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Expression of autophosphorylating protein kinase 500 in normal and neoplastic rat cells
(RIA/normal and tumor expression)
GIPSY MAJUMDAR*, MICHAEL CASHEL+, AND RAMESHWAR K. SHARMA ** #
*Department of Biochemistry, University of Tennessee, Center for the Health Sciences, Memphis, TN 38163; and tLaboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20205
Communicated by Van Rensselaer Potter, April 18, 1985
ABSTRACT Autophosphorylating protein kinase 500 (AUT-PK 500) is a unique serine protein kinase that was originally purified and characterized from the rat adrenocorti- cal carcinoma. A specific RIA with an assay sensitivity of 10 ng (0.02 pmol) was developed for AUT-PK 500 and applied to normal, embryonic, fetal, neonatal, immortal, and neoplastic tissues and cultured cells. As compared to normal rat tissues, the expression of AUT-PK 500 is elevated 100-fold in sponta- neously occurring adrenocortical carcinoma 494, 50- to 60-fold in four chemically induced, rapidly growing hepatomas, 30- fold in the chemically induced mammary carcinoma, 20-fold in the cultured hepatoma cell line, and 4-fold in the Rat I and Rat II established tissue culture cell lines. There was also a 5-fold increase in the enzyme when freshly cultured rat skin epithelial- like cells were established. Furthermore, in vivo studies showed that when the rat liver was chemically transformed into its premalignant altered foci, there was a 7-fold elevation of AUT-PK 500. Embryonic cells and fetal and neonatal tissues contained barely detectable (<0.22 µg/mg of protein) amounts of the protein kinase. These results suggest that AUT-PK 500 is not involved in the differentiation process during fetal development but may be elevated during early steps of carcin- ogenesis and is further elevated during later stages.
Recently, we reported (1) on the purification and character- ization of a unique protein kinase, autophosphorylating protein kinase 500 (AUT-PK 500), from the rat adrenocortical carcinoma. The M, of AUT-PK 500 is 481,400. It is composed of two identical subunits, each with a M, of 250,000. The enzyme is acidic with a pI of 4.6. AUT-PK 500 is unique. In contrast to the previously characterized cyclic nucleotide- dependent, or -independent, protein kinases, it is not regu- lated by the cyclic nucleotides and is not stimulated by calcium or calcium/calmodulin. It is also distinct from other cyclic nucleotide-independent protein kinases such as casein kinases I and II (2, 3), the hemin-controlled repressor (4-8), the double-stranded RNA-activated inhibitor (4, 9, 10), protein kinase 380 (11), protease-activated kinases I and II (12, 13), and tyrosine kinases, since it does not catalyze the phosphorylation of substrates such as casein, eukaryotic initiation factor 2, histones, and its own tyrosine residue(s). Instead, the enzyme catalyzes the phosphorylation of its own serine residue and is specific for the phosphorylation of M. 31,000 ribosomal protein (1).
In this paper we describe the development of a specific RIA for AUT-PK 500 and show that the elevation of the enzyme can be associated with one or more steps of malignancy.
MATERIALS AND METHODS
Materials. Na125I (17.4 Ci/mg; 1 Ci = 37 GBq) was purchased from New England Nuclear and [ y-32P]ATP (3000 Ci/mmol) was from Amersham. Goat anti-rabbit IgG was obtained from Cappel Laboratories. All other reagents were analytical grade and were obtained commercially.
Preparation of Protein Kinases. The catalytic subunit of cyclic AMP-dependent protein kinase, and the type I and II regulatory subunits of cyclic AMP-dependent protein kinase, from rabbit skeletal muscle and bovine heart, were prepared by the methods of Beavo et al. (14) and Dills et al. (15), respectively. Cyclic GMP-dependent protein kinase was purified from bovine adrenal cortical tissue as described (16). Protein kinase 380 was purified from bovine adrenal glands according to the procedure of Kuroda and Sharma (17).
Tumor and Cell Lines. The fast-growing rat hepatomas 5132 TC, 7800, 7777, and 3924 A were obtained from Wayne Criss, under contract from the National Cancer Institute. Estab- lished rat hepatoma as well as established, Rat I, Rat II, and pituitary cell lines (18) were obtained from Patricia Earl (National Cancer Institute). Dimethylbenz[a]anthracene-in- duced rat mammary carcinoma cultured cell lines were obtained from Y. Cho-Chung (National Cancer Institute).
The adrenocortical carcinoma 494 cell line is a cloned cell line maintained in our laboratory (1). The skin and embryonic cells were adapted to continuous culture. To initiate the rat skin cultures, normal 5-week-old Sprague-Dawley rats were decapitated and the abdominal skin was removed, trimmed free of hair, washed in Dulbecco’s modified Eagle’s medium, and finely minced. Epithelial-like cell monolayers developed from the minces were incubated in stationary culture in Dulbecco’s modified Eagle’s medium with 10% calf serum at 37℃. These have been passaged weekly with the use of 0.05% trypsin at transfer ratios of 1:12. To initiate the rat embryonic cultures, embryos of =2 weeks’ gestation were used. Soft internal tissue, principally liver, was removed and dispersed by consecutive passage through 16-, 18-, and 21-gauge needles. The resulting cell suspension formed monolayers of polygonal-shaped cells in stationary cultures in Dulbecco’s modified Eagle’s medium/10% calf serum. These cultures have been passaged weekly at transfer ratios of 1:4.
Preparation of Tissue Extracts. Fresh rat and tumor tissues were homogenized in 20 mM Tris.HCI buffer (pH 7.5) containing 10 mM benzamidine hydrochloride. The homog- enization was carried out in a Brinkmann Polytron for two 30-sec periods at a setting of 6, followed by centrifugation at 10,000 x g for 40 min. The postmitochondrial supernatant fraction was concentrated by the addition of solid ammonium
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Abbreviations: AUT-PK 500, autophosphorylating protein kinase 500; NP-40, Nonidet P-40.
To whom reprint requests should be addressed.
sulfate to 60% saturation. The precipitate was dissolved in 20 mM Tris.HCI buffer (pH 7.5) with 10 mM benzamidine hydrochloride and dialyzed overnight against the same buffer before assaying for AUT-PK 500 and protein content.
Preparation of Cell Extracts. The cells were removed from the cultures with a rubber policeman and the cell suspensions were centrifuged at 800 x g for 15 min. The cell pellets were washed twice with 2 ml of 0.15 M NaCl and resuspended in 2 ml of 20 mM Tris.HCI buffer (pH 7.5) containing 10 mM benzamidine hydrochloride. The suspended cells were son- icated on ice twice for 10 sec in a bath-type sonicator and were centrifuged at 400 x g for 15 min. The supernatants were removed and saved for RIA and protein determination.
Subcellular Fractionation. Adrenocortical carcinoma tissue (30 g) was homogenized in 5 vol of ice-cold 50 mM Tris.HCI buffer (pH 7.4) containing 0.25 M sucrose, 25 mM KCI, and 10 mM MgCl2 for four 30-sec periods in a Brinkman Polytron at a setting of 6. After filtration through a double layer of guaze, the homogenate was centrifuged at 600 x g for 10 min at 4℃ to yield a crude nuclear pellet. The pellet was resuspended in 50 mM Tris.HCI buffer supplemented with 1.8 M sucrose, 25 mM KCI, and 10 mM MgCl2 and was centrifuged at 4℃ for 20 min at 55,000 x g. The resulting pellet was washed three times with 10 ml of the same buffer containing 0.25 M sucrose and was centrifuged again for 5 min at 600 x g.
The final pellet represented the nuclear fraction. The postnuclear supernatant was centrifuged at 28,000 x g for 10 min at 4℃. The resulting pellet was washed twice with 10 ml of 20 mM Tris.HCI buffer (pH 7.5) with 10 mM benzamidine hydrochloride and was recentrifuged at 28,000 x g for 10 min to obtain the membrane fraction. The supernatant represent- ed the cytosol. The particulate fractions were resuspended in 3-5 vol of 50 mM Tris.HCI buffer (pH 7.5) containing 0.3% NaDodSO4. Each fraction was homogenized and then incu- bated in a boiling water bath for 10 min. Traces of unsolubil- ized tissues were removed by centrifugation at 15,000 x g for 10 min at 4℃. The supernatants were saved for AUT-PK 500 and protein content. The cytosol fraction was dialyzed against 20 mM Tris.HCI (pH 7.5) with 10 mM benzamidine hydrochloride before use for RIA and protein determination.
Phosphorylation of AUT-PK 500. The enzyme autophos- phorylates. The reaction mixture (50 ul) consisted of 20 mM Tris.HCI (pH 7.5), 10 mM MgCl2, 0.25 mM [y-32P]ATP (4-8 × 103 cpm/pmol), and appropriate amount of AUT-PK 500 or tumor tissue extract. Incubation was carried out at 37℃ for 10 min. The reaction was terminated by the addition of 17 pl of 20 mM Tris.HCI (pH 7.5) with 5% NaDodSO4 and the sample was heated at 90℃ for 2 min.
Radioiodination of AUT-PK 500. AUT-PK 500 was iodi- nated by chloramine-T according to the method of Hunter and Greenwood (19). To 8 µg of the enzyme, in the reaction mixture (50 pl), 0.5 mCi of Na125I was added, followed by the addition of 50 µg of chloramine-T. After 40 sec, the reaction was stopped by the addition of 125 µg of sodium metabisulfite and 200 µg of KI. 125I-labeled AUT-PK 500 (125I-AUT-PK 500) and unreacted Na125I were separated by filtration on a 2-g Sephadex G-25 column presaturated with 1% bovine serum albumin in 0.05 M sodium phosphate buffer (pH 7.5).
Gel Electrophoresis. The labeled enzyme was analyzed by NaDodSO4/PAGE to verify its authenticity. Polyacrylamide disc gels (0.5 x 12 cm) containing 0.1% NaDodSO4 were prepared as described (20) with some modifications (21). The phosphorylated and the iodinated enzyme samples, after mixing with sample buffer (21), were electrophorsed in the presence of Tris/glycine buffer, pH 8.3. Gels were sliced (1.5 mm) and assayed for radioactive 32P and 125I in liquid and y scintillation counters, respectively. Parallel sets of gels were sliced longitudinally, dried, and subjected to autoradiography (Kodak NS-2T film).
RIA of AUT-PK 500. The antibodies against AUT-PK 500 were raised in rabbits and anti-AUT-PK 500 IgG was used for RIA (1). Double-antibody AUT-PK 500 RIA was conducted in 12 x 75 mm polystyrene tubes (Falcon) in a final volume of 250 pl. All dilutions were made in 0.15 M NaCl/0.05 M potassium phosphate buffer, pH 7.2 (Pi/NaCl), containing 5 mg of bovine serum albumin per ml and 10 mM benzamidine hydrochloride. Varying quantities of standard AUT-PK 500 (1.8-3800 ng) or samples to be analyzed (3-60 µg of protein) were placed in each tube and sufficient Pi/NaCl with 0.5% bovine serum albumin was added to bring the volume to 100 pl. In the reaction tube, 100 ul of anti-AUT-PK 500 IgG (1:2400 in Pi/NaCl with 0.5% bovine serum albumin contain- ing 1% normal rabbit serum to give a final dilution of 1:6000) and 50 ul of 125I-AUT-PK 500 (10,000 cpm) were added to each tube and incubated for 24 hr at 4℃. Finally, 10 ul of undiluted goat anti-rabbit gamma globulin was added to all of the tubes and the mixture was incubated for another 16 hr at 4°C.
The precipitate was collected by centrifugation at 3000 rpm for 20 min and the radioactivity was counted in an automatic y counter. The RIA was slightly modified to quantitate AUT-PK 500 in the presence of 0.1% NaDodSO4. Since the antigen-antibody complex is disrupted in 0.1% NaDodSO4 alone, a nonionic detergent, Nonidet P-40 (NP-40), was added in a concentration in the assay system so as to maintain a ratio of NP-40 to NaDodSO4 in excess of 5:1 to stabilize the complex (22). The final reaction volume of the RIA was 250 ul and all dilutions were made in Pi/NaCl with 0.5% bovine serum albumin containing 0.1% NaDodSO4/0.5% NP-40. Samples were adjusted to a 100-ul volume by adding Pi/NaCl with 0.5% bovine serum albumin containing 0.1% NaDod- SO4/0.5% NP-40 in a final concentration and were assayed as described. The precipitate was collected and radioactivity was counted. The percentage of bound radioactivity was calculated as a function of total radioactivity, by dividing the radioactivity precipitated with antibody minus radioactivity precipitated with nonimmune serum by total radioactivity added to the incubation.
Assay Specificity. Assay specificity was evaluated by com- paring the dose-response curve of adrenocortical carcinoma tissue extract with the standard AUT-PK 500 curve and by studying the crossreactivity with some protein kinases, such as protein kinase 380 and cyclic AMP- and cyclic GMP- dependent protein kinases, in the assay system by using anti-AUT-PK 500 IgG. In addition, to check the specificity of the precipitation step of the assay, tumor extract (40 µg of protein) was phosphorylated and immunoprecipitated with antiserum or preimmune serum under the same conditions as used in the modified RIA procedure (125I-AUT-PK 500 was excluded). The immunoprecipitate was dissolved in sample buffer, applied on a 6% NaDodSO4/polyacryamide gel (23), and analyzed by autoradiography.
Protein Determination. Protein was determined by the method of Bradford (24) using the Bio-Rad reagent with bovine serum albumin as a standard.
RESULTS
RIA of AUT-PK 500. AUT-PK 500 was iodinated without any dissociation of the enzyme, as revealed by the autora- diographic analysis of the NaDodSO4/polyacrylamide gel that shows a single iodinated polypeptide band that comi- grates with the authentic phosphorylated enzyme band (Fig. 1). The protein retained immunoreactivity after this modifi- cation, with 75-80% of the total incorporated radioactivity being immunoprecipitable.
The binding of 125I-AUT-PK 500 to various dilutions of anti-AUT-PK 500 IgG was linear on a logarithmic scale between 1:1000 and 1:16,000 (data not shown). A dilution of
32P INCORPORATED cpm × 10-3
15
30
45
0
TOP
TOP
D
240
D
10
D
SLICE NUMBER
20
30
40
DYE
DYE
1
2
3
4
125
INCORPORATED, cpm x 10-3
1:6000 bound 30-35% of the labeled enzyme. By using this dilution of anti-AUT-PK 500 IgG, a standard curve for the RIA was constructed (Fig. 2). The binding of 125I-AUT-PK 500 to its antibody was progressively inhibited by increasing amounts of nonlabeled AUT-PK 500. Approximately 60 ng of nonlabeled AUT-PK 500 displaced 50% of the bound 1251- AUT-PK 500. The least detectable dose, expressed as the minimal concentration of AUT-PK 500 that caused linear displacement in the assay, that could be measured by the RIA
was 10 ng or 0.02 pmol, with intra- and inter-assay variabil- ities of <3% and <6%, respectively. Competition curves obtained with all rat tissues and cell lines exhibited parallel- ism with the standard curve, showing the applicability of the RIA to rat tissues (data not shown). All data were calculated with dilutions of samples that fell in the useful range of the RIA-i.e., 10-240 ng in the assay.
The RIA described above was slightly modified to allow measurement of AUT-PK 500 in the subcellular fractions that were treated with the detergent NaDodSO4. The standard curve obtained with the RIA is shown in Fig. 2. The presence of NaDodSO4 slightly increased the sensitivity of the assay by shifting the curve to the left. The concentration of protein displacing 50% of the iodinated antigen is 45 ng in this assay system. The sensitivity of the modified RIA is 7.5 ng.
Assay Specificity. The antibody is specific for the enzyme AUT-PK 500 purified from rat adrenocortical carcinoma, since strong parallelism existed between the dose-response curves obtained with multiple volumes of adrenocortical tumor extracts and that of the standard curve as depicted in Fig. 2. This suggests that the anti-AUT-PK 500 antibodies could recognize the endogenous AUT-PK 500 in the crude extracts of the tumor. To determine the specificity of binding, the effects of some cyclic nucleotide-dependent and -indepen- dent protein kinases were investigated on the 125I-AUT-PK 500 binding to anti-AUT-PK 500 IgG in the immunoassay (Fig. 2). The various protein kinases did not show any competition with 125I-AUT-PK 500 up to 10,000-fold greater concentrations. Moreover, the specificity of the immuno- precipitation of AUT-PK 500 from a mixture of phosphoryl- ated proteins in the tumor extract was checked. As seen in Fig. 3, AUT-PK 500 was the only 32P-labeled protein pre- cipitated by the anti-AUT-PK 500 antibody. No labeled protein was precipitated by the preimmune IgG, suggesting that under the conditions of the assay only specific precipi- tation occurred. The specificity of the antibody against AUT-PK 500 was further assured by the electrophoretic transfer blot analysis in which only a single AUT-PK 500 protein band with M, = 250,000 was obtained (data not shown).
Quantitation of AUT-PK 500. Using this specific RIA, we measured the level of AUT-PK 500 in a variety of tissues and cultured cells. The data in Table 1 show that the adrenocorti- cal carcinoma contained a high concentration of AUT-PK 500, which was at least 100-fold in excess when compared to that in normal adrenal glands. The subcellular fractionation
V
T
V
125|-AUT-PK 500 BOUND (%)
100
٥٠
6
80
o
6
60
4
o
40
A
o
20
O
6
4
O.
o
A
0
1
10
102
103
104
105
106
0
10
102
103
PROTEIN (ng)
1 2 3 Mr X 10-3
..
.- 240
studies (Table 2) revealed that the bulk of the enzyme was located in the cytosolic fraction. Though a detectable quan- tity could be extracted from the membranes, the nuclear materials demonstrated negligible crossreactivity in the as- say. Extracts of other normal tissue and cells also showed the presence of AUT-PK 500 (Table 1). Interestingly, the AUT-
| Source | AUT-PK 500, µg/mg of protein |
|---|---|
| Adult tissue | |
| Liver | 0.24 ± 0.04 |
| Kidney | 0.23 ± 0.05 |
| Spleen | 0.24 ± 0.04 |
| Ovary | 0.24 ± 0.07 |
| Testis | 0.25 ± 0.06 |
| Adrenal gland | 0.22 ± 0.06 |
| Heart | 0.24 ± 0.04 |
| Lungs | 0.26 ± 0.05 |
| Pituitary | 0.25 ± 0.03 |
| Mammary gland | 0.25 ± 0.03 |
| Pancreas | 0.26 ± 0.05 |
| Neonatal tissue | |
| Liver | 0.31 ± 0.02 |
| Spleen | 0.29 ± 0.04 |
| Adrenal gland | 0.28 ± 0.02 |
| Fetal tissue | |
| Liver | 0.25 ± 0.03 |
| Spleen | 0.29 ± 0.03 |
| Cultured cells | |
| Spleen | 0.21 ± 0.02 |
| Skin | 0.21 ± 0.04 |
| Embryonic | 0.16 ± 0.05 |
An appropriate volume (70-80 pl) of each tissue or cell extract was assayed for AUT-PK 500 by RIA. Neonatal and fetal tissues were obtained from 48-hr newborn and 18-day pregnant rats, respectively. Data are mean + SD from three animals or three separate estimates. Each assay was done in duplicate and was repeated at least three times.
| Source | AUT-PK 500, ug/mg of protein |
|---|---|
| Tissue | |
| Adrenocortical carcinoma | 21.9 ± 3.4 |
| Plasma membrane | 0.20 ± 0.02 |
| Nucleus | 0.03 ± 0.004 |
| Cytosol | 17.80 ± 1.6 |
| Hepatoma | |
| 3924 A | 12.40 ± 1.94 |
| 5132 TC | 13.50 ± 1.4 |
| 7800 | 10.10 ± 2.4 |
| 7777 | 15.11 ± 2.6 |
| Mammary carcinoma | 6.3 ± 0.42 |
| Cultured cell lines | |
| Hepatoma | 4.2 ± 1.08 |
| Pituitary (tumor) | 6.45 ± 1.53 |
| Adrenocortical carcinoma 494 | 14.1 ± 2.6 |
The subcellular fractions (plasma membrane, nucleus, cytosol) were prepared and solubilized in 0.3% NaDodSO4. The samples were diluted and adjusted to contain 0.1% NaDodSO4/0.5% NP-40 in final concentrations and then appropriate aliquots (5-80 pl) were assayed by the modified RIA. The level of AUT-PK 500 in subcellular fractions is expressed in terms of total protein. An appropriate volume (5-10 ml) of other malignant tissues or cell extracts was assayed by the RIA. Data are mean + SD from three animals or three separate estimates.
PK 500 content of the cultured embryonic cells and tissues of fetuses and neonates did not show any significant deviation from the normal adult values. On the other hand, in rat liver chemically transformed into its premalignant altered foci or in immortalized liver cells (Rat I and Rat II) and established rat skin epithelial-like cells, the AUT-PK 500 levels increased by severalfold when compared to their normal counterparts (Table 3). A further rise of 5- to 10-fold in the AUT-PK 500 concentration was seen in malignant tissues, such as the different fast-growing hepatomas and mammary carcinoma, and in cultured hepatoma and pituitary tumor cell lines (Table 2).
DISCUSSION
For the quantitative measurement of AUT-PK 500 in various tissues and cells, we have developed a double-antibody RIA using radioiodinated AUT-PK 500 of high specific activity. The RIA is specific for AUT-PK 500 and can detect as low as 10 ng of the protein. The intra- and inter-assay variabilities are <3% and <6%, respectively. The RIA was slightly modified to include the detergent NaDodSO4, which allows measurement of the enzyme in particulate fractions. Appli- cation of the modified RIA to the subcellular fractions
| Source | AUT-PK 500, ug/mg of protein |
|---|---|
| Premalignant foci of rat liver | 1.6 ± 0.12 |
| Skin cells cultured in excess of 200 passages | 1.1 ± 0.15 |
| Rat I* | 0.78 ± 0.05 |
| Rat II* | 0.84 ± 0.05 |
An appropriate volume (15-50 pl) of tissue or cell extract was assayed for AUT-PK 500 by RIA. Data are mean + SD from three separate determinations. Each assay was done in duplicate. *Established rat liver cell lines.
revealed that AUT-PK 500 resides mainly in the cytosolic part of the cell, confirming earlier cytochemical studies (1).
RIA of AUT-PK 500 in the various rat tissues and cells indicates that the adult tissues such as liver, spleen, testis, kidney, ovary, adrenal gland, pancreas, heart, lung, pitu- itary, and mammary gland contain minute but detectable amounts (0.2 µg/mg of protein) of this enzyme. In contrast, there is a 100-fold elevation of the enzyme in the adrenocorti- cal carcinoma, a spontaneous tumor, a 50- to 60-fold eleva- tion in four chemically induced, rapidly growing hepatomas, a 30-fold increase in a chemically induced mammary carci- noma, a 20-fold elevation in the cultured hepatoma cell line, and a 30-fold elevation in the pituitary tumor. It is noteworthy that so far in all of the malignant rat tissues tested, the enzyme is strikingly elevated, indicating the association of the incre- ment of the enzyme with malignancy. We are assured that antibody reactivity scored in normal and tumor cells resides in AUT-PK 500, rather than crossreactive material, from electrophoretic transfer blot analyses of extracts of both cell types (unpublished). There is no increment in the expression of AUT-PK 500 in the fetal tissue or cultured embryonic cells, indicating that its elevation seems unrelated to differentiation or development.
In the multistep process of carcinogenesis, the initial important step is the immortalization of the cells (25, 26). The immortalized cells, Rat I and Rat II (18), both contained 4-fold elevated levels of protein kinases; freshly cultured skin epithelial-like cells, representative of nonestablished cells, contained only basal amounts of the enzyme. Of particular significance is the finding that when normal rat liver, in vivo, is chemically transformed to premalignant altered foci, there is a 7-fold increment in the enzyme level and a further 7-fold increment in the terminal stage of malignancy. The rat adrenocortical tumor cell line, which serves as a biochemical source for AUT-PK 500, has exceedingly high amounts of the enzyme, which approach 1-2% of total cellular proteins.
There are a number of intriguing aspects to our observa- tions that AUT-PK 500 seems progressively elevated in immortal cells, premalignant foci, and malignant cells. Im- mortalization itself is probably related to the ability of cells to overcome cell cycle barriers (27) and requires the coop- erative interactions of altered expression of more than one protooncogene-for example, c-myc and c-ras in primary embryo fibroblasts (28). Mouse adrenocortical tumor cells do show both amplification and overexpression of at least one oncogene (c-Ki-ras) (29). Furthermore, protooncogene (c- myc) expression has been shown to be regulated in a cell cycle-dependent fashion by mitogens, including platelet- derived growth factor (30). Considerable interest surrounds the mechanisms of growth factor function, their relationship to tumor promoters (31), and their oncogene-like ability to stimulate phosphorylation of tyrosine residues in protein (32-34). Yet, unlike oncogene products and membrane growth factor receptors, AUT-PK 500 is a cytoplasmic, serine-specific protein kinase. It possesses a sharply restrict- ed range of substrates, itself and a M, 31,000 ribosomal protein, probably S6 (1). Ribosomal protein S6 phosphoryl- ation has also been associated with cell cycle transitions (35).
We do not know whether AUT-PK 500 overexpression documented here is causally or coincidentally related to either cellular immortalization or carcinogenesis. We antic- ipate that cloning of the AUT-PK 500 gene will provide information on both the regulation of hyperexpression and its consequences.
We thank Dr. Henry C. Pitot, McArdle Laboratories, for providing us with premalignant altered liver foci, Dr. Y. Cho-Chung (National
Cancer Institute) for dimethylbenz[a]anthracene-induced rat mam- mary cell carcinoma cultured cell lines, and Dr. Patricia Earl (National Cancer Institute) for Rat I, Rat II, and pituitary cell lines. This investigation was supported by Grant CA-16091 from the National Cancer Institute.
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