Adrenocortical Carcinoma Protein Kinase, Autophosphorylating Cyclic AMP-binding Protein Kinase 134 PURIFICATION AND CHARACTERIZATION*
(Received for publication, September 7, 1979)
Gouri Shanker and Rameshwar K. Sharma From the Department of Biochemistry, University of Tennessee, Center for the Health Sciences, Memphis, Tennessee 38163
Recently, investigations in this laboratory revealed an autophosphorylating cyclic AMP-binding protein kinase, autophosphorylating cyclic AMP-binding pro- tein kinase 85, in adrenocortical carcinoma (Shanker, G., Ahrens, H., and Sharma, R. K. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 66-70). We describe here the purification to apparent homogeneity of another pro- tein kinase (designated autophosphorylating cyclic AMP-binding protein kinase 134) from adrenocortical carcinoma 494, as evidenced by sodium dodecyl sulfate- polyacrylamide gel electrophoresis.
Autophosphorylating cyclic AMP-binding protein ki- nase 134 binds cyclic AMP (cAMP) and autophosphor- ylates, but it does not use histone, phosphorylase b, casein, or polysomes as a substrate in the presence or absence of cAMP. Stoichiometry of phosphate incor- poration was 0.72 mol/mol of enzyme. The enzyme was found to have a molecular weight of 134,000 based on gel filtration. The protein was composed of polypep- tides having the same molecular weight, 67,000, and thus it appears to consist of two subunits of equal size. The enzyme bound 2 cAMP molecules, indicating that each subunit binds 1 molecule of cAMP. The homoge- neous enzyme did not inhibit the protein kinase activity of the free catalytic subunit of normal adrenal cAMP- dependent protein kinase under experimental condi- tions that allowed recombination with the free regula- tory subunit. cAMP bound specifically to the enzyme with an apparent dissociation constant (Ka) of 1.2 x 10-8 M. Scatchard plot data indicated one type of bind- ing site for cAMP. The enzyme did not bind adenosine. This novel class of autophosphorylating, cAMP-bind- ing, protein kinases may be a characteristic of certain adrenal neoplasms.
Studies with adrenocortical carcinoma 494 (1) have dem- onstrated that in contrast to that which occurs in the normal adrenal cortex, neither corticotropin nor cAMP stimulate the formation of corticosterone (for a review, see Ref. 2). Molec- ular studies designed to investigate the causes of this abnor- mality have revealed that the steroidogenic route (20 S)-20- hydroxycholesterol → pregnenolone → progesterone -+ deox- ycorticosterone → corticosterone, is at least partly intact in the tumor cell (3-7). Based on the premise that a cAMP- dependent protein kinase (8) is involved in cAMP-activated
adrenal steroidogenesis, it was postulated (3-5) that the defect in the tumor may be due to a defect in the cAMP-dependent protein kinase system. The presence of a crude protein kinase that specifically bound cAMP, but was unable to phosphoryl- ate exogenous histone in the presence or absence of the nucleotide, was demonstrated (9). Recently, we purified to apparent homogeneity, a self-phosphorylating cAMP-binding protein kinase, AUT-PK’ 85, from adrenocortical carcinoma (10). This novel protein kinase could be differentiated from the known cAMP-binding protein kinase on the basis of the following properties: (a) it had a different molecular weight; (b) it did not possess different regulatory and catalytic sub- units; (c) it did not phosphorylate the exogenous substrates such as histone, casein, or phosphorylase b in the presence or absence of cAMP; and (d) it self-phosphorylated. The present report describes the purification to homogeneity and charac- terization of another enzyme, designated AUT-PK 134, from adrenocortical carcinoma. Except for its molecular weight, AUT-PK 134 has almost identical kinetic properties to AUT- PK 85.
EXPERIMENTAL PROCEDURES
Animals-Adrenocortical carcinoma 494, a spontaneously occur- ring tumor discovered by Snell and Stewart (1) and maintained in our laboratory (11), was used for purification of the enzyme.
Materials-Cyclic nucleotides, phosphorylase b, calf thymus his- tone (type IIA), and Tris-base were from Sigma Chemical Co .; bovine serum albumin (mixture of dimer and trimer) was from Miles Labo- ratories, Inc .; bovine serum albumin (monomer), aldolase, ovalbumin, and ribonuclease were from Pharmacia Fine Chemicals, Inc .; and Escherichia coli RNA-polymerase was a gift from Dr. R. R. Burgess, McArdle Laboratories, Madison, Wisc. Ammonium sulfate (enzyme grade) was from Schwarz/Mann; DEAE-cellulose (DE 52) and GF/C glass fiber microfilters were from Whatman Biochemicals Ltd .; Ultra- gel ACA-34 was from LKB Instruments, Inc .; reagents for polyacryl- amide gel electrophoresis were from Bio-Rad Laboratories; and tol- uene (scintillation grade) was from Fisher Scientific Co. [y-312PJATP (3000 Ci/mmol) was from Amersham Corp .; and cyclic [3H]GMP (40 Ci/mmol), cyclic [H]AMP (40 Ci/mmol) and Omnifluor were from New England Nuclear. All other chemicals were reagent grade and were obtained commercially.
The cAMP-binding and protein kinase activities were determined as described previously (10). All assays were performed in duplicate and the data were corrected for background radioactivity determined from samples of the complete reaction mixture without enzyme.
Protein concentrations were measured by the method of Lowry et al. (12) or by the fluorescamine assay (13).
For electrophoresis in a nondenaturing system, polyacrylamide gels were prepared essentially as described (14, 15) with some modification (10). Denaturing polyacrylamide gels containing 0.1%, sodium dodecyl sulfate were prepared by the method of Favre and Laemli (16) as modified by Engbalk et al. (17).
* This investigation was supported by Grant CA 16091 from the National Cancer Institute and Grant PCM 7800860 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
’ The abbreviation used is: AUT-PK, autophosphorylating cyclic AMP-binding protein kinase.
Photoaffinity labeling of cAMP with AUT-PK 134 was performed as previously described (10, 18). The reaction mixture was analyzed by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for determination of the molecular weight and subunit composition of the enzyme.
Purification-All procedures were carried out at 0-4℃. The steps of purification were essentially the same as described for AUT-PK 85 (10).
Adrenocortical carcinoma tissue was homogenized with 200 ml of 20 mM Tris-HCI, pH 7.5, containing 5 mM 2-mercaptoethanol and 0.2 M EDTA (TME buffer) in a Waring Blendor for 2 to 3 min. The homogenate was centrifuged for 45 min at 30,900 x g, and the supernatant was brought to 50% saturation by the addition of solid ammonium sulfate. The precipitate was centrifuged at 30,000 x g for 45 min, then dissolved in 10% (w/v) glycerol in TME buffer (TMGE buffer) and extensively dialyzed against TMGE buffer. The dialyzed sample was fractionated on a DEAE-cellulose column with a linear NaCl gradient (0.04 to 0.25 M), and the fractions were assayed for binding and cAMP-dependent kinase activity (Fig. 1, top). Two dis- tinct cAMP-binding peaks were obtained: one eluting at 0.10 M NaCl, and the other eluting at 0.17 M NaCl. The former was used for further purification. No cAMP-dependent kinase activity was detected in any of the column fractions.
The cAMP-binding peak fractions eluted with 0.10 M NaCl were chromatographed on hexyl-Sepharose (19). This removed most of the high molecular weight and hydrophilic proteins (Fig. 1, bottom). The cAMP-binding peak fractions were pooled and dialyzed against TMGE buffer. cAMP-dependent kinase activity, as assessed by the phosphorylation of histones, was not detected in any of these frac- tions. Complete purification of the tumor enzyme was achieved when the dialyzed enzyme activity from the hydrophobic chromatography
DEAE-CELLULOSE
[32P] INCORPORATED (pmol /50ul) ++
100
IO
25
250
80
8
20
200
60
6
5
150
40
CYCLIC [3H] AMP BOUND (cpm x 10”3/50ul)
4
1.O
ABSORBANCE AT 280 nm
100
20
2
05
50
NaCl(mM)
HEXYL -SEPHAROSE
3
200
2
04
160
03
120
02
80
01
40
20
40
60
80
100
FRACTION NUMBER
| TABLE I Purification of AUT-PK 134 | ||||
|---|---|---|---|---|
| Step | Protein | cAMP-binding ac- tivity | Purifi- cation | |
| Total | Specific | |||
| mg | nmol | nmol/mg | -fold | |
| 30,900 × g supernatant | 2500 | 7.50 | 0.003 | 1 |
| Ammonium sulfate pellet | 410 | 5.85 | 0.014 | 5 |
| DEAE-Cellulose | 23.33 | 2.10 | 0.090 | 30 |
| Hexyl-Sepharose | 1.35 | 1.35 | 0.996 | 332 |
| Affinity | 0.034 | 0.47 | 13.980 | 4660 |
A
B
Mx 10-3
SLICE NUMBER
160
10
AUT-PK 134
B.B
σ
BSA
90
20
68
AUT-PK 67
30
a
40
40
2
DYE
O
2
4
6
8
CYCLIC [3H] AMP BOUND
(cpm × 10-2)
was adsorbed to a 10-ml N6-(2-aminoethyl) amino-cAMP-Sepharose column (20). Neither binding nor kinase activity could be detected in the column effluent.
To remove all impurities, the cAMP-Sepharose resin was washed extensively with TMGE buffer containing 0.1, 0.5, 1.0, and 2.0 M NaCl, and the tumor protein kinase was recovered by incubating the resin with 50 ml of 20 mM cAMP in TMGE buffer at room temperature. The extracts were concentrated by adsorption to a 5-ml DEAE- cellulose column followed by a step-off elution with TMGE containing 0.5 M NaCl. The eluate was dialyzed extensively against TMGE buffer. A dialysis bag containing charcoal was suspended in the final buffer. Our preliminary experiments with the adrenal cAMP-depend- ent protein kinase and regulatory subunit indicated that this method removes essentially all bound cAMP. This conclusion was based on the fact that the treated and untreated holoenzyme showed the same capacity of cAMP binding and stimulation of kinase activity by the nucleotide. Furthermore, when the experiment was conducted with [3H]cAMP bound to the receptor the dialyzed preparation did not show any radioactivity indicating that all cAMP had dissociated from the regulatory subunit. Table I summarizes the purification proce- dure.
RESULTS
Molecular Weight and Purity-The AUT-PK 134 isolated by affinity chromatography yielded a single stained band of 67,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating apparent homogeneity (Fig. 2B). On a Bio-Gel P-150 gel filtration column, AUT-PK 134 eluted in the fraction corresponding to 134,000 daltons, indicating that the enzyme exists as a dimer (Fig. 3).
To ascertain that the stained band obtained after electro- phoresis was the protein that binds cAMP, the 3H-labeled nucleotide was incorporated covalently by photolysis (10, 18) in the purified AUT-PK 134. The radioactivity was associated with a protein band at 67,000 daltons (Fig. 2A), confirming the molecular weight and establishing that the protein isolated
by affinity chromatography was the cAMP-binding protein. In accord with the previous results (10), analysis of the regu- latory subunit from the normal adrenal cortex enzyme showed that the label was associated with a protein band at 49,000 daltons. No 67,000-dalton cAMP band was detected in the normal adrenal extract.
cAMP Binding Characteristics-Studies of the stoichi- ometry of cAMP binding with the homogeneous preparation of AUT-PK 134 showed that this enzyme bound 2 mol of
P-150 COLUMN
20
15
Vr
. BSA
AUT-PK
₹
MOLECULAR WEIGHT, x 10
10
R
5
OVALBUMIN
4
3
2
RNase
1
Q
20
40
60
80
FRACTION NUMBER
SCATCHARD - PLOT
3.0
CYCLIC[ H]AMP BOUND, x 108 M
2.5
Kg* 1.05 x 10 8 M
2.0
1.5
1.0
0.5
0
0.5
1.0
1.5
2.0
2.5
3.0
B
F
CYCLIC[ HJAMP BOUND (cpm x 10
4-4-[32p]INCORPORATED (cpm x 10”2)
10
10
8
8
6
6
4
4
2
2
0
10
20
30
40
50
60
70
SLICE NUMBER
cAMP/mol of the enzyme, indicating that each subunit binds cAMP in a 1:1 mole ratio.
Scatchard plot representation of cAMP binding yielded a straight line, indicating a single set of binding sites (Fig. 4). The apparent dissociation constant, Ka, calculated from the slope was 1.05 × 10-8 M.
The receptor of AUT-PK 134 exhibited high specificity for cAMP. Only cyclic IMP (cIMP) competed to a small extent (20%) when the binding of radioactive cAMP was assayed in the presence of a 500-fold excess of unlabeled cyclic GMP (cGMP), cIMP, cyclic UMP (cUMP), or cyclic CMP (cCMP). The binding of [3H]adenosine with the enzyme also was eval- uated, but no affinity for the nucleoside was found. A study of the kinetics of cAMP-binding showed that half of the binding sites became saturated within 5 min and complete saturation occurred within 30 min (data not shown).
The pH range for effective cAMP binding by AUT-PK 134 was between 4 and 7.
Phosphorylation of Histones-It is well established that cAMP-dependent protein kinases from different sources phos- phorylate histones (20-22). However, with AUT-PK 134, the amount of 32P incorporated in the presence or absence of histones, casein, phosphorylase b, or adrenal polysomes was the same, indicating that these proteins did not serve as substrates. This property of the enzyme is identical with that of AUT-PK 85 (10).
Autophosphorylation Studies-Our previous study de- scribed the property of self-phosphorylation of AUT-PK 85 (10). To investigate if this property is shared by AUT-PK 134, a hexyl-Sepharose fraction of the enzyme was preincubated with either [H]cAMP of [y-32P]ATP and analyzed by non- denaturing electrophoresis. The tumor protein that bound cAMP co-migrated with the phosphorylated protein, indicat- ing that the enzyme underwent autophosphorylation (Fig. 5). When purified AUT-PK 134 was subjected to nondenaturing electrophoresis the enzyme extracted from the gel showed cAMP-binding and kinase activity thus supporting the evi- dence for self-phosphorylation of the enzyme. It is noteworthy that protein kinase from normal adrenal cortex does not autophosphorylate (10).
Fig. 6 shows the time course for autophosphorylation of the homogeneous AUT-PK 134. Addition of cAMP had no effect on the rate of phosphorylation or on the amount of P incorporated. Stoichiometry of phosphate incorporation was
[32p]INCORPORATED (pmoles)
180
AL-CAMP
150
120
90
60
30
+cAMP
0
5
10
15
20
25
30
MINUTES
12
AUT-PK
.
[32P] INCORPORATED
8
(cpm x 10-2 )
NORMAL “R
4
.
·
0
0.01
0.10
CAMP (PM)
1.0
5
10
0.72 mol/mol of the enzyme. When AUT-PK 134 was incu- bated with cAMP in the presence of cAMP-dependent protein kinase inhibitor (23), no decrease in the rate of phosphoryla- tion was observed (Fig. 6). Furthermore, the rate of phospho- rylation was unaffected by different dilutions of the enzyme. This proved that self-phosphorylation was an intrinsic prop- erty of the enzyme. Analogous to the terminology used for AUT-PK 85, we have named the enzyme “autophosphorylat- ing cAMP-binding protein kinase, AUT-PK 134.”
Reconstitution Studies-To examine if AUT-PK 134 has any affinity for binding with the catalytic subunit of normal adrenal cortex protein kinase, reconstitution with the catalytic subunit was attempted. Incubation of the free catalytic sub- unit with AUT-PK 134 in the absence and presence of varying concentrations of cAMP had no effect on the amount of 32P incorporated into histones. This indicated that the tumor kinase cannot reconstitute with the normal catalytic subunit to form an inactive holoenzyme. In contrast, a cAMP-respon- sive holoenzyme could be readily reconstituted from the nor- mal regulatory and catalytic subunit (Fig. 7). This property of AUT-PK 134 again is identical with that of AUT-PK 85 (10).
DISCUSSION
This paper describes the presence of a self-phosphorylating cAMP-binding protein kinase, AUT-PK 134, in adrenocortical carcinoma. This enzyme has been purified 5000-fold to appar- ent homogeneity as evidenced by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. Except for the differences in molecular weight, AUT-PK 134 has almost identical kinetic properties to AUT-PK 85, another enzyme isolated from this neoplastic tissue (10).
AUT-PK 134 appears to be a dimeric molecule with a
molecular weight of 134,000. The stoichiometry of cAMP binding with the enzyme showed that it binds 2 mol of cAMP/ mol of protein. This indicates that each receptor chain binds 1 mol of cAMP.
The binding of cAMP with the tumor enzyme was specific and exhibited one type of binding site as evidenced by the Scatchard plot. Analogous to AUT-PK 85, but in contrast to the normal adrenal protein kinase, the tumor enzyme lacked cAMP-dependent protein kinase activity in the phosphoryla- tion of histones. The enzyme also was unable to phosphorylate other exogenous substrates, such as casein or phosphorylase b, in the presence or absence of cAMP. On the other hand, AUT-PK 134 self-phosphorylates and this process is not in- hibited by cAMP-dependent protein kinase inhibitor (23). This indicates that autophosphorylation is the intrinsic prop- erty of the enzyme. The stoichiometry of phosphate incorpo- ration was 0.72 mol/mol of the enzyme.
AUT-PK 134, similar to AUT-PK 85, does not inhibit the phosphotransferase activity of the free catalytic subunit of normal cAMP-dependent protein kinase. This property of the tumor enzyme, along with the other properties discussed above, differentiate it from the normal cAMP-dependent pro- tein kinase.
The demonstration of the presence of AUT-PK 85 (10) and AUT-PK 134 in adrenocortical carcinoma, establishes a new class of cAMP-binding protein kinases. Inclusion of the AUT- PKs with the already known cAMP-, cGMP-, calcium-de- pendent, and cyclic nucleotide-independent casein protein kinases would constitute the existence of five types of kinases in nature. The properties of these kinases are different from each other. cAMP-dependent protein kinase is a holoenzyme that consists of one regulatory subunit dimer and two catalytic subunit monomers (24-28). The enzyme is activated by bind- ing with 2 mol of the cyclic nucleotide, although there is recent evidence that 4 mol of cAMP bind with 1 mol of the enzyme (29, 30). On the other hand, cGMP-dependent protein kinase is not dissociated by cGMP, and the enzyme is com- posed of two nondissociable subunits (31-34). The third cal- cium-dependent protein kinase is neither stimulated nor in- hibited by cyclic nucleotides (35). Instead, this enzyme ap- pears to be selectively activated by a heat-stable membrane- associated macromolecule. Another type of calcium-depend- ent protein kinase that is activated by calmodulin (36) also has been reported (37, 38). A fourth type of cyclic nucleotide- independent casein kinase is markedly stimulated by basic polypeptides such as polylysine, polyarginine, or histone in the phosphorylation of endogenous cytoplasmic proteins (39). These four classes of kinases are able to phosphorylate various exogenous substrates including histones, whereas the AUT- PK enzymes use themselves as substrates. At this time, it is not known whether the neoplastic cell has two separate genes or a single gene for these enzymes. In the latter case, the tumor would have the ability to produce AUT-PK 85 and AUT-PK 134 by modifying either the mRNA or the protein whose sequences are specified by the common gene.
At present, we cannot assign any physiological role to the AUT-PK enzymes. Therefore, the relationship of these pro- tein kinases with the adrenal neoplasia is not clear. Before such a correlation can be established, it would be important to demonstrate the complete absence of these enzymes in the normal adrenal cortex. To date, we have been unable to detect these enzymes in postmitochondrial supernatant fractions of this tissue. Furthermore, we have been unable to detect nor- mal cAMP-dependent protein kinase in the postmitochondrial cytosolic fraction of the tumor. In order to completely rule out the presence of the AUT-PK enzymes in the normal adrenal cortex, it will be necessary to verify their absence by
other sensitive techniques such as radioimmunoassay. If in- deed this enzyme is unique to neoplasia, it is tempting to speculate that the biochemical lesion responsible for the lack of cAMP-activated corticosterone synthesis in the tumor is due to the presence of a class of variant protein kinase enzymes such as AUT-PK 85 and AUT-PK 134. This would represent a new control system that is unique to the tumor.
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