Alteration of . epidermal growth factor-dependent phosphorylation during rat liver regeneration ( partial hepatectomy / growth / protein ldinase / phosphotyrosine )

Epidermal growth factor (EGF) stimulates membrane protein phosphorylation in a human cell line, A-431. The known hepatic mitogenic action ofEGF and the reduction in EGF receptor number that occurs during liver regeneration led us to study whether EGF-dependent protein kinase activity was present in rat liver and whether its activity was altered after partial hepatectomy. Liver membranes, preincubated with or without EGF, were phosphorylated (0C, 15 sec) and subjected to NaDodSOd polyacrylamide gel electrophoresis and autoradiography. In microsomal fractions, EGF at 5-2000 ng/ml produced a dose-related stimulation of 32p incorporation into a single 170,000-dalton protein (p170). In plasma membranes, a similar EGF-dependent phosphorylation was present and was substantially enriched relative to the microsomal fraction. Acid hydrolysis of labeled microsomal fraction followed by ehosphoamino acid determination revealed that EGF stimulated 2p incorporation into phosphotyrosine residues. The EGF-dependent phosphorylation ofp170 was compared in microsomal fractions isolated from rats 36 hr after partial hepatectomy or sham operation. In the absence of EGF, in vitro labeling of p170 was similar. EGF stimulated the labeling of p170 in both groups, but the response was clearly diminished after partial hepatectomy. In the presence of EGF, the labeling of p170 in microsomal fraction from regenerating livers was only 47 ± 6% of that observed in membranes from sham-operated rats (P < 0.005). Reduction ofEGF-dependent phosphorylation during liver regeneration paralleled the loss of binding of '2I-labeled EGF. An increase in the EGF-independent phosphorylation of a 130,000-dalton protein was also observed after partial hepatectomy. The increase in the amount of this phosphoprotein was roughly equal to the loss ofEGF-stimulated p170 phosphorylation. Several additional proteins showed increased phosphorylation in membranes from partially hepatectomized rats. These findings indicate that alterations in membrane tyrosine residue phosphorylation occur during regulated growth in viva Epidermal growth factor (EGF) is mitogenic in many cultured cell lines (1) and stimulates DNA synthesis in hepatocytes, both in vitro (2) and in vivo (3). The plasma membrane receptor for EGF, a 150,000to 180,000-dalton glycoprotein, binds EGF with high affinity and is subsequently internalized (1, 4). EGF rapidly alters ion and nutrient flux, but more prolonged exposure is needed to stimulate DNA synthesis (5). The mechanism by which EGF exerts these actions has not been established, but Carpenter et al (6) have shown that EGF stimulates the phosphorylation ofa number ofproteins in membranes from a human epidermoid carcinoma cell line, A-431. Cohen et at (7) have shown that the major protein phosphorylated in the EGF-dependent manner copurifies with the EGF receptor itself. The EGF-stimulated phosphorylation ofthe EGF receptor occurs in part on tyrosine residues both in cell membranes (8) and in cultured cells (9). Protein phosphorylation on tyrosine residues has recently been reported by Hunter and Sefton (10), Witte et al. (11), and Erickson et at (12) in studies of protein kinase activities associated with transforming viruses. Increases in cellular tyrosine residue phosphorylation correlate with the transformed phenotype and rapid growth. The viral enzymes appear to be homologues ofnormal cellular protein kinases which may regulate growth and development (13). Tyrosine residue kinase activity has also been observed in Ehrlich ascites tumor cells (14). Rat liver regeneration is a well-characterized in vivo model of growth regulation. The signal for growth after partial hepatectomy is humoral, but not all of its constituents have been identified (15). A role for insulin and glucagon has been demonstrated (16), and we have recently shown that EGF receptor number is markedly decreased after partial hepatectomy (17). This indicates that EGF, or humoral factors which are capable of modulating the EGF receptor number (18, 19), may participate in the proliferative response. The present studies were designed to investigate whether EGF-dependent protein kinase is present in liver membranes and whether its activity is altered during liver regeneration. The data demonstrate that rat liver membranes exhibit EGF-dependent phosphorylation of a single 170,000-dalton membrane protein. This phosphorylation occurs at least in part on tyrosine residues. The amount of EGF-dependent phosphorylation decreases during liver regeneration, as does the EGF receptor number. In addition, membranes from regenerating liver contain several proteins whose phosphorylation significantly increases after partial hepatectomy. MATERIALS AND METHODS Membrane Preparation and Assays. Male Sprague-Dawley rats (150-200 g) from Charles River Breeding Laboratories had access to food and water ad lib. When indicated, the median and left hepatic lobes were excised (66-70% hepatectomy) or manipulated and returned to the peritoneal cavity (sham operation). Control, sham-operated, or partially hepatectomized rats were killed between 0800 and 0900 hours. The livers were homogenized in 0.25 M sucrose/10 mM Tris'HCl, pH 8.0 with a Brinkman Polytron apparatus. Homogenates were centrifuged at 30,000 X g for 7.5 min. The supernatant was centrifuged at 105,000 X g to obtain the microsomal fraction. Plasma membranes were made by the method of Touster et al. (20). The microsomal fraction was centrifuged for 16 hr in a discontinuous sucrose gradient. The plasma membranes were collected and repelleted at 105,000 Abbreviations: EGF, epidermal growth factor; p170, 170,000-dalton protein whose phosphorylation is stimulated by EGF. 776 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. NatL Acad. Sci. USA 79 (1982) 777 x g. Protein determinations were done by the method ofLowry et aL (21). 5'-Nucleotidase, a plasma membrane marker enzyme, was assessed by published methods (22). l"I-Labeled EGF ('25I-EGF) binding was determined with iodinated mouse EGF purified to homogeneity by the method of Savage and Cohen (23). Nonspecific binding was assessed in the presence of 1 ,uM native EGF. Details of the assay have been published (17). In Vitro Phosphorylation. The phosphorylation reaction was performed at 00C in a total volume of 50 Aul, containing 50 Ag of membrane protein, 50 mM Pipes at pH 7.0, 30 mM MgCl2, 10 mM 2-mercaptoethanol, and 0.32 mM EGTA. The diluent for EGF, 10 u1 of phosphate-buffered saline containing 0.1% bovine serum albumin, was added to each control tube. After 15-min preincubation at 00C with or without EGF, the reaction was started by the addition of 15 pACi (1 Ci = 3.7 X 1010 becquerels) of [y-32P]ATP (ICN) to a final concentration of 2 uM. It was stopped after 15 sec with 25 1ul of 9% NaDodSOJ0.05% bromphenol blue/15% (vol/vol) glycerol/6% 2-mercaptoethanol/30 mM Tris, pH 7.8. Tubes were corked and heated to 1000C (5 min). Aliquots (50 ,ul) were electrophoresed on NaDodSOJ8% polyacrylamide slab gels with a 3% stacking gel, essentially as described by Rudolph and Krueger (24). Gels were stained with Coomassie brilliant blue, destained, dried, and autoradiographed on Kodak XAR-5 film. Exposure time varied from 12 hr to 7 days. Autoradiograms were scanned with a Perkin-Elmer densitometer; peak integration was performed on-line by the microprocessor of a Varian 8000 high-pressure liquid chromatography instrument. Phosphoamino Acid Determination. Freshly prepared liver microsomes (1.5 mg of protein) were phosphorylated as above with or without EGF at 200 ng/ml. The ATP concentration was 15 ,uM (90 ,uCi of [y-32P]ATP per sample) and the 0°C incubation was prolonged to 5 min. The membranes were precipitated and washed sequentially with trichloroacetic acid and ether/ethanol as described by Ushiro and Cohen (8). The hydrolysis (6 M HCl, 1100C, 3 hr) and phosphoamino acid separation were performed by the technique of Hunter and Sefton (10). After lyophilization, the residue was resuspended in H20 with marker phosphoamino acids (phosphoserine and phosphothreonine from Sigma; phosphotyrosine was generously provided by J. Larner and C. Schwartz). Separation on cellulose thin-layer plates was accomplished by electrophoresis (900 V) atpH 1.9 for 60 min and subsequent ascending chromatography in the second dimension [isobutyric acid/0.5 M NH40H, 5:3, (vol/vol)]. The plates were dried, standard phosphoamino acids were detected with ninhydrin, and autoradiography was performed.