360
Biochimica et Biophysica Acta, 585 ( 1 9 7 9 ) 3 6 0 - - 3 7 3 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28921
P H O S P H O P R O T E I N PHOSPHATASE ACTIVITY AT THE O U T E R S U R F A C E OF INTACT N O R M A L AND T R A N S F O R M E D 3T3 F I B R O B L A S T S
N I Z A R R. M A K A N
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Wing Hall, Ithaca, N Y 14853 (U.S.A.) (Received October 30th, 1978)
Key words: Phosphoprotein phosphatase; Cell surface; Ectoenzyme; (3T3 cell)
Summary Using 32P-labeled phosphocasein or phosphohistones as exogenous substrates it was possible to detect a phosphoprotein phosphatase activity on the outer surface of intact normal and transformed 3T3 fibroblasts. Incubation of monolayers of intact cells in buffered salt solution with the radioactively labeled substrate resulted in the release of alkali-labile 32p counts into the surrounding medium. The reaction was: (a) linear with time (at least up to 20 min); (b) proportional to the cell density; (c) dependent on the temperature and pH of the incubation medium; (d) stimulated by K*; and (e) inhibited by sodium fluoride, inorganic pyrophosphate, zinc chloride and relatively impermeant sulfhydryl reagents. Less than 2% of the externally located phosphoprotein phosphatase activity was detectable in pooled cell-free washings of the intact cell monolayer. Phosphocasein did not cause any detectable leakage of intracellular lactate dehydrogenase or soluble phosphoprotein phosphatase activity into the external medium; incubation of the cells with phosphohistones, on the other hand, resulted in appreciable leakage of both these cytoplasmic activities. Neoplastic transformation was associated with a nearly two-fold decrease in the activity of the surface phosphoprotein phosphatase. Addition of serum to either non-transformed 3T3 or spontaneously transformed 3T6 cells resulted in a rapid and remarkable drop in the cell surface dephosphorylating activity. Acrylamide gel electrophoresis of the dephosphorylated casein or histone substrate revealed no proteolytic degradation or change in electrophoretic mobility. The intact cells showed no damage u p o n microscopic examination as a result of exposure to phosphocasein or phosphohistones. Abbreviations: 3T6, a cell line derived from Swiss mouse 3T3 cells by spontaneous transformation; SV3T3, 3T3 cells transformed by Simian vacuolating virus 40, wild type.
361
Introduction The existence of membrane-bound protein kinases in normal and neoplastic cells has already been amply demonstrated [1--6]. Using extrinsic acceptor proteins, it has been shown that at least part of this plasma membrane activity is located at the external surface [7--10]. Mastro and Rozengurt [8] have reported evidence for increased protein kinase activity at the outer surface of 3T3 fibroblasts following oncogenic transformation. Glia cells [9] and dermal fibroblasts [10], on the other hand, have been reported to have a higher protein kinase activity on their outer surfaces than their respective malignant derivatives, the glioma and fibrosarcoma cells. In any case, the significance of this enzymatic activity on the outer cell surface remains unclear. I have recently [11] reported data which are consistent with the existence on the transformed cell surface of an ATP-requiring protein kinase and a fluorideinhibitable phosphoprotein phosphatase, which could control the changes in passive membrane permeability reported earlier in transformed cells treated with exogenous ATP [12,13]. Reports are appearing with increasing frequency concerning the existence of phosphorylation-dephosphorylation mechanisms and their role in controlling various cellular functions [14--19]. The demonstration of protein kinase activity on the external surfaces of various normal and transformed intact cells suggested the possibility that a dephosphorylating activity might similarly exist on the outer cell surface. This paper describes the existence and properties of such a dephosphorylating activity at the outer surface of intact normal and transformed 3T3 fibroblasts. It is shown that this activity decreases in response to activation of cell growth by serum. Materials and Methods Chemicals. Calf t h y m u s histone, t y p e IIA (histone mixture) and casein were purchased from Sigma Chemical Company. The catalytic subunit of cyclic AMP-dependent protein kinase from bovine heart [20] was a gift of Dr. E d m o n d H. Fischer (University of Washington, Seattle). [~/32P]ATP was prepared either b y a modification of the m e t h o d of Avron [21] or according to the m e t h o d of Glynn and Chappell [22] and was generously provided by the laboratories of Drs. E. Racker and R. Wu. Chemicals used for polyacrylamide gel electrophoresis were purchased from BioRad. Serum, from various animal sources, was purchased from Grand Island Biological Company (GIBCO). All other chemicals were of analytical grade. Cell lines. Stock cultures of Swiss mouse 3T3 [23] were grown in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% calf serum (GIBCO), 1 0 0 u n i t s / m l penicillin, 1 0 0 p g / m l streptomycin in a humidified atmosphere of 10% CO2 and air at 37°C. Stock cultures of 3T6 [23] and SV3T3 cells were propagated similarly. The 3T3 cells were maintained subconfluent to avoid the selection of spontaneous transformants. Cells were subcultured into 30-mm Falcon plastic dishes with medium containing 10% calf serum and used 2--3 days after plating when the cultures approached confluency. For purposes of studying the effect of serum on cell surface dephos-
362 phorylating activity 3T3 and 3T6 cells were plated in medium containing 5% and 0.5% serum, respectively; 3--4 days after plating, the medium was removed and replaced with serum-free Dulbecco's modified Eagle's medium and the cultures incubated in this medium for 48 h. Routine testing of the cell cultures on mycoplasma agar [24] revealed no detectable mycoplasma contamination. Preparation of phosphorylated substrates. [32p]Histone was prepared by incubation of histone IIA with [~/-32p]ATP in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine heart [20]. I ml of incubation mixture contained 20 mg of histone, 16.6 pmol of potassium phosphate buffer (pH 7.0), 6 pmol of magnesium acetate, 1 pmol of dithiothreitol, 10 pmol of sodium fluoride, and 0.3 pmol of [~,-32P]ATP (17--34 • 106 cpm). The reaction was initiated by the addition of 38 pg of the catalytic subunit of protein kinase which was suspended in a buffer (pH 6.7) containing 30 mM KH2PO4, 0.1 mM EDTA, 0.25 M KC1, 1 mM dithiothreitol and 1 mg/ml bovine serum albumin. The incubation was carried out at 30°C for 4 5 - 6 0 min and the reaction terminated b y the addition of 20% trichloroacetic acid (final concentration). The resultant precipitate was centrifuged and treated as described b y Maeno and Greengard [25]; in addition [32p]histone was converted to its hydrochloride by precipitation with a mixture of acetone/1 N HC1 (98 : 2, v/v) [26]. [32p]Casein was prepared essentially as described above for histone except that the incubation mixture contained 20 pmol of Tris-HC1 buffer (pH 7.0) and 6 pmol of MgC12. Characterization of the phosphorylated substrates. To verify that the [32p]_ phosphate in [32P]casein and [32P]histone was present in phosphoserine and/or phosphothreonine linkage with the protein, the phosphorylated substrates were subjected to acid hydrolysis for 6 h at l l 0 ° C in ampoules sealed under vacuum. 6 N HC1 was used to hydrolyze phosphohistone and concentrated HC1 was used for phosphocasein. After drying in vacuo, the samples were dissolved in a small volume of water and samples of the hydrolysate applied onto a thin-layer chromatographic plate (Eastman Kodak). [32P]Orthophosphate, genuine phosphoserine and phosphothreonine were used as markers. The samples were subjected to high voltage electrophoresis in 2.5% formic acid/7.8% acetic acid (pH 1.9) at 1000 V for 45 min. Whatmann No. 3 MM paper was used as a wick and attached to both ends of the chromatographic plate and allowed to dip in the buffer. The phosphorylated amino acids were detected with 0.2% ninhydrin/ pyridine stain and radioactivity located by autoradiography using Kodak X-omat R film (XR-5). Screening of various sera for ability to hydrolyze [32P]histone. In order to be sure that the phosphoprotein phosphatase activity assayed on the outer cell surface was not derived from the surrounding serum, sera from numerous sources (fetal calf, newborn calf, adult calf, chick, goat, horse, lamb and rabbit) were screened for intrinsic phosphoprotein phosphatase activity using [32p]_ histone as a substrate. In each case, the serum was diluted to a final concentration of 1% in order to reduce competition of serum phosphoprotein with [3~p]_ histone as substrate for dephosphorylation. Of the indicated sera, only fetal calf serum contained this enzymatic activity. Moreover, the absence of phos-
363 phoprotein phosphatase activity in b o t h newborn and adult calf serum indicated a probable placental origin of this enzyme. Consequently, for purposes of experiments outlined in this paper, all the cell lines were propagated strictly in growth m e d i u m containing adult calf serum.
Identification of phosphoprotein phosphatase activity as Ectoenzyme. Monolayers of intact normal or transformed cells were washed four times with 2 ml of isotonic saline containing 50 pM CaC12 and incubated with 100 pg of [32P]casein or [32P]histone (5--12 • 104 cpm) in 0.5 ml medium A (pH 7.2) containing 50 pmol Tris/acetic acid, 25 pmol NaC1, 25 nmol CaC12 and 1 pmol NaPi. Incubation was performed at 37°C for 10 min (unless stated otherwise) at the end of which the supernatant was p r o m p t l y removed with the aid of a Pasteur pipette and precipitated with ice-cold 20% trichloroacetic acid (final concentration). Bovine serum albumin (0.5%) was added as a carrier and protein was removed by centrifugation. [32p]Orthophosphate was extracted and measured as described b y Maeno and Greengard [25]. To correct for nonenzymatic hydrolysis of [32P]casein or [32P]histone, dishes without cells were always run in parallel. The amount of substrate labeled with 32p and the a m o u n t of 32p released were calculated from the specific activity of the radioactive ATP used in the preparation of the 32P-labeled substrate. The activity of the surface-bound phosphoprotein phosphatase is expressed as pmol of 32p released/mg of cell protein or per cell culture dish under the described standard assay conditions. In order to reduce influx into the cells of free [32P]orthophosphate released during the assay, 2 mM cold Pi was routinely included in the incubation medium. While higher concentrations of Pi inhibited, this level of unlabeled orthophosphate was determined to have no inhibitory effect on either histone or casein phosphatase activities. Such inclusion of the unlabeled phosphate, however, did not increase appreciably the radioactivity of the deproteinized supernatant suggesting that no significant loss into the cells of free [32P]orthophosphate occurred during the period of incubation. Indeed, the radioactivity introduced into the incubation mixture was recovered, after 10 min, as follows: enzymatically released [32P]orthophosphate (9--11%); residual substrate radioactivity (8 5--90%); acid-soluble radioactivity of the cell layer (1--2%); and acid-insoluble radioactivity of the cell layer (
Biochimica et Biophysica Acta, 585 ( 1 9 7 9 ) 3 6 0 - - 3 7 3 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28921
P H O S P H O P R O T E I N PHOSPHATASE ACTIVITY AT THE O U T E R S U R F A C E OF INTACT N O R M A L AND T R A N S F O R M E D 3T3 F I B R O B L A S T S
N I Z A R R. M A K A N
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Wing Hall, Ithaca, N Y 14853 (U.S.A.) (Received October 30th, 1978)
Key words: Phosphoprotein phosphatase; Cell surface; Ectoenzyme; (3T3 cell)
Summary Using 32P-labeled phosphocasein or phosphohistones as exogenous substrates it was possible to detect a phosphoprotein phosphatase activity on the outer surface of intact normal and transformed 3T3 fibroblasts. Incubation of monolayers of intact cells in buffered salt solution with the radioactively labeled substrate resulted in the release of alkali-labile 32p counts into the surrounding medium. The reaction was: (a) linear with time (at least up to 20 min); (b) proportional to the cell density; (c) dependent on the temperature and pH of the incubation medium; (d) stimulated by K*; and (e) inhibited by sodium fluoride, inorganic pyrophosphate, zinc chloride and relatively impermeant sulfhydryl reagents. Less than 2% of the externally located phosphoprotein phosphatase activity was detectable in pooled cell-free washings of the intact cell monolayer. Phosphocasein did not cause any detectable leakage of intracellular lactate dehydrogenase or soluble phosphoprotein phosphatase activity into the external medium; incubation of the cells with phosphohistones, on the other hand, resulted in appreciable leakage of both these cytoplasmic activities. Neoplastic transformation was associated with a nearly two-fold decrease in the activity of the surface phosphoprotein phosphatase. Addition of serum to either non-transformed 3T3 or spontaneously transformed 3T6 cells resulted in a rapid and remarkable drop in the cell surface dephosphorylating activity. Acrylamide gel electrophoresis of the dephosphorylated casein or histone substrate revealed no proteolytic degradation or change in electrophoretic mobility. The intact cells showed no damage u p o n microscopic examination as a result of exposure to phosphocasein or phosphohistones. Abbreviations: 3T6, a cell line derived from Swiss mouse 3T3 cells by spontaneous transformation; SV3T3, 3T3 cells transformed by Simian vacuolating virus 40, wild type.
361
Introduction The existence of membrane-bound protein kinases in normal and neoplastic cells has already been amply demonstrated [1--6]. Using extrinsic acceptor proteins, it has been shown that at least part of this plasma membrane activity is located at the external surface [7--10]. Mastro and Rozengurt [8] have reported evidence for increased protein kinase activity at the outer surface of 3T3 fibroblasts following oncogenic transformation. Glia cells [9] and dermal fibroblasts [10], on the other hand, have been reported to have a higher protein kinase activity on their outer surfaces than their respective malignant derivatives, the glioma and fibrosarcoma cells. In any case, the significance of this enzymatic activity on the outer cell surface remains unclear. I have recently [11] reported data which are consistent with the existence on the transformed cell surface of an ATP-requiring protein kinase and a fluorideinhibitable phosphoprotein phosphatase, which could control the changes in passive membrane permeability reported earlier in transformed cells treated with exogenous ATP [12,13]. Reports are appearing with increasing frequency concerning the existence of phosphorylation-dephosphorylation mechanisms and their role in controlling various cellular functions [14--19]. The demonstration of protein kinase activity on the external surfaces of various normal and transformed intact cells suggested the possibility that a dephosphorylating activity might similarly exist on the outer cell surface. This paper describes the existence and properties of such a dephosphorylating activity at the outer surface of intact normal and transformed 3T3 fibroblasts. It is shown that this activity decreases in response to activation of cell growth by serum. Materials and Methods Chemicals. Calf t h y m u s histone, t y p e IIA (histone mixture) and casein were purchased from Sigma Chemical Company. The catalytic subunit of cyclic AMP-dependent protein kinase from bovine heart [20] was a gift of Dr. E d m o n d H. Fischer (University of Washington, Seattle). [~/32P]ATP was prepared either b y a modification of the m e t h o d of Avron [21] or according to the m e t h o d of Glynn and Chappell [22] and was generously provided by the laboratories of Drs. E. Racker and R. Wu. Chemicals used for polyacrylamide gel electrophoresis were purchased from BioRad. Serum, from various animal sources, was purchased from Grand Island Biological Company (GIBCO). All other chemicals were of analytical grade. Cell lines. Stock cultures of Swiss mouse 3T3 [23] were grown in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% calf serum (GIBCO), 1 0 0 u n i t s / m l penicillin, 1 0 0 p g / m l streptomycin in a humidified atmosphere of 10% CO2 and air at 37°C. Stock cultures of 3T6 [23] and SV3T3 cells were propagated similarly. The 3T3 cells were maintained subconfluent to avoid the selection of spontaneous transformants. Cells were subcultured into 30-mm Falcon plastic dishes with medium containing 10% calf serum and used 2--3 days after plating when the cultures approached confluency. For purposes of studying the effect of serum on cell surface dephos-
362 phorylating activity 3T3 and 3T6 cells were plated in medium containing 5% and 0.5% serum, respectively; 3--4 days after plating, the medium was removed and replaced with serum-free Dulbecco's modified Eagle's medium and the cultures incubated in this medium for 48 h. Routine testing of the cell cultures on mycoplasma agar [24] revealed no detectable mycoplasma contamination. Preparation of phosphorylated substrates. [32p]Histone was prepared by incubation of histone IIA with [~/-32p]ATP in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine heart [20]. I ml of incubation mixture contained 20 mg of histone, 16.6 pmol of potassium phosphate buffer (pH 7.0), 6 pmol of magnesium acetate, 1 pmol of dithiothreitol, 10 pmol of sodium fluoride, and 0.3 pmol of [~,-32P]ATP (17--34 • 106 cpm). The reaction was initiated by the addition of 38 pg of the catalytic subunit of protein kinase which was suspended in a buffer (pH 6.7) containing 30 mM KH2PO4, 0.1 mM EDTA, 0.25 M KC1, 1 mM dithiothreitol and 1 mg/ml bovine serum albumin. The incubation was carried out at 30°C for 4 5 - 6 0 min and the reaction terminated b y the addition of 20% trichloroacetic acid (final concentration). The resultant precipitate was centrifuged and treated as described b y Maeno and Greengard [25]; in addition [32p]histone was converted to its hydrochloride by precipitation with a mixture of acetone/1 N HC1 (98 : 2, v/v) [26]. [32p]Casein was prepared essentially as described above for histone except that the incubation mixture contained 20 pmol of Tris-HC1 buffer (pH 7.0) and 6 pmol of MgC12. Characterization of the phosphorylated substrates. To verify that the [32p]_ phosphate in [32P]casein and [32P]histone was present in phosphoserine and/or phosphothreonine linkage with the protein, the phosphorylated substrates were subjected to acid hydrolysis for 6 h at l l 0 ° C in ampoules sealed under vacuum. 6 N HC1 was used to hydrolyze phosphohistone and concentrated HC1 was used for phosphocasein. After drying in vacuo, the samples were dissolved in a small volume of water and samples of the hydrolysate applied onto a thin-layer chromatographic plate (Eastman Kodak). [32P]Orthophosphate, genuine phosphoserine and phosphothreonine were used as markers. The samples were subjected to high voltage electrophoresis in 2.5% formic acid/7.8% acetic acid (pH 1.9) at 1000 V for 45 min. Whatmann No. 3 MM paper was used as a wick and attached to both ends of the chromatographic plate and allowed to dip in the buffer. The phosphorylated amino acids were detected with 0.2% ninhydrin/ pyridine stain and radioactivity located by autoradiography using Kodak X-omat R film (XR-5). Screening of various sera for ability to hydrolyze [32P]histone. In order to be sure that the phosphoprotein phosphatase activity assayed on the outer cell surface was not derived from the surrounding serum, sera from numerous sources (fetal calf, newborn calf, adult calf, chick, goat, horse, lamb and rabbit) were screened for intrinsic phosphoprotein phosphatase activity using [32p]_ histone as a substrate. In each case, the serum was diluted to a final concentration of 1% in order to reduce competition of serum phosphoprotein with [3~p]_ histone as substrate for dephosphorylation. Of the indicated sera, only fetal calf serum contained this enzymatic activity. Moreover, the absence of phos-
363 phoprotein phosphatase activity in b o t h newborn and adult calf serum indicated a probable placental origin of this enzyme. Consequently, for purposes of experiments outlined in this paper, all the cell lines were propagated strictly in growth m e d i u m containing adult calf serum.
Identification of phosphoprotein phosphatase activity as Ectoenzyme. Monolayers of intact normal or transformed cells were washed four times with 2 ml of isotonic saline containing 50 pM CaC12 and incubated with 100 pg of [32P]casein or [32P]histone (5--12 • 104 cpm) in 0.5 ml medium A (pH 7.2) containing 50 pmol Tris/acetic acid, 25 pmol NaC1, 25 nmol CaC12 and 1 pmol NaPi. Incubation was performed at 37°C for 10 min (unless stated otherwise) at the end of which the supernatant was p r o m p t l y removed with the aid of a Pasteur pipette and precipitated with ice-cold 20% trichloroacetic acid (final concentration). Bovine serum albumin (0.5%) was added as a carrier and protein was removed by centrifugation. [32p]Orthophosphate was extracted and measured as described b y Maeno and Greengard [25]. To correct for nonenzymatic hydrolysis of [32P]casein or [32P]histone, dishes without cells were always run in parallel. The amount of substrate labeled with 32p and the a m o u n t of 32p released were calculated from the specific activity of the radioactive ATP used in the preparation of the 32P-labeled substrate. The activity of the surface-bound phosphoprotein phosphatase is expressed as pmol of 32p released/mg of cell protein or per cell culture dish under the described standard assay conditions. In order to reduce influx into the cells of free [32P]orthophosphate released during the assay, 2 mM cold Pi was routinely included in the incubation medium. While higher concentrations of Pi inhibited, this level of unlabeled orthophosphate was determined to have no inhibitory effect on either histone or casein phosphatase activities. Such inclusion of the unlabeled phosphate, however, did not increase appreciably the radioactivity of the deproteinized supernatant suggesting that no significant loss into the cells of free [32P]orthophosphate occurred during the period of incubation. Indeed, the radioactivity introduced into the incubation mixture was recovered, after 10 min, as follows: enzymatically released [32P]orthophosphate (9--11%); residual substrate radioactivity (8 5--90%); acid-soluble radioactivity of the cell layer (1--2%); and acid-insoluble radioactivity of the cell layer (
