Biochimica et Biophysica Acta, 4 0 7 ( 1 9 7 5 ) 1 1 4 - - 1 1 9 © Elsevier Scientific Publishing Company,
A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 91417
NUCLEAR PHOSPHOPROTEIN KINASE ACTIVITIES IN NORMAL AND NEOPLASTIC TISSUES
JUDITH A. T H O M S O N , JEN-FU CHIU* and L U B O M I R S. HNILICA* Department of Biochemistry, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute,Houston, Texas 77025 (U.S.A.) (Received June 20th, 1975)
Summary Nuclear phosphoprotein kinases from normal rat liver and transplantable neoplasms were fractionated and compared. A phosphoprotein kinase fraction activated by Mn 2+ was found to be present only in the neoplasms. This nuclear protein kinase phosphorylated nuclear proteins represented by one major and several minor bands as determined by polyacrylamide gel electrophoresis (M ~ 50 000). Nonhistone chromosomal proteins, particularly the nuclear phosphoprotein fraction, exhibit many properties expected of gene regulatory macromolecules [1--5]. For example, phosphoproteins alter the rate of RNA synthesis in vitro [6--9], they are heterogenous and tissue specific [6,10], and they bind specifically to homologous DNA [6,9,11,12]. Changes in the phosphorylation of chromosomal nonhistone proteins could be correlated with activation of gene activity in a variety of systems [13--18]. In order to explore the possible regulatory function of the phosphorylated nonhistone proteins during neoplastic growth, we have investigated the changes in their phosphorylation during hepatocarcinogenesis [ 15] and in regenerating rat liver [16,17], and kidney [18]. The results of these experiments suggest that phosphorylated nonhistone proteins may be involved in effecting changes associated with neoplastic growth. It was shown that the enzymes responsible for nuclear protein phosphorylation, i.e. nuclear protein kinases are quite heterogenous [19] and their activities increase in response to genetic activation [15--18]. It appears that both the nuclear phosphoproteins and their kinases play an active role in gene regulation. The purpose of this investigation was to determine if any differences could be observed in fractionated protein kinase activities or protein kinase species between normal and neoplastic tissues. We report here that a * Present address: D e p a r t m e n t o f B i o c h e m i s t r y , Vanderbilt University S c h o o l o f M e d i c i n e , Nashville, Tenn. 37232. R e p r i n t r e q u e s t s s h o u l d b e m a i l e d t o this address.
115
tumor specific protein kinase has been characterized in Novikoff hepatoma and Ehrlich ascites nuclei. Liver nuclei from adult Sprague-Dawley rats {about 150 g) were prepared by the method of Chauveau et al. [20]. The nuclei of Novikoff hepatoma and Ehrlich ascites were isolated by the method of Wilhelm et al. [21]. Phosphoproteins and phosphoprotein kinases were isolated by the method described by Kish and Kleinsmith [19]. Briefly, isolated nuclei were homogenized in 0.14 M NaC1 to remove cytoplasmic contaminants and nuclear sap. The nonhistone proteins were extracted by homogenizing the nuclear pellets in equal vol. of 2 M NaC1, 0.05 M Tris, pH 7.5. The resulting solution was mixed with 1.5 vol. of 0.02 M Tris, pH 7.5, and the precipitated nucleohistone and nuclear debris were removed by centrifugation at 81 500 ×g for 1 h. The nuclear phosphoproteins and kinases were further purified by Bio-Rex 70 chromatography and calcium phosphate gel absorption as described by Kish and Kleinsmith [19]. Phosphoproteins and kinases isolated from nuclei were dialyzed overnight against 100 vol. of 0.1 M NaC1, 0.05 M Tris, pH 7.5, with 3 changes. Approximately 3 mg of protein were applied to a phosphocellulose column (14 × 1.0 cm). Phosphoprotein kinases were eluted by steps of 0.1, 0.3, 0.5, 0.7 and 0.9 M NaC1 in 0.05 M Tris buffer (pH 7.5). The protein kinase Pk4 Pkl
12 Pk3 10
O3
(17
0.5
(19
8 X
.¢_ E 6
0
4 Pk2 f,
i!
~ab',
", 20
'~
,.%1
40
Pk5
',,~...Jl/~', 60
80
""-,/Ilk",, 100
120
Fraction number F i ¢ l . Nuclear protein kinase activities eluted from phosphocellulose column. Three mg of phosphoprot e i n f r a c t i o n f r o m n o r m a l rat liver ( ), N o v i k o f f h e p a t o m a ( . . . . . ) o r E h r l i c h a s c i t e s n u c l e i ( ° - - * ) w e r e a p p l i e d t o a p h o s p h o c e l l u l o s e c o l u m n a n d e l u t e d w i t h 0.1 M, 0.3 M, 0 . 5 M, 0.7 M a n d 0.9 M NaCI as i n d i c a t e d b y t h e a r r o w s . T h e f l o w r a t e w a s 0 . 5 - - 0 . 5 5 m l / m i n . O n e m l f r a c t i o n s w e r e c o l l e c t e d a n d t h e i r p r o t e i n k i n a s e a c t i v i t y w a s a s s a y e d as d e s c r i b e d in t h e t e x t .
116 activity was assayed in a reaction mixture which contained 30 pl of 0.43 M Tris (pH 7.5), 10 pl of 0.75 M MgC12, 50 pl of H20 and 200 pl of the phosphocellulose column fraction. 10 pl of [~.32p] ATP (0.6 pM/ml, 0.5 Ci/mM ATP) was added to each assay to start the reaction and the tubes were incubated at 30 °C for 10 min. The separation of phosphoproteins into five protein kinase fractions from normal rat liver is shown in Fig.1. This separation was repeated several times and gave extremely reproducible results. In order to compare the protein kinases from normal rat liver and neoplastic tissues, the Novikoff hepatoma and Ehrlich ascites enzymes were separated according to the described procedure (Fig.l). An additional enzymatic activity which eluted at the later part of the 0.3 M NaC1 elution step was found in both Novikoff h e p a t o m a and Ehrlich ascites phosphoproteins. A Similar peak of nuclear protein kinase activity was also obtained from Walker carcinosarcoma (a solid tumor). The t u m o r associated phosphoprotein kinase was n o t detected in the cytosol of either normal rat liver or neoplastic tissues, implying that this enzyme fraction is a true nuclear protein kinase and n o t a cytoplasmic contaminant. Because of the stepwise elution, the question arises if the five kinase fractions from normal rat liver and the additional enzyme from tumors do indeed represent individual protein kinase enzymes. There are three lines of evidence (details will be published elsewhere) which indicate that these fractions are distinctly different. First, each fraction requires different optimal concentration of Mg 2+ and Mn 2÷ (Table I). Second, they differ in their phosphorylation of total nonhistone proteins, histones and casein (Table I). Third, they phosphorylate different species of chromosomal nonhistone proteins as can be demonstrated by polyacrylamide gel electrophoresis. The additional enzyme fraction (phosphoprotein kinase 2b) isolated from neoplastic tissues eluted from the phosphocellulose column in almost identical position for each t u m o r and represented 3--12% of the total kinase activity. The activity of this enzyme fraction was greatly stimulated {138--400% higher) when assayed in the presence of Mn 2+ instead of Mg 2÷. Conversely, there was only 30--60% of the original activity retained when Mg 2+ was replaced in the other five fractions of phosphoprotein kinases from normal or neoplastic tissues (Table I). Although the Mn 2+ stimulated kinase present in neoplastic tissues has not been reported previously, phosphorylation of specific protein spots detectable b y t w o dimensional gel electrophoresis of Novikoff h e p a t o m a nuclear proteins was described when the phosphorylation was performed in the presence of Mn 2÷ [ 2 2 ] . When the t u m o r specific kinase phosphoprotein kinase 2b isolated from Ehrlich ascites was assayed in the presence of endogenous substrate (heatinactivated phosphoprotein) from Ehrlich ascites its activity was 950--1500% higher than when assayed in the presence of endogenous substrate alone. On the other hand when the phosphoprotein kinase 2b enzyme was assayed in the presence of heat-inactivated phosphoproteins from normal rat liver, the enzyme activity was only 96% of that assayed in the presence of endogenous* *i.e. e n z y m e and h o m o l o g o u s p h o s p h o p r o t e i n s .
I
OF NUCLEAR
PROTEIN
KINASE
(PK) FRACTIONS
FROM
PHOSPHOCELLULOSE
COLUMN
146 2145 1930
102 373 298
. 275 359
246 750 543
306 360 415
134 212 149
NRL NH EA
NRL NH EA
NRL NH EA
NRL NH EA
NRL NH EA
PK 2
PK~
PK 3
PK 4
PK s
Histories
.
143 152 201
400 301 210
294 181 475
950 1500
90 122 138
103 308 345
. -64
539 -304
1161 -469
156 -253
.
170 -499
160 -156
Casein
activity with
Nonhistones
(a) % of endogenous addition of
NRL NH EA
Tissue
PK,
Enzyme peak
10 25 10
25 25 20
25 10 20
. 20 20
20 10 25
10 10 10
(b) OPtimal M g 2+ c o n c n
.
0.6 0.9 --
1.6 1.0 --
0.8 0.7 --
1.4 1.4
1.6 1.2 --
0.8 1.0 --
(c) Optimal M n 2+ c o n c h
34 44 60
42 42 25
53 111 99
210 269
66 113 115
49 56 61
Average ratio of activity at optimal M n 2÷ a n d M g 2+ c o n c n s (Mn/Mg)
The conditions for phosphoprotein kinase assays described in the text, except that inactivated exogenous substrates (a), or d i f f e r e n t c o n c . o f M g 2+ ( b ) o r M n 2÷ ( c ) w e r e u s e d . T h e v a l u e s a r e a v e r a g e s o f 2 - - 4 e x p e r i m e n t s . N R L , n o r m a l r a t l i v e r ; NH, Novikoff hepatoma; EA, Ehriich s~cites.
CHARACTERIZATION
TABLE
118
10001
750
a
b
t
1
d
e
c-
o
250
Gel
length(cm )
Fig.2. The radioactivity profiles in p o l y a c r y l a m i d e gels o f h e a t e d rat liver p h o s p h o p r o t e i n s (. . . . -7 and o f h e a t e d Ehrlich ascites p h o s p h o p r o t e i n s ( ) w h i c h have b e e n p h o s p h o r y l a t e d w i t h the p r o t e i n kinase fraction PK 2b isolated f r o m Ehrlich ascites nuclei. 200/~l o f the p r o t e i n kinase PK 2b w e r e i n c u b a t e d w i t h 120 mg o f h e a t i n a c t i v a t e d p h o s p h o p r o t e i n , 43 mM Tris, 1.4 mM MnCI~ and 10 ~l of [T-32P] ATP s o l u t i o n (0.6/~M/ml, 0,5 Ci/mM) in final v o l u m e 300 ~tl. After 10 min at 30 C, the reaction was t e r m i n a t e d b y the a d d i t i o n of ultra-pure urea to the final c o n c e n t r a t i o n o f 4 M. T h e t u b e s w e r e well mixed and the samples d i a l y z e d against e l e c t r o p h o r e t i c sample b u f f e r (0.01 M s odi um p h o s p h a t e buffer, pH 7.0, 8 M urea, 1.2% sodium dodecyl sulfate, 10% ~ - m e r c a p t o e t h a n o l ) . T h e samples were s u b j e c t e d to p o l y a c r y l a m i d e gel electrophoresis [ 2 3 ] . The m o l e c u l a r w e i g h t markers are indicated b y the arrows (a--c). a, h e m o g l o b i n (160 000); h, b o v i n e s e r u m a l b u m i n (67 000); c, ovalbumin (45 000); d, c hymotrypsinogen (25 0007; e, c y t o c b r o m e c (12 4007.
substrate. After incubation with the phosphoprotein kinase 2b fraction from Ehrlich ascites tumor, the heat-inactivated phosphoproteins from Ehrlich ascites nuclei showed phosphorylation of one major protein region detectable by polyacrylamide gel electrophoresis (Fig.2). There was virtually no phosphorylation of heat-inactivated phosphoproteins from normal rat liver by this enzyme. When heat-inactivated phosphoproteins from Ehrlich ascites were incubated with the individual protein kinase fractions from normal rat liver, no phosphorylation of the Ehrlich ascites phosphoproteins could be detected in the region where the protein peak phosphorylated by the fraction phosphoprotein kinase 2b was found. Based on the experiments described here we conclude that a unique nuclear protein kinase fraction and a specific phosphoprotein substrate are present in neoplastic tissue nuclei which cannot be found in normal rat liver. Further characterization of this specific phosphoprotein kinase will be described in a full paper. Research described in this report was supported by the Grants from the U.S. Public Health Service (CA 07746) and the Eobert A. Welch Foundation
(G 138).
-
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Paul, J. (1970) Curt. Topics Develop. Biol. 5, 317--352. Stein, G.S., Spelsberg, T.C. an d IGeinsmith, L.J. (1974) Science 183, 817--824. Speisberg, T.C., Wilhelm, J.A. and Hnilica, L.S. (1972) Sub-cell Biochem. 1 , 1 0 7 - - 1 4 5 . Hnilica, L.S. (1972) The Structure and F u n c t i o n of Histone, pp.151--167. Baserga, P~. and Stein, G. (1971) Fed. Proc. 30, 1752--1759. Teng, C.S., Teng, C.T. and Allfrey, V.G. (1971) J. Biol. Chem. 246, 3597--3609. Kamiy ama, M., Dastugue, B. and Kruh, J. (1971) Biochim. Biophys. Res. C ommun. 44, 1345--1350. Kamiyama, M. an d Wang, T.Y. (1971) Biochim. Biophys. Acta 228, 563--576. Shea, M. and Kleinsmith, L.J. (1973) Biochem. Binphys. Res. Commun. 50, 473--477. Platz, R.D., Kish, V.M. and Kleinsmith, L.J. (1970) FEBS Lett. 12, 38--40. Kleinsmith, L.J., Heidema, J. and Carroll, A. (1970) Nature 226, 1025---1026. Wakabayashi, K., Wang, S., Hord, G. and Hnilica, L.S. (1973) FEBS Lett. 32, 46--48. Allfrey, V.G., Johnson, E.M., Karn, J. and Vidali, G. (1973) in Protein P h o s p h o r y l a t i o n in Control Mechanisms, (Juijing, F. and Lee, E.Y.C., eds), Vol. 5, pp.217--232, Miami Winter Symposia, Acad. Press. J u n g m a n n , R.A. and Schweppe, J.S. (1972) J. Biol. Chem. 247, 5535--5542. Chin, J.F., Craddock, C., Getz, S. and Hnilica, L.S. (1973) FEBS Lett. 33, 247--250. Chiu, J.F., Brade, W.P., Thomson, J., Tsai, Y.H. and Hnilica, L.S. (1975) Exp. Cell Res. 91, 200--206. Brade, W.P., Chin, J.F. and Hnilica, L.S. (1974) Mol. Pharmacol. 10, 398--405. Brade, W.P., Thomson, J.A., Chin, J.F. and Hni~ica, L.S. (1974) Exp. Cell Res. 84, 183--190 Kish, V.M. and Kleinsmith, L.J. (1974) J. Biol. Chem. 249, 750--760. Chauveau, J., Moule, Y. and Rouiller, C. (1956) Exp. Cell Res. 1 1 , 3 1 7 - - 3 2 1 . Wilhelm, J.A., Groves, C.M. an d Hnilica, L.S. (1971) Biochim. Biophys. Acta 238, 295--302. Kang, Y.J., Olson, M.O.J. and Busch, H. (1974) J. Biol. Chem. 249, 5580--5585. Wilhelm, J.A., Ansevin, A.T., Johnson, A.W. and Hnilica, L.S. (1972) Biochim. Biophys. Acta 272, 220--230.
A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 91417
NUCLEAR PHOSPHOPROTEIN KINASE ACTIVITIES IN NORMAL AND NEOPLASTIC TISSUES
JUDITH A. T H O M S O N , JEN-FU CHIU* and L U B O M I R S. HNILICA* Department of Biochemistry, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute,Houston, Texas 77025 (U.S.A.) (Received June 20th, 1975)
Summary Nuclear phosphoprotein kinases from normal rat liver and transplantable neoplasms were fractionated and compared. A phosphoprotein kinase fraction activated by Mn 2+ was found to be present only in the neoplasms. This nuclear protein kinase phosphorylated nuclear proteins represented by one major and several minor bands as determined by polyacrylamide gel electrophoresis (M ~ 50 000). Nonhistone chromosomal proteins, particularly the nuclear phosphoprotein fraction, exhibit many properties expected of gene regulatory macromolecules [1--5]. For example, phosphoproteins alter the rate of RNA synthesis in vitro [6--9], they are heterogenous and tissue specific [6,10], and they bind specifically to homologous DNA [6,9,11,12]. Changes in the phosphorylation of chromosomal nonhistone proteins could be correlated with activation of gene activity in a variety of systems [13--18]. In order to explore the possible regulatory function of the phosphorylated nonhistone proteins during neoplastic growth, we have investigated the changes in their phosphorylation during hepatocarcinogenesis [ 15] and in regenerating rat liver [16,17], and kidney [18]. The results of these experiments suggest that phosphorylated nonhistone proteins may be involved in effecting changes associated with neoplastic growth. It was shown that the enzymes responsible for nuclear protein phosphorylation, i.e. nuclear protein kinases are quite heterogenous [19] and their activities increase in response to genetic activation [15--18]. It appears that both the nuclear phosphoproteins and their kinases play an active role in gene regulation. The purpose of this investigation was to determine if any differences could be observed in fractionated protein kinase activities or protein kinase species between normal and neoplastic tissues. We report here that a * Present address: D e p a r t m e n t o f B i o c h e m i s t r y , Vanderbilt University S c h o o l o f M e d i c i n e , Nashville, Tenn. 37232. R e p r i n t r e q u e s t s s h o u l d b e m a i l e d t o this address.
115
tumor specific protein kinase has been characterized in Novikoff hepatoma and Ehrlich ascites nuclei. Liver nuclei from adult Sprague-Dawley rats {about 150 g) were prepared by the method of Chauveau et al. [20]. The nuclei of Novikoff hepatoma and Ehrlich ascites were isolated by the method of Wilhelm et al. [21]. Phosphoproteins and phosphoprotein kinases were isolated by the method described by Kish and Kleinsmith [19]. Briefly, isolated nuclei were homogenized in 0.14 M NaC1 to remove cytoplasmic contaminants and nuclear sap. The nonhistone proteins were extracted by homogenizing the nuclear pellets in equal vol. of 2 M NaC1, 0.05 M Tris, pH 7.5. The resulting solution was mixed with 1.5 vol. of 0.02 M Tris, pH 7.5, and the precipitated nucleohistone and nuclear debris were removed by centrifugation at 81 500 ×g for 1 h. The nuclear phosphoproteins and kinases were further purified by Bio-Rex 70 chromatography and calcium phosphate gel absorption as described by Kish and Kleinsmith [19]. Phosphoproteins and kinases isolated from nuclei were dialyzed overnight against 100 vol. of 0.1 M NaC1, 0.05 M Tris, pH 7.5, with 3 changes. Approximately 3 mg of protein were applied to a phosphocellulose column (14 × 1.0 cm). Phosphoprotein kinases were eluted by steps of 0.1, 0.3, 0.5, 0.7 and 0.9 M NaC1 in 0.05 M Tris buffer (pH 7.5). The protein kinase Pk4 Pkl
12 Pk3 10
O3
(17
0.5
(19
8 X
.¢_ E 6
0
4 Pk2 f,
i!
~ab',
", 20
'~
,.%1
40
Pk5
',,~...Jl/~', 60
80
""-,/Ilk",, 100
120
Fraction number F i ¢ l . Nuclear protein kinase activities eluted from phosphocellulose column. Three mg of phosphoprot e i n f r a c t i o n f r o m n o r m a l rat liver ( ), N o v i k o f f h e p a t o m a ( . . . . . ) o r E h r l i c h a s c i t e s n u c l e i ( ° - - * ) w e r e a p p l i e d t o a p h o s p h o c e l l u l o s e c o l u m n a n d e l u t e d w i t h 0.1 M, 0.3 M, 0 . 5 M, 0.7 M a n d 0.9 M NaCI as i n d i c a t e d b y t h e a r r o w s . T h e f l o w r a t e w a s 0 . 5 - - 0 . 5 5 m l / m i n . O n e m l f r a c t i o n s w e r e c o l l e c t e d a n d t h e i r p r o t e i n k i n a s e a c t i v i t y w a s a s s a y e d as d e s c r i b e d in t h e t e x t .
116 activity was assayed in a reaction mixture which contained 30 pl of 0.43 M Tris (pH 7.5), 10 pl of 0.75 M MgC12, 50 pl of H20 and 200 pl of the phosphocellulose column fraction. 10 pl of [~.32p] ATP (0.6 pM/ml, 0.5 Ci/mM ATP) was added to each assay to start the reaction and the tubes were incubated at 30 °C for 10 min. The separation of phosphoproteins into five protein kinase fractions from normal rat liver is shown in Fig.1. This separation was repeated several times and gave extremely reproducible results. In order to compare the protein kinases from normal rat liver and neoplastic tissues, the Novikoff hepatoma and Ehrlich ascites enzymes were separated according to the described procedure (Fig.l). An additional enzymatic activity which eluted at the later part of the 0.3 M NaC1 elution step was found in both Novikoff h e p a t o m a and Ehrlich ascites phosphoproteins. A Similar peak of nuclear protein kinase activity was also obtained from Walker carcinosarcoma (a solid tumor). The t u m o r associated phosphoprotein kinase was n o t detected in the cytosol of either normal rat liver or neoplastic tissues, implying that this enzyme fraction is a true nuclear protein kinase and n o t a cytoplasmic contaminant. Because of the stepwise elution, the question arises if the five kinase fractions from normal rat liver and the additional enzyme from tumors do indeed represent individual protein kinase enzymes. There are three lines of evidence (details will be published elsewhere) which indicate that these fractions are distinctly different. First, each fraction requires different optimal concentration of Mg 2+ and Mn 2÷ (Table I). Second, they differ in their phosphorylation of total nonhistone proteins, histones and casein (Table I). Third, they phosphorylate different species of chromosomal nonhistone proteins as can be demonstrated by polyacrylamide gel electrophoresis. The additional enzyme fraction (phosphoprotein kinase 2b) isolated from neoplastic tissues eluted from the phosphocellulose column in almost identical position for each t u m o r and represented 3--12% of the total kinase activity. The activity of this enzyme fraction was greatly stimulated {138--400% higher) when assayed in the presence of Mn 2+ instead of Mg 2÷. Conversely, there was only 30--60% of the original activity retained when Mg 2+ was replaced in the other five fractions of phosphoprotein kinases from normal or neoplastic tissues (Table I). Although the Mn 2+ stimulated kinase present in neoplastic tissues has not been reported previously, phosphorylation of specific protein spots detectable b y t w o dimensional gel electrophoresis of Novikoff h e p a t o m a nuclear proteins was described when the phosphorylation was performed in the presence of Mn 2÷ [ 2 2 ] . When the t u m o r specific kinase phosphoprotein kinase 2b isolated from Ehrlich ascites was assayed in the presence of endogenous substrate (heatinactivated phosphoprotein) from Ehrlich ascites its activity was 950--1500% higher than when assayed in the presence of endogenous substrate alone. On the other hand when the phosphoprotein kinase 2b enzyme was assayed in the presence of heat-inactivated phosphoproteins from normal rat liver, the enzyme activity was only 96% of that assayed in the presence of endogenous* *i.e. e n z y m e and h o m o l o g o u s p h o s p h o p r o t e i n s .
I
OF NUCLEAR
PROTEIN
KINASE
(PK) FRACTIONS
FROM
PHOSPHOCELLULOSE
COLUMN
146 2145 1930
102 373 298
. 275 359
246 750 543
306 360 415
134 212 149
NRL NH EA
NRL NH EA
NRL NH EA
NRL NH EA
NRL NH EA
PK 2
PK~
PK 3
PK 4
PK s
Histories
.
143 152 201
400 301 210
294 181 475
950 1500
90 122 138
103 308 345
. -64
539 -304
1161 -469
156 -253
.
170 -499
160 -156
Casein
activity with
Nonhistones
(a) % of endogenous addition of
NRL NH EA
Tissue
PK,
Enzyme peak
10 25 10
25 25 20
25 10 20
. 20 20
20 10 25
10 10 10
(b) OPtimal M g 2+ c o n c n
.
0.6 0.9 --
1.6 1.0 --
0.8 0.7 --
1.4 1.4
1.6 1.2 --
0.8 1.0 --
(c) Optimal M n 2+ c o n c h
34 44 60
42 42 25
53 111 99
210 269
66 113 115
49 56 61
Average ratio of activity at optimal M n 2÷ a n d M g 2+ c o n c n s (Mn/Mg)
The conditions for phosphoprotein kinase assays described in the text, except that inactivated exogenous substrates (a), or d i f f e r e n t c o n c . o f M g 2+ ( b ) o r M n 2÷ ( c ) w e r e u s e d . T h e v a l u e s a r e a v e r a g e s o f 2 - - 4 e x p e r i m e n t s . N R L , n o r m a l r a t l i v e r ; NH, Novikoff hepatoma; EA, Ehriich s~cites.
CHARACTERIZATION
TABLE
118
10001
750
a
b
t
1
d
e
c-
o
250
Gel
length(cm )
Fig.2. The radioactivity profiles in p o l y a c r y l a m i d e gels o f h e a t e d rat liver p h o s p h o p r o t e i n s (. . . . -7 and o f h e a t e d Ehrlich ascites p h o s p h o p r o t e i n s ( ) w h i c h have b e e n p h o s p h o r y l a t e d w i t h the p r o t e i n kinase fraction PK 2b isolated f r o m Ehrlich ascites nuclei. 200/~l o f the p r o t e i n kinase PK 2b w e r e i n c u b a t e d w i t h 120 mg o f h e a t i n a c t i v a t e d p h o s p h o p r o t e i n , 43 mM Tris, 1.4 mM MnCI~ and 10 ~l of [T-32P] ATP s o l u t i o n (0.6/~M/ml, 0,5 Ci/mM) in final v o l u m e 300 ~tl. After 10 min at 30 C, the reaction was t e r m i n a t e d b y the a d d i t i o n of ultra-pure urea to the final c o n c e n t r a t i o n o f 4 M. T h e t u b e s w e r e well mixed and the samples d i a l y z e d against e l e c t r o p h o r e t i c sample b u f f e r (0.01 M s odi um p h o s p h a t e buffer, pH 7.0, 8 M urea, 1.2% sodium dodecyl sulfate, 10% ~ - m e r c a p t o e t h a n o l ) . T h e samples were s u b j e c t e d to p o l y a c r y l a m i d e gel electrophoresis [ 2 3 ] . The m o l e c u l a r w e i g h t markers are indicated b y the arrows (a--c). a, h e m o g l o b i n (160 000); h, b o v i n e s e r u m a l b u m i n (67 000); c, ovalbumin (45 000); d, c hymotrypsinogen (25 0007; e, c y t o c b r o m e c (12 4007.
substrate. After incubation with the phosphoprotein kinase 2b fraction from Ehrlich ascites tumor, the heat-inactivated phosphoproteins from Ehrlich ascites nuclei showed phosphorylation of one major protein region detectable by polyacrylamide gel electrophoresis (Fig.2). There was virtually no phosphorylation of heat-inactivated phosphoproteins from normal rat liver by this enzyme. When heat-inactivated phosphoproteins from Ehrlich ascites were incubated with the individual protein kinase fractions from normal rat liver, no phosphorylation of the Ehrlich ascites phosphoproteins could be detected in the region where the protein peak phosphorylated by the fraction phosphoprotein kinase 2b was found. Based on the experiments described here we conclude that a unique nuclear protein kinase fraction and a specific phosphoprotein substrate are present in neoplastic tissue nuclei which cannot be found in normal rat liver. Further characterization of this specific phosphoprotein kinase will be described in a full paper. Research described in this report was supported by the Grants from the U.S. Public Health Service (CA 07746) and the Eobert A. Welch Foundation
(G 138).
-
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
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