Im. J. Bioehem. Printed in Great
Vol.24, Britain.
No. 7, pp. 1057-1064, All rights reserved
1992
Copyright
0020-71 IX/92 $5.00 + 0.00 Q 1992 PergamonPressLtd
CHARACTERIZATION OF 130 kDa PROTEIN FROM CEREBELLUM SYNAPTOSOMAL MEMBRANES PHOSPHORYLATED BY PKC
RAT
LUDMLYA ~YLI&SKAand LILLALACHOWICZ II Department of Biochemistry, Institute of Physiology and Bi~hemistry, School of Medicine, Lindleya 6, 90- 131 Wdi, Poland (Received 1 November 1991)
Abstract-l. The effect of endogenous PMA-stimulated phosphorylation of the protein in the molecular weight range of 130 kDa in rat cerebellum synaptosomal membranes was examined. 2. The SO% inhib~tjon of the phosphorylation of 130 kDa protein by 5 !l’M polymyxin B was observed after 6 min of preincubation. 3. The sensitivity of 130 kDa protein for phosphorylation in the presence of exogenous protein kinase C suggests, that this protein could serve as a physiological substrate of protein kinase C. 4. Partial characterization of 130 kDa protein was performed. Upon incubation with [>j-jZP]ATP the 130 kDa protein formed Ca’+-dependent, hydroxylamine-sensitive phosphointermediate, which was inhibited by 50 PM vanadate, but not 0.5 mM vanadyl. 5. One-dimensional peptide mapping by proteolysis of 130 kDa protein with V8 protease was obtained.
INTRODUCTION
MATERIALSANDMETHODS
The phosphorylation of proteins by protein kinases C (PKC) play an important role in the regulation of neuronal functions (Routtenberg, 1986; Linden and Routtenberg, 1989). Most of the brain PKC is associated with synaptic membranes, whereas in most other tissues the enzyme is present mainly in the soluble fraction as an inactive form (De Graan et nl., 1990) The PKC system therefore appears to be an important regulator of [Ca*+],, and has been shown to be involved in stimulation of the plasma membrane Ca2+-pumps (Rasmussen, 1990). Stimulation of the Ca*+-pump in’human erythrocyte (and also of the purified pump) by protein kinase C was recently reported by Smallwood et al. (1988). The data obtained by Fukuda et aE. (1990) suggest that PKC activation stimulates the activity of the sarcolemmal Ca2+-pump from bovine aortic smooth muscle. They concluded that the PKC system provides an important negative feedback mechanism for the control of [Ca’+], by accelerating restoration of elevated [Ca*+ Ii to the resting level, but the precise mechanism of its action remained unknown. The developmental expression of PKC isozymes in rat cerebellum is under separate control. Their localization suggest their unique function and a regulatory role of cerebellum PKC in the neuronal signal transduction (Huang et al., 1990). In our study we tried to determine the characteristics of PKC-phosphorylated 130 kDa protein in synaptosomal membranes of rat cerebellum.
Polymyxin B, PMA (phorbol 12-myristate 13-acetate)and S. aureu~V8 protease were purchased from Sigma. PKC and Molecular Marker Kit was obtained from Calbiochem. DTT (dithiothreitol) was from Merck. All other chemicals were of analytical grade. Adult Wistar rats were decapitated without anaesthesia. The cerebellar lobes were isolated according to Glowinski and fversen method (1966). Synaptosomal membranes were prepared by the procedure of Booth and Clark (1978). Protein was determined by the method of Ohnishi and Barr (1978). Phosphorylation of synaptosomal plasma membranes
The standard phosphoryiation assay medium (in final volume 20~1) contained 20mM Tri-HCI, pH = 7.4, 10 mM MgCI,, 1mM DTT, 0.5 mM CaCI,, f pM PMA, 1 mM vanadyl sulphate-phosphatase inhibitor (Ziai and Ferrone, 1989). In brief, 20 pg of membrane proteins were preincubated for 0, 3 and 6min at 30°C with concentrations of 1.3, 3.3 and 5 pM polymyxin B as indicated. Control samples were run with the same p~incubation step, in the absence of polymyxin B. In assay performed with exogenous PKC, 1 ~1 enzyme was added to 20 pg membrane proteins (control) or to 20 pg denaturated membrane proteins in standard reaction mixture, and preincubated for 0 and 3 mm. The reaction was started by the addition of 9 p M [y32P]ATP (5 pCi) and was stopped after 2 min by addition of 20 mM Tris-HCl, pH = 6.8, containing 6% (w/v) SDS, 20% (v/v) glycerol, 2.5% /?-mercaptoethanol, 0.002% bromophenol blue and heating at 100°C for 3 min. One-dimensional SDS-PAGE was performed according to the procedure of Laemmli (1970) on 7.5% polyacrylamide gel. After electrophoresis gels were silver-stained
to57
1058
LUDM~A ZYLIY&KA and LILLA LACHOWICZ
(Wray et al., 1981) and autoradiographed with an intensify-
ing screen. The proteins was measured phosphorylation samples, taken were estimated kit.
-
130 kDa ----__
were excised from gel and 12P incorporation by counting for Cerenkov radiation. The was expressed with regard to control as 100%. Molecular weight of the proteins by the comparison with molecular marker
T
Determination of the phosphorylated intermediate
Phosphorylation of the synaptosomal membranes (20 pg) was conducted in 30 ~1 of 50 mM imidazole buffer pH = 6.0 containing 2.8 PM [y-‘*P]ATP (8 PCi) and 0.2 mM CaCI,, 2 mM MgCl,, 0.5 mM vanadyl sulphate or 50 p M vanadate as indicated. The incubation was performed at 0°C for 15 set and stopped by adding ice-cold 10% TCA. After centrifugation for 5 min the pellet was washed twice with 10% TCA and once with water, and resuspended for gel electrophoresis in buffer containing 10 mM imidazole pH = 6.0, 1% SDS, 10% glycerol, 2.5% /I-mercaptoethanol and 0.02% bromophenol blue. SDS-PAGE of the ‘*Plabelled proteins on 7.5% gels at pH = 6.0 was done. The effect of hyroxylamine was studied as described by Wuytack ef al. (1982). The pellets of precipitated phosphorylated proteins were incubated for 10 min at room temperature with 200 ~1 of solution containing 150 mM hydroxylamine and 150 mM sodium acetate buffer brought to pH = 6.0 with Tris. Controls were treated likewise with a solution of 150 mM Tris-HCI, 150 mM sodium acetate buffer at pH = 6.0. After incubation the samples were treated as described above. ‘*P-Labelled proteins were detected by autoradiography with X-ray film. Peptide mapping by limired proteolysis with S. aureus V8 prolease
The synaptosomal membrane samples (100 pg) were fractionated by SDS-PAGE (7.5%) and stained with Coomassie Brilliant Blue. The band of 130 kDa protein was excised. Gel slices were overlaid with 2 pugof V8/well in 3% stacking gel and digested for 30 min at room temperature. The generated peptides were fractionated by 12.5% SDS-PAGE according to the procedure of Cleveland et al. (1977). The peptides were located by silver staining. ,,o._-_---‘“?_____
PB
l.ljbM
3.3pM
S.O/1M
Fig. 2. Effect of varying polymyxin B concentration on the 130 kDa protein phosphorylation. Membranes were treated as described under Materials and Methods. Data are given as means & SEM (N = 4) of radioactivity incorporated as a percentage of the control (100%). Statistically significant: P < 0.001, +P < 0.01.
0’
1 3’
Fig. 3. Effect of exogenous PKC on the phosphorylation of 130 kDa protein. Membranes were treated as described under Materials and Methods. The data are expressed as percentage relative to control (100%). The results are means + SEM (N = 4). Statistically significant: P < 0.001. RESULTS AND DISCUSSION
In order to demonstrate that synaptosomal membrane protein of rat cerebellum in our experimental conditions is the substrate for endogenous PKC, the phosphorylation was stimulated with 0.5 mM Ca*+ and 1 PM PMA. PMA is known to be a good probe for PKC function (Furukawa et al., 1989). We observed the phosphorylation of several proteins, particularly of protein, whose molecular weight was estimated to be 130 f 3 kDa (Fig. 1). We also examined the effect of polymyxin B-a cyclic, polycationic peptide antibiotic-the potent PKC inhibitor, on phosphorylation of 130 kDa protein (Tamaoki and Nakano, 1990). It is known that polymyxin B inhibits also CaMdependent kinase, but not cyclic nucleotide-dependent kinase (Linden and Routtenberg, 1989). In our experiments we observed the dose-dependent decrease of phosphorylation up to 50% for 5 PM polymyxin B (Fig. 2). It was interesting to note, that after 6 min of preincubation with 1.3 and 3.3 PM the inhibition was less effective. It may be due to peptidase activity. To confirm, that 130 kDa protein was in vitro the substrate for protein kinase C, experiments were also carried out using exogenous PKC. The data in Fig. 3 show that after 3 min of preincubation the phosphorylation was about 80% of control value. This suggests that 130 kDa protein in our experimental conditions may be the substrate not only for protein kinase C, but also for other kinases. In the next series of experiments we tried to determine the characteristics of the 130 kDa protein. The Ca2+-dependent, vanadate and hydroxylamine-sensitive phosphorylation with [r -32P]ATP of proteins can be considered to be a marker for P-type
Rat cerebellum
synaptosomal
membrane
130 kDa protein
67
43
Fig. 1. Phosphorylation of 130 kDa protein by endogenous PKC. Synaptosomal membranes from rat cerebellum (20 pg) were preincubated for 3 min in a standard reaction mixture and incubated as described under Materials and Methods. (A) SDS-PAGE analysis and silver staining of gel (7.5%). (B) Autoradiogram. Molecular weight standards (x 10-j) are indicated. Position of the protein 130 kDa indicated.
1059
1060
LUDMIEA~YLIP;ISKA and LILLA LACHOWICZ
130
Fig. 4. Formation of phosphorylated intermediate. The synaptosomal membranes (2Opg) were phosphorylated at 0°C for 15 set in a medium containing 50mM imidazole buffer pH = 6.0 and 2.8 PM [y-‘*P]ATP (8 PCi) in the presence: lane I-no addition; lane 2-CaC1,; iane 3-MgCI,; lane 4-CaCl, + MgCl,; lane S-CaCl, + MgCl, + vanadyl; lane 6--CaCI, + MgCI, + vanadate. After electrophoresis at pH = 6.0 gels were autoradiographed.
Rat cerebellum synaptosomal membrane 130 kDa protein
MW x 10-3
t-
94
-
Fig. 5. Effect of hydroxylamine. The samples were treated as described under Materials and Methods. After el~tropboresis at pH = 6.0 gels were autorad~ograph~. Lane I-phosphorylation in Ca:+ and with 150 mM Tris-HCI, 1.50mM sodium acetate buffer Mg*+, containing medium; lane 2-incubation pH = 6.0 (control); lane 3-incubation with 150mM hydroxylamine, 150mM sodium acetate buffer pH = 6.0.
1061
1062
LUDMIZA~~YLI~~SKA and LILLALACHOWICZ
MW x1o-3
67 -
57
-
47
-
41
43 !
va
-
23
-
15
20
Fig. 6. Peptide mapping analysis by S. aureus V8 proteolysis of 130 kDa protein. The position of V8 and of molecular weight markers are indicated. The peptides were located by silver staining.
Rat cerebellum synaptosomal membrane 130 kDa protein ATPases (Pedersen results demonstrate
and Carafoli, 1987a, b). Our (Fig. 4) that the 130 kDa phos-
phorylated intermediate is formed, when the synaptosomal membranes are incubated at 0°C with [Y-~~P]ATP and 0.2 mM CaCl,. No phosphorylation was observed after incubation the synaptosomal membranes with [Y-~‘P]TP or [Y-~~P]ATP and 2 mM MgCl,. Since our experimental conditions were not optimal for phosphorylation mediated by protein kinases, we concluded that 130 kDa protein was acylphosphoprotein. This form was unstable in the presence of 150mM hydroxylamine (Fig. 5). It should be pointed out that the second major phosphoprotein band, whose molecular weight was estimated as - 110 kDa, was also visible. Recent work of the effect of the intracellular Ca2+-dependent protease calpain indicates that calpain reduces the apparent molecular weight of the erythrocyte membrane Ca2+-ATPase to - 124 kDa followed by 85 kDa (Wang et al., 1988). The calpaindigested fragments were phosphorylated in the presence of Ca*+ and they were sensitive to hydroxylamine treatment. We can assume that 110 kDa protein in our experiments is a proteolytic product of 130 kDa. As mentioned above, the Ca*+-pumps showed inhibitory effect of vanadate on ATPase activity. In the physiological pH, vanadium ions may exist in the form of pentavalent vanadate anion and tetravalent vanadyl cation (Krivanek, 1988). Vanadate inhibits by binding to the amino acid residue that forms the phosphorylated intermediate at active site, and acts as a noncompetitive inhibitor against ATP (Carafoli, 1991). When the synaptosomal membranes were incubated in the presence of 50 PM vanadate, the formation of phosphointermediate was significantly reduced. Vanadyl cations stimulated phosphorylation of 130 kDa protein suggests different mechanisms by which vanadium ions may exert their physiological effects. The absence of Mg2’ is required to prolong the lifetime of the phosphorylated conformational state of Ca*+-pump (Inesi et al., 1990; Villalobo, 1990). On the other hand, Mg*+ ions are also required for the dephosphorylation step of the ATPase reaction cycle (Carafoli, 1991). Our analysis indicated that 2 mM MgClz decreased the level of phosphorylated intermediate at pH = 6.0. We concluded that ions Mg?+ in our experimental conditions increased dephosphorylation step of ATPase reaction. We also studied the fragmentation pattern of the 130 kDa protein produced by S. aureus V8 protease
(Fig. 6). The fragments have relative molecular masses of 110, 57, 47, 41 kDa and smaller components of 23 and 15 kDa. CONCLUSION
There have been several recent studies, which suggest that modulation of the activities of membrane
1063
Ca2+-transport system is mediated via activation of protein kinase C (Fukuda et al., 1990; Furukawa et uf., 1989). These results suggest that PKC stimulation of Ca2+-pump may be a general phenomenon. Our results demonstrate that 130 kDa protein may be functionally and structurally related Ca2+-pumps from other tissues, as obtained by the demonstration of phospho-intermediate hydroxylamine-sensitive and inhibited by vanadate. These studies indicate the presence of PKC-phosphorylated Ca2+-ATPase-like protein in synaptosomal membranes of rat cerebellum. wish to thank Mrs Krystyna Marciniak for excellent technical assistance. The study was financially supported by a grant from the School of Medicine of Mdi under contract No. 502-I l-021. Acknowledgements-We
REFERENCES
Booth R. F. G. and Clark J. B. (1978) A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain. Biochem. J. 176, 365-370.
Carafoli E. (1991) Calcium pump of the plasma membrane. Physiol. Rev. 71, 129-153.
Cleveland D. W., Fischer S. G., Kirshner M. W. and Laemmli U. K. (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J. biol. Chem. 252, 1102-I 106. De Graan P. N. D., Schotman P. and Versteeg D. H. G. (1990) Neural mechanisms of action of neuropeptides: macromolecules and neurotransmitters. Neuropeptides: Basics and Perspecrives, (Edited by De Wied), pp. 139-l 74. Elsevier Science, Amsterdam. Fukuda T., Ogurusu T., Furukawa K.-I. and Shigekawa M. (1990) Protein kinase C-dependent phosphorylation of sarcolemma Ca2+-ATPase isolated from bovine aortic smooth muscle. J. Biochem. 108, 629434. Furukawa K. J., Tawada Y. and Shigekawa M. (1989) Protein kinase C activation stimulates plasma membrane Caz+ pump in cultured vascular smooth muscle cells. J. biol. Chem. 264, 48484849.
Glowinski J. and Iversen L. (1966) Regional studies of catecholamines in the rat brain. I. The disposition of [‘Hlnorepinephrine, [3Hldopamine and [3H]DOPA in various regions of the brain. J. Neurochem. 13, 655-669. Huang F. L., Scott Young W. III, Yoshida Y. and Huang K. P. (1990) Developmental expression of protein kinase C isozymes in rat cerebellum. Dev. Bruin Res. 52, 121-130.
Inesi G., Sumbilla C. and Kirtley M. E. (1990) Relationships of molecular structure and function in Ca*+-transport ATPase. Physiol. Rev. 70, 749-760. James P., Vorherr T., Krebs J., Morelli A., Caste110 G., McCormick D. J., Penniston J. T., DeFlora A. and Carafoli E. (1989) Modulation of erythrocyte Ca*+ ATPase by selective calpain cleavage of the calmodulin binding domain. J. biol. Chem. 264, 8289-8296. Krivanek J. (1988) Do vanadium ions exert any specific effect on brain protein phoshorylation. Neurochem. Res. 13, 395401.
Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature 227, 680-685.
1064
LUDMIWZYLI~SKAand LILLALACHOWICZ
Linden D. J. and Routtenberg A. (1989) The role of protein kinase C in long-term potentiation: A testable model. Brain Res. Rev. 14, 219-296.
Ohnishi S. T. and Barr J. T. (1978) A simplified method of quantitating protein using the biuret and phenol reagent. Analyf. Biochem. 86, 193-200.
Pedersen P. L. and Carafoli E. (1987a) Ion motive ATPases. Trends Biochem. Sci. 12, 146-150.
Pedersen P. L. and Carafoli E. (1987b) Ion motive ATPases. Trends Biochem. Sci. 12, 186-189.
Rasmussen H. (1990) The complexities of intracellular Ca2+ signalling. Biol. Chem. Hoppe-Seyler 371, 191-206. Routtenberg A. (1986) Synaptic plasticity and protein kinase C. Prog. Brain Res. 69, 211-234. Smallwood J. I., Gugi B. and Rasmussen H. (1988) Regulation of erythrocyte CaZ+ pump activity by protein kinase C. J. biol. Chem. 263, 2195-2202. Tamaoki T. and Nakano H. (1990) Potent and specific inhibitors of protein kinase C of microbial origin. Biotechnology 8, 132-735.
Villalobo A. (1990) Reconstitution of ion-motive transport ATPases in artificial lipid membranes. Biochim. biophys. Acfa 1017, l-48.
Wang K. K. W., Roufogalis B. D. and Villalobo A. (1988) Further characterization of calpain-mediated proteolysis of the human erythrocyte plasma membrane Ca*+ATPase. Archs Biochem. Biophys. 267, 311-321. Wray W., Boulikas T., Wray V. P. and Hancock R. (1981) Silver staining of proteins in polyacrylamide gels. Analyr. Biochem. 118, 197-203.
Wuytack F., Raeymaekers L., De Schutter G. and Casteels R. (1982) Demonstration of the phosphorylated intermediates of the Ca2+-transport ATPase in a microsomal fraction and in a (Ca’+ + Mg2+)-ATPase purified from smooth muscle by means of calmodulin affinity chromatography. Biochim. biophys. Acta 693, 45-52. Ziai M. R. and Ferrone S. (1989) Specific phosphorylation by calcium-EGTA complex of a 75 kDa human tumor plasma membrane protein (pp. 75). Int. J. Biochem. 21, 731-738.
Vol.24, Britain.
No. 7, pp. 1057-1064, All rights reserved
1992
Copyright
0020-71 IX/92 $5.00 + 0.00 Q 1992 PergamonPressLtd
CHARACTERIZATION OF 130 kDa PROTEIN FROM CEREBELLUM SYNAPTOSOMAL MEMBRANES PHOSPHORYLATED BY PKC
RAT
LUDMLYA ~YLI&SKAand LILLALACHOWICZ II Department of Biochemistry, Institute of Physiology and Bi~hemistry, School of Medicine, Lindleya 6, 90- 131 Wdi, Poland (Received 1 November 1991)
Abstract-l. The effect of endogenous PMA-stimulated phosphorylation of the protein in the molecular weight range of 130 kDa in rat cerebellum synaptosomal membranes was examined. 2. The SO% inhib~tjon of the phosphorylation of 130 kDa protein by 5 !l’M polymyxin B was observed after 6 min of preincubation. 3. The sensitivity of 130 kDa protein for phosphorylation in the presence of exogenous protein kinase C suggests, that this protein could serve as a physiological substrate of protein kinase C. 4. Partial characterization of 130 kDa protein was performed. Upon incubation with [>j-jZP]ATP the 130 kDa protein formed Ca’+-dependent, hydroxylamine-sensitive phosphointermediate, which was inhibited by 50 PM vanadate, but not 0.5 mM vanadyl. 5. One-dimensional peptide mapping by proteolysis of 130 kDa protein with V8 protease was obtained.
INTRODUCTION
MATERIALSANDMETHODS
The phosphorylation of proteins by protein kinases C (PKC) play an important role in the regulation of neuronal functions (Routtenberg, 1986; Linden and Routtenberg, 1989). Most of the brain PKC is associated with synaptic membranes, whereas in most other tissues the enzyme is present mainly in the soluble fraction as an inactive form (De Graan et nl., 1990) The PKC system therefore appears to be an important regulator of [Ca*+],, and has been shown to be involved in stimulation of the plasma membrane Ca2+-pumps (Rasmussen, 1990). Stimulation of the Ca*+-pump in’human erythrocyte (and also of the purified pump) by protein kinase C was recently reported by Smallwood et al. (1988). The data obtained by Fukuda et aE. (1990) suggest that PKC activation stimulates the activity of the sarcolemmal Ca2+-pump from bovine aortic smooth muscle. They concluded that the PKC system provides an important negative feedback mechanism for the control of [Ca’+], by accelerating restoration of elevated [Ca*+ Ii to the resting level, but the precise mechanism of its action remained unknown. The developmental expression of PKC isozymes in rat cerebellum is under separate control. Their localization suggest their unique function and a regulatory role of cerebellum PKC in the neuronal signal transduction (Huang et al., 1990). In our study we tried to determine the characteristics of PKC-phosphorylated 130 kDa protein in synaptosomal membranes of rat cerebellum.
Polymyxin B, PMA (phorbol 12-myristate 13-acetate)and S. aureu~V8 protease were purchased from Sigma. PKC and Molecular Marker Kit was obtained from Calbiochem. DTT (dithiothreitol) was from Merck. All other chemicals were of analytical grade. Adult Wistar rats were decapitated without anaesthesia. The cerebellar lobes were isolated according to Glowinski and fversen method (1966). Synaptosomal membranes were prepared by the procedure of Booth and Clark (1978). Protein was determined by the method of Ohnishi and Barr (1978). Phosphorylation of synaptosomal plasma membranes
The standard phosphoryiation assay medium (in final volume 20~1) contained 20mM Tri-HCI, pH = 7.4, 10 mM MgCI,, 1mM DTT, 0.5 mM CaCI,, f pM PMA, 1 mM vanadyl sulphate-phosphatase inhibitor (Ziai and Ferrone, 1989). In brief, 20 pg of membrane proteins were preincubated for 0, 3 and 6min at 30°C with concentrations of 1.3, 3.3 and 5 pM polymyxin B as indicated. Control samples were run with the same p~incubation step, in the absence of polymyxin B. In assay performed with exogenous PKC, 1 ~1 enzyme was added to 20 pg membrane proteins (control) or to 20 pg denaturated membrane proteins in standard reaction mixture, and preincubated for 0 and 3 mm. The reaction was started by the addition of 9 p M [y32P]ATP (5 pCi) and was stopped after 2 min by addition of 20 mM Tris-HCl, pH = 6.8, containing 6% (w/v) SDS, 20% (v/v) glycerol, 2.5% /?-mercaptoethanol, 0.002% bromophenol blue and heating at 100°C for 3 min. One-dimensional SDS-PAGE was performed according to the procedure of Laemmli (1970) on 7.5% polyacrylamide gel. After electrophoresis gels were silver-stained
to57
1058
LUDM~A ZYLIY&KA and LILLA LACHOWICZ
(Wray et al., 1981) and autoradiographed with an intensify-
ing screen. The proteins was measured phosphorylation samples, taken were estimated kit.
-
130 kDa ----__
were excised from gel and 12P incorporation by counting for Cerenkov radiation. The was expressed with regard to control as 100%. Molecular weight of the proteins by the comparison with molecular marker
T
Determination of the phosphorylated intermediate
Phosphorylation of the synaptosomal membranes (20 pg) was conducted in 30 ~1 of 50 mM imidazole buffer pH = 6.0 containing 2.8 PM [y-‘*P]ATP (8 PCi) and 0.2 mM CaCI,, 2 mM MgCl,, 0.5 mM vanadyl sulphate or 50 p M vanadate as indicated. The incubation was performed at 0°C for 15 set and stopped by adding ice-cold 10% TCA. After centrifugation for 5 min the pellet was washed twice with 10% TCA and once with water, and resuspended for gel electrophoresis in buffer containing 10 mM imidazole pH = 6.0, 1% SDS, 10% glycerol, 2.5% /I-mercaptoethanol and 0.02% bromophenol blue. SDS-PAGE of the ‘*Plabelled proteins on 7.5% gels at pH = 6.0 was done. The effect of hyroxylamine was studied as described by Wuytack ef al. (1982). The pellets of precipitated phosphorylated proteins were incubated for 10 min at room temperature with 200 ~1 of solution containing 150 mM hydroxylamine and 150 mM sodium acetate buffer brought to pH = 6.0 with Tris. Controls were treated likewise with a solution of 150 mM Tris-HCI, 150 mM sodium acetate buffer at pH = 6.0. After incubation the samples were treated as described above. ‘*P-Labelled proteins were detected by autoradiography with X-ray film. Peptide mapping by limired proteolysis with S. aureus V8 prolease
The synaptosomal membrane samples (100 pg) were fractionated by SDS-PAGE (7.5%) and stained with Coomassie Brilliant Blue. The band of 130 kDa protein was excised. Gel slices were overlaid with 2 pugof V8/well in 3% stacking gel and digested for 30 min at room temperature. The generated peptides were fractionated by 12.5% SDS-PAGE according to the procedure of Cleveland et al. (1977). The peptides were located by silver staining. ,,o._-_---‘“?_____
PB
l.ljbM
3.3pM
S.O/1M
Fig. 2. Effect of varying polymyxin B concentration on the 130 kDa protein phosphorylation. Membranes were treated as described under Materials and Methods. Data are given as means & SEM (N = 4) of radioactivity incorporated as a percentage of the control (100%). Statistically significant: P < 0.001, +P < 0.01.
0’
1 3’
Fig. 3. Effect of exogenous PKC on the phosphorylation of 130 kDa protein. Membranes were treated as described under Materials and Methods. The data are expressed as percentage relative to control (100%). The results are means + SEM (N = 4). Statistically significant: P < 0.001. RESULTS AND DISCUSSION
In order to demonstrate that synaptosomal membrane protein of rat cerebellum in our experimental conditions is the substrate for endogenous PKC, the phosphorylation was stimulated with 0.5 mM Ca*+ and 1 PM PMA. PMA is known to be a good probe for PKC function (Furukawa et al., 1989). We observed the phosphorylation of several proteins, particularly of protein, whose molecular weight was estimated to be 130 f 3 kDa (Fig. 1). We also examined the effect of polymyxin B-a cyclic, polycationic peptide antibiotic-the potent PKC inhibitor, on phosphorylation of 130 kDa protein (Tamaoki and Nakano, 1990). It is known that polymyxin B inhibits also CaMdependent kinase, but not cyclic nucleotide-dependent kinase (Linden and Routtenberg, 1989). In our experiments we observed the dose-dependent decrease of phosphorylation up to 50% for 5 PM polymyxin B (Fig. 2). It was interesting to note, that after 6 min of preincubation with 1.3 and 3.3 PM the inhibition was less effective. It may be due to peptidase activity. To confirm, that 130 kDa protein was in vitro the substrate for protein kinase C, experiments were also carried out using exogenous PKC. The data in Fig. 3 show that after 3 min of preincubation the phosphorylation was about 80% of control value. This suggests that 130 kDa protein in our experimental conditions may be the substrate not only for protein kinase C, but also for other kinases. In the next series of experiments we tried to determine the characteristics of the 130 kDa protein. The Ca2+-dependent, vanadate and hydroxylamine-sensitive phosphorylation with [r -32P]ATP of proteins can be considered to be a marker for P-type
Rat cerebellum
synaptosomal
membrane
130 kDa protein
67
43
Fig. 1. Phosphorylation of 130 kDa protein by endogenous PKC. Synaptosomal membranes from rat cerebellum (20 pg) were preincubated for 3 min in a standard reaction mixture and incubated as described under Materials and Methods. (A) SDS-PAGE analysis and silver staining of gel (7.5%). (B) Autoradiogram. Molecular weight standards (x 10-j) are indicated. Position of the protein 130 kDa indicated.
1059
1060
LUDMIEA~YLIP;ISKA and LILLA LACHOWICZ
130
Fig. 4. Formation of phosphorylated intermediate. The synaptosomal membranes (2Opg) were phosphorylated at 0°C for 15 set in a medium containing 50mM imidazole buffer pH = 6.0 and 2.8 PM [y-‘*P]ATP (8 PCi) in the presence: lane I-no addition; lane 2-CaC1,; iane 3-MgCI,; lane 4-CaCl, + MgCl,; lane S-CaCl, + MgCl, + vanadyl; lane 6--CaCI, + MgCI, + vanadate. After electrophoresis at pH = 6.0 gels were autoradiographed.
Rat cerebellum synaptosomal membrane 130 kDa protein
MW x 10-3
t-
94
-
Fig. 5. Effect of hydroxylamine. The samples were treated as described under Materials and Methods. After el~tropboresis at pH = 6.0 gels were autorad~ograph~. Lane I-phosphorylation in Ca:+ and with 150 mM Tris-HCI, 1.50mM sodium acetate buffer Mg*+, containing medium; lane 2-incubation pH = 6.0 (control); lane 3-incubation with 150mM hydroxylamine, 150mM sodium acetate buffer pH = 6.0.
1061
1062
LUDMIZA~~YLI~~SKA and LILLALACHOWICZ
MW x1o-3
67 -
57
-
47
-
41
43 !
va
-
23
-
15
20
Fig. 6. Peptide mapping analysis by S. aureus V8 proteolysis of 130 kDa protein. The position of V8 and of molecular weight markers are indicated. The peptides were located by silver staining.
Rat cerebellum synaptosomal membrane 130 kDa protein ATPases (Pedersen results demonstrate
and Carafoli, 1987a, b). Our (Fig. 4) that the 130 kDa phos-
phorylated intermediate is formed, when the synaptosomal membranes are incubated at 0°C with [Y-~~P]ATP and 0.2 mM CaCl,. No phosphorylation was observed after incubation the synaptosomal membranes with [Y-~‘P]TP or [Y-~~P]ATP and 2 mM MgCl,. Since our experimental conditions were not optimal for phosphorylation mediated by protein kinases, we concluded that 130 kDa protein was acylphosphoprotein. This form was unstable in the presence of 150mM hydroxylamine (Fig. 5). It should be pointed out that the second major phosphoprotein band, whose molecular weight was estimated as - 110 kDa, was also visible. Recent work of the effect of the intracellular Ca2+-dependent protease calpain indicates that calpain reduces the apparent molecular weight of the erythrocyte membrane Ca2+-ATPase to - 124 kDa followed by 85 kDa (Wang et al., 1988). The calpaindigested fragments were phosphorylated in the presence of Ca*+ and they were sensitive to hydroxylamine treatment. We can assume that 110 kDa protein in our experiments is a proteolytic product of 130 kDa. As mentioned above, the Ca*+-pumps showed inhibitory effect of vanadate on ATPase activity. In the physiological pH, vanadium ions may exist in the form of pentavalent vanadate anion and tetravalent vanadyl cation (Krivanek, 1988). Vanadate inhibits by binding to the amino acid residue that forms the phosphorylated intermediate at active site, and acts as a noncompetitive inhibitor against ATP (Carafoli, 1991). When the synaptosomal membranes were incubated in the presence of 50 PM vanadate, the formation of phosphointermediate was significantly reduced. Vanadyl cations stimulated phosphorylation of 130 kDa protein suggests different mechanisms by which vanadium ions may exert their physiological effects. The absence of Mg2’ is required to prolong the lifetime of the phosphorylated conformational state of Ca*+-pump (Inesi et al., 1990; Villalobo, 1990). On the other hand, Mg*+ ions are also required for the dephosphorylation step of the ATPase reaction cycle (Carafoli, 1991). Our analysis indicated that 2 mM MgClz decreased the level of phosphorylated intermediate at pH = 6.0. We concluded that ions Mg?+ in our experimental conditions increased dephosphorylation step of ATPase reaction. We also studied the fragmentation pattern of the 130 kDa protein produced by S. aureus V8 protease
(Fig. 6). The fragments have relative molecular masses of 110, 57, 47, 41 kDa and smaller components of 23 and 15 kDa. CONCLUSION
There have been several recent studies, which suggest that modulation of the activities of membrane
1063
Ca2+-transport system is mediated via activation of protein kinase C (Fukuda et al., 1990; Furukawa et uf., 1989). These results suggest that PKC stimulation of Ca2+-pump may be a general phenomenon. Our results demonstrate that 130 kDa protein may be functionally and structurally related Ca2+-pumps from other tissues, as obtained by the demonstration of phospho-intermediate hydroxylamine-sensitive and inhibited by vanadate. These studies indicate the presence of PKC-phosphorylated Ca2+-ATPase-like protein in synaptosomal membranes of rat cerebellum. wish to thank Mrs Krystyna Marciniak for excellent technical assistance. The study was financially supported by a grant from the School of Medicine of Mdi under contract No. 502-I l-021. Acknowledgements-We
REFERENCES
Booth R. F. G. and Clark J. B. (1978) A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain. Biochem. J. 176, 365-370.
Carafoli E. (1991) Calcium pump of the plasma membrane. Physiol. Rev. 71, 129-153.
Cleveland D. W., Fischer S. G., Kirshner M. W. and Laemmli U. K. (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J. biol. Chem. 252, 1102-I 106. De Graan P. N. D., Schotman P. and Versteeg D. H. G. (1990) Neural mechanisms of action of neuropeptides: macromolecules and neurotransmitters. Neuropeptides: Basics and Perspecrives, (Edited by De Wied), pp. 139-l 74. Elsevier Science, Amsterdam. Fukuda T., Ogurusu T., Furukawa K.-I. and Shigekawa M. (1990) Protein kinase C-dependent phosphorylation of sarcolemma Ca2+-ATPase isolated from bovine aortic smooth muscle. J. Biochem. 108, 629434. Furukawa K. J., Tawada Y. and Shigekawa M. (1989) Protein kinase C activation stimulates plasma membrane Caz+ pump in cultured vascular smooth muscle cells. J. biol. Chem. 264, 48484849.
Glowinski J. and Iversen L. (1966) Regional studies of catecholamines in the rat brain. I. The disposition of [‘Hlnorepinephrine, [3Hldopamine and [3H]DOPA in various regions of the brain. J. Neurochem. 13, 655-669. Huang F. L., Scott Young W. III, Yoshida Y. and Huang K. P. (1990) Developmental expression of protein kinase C isozymes in rat cerebellum. Dev. Bruin Res. 52, 121-130.
Inesi G., Sumbilla C. and Kirtley M. E. (1990) Relationships of molecular structure and function in Ca*+-transport ATPase. Physiol. Rev. 70, 749-760. James P., Vorherr T., Krebs J., Morelli A., Caste110 G., McCormick D. J., Penniston J. T., DeFlora A. and Carafoli E. (1989) Modulation of erythrocyte Ca*+ ATPase by selective calpain cleavage of the calmodulin binding domain. J. biol. Chem. 264, 8289-8296. Krivanek J. (1988) Do vanadium ions exert any specific effect on brain protein phoshorylation. Neurochem. Res. 13, 395401.
Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature 227, 680-685.
1064
LUDMIWZYLI~SKAand LILLALACHOWICZ
Linden D. J. and Routtenberg A. (1989) The role of protein kinase C in long-term potentiation: A testable model. Brain Res. Rev. 14, 219-296.
Ohnishi S. T. and Barr J. T. (1978) A simplified method of quantitating protein using the biuret and phenol reagent. Analyf. Biochem. 86, 193-200.
Pedersen P. L. and Carafoli E. (1987a) Ion motive ATPases. Trends Biochem. Sci. 12, 146-150.
Pedersen P. L. and Carafoli E. (1987b) Ion motive ATPases. Trends Biochem. Sci. 12, 186-189.
Rasmussen H. (1990) The complexities of intracellular Ca2+ signalling. Biol. Chem. Hoppe-Seyler 371, 191-206. Routtenberg A. (1986) Synaptic plasticity and protein kinase C. Prog. Brain Res. 69, 211-234. Smallwood J. I., Gugi B. and Rasmussen H. (1988) Regulation of erythrocyte CaZ+ pump activity by protein kinase C. J. biol. Chem. 263, 2195-2202. Tamaoki T. and Nakano H. (1990) Potent and specific inhibitors of protein kinase C of microbial origin. Biotechnology 8, 132-735.
Villalobo A. (1990) Reconstitution of ion-motive transport ATPases in artificial lipid membranes. Biochim. biophys. Acfa 1017, l-48.
Wang K. K. W., Roufogalis B. D. and Villalobo A. (1988) Further characterization of calpain-mediated proteolysis of the human erythrocyte plasma membrane Ca*+ATPase. Archs Biochem. Biophys. 267, 311-321. Wray W., Boulikas T., Wray V. P. and Hancock R. (1981) Silver staining of proteins in polyacrylamide gels. Analyr. Biochem. 118, 197-203.
Wuytack F., Raeymaekers L., De Schutter G. and Casteels R. (1982) Demonstration of the phosphorylated intermediates of the Ca2+-transport ATPase in a microsomal fraction and in a (Ca’+ + Mg2+)-ATPase purified from smooth muscle by means of calmodulin affinity chromatography. Biochim. biophys. Acta 693, 45-52. Ziai M. R. and Ferrone S. (1989) Specific phosphorylation by calcium-EGTA complex of a 75 kDa human tumor plasma membrane protein (pp. 75). Int. J. Biochem. 21, 731-738.
