation of MEK kinase activity, present in multiple peaks (Fig. 5). EGF-inducible MEK kinase activity was also present in the adult, albeit at a lower level than in the fetus and as a single chromatographic peak (Fig. 5). Although we have not yet characterized the multiple MEK kinase activity peaks present in liver from EGF-injected fetuses, the response to EGF was taken as evidence for intact signaling at this step.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   In vivo stimulation of Raf and MEK activity in fetal and adult liver by EGF. Fetal (E19) and adult rat livers were harvested 15 min after intraperitoneal injection of EGF (solid circles) or phosphate-buffered saline as control (open circles). Homogenates were prepared and equal amounts of protein (5 mg) were fractionated by Mono Q chromatography. Column fractions were assayed for Raf activity (top two graphs) using recombinant kinase-deficient MEK-1 as substrate or for MEK activity (bottom two graphs) using recombinant ERK-1 as substrate. The results shown represent densitometric analysis of the resulting autoradiograms.

In Vitro Activation of Fetal MAP Kinase-- The results to this point were interpreted as indicating that the ability of EGF to only minimally activate hepatic MAP kinase in vivo resided at the level of MAP kinase phosphorylation by MEK. The first hypothetical mechanism that was investigated was that MAP kinase and/or MEK were altered in fetal liver such that they did not interact normally. This possibility had not been addressed in the experiments utilizing recombinant kinases as substrates.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   In vitro activation of partially purified rat liver MAP kinase. Fetal and adult liver homogenates from rats injected intraperitoneally with EGF (0.5 µg/g body weight) were fractionated on a gel filtration column to separate active MEK proteins. Each gel filtration fraction was mixed with either fetal rat liver MAP kinase from an uninjected animal (basal activity) partially purified on a Mono Q column (closed circles), recombinant human ERK-1 (squares), or Mono Q buffer A (open circles, control) as substrate. MAP kinase activity was measured as MBP phosphorylation.

View larger version (53K): [in this window] [in a new window]   Fig. 7.   MEK and phospho-MEK content in fetal and adult rat liver after EGF stimulation. Fractions 30-37 from the fetal and adult gel filtration columns shown in Fig. 6 were separated on a 10% SDS gel, transferred to nitrocellulose, and sequentially immunoblotted with the antibody to dually phosphorylated MEK-1/2, to MEK-1, and to MEK-2. Numbers to the right of the immunoblots represent the position of the 45-kDa marker.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   In vitro activation of fetal and adult rat liver MAP kinase by partially purified active MEK. Activated MEK from an EGF-injected adult rat was partially purified (see "Experimental Procedures"). Individual fractions obtained by Mono Q chromatography of control fetal (left) or adult (right) rat liver homogenates (7) were then combined with the partially purified MEK (solid circles) or buffer as control (open circles). MAP kinase activity was measured as MBP phosphorylation. Thus, the resulting chromatograms represent the in vitro activation of MAP kinase contained in the individual column fractions.

Uncoupling of MAP Kinase Activation in Fetal Hepatocytes-- We had shown previously that 24 h exposure of fetal hepatocytes in primary culture to TGF- or hepatocyte growth factor resulted in desensitization of MAP kinase to activation by either growth factor (11). We hypothesized that this desensitization might be analogous to the uncoupling seen in vivo. To examine this, E19 fetal hepatocytes were cultured in the absence or presence of TGF- for 24 h and then stimulated with additional TGF- for 10 min. Shc immunoprecipitation showed an increase in tyrosine phosphorylation as well as Shc-Grb2 complex formation upon acute TGF- stimulation. This was not affected by 24 h preincubation with TGF- (Fig. 9). This was interpreted as indicating that tonic growth factor exposure for 24 h had not resulted in sufficient down-regulation of EGF receptor number or tyrosine kinase activity to cause an attenuation of proximal signaling. Given that this result was consistent with the in vivo findings, we went on to examine downstream signaling.

View larger version (47K): [in this window] [in a new window]   Fig. 9.   Shc tyrosine phosphorylation and Shc-Grb2 complex formation in fetal rat hepatocytes. Cultured fetal rat hepatocytes (E19) were incubated for 24 h under defined conditions in the absence (none) or presence (24 h) of 1.7 nM TGF-. The cells were then stimulated with the re-addition of TGF- for 10 min (11). Samples were immunoprecipitated with immobilized Shc antibody followed by sequential Western immunoblotting with antibodies to Shc, phosphotyrosine (PY), and Grb2. These results were replicated in several additional experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 10.   Induction of the uncoupling of MAP kinase activation from MEK activation in fetal hepatocytes. E19 fetal hepatocytes were cultured in the absence (closed circles) or presence (open circles) of 1.7 nM TGF- for 24 h. The cells were then stimulated with the re-addition of TGF- for 10 min (11). MEK activity was measured as the increase in the activity of MAP kinase (phosphorylation of MBP), and MAP kinase was measured directly using MBP as substrate.

Investigation of a Potential Role for MAP Kinase Phosphatases in MAP Kinase/MEK Uncoupling-- Recently cloned dual specificity phosphatases like MKP-1 (MAP kinase phosphatase 1, encoded by the murine gene 3CH134 (12)) or CL100, the human homologue of MKP-1 (13), exhibit dual catalytic activity toward phosphotyrosine and phosphothreonine, thus providing a mechanism for the inactivation of MAP kinase. Based on the above studies, we treated cultured (E19) fetal hepatocytes incubated with or without TGF- for 24 h with either the tyrosine phosphatase inhibitor, sodium orthovanadate, or the serine-threonine phosphatase inhibitor, okadaic acid. Phosphatase inhibitors were added for 30 min prior to restimulation with TGF-. In both cases, suppression of MAP kinase activation remained intact, that is MEK activation was unaffected by growth factor preincubation while MAP kinase activation was attenuated in a manner indistinguishable from that shown in Fig. 10.

Investigation into the Presence of an Inhibitory MAP Kinase Binding Protein-- An alternative hypothesis to explain the dissociation of MAP kinase and MEK activation in fetal liver could involve a protein which binds to MAP kinase, thereby preventing its activation in vivo. To obtain an indicator of the relative size of fetal and adult liver MAP kinases, fetal and adult rat liver homogenates, prepared using conditions aimed at minimizing disruption of protein-protein interactions, were analyzed using a Superose 6 gel filtration column (Pharmacia). Results (not shown) were consistent with the elution of MAP kinases from both fetal and adult rat liver as soluble monomers.

	DISCUSSION

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Our prior observation that intraperitoneal injection of late gestation fetal rats in situ with EGF showed only minimal activation of hepatic MAP kinases (8) was initially interpreted as indicating a probable block at the level of Ras activation. This was based on observations from several laboratories indicating that growth factor activation of the MAP kinase pathway can lead to phosphorylation of the Ras guanyl nucleotide exchange protein SOS, thereby down-regulating the MAP kinase pathway (14-19). Such a mechanism might pertain to negative feedback inhibition of hepatic MAP kinase activation in the growth factor-rich fetal environment. The ability of EGF to activate hepatic MAP kinases in vivo was recovered during the early phase of postnatal development, a period when hepatocyte proliferation is slowing and fetal hepatocytes are making the transformation to an adult hepatocyte phenotype. However, the observation that Raf and MEK activation were intact in the absence of MAP kinase activation indicated an alternative regulatory mechanism.

The conclusion that in situ EGF injection of a late gestation fetal rat could produce MEK activation with minimal MAP kinase activation is consistent with the entirety of the experiments we performed. In addition to activity measurements, Western immunoblotting confirmed the presence of MAP kinase but the absence of its phosphorylation in fetal liver. Furthermore, the lack of activation was accompanied by the lack of nuclear translocation. Gonzalez et al. (20) have reported that the majority of the MAP kinase in quiescent cells is in the cytoplasm. Upon activation of the cells with serum, cytoplasmic MAP kinase translocates into the nucleus. Similar translocation could be detected in subcellular fractions from adult, but not fetal, liver. The evidence for intact MEK activation included not only activity measurements but also Western immunoblotting for the active forms of MEK-1 and -2.

The physiologic in vivo uncoupling of MAP kinase activation at the level of MAP kinase phosphorylation by MEK has not been reported previously. However, several reports have suggested similar uncoupling of MAP kinase activation in vitro. By using a mouse myoblast cell line, Campbell et al. (21) showed that following withdrawal from serum and bFGF for 3 h bFGF stimulated MAP kinase kinase activity but that MAP kinase and S6 peptide kinase activities were not detected. When serum and bFGF were withdrawn for 10 h, the activities of MAP kinase kinase, MAP kinase, and S6 peptide kinase were coordinately stimulated by bFGF. Similar results were seen using EGF. Furthermore, it was demonstrated that incubation with the tyrosine phosphatase inhibitor, sodium orthovanadate, restored MAP kinase activation. Microcystin, a serine-threonine phosphatase inhibitor, had little effect.

Samuels et al. (22), using retroviruses encoding a fusion protein consisting of an oncogenic form of human p74^raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER), have studied MAP kinase regulation in rat1a cells conditionally transformed by hrafER. In these cells, estradiol caused the activation of MAP kinase kinase but not activation of p42/p44 MAP kinases or phosphorylation of p74^raf-1. Estradiol-dependent activation of MAP kinase and phosphorylation of p74^raf-1 was observed when cells were pretreated with the serine-threonine phosphatase inhibitor okadaic acid. Sodium vanadate alone had little effect. It has also been reported that in v-raf-infected macrophages, MEK activation occurs normally after growth factor treatment, whereas MAP kinase shows no activation correlating with lack of tyrosine phosphorylation of this enzyme (23). Suppression of MAP kinase activation was reversed in these cells by treating with sodium orthovanadate.

We did not observe an effect of phosphatase inhibitors on either the in vivo or in vitro uncoupling of MAP kinase activation from MEK activation. Although these findings do not rule out a fetal hepatic MAP kinase phosphatase as the factor required for uncoupling of MAP kinase activation from MEK activation, they also do not support this hypothesis. A responsible dual function phosphatase would have to be insensitive to vanadate and okadaic acid. In addition, there appears to be a MAP kinase phosphatase expressed in fetal hepatocytes maintained under non-growth factor-stimulated conditions. Thus, induction of a phosphatase would have to modify the effects of one already present. Nonetheless, given that the transcription of the dual specificity phosphatases has been shown to be stimulated by serum and growth factors (12, 24, 25), this remains a viable hypothesis.

Our hypothesis that MAP kinase activation by MEK could be blocked by a MAP kinase binding protein is not without precedent. It has recently been reported that the protein p21^{WAF1/cip1/Sdi1}, a DNA damage-inducible cell cycle inhibitor, acts as an inhibitor of the stress-activated protein kinases, also called the c-Jun amino-terminal kinases (JNKs). Dickens et al. (26) have described an additional mechanism for attenuating effects mediated by the JNK pathway. These investigators have cloned a cytoplasmic inhibitor of the JNK pathway, JIP-1, that binds specifically to JNK. JIP-1 causes cytoplasmic retention of JNK and inhibition of JNK-related gene expression. Given that the JNK enzymes represent a subfamily of the MAP kinases (27), we considered an analogous mechanism. However, we were unable to obtain evidence for fetal hepatic MAP kinases being present in a form other than soluble monomers. In addition, our data indicated a lack of MAP kinase phosphorylation, an effect not attributed to JIP-1.

In summary, our results demonstrate that hepatic, EGF-induced MAP kinase activation is suppressed in late gestation through the early postnatal period in the developing rat. Western immunoblotting indicated that the absence of MAP kinase activity correlates with the absence of MAP kinase phosphorylation. However, activation of MEK, the kinase which phosphorylates and activates MAP kinase, remains intact upon acute stimulation with EGF. Furthermore, the uncoupling of MAP kinase from the signaling cascade can be induced in vitro by the incubation of fetal hepatocytes for 24 h with growth factors. These findings may define a mechanism for attenuating MAP kinase activation under conditions in which cells are subject to tonic growth factor stimulation.