Int. J. Cancer: 52,399-403 (1992) 0 1992 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication de I'Union Internationale Contre le Cancer
INDUCTION OF PROTEIN KINASE C DOWN-REGULATION BY THE PHORBOL ESTER TPA IN A CALPAIN/PROTEIN KINASE C COMPLEX Michel SAVART',Pascale LETARD,Sandrine BULTELand AndrC DUCASTAING ISTAB, Universiti!Bordeaux I, Avenue des Facultis, 33405 Talence Cedex, France. Using a calpain/protein kinase C (PKC) complex, we were able to reproduce, in vitro, the induction of PKC downregulation by the phorbol ester 12-0-tetradecanoyl-phorbol-13acetate (TPA) which had been previously observed in cells. We show that TPA initiates this phenomenon by promoting a calpain-dependentconversion of PKC to the Ca2+phospholipidindependent protein kinase M (PKM), at physiological calcium concentrations. This effect of TPA was dependent upon the presence of phosphatidylserine and was observed only when PKC was the substrate for the protease, inactivation of calpain by autolysis not being modified by the presence of TPA. Moreover, PKM generated from the calpain-PKC complex was resistant to calpain, even after addition of TPA. These results suggest that TPA induces a conformational change in PKC, increasingthe affinity of the kinase for calpain and consequently permitting its proteolysis for the basal level of calcium in cells.
o 1992 Wiley-Lcss, Inc. Prolonged incubation of cells with the phorbol ester TPA, one of the most potent tumor promoters known, induces a down-regulation of PKC, which results in a decrease in PKC activity (Jaken et al., 1981; Solanki et al., 1981). It has been established that this TPA-dependent decrease in PKC activity is due to an increased rate of proteolysis, with no change in the amount of mRNA or in the rates of polypeptide synthesis (Mizuguchi et a/., 1988; Young et al., 1987). It has also been observed that intramolecular PKC autophosphorylation is not a prerequisite for its down-regulation (Pears and Parker, 1991). It first appeared that this PKC proteolysis was mediated by non-lysosomal proteases (Chida et al., 1986). Then, Pontremoli eta/. (1988) reported that PKC proteolysis induced by TPA in neutrophils was significantly decreased following uptake of a monoclonal anti-calpain antibody by these cells. In addition, calpain inhibitors such as calpeptin or calpastatin (the endogenous inhibitor of calpain) block TPA-dependent downregulation of PKC (Adachi et al., 1990).Zn vitro, calpains cleave PKCs in the V3 region and this proteolysis generates a 36-kDa regulatory fragment and a Ca2+ phospholipid-independent protein kinase of 45-49 kDa, protein kinase M (PKM) (Kishimot0 et al., 1989). Until now, data concerning PKC downregulation have been obtained from cells treated with TPA, but the mechanism by which this tumor promoter induces PKC proteolysis has not yet been elucidated. Taking into consideration our previous demonstration of a close association between calpains and PKCs (Savart et al., 1987, 1991) and the fact that the regulatory domain of PKC binds TPA with high affinity (Burns and Bell, 1991), we have attempted to reproduce in vitro the induction of PKC down-regulation by TPA. For this purpose, we have isolated a PKC-calpain complex devoid of calpastatin and have studied the effect of TPA on the Ca'+-dependent proteolysis of PKC within this complex at physiological calcium concentrations. MATERIAL AND METHODS
Material Casein was obtained from Merck, Darmstadt, Germany (art. 2244). y31PP-ATP(3000 Ci/mmol) was purchased from NEN (Boston, MA). Phenyl-Sepharose CL-4B was supplied by Pharmacia-LKL3 (Uppsala, Sweden). L-a-Phosphatidyl-Lserine, histone type 111s from calf thymus, phenylmethylsulphonyl fluoride (PMSF), leupeptin, TPA and fura-2 (pen-
tapotassium salt) were obtained from Sigma (St Louis, MO). Ecoscint 0 was from National Diagnostics, Manville, NJ. Subcellularfiactionation
All operations were conducted at 4°C. Muscles from adult, male New Zealand rabbits, killed by cervical dislocation, were used as the starting material. These tissues were rapidly dissected and homogenized in a Waring blendor, for 30 sec at low speed followed by 30 sec at high speed, with 5 vol of extraction buffer (30 mM Tris/HCI, pH 7.4, containing 250 mM sucrose, 0.5 mM PMSF, 10 mM EGTA, 2 mM EDTA and 1 mM NaN3). The homogenate was centrifuged at 12,OOOgfor 15 min. The supernatant was filtered through cheese-cloth to remove free-floating fat and centrifuged at 100,000 g for 60 min. The resulting supernatant is defined as the cytosol.
Preparation of the calpain-PKC complex The cytosol, whose pH was adjusted to 7.5 with NaOH, was centrifuged at 200,000 g for 16 hr. The 200,000-g supernatant obtained was brought to a final concentration of 1 M NaCl by addition of solid salt. The saline solution was then readjusted to pH 7.5 and filtered on Whatman 1 paper. This sample was loaded on a phenyl-Sepharose CL-4B column (2.6 x 10 cm) previously equilibrated with 20 mM Tris/HC1 buffer (pH 7.5) containing 1 mM EDTA, 1 mM EGTA, 1 mM NaN3 and 1 M NaCl (buffer A). The column was washed at a flow rate of 60 ml/h with buffer A and the bound proteins were eluted at the same rate with 20 mM Tris/HCI pH 7.5, containing 1 mM EDTA, 1 mM NaN3 and 1% ethylene glycol (v/v). Fractions of 6 ml were collected and used for subsequent enzyme assays. Enzyme assays Calpain activities were assayed by using casein as substrate and measuring the absorbance of a 10% trichloroacetic acid supernatant at 280 nm as described previously (Savart et al., 1987). One unit of calpain activity was defined as the amount of enzyme which catalyzed an increase of 0.001 absorbance unit at 280 nm after 1 rnin incubation. PKC activity was measured in a reaction mixture (100 ~ 1 ) containing 30 mM Tris-HCI (pH 7.9, 5 mM magnesium acetate, 200 kg/ml histone type IIIS, 10 kM ATP (0.8 x 106 cpm), 0.2 mM EGTA, 0.2 mM EGTA (from enzyme fraction), 1mM CaCI2,20 Fg/ml phosphatidylserine and 20 k1 of enzyme preparation. After incubation for 3 rnin at 30"C, aliquots of 40 ~1 were spotted on 3 x 3 cm squares of phosphocellulose paper (Whatman P 81). The sheet of paper was rinsed twice for 7 rnin with 10% (w/v) trichloroacetic acid containing 15 mM Na4P207,then washed for 40 min under running tap water. Finally, the sheet was dried with a stream of hot air and each paper square was cut out of the matrix and counted for radioactivity in 4 ml of a biodegradable scintillation solution (Ecoscint 0, Manville, NJ). The Ca2+ and phospholipidindependent phosphorylation (PKM) determined in the presence of 1.2 mM EGTA instead of Ca2+and phospholipids, was subtracted from the total amount of 32Pincorporated in order to determine the total PKC activity. 'To whom correspondence and reprint requests should be sent.
Received: February 27,1992 and in revised form June 1,1992.
400
SAVART E T A L .
TPA was prepared as a 0.32-mM stock solution in 100% ethanol and stored at 4°C. Appropriate dilutions were used so that the final ethanol concentration was
Publication of the International Union Against Cancer Publication de I'Union Internationale Contre le Cancer
INDUCTION OF PROTEIN KINASE C DOWN-REGULATION BY THE PHORBOL ESTER TPA IN A CALPAIN/PROTEIN KINASE C COMPLEX Michel SAVART',Pascale LETARD,Sandrine BULTELand AndrC DUCASTAING ISTAB, Universiti!Bordeaux I, Avenue des Facultis, 33405 Talence Cedex, France. Using a calpain/protein kinase C (PKC) complex, we were able to reproduce, in vitro, the induction of PKC downregulation by the phorbol ester 12-0-tetradecanoyl-phorbol-13acetate (TPA) which had been previously observed in cells. We show that TPA initiates this phenomenon by promoting a calpain-dependentconversion of PKC to the Ca2+phospholipidindependent protein kinase M (PKM), at physiological calcium concentrations. This effect of TPA was dependent upon the presence of phosphatidylserine and was observed only when PKC was the substrate for the protease, inactivation of calpain by autolysis not being modified by the presence of TPA. Moreover, PKM generated from the calpain-PKC complex was resistant to calpain, even after addition of TPA. These results suggest that TPA induces a conformational change in PKC, increasingthe affinity of the kinase for calpain and consequently permitting its proteolysis for the basal level of calcium in cells.
o 1992 Wiley-Lcss, Inc. Prolonged incubation of cells with the phorbol ester TPA, one of the most potent tumor promoters known, induces a down-regulation of PKC, which results in a decrease in PKC activity (Jaken et al., 1981; Solanki et al., 1981). It has been established that this TPA-dependent decrease in PKC activity is due to an increased rate of proteolysis, with no change in the amount of mRNA or in the rates of polypeptide synthesis (Mizuguchi et a/., 1988; Young et al., 1987). It has also been observed that intramolecular PKC autophosphorylation is not a prerequisite for its down-regulation (Pears and Parker, 1991). It first appeared that this PKC proteolysis was mediated by non-lysosomal proteases (Chida et al., 1986). Then, Pontremoli eta/. (1988) reported that PKC proteolysis induced by TPA in neutrophils was significantly decreased following uptake of a monoclonal anti-calpain antibody by these cells. In addition, calpain inhibitors such as calpeptin or calpastatin (the endogenous inhibitor of calpain) block TPA-dependent downregulation of PKC (Adachi et al., 1990).Zn vitro, calpains cleave PKCs in the V3 region and this proteolysis generates a 36-kDa regulatory fragment and a Ca2+ phospholipid-independent protein kinase of 45-49 kDa, protein kinase M (PKM) (Kishimot0 et al., 1989). Until now, data concerning PKC downregulation have been obtained from cells treated with TPA, but the mechanism by which this tumor promoter induces PKC proteolysis has not yet been elucidated. Taking into consideration our previous demonstration of a close association between calpains and PKCs (Savart et al., 1987, 1991) and the fact that the regulatory domain of PKC binds TPA with high affinity (Burns and Bell, 1991), we have attempted to reproduce in vitro the induction of PKC down-regulation by TPA. For this purpose, we have isolated a PKC-calpain complex devoid of calpastatin and have studied the effect of TPA on the Ca'+-dependent proteolysis of PKC within this complex at physiological calcium concentrations. MATERIAL AND METHODS
Material Casein was obtained from Merck, Darmstadt, Germany (art. 2244). y31PP-ATP(3000 Ci/mmol) was purchased from NEN (Boston, MA). Phenyl-Sepharose CL-4B was supplied by Pharmacia-LKL3 (Uppsala, Sweden). L-a-Phosphatidyl-Lserine, histone type 111s from calf thymus, phenylmethylsulphonyl fluoride (PMSF), leupeptin, TPA and fura-2 (pen-
tapotassium salt) were obtained from Sigma (St Louis, MO). Ecoscint 0 was from National Diagnostics, Manville, NJ. Subcellularfiactionation
All operations were conducted at 4°C. Muscles from adult, male New Zealand rabbits, killed by cervical dislocation, were used as the starting material. These tissues were rapidly dissected and homogenized in a Waring blendor, for 30 sec at low speed followed by 30 sec at high speed, with 5 vol of extraction buffer (30 mM Tris/HCI, pH 7.4, containing 250 mM sucrose, 0.5 mM PMSF, 10 mM EGTA, 2 mM EDTA and 1 mM NaN3). The homogenate was centrifuged at 12,OOOgfor 15 min. The supernatant was filtered through cheese-cloth to remove free-floating fat and centrifuged at 100,000 g for 60 min. The resulting supernatant is defined as the cytosol.
Preparation of the calpain-PKC complex The cytosol, whose pH was adjusted to 7.5 with NaOH, was centrifuged at 200,000 g for 16 hr. The 200,000-g supernatant obtained was brought to a final concentration of 1 M NaCl by addition of solid salt. The saline solution was then readjusted to pH 7.5 and filtered on Whatman 1 paper. This sample was loaded on a phenyl-Sepharose CL-4B column (2.6 x 10 cm) previously equilibrated with 20 mM Tris/HC1 buffer (pH 7.5) containing 1 mM EDTA, 1 mM EGTA, 1 mM NaN3 and 1 M NaCl (buffer A). The column was washed at a flow rate of 60 ml/h with buffer A and the bound proteins were eluted at the same rate with 20 mM Tris/HCI pH 7.5, containing 1 mM EDTA, 1 mM NaN3 and 1% ethylene glycol (v/v). Fractions of 6 ml were collected and used for subsequent enzyme assays. Enzyme assays Calpain activities were assayed by using casein as substrate and measuring the absorbance of a 10% trichloroacetic acid supernatant at 280 nm as described previously (Savart et al., 1987). One unit of calpain activity was defined as the amount of enzyme which catalyzed an increase of 0.001 absorbance unit at 280 nm after 1 rnin incubation. PKC activity was measured in a reaction mixture (100 ~ 1 ) containing 30 mM Tris-HCI (pH 7.9, 5 mM magnesium acetate, 200 kg/ml histone type IIIS, 10 kM ATP (0.8 x 106 cpm), 0.2 mM EGTA, 0.2 mM EGTA (from enzyme fraction), 1mM CaCI2,20 Fg/ml phosphatidylserine and 20 k1 of enzyme preparation. After incubation for 3 rnin at 30"C, aliquots of 40 ~1 were spotted on 3 x 3 cm squares of phosphocellulose paper (Whatman P 81). The sheet of paper was rinsed twice for 7 rnin with 10% (w/v) trichloroacetic acid containing 15 mM Na4P207,then washed for 40 min under running tap water. Finally, the sheet was dried with a stream of hot air and each paper square was cut out of the matrix and counted for radioactivity in 4 ml of a biodegradable scintillation solution (Ecoscint 0, Manville, NJ). The Ca2+ and phospholipidindependent phosphorylation (PKM) determined in the presence of 1.2 mM EGTA instead of Ca2+and phospholipids, was subtracted from the total amount of 32Pincorporated in order to determine the total PKC activity. 'To whom correspondence and reprint requests should be sent.
Received: February 27,1992 and in revised form June 1,1992.
400
SAVART E T A L .
TPA was prepared as a 0.32-mM stock solution in 100% ethanol and stored at 4°C. Appropriate dilutions were used so that the final ethanol concentration was
