Proc. Nati. Acad. Sci. USA Vol. 87, pp. 3816-3820, May 1990 Biochemistry
Activated type I phosphatidylinositol kinase is associated with the epidermal growth factor (EGF) receptor following EGF stimulation (tyrosine kinase/signal transduction)
JEFFREY D. BJORGE*, TUNG-ON CHAN*, MICHAEL ANTCZAKt, HSING-JIEN KUNGt, AND DONALD J. FUJITA** *Cell Regulation Group, Department of Medical Biochemistry, University of Calgary, Calgary, AB T2N 4N1, Canada; and tDepartment of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106
Communicated by Peter Vogt, February 20, 1990 (received for review December 27, 1989)
contain an associated PI kinase activity (23) and EGF treatment of some cell types (e.g., the human epidermoid carcinoma cell line A431) results in PI kinase activation (24). In this report, we demonstrate that EGF mediates an enhancement or activation of a type I PI kinase activity that is found in association with the EGF receptor. Unlike the PI 4' kinases (type II) described in previous reports concerning the EGF receptor (23, 24), this PI kinase possesses properties characteristic of PI 3' kinases (type I), typified by the ability to phosphorylate PI on the 3' position of the inositol ring (25, 26).
We have shown that a type I phosphatidylABSTRACT inositol (PI) kinase activity is associated with the epidermal growth factor (EGF) receptor in a mouse fibroblast cell line expressing human EGF receptors (NRHER5) and that this activity increases dramatically upon treatment of cells with physiologically relevant concentrations of EGF. EGF stimulated a time-dependent increase in EGF receptor-associated PI kinase activity measured in EGF receptor immunoprecipitates. Activation was detected 15 min after the addition of EGF, and it peaked between 1 and 2 hr. Activation of PI kinase was detected with EGF concentrations as low as 10 pM and maximal stimulation occurred at -1 nM. Analysis of deacylated PI phosphate products, and inhibition of the PI kinase activity by nonionic detergent, indicated that the PI kinase described here was type I or PI 3' kinase. These results demonstrate the regulation of a type I PI kinase by EGF and suggest a potential role in the EGF receptor signal transduction pathway.
MATERIALS AND METHODS Cell Culture. NR6 cells lacking detectable EGF receptors (27) and NR6 cells expressing the construct pCO12 (28) containing the normal human EGF receptor (NRHER5 cells) were cultured in Dulbecco's modified Eagle's medium containing low glucose supplemented with 8% (vol/vol) fetal bovine serum (GIBCO). NRHER5 cells were clonally isolated from a population of NR6 cells infected with viruses carrying the human EGF receptor gene (in the form of the pCO12 construct). The viruses were derived from Psi-2 cells (29) transfected with the construct pCO12. Cells from the clone NRHER5 displayed a low background level of human EGF receptor activity under normal growth conditions and responded to very low levels of exogenous ligand (EGF or transforming growth factor type a). The NRHER5 clone was found to express 106 human EGF receptor molecules per cell. For soft agar analysis, 1 x 105 cells in 4.0 ml of 0.35% Bacto agar (Difco) were plated onto prehardened 0.5% Bacto agar bottoms in 60-mm plates; 2-4 days after initial seeding, an additional 3.0 ml of top agar was added to each dish containing sufficient ligand levels to bring the total volume in each plate to the desired ligand concentration. Only a single ligand addition was used in each case. All agar solutions were made up in complete growth medium. Cell Lysis and Immunoprecipitation of the EGF Receptor. Cells on 10-cm plates were placed on ice and rinsed twice with 4.0 ml of ice-cold phosphate-buffered saline (PBS) containing 5 mM EDTA; 0.5 ml of ice-cold lysing buffer [0.15 M NaCl/20 mM Tris HCl, pH 8.0/5 mM EDTA/1% Nonidet P-40/2% (vol/vol) glycerol/150 ,M Na3VO4/50 ,ug of leupeptin per ml/3.75 mg ofp-nitrophenylphosphate per ml] was added and lysates were scraped into a microcentrifuge tube [method adapted from Whitman et al. (30)]. After 30 min on ice followed by centrifugation at 13,000 x g for 15 min, aliquots of supernatant containing 300 ,ug of total cellular protein (as determined by protein assay; Bio-Rad) were
In recernt years, several steps involved in signal transduction pathways mediated by epidermal growth factor (EGF) have been elucidated. Early responses (1 hr) include the stimulation of DNA synthesis and cell proliferation (9, 10). Although many of the effects of receptor activation have been carefully documented, it appears that additional important information is required to establish which of these documented responses are critical for late events such as cell division. In addition, there most certainly are important cellular events that remain to be uncovered. One family of growth factor-activated signals that has drawn a large amount of interest recently includes those derived from the PI pathway. These include the second messengers inositol 1,4,5-trisphosphate, which has been implicated in the mobilization of intracellular calcium (11, 12), and diacylglycerol, which activates the calcium- and phospholipid-dependent kinase protein kinase C (13, 14). In addition, a number of other second messengers, including prostaglandins, PI polyphosphates, and inositol polyphosphates, are generated, which may have important roles in cell growth (for review, see ref. 15). EGF has been shown to stimulate PI turnover in a variety of cell types (7, 16-18), presumably through phosphorylation and activation of phospholipase C (19-22). In addition, partially purified EGF receptor preparations have been found to
Abbreviations: EGF, epidermal growth factor; PI, phosphatidylinositol; PDGF, platelet-derived growth factor; mAb, monoclonal antibody; PI4P, PI 4-phosphate; PI3P, PI 3-phosphate; PIP, PI phosphate; MTAg, middle-sized tumor antigen. 4To whom reprint requests should be addressed.
The publication costs of this 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.
3816
Biochemistry: Bjorge et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
immunoprecipitated for 1 hr on ice with 5 Ag of monoclonal antibody (mAb) B1D8 (31) specific for the EGF receptor. After incubation with 60 ul of fixed Staphylococcus aureus (Bethesda Research Laboratories) for an additional 30 min, the immune complexes were washed as follows: twice with PBS containing 1% Nonidet P-40, and once each with PBS, 0.5 M LiCJ in 100 mM Tris HCl (pH 7.4), H20, and 10 mM Tris-HCI (pH 7.4) containing 100 mM NaCl and 1 mM EDTA. PI Kinase Assay. PI kinase activity was measured by the method of Whitman et al. (26). Briefly, the washed immunoprecipitates were resuspended in 50 1.d of kinase buffer [20 mM Tris HCl, pH 7.6/0.10 M NaCJ/10 mM MgCl2/10 ,uCi of [y-32P]ATP (3000 Ci/mmol; 1 Ci = 37 GBq; New England Nuclear)/0.2 mg of sonicated PI (Serdary Research Laboratories, London, ON, Canada) per ml/0.01 mM ATP]. Reaction mixtures were incubated for 5 min at 30°C and stopped by the addition of 100 ,ul of 1 M HCl. Lipids were extracted with 200 ,ul of chloroform/methanol (1:1) and chromatographed on thin-layer silica plates (G-25; Macherey & Nagel) using a chloroform/methanol/4.0 M ammonium hydroxide (9:7:2) developing solvent. Radioactive spots were visualized by autoradiography, identified by comparison with lipid standards (Sigma) stained with iodine vapor, and quantitated by liquid scintillation counting of the scraped spot. PI 4phosphate (PI4P) was used to identify the position of the type I PI kinase product because PI 3-phosphate (PI3P) is not commercially available, and the two lipids have been reported to migrate with similar Rf values, with PI3P migrating very slightly slower than PI4P, under the chromatographic conditions used here (26). Deacylation and HPLC Analysis of the PI Kinase Products. Radioactive spots identified as PIP were scraped from the TLC plate, treated with methylamine to remove fatty acyl side chains, and processed in preparation for HPLC exactly as described (26). HPLC analysis of the deacylated products (glycerophosphoinositol phosphates) was carried out on a strong anion-exchange column (Partisphere SAX column; Whatman). Samples were loaded in water and eluted with 0.42 M (NH4)2HP04 (pH 3.8) at 1.0 ml/min with a linear gradient of 0-0.109 M over 40 min, followed by 0.109-0.42 M over 10 min.
1 2 3 4 5 6 7
a a
0 *
,
*
0
Immunoblotting with Anti-EGF Receptor and Anti-Phos-
photyrosine Antibodies. Immunoprecipitated EGF receptor preparations were divided into two aliquots: one for PI kinase assay and one for immunoblotting. Samples for immunoblotting were electrophoresed on 7.5% SDS/polyacrylamide gel and transferred to nitrocellulose as described (32). The blot was blocked in a solution containing 50 mM Tris HCl (pH 8.0), 2% bovine serum albumin, 2% polyvinylpyrrolidone (Mr 360,000), and 2% Ficoll (Mr 400,000) for 1 hr. The blot was initially probed with mAb H9B4 (32) directed against the EGF receptor (1 ,ug/ml in PBS containing 5% bovine serum albumin for 2 hr at 20°C). After five rapid rinses in PBS containing 0.1% bovine serum albumin, bound antibody was visualized by sequential incubations with affinity-purified rabbit anti-mouse IgG and 1251I-labeled protein A (87 ACi/,g; New England Nuclear), followed by autoradiography. The same blot was then stripped of bound antibodies by incubation for 90 min in 0.1 M glycine, pH 1.8/20 mM MgOAc/0.05 M KCl, reblocked, and probed with 1 ,g of rabbit antiphosphotyrosine antibody per ml [prepared as described (33)]. The bands were visualized by incubation with 125I. labeled protein A followed by autoradiography.
RESULTS Activated PI Kinase Activity Is Associated with the EGF Receptor upon Ligand Stimulation. NRHER5 cells were treated with 10 nM EGF for various amounts of time and PI kinase activity was measured in anti-EGF receptor immunoprecipitates. As shown in Fig. la, a small but significant amount of PI kinase activity was specifically associated with the "unactivated" immunoprecipitated EGF receptor. Upon EGF treatment, the amount of associated activity increased in a time-dependent manner, peaking between 1 and 2 hr after EGF addition. Quantitation of the radioactive spots adjacent to the PIP standard and correction for PI kinase activity associated with nonimmune immunoprecipitates (Fig. lb) revealed an 8- to 9-fold enhancement of EGF receptorassociated activity. The EGF-mediated enhancement of PI kinase activity appeared dependent on the presence of EGF receptors in the immunoprecipitates because immunoprecip-
b
8 9 10 -
pI p
LL
0
>o 10
z>
8
°
Activated type I phosphatidylinositol kinase is associated with the epidermal growth factor (EGF) receptor following EGF stimulation (tyrosine kinase/signal transduction)
JEFFREY D. BJORGE*, TUNG-ON CHAN*, MICHAEL ANTCZAKt, HSING-JIEN KUNGt, AND DONALD J. FUJITA** *Cell Regulation Group, Department of Medical Biochemistry, University of Calgary, Calgary, AB T2N 4N1, Canada; and tDepartment of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106
Communicated by Peter Vogt, February 20, 1990 (received for review December 27, 1989)
contain an associated PI kinase activity (23) and EGF treatment of some cell types (e.g., the human epidermoid carcinoma cell line A431) results in PI kinase activation (24). In this report, we demonstrate that EGF mediates an enhancement or activation of a type I PI kinase activity that is found in association with the EGF receptor. Unlike the PI 4' kinases (type II) described in previous reports concerning the EGF receptor (23, 24), this PI kinase possesses properties characteristic of PI 3' kinases (type I), typified by the ability to phosphorylate PI on the 3' position of the inositol ring (25, 26).
We have shown that a type I phosphatidylABSTRACT inositol (PI) kinase activity is associated with the epidermal growth factor (EGF) receptor in a mouse fibroblast cell line expressing human EGF receptors (NRHER5) and that this activity increases dramatically upon treatment of cells with physiologically relevant concentrations of EGF. EGF stimulated a time-dependent increase in EGF receptor-associated PI kinase activity measured in EGF receptor immunoprecipitates. Activation was detected 15 min after the addition of EGF, and it peaked between 1 and 2 hr. Activation of PI kinase was detected with EGF concentrations as low as 10 pM and maximal stimulation occurred at -1 nM. Analysis of deacylated PI phosphate products, and inhibition of the PI kinase activity by nonionic detergent, indicated that the PI kinase described here was type I or PI 3' kinase. These results demonstrate the regulation of a type I PI kinase by EGF and suggest a potential role in the EGF receptor signal transduction pathway.
MATERIALS AND METHODS Cell Culture. NR6 cells lacking detectable EGF receptors (27) and NR6 cells expressing the construct pCO12 (28) containing the normal human EGF receptor (NRHER5 cells) were cultured in Dulbecco's modified Eagle's medium containing low glucose supplemented with 8% (vol/vol) fetal bovine serum (GIBCO). NRHER5 cells were clonally isolated from a population of NR6 cells infected with viruses carrying the human EGF receptor gene (in the form of the pCO12 construct). The viruses were derived from Psi-2 cells (29) transfected with the construct pCO12. Cells from the clone NRHER5 displayed a low background level of human EGF receptor activity under normal growth conditions and responded to very low levels of exogenous ligand (EGF or transforming growth factor type a). The NRHER5 clone was found to express 106 human EGF receptor molecules per cell. For soft agar analysis, 1 x 105 cells in 4.0 ml of 0.35% Bacto agar (Difco) were plated onto prehardened 0.5% Bacto agar bottoms in 60-mm plates; 2-4 days after initial seeding, an additional 3.0 ml of top agar was added to each dish containing sufficient ligand levels to bring the total volume in each plate to the desired ligand concentration. Only a single ligand addition was used in each case. All agar solutions were made up in complete growth medium. Cell Lysis and Immunoprecipitation of the EGF Receptor. Cells on 10-cm plates were placed on ice and rinsed twice with 4.0 ml of ice-cold phosphate-buffered saline (PBS) containing 5 mM EDTA; 0.5 ml of ice-cold lysing buffer [0.15 M NaCl/20 mM Tris HCl, pH 8.0/5 mM EDTA/1% Nonidet P-40/2% (vol/vol) glycerol/150 ,M Na3VO4/50 ,ug of leupeptin per ml/3.75 mg ofp-nitrophenylphosphate per ml] was added and lysates were scraped into a microcentrifuge tube [method adapted from Whitman et al. (30)]. After 30 min on ice followed by centrifugation at 13,000 x g for 15 min, aliquots of supernatant containing 300 ,ug of total cellular protein (as determined by protein assay; Bio-Rad) were
In recernt years, several steps involved in signal transduction pathways mediated by epidermal growth factor (EGF) have been elucidated. Early responses (1 hr) include the stimulation of DNA synthesis and cell proliferation (9, 10). Although many of the effects of receptor activation have been carefully documented, it appears that additional important information is required to establish which of these documented responses are critical for late events such as cell division. In addition, there most certainly are important cellular events that remain to be uncovered. One family of growth factor-activated signals that has drawn a large amount of interest recently includes those derived from the PI pathway. These include the second messengers inositol 1,4,5-trisphosphate, which has been implicated in the mobilization of intracellular calcium (11, 12), and diacylglycerol, which activates the calcium- and phospholipid-dependent kinase protein kinase C (13, 14). In addition, a number of other second messengers, including prostaglandins, PI polyphosphates, and inositol polyphosphates, are generated, which may have important roles in cell growth (for review, see ref. 15). EGF has been shown to stimulate PI turnover in a variety of cell types (7, 16-18), presumably through phosphorylation and activation of phospholipase C (19-22). In addition, partially purified EGF receptor preparations have been found to
Abbreviations: EGF, epidermal growth factor; PI, phosphatidylinositol; PDGF, platelet-derived growth factor; mAb, monoclonal antibody; PI4P, PI 4-phosphate; PI3P, PI 3-phosphate; PIP, PI phosphate; MTAg, middle-sized tumor antigen. 4To whom reprint requests should be addressed.
The publication costs of this 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.
3816
Biochemistry: Bjorge et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
immunoprecipitated for 1 hr on ice with 5 Ag of monoclonal antibody (mAb) B1D8 (31) specific for the EGF receptor. After incubation with 60 ul of fixed Staphylococcus aureus (Bethesda Research Laboratories) for an additional 30 min, the immune complexes were washed as follows: twice with PBS containing 1% Nonidet P-40, and once each with PBS, 0.5 M LiCJ in 100 mM Tris HCl (pH 7.4), H20, and 10 mM Tris-HCI (pH 7.4) containing 100 mM NaCl and 1 mM EDTA. PI Kinase Assay. PI kinase activity was measured by the method of Whitman et al. (26). Briefly, the washed immunoprecipitates were resuspended in 50 1.d of kinase buffer [20 mM Tris HCl, pH 7.6/0.10 M NaCJ/10 mM MgCl2/10 ,uCi of [y-32P]ATP (3000 Ci/mmol; 1 Ci = 37 GBq; New England Nuclear)/0.2 mg of sonicated PI (Serdary Research Laboratories, London, ON, Canada) per ml/0.01 mM ATP]. Reaction mixtures were incubated for 5 min at 30°C and stopped by the addition of 100 ,ul of 1 M HCl. Lipids were extracted with 200 ,ul of chloroform/methanol (1:1) and chromatographed on thin-layer silica plates (G-25; Macherey & Nagel) using a chloroform/methanol/4.0 M ammonium hydroxide (9:7:2) developing solvent. Radioactive spots were visualized by autoradiography, identified by comparison with lipid standards (Sigma) stained with iodine vapor, and quantitated by liquid scintillation counting of the scraped spot. PI 4phosphate (PI4P) was used to identify the position of the type I PI kinase product because PI 3-phosphate (PI3P) is not commercially available, and the two lipids have been reported to migrate with similar Rf values, with PI3P migrating very slightly slower than PI4P, under the chromatographic conditions used here (26). Deacylation and HPLC Analysis of the PI Kinase Products. Radioactive spots identified as PIP were scraped from the TLC plate, treated with methylamine to remove fatty acyl side chains, and processed in preparation for HPLC exactly as described (26). HPLC analysis of the deacylated products (glycerophosphoinositol phosphates) was carried out on a strong anion-exchange column (Partisphere SAX column; Whatman). Samples were loaded in water and eluted with 0.42 M (NH4)2HP04 (pH 3.8) at 1.0 ml/min with a linear gradient of 0-0.109 M over 40 min, followed by 0.109-0.42 M over 10 min.
1 2 3 4 5 6 7
a a
0 *
,
*
0
Immunoblotting with Anti-EGF Receptor and Anti-Phos-
photyrosine Antibodies. Immunoprecipitated EGF receptor preparations were divided into two aliquots: one for PI kinase assay and one for immunoblotting. Samples for immunoblotting were electrophoresed on 7.5% SDS/polyacrylamide gel and transferred to nitrocellulose as described (32). The blot was blocked in a solution containing 50 mM Tris HCl (pH 8.0), 2% bovine serum albumin, 2% polyvinylpyrrolidone (Mr 360,000), and 2% Ficoll (Mr 400,000) for 1 hr. The blot was initially probed with mAb H9B4 (32) directed against the EGF receptor (1 ,ug/ml in PBS containing 5% bovine serum albumin for 2 hr at 20°C). After five rapid rinses in PBS containing 0.1% bovine serum albumin, bound antibody was visualized by sequential incubations with affinity-purified rabbit anti-mouse IgG and 1251I-labeled protein A (87 ACi/,g; New England Nuclear), followed by autoradiography. The same blot was then stripped of bound antibodies by incubation for 90 min in 0.1 M glycine, pH 1.8/20 mM MgOAc/0.05 M KCl, reblocked, and probed with 1 ,g of rabbit antiphosphotyrosine antibody per ml [prepared as described (33)]. The bands were visualized by incubation with 125I. labeled protein A followed by autoradiography.
RESULTS Activated PI Kinase Activity Is Associated with the EGF Receptor upon Ligand Stimulation. NRHER5 cells were treated with 10 nM EGF for various amounts of time and PI kinase activity was measured in anti-EGF receptor immunoprecipitates. As shown in Fig. la, a small but significant amount of PI kinase activity was specifically associated with the "unactivated" immunoprecipitated EGF receptor. Upon EGF treatment, the amount of associated activity increased in a time-dependent manner, peaking between 1 and 2 hr after EGF addition. Quantitation of the radioactive spots adjacent to the PIP standard and correction for PI kinase activity associated with nonimmune immunoprecipitates (Fig. lb) revealed an 8- to 9-fold enhancement of EGF receptorassociated activity. The EGF-mediated enhancement of PI kinase activity appeared dependent on the presence of EGF receptors in the immunoprecipitates because immunoprecip-
b
8 9 10 -
pI p
LL
0
>o 10
z>
8
°
