228
P U R I F I C A T I O N OF P R O T E I N KINASES
[18]
[18] E x p r e s s i o n , Separation, and A s s a y o f P r o t e i n Kinase C Subspecies
By KouJI OGITA, YOSHITAKA ONO, USHIO KIKKAWA, and YASUTOMI NISHIZUKA
A large number of extracellular signals which stimulate inositol phospholipid hydrolysis appear to operate through a bifurcating intracellular signal pathway which involves Ca z÷ mobilization and protein kinase C activation. 1 Molecular cloning analysis has revealed that protein kinase C exists as a family of multiple subspecies. 2-1~ Initially, four cDNA clones designated a, flI, 1311, and 3~were isolated, 12and these clones were shown to be conserved among different mammalian species such as human, bovine, rat, and rabbit. 2-6 These subspecies have closely related structures with several highly conserved regions in this protein kinase family. This heterogeneity comes from different genes as well as from alternative splicing of a single RNA transcript. 3'~3 Later, another group of cDNA clones I y. Nishizuka, Nature (London) 334, 661 (1988). P. J. Parker, L. Coussens, N. Totty, L. Rhee, S. Young, E. Chen, S. Stabel, M. D. Waterfield, and A. Ullrich, Science 233, 853 (1986). 3 L. Coussens, P. J. Parker, L. Rhee, T. L. Yang-Feng, E. Chen, M. D. Waterfield, U. Francke, and A. Ullrich, Science 233, 859 (1986). 4 y. Ono, T. Kurokawa, T. Fujii, K. Kawahara, K. Igarashi, U. Kikkawa, K. Ogita, and Y. Nishizuka, FEBS Lett. 206, 347 (1986). 5 j. L. Knopf, M.-H. Lee, L. A. Sultzman, R. W. Kriz, C. R. Loomis, R. M. Hewick, and R. M. Bell, Cell (Cambridge, Mass.) 46, 491 (1986). 6 S. Ohno, H. Kawasaki, S. Imajoh, K. Suzuki, M. Inagaki, H. Yokokura, T. Sakoh, and H. Hidaka, Nature (London) 325, 161 (1987). 7 y. Ono, T. Fujii, K. Igarashi, U. Kikkawa, K. Ogita, and Y. Nishizuka, Nucleic Acids Res. 16, 5199 (1988). s G. M. Housey, C. A. O'Brian, M. D. Johnson, P. Kirschmeier, and I. B. Weinstein, Prof. Natl. Acad. Sci. U.S.A. 84, 1065 (1987). 9 y. Ono, T. Fujii, K. Igarashi, U. Kikkawa, K. Ogita, and Y. Nishizuka, J. Biol. Chem. 263, 6927 (1988). to S. Ohno, Y. Akita, Y. Konno, S. Imajoh, ad K. Suzuki, Cell (Cambridge, Mass.) 53, 731 (1988). IID. Schaap, P. J. Parker, A. Bristol, R. Kriz, and J. Knopf, FEBS Lett. 243, 351 (1989). 12The nomenclature of the cDNA clones used in this chapter is as described by Nishizuka.l The c~, /31, /311, and ~ isolated from different species encode 672, 671,673, and 697 amino acids, respectively. 13y. Ono, U. Kikkawa, K. Ogita, T. Fujii, T. Kurokawa, Y. Asaoka, K. Sekiguchi, K. Ase, K. Igarashi, and Y. Nishizuka, Science 236, 1116 (1987). Copyright © 1991 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 200
All fights of reproduction in any form reserved.
[18]
EXPRESSION OF PROTEIN KINASE C SUBSPECIES
229
was obtained that has structures similar to but distinct from the previous four e D N A clone s. 8-1~Differential tis sue and cellular expression and different enzymatic activity of these subspecies has been o b s e r v e d ) Thus, it is important to isolate each subspecies and study the properties of this enzyme family. This chapter describes a method for the transient expression of protein kinase C subspecies, a,/3I,/311, and % in COS-7 cells using the c D N A clones isolated from rat brain. 4,7,~3The methods for the separation and assay o f these subspecies are also described.
Construction of Expression Plasmids The expression vector designated pTB701 isemployed for the construction of expression plasmids of protein kinase C.9'13A4The expression vector is constructed from Okayama and Berg vectors15and has a single cloning site of EcoRI downstream from the Abelson murine leukemia virus long terminal repeat,16 simian virus 40 (SV40) origin of D N A replication, and SV40 early promoter regions. The whole insert of the protein kinase C c D N A clones is incorporated into the EcoRI site of the pTB701. Thc resulting expression plasmids for a,/3I,/3II,and y contain inserts of 3305 nucleotides from clone hCKRo~5, 7 3190 nucleotidcs from clones hCKRI52 and hCKRI08, 4'13 3406 nucleotides from clones hCKR152 and hCKRI07, 4,13and 3025 nucleotides from clone hCKRTI,7 rcspectively. Transfection COS-7 cells are transfected by the calcium phosphate coprecipitation technique 17 with glycerol shock, la Fresh monolayers of COS-7 cells, in 10-cm diameter plates (50 plates) each containing 10 ml of Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal calf serum (FCS), are transfected with 30 ~g each of the plasmid DNA. After a transfection period of 3.5 hr, the cells are shocked with glycerol for 3 min at room temperature, and fresh medium is added to each plate. After incubation for 18 hr at 37°, the medium is replaced by 15 ml of fresh Dulbecco's modified Eagle's medium containing 5% fetal calf serum. Cells are harvested after an additional 48-hr incubation at 37 °. 14U. Kikkawa, Y. Ono, K. Ogita, T. Fujii, Y. Asaoka, K. Sekiguchi,Y. Kosaka, K. Igarashi, and Y. Nishizuka, FEBS Lett. 217, 227 (1987). 15H. Okayama and P. Berg, Mol. Cell. Biol. 3, 280 (1983). 16E. P. Reddy, M. J. Smith, and A. Srinivasan, Proc. Natl. Acad. Sci. U.S.A. 80, 3623 (1983). x7F. L. Graham and A. J. van der Eb, Virology 52, 456 (1973). is C. Gorman, R. Padmanabhan, and B. H. Howard, Science 221, 551 (1983).
230
PURIFICATION OF PROTEIN KINASES
[18]
Separation Protein kinase C expressed in COS-7 cells is separated into each subspecies by hydroxyapatite column chromatography using a fast protein liquid chromatography (FPLC) system (Pharmacia, Piscataway, N J). '3,~4 To obtain good resolution by the hydroxyapatite column, it is necessary to partially purify the protein kinase C from the COS-7 cell extracts before this column chromatography. For this purpose, it is possible to use an ionexchange column chromatography by Mono Q column (Pharmacia). The following steps are carried out at 0 to 4°. The COS-7 cells (approximately 3 × l08 cells from 50 plates) are homogenized by sonication (Kontes, Vincland, NJ; sonificr or equivalent) for l min in 5 ml of 20 mM TrisHCI at pH 7.5, containing 0.25 M sucrose, 10 mM ethylene glycol bis(~aminocthyl ester)-N,N,N',N'-tetraacetic acid (EGTA), 2 mM cthylencdiaminetetraacetic acid (EDTA), and 20 ~g/ml lcupcptin. The homogenatc is centrifuged for 50 rain at 100,000 g, and the supernatant is diluted with 6 vol of 20 mM Tris-HC1 at pH 7.5, containing 0.5 mM EGTA, 0.5 mM EDTA, and l0 mM 2-mercaptoethanol (buffer A), filtered through a membrane filter (pore size 0.22 ~m, Millex-GS, Millipore, Bedford, MA, or equivalent), and applied to a Mono Q column (1 × 10 cm, Pharmacia HR10/10) that is connected to a Pharmacia FPLC system and equilibrated with buffer A. Protein kinase C is eluted by application of a 164-ml linear concentration gradient of NaCl (0 to 0.6 M) in buffer A at a flow rate of 2 ml/min. Fractions (4 ml each) arc collected. Protein kinase C activity appears at fractions 8 to 12 at 0.18 MNaCI. The protein kinase fractions are pooled and diluted with an equal volume of 20 mM potassium phosphate at pH 7.5, containing 0.5 mM EGTA, 0.5 mM EDTA, 10% (w/v) glycerol, and l0 mM 2-mcrcaptoethanol (buffer B), and applied to a packed hydroxyapatite column (0.78 × l0 cm, type S; Koken, Tokyo, Japan) which is connected to the Pharmacia FPLC system and equilibrated with buffer B. The protein kinase is eluted by application of an 84-ml linear concentration gradient of potassium phosphate (20 to 250 raM) in buffer B at a flow rate of 0.4 ml/min, and fractions (1 ml each) are collected. Active fractions are pooled and ~lialyzcd against buffer A containing 10% (w/v) glycerol. This enzyme preparation can be stored at least for several months in the presence of 0.05% (w/v) Triton X-100 at - 8 0 °. Rat brain protein kinasc C as a control is purified from its cytosol fraction by DEAE-cellulose (DE-52; Whatman, Milford, MA), threoninc-Sepharose, and TSK phenyl-5PW (Toyo Soda, Tokyo, Japan) column chromatographies as described,19 and 19 U. Kikkawa, M. Go, J. Koumoto, and Y. Nishizuka, Biochem. Biophys. Res. Commun. 135, 636 (1986).
[18]
EXPRESSION OF PROTEIN KINASE C SUBSPECIES
231
resolved into distinct fractions by the hydroxyapatite column chromatography as described above.
Assay Protein kinase C is assayed by measuring the incorporation of [32P]P i into calf thymus H1 histone from [y-aEp]ATP, essentially as described previously. 19The reaction mixture (0.25 ml) contains 20 mM Tris-HCl at pH 7.5, 5 mM magnesium acetate, 10 ~M [y-32p]ATP(50 to 100 cpm/pmol), 200 /~g/ml of histone H1, 8 /~g/ml of phosphatidylserine, 0.8 /xg/ml of diacylglycerol (1,2-diolein) or 10 ng/ml of 12-O-tetradecanoylphorbol 13acetate (TPA), various concentrations of C a C I 2 , and the enzyme fraction. Phosphatidylserine and diacylglycerol, stored separately as chloroform solutions at - 2 0 °, are mixed first in chloroform, dried, suspended in 20 mM Tris-HC1 at pH 7.5, sonicated, and then added to the reaction mixture as lipid micelles as described. 19TPA, dissolved in dimethyl sulfoxide, is diluted with distilled water and added directly to the reaction mixture with sonicated phosphatidylserin. The final concentrations of dimethyl sulfoxide should be kept less than 0.01% (v/v), because dimethyl sulfoxide at high concentrations sometimes varies protein kinase activity. Basal activity is measured in the presence of 0.5 mM EGTA, instead of CaC12 . It is convenient to prepare a mixed solution of Tris-HCl, magnesium acetate, [y-32p]ATP, and histone H 1. The reaction is started by the addition of this solution to the assay tube that contains CaC12, lipid micelles, and the enzyme fraction. After 3 min at 30°, the reaction is stopped by the addition of 3 ml of 25% trichloroacetic acid (TCA). Acid-precipitable materials are collected on a nitrocellulose membrane filter (pore size 0.45 /.~m; Toyo Roshi, Tokyo, Japan or equivalent) in a suction apparatus. The membrane filter is washed three times with 25% TCA, and the radioactivity is quantitated by Cerenkov counting. Comments
The protein kinase C activity recovered from the transfected cells by Mono Q column chromatography is severalfold higher than that from the control cells. Figure 1 shows the separation of the protein kinase C subspecies from the transfected cells by hydroxyapatite column chromatography. The rat brain protein kinase C was first reported to be resolved into three distinct fractions on hydroxyapatite column chromatography by K.-P. Huang et al. 2° These fractions are called Type I, II, and H1 according ~0 K.-P. Huang, H. Nakabayashi, and F. L. Huang, Proc. Natl. Acad. Sci. U.S.A. 83, 8535 (1986).
I 4O
|
I
~
- O.2
20
0 1
0
0
20
0.i
o -
................. I
o
40
0.2
20
0.i
m H
cn
< z
0
z
40
0.2
20
0.i
E--t O
~:
0
0
0 •-
l
40 ~ ~ 20
~n
~
0.2
~
0.i
0
0 20
40
60
FRACTION NUMBER FIG. 1. Separation of protein kinase C subspecies by hydroxyapatite column chromatography. Protein kinase C subspecies expressed in COS-7 cells and the enzyme preparation purified from rat brain were applied to hydroxyapatite column as described in the text. Enzyme activity was assayed in the presence of phosphatidylserine, diacylglycerol, and 0.5 mM CaCI2 (O), or in the presence of 0,5 mM EGTA instead of phosphatidylserine, diacylglycerol, and CaCl2 (O). Solid line indicates the concentrations of potassium phosphate. (A) Rat brain protein kinase C. (B)-(D) Protein kinase C from COS-7 cells transfected with the expression plasmid of protein kinase C of o~, fill, and y, respectively. (E) Protein kianse C from control COS-7 cells. (Taken from Ref. 14.)
[18]
EXPRESSION OF PROTEIN KINASE C SUBSPECIES
-
V]
®
233
D
i00
P U R I F I C A T I O N OF P R O T E I N KINASES
[18]
[18] E x p r e s s i o n , Separation, and A s s a y o f P r o t e i n Kinase C Subspecies
By KouJI OGITA, YOSHITAKA ONO, USHIO KIKKAWA, and YASUTOMI NISHIZUKA
A large number of extracellular signals which stimulate inositol phospholipid hydrolysis appear to operate through a bifurcating intracellular signal pathway which involves Ca z÷ mobilization and protein kinase C activation. 1 Molecular cloning analysis has revealed that protein kinase C exists as a family of multiple subspecies. 2-1~ Initially, four cDNA clones designated a, flI, 1311, and 3~were isolated, 12and these clones were shown to be conserved among different mammalian species such as human, bovine, rat, and rabbit. 2-6 These subspecies have closely related structures with several highly conserved regions in this protein kinase family. This heterogeneity comes from different genes as well as from alternative splicing of a single RNA transcript. 3'~3 Later, another group of cDNA clones I y. Nishizuka, Nature (London) 334, 661 (1988). P. J. Parker, L. Coussens, N. Totty, L. Rhee, S. Young, E. Chen, S. Stabel, M. D. Waterfield, and A. Ullrich, Science 233, 853 (1986). 3 L. Coussens, P. J. Parker, L. Rhee, T. L. Yang-Feng, E. Chen, M. D. Waterfield, U. Francke, and A. Ullrich, Science 233, 859 (1986). 4 y. Ono, T. Kurokawa, T. Fujii, K. Kawahara, K. Igarashi, U. Kikkawa, K. Ogita, and Y. Nishizuka, FEBS Lett. 206, 347 (1986). 5 j. L. Knopf, M.-H. Lee, L. A. Sultzman, R. W. Kriz, C. R. Loomis, R. M. Hewick, and R. M. Bell, Cell (Cambridge, Mass.) 46, 491 (1986). 6 S. Ohno, H. Kawasaki, S. Imajoh, K. Suzuki, M. Inagaki, H. Yokokura, T. Sakoh, and H. Hidaka, Nature (London) 325, 161 (1987). 7 y. Ono, T. Fujii, K. Igarashi, U. Kikkawa, K. Ogita, and Y. Nishizuka, Nucleic Acids Res. 16, 5199 (1988). s G. M. Housey, C. A. O'Brian, M. D. Johnson, P. Kirschmeier, and I. B. Weinstein, Prof. Natl. Acad. Sci. U.S.A. 84, 1065 (1987). 9 y. Ono, T. Fujii, K. Igarashi, U. Kikkawa, K. Ogita, and Y. Nishizuka, J. Biol. Chem. 263, 6927 (1988). to S. Ohno, Y. Akita, Y. Konno, S. Imajoh, ad K. Suzuki, Cell (Cambridge, Mass.) 53, 731 (1988). IID. Schaap, P. J. Parker, A. Bristol, R. Kriz, and J. Knopf, FEBS Lett. 243, 351 (1989). 12The nomenclature of the cDNA clones used in this chapter is as described by Nishizuka.l The c~, /31, /311, and ~ isolated from different species encode 672, 671,673, and 697 amino acids, respectively. 13y. Ono, U. Kikkawa, K. Ogita, T. Fujii, T. Kurokawa, Y. Asaoka, K. Sekiguchi, K. Ase, K. Igarashi, and Y. Nishizuka, Science 236, 1116 (1987). Copyright © 1991 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 200
All fights of reproduction in any form reserved.
[18]
EXPRESSION OF PROTEIN KINASE C SUBSPECIES
229
was obtained that has structures similar to but distinct from the previous four e D N A clone s. 8-1~Differential tis sue and cellular expression and different enzymatic activity of these subspecies has been o b s e r v e d ) Thus, it is important to isolate each subspecies and study the properties of this enzyme family. This chapter describes a method for the transient expression of protein kinase C subspecies, a,/3I,/311, and % in COS-7 cells using the c D N A clones isolated from rat brain. 4,7,~3The methods for the separation and assay o f these subspecies are also described.
Construction of Expression Plasmids The expression vector designated pTB701 isemployed for the construction of expression plasmids of protein kinase C.9'13A4The expression vector is constructed from Okayama and Berg vectors15and has a single cloning site of EcoRI downstream from the Abelson murine leukemia virus long terminal repeat,16 simian virus 40 (SV40) origin of D N A replication, and SV40 early promoter regions. The whole insert of the protein kinase C c D N A clones is incorporated into the EcoRI site of the pTB701. Thc resulting expression plasmids for a,/3I,/3II,and y contain inserts of 3305 nucleotides from clone hCKRo~5, 7 3190 nucleotidcs from clones hCKRI52 and hCKRI08, 4'13 3406 nucleotides from clones hCKR152 and hCKRI07, 4,13and 3025 nucleotides from clone hCKRTI,7 rcspectively. Transfection COS-7 cells are transfected by the calcium phosphate coprecipitation technique 17 with glycerol shock, la Fresh monolayers of COS-7 cells, in 10-cm diameter plates (50 plates) each containing 10 ml of Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal calf serum (FCS), are transfected with 30 ~g each of the plasmid DNA. After a transfection period of 3.5 hr, the cells are shocked with glycerol for 3 min at room temperature, and fresh medium is added to each plate. After incubation for 18 hr at 37°, the medium is replaced by 15 ml of fresh Dulbecco's modified Eagle's medium containing 5% fetal calf serum. Cells are harvested after an additional 48-hr incubation at 37 °. 14U. Kikkawa, Y. Ono, K. Ogita, T. Fujii, Y. Asaoka, K. Sekiguchi,Y. Kosaka, K. Igarashi, and Y. Nishizuka, FEBS Lett. 217, 227 (1987). 15H. Okayama and P. Berg, Mol. Cell. Biol. 3, 280 (1983). 16E. P. Reddy, M. J. Smith, and A. Srinivasan, Proc. Natl. Acad. Sci. U.S.A. 80, 3623 (1983). x7F. L. Graham and A. J. van der Eb, Virology 52, 456 (1973). is C. Gorman, R. Padmanabhan, and B. H. Howard, Science 221, 551 (1983).
230
PURIFICATION OF PROTEIN KINASES
[18]
Separation Protein kinase C expressed in COS-7 cells is separated into each subspecies by hydroxyapatite column chromatography using a fast protein liquid chromatography (FPLC) system (Pharmacia, Piscataway, N J). '3,~4 To obtain good resolution by the hydroxyapatite column, it is necessary to partially purify the protein kinase C from the COS-7 cell extracts before this column chromatography. For this purpose, it is possible to use an ionexchange column chromatography by Mono Q column (Pharmacia). The following steps are carried out at 0 to 4°. The COS-7 cells (approximately 3 × l08 cells from 50 plates) are homogenized by sonication (Kontes, Vincland, NJ; sonificr or equivalent) for l min in 5 ml of 20 mM TrisHCI at pH 7.5, containing 0.25 M sucrose, 10 mM ethylene glycol bis(~aminocthyl ester)-N,N,N',N'-tetraacetic acid (EGTA), 2 mM cthylencdiaminetetraacetic acid (EDTA), and 20 ~g/ml lcupcptin. The homogenatc is centrifuged for 50 rain at 100,000 g, and the supernatant is diluted with 6 vol of 20 mM Tris-HC1 at pH 7.5, containing 0.5 mM EGTA, 0.5 mM EDTA, and l0 mM 2-mercaptoethanol (buffer A), filtered through a membrane filter (pore size 0.22 ~m, Millex-GS, Millipore, Bedford, MA, or equivalent), and applied to a Mono Q column (1 × 10 cm, Pharmacia HR10/10) that is connected to a Pharmacia FPLC system and equilibrated with buffer A. Protein kinase C is eluted by application of a 164-ml linear concentration gradient of NaCl (0 to 0.6 M) in buffer A at a flow rate of 2 ml/min. Fractions (4 ml each) arc collected. Protein kinase C activity appears at fractions 8 to 12 at 0.18 MNaCI. The protein kinase fractions are pooled and diluted with an equal volume of 20 mM potassium phosphate at pH 7.5, containing 0.5 mM EGTA, 0.5 mM EDTA, 10% (w/v) glycerol, and l0 mM 2-mcrcaptoethanol (buffer B), and applied to a packed hydroxyapatite column (0.78 × l0 cm, type S; Koken, Tokyo, Japan) which is connected to the Pharmacia FPLC system and equilibrated with buffer B. The protein kinase is eluted by application of an 84-ml linear concentration gradient of potassium phosphate (20 to 250 raM) in buffer B at a flow rate of 0.4 ml/min, and fractions (1 ml each) are collected. Active fractions are pooled and ~lialyzcd against buffer A containing 10% (w/v) glycerol. This enzyme preparation can be stored at least for several months in the presence of 0.05% (w/v) Triton X-100 at - 8 0 °. Rat brain protein kinasc C as a control is purified from its cytosol fraction by DEAE-cellulose (DE-52; Whatman, Milford, MA), threoninc-Sepharose, and TSK phenyl-5PW (Toyo Soda, Tokyo, Japan) column chromatographies as described,19 and 19 U. Kikkawa, M. Go, J. Koumoto, and Y. Nishizuka, Biochem. Biophys. Res. Commun. 135, 636 (1986).
[18]
EXPRESSION OF PROTEIN KINASE C SUBSPECIES
231
resolved into distinct fractions by the hydroxyapatite column chromatography as described above.
Assay Protein kinase C is assayed by measuring the incorporation of [32P]P i into calf thymus H1 histone from [y-aEp]ATP, essentially as described previously. 19The reaction mixture (0.25 ml) contains 20 mM Tris-HCl at pH 7.5, 5 mM magnesium acetate, 10 ~M [y-32p]ATP(50 to 100 cpm/pmol), 200 /~g/ml of histone H1, 8 /~g/ml of phosphatidylserine, 0.8 /xg/ml of diacylglycerol (1,2-diolein) or 10 ng/ml of 12-O-tetradecanoylphorbol 13acetate (TPA), various concentrations of C a C I 2 , and the enzyme fraction. Phosphatidylserine and diacylglycerol, stored separately as chloroform solutions at - 2 0 °, are mixed first in chloroform, dried, suspended in 20 mM Tris-HC1 at pH 7.5, sonicated, and then added to the reaction mixture as lipid micelles as described. 19TPA, dissolved in dimethyl sulfoxide, is diluted with distilled water and added directly to the reaction mixture with sonicated phosphatidylserin. The final concentrations of dimethyl sulfoxide should be kept less than 0.01% (v/v), because dimethyl sulfoxide at high concentrations sometimes varies protein kinase activity. Basal activity is measured in the presence of 0.5 mM EGTA, instead of CaC12 . It is convenient to prepare a mixed solution of Tris-HCl, magnesium acetate, [y-32p]ATP, and histone H 1. The reaction is started by the addition of this solution to the assay tube that contains CaC12, lipid micelles, and the enzyme fraction. After 3 min at 30°, the reaction is stopped by the addition of 3 ml of 25% trichloroacetic acid (TCA). Acid-precipitable materials are collected on a nitrocellulose membrane filter (pore size 0.45 /.~m; Toyo Roshi, Tokyo, Japan or equivalent) in a suction apparatus. The membrane filter is washed three times with 25% TCA, and the radioactivity is quantitated by Cerenkov counting. Comments
The protein kinase C activity recovered from the transfected cells by Mono Q column chromatography is severalfold higher than that from the control cells. Figure 1 shows the separation of the protein kinase C subspecies from the transfected cells by hydroxyapatite column chromatography. The rat brain protein kinase C was first reported to be resolved into three distinct fractions on hydroxyapatite column chromatography by K.-P. Huang et al. 2° These fractions are called Type I, II, and H1 according ~0 K.-P. Huang, H. Nakabayashi, and F. L. Huang, Proc. Natl. Acad. Sci. U.S.A. 83, 8535 (1986).
I 4O
|
I
~
- O.2
20
0 1
0
0
20
0.i
o -
................. I
o
40
0.2
20
0.i
m H
cn
< z
0
z
40
0.2
20
0.i
E--t O
~:
0
0
0 •-
l
40 ~ ~ 20
~n
~
0.2
~
0.i
0
0 20
40
60
FRACTION NUMBER FIG. 1. Separation of protein kinase C subspecies by hydroxyapatite column chromatography. Protein kinase C subspecies expressed in COS-7 cells and the enzyme preparation purified from rat brain were applied to hydroxyapatite column as described in the text. Enzyme activity was assayed in the presence of phosphatidylserine, diacylglycerol, and 0.5 mM CaCI2 (O), or in the presence of 0,5 mM EGTA instead of phosphatidylserine, diacylglycerol, and CaCl2 (O). Solid line indicates the concentrations of potassium phosphate. (A) Rat brain protein kinase C. (B)-(D) Protein kinase C from COS-7 cells transfected with the expression plasmid of protein kinase C of o~, fill, and y, respectively. (E) Protein kianse C from control COS-7 cells. (Taken from Ref. 14.)
[18]
EXPRESSION OF PROTEIN KINASE C SUBSPECIES
-
V]
®
233
D
i00
