Suppression of Epidermal Growth Factor–Induced Phospholipase C Activation Associated With Actin Rearrangement in Rat Hepatocytes in Primary Culture

Suppression of Epidermal Growth Factor–Induced Phospholipase C Activation Associated With Actin Rearrangement in Rat Hepatocytes in Primary Culture SHUNSUKE NOJIRI* AND JAN B. HOEK

Hepatocytes maintained in primary culture for periods of 1 to 24 hours exhibited a rapid decline in epidermal growth factor (EGF)-induced activation of phospholipase C (PLC), as was evident in a loss of EGF-induced inositol 1,4,5trisphosphate (IP3) formation and mobilization of Ca2ⴙ from intracellular Ca2ⴙ stores. The loss of PLC activation was not the result of a decrease in EGF receptor or phospholipase C-␥1 (PLC␥1) protein levels, nor the result of a loss of tyrosine phosphorylation of these proteins, but was associated with a decrease in EGF-induced translocation of PLC␥1 to the Triton-insoluble fraction, presumably reflecting binding to the actin cytoskeleton. Disruption of F-actin by treatment of cultured hepatocytes with cytochalasin D recovered the EGF-induced IP3 formation and Ca2ⴙ mobilization to the same level and with the same dose-response relationship as was obtained in freshly isolated cells. Analysis of PLC␥1 colocalization with F-actin by confocal microscopy showed that PLC␥1 was mostly distributed diffusely in the cytosol, both in freshly plated cells and in cells in culture for 24 hours, despite marked differences in actin structures. EGF stimulation caused a modest redistribution of PLC␥1 and a detectable increase in colocalization with cortical actin structures in freshly plated cells or in cytochalasin D-treated cells, but in cells that had been maintained and spread in culture only a limited PLC␥1 relocation was detected to specific actin-structure associated with lamellipodia and membrane ruffles. We conclude that actin cytoskeletal structures can exert negative control over PLC␥1 activity in hepatocytes and the interaction of the enzyme with specific actin structures dissociates PLC␥1 tyrosine phosphorylation from activation of its enzymatic activity. (HEPATOLOGY 2000;32:947-957.) Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; SH2, src homology-2; PLC␥1, phospholipase C-␥1; PIP2, phosphatidylinositol 4,5bisphosphate; IP3, inositol 1,4,5-trisphosphate; [Ca2⫹]i, intracellular Ca2⫹ concentration; PITP, phosphatidylinositol transfer protein; CD, cytochalasin D; BSA, bovine serum albumin; FCS, fetal calf serum; FITC, fluorescein isothiocyanate. From the Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA. Received May 15, 2000; accepted August 7, 2000. Supported by NIH Grants AA07186, AA10968, AA08714, and AA11689 from the National Institute of Alcohol Abuse and Addiction. Address reprint requests to: Jan B. Hoek, Ph.D., Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, JAH Rm. 269, 1020 Locust Street, Philadelphia, PA 19107. E-mail: [email protected]; fax: 215-923-2218. *Current address: First Department of Internal Medicine, Nagoya City University Medical School, Nagoya, Japan. Copyright © 2000 by the American Association for the Study of Liver Diseases. 0270-9139/00/3205-0011$3.00/0 doi:10.1053/jhep.2000.18662

Binding of the epidermal growth factor (EGF) to its receptor (EGFR) initiates dimerization of the receptor protein and activation of its intrinsic tyrosine kinase, resulting in autophosphorylation of multiple tyrosine residues on the cytoplasmic domain of the receptor.1-3 These phosphotyrosine residues act as specific docking sites for target proteins that contain characteristic protein domains, such as the src homology-2 (SH2) domain.4,5 Activation of different target proteins initiates a branching network of signaling reactions in the cell, resulting in the appropriate biological response patterns.6 One of the intracellular signaling pathways activated by EGF involves phospholipase C-␥1 (PLC␥1), a member of the phosphoinositide-specific phospholipase C family of proteins, which, in addition to the catalytic domain and Ca2⫹-binding domains shared with other PLC isoforms, contains several tyrosine phosphorylation sites (Tyr771, Tyr783, and Tyr 1254) that are targets for the tyrosine kinase activity of growth-factor receptors, two SH2 domains that bind to autophosphorylation sites on the receptor, an SH3 domain that interacts with proline-rich regions in proteins, and a pleckstrin homology (PH) domain that is thought to help target the protein to the membrane.7,8 Activation of PLC␥1 induces hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) resulting in the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. These second messengers increase the intracellular Ca2⫹ concentration ([Ca2⫹]i) and activate the serine/threonine-specific protein kinase C, respectively.9 Tyrosine phosphorylation of PLC␥1 is required for its activation in intact cells, and the expression of mutant PLC␥1 proteins that lack tyrosine phosphorylation sites or have dysfunctional SH2 domains do not exhibit EGF-induced IP3 formation and [Ca2⫹]i changes.10-13 However, the specific significance of the tyrosine phosphorylation of PLC␥1 is not well defined. Tyrosine phosphorylation does not increase the intrinsic enzymatic activity of PLC␥1 with micellar substrates, although it may enhance the affinity for its substrate PIP2 or promote the enzyme’s interaction with PIP2 in the membrane surface.14,15 It has been suggested that tyrosine phosphorylation promotes access of PLC␥1 to its substrate PIP2 in the intact cell by enhancing competition with other PIP2 binding proteins, such as the actin-binding protein profilin.16 However, there is evidence that substrate-supply to PLC␥1 is controlled to a large extent by colocalization of the enzyme with phosphatidylinositol transfer protein (PITP), phosphatidylinositol-4-kinase, and phosphatidylinositol-4-P 5-kinase, which generate PIP2 from its precursor phospholipid.17 PLC␥1 activity may also be regulated by interacting with other phosphoinositide lipids, e.g., with products of phosphatidylinositol 3-kinase.18-20

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The mechanism of EGF-induced PLC␥1 activation and the functional significance of tyrosine phosphorylation of the enzyme in liver cells have been particularly controversial. Liang and Garrison21 reported that tyrosine phosphorylation occurred only in a small fraction of the total PLC-␥1 available in hepatocytes after stimulation with EGF. Kholodenko et al.6 found that stimulation of isolated hepatocytes with maximally effective EGF concentrations resulted in a transient tyrosine phosphorylation of PLC␥1 that even at its peak represented only 10% to 15% of the total PLC␥1 protein. Nevertheless, several groups reported a significant EGF-induced formation of IP3 with an associated increase in [Ca2⫹]i both in freshly isolated hepatocytes and in hepatocytes in primary culture, although the reported magnitude of the response and the EGF dose-dependency varied considerably between different groups.6,21-28 Thus, it appears that even a modest degree of tyrosine phosphorylation of PLC␥1 is sufficient to activate the enzymatic activity and bring about formation of IP3 and increase of [Ca2⫹]i, but the factors that determine the magnitude of the response may be subject to other regulatory factors that are poorly characterized. Interaction of the EGFR/PLC␥1 signaling system with the actin cytoskeleton may contribute to this regulation.29-32 Substantial evidence indicates that EGFR signaling is interlinked with actin cytoskeletal structures. EGF stimulation of A431 cells induces substantial changes in actin cytoskeletal structures, including a degradation of actin stress fibers and an increase in membrane ruffling.33-35 Actin binds directly to the EGFR through an actin binding domain on the receptor located between residues 986-999.36,37 The actin-binding domain of EGFR is required for tumor invasion of transformed cells,38 and recent evidence suggests that PLC␥1 activation is required for tumor invasiveness in breast and prostate carcinomas through its effects on cell motility.39,40 PLC␥1 also binds to actin through its carboxyterminal SH2 domain,41 and translocation of the enzyme following stimulation with EGF has been reported in association with the onset of membrane ruffling in A431 cells.34 Importantly, even the PITP and phosphoinositide-kinases that generate PIP2 for use by PLC␥1 have been found to be associated with the actin cytoskeleton.30 It is not clear how significant these associations are for PLC␥1 function, or how much control these different factors exert over PLC␥1 activation by growth factors. Isolated hepatocytes in primary culture develop an extensive network of actin microfilaments over several days.42 Translocation of PLC␥1 to actin has been reported to enhance the enzymatic activity of the enzyme in immunoprecipitates obtained from EGF-stimulated hepatocytes in primary culture, using an assay with micellar substrate,31 although the mechanism responsible for the enhanced enzymatic activity and its relationship to tyrosine phosphorylation of PLC␥1 was not clear in these studies. Our studies are aimed at understanding the factors that control the interaction of PLC␥1 with the activated EGFR and its role in the EGF-mediated signaling network in hepatocytes.6,27 The present investigation was prompted by the finding that hepatocytes rapidly lost their capacity to generate a Ca2⫹ response during relatively brief periods in culture, even though other EGF-dependent responses were active, suggesting that EGF-induced activation of PLC␥1 was selectively suppressed under these conditions. Surprisingly, disruption of the actin cytoskeleton with cytochalasin D (CD) resulted in

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complete recovery of the Ca2⫹ response. We localize the defect in PLC␥1 activation at a site distal to EGF-induced PLC␥1 phosphorylation and conclude that actin restructuring during cell culture exerts negative control over the hydrolysis of PIP2 through PLC␥1 in liver cells. The data suggest that PLC␥1 translocation and activation of its enzymatic activity are regulated independently from its tyrosine phosphorylation in liver cells and may serve other functions than the formation of the second messenger molecules IP3 and diacylglycerol. MATERIALS AND METHODS Materials. Fetal bovine serum, Williams E medium, and antibiotics for cell culture were obtained from Gibco-BRL-Life Technologies (Grand Island, NY). Fura 2-AM was purchased from Molecular Probes (Eugene, OR). Antibodies against EGFR (sheep polyclonal) and PLC␥ (mixed mouse monoclonals) were obtained from Upstate Biotechnology Inc., (Lake Placid, NY), and anti-phosphotyrosineHRPO (type RC20H) from Transduction Laboratories (Lexington, KY). Secondary rabbit anti-mouse IgG was purchased from GibcoBRL-Life Technologies (Grand Island, NY). Antiphosphotyrosineagarose conjugates (mouse monoclonal) and tetramethylrhodamine isothiocyanate-labeled phalloidin were obtained from Sigma Chemical Co. (St. Louis, MO). Anti-IgG-HRPO and antimouse IgG-fluorescein isothiocyanate conjugates were from Pierce (Rockford, IL). Gradient gels and nitrocellulose membranes were from Bio-Rad (Hercules, CA), and detection of the Westerns was done by chemiluminescence using Supersignal reagent (Pierce, Rockford, IL). Collagenase type I was from Worthington (Freehold, NJ), BSA fraction V and the Complete protease inhibitor cocktail were obtained from Boehringer Mannheim (Indianapolis, IN). EGF (receptor grade) and protein G-sepharose were from Sigma Chemical Co. (St. Louis, MO). Insotiol-1,4,5-trisphosphate [3H] radioreceptor kits were obtained from NEN Life Science Products (Boston, MA). Other chemicals and biochemicals were obtained from Sigma Chemical Co. or Fisher Scientific Co. (Pittsburgh, PA). Cell culture plates were from CorningCostar (Oneonta, NY). Cell Preparations and Culture Conditions. Male Sprague-Dawley rats (180 g-200 g) were used to obtain isolated hepatocytes by collagenase perfusion as described previously.23 Animals were housed in approved animal facilities and treated in accordance with NIH guidelines. All animal protocols were approved by the Institutional Animal Care and Use Committee. Cell preparations were suspended in a modified Krebs-Ringer’s bicarbonate (KRB) buffer (127 mmol/L NaCl, 25 mmol/L NaHCO3, 4 mmol/L KCl, 1.2 mmol/L MgCl2, 1.2 mmol/L potassium phosphate, 10 mmol/L HEPES (pH7.4), and 1 mmol/L CaCl2). The medium was gassed with 5% CO2/95% O2. Cell preparations were stored on ice until use. Isolated hepatocytes were washed and resuspended in Williams E medium containing 2 mmol/L glutamine, 2% bovine serum albumin (BSA) or 10% fetal calf serum (FCS), 100 units/mL penicillin G, 0.1 mg/mL streptomycin and other additions as indicated, and cultured in the same medium as monolayers on glass coverslips, which were previously coated with laminin (or other substrata as indicated) and placed in a 60-mm dish at a cell density of 5 ⫻ 105 cells/dish. The medium was changed once after 3 hours of plating, and cells were incubated in the same medium for different periods up to 24 hours. For short-term culture (1-3 hours), cells were washed with KRB buffer at the end of the culture period, loaded with Ca2⫹ indicator when required (see later), and stimulated with different concentrations of EGF as described later. In some experiments, cells were incubated with 4 ␮mol/L CD for 1 to 3 hours before stimulation. Immunoprecipitation Conditions, Gel Electrophoresis, and Western Blotting. For the analysis of protein phosphorylation, cells were stimu-

lated by the addition of EGF dissolved in incubation buffer, and reactions were stopped at the appropriate times by addition of lysis buffer containing (final concentrations) 25 mmol/L HEPES (pH7.4), 75 mmol/L NaCl, 5 mmol/L EGTA, 10% glycerol, 0.5% Triton X-100, 50 mmol/L NaF, 100 ␮mol/L Na-o-vanadate, 5mmol/L Na-pyrophos-

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phate, and protease inhibitor cocktail. After 10 minutes on ice, lysates were centrifuged in the cold for 5 minutes and either used immediately or stored at ⫺70°C until use. In some experiments, the Triton-insoluble fraction was further extracted with hot Laemmli buffer (20% glycerol, 3% sodium dodecyl sulfate, 3% ␤-mercaptoethanol, 10 mmol/L EDTA, and 0.05% bromophenol blue) to recover proteins from this fraction. Immunoprecipitations of proteins from Triton X-100 lysates were conducted following standard procedures, essentially as described earlier.27 Tyrosine phosphorylated proteins were immunoprecipitated from protein-matched lysate samples during a 4-hour incubation with 15 ␮L antiphosphotyrosine-agarose conjugate (mouse monoclonal). Immunoprecipitates were washed extensively with HNTG buffer (containing 20 mmol/L HEPES [pH7.5], 150 mmol/L NaCl, 0.1% TritonX-100, 10% glycerol, 0.2 mmol/L sodium-o-vanadate, 10 mmol/L NaF, and protease inhibitor cocktail), dissolved in Laemmli buffer, and placed in a boiling waterbath for 5 minutes. Equal volumes of each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12.5% gels and transferred to nitrocellulose membranes. After blocking with 1% BSA in TBST (10 mmol/L Tris [pH8.0], 150 mmol/L NaCl, and 0.05% Triton X-100) for 1 hour at room temperature, antiphosphotyrosine immunoprecipitates were probed with anti-PLC␥ antibody (mouse mixed monoclonal, 0.25 ␮g/mL in TBST, 60 minutes), followed by antimouse IgG-horse radish peroxidase (HRPO) (1:20,000, 30 minutes), or with anti-EGFR antibody (sheep polyclonal, 0.5 ␮g/mL in TBST, 60 minutes), followed by antisheep IgG-HRPO (1:20,000, 30 minutes). Total cell lysates were directly diluted in Laemmli buffer, boiled, and analyzed by gel electrophoresis. After extensive washing with TBST, membranes were treated with enhanced chemiluminescence reagent (Supersignal enhanced chemiluminescence reagent, Pierce, Rockford, IL) for 1 minute, drained, wrapped in cling foil, and exposed to autoradiography film. Bands were quantified densitometrically. Where required, the density of the phosphorylated bands was normalized by comparison to the EGFR protein bands measured in the same experiment. Immunocytochemistry. For immunolocalization of PLC␥1 and Factin, cells were fixed for 30 minutes with 3.7 % paraformaldehyde in PBS (150 mmol/L NaCl, 10 mmol/L potassium phosphate, pH 7.4) on ice. After washing 3 times with PBS, cells were permeabilized for 5 minutes on ice with 0.1% Triton X-100 in PBS, washed once again with PBS, and blocked for 30 minutes at room temperature with 3% BSA in PBS. Cells were incubated overnight at 4°C with anti-PLC␥1 antibody (5 ␮g/mL mouse mixed monoclonal in PBS). After washing twice with PBS, cells were incubated for 1.5 hours at room temperature with fluorescein isothiocyanate (FITC)-conjugated secondary goat antimouse IgG (1:200 in PBS) with 1% BSA, washed 3 times with PBS, and incubated for 30 minutes with tetramethylrhodamine isothiocyanate-labeled phalloidin (1 ␮g/mL in PBS). Cover slips were again washed 3 times with PBS and mounted on a slide. Cells were viewed with a confocal laser scanning microscope (Bio-Rad MRC 1024ES, Hercules, CA). Four to six optical sections were recorded per sample. For quantitation of colocalization, pixel-by-pixel fluorescence intensities for line scans through individual cells in selected optical sections were normalized to the total fluorescence intensity over each cell. Calcium Measurements. After plating for different periods, the cells were loaded with fura-2 by incubation with fura-2-penta-acetoxymethylester (fura 2-AM, 2 ␮mol/L in dimethyl sulfoxide containing 0.03% Pluronic F-127) for 30 minutes at 37°C in HEPES-buffered Hanks’ medium, containing 147 mmol/L NaCl, 4 mmol/L KCl, 1.2 mmol/L MgCl2, 1.2 mmol/L potassium phosphate, 10 mmol/L HEPES (pH7.4), 1 mmol/L CaCl2, with 2% BSA and 200 ␮mol/L sulfinpyrazone. Fluorescence ratio images were obtained as described in detail by Rooney et al.43 After dye loading of the cells, coverslips were transferred into a chamber with 1 mL of Hanks’ medium and mounted on the stage of an inverted microscope. The stage, 16⫻ oil immersion objective, and chamber were thermostatically regulated at 37°C. A charge-coupled device camera (Photomet-

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rics Limited, Tucson, AZ) was used as the imaging device. Images were digitized at 12-bit resolution and stored and analyzed with a Macintosh computer (Apple Co., Cupertino, CA). Fluorescence images were collected alternately at excitation wavelengths of 340 nm and 380 nm (10-nm band width) with an emission wavelength of 460 nm to 640 nm. The integration time for each image was 250 ms. [Ca2⫹] was calculated from the fluorescence measurements using the ratio method after correction for background fluorescence and compartmentalized dye, as described by Rooney et al.43 IP3 Assays. After stimulation with EGF as described earlier, reactions were terminated with 16.7% trichloroacetic acid, and IP3 was measured in the extract by a binding assay using a kit from NEN Life Sciences (Boston, MA). Statistical significance was calculated by Student’s t test. Results are expressed as the mean ⫾ SEM for 3 or more individual experiments. RESULTS Rapid Decline in EGF-Induced Ca2ⴙ Responses During Short-Term Culture of Rat Hepatocytes. In freshly isolated hepatocytes, EGF

elicits a transient elevation of [Ca2⫹]i that is mediated by IP3 formed from PIP2 by PLC␥1.6,21,27 As shown in Fig. 1A, when hepatocytes were maintained in primary culture over a 24hour period, the EGF-induced [Ca2⫹]i signal rapidly declined. Cells that were plated for 1 hour or less exhibited a Ca2⫹ response pattern comparable with that of freshly isolated cells in its dose-response relationship, with 100% of the cells responding to a saturating concentration of EGF (20 nmol/L) and a 50% response frequency at 2 nmol/L EGF. This doseresponse relationship is comparable with that obtained for EGFR autophosphorylation in freshly isolated hepatocytes in suspension, but shifted somewhat to the left relative to PLC␥1 tyrosine phosphorylation, which reaches half-maximal levels at EGF concentrations of 5 to 10 nmol/L (Saso et al.,27 and Moehren G, Hoek JB, unpublished data, 1999). The Ca2⫹ responses in individual cells characteristically occurred after a delay of 1 to 2 minutes, and the majority of responsive cells exhibited a single broad, irregular Ca2⫹ peak, lasting for 2 to 3 minutes. The peak [Ca2⫹]i of 466 ⫾ 15 nmol/L was comparable with that obtained after release of intracellular Ca2⫹ stores with thapsigargin (which inhibits the endoplasmic reticular Ca2⫹ pump), in agreement with the interpretation that release from IP3-sensitive Ca2⫹ stores accounts for the EGF-induced Ca2⫹ elevation (Fig. 1B and Table 1). Characteristically, the duration of the Ca2⫹ transient and the peak [Ca2⫹]i in individual cells were independent of the EGF concentration used for stimulation; the dose dependency of the Ca2⫹ response was accounted for entirely by changes in the number of cells that generated a Ca2⫹ transient within the assay period. This response pattern is compatible with the transient nature of PLC␥1 activation in hepatocytes6 and suggests a threshold mechanism for IP3-induced Ca2⫹ release from intracellular storage sites. After hepatocytes had been cultured for 3 hours or 24 hours, a substantial decline in Ca2⫹ response occurred. At 3 hours, the decreased response was evident primarily as an increase in the EC50 for EGF to approximately 10 nmol/L. However, after a 24-hour culture period, even saturating concentrations of EGF no longer generated a consistent elevation of [Ca2⫹]i (Fig. 1A). Higher concentrations of EGF (up to 100 nmol/L) did not overcome the lack of response, suggesting that the suppression was not the result of a shift in the EGFR binding characteristics (see later). There was no significant difference in basal [Ca2⫹]i between those cells that lost the

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FIG. 1. Decline of EGF-induced Ca2⫹ response capacity in cultured hepatocytes as a function of time in culture. Isolated hepatocytes were plated on laminin-coated coverslips as described in Materials and Methods for periods up to 24 hours. Cells were washed and loaded with fura 2, and the EGF-induced Ca2⫹ response was analyzed by fluorescence microscopy imaging with different concentrations of EGF. (A) Fraction of cells exhibiting a significant Ca2⫹ elevation in response to different concentrations of EGF. Each point represents the mean ⫾ SEM of 3 to 5 separate experiments. (B) Representative Ca2⫹ traces of individual cells in a population of cells stimulated with 2 nmol/L EGF, followed by addition of thapsigargin (1 ␮mol/L) to determine maximal Ca2⫹ elevation in response to store depletion.

capacity to exhibit a Ca2⫹ response to EGF and those that retained their response. Also, in cells that did retain EGFinduced Ca2⫹ response capacity, the characteristics of the Ca2⫹ transient were indistinguishable from those detected immediately after plating, with identical peak Ca2⫹ levels and peak width (see Table 1). Moreover, even in cells that did not respond to EGF, treatment with thapsigargin induced an elevation of [Ca2⫹]i of the same magnitude as in freshly plated cells, suggesting that the different response characteristics were not the result of a depletion of intracellular Ca2⫹ stores. This conclusion is also supported by the observation that stimulation with phenylephrine (20 ␮mol/L) or vasopressin TABLE 1. Ca2ⴙ Response Characteristics in EGF-Stimulated Hepatocytes From Control and CD-Treated Cells After Different Times in Culture Time in Culture

1h

Basal [Ca2⫹]i (nmol/L) Peak Ca2⫹ increase over basal in responsive cells (nmol/L) Thapsigargin-induced Ca2⫹ increase over basal (nmol/L) Latency period (sec)

3h

24h

24h CDTreated

157 ⫾ 15 143 ⫾ 13 146 ⫾ 17 148 ⫾ 18 309 ⫾ 15 271 ⫾ 25 296 ⫾ 30 319 ⫾ 29 343 ⫾ 35 306 ⫾ 27 354 ⫾ 20 380 ⫾ 31 101 ⫾ 12 98 ⫾ 11 108 ⫾ 15 99 ⫾ 13

NOTE. Isolated hepatocytes were maintained in culture for the periods indicated and loaded with fura-2 for analysis of Ca2⫹ responses by fluorescence microscopic imaging as shown in Fig. 1. Data from 6 separate experiments were used for the statistical analysis, and in each experiment 35 to 80 cells were analyzed. Results are presented as means ⫾ SEM. Basal Ca2⫹ concentrations were not significantly different between responsive and nonresponsive cells. Other parameters were analyzed in responsive cells only. No significant differences were obtained in these parameters between different treatment conditions.

(10 nmol/L) induced characteristic Ca2⫹ oscillation patterns, even after 24 hours of culture (not shown, see also Rooney et al.,43). The decline in EGF-induced Ca2⫹ response occurred under a wide variety of culture conditions, including various substrata (laminin, collagen-I, fibronectin, polylysine, Matrigel, Cell-Tak, Becton Dickinson Co. Franklin Lakes, NJ) and additions to the culture medium. Our standard culture conditions for 24-hour culture included an initial 3-hour plating period in Williams E medium with 10% FCS, followed by overnight plating with 10% FCS or 2% BSA. The decline in EGF-induced Ca2⫹ response was comparable under either condition. Also, the early decline in EGF-induced Ca2⫹ response was similar, whether cells were plated in the presence of FCS or with BSA. In some experiments, insulin (160 nmol/L) was included in the culture medium to promote cell attachment. EGF-induced Ca2⫹ responses declined somewhat more rapidly in insulin-containing medium than in media without insulin (data not shown). Inclusion of dexamethasone (1 mmol/L) in the culture medium had no effect on the decline in Ca2⫹ response capacity. Thus, despite screening a wide variety of culture conditions, we were unable to overcome the decline in EGF-induced Ca2⫹ response capacity during short-term culture of the hepatocytes by these treatments. The Block in EGF-Induced Ca2ⴙ Response Induced by Short-Term Culture is Downstream From EGFR Activation and PLC␥1 Tyrosine Phosphorylation. Previous studies on Ca2⫹ responses induced

by stimuli acting through G protein-coupled receptors43 had shown that a loss of V1a receptor and ␣1-adrenergic receptor content occurred in hepatocytes during overnight culture, which accounted for a shift in the dose-response relationships of Ca2⫹ stimulation involving these receptors. By contrast, as shown in Fig. 2A, the cellular content of EGFR and PLC␥1

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FIG. 2. EGF-induced receptor autophosphorylation and PLC␥1 tyrosine phosphorylation after different times in culture. Hepatocytes cultured for different periods were stimulated with EGF (5 nmol/L) for 1 minute and extracted with Triton X-100 lysate buffer. Control: unstimulated cells. (A) Total EGFR protein and PLC␥1 protein were determined in EGFR and PLC␥1 immunoprecipitates by Western blotting. (B) Tyrosine phosphorylated EGFR and PLC␥1 were determined in antiphosphotyrosine immunoprecipitates and normalized to EGFR or PLC␥1 protein levels in the same lysates. Bottom panels show means ⫾ SEM for 4 to 5 experiments.

protein was not significantly affected during overnight culture of hepatocytes. Moreover, EGFR autophosphorylation and PLC␥1 tyrosine phosphorylation were equally effective, irrespective of the period in culture (Fig. 2B). The dose dependency of EGFR autophosphorylation and PLC␥1 tyrosine phosphorylation in cells after 1-hour and 24-hour culture was comparable (Fig. 3). Also, PLC␥1 tyrosine phosphorylation occurred with a comparable time course with that obtained in freshly isolated cells, with an early peak in phosphorylation (occurring between 30 seconds and 1 minute after stimulation in cells in culture), followed by a decline to a low sustained level of phosphorylation (Fig. 4). Thus, the decline in EGFinduced Ca2⫹ response was not the result of a loss or inactivation of EGFR or a lack of tyrosine phosphorylation of PLC␥1. Figure 5 shows the time course of EGF-induced IP3 formation in hepatocytes maintained in culture for 1 hour and 24 hours. An early activation of IP3 formation is evident in the cells plated for 1 hour, reaching a peak between 1 to 2 minutes after EGF addition and declining after longer periods. This response pattern is comparable with that reported previously with freshly isolated cells.27 By contrast, in cells that had been

FIG. 3. Dose-dependency of EGF-induced receptor autophosphorylation and PLC␥1 tyrosine phosphorylation in hepatocytes maintained in culture for 1 hour or 24 hours. Cells were stimulated with different concentrations of EGF for 1 minute and lysed for immunoprecipitation and analysis of tyrosine phosphorylation and protein levels of EGFR and PLC␥1 as described for Fig. 2. Results are means ⫾ SEM for 4 separate experiments.

in culture for 24 hours, EGF-induced IP3 formation was undetectable. These data show that the inhibition of EGF-induced Ca2⫹ response was the result of a defect in the activation of PLC␥1 enzymatic activity, despite a normal tyrosine phosphorylation pattern of the enzyme. EGF-Induced Ca2ⴙ Mobilization Is Recovered by Disruption of Actin Cytoskeleton. Because EGFR and PLC␥1 are known to in-

teract with the actin cytoskeleton,33 we considered the possibility that the cytoskeletal rearrangements associated with cell attachment and spreading affected the EGF-induced signaling machinery leading to PLC␥1 activation. Figure 6 shows that treatment of cells with CD (4 ␮mol/L), which disrupts the actin cytoskeleton, enabled the cells to retain their Ca2⫹ response capacity on stimulation with EGF throughout the 24hour culture period. Moreover, when hepatocytes cultured overnight were treated with CD for 3 hours, the Ca2⫹ response capacity was recovered to the same level as in freshly plated cells and with a similar dose-response curve to EGF. Furthermore, CD treatment recovered the EGF-induced IP3-formation in 24-hour cultured cells (28% ⫾ 6% increase after 1 minute stimulation, compared with 36% ⫾ 7 % in cells that were plated for 1 hour) that had no detectable IP3 response before this treatment (compare Figs. 5 and 7). In agreement with the interpretation that the recovery of the Ca2⫹ response by disruption of the cytoskeleton was exerted at the level of the control of PLC activity, CD treatment had no effect on the characteristics of EGF-induced Ca2⫹ transients in individual cells (Fig. 6B and Table 1). However, CD at these concentrations exerted a modest (approximately 20%) inhibitory effect on EGFinduced Ca2⫹ release, which was independent of cell culture time and which was evident even in cells that had been plated only briefly, merely to attach the cells to the coverslip (10-30 min, using Cell-Tak, Becton Dickinson). This inhibitory effect was probably exerted at the level of EGFR function, because EGFR autophosphorylation and PLC␥1 tyrosine phosphorylation also were suppressed by 30% to 40% (not shown). EGF Stimulation Causes Translocation of PLC␥1 to Cortical Actin Structures in Freshly Plated Hepatocytes, but not in Hepatocytes Cul-

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FIG 4. Time course of EGF-induced PLC␥1 tyrosine phosphorylation in freshly isolated hepatocytes in suspension (A) and in cells maintained in culture for 1 hour (B) or 24 hours (C). Cells were stimulated with EGF (20 nmol/L) and lysed after different reaction periods for immunoprecipitation and analysis of tyrosine phosphorylation and protein levels of PLC␥. Results are representative for 3 separate experiments.

tured for 24 Hours. Multiple studies have shown actin binding

of PLC␥1, presumably mediated through the carboxyterminal SH2 domains of the protein.31,34,35,41 Translocation of PLC␥1 to actin is stimulated by EGF and is thought to play a role in activation of the enzyme. However, it is not clear how this

FIG. 5. Time course of EGF-induced IP3 formation in hepatocytes maintained in culture for 1 hour or 24 hours. Cells were cultured for 1 hour or 24 hours and stimulated with EGF (20 nmol/L) for 1 minute, followed by TCA extraction and analysis of IP3 concentrations. Basal IP3 levels were 2.5 ⫾ 0.5 pmol/mg protein and were not significantly different between cells maintained in culture for 1 hour or 24 hrs. *P ⬍ .05, **P ⬍ .01, ***P ⬍ .001.

interaction affects its phospholipase activity or at which step(s) in the activation process actin binding is involved. In the experiments of Fig. 8 the effect of EGF stimulation on PLC␥1 association with the Triton-insoluble cell fraction was determined after maintaining cells in culture for periods of 1 hour or 24 hours. In freshly isolated hepatocytes, the predominant fraction (⬎85%) of PLC␥1 is recovered in the soluble fraction and can be released from the cells even by permeabilization of the plasma membrane with digitonin.6 However, after attaching the cells to laminin-coated coverslips for a 1-hour period, a significant percentage (⬎30%) of total PLC␥1 protein remained attached to the particulate cell fraction following solubilization with 1% Triton-X100, suggesting its interaction with the cytoskeletal fraction (or other Triton-insoluble cell components) (Fig. 8A). A 1-minute EGF treatment increased the percentage associated with the Triton-insoluble fraction by approximately 14%. By contrast, after 24 hours of culture, EGF did not induce a detectable enhancement of the total PLC␥1 protein association with the Triton-insoluble fraction. However, treatment of these cells with CD for 3 hours recovered the EGF-induced enhancement of PLC␥1 binding to the Triton-insoluble fraction. CD treatment had no significant effect on the association of PLC␥1 protein with the Triton-insoluble fraction in cells that were maintained in culture for only 1 hour. Surprisingly, tyrosine-phosphorylation did not appear to enhance association with the Triton-insoluble fraction, because less than 20% of tyrosine phosphorylated PLC␥1 was associated with this frac-

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FIG. 6. Recovery of EGF-induced Ca2⫹ responses in hepatocytes in culture by treatment with CD. Conditions were similar to those described for Fig. 1, except that CD (4 ␮mol/L) was added at the time of plating for cells maintained in culture for 1 hour or 3 hours and 3 hours before analysis for cells maintained in culture for 24 hours. (A) Response frequency after different times in culture. (B) Characteristics of EGF-induced Ca2⫹ transients in individual hepatocytes after different times in culture.

tion in cells plated for 1 hour and less than 5% in cells cultured for 24 hours, both under control and EGF-stimulated conditions (Fig. 8B). CD treatment did not significantly affect the association of phosphorylated PLC␥1 with the Triton-insoluble fraction in either the 1-hour cultures or the 24-hour cultured cells. These data suggest that the EGF-induced association of PLC␥1 with the actin cytoskeleton (or other

FIG. 7. Effect of CD treatment on EGF-induced IP3 formation in hepatocytes after 24 hours in culture. Cells were cultured with or without CD as described for Fig. 6 and extracted 1 minute after stimulation with EGF for assay of IP3. *P ⬍ .001.

components of the Triton-insoluble fraction) may not be related to the stimulation of phospholipase activity that occurs under these conditions. A more detailed illustration of the effect of cell culture on the changes in PLC␥1 association with the actin cytoskeleton was obtained by confocal microscopy, as shown in Fig. 9. In these experiments, hepatocytes were cultured for 1 hour or 24 hours as described earlier and after appropriate treatments as indicated, fixed for detection of F-actin with rhodamine-phalloidin and of PLC␥1 with anti-PLC␥1 antibody and a FITClabeled secondary antibody. As shown in Figs. 9A and B, cells in culture for 1 hour maintained their rounded shape with a layer of cortical actin located immediately below the plasma membrane. In agreement with earlier reports,34 PLC␥1 was distributed more or less evenly throughout the cell, though being largely excluded from the nuclear matrix. Only a modest degree (approximately 20%) of colocalization of the enzyme with actin was apparent both from the intensity of yellow color in the overlay picture (Figs. 9A and B) and from the quantitative colocalization analysis (Fig. 10). Stimulation of the cells with EGF (20 nmol/L) for 1 minute significantly enhanced the apparent colocalization of PLC␥1 with the cortical actin layer (Figs. 9B and 10), even though in EGF-stimulated cells, the predominant fraction of PLC␥1 remained distributed throughout the cytosolic space, in agreement with our previous studies on hepatocytes.6 After 24 hours in culture, cells had spread extensively and formed islands of contiguous monolayers. Visualization of the actin cytoskeleton showed the presence of stress fibers, lamellipodia, and cortical actin layers associated with membranes separating adjoining cells (Fig. 9C and D). PLC␥1 was still primarily distributed almost uniformly throughout the cyto-

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After treatment with CD (Figs. 9E and F), some of the remaining actin structures appeared in the form of small clusters surrounding the cell membrane, presumably derived from the cortical cytoskeleton.44 In addition, a large perinuclear mass of F-actin was evident. Interestingly, the subplasmalemmal actin clusters colocalized substantially with PLC␥1 (e.g., Fig. 9F, arrowhead), and this association was significantly enhanced after stimulation with EGF (Fig. 9F and 10). No such association with the perinuclear F-actin was evident, suggesting that PLC␥1 association with the actin fibers was selectively in favor of cortical actin structures. Thus, these data support the interpretation that actin reorganization associated with plating and culture and with its disruption by CD affect the relocation of PLC␥1 induced by EGF. DISCUSSION

FIG. 8. Effect of CD and time in culture on association of PLC␥1 with the Triton-insoluble fraction in hepatocytes. Cells were cultured with or without CD as described for Fig. 6 and stimulated with EGF (20 nmol/L) for 1 minute before lysis with Triton X-100 lysis buffer. Particulate and soluble cell fractions were separated immediately by microfuge centrifugation and solubilized with Laemmli buffer for analysis of PLC␥1 protein (A) and tyrosine phosphorylated PLC␥1 (B). Supernatant ( ) and particulate ( ) fractions were analyzed in parallel in the same gels and normalized to 100%. Results are expressed as means ⫾ SEM for 3 independent experiments. *P ⬍ .05; **P ⬍ .01.

solic space with some colocalization with cortical actin layers. However, areas containing stress fibers were generally less dense with PLC␥1. Specifically, in a z-scan of the cells, the basal layer where cells were attached to the substratum was almost completely devoid of PLC␥1 (not shown). Colocalization analysis detected a significantly enhanced occurrence of pixels with both actin and PLC␥1 fluorescence (Fig. 10), but this could probably in large part be attributed to the higher density of F-actin in these cells. EGF stimulation of the 24-hour cultures did not induce a redistribution of the PLC␥1 that was readily quantifiable by colocalization analysis on a cell-by-cell basis (Fig. 10). However, on close scrutiny of the EGF-stimulated cells, specific colocalization of PLC␥1 was evident with actin bundles associated with lamellipodia extending from EGF-stimulated cells (e.g., Fig. 9D, arrowhead). This is in agreement with earlier reports34,35 that EGF treatment of A431 cells induced a rapid and transient relocation of PLC␥1 to the actin structures associated with cell spreading.

PLC␥ isoforms constitute a subgroup of the large phospholipase C family that are excellent substrates for tyrosine kinases.13,45 Although tyrosine phosphorylation of the enzyme is generally required for stimulation of its enzymatic activity, the specific role of tyrosine phosphorylation is poorly characterized.10,46,47 Tyrosine phosphorylation does not have marked effects on the enzymatic activity in vitro, other than causing a modest shift in Km for its substrate PIP2.15 Goldschmidt-Clermont et al.16 suggested that tyrosine phosphorylation may promote competition of PLC␥1 for access to its substrate by displacing the actin binding protein profilin. Multiple proteins interact with PIP2 and access of PLC␥1 to its substrate may, therefore, be a point of control, either by direct competition, or by selective regulation of the interaction of proteins with PIP2. PIP2-binding proteins are generally dependent on delivery of the substrate through PITP and the phosphoinositide kinases that phosphorylate the 4- and 5-positions of the inositol ring.17 PITP is required for EGF-induced PLC␥1 activation in A431 cells, and EGF promotes the formation of a complex between these enzymes that requires tyrosine phosphorylation of PLC␥1.17 In liver cells, EGF and other growth factors induce an elevation of [Ca2⫹]i, but this response is associated with only a modest degree of tyrosine phosphorylation of PLC␥1,6,21 and some authors have questioned the role of IP3 formation in the EGF-induced Ca2⫹ response.25 Although we and others have reported EGF-induced PLC␥1 tyrosine phosphorylation, IP3 formation and [Ca2⫹]i elevation in isolated hepatocytes,6,21,27 including cells in culture,26,28 the factors controlling the strength of this response are not clear. It is not clear why the hepatocytes used in our studies were more strongly suppressed during overnight culture than the Ca2⫹ response to EGF in hepatocytes used by some other groups24,26,28 or, for that matter, the EGF-induced PLC␥1 activation in A431 cells or other cell lines. A variety of different culture conditions were tested to assess the possible role of specific treatments in the suppression of PLC␥1 activation, but there was no systematic correlation with a particular pretreatment that could explain these differences. It should be pointed out, however, that in previous studies the Ca2⫹ response capacity of hepatocytes appeared to vary considerably between different investigators, as determined by the EGF concentrations used for stimulation and the fraction of cells responding to saturating EGF levels (e.g., compare Tanaka et al.,24 Yang et al.,26 and Zhang et al.28). The rate and extent of formation of actin microfilaments probably varies with the culture conditions.

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FIG. 9. Confocal microscopic imaging of actin cytoskeleton and PLC␥1 in hepatocytes in culture for 1 hour or 24 hours. Red: actin filaments, detected with rhodamine-phalloidin; green: PLC␥1 detected with mouse anti-PLC␥1 antibody and FITC-labeled antimouse secondary antibody; yellow: green-red overlay indicating colocalization. (A) Unstimulated 1-hour cultures. (B) EGF-stimulated 1-hour cultures (20 nmol/L, 1 min). (C) Unstimulated 24-hour cultures. (D) EGF-stimulated 24-hour cultures. (E) CD-treated unstimulated 24-hour cultures. (F) CD-treated EGF-stimulated 24 hours. Bars indicate 20 ␮m.

For instance, Mak et al.,42 reported that hepatocytes that were cultured on floating collagen gels took several days before actin microfilaments started to form. It is possible that apparent variability in Ca2⫹ response characteristics between different studies reflect such variations in the actin cytoskeleton formation that were not recorded in those studies. Several authors have provided evidence that EGF stimulation of liver cells and other cell types stimulates PLC␥1 binding to the actin cytoskeleton, and suggested that actin binding may be required for its activation.31,35 By contrast, our data suggest that the regulation of PLC␥1 activity by interaction with filamentous actin is more complex. Specifically, the extensive formation of stress fibers associated with maintenance of the cells in culture resulted in an effective suppression of EGF-induced IP3 formation (Fig. 5) and the associated Ca2⫹ response (Fig. 1). Characteristically, the disruption of the actin filaments with CD effectively recovered both the IP3 formation and the changes in [Ca2⫹]i in cells that were stimulated with EGF (Figs. 6 and 7). CD preferentially binds to the barbed end of actin filaments and disrupts the integrity of the cytoskeletal network,42,48 with the remaining F-actin forming clusters around the plasma membrane and in the perinuclear region (Figs. 9E and F). These findings strongly suggest that the extensive network of actin stress fibers in the cultured hepatocytes contributed to the suppression of the EGF-induced PLC␥1 activation.

The mechanism responsible for the inhibition of PLC␥1 activity may relate to its access to substrate. The observation that EGFR autophosphorylation and PLC␥1 tyrosine phosphorylation were unaffected by maintaining cells in culture shows that the loss of Ca2⫹ response was not the result of a decline in EGFR or PLC␥1 protein levels, or in a loss of tyrosine kinase activity of the receptor or its ability to interact with PLC␥1. Moreover, the intracellular Ca2⫹ stores remained intact and were readily mobilized by other agents that generate IP3, including vasopressin or ␣1-adrenergic agonists. These findings, and the observation that IP3 formation is suppressed, suggest that the defect is related to factors that control the activity of the tyrosine phosphorylated PLC␥1. Interestingly, the loss of EGF-induced IP3 formation was correlated with an apparent lack of translocation of PLC␥1 to the Tritoninsoluble fraction, which was recovered after CD treatment of the cultured cells (Fig. 8). Translocation of PLC␥1 could also be detected by confocal microscopy in EGF-stimulated cells immediately after plating, which appeared to be lost after overnight culture but was recovered following CD treatment (Figs. 9 and 10). However, the functional significance of this translocation for the EGF-induced Ca2⫹ response is not well defined, because, in our hands, the EGF-induced association with the Triton-insoluble fraction did not appear to involve the tyrosine phosphorylated enzyme (Fig. 8).

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FIG. 10. Quantitative estimation of PLC␥1-actin colocalization. Images of different preparations, unstimulated or treated with 20 nmol/L EGF for 1 minute, as shown in Fig. 9, were analyzed by recording the PLC␥1 or actin fluorescence intensity (arbitrary units) per pixel through a cross-section of the cell. Colocalization was determined as PLC␥1 fluorescence in pixels coincident with significant actin fluorescence, expressed as a fraction of total PLC␥1 fluorescence intensity in the cell. Fifteen representative cells from three separate experiments were analyzed for each condition. Asterisks indicate significant differences between EGF-stimulated and unstimulated cells (*P ⫽ .02; **P ⫽ .05); NS, not significant.

It is noteworthy that in the CD-treated cells PLC␥1 appeared to colocalize selectively with the actin clusters that ringed the plasma membrane, but was almost entirely excluded from the perinuclear actin structures (Figs. 9E and F). It is conceivable that the sublamellar actin clusters derive from cortical actin, whereas the perinuclear actin may represent partially degraded stress fibers. Diakonova et al.,35 reported previously that PLC␥1 translocated preferentially to cortical actin structures and was excluded from stress fibers in EGF-stimulated A431 cells. A preferential interaction of PLC␥1 with cortical actin and an exclusion from stress-fiber structures would also be compatible with the observation that the extensive cytoskeletal network of stress fibers and focal adhesion contacts in the basal region of cultured hepatocytes was essentially devoid of PLC␥1 in our studies, whereas notable colocalization was evident in the cortical actin filaments in freshly plated cells. Although studies by Williamson and coworkers31,41 had emphasized the direct binding of PLC␥1 to F-actin through the carboxy-terminal SH2-domain, the selectivity of the interaction for certain cellular actin structures would suggest that specific actin binding proteins may be major determinants of the interaction of the enzyme with the actin cytoskeleton. Previous studies of the EGF-induced PLC␥1 translocation to the actin cytoskeleton in A431 cells30,34 had reported evidence that the enzyme colocalizes with actin structures associated with membrane ruffles. Interestingly, even in the hepatocytes that were maintained in overnight culture, a modest but distinct colocalization of PLC␥1 with actin bundles in membrane ruffles was detectable (Fig. 9D), though it was impossible to quantify any translocation against the large background of diffusely distributed enzyme. In view of the almost complete suppression of the EGF-induced Ca2⫹ re-

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sponse in these cells, this observation raises the question of the functional significance of this colocalization. It is conceivable, of course, that a local stimulation of the enzymatic activity of PLC␥1 occurs in such structures, which may not have been detectable in our analysis of [Ca2⫹]i changes in the cell body. However, in other studies we were not able to detect such a localized [Ca2⫹]i change in membrane ruffles (Shimbo H and Hoek JB, unpublished observations, 2000). An alternative possibility is that PLC␥1 fulfills other functions in these structures that are not necessarily dependent on its enzymatic activity, but that would relate to its capacity to interlink polyphosphoinositide-enriched membrane structures and phosphotyrosine residues or polyproline domains on proteins through its complement of PH, SH2, and SH3 domains. The potential for such alternative functions of PLC␥1 would provide a new perspective on the finding that, in many instances, the capacity for IP3 formation in response to growth factors is extremely modest and seems out of order with the large quantities of protein that are present in many cells, including hepatocytes. To what extent the findings reported here relate to the role of PLC␥1 in the normal liver remains to be elucidated. It is likely that a preferential association of the enzyme with cortical actin structures may be relevant in the intact tissue, where actin stress fibers are largely absent. Actin filaments are also involved in the regulation of bile canalicular function, which is at least in part controlled by Ca2⫹ and PKC signals.49 Furthermore, extensive restructuring of the cytoskeletal structure and its interaction with extracellular matrix molecules is associated with the process of liver regeneration50 that is at least in part controlled by growth factors, such as EGF, transforming growth factor-␣, and hepatocyte growth factor,51 which activate PLC␥1 in hepatocytes. To what extent the activation of PLC␥1 and the associated signaling responses through the DAG/PKC and IP3/[Ca2⫹]i signaling branches are involved in these cytoskeletal alterations and its interaction with the extracellular matrix (or in other aspects of liver regeneration) remains to be determined. REFERENCES 1. Schlessinger J, Bar-Sagi D. Activation of Ras and other signaling pathways by receptor tyrosine kinases. Cold Spring Harb Symp Quant Biol 1994; 59:173-179. 2. Brown KD. The epidermal growth factor/transforming growth factor-␣ family and their receptors. Eur J Gastroenterol Hepatol 1995;7:914-922. 3. Carpenter G. Receptor tyrosine kinase substrates: src homology domains and signal transduction. FASEB J 1992;6:3283-3289. 4. Schlessinger J. SH2/SH3 signaling proteins. Curr Opin Genet Dev 1994; 4:25-30. 5. Bork P, Margolis B. A phosphotyrosine interaction domain [letter]. Cell 1995;80:693-694. 6. Kholodenko BN, Demin OV, Moehren G, Hoek JB. Quantification of short-term signaling by the epidermal growth factor receptor: a kinetic approach. J Biol Chem 1999;274:30169-30181. 7. Rhee SG, Choi KD. Multiple forms of phospholipase C isozymes and their activation mechanisms. Adv Second Messenger Phosphoprotein Res 1992;26:35-61. 8. Carpenter G, Ji Q. Phospholipase C-␥ as a signal-transducing element. Exp Cell Res 1999;253:15-24. 9. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature 1993; 361:315-325. 10. Meisenhelder J, Suh PG, Rhee SG, Hunter T. Phospholipase C-␥ is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell 1989;57:1109-1122. 11. Kim HK, Kim JW, Zilberstein A, Margolis B, Kim JG, Schlessinger J, Rhee SG. PDGF stimulation of inositol phospholipid hydrolysis requires PLC-␥1 phosphorylation on tyrosine residues 783 and 1254. Cell 1991; 65:435-441.

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