0 1984 by The American Society of Biological Chemists, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 259, No. 9, Issue of May 10, pp. 5959-5969, 1984 Printed m L1.S.A.

Rat Adrenocortical Carcinoma 494 Autophosphorylating Protein Kinase, Autophosphorylating Protein Kinase 500 PURIFICATION, BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERIZATION, AND SUBSTRATE SPECIFICITY*

(Received for publication, June 20, 1983)

Chhanda Ganguly, Audrey N. Roberts, Yoshikazu KurodaS, and Rameshwar K. SharmaS From the Laboratories of Basic Hormone Research and the Departments of Biochemistry, Microbiology and Immunology, University of Tennessee, Center for the Health Sciences, Memphis, Tennessee 38163

A novel autophosphorylating protein kinase, auto- phosphorylating protein kinase 500, independent of cyclic AMP, cyclic GMP, calcium, and calmodulin was purified from rat adrenocortical carcinoma 494 by ammonium sulfate fractionation followed by the chro- matographic steps of DEAE-cellulose, gel filtration, cyclic AMP-epoxy Sepharose, and phosphocellulose. Sometimes two additional chromatographic purifica- tion steps of chromatofocusing and gel filtration were necessary for complete purification. The enzyme was homogeneous as evidenced by one- and two-dimen- sional sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis. Sucrose density sedimentation studies in- dicated that M, of the enzyme was 490,000, while ultracentrifugal analysis demonstrated a value of 481,400 (27%). The protein was composed of two iden- tical subunits each with M, = 250,000. The enzyme molecule was slightly asymmetric with frictional and sedimentation coefficients of 1.Z08 and 18.20, respec- tively, and a Stokes radius of 66 A. Isoelectric focusing electrophoresis revealed a single peak with PI 4.6, indicating acidity of the protein. The enzyme self phos- phorylated one or more of its serine residues. The reaction utilized the terminal phosphate of ATP; GTP was inactive. Divalent cations (5 mM Mn2+ or 10 mM Mg2+) were essential for optimum activity. Autophos- phorylating protein kinase 500 did not phosphorylate the commonly used exogenous substrates such as his- tones, casein, phosvitin, or protamine.

Analysis of autophosphorylating protein kinase 500 with rabbit anti-autophosphorylating protein kinase 500 IgG by immunoelectrophoresis and crossed im- mune electrophoresis demonstrated single arcs of pre- cipitation, confirming the biochemical demonstration of enzyme purification and homogeneity. Indirect im- munofluorescence studies revealed an intracyto- plasmic localization of the enzyme in cultured and freshly isolated adrenocortical carcinoma 494 cells. Both cell types revealed an intensity of perinuclear enzyme fluorescence, but an absence of the enzyme in the nuclei or nucleoli. The anti-autophosphorylating protein kinase 500 IgG blocked the self-catalyzed

.” ~~ ~~

* This investigation was supported by Grant CA-16091 from the National Cancer Institute and Grant PCM80-0873 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.

$Present address, Department of Surgery, University of Kobe, Japan.

§ To whom reprint requests should be addressed.

phosphorylation of autophosphorylating protein ki- nase 500, providing immunological support of the bio- chemical results that autophosphorylation is an intrin- sic characteristic of the enzyme. When autophosphor- ylating protein kinase 500 was incubated with mem- brane-bound ribosomes, it phosphorylated a M, = 31,000 protein. This phosphorylation was blocked by the anti-autophosphorylating protein kinase 500 IgG.

Since the substrate of autophosphorylating protein kinase 500 resides in the membrane-bound ribosomes, the possibility exists that this protein kinase might be involved in the translational control of the neoplastic cell.

Covalent modification of a polypeptide by the process of phosphorylation is an important cellular control mechanism (1, 2). The protein kinases which modulate the activity of phosphopeptides act as mediators for effector molecules such as cyclic nucleotides or noncyclic nucleotides, and are termed cyclic nucleotide-dependent or cyclic nucleotide-independent protein kinases. While a great deal is known about the struc- ture, mechanism of activation, and in certain cases the bio- logical function of cyclic AMP- and cyclic GMP-dependent protein kinases ( 3 ) , it is only recently that the importance of cyclic nucleotide-independent protein kinases in cellular reg- ulation has begun to be appreciated. A variety of enzymes such as phosphorylase kinase (4 ,5) , myosin light chain kinase (6-lo), and glycogen synthase kinase (11) are now known to be regulated by calcium-calmodulin, and therefore are termed as calcium-calmodulin-dependent protein kinases. A new spe- cies of protein kinase, termed C-kinase, that is calmodulin- independent, but calcium-phospholipid-dependent, has also been reported (12, 13). In addition to these protein kinases which regulate the activity of three recognized general media- tors of hormone action, cyclic AMP, cyclic GMP, and calcium, other protein kinases specifically regulating the activity of more specialized molecules such as hemin, double-stranded RNA, and epidermal growth factor have also been character- ized and are named as hemin-controlled repressor (14-18), dsRNA-activated inhibitor (14, 19, 20), and epidermal growth factor receptor protein kinase (21, 22), respectively. The in- teresting aspect of the latter enzyme is that the epidermal growth factor may itself be a protein kinase that catalyzes the phosphorylation of tyrosine residue(s) in the epidermal growth factor receptor (23). Other tyrosine protein kinases, that apparently regulate the activity of various RNA tumor viruses, also have been characterized (24-26). There are ad- ditional protein kinases such as casein kinases I and I1 (27,

5959

by guest on June 26, 2020http://w

ww

.jbc.org/D

ownloaded from

5960 Adrenocortical Carcinoma Autophosphorylating Protein Kinase 500

28) and protease-act ivated kinases I (29) and I1 (30), whose primary regulators and biological functions are unknown. Similarly, neither the primary regulator of another self-phos- phorylating hist idine protein kinase, self phosphorylating his- t idine protein kinase 380, nor i t s biological functions are known (31) although the partially purified enzyme phospho- rylates the N subuni t of the eukaryotic init iation factor 2 in vitro (32). Before a complete understanding of the biological role of any protein kinase in the regulation of a particular cellular event can be achieved, it is vital to purify the enzyme in quest ion and study its biochemical characteristics.

In this paper we describe the purification to homogeneity and characterization of a novel autophosphorylating protein kinase, AUT-PK 500,' f rom the rat adrenocortical carcinoma. These biochemical studies are complemented with an immu- nological approach to further characterize the enzyme, dem- onstrate i ts presence in freshly isolated and cultured adreno- cortical carcinoma cells, and identify its endogenous sub- strate(s), a M, = 31,000 protein that resides in membrane- bound ribosomes.

EXPERIMENTAL PROCEDURES

Materials~DEAE-cellulose, cyclic nuckotides, histone (type IIA), casein, phosvitin, and protamine were purchased from Sigma; Ultro- gel ACA 34 and ampholine (pH 3.5-10) from LKB; [Y-~'P]ATP (3,000 Ci/mmol) and [3H]cAMP (40 Ci/mmol) from Amersham Corp. Phos- phocellulose, all of the reagents for SDS-polyacrylamide gel electro- phoresis, and immunoelectrophoresis were obtained from Bio-Rad. Dulbecco's modified Eagle's medium was from Gibco Laboratories, and fluorescein-conjugated goat anti-rabbit IgG was from N. L. Cap- pel Laboratories Inc. All other reagents were analytical grade and were obtained commercially.

Cyclic AMP-Sepharose was synthesized by coupling cyclic AMP with epoxy-activated Sepharose and cyclic AMP-dependent protein kinase inhibitor was prepared according to the method of Walsh et al. (33). Calmodulin was a kind gift from Dr. W. Y. Cheung, St. Jude Hospital and the Department of Biochemistry, University of Tennes- see Center for Health Sciences.

Protein Kinase Assay-Protein kinase activity was measured by the incorporation of 32P from [Y-~'P]ATP into an appropriate sub- strate. The 250-pl reaction mixture contained 20 mM Tris-HC1 (pH 7.5), 10 mM Mg'+, 2.5 X M [Y-~'P]ATP (400-600 cpm/pmol), and an appropriate amount of the enzyme. The reaction was incubated at 37 "C for 1 min or as specified, and was terminated by the addition of 2 ml of ice-cold 10% trichloroacetic acid followed by the addition of 500 p g of bovine serum albumin. The precipitate was immediately filtered on a GF/C filter and washed three times with 2 ml of ice-cold 10% trichloroacetic acid. The filter was dried and counted in a mixture of Omnifluor in toluene.

Cyclic Nucleotide Binding Assay-Cyclic nucleotide binding assay was performed as previously described (34). An appropriate aliquot of the enzyme was incubated at 0 "C for 60 min in 50 mM sodium acetate buffer (pH 4.0), 2 mM EDTA, and 3 pmol of [3H]cAMP (1 X lo5 cpm/pmol) in a final volume of 100 pl. The reaction was initiated by the addition of AUT-PK 500 and was terminated with 2 ml of ice- cold 70% saturated ammonium sulfate solution. The precipitate was collected on GF/C filters, washed three times with 25% saturated ammonium sulfate solution, and the filters were dried and counted for radioactivity in 5 ml of Omnifluor mixture.

Autophosphorylation and Purity of the Enzyme-Autophosphory- lation of AUT-PK 500 was evaluated by phosphorylating 40 pg of enzyme with [y-32P]ATP, followed by its examination with two- dimensional gel electrophoresis according to the method of O'Farrell (35), using nonequilibrium pH gradient (pH 3.5-10) electrophoresis in the first dimension. The isoelectric focusing gel contained 9 M urea, 6% acrylamide, 0.2% bis, 2% NP40, 2% Ampholines (pH 3.5- lo), 0.02% ammonium persulfate, and 0.007% TEMED. In the second

The abbreviations used are: AUT-PK 500, autophosphorylating protein kinase 500; SDS, sodium dodecylsulfate; TEMED, N,N,N',N'-tetramethylethylenediamine; EGTA, ethylene glycol bis(6-aminoethyl ether)-N,N,N',N'-tetraacetic acid eIF, eukaryotic initiation factor.

~ ~~ .- ~ ~ ~~ _____

dimension, SDS-polyacrylamide gel (0.1% SDS, 6% acrylamide, and 0.3% bis) electrophoresis was conducted in the presence of 20 mM Tris and 154 mM glycine (pH 8.3) (36). The gel was stained with 0.05% Coomassie brilliant blue and destained with 7.5% acetic acid. The stained gel was dried and subjected to autoradiography (37) with the use of Kodak No-screen film (NS-2T).

Autophosphorylation of AUT-PK 500 was also analyzed by one- dimensional SDS-polyacrylamide gel electrophoresis. The enzyme was incubated in the presence of [y-32P]ATP. The reaction was terminated with sample buffer (38) containing 10% glycerol, 5.6% 2- mercaptoethanol, 3% SDS, and 0.0625 M Tris-HC1 (pH 6.8), in the presence of 0.04% SDS. The phosphorylated sample was applied to 6% acrylamide containing 0.1% SDS and 0.3% bis. Electrophoresis was conducted in the presence of Tris glycine buffer (pH 8.3). The gel was stained with Coomassie brilliant blue, destained with 7.5% acetic acid, dried, and subjected to autoradiography (37).

The purity of the enzyme was checked by both two-dimensional gel electrophoresis and one-dimensional SDS-polyacrylamide gel elec- trophoresis. For two-dimensional gel electrophoresis, up to 40 pg of AUT-PK 500 was subjected to isoelectric focusing in the first dimen- sion followed by SDS-polyacrylamide gel electrophoresis in the sec- ond dimension. The gel was stained in 0.005% Coomassie brilliant blue, 50% alcohol, 7% acetic acid and destained with 7% acetic acid. For SDS-polyacrylamide gel electrophoresis, disc gels (0.5 X 12 cm) were prepared (38, 39) and electrophoresis was conducted in Tris glycine buffer (pH 8.3). The gels were stained with 0.05% Coomassie brilliant blue for 2 h and destained in 7.5% acetic acid.

Isoelectric Focusing-The isoelectric point of AUT-PK 500 was determined under nondenaturing conditions as previously described (31). 5% polyacrylamide (39, 40) in 2% ampholite with a pH range of 3.0-10.0 was photopolymerized with riboflavin (41) in gel tubes (0.5 X 12 cm). Isoelectric focusing was carried out at 4 "C for 15 h a t 450 V with 10 mM HaPo1 as the anode electrode solut,ion in the lower reservior chamber and 20 mM NaOH as the cathode electrode solution in the upper reservoir chamber (35). The gels were cut into 1.5-mm slices, extracted with 500 g1 of deionized, degassed water, and assayed for pH and protein kinase activity.

Determination of Molecular Weight, Stokes Radius, and Frictional Coefficient-The Stokes radius, frictional ratio, and M , of AUT-PK 500 were determined by gel filtration column chromatography using ACA-34 by the method of Siege1 and Monty (42). The protein stand- ards used for the calibration of the column were thyroglobulin (80 A), phosphorylase a (63 A), and catalase (52 A).

Sucrose density gradient was performed according to the method of Martin and Ames (431, using thyroglobulin (19.4 S), apoferritin (17.6 S), and catalase (11.2 S) as standards.

Preparation of Membrane-bound Ribosomes-Male Holtzman rats were fed ad libitum until 5:OO p.m. on the day preceding the isolation of membrane-bound ribosomes. At 5:OO p.m. the food was removed. The following morning the rats were decapitated and the livers were rapidly removed and chilled in double-distilled water a t 4 "C. All subsequent procedures were performed a t 4 "C.

Membrane-bound ribosomes were prepared from postmitochon- drial supernatants as described by Ikehara and Pitot (45). The iso- lated free and membrane-bound ribosomal solutions (70-250 A,,,, units/ml) were stored for as long as 6 months a t -70 "C in the presence of 5 mM 2-mercaptoethanol in 20 mM Tris-HC1 (pH 7.4).

Phosphorylation of Membrane- bound Ribosomes by A UT-PK 500- 2-3 pg of AUT-PK 500 were incubated in the presence of 20-30 fig of membrane-bound ribosomes and 2.5 X M [y3'P]ATP. The reaction volume (50 p l ) also contained 20 mM Tris-HC1 (pH 7.5) and 10 mM Mg2f. The reaction was carried out a t 37 "C for 15 min and terminated by the addition of 17 pI of 4 x sample buffer. The phosphorylated proteins were analyzed by SDS-polyacrylamide gel (0.3% bis, 6 or 10% acrylamide, and 0.1% SDS) electrophoresis (36). Electrophoresis was performed in Tris glycine buffer (pH 8.3) (20 mM Tris and 153 mM glycine). The gel was stained with Coomassie brilliant blue, destained with 7.5% glacial acetic acid, and dried. The dried gel was subjected to autoradiography (37) with the use of Kodak NS-2T film.

The phosphorylation of membrane-bound ribosomal proteins also was performed in the presence of 5 pg of cyclic AMP-dependent protein kinase inhibitor (33) to rule out the AUT-PK 500-dependent phosphorylation of ribosomal protein by the catalytic subunit of cyclic AMP-dependent protein kinase potentially present as a contaminant in the reaction mixture.

Blockage of Autophosphorylation of AUT-PK 500 and of AUT-PK

by guest on June 26, 2020http://w

ww

.jbc.org/D

ownloaded from

Adrenocortical Carcinoma Autophosphorylating Protein Kinase 500 5961

500-dependent Phosphorylation of Membrane-bound Ribosomal M, = 31,000 Protein in the Presence of Anti-AUT-PK 500 IgG-2-3 pg (10

of AUT-PK 500 were incubated in the presence of 10 p1 of rabbit anti-AUT-PK 500 IgG for 15 min a t 4 "C. As a control, 10 pl of preimmune rabbit serum IgG was substituted for the specific immune IgG. Membrane-bound ribosomes (20-30 pg) were added to these mixtures, and the reactions were initiated by the addition of 30 pl of a solution containing 20 mM Tris-HC1 (pH 7.5), 10 mM M$+, and 2.5 X M [Y-~'P]ATP. The reactions were incubated at 37 "C for 15 min, then terminated with 4 X sample buffer. Aliquots of each reaction mixture were analyzed by one dimensional SDS-polyacryl- amide gel electrophoresis and autoradiography as described above.

Preparation of Rabbit Anti-AUT-PK 500 IgG-Rabbits were im- munized with purified AUT-PK 500 emulsified in complete Freund's adjuvant (Gibco Laboratories, Grand Island, NY) to give a final protein concentration of 150 pg/ml. 1 ml of the emulsion was distrib- uted subcutaneously into five sites in the nuchal region, and 1.0 ml was injected intramuscularly into each hind leg muscle. The intra- muscular injections were repeated a t weekly intervals for 2 weeks. After 8 weeks, the rabbits were boosted with intramuscular injections consisting of SDS-polyacrylamide gel electrophoresis slices contain- ing the isolated AUT-PK 500 emulsified in incomplete Freund's adjuvant, and blood samples were collected from the ear veins a t 3- week intervals thereafter. The sera were pooled, heat-inactivated (56 "C, 30 min), and stored at -20 "C. Booster injections were contin- ued at 8-week intervals. IgG fractions were prepared from serum pools by 3 X precipitation with (NH,),SO, followed by DEAE-cellulose chromatography (46). These IgG fractions from immune and normal serum pools were used for all experiments.

Immunoelectrophoresis-Techniques for immunoelectrophoresis and crossed immune electrophoresis have been described (46,47). All tests were performed in 1.0% agarose and 0.1 M barbital buffer (pH 8.6) (48). Immunoelectrophoresis patterns were dried and stained with acid fuchsin. The crossed immune electrophoresis patterns were stained with Coomassie blue.

Preparation of Tumor Cell Cultures and Suspensions for Immuno- fluorescence-Studies on the intracellular localization of AUT-PK 500 employed coverglass monolayers in logarithmic growth prepared from a cloned, established tumor cell line, RADC,, that has been cultured in excess of 220 serial passages. The coverglass cultures were fixed in acetone a t 0 "C for 10 min and stored a t -20 "C. Freshly isolated tumor cells also were examined for the distribution of AUT- PK 500. For these observations 4 week tumors were removed, trimmed of fibrous membranes, sliced into quarters, and washed free of the central necrotic tissue in Dulbecco's modified Eagle's medium con- taining penicillin (50 units/ml) and streptomycin (50 rg/ml). The quarters then were minced and stirred rapidly for 60 min a t room temperature. The tumor cells released by agitation were harvested by centrifugation, and the pelleted cells were resuspended, layered on slides, and allowed to air dry. These preparations were fixed in acetone at 0 "C for 10 min and stored at -20 "C. Assays for viable cell membrane localization of AUT-PK 500 used RADC, cell suspensions released from monolayer cultures with the use of a rubber policeman.

Immunofluorescence-Indirect immunofluorescence was used to detect the reaction of the specific rabbit anti-AUT-PK 500 IgG with the tumor cells. Fluorescein-conjugated goat anti-rabbit IgG (H and L chains specific) was used to reveal specific binding. The procedures for cytoplasmic studies, viable membrane assays, and for incubation with immunoglobulin have been described (49). Controls to rule out possible nonspecific globulin binding included (a) indirect assays with the preimmune rabbit serum IgG substituted for the test IgG in the primary incubation step, and (b ) direct tests with the fluorescein- conjugated goat anti-rabbit IgG.

Protein Determination-Protein concentrations were determined by the method of Bradford (44) using the Bio-Rad reagent and bovine serum albumin as a standard.

Characterization of the Phosphorylated Amino Acid-To character- ize the amino acid residue modified by autophosphorylation of AUT- PK 500, the enzyme (100 pg) was incubated for 30 min with [y-"P] ATP under standard conditions. The protein was precipitated with acetone and extensively washed with the same solvent, lyophilized, then hydrolyzed with 0.5 ml of 6 M HCI a t 110 "C for 3 h in a vacuum sealed tube. The hydrolysate was lyophilized and dissolved in 30 pl of deionized water containing 30 pg each of authentic samples of phosphotyrosine, phosphoserine, or pbosphothreonine; the amino acids were resolved by high voltage paper electrophoresis (Whatman 3MM, 1200 volts, 2 h, pH 2.0) using glacial acetic acid/formic acid/

water (12:3:85, v/v) as an ionizing buffer. The paper was dried, sprayed with ninhydrin to detect amino acid spots, and autoradi- ographed with Kodak NS-2T film.

RESULTS

Purification of A UT-PK 500

Unless otherwise indicated, all procedures were carried out at 0-4 "C using deionized water.

Step 1: Homogenization and Ammonium Sulfate Precipita- tion-Fresh rat adrenocortical carcinoma 494 (50) was col- lected into 0.9% NaCl containing 8 mM benzamidine hydro- chloride. The tumor tissue (40 g) was cleaned of necrotic debris and surrounding fibrous tissue, then homogenized with 80 ml of 20 mM Tris-HC1 buffer containing 6 mM 2-mercap- toethanol, 2 mM EDTA, and 10 mM benzamidine hydrochlo- ride (pH 7.5) for 2 min. The homogenate was centrifuged a t 10,000 x g for 60 min. Solid ammonium sulfate was added to the supernatant to 60% saturation. The precipitate was dis- solved in 20 mM Tris-HC1 containing 6 mM 2-mercaptoetha- nol, 2 mM EDTA, 10 mM benzamidine hydrochloride, and 10% glycerol (buffer A, pH 7.5), and dialyzed overnight against the same buffer.

Step 2-The dialyzed fraction was applied to a DEAE- cellulose column (2.8 X 14 cm) pre-equilibrated with buffer A. The column was washed with 400 ml of buffer A and eluted with 1 liter of a linear 0-400 mM NaCl gradient in buffer A. The peak AUT-PK 500 activity emerged at 60 mM NaCl (Fig. 1) and was distinct from the cyclic AMP-binding peak. The AUT-PK 500 peak fractions were pooled, and ammonium sulfate was added to give a final concentration of 60% satu- ration. The precipitate was dissolved in 20 mM potassium phosphate buffer (pH 7.0), 6 mM 2-mercaptoethanol, 2 mM EDTA, and 8 mM benzamidine hydrochloride (buffer B), and dialyzed overnight against the same buffer.

DEAE - CELLULOSE I I

FRACTION NUMBER

FIG. 1. DEAE-cellulose column chromatography of AUT- PK 500 from rat adrenocortical carcinoma 494. 1300 mg of protein from 60% saturated ammonium sulfate precipitation of post- mitochondrial supernatant were applied to a DEAE-cellulose column (2.8 X 14 cm) which was equilibrated with 20 mM Tris-HC1 (pH 7.5), 6 mM 2-mercaptoethanol, 2 mM EDTA, and 10% glycerol. The column was washed with 5 column volumes of the same buffer and eluted with a linear gradient of NaCl(0-0.4 M ) in this buffer. Fractions were analyzed for protein kinase (0) and cyclic AMP binding (0) as described under "Experimental Procedures."

by guest on June 26, 2020http://w

ww

.jbc.org/D

ownloaded from

5962 Adrenocortical Carcinoma Autophosphorylating Protein Kinase 500

GEL FILTRATION ( A c A - ~ L )

30 - 0

I 0

7 25 g s - T

- x

X

!5 20- - 5 n

8 15- 4

z 10-

a

a ‘ U

W

I 0

d z

8 - f

V * 0- V N

5 -

50 70 90 110 130 150 FRACTION NUMBER

FIG. 2 . Ultrogel ACA 34 column chromatography of AUT- PK 500. 40 mg of protein obtained by elution from DEAE-cellulose were applied to the column (1.5 X 68 cm) equilibrated with 20 mM potassium phosphate buffer (pH 7.0), 6 mM 2-mercaptoethanol, 2 mM EDTA, and 8 mM benzamidine hydrochloride (buffer B). The enzyme was eluted with the same buffer. Kinase assays (0) and cyclic AMP binding assays (0) were performed on every other fraction (1.0 ml) as described under “Experimental Procedures.”

TABLE I Purification of AUT-PK 500 from rat adrenocortical carcinoma

Purification of AUT-PK 500 was performed as described under “Results.” The assay procedure used was described under “Experi- mental Procedures.”

Fractions

. ~~~ ~

Total Specific Total Purifi- protein activity activity” cation

rng unitslmg units -fold 1. 60% Ammonium sulfate 1300 2 2660 1

. ~

precipitation of postmitochondrial supernatant

2. DEAE-cellulose 40 56 2231 28 3. ACA-gel filtration 1 1350 1350 675 4. Cyclic AMP-Sepharose 0.66 1400 960 700 5. Phosphocellulose 0.33 1900 633 950

The amount of enzyme which catalyzed the transfer of 1 pmol of inorganic phosphate from [y-32P]ATP into endogenous substrate/ min at 37 “C was taken as 1 unit.

~~ ~ . ~ ~~ - ~~~~~ _~

Step 3: Gel Filtration Chromatography-The AUT-PK 500 fraction (40 mg) was applied to a 130-ml AcA 34 column (1.5 x 68 cm) previously equilibrated with buffer B, and eluted with the same buffer. The peak activity fractions were pooled (Fig. 2).

Step 4: Cyclic AMP Affinity Chromatography-In order to ensure the removal of any contaminant cyclic AMP-binding protein, the pooled fractions from Step 3 were equilibrated with 5 ml of cyclic AMP-Sepharose for 1 h at 0 “C. The resin was transferred to a column and washed with 5 ml of buffer B. The flow-through fraction contained AUT-PK 500.

Step 5: Phosphocellulose Chromatography-The flow- through fraction of AUT-PK 500 (Step 4) was dialyzed against buffer B, and applied to a phosphocellulose column (0.9 x 2.5 cm) pre-equilibrated with buffer B. The enzyme did not bind to the resin. Flow-through fractions containing enzyme activ-

ity were pooled and concentrated by Amicon ultrafiltration

80% of the preparations obtained from these purification steps demonstrated homogeneous AUT-PK 500 (Table I; purification 950-fold). In the remaining 20%, the purity was 85-89%, ascertained by densitometer scanning of the enzyme resolved by SDS-polyacrylamide gel electrophoresis. In these instances, additional purification steps were performed.

Step 6: Chromatofocusing-The AUT-PK 500 obtained in Step 5 was further purified by the technique of chromatofo- cusing (51-53); the purification directions were strictly fol- lowed as described in the instruction manual provided with the Pharmacia Fine Chemicals Chromatofocusing kit. In this technique the protein to be fractionated is absorbed onto an ion exchanger, PBE 94, a pH gradient is developed with appropriate polyamine buffer, and proteins are eluted in the order of their isoelectric points.

AUT-PK 500 was applied to the chromatofocusing gel column (0.9 X 15 cm) previously equilibrated with 0.25 M imidazole-HC1 buffer (pH 7.4). The column was eluted with polybuffer 74 (pH 4.0). AUT-PK 500 peak activity appeared at pH 5.0. The peak activity fractions were pooled, precipi- tated by the addition of solid ammonium sulfate to 60% saturation, dissolved in 20 mM potassium phosphate buffer containing 30 mM 2-mercaptoethanol, 0.2 mM EDTA, and 8 mM benzamidine hydrochloride, and dialyzed overnight against the same buffer.

Step 7: Gel Filtration Chromatography-AUT-PK 500 (Step 6) was subjected to the gel filtration chromatographic step exactly as described in Step 3.

(PM-10).

Purity and Autophosphorylation

When homogeneous AUT-PK 500 was subjected to either one-dimensional or two-dimensional SDS-polyacrylamide gel electrophoresis, a single stained band corresponding to M , = 250,000 was obtained (Fig. 3). The M , = 250,000, based on SDS-polyacrylamide gel, remains unchanged when the en- zyme is analyzed under harsher denaturing conditions such as boiling the protein in 6 M guanidine hydrochloride and the adding of a reducing agent such as P-mercaptoethanol (data not shown). Incubation of the enzyme with [Y-~’P]ATP showed an autophosphorylated band that co-migrated with the Coomassie brilliant blue-stained band (Fig. 4). These results demonstrated the homogeneity and autophosphorylat- ing property of the enzyme.

Physical Properties of the Enzyme

A Stokes radius of 66 A was determined by gel filtration on ACA-34 (Fig. 5), and ~ ~ 0 . ~ determined by sucrose density gradient centrifugation (Fig. 6 ) was 18.20. The molecular weight calculated from these parameters (42) was 490,000. Sedimentation equilibrium studies using the photoelectric scanner of the ultracentrifuge at 230 nm revealed a M , = 481,400 (+7%). Since the enzyme showed M , = 250,000 by SDS-polyacrylamide gel electrophoresis, this indicated that AUT-PK 500 is composed of two subunits of identical size. The calculated frictional coefficient was 1.28, indicating that the molecule is slightly asymmetric.*

To determine the PI value of the enzyme, AUT-PK 500 was -~

‘The Stokes radius was estimated from

uersus the elution volume (Fig. 5, inset); f/fo

.. ~~

Stokes radius, u = partial specific volume of 0.73 for an average protein; M = molecular weight, and N = Avogadro’s number.

by guest on June 26, 2020http://w

ww

.jbc.org/D

ownloaded from

Adrenocortical Carcinoma Autophosphorylating Protein Kinase 500

PK - 500 ISOELECTRIC FOCUSING

9.8 PH L.6 3;s

A- B 1 SDS-PAGE

F w (3

a Q

In 0 m

I

M x 10-3 r TOP

L ” 4 3 “DYE

PROTEIN STAINING PATTERN FIG. 3. Purity of AUT-PK 500. A, 6 pg of AUT-PK 500 were

suhjected to isoelectric focusing under denaturing conditions as de- scribed under “Experimental Procedures.” The gel was stained with Coomassie blue and destained with acetic acid. R, one dimensional SDS-polyacrylamide gel electrophoresis of AUT-PK 500 (30 pg) was performed as described under “Experimental Procedures.” The marker proteins used were thyroglobulin (330,000), myosin (200,000), e-galactosidase (116,000), phosphorylase b (97,333). bovine serum alhumin (68,000), and ovalbumin (43,000). C, 40 pg of AUT-PK 500 was subjected to two-dimensional gel electrophoresis as described under “Experimental Procedures.” The marker proteins used were the same as R.

PK- 500 ISOELECTRIC FOCUSING

9.8 PH L.6 3;5

A -

F W (3

8 4 In 0 In

1 0 7 SDS-PAGE

1 M X 10-3

r

1 1 6 9 L 6 8

AUTORADIOGRAPHY FIG. 4. Autophosphorylation of AUT-PK 500. A, 6 pg of

AUT-PK 500 were phosphorylated in the presence of [y-’*P]ATP. The phosphorylated enzyme was subjected to isoelectric focusing (pH 35-10) under denaturing conditions as described under “Experimen- tal Procedures.” The gel was stained with Coomassie blue, destained with 7.5% acetic acid, and subjected to autoradiography (Kodak NS- 2T film). B, 5 pg of AUT-PK 500 were incubated in the presence of [y3’P]ATP. The phosphorylated enzyme was subjected to one-di- mensional SDS-polyacrylamide gel electrophoresis. The gel was stained, destained, and autoradiographed as described under “Exper- imental Procedures.” C, 15 pg of AUT-PK 500 from the phosphocel- lulose step were phosphorylated in the presence of [y-“PIATP. The phosphorylated sample was suhjected to isoelectric focusing (pH 3.5- 10) in the first dimension and SDS-polyacrylamide gel electrophoresis containing6% polyacrylamide, 0.10% SDS, and 0.3% bis in the second dimension as described under “Experimental Procedures.” The gel was stained, destained, dried, and subjected to autoradiography.

- .-.-+-. . 50 70 90 110 130 150

FRACTION NUMBER

5963

“t 1

FIG. 5. Determination of Stokes radius and molecular weight. The enzyme from the phosphocellulose step (40 pg) was chromatographed on an Ultrogel AcA 34 column (1.5 X 68 cm) equilibrated with buffer B and the enzyme was eluted with the same buffer. The flow rate was 15 ml/h and 1.0-ml fractions were collected. An aliquot of 50 pl was assayed for endogenous kinase activity as described under “Experimental Procedures.” For the determination of Stokes radius, shown in the inset, the column was calibrated with thyroglobulin, 80 A (1); phosphorylase, 63 A (2). and catalase, 52 A (3). The marker proteins used for M, determination were: blue dextran (Vo); thyroglobulin, 660,000 ( I ) ; phosphorylase a, 289,648 (2); cata- lase, 248,000 (3) , and methylene blue ( Vt) .

subjected to isoelectric focusing (Fig. 7). As indicated by its PI of 4.6, the protein is acidic.

Identification of the Phosphorylated Amino Acid The phosphate bond in self-phosphorylated AUT-PK 500

is acid-stable and alkali-labile, excluding the nature of the phosphorylated amino acids to be histidine or lysine. Tyrosine and serine are two other major amino acids that are phospho- rylated in protein kinase reactions (3) and their phosphate bonds are not acid labile. In order to evaluate whether any of these amino acids is phosphorylated in the AUT-PK 500 autophosphorylation reaction, phosphorylated AUT-PK 500 containing “P was acid hydrolyzed; authentic phosphoserine and phosphotyrosine were added to the hydrolyzed product and the resultant mixture was subjected to high voltage paper electrophoresis. Radioautographic analysis of the two ninhy- drin stained spots revealed radioactivity only in phosphoser- ine (Fig. 8), indicating that the phosphorylated amino acid residue is serine.

In a separate experiment, the phosphorylation of a threo- nine residue(s) by the similar technique was evaluated, the results indicated that this amino acid was not phosphorylated in the autophosphorylation reaction (data not shown).

Demonstration that A UT-PK 500 Is a Novel Protein Kinase

Most of the known cyclic nucleotide-dependent or -inde- pendent protein kinases catalyze the phosphorylation of one or more of the commonly used exogenous substrates such as histone, casein, protamine, and phosvitin. None of these proteins affected the phosphorylation of AUT-PK 500 (Table II), suggesting that none of them was the substrate of the enzyme. In order to confirm this conclusion, AUT-PK 500 was incubated with [y-”PJATP and histone in the presence or absence of cyclic AMP, then subjected to SDS-polyacryl-

by guest on June 26, 2020http://w

ww

.jbc.org/D

ownloaded from

5964 Adrenocortical Carcinoma Autophosphorylating Protein Kinase 500 SUCROSE DENSITY GRADIENT

L 3

I . ‘.e i! “4-L 10 20 30

FRACTIW NUMBER

FIG. 6. Sucrose density gradient of AUT-PK 500. A, 10 pg of AUT-PK 500 from the phosphocellulose column were centrifuged for 14 h at 186,000 X g in a linear 5-20% sucrose gradient in 20 mM Tris-HCI (pH 7.5), 8 mM 2-mercaptoethanol, 2 mM EDTA, 6 mM benzamidine hydrochloride according to the method of Martin and Ames (43). Polyallomer tubes containing the sucrose gradient were pierced at the bottom and fractions were collected. The standard proteins used were: thyroglobulin, 19.4 S (a); apoferritin, 17.6 S (b); catalase, 11.3 S (c); and were treated identically as the enzyme. B, each fraction was assayed for endogenous kinase activity as described under “Experimental Procedures.” Arrow marks the position of AUT- PK 500.

2 m E*\-. 1’ 0 0 10 20 30 40

FRACTION NUMBER

FIG. 7. Isoelectric focusing of AUT-PK 500. 10 pg of enzyme from the phosphocellulose step were applied to an isoelectric focusing gel (0.5 X 8 cm). The focused gel was sliced into 2-mm pieces and kinase activity (0) and pH gradient (A) were measured on each fraction (500 pl) as described under “Experimental Procedures.”

amide gel electrophoresis and analyzed by radioautography. There was no radioactivity corresponding to the histone band (data not shown), indicating that histone is not the substrate

The two well characterized cyclic nucleotide-dependent protein kinases are cyclic AMP-dependent and cyclic GMP- dependent protein kinases (2,3). Their activities are depend- ent on cyclic AMP and cyclic GMP, respectively. In addition, these two protein kinases can be differentiated from each

of AUT-PK 500.

FIG. 8. Characterization of the phosphorylated amino acid. 100 pg of AUT-PK 500 were endogenously phosphorylated in the presence of [y3’P]ATP. The phosphorylated protein was precipitated with acetone and washed extensively with the same solvent. The precipitate was hydrolyzed in the presence of 6 M HCI and subjected to high voltage paper electrophoresis at pH 2.0 as described under “Experimental Procedures.” Phosphoserine and phosphotyrosine were used as markers. The phosphorylated amino acids were stained with ninhydrin and the paper was subjected to autoradiography (0, origin). RFof phosphoserine and phosphotyrosine were 0.17 and 0.13, respectively.

TABLE I1 Substrate specificity of A UT-PK 500 and its cyclic nucleotide

independence 1 pg of enzyme was assayed for autophosphorylation in the presence

of different substrates, cyclic nucleotides, and cyclic AMP-dependent protein kinase inhibitor, as indicated below. The assay procedure was described under “Experimental Procedures.”

Additions Incorporated

CPm 1. Enzyme 16,000 2. Enzyme + casein (10 pg) 10,000 3. Enzyme + phosvitin (10 pg) 15,000 4. Enzyme + protamine (10 pg) 12,000 5. Enzyme + histone (10 pg) 15,400 6. Enzyme + cyclic AMP (1 p ~ ) 15,500 7. Enzyme + histone + cyclic AMP 16,300 8. Enzyme + cyclic AMP protein 16,100

9. Enzyme + cyclic GMP (1 PM) 16,300 10. Enzyme + cyclic GMP + histone 15,400

kinase inhibitor (10 pg)

~~

TABLE Ill Effect of calcium, calmodulin, and EGTA on autophosphorylation of

1 pg of enzyme was assayed for autophosphorylation of AUT-PK 500 in the presence of different effector molecules. The assay proce- dure was described under “Experimental Procedures.”

A UT- PK 500

~ ~~

Additions ”P incorporated ”

CPm Enzyme 18,500 Enzyme + EGTA (0.8 mM) 14,200 Enzyme + Ca’+ (0.4 mM) 17,000 Enzyme + calmodulin (100 pg/ml) 17,400 Enzyme + Ca2+ + calmodulin 16,300

other, since only cyclic AMP-dependent protein kinase activ- ity is blocked by the heat-stable inhibitor (54) and only cyclic GMP-dependent protein kinase phosphorylation of histones is stimulated by calmodulin or troponin (34). The AUT-PK 500 activity was independent of cyclic AMP and cyclic GMP and not blocked by the heat-stable inhibitor (Table 11). In addition, since AUT-PK 500 did not phosphorylate histone or casein, these results together demonstrate that AUT-PK 500 is not a cyclic nucleotide-dependent protein kinase.

As the names indicate, calcium- and calcium-calmodulin- dependent protein kinases are regulated by calcium and cal- cium-calmodulin. AUT-PK 500 activity was not affected by either calcium or calcium-calmodulin (Table 111); there was a

by guest on June 26, 2020http://w

ww

.jbc.org/D

ownloaded from

Adrenocortical Carcinoma Autophosphorylating Protein Kinase 500 5965

small (23.2%) inhibition by EGTA, which could be explained by partial chelation of Mg” used in the protein kinase buffer. These results indicate that AUT-PK 500 is a calcium-, cal- cium-calmodulin-independent protein kinase.

Reticulocytes contain several cyclic nucleotide-independent protein kinases such as casein kinases I and I1 (27, 28), the hemin-controlled repressor (14-18), the dsRNA-activated in- hibitor (14, 19, 20), protease-activated kinase I (29), and protease-activated kinase I1 (30). The substrates for these enzymes are casein and eIF-2P for casein kinases I and 11; eIF-2n for hemin-controlled repressor; eIF-2P and histones for dsRNA; casein for protease-activated kinase I; casein and eIF-2P for protease-activated kinase I1 (55). AUT-PK 500 did not phosphorylate any of these substrates (data not shown), indicating that it is distinct from these protein kinase en- zymes.

AUT-PK 500 is also distinct from tyrosine kinases (21-26), since it did not phosphorylate its tyrosine residue (Fig. 8). Another protein kinase, self phosphorylating histidine protein kinase 380, self phosphorylates its histidine residue (31) and the partially purified enzyme catalyzes the phosphorylation of the serine residue residing in eIF-2n (32). Since AUT-PK 500 self phosphorylated its serine(s) residue and did not catalyze the phosphorylation of eIF-2cu, it is obvious that this enzyme is different from self phosphorylating histidine pro- tein kinase 380. We therefore conclude that AUT-PK 500 is a novel protein kinase.

Time Course of Self Phosphorylation of AUT-PK 500 To study the time course of AUT-PK 500 self phosphory-

lation, the incorporation of [y-”P] from [y3’P]ATP into the 250,000-dalton subunit was measured (Fig. 9). The initial rate was rapid and linear through 10 s, and almost completed in 30 s. The stoichiometry of phosphate incorporation was 0.3 mol/mol of holoenzyme. This may not reflect true stoichi- ometry of phosphate in the holoenzyme, since the original enzyme could exist in a partially phosphorylated state.

The autophosphorylation of AUT-PK 500 was determined over a range of ATP concentrations. The reaction rate was hyperbolic and was dependent on ATP concentration in a saturable manner.

A T P Specificity of A UT-PK 500 When [y:”P]GTP was used as phosphate donor, no incor-

poration of radioactivity was observed, indicating a specificity

TIME COURSE

ru m a

, , I , ,

10 20 30 40 50 60 TIME( Sec )

FIG. 9. Time course of autophosphorylation of AUT-PK 500. 3 pg of AUT-PK 500 from the phosphocellulose step were assayed as described under “Experimental Procedures” for the incu- bation times indicated in the figure.

for ATP. Furthermore, GTP did not affect the ATP-depend- ent rate of self phosphorylation (data not shown).

Effect of Polyamines on the Autophosphorylation of AUT-PK 500

No effect of the polyamines putrescine, spermine, and sper- midine each a t 1 mM concentration, or of polylysine and polyarginine in concentrations of up to 200 pg/ml, on the autophosphorylation of AUT-PK 500 was detected either by filter assay or by radioautographic analysis of the SDS-poly- acrylamide gel resolved enzyme (data not shown).

Effect of Divalent Cations Each of the divalent cations, Mg2+, Mn”, Sr2+, and Co2+ at

2.5 mM concentration resulted in half-maximal stimulation of AUT-PK 500. However, peak activity of the enzyme was obtained with 5 mM Mn2+ and Co2+, 10 mM M P , 20 mM Ca2+, and 50 mM Sr2+. The maximal activation by Co2+ was 40% and by CaZ+, 60%, of that obtained by Mn2+ or M e . The maximal activation by Sr2+ was of the same order as that by Mnz+ or Mg2+ (Fig. 10).

Monovalent cations, such as sodium, potassium, and lith- ium, in concentrations of up to 100 mM, showed no significant effect on the AUT-PK 500 activity (data not shown).

Other Properties of AUT-PK 500 The optimum pH for AUT-PK 500 activity was 7.5 (data

not shown). N-Ethylmaleimide and p-chloromercuriphenyl-

D I W N T CATION ( mM 1 FIG. 10. Effect of divalent cations on autophosphorylation

of AUT-PK 500. The enzyme from the phosphocellulose step was dialyzed against 20 mM Tris-HC1 (pH 7.5), containing 6 mM 2- mercaptoethanol. 5 of the dialyzed enzyme were assayed for autophosphorylation in the presence of different concentrations of Mgz+ (A), Mn2+ (A), Ca2+ (O), Co2+ (O), and Sr2+ (0) as described under “Experimental Procedures.” All of the metals used were in chloride form.

TABLE IV Effect of “ S H group inhibitors on autophosphorylation of A UT-PK

500 1 pg of enzyme was assayed in the presence of different “ S H

group inhibitors as indicated below. The enzyme activity was mea- sured under standard conditions.

.”

’*F incorporated ~- ..”

cpm Enzyme 18,500 Enzyme + N-ethylmaleimide ( 5 mM) 18,000 Enzyme + iodoacetamide (5 mM) 18,600 Enzyme + p-chloromercuriphenyl- 18,300

sulfonic acid (5 mM) ___”

by guest on June 26, 2020http://w

ww

.jbc.org/D

ownloaded from

5966 Adrenocortical Carcinoma Autophosphorylating Protein Kinase 500

FIG. 11. Immunoelectrophoretic purity of AUT-PK 500. A, 50 pg of AUT-PK 500 were subjected to electrophoresis in agarose gel and analyzed by immunodiffusion precipitation with rabbit anti- AUT-PK 500 IgG as described under “Experimental Procedures.” B 10 pg of enzyme were subjected to electrophoresis in agarose gel, followed by crossed immune electrophoresis in gel containing a 1:20 dilution of rabbit anti-AUT-PK 500 IgG as described under “Exper- imental Procedures.”

sulfonic acid, which inhibit the activity of enzymes dependent on the structural requirement of sulfhydryl groups, did not influence AUT-PK 500 activity (Table IV).

Immunoelectrophoretic Purity of A UT-PK 500

Immunoelectrophoresis and crossed immune electrophore- sis analyses of the purified AUT-PK 500 with the use of the rabbit anti-AUT-PK 500 IgG both demonstrated single arcs of precipitation (Fig. 11). Control tests in which normal rat serum was substituted for AUT-PK 500 in reactions with the specific immune IgG and in which preimmune rabbit I g G was substituted for the specific immune IgG in reactions with the enzyme were negative (not shown). These results were indic- ative of the homogeneity of the enzyme and demonstrated the specificity of the anti-AUT-PK 500 IgG. In other experiments (data not shown), the enzyme was either phosphorylated with [r-:”P]ATP or isotopically labeled with ’‘‘I, immunoprecipi- tated with the specific anti-AUT-PK 500 IgG, and then re- solved by SDS-polyacrylamide gel electrophoresis. Ftadioau- tographic analysis of the gels showed a single phosphorylated or iodinated protein which co-migrated identically with the Coomassie blue-stained band, thus providing additional evi- dence for purity of the enzyme.

Immunofluorescence Localization of A UT-PK 500

Indirect immunofluorescence observations with anti-AUT- PK 500 IgG revealed an intracytoplasmic localization of the enzyme in the fixed RADC, cell monolayers and in the freshly isolated tumor cells. The patterns of localization differed, however, with the enzyme concentrated in densely fluorescent vesicles of the cultured cells (Fig. 12A), while demonstrating a smooth, homogeneous cytoplasmic distribution in the freshly isolated cells (Fig. 12B). Cells from both sources presented an intensity of perinuclear enzyme fluorescence, but AUT-PK 500 was not apparent in the nuclei or nucleoli. Studies of viable RADC, cell membranes were negative (not shown) indicative that if expressed on the membrane, the AUT-PK 500 was in concentrations too low for immunoflu- orescence detection. Control assays with preimmune rabbit serum IgG and direct tests with fluorescein-conjugated goat anti-rabbit IgG were negative, supporting the specificity of the immunofluorescence observations (not shown).

FIG. 12. Immunofluorescent localization of AUT-PK 500 in adrenocortical carcinoma 494 cells. A. indirect immunofluores- cence reaction of rahhit anti-AUT-PK 500 IgC with a fixed monolayer of the established RADC, tumor cell line revealing the vesicular cytoplasmic and perinuclear distribution of the enzyme X 1000. R, indirect immunofluorescence reaction of rabbit anti-AUT-PK 500 IgG with fixed cells that were freshly isolated from a rat adrenocor- tical carcinoma 494, demonstrating the perinuclear concentration of the enzyme as well as its homogeneous cytoplasmic distribution X 1000.

Immunological Evidence for the Autophosphorylation Characteristic of A UT-PK 500

When AUT-PK 500 was incubated in the presence of anti- AUT-PK 500 IgG and [y3’P]ATP, analysis by one-dimen- sional SDS-polyacrylamide gel electrophoresis followed by radioautography demonstrated that the antibody blocked the self-phosphorylation reaction of the enzyme (Fig. 13).

Evidence That Membrane-bound M, = 31,000 Protein Is a Substrate of A UT-PK 500

In the presence of [r-”P]ATP, AUT-PK 500 phosphoryl- ated a M, = 31,000 membrane-bound ribosomal protein. The reaction was insensitive to the cyclic AMP-dependent protein kinase inhibitor (Fig. 14). The specific anti-AUT-PK 500 IgG blocked the AUT-PK 500-dependent phosphorylation of the M, = 31,000 protein (Fig. 15).

Phosphorylation of Membrane-bound Ribosomal M, = 31,000 Protein by the Catalytic Subunit of Cyclic AMP-dependent

Protein Kinase The catalytic subunit of cyclic AMP-dependent protein

kinase phosphorylated the M, = 31,000 membrane-bound

by guest on June 26, 2020http://w

ww

.jbc.org/D

ownloaded from

Adrenocortical Carcinoma Autophosphorylating Protein Kinase 500 5967

a b c Mr x

r TOP 330 - t- 210

- 200

- 116 - 91

- 68

- DYE

FIG. 13. Blockage of AUT-PK 500 autophosphorylation by rabbit anti-AUT-PK 500 IgC. AUT-PK 500 (2-3 pg) was incu- hated in the presence and absence of anti-AUT-PK 500 IgC (10 pl ) and [y-”PIATP (2.5 X M). Preimmune rabbit serum IgC was used as a negative control. The treated samples were analyzed by one-dimensional SDS-polyacrylamide gel electrophoresis and auto- radiography as described under “Experimental Procedures.” a; AUT- PK 500; b, AUT-PK 500 and preimmune serum IgC; c, AUT-PK 500 and anti-AUT-PK 500 IgC.

t 9L

- 00 - - tD:: Flc. 14. Phosphorylation of membrane-bound ribosomal

proteins by AUT-PK 500. The enzyme (2-3 pg) was incubated in the presence of [yR‘P]ATP (2.5 X M), membrane-bound ribo- somes (20-30 pg) , and cyclic AMP-dependent protein kinase inhibitor (5 pg). The phosphorylated samples were subjected to one-dimen- sional SDS-polyacrylamide gel electrophoresis and autoradiography as described under “Experimental Procedures.” a, AUT-PK 500; b, membrane-bound ribosomal proteins; c, AUT-PK 500 and membrane- bound ribosomal proteins; d; AUT-PK 500, membrane-bound ribo- somal proteins and cyclic AMP-dependent protein kinase inhibitor.

ribosomal protein and this phosphorylation was blocked by the cyclic AMP-dependent protein kinase inhibitor (Fig. 16).

DISCUSSION

In this paper we describe the purification to homogeneity and characterization of a novel autophosphorylating protein kinase from the rat adrenocortical carcinoma. The criteria for homogeneity and autophosphorylation are: (a) one- and two- dimensional polyacrylamide gel electrophoresis of as much as 40 pg of the enzyme under denaturing conditions reveals a single stained band; (b) analyses by immunoelectrophoresis and crossed immune electrophoresis with an immunoglobulin produced against the enzyme demonstrate a single precipitat- ing band; (c) when the enzyme is either phosphorylated with [y-’*P]ATP or iodinated and then immunoprecipitated with specific anti-AUT-PK 500 IgG, resolution of the protein by SDS-polyacrylamide gel electrophoresis followed by radioau- tographic analysis reveals a single 250,000-dalton phospho-

a b c d e Mr x

“

0- -. - t 31

FIG. 15. Blockage of AUT-PK 500-dependent phosphoryl- ation of membrane-bound ribosomal M . = 31,000 protein in the presence of rabbit anti-AUT-PK 500 I@. AUT-PK 500 (2- 3 pg) was incubated in the presence of membrane-hound ribosomes (20-30 pg) and anti-AUT-PK 500 I& (10 pl ) with [y-“PIATP (2.5 X M). The samples were analyzed by one-dimensional SDS- polyacrylamide gel electrophoresis and autoradiography as described under “Experimental Procedures.” a, AUT-PK 500; b, membrane- bound ribosomes; c, AUT-PK 500 and membrane-hound ribosomes; d , AUT-PK 500, membrane-hound ribosomes, and preimmune serum IgC; e, AUT-PK 500, membrane-bound ribosomes, and anti-AUT- PK 500 IgC.

a b c Mr x 10-3

3. I

Ir a i DYE

FIG. 16. Phosphorylation of membrane-bound ribosomal M. = 31,000 protein by catalytic subunit of cyclic AMP-depend- ent protein kinase. Membrane bound ribosomes (20-30 pg) were incubated with catalytic subunit of cyclic AMP-dependent protein kinase (2 pg) in the presence or absence of the cyclic AMP-dependent protein kinase inhibitor (5 pg), and [y3*P]ATP (2.5 X M). The phosphorylation of M, = 31,000 membrane-bound ribosomal protein was examined as described under “Experimental Procedures.” a, membrane-bound ribosomes; b, membrane-bound ribosomes and cat- alytic subunit of cyclic AMP-dependent protein kinase; c, membrane- bound ribosomes, catalytic subunit of cyclic AMP-dependent protein kinase, and cyclic AMP-dependent protein kinase inhibitor.

rylated or iodinated polypeptide that coincides with the Coo- massie blue-stained band.

AUT-PK 500 is unique with respect to the previously characterized cyclic nucleotide-dependent or -independent protein kinases, since it is not regulated by the cyclic nucleo- tides, calcium or calcium-calmodulin. It is also distinct from other cyclic nucleotide-independent protein kinases such as casein kinases I and I1 (27,28), the hemin-controlled repressor (14-18), the double stranded RNA-activated inhibitor (14,19, 20), and protease-activated kinases I and I1 (29, 30), since it does not catalyze the phosphorylation of substrates such as casein, eukaryotic initiation factor 2, and histones.

AUT-PK 500 catalyzes the phosphorylation of its serine residue(s); the terminal phosphate of ATP is specifically used

by guest on June 26, 2020http://w

ww

.jbc.org/D

ownloaded from

5968 Adrenocortical Carcinoma Autophosphorylating Protein Kinase 500

in this reaction. There are three interesting features of the self phosphorylation reaction. First, the reaction is extremely rapid with half ofthe phosphorylation sites saturated in about 5 s. Second, although Mn2+ and Mg2+ are the most potent cations, other divalent cations such as Co2+, Ca2+, and ST2+ are able to substitute for Mn2+ or Mgz+. Third, polyarginine, which catalyzes the autophosphorylation of cyclic AMP-de- pendent protein kinase type I1 (56), cyclic GMP-dependent protein kinase (57), and the 120,000-dalton peptide of adre- nocortical self phosphorylating protein kinase (31), does not affect the self phosphorylation of AUT-PK 500. From these limited studies, it is premature to draw conclusions on the common mechanisms of regulation of autophosphorylation reactions of different protein kinases. The possibility exists, however, that the autophosphorylation characteristic of the protein kinase may in fact be an important contributory factor to the total catalytic activity of the enzyme.

Indirect immunofluorescence observations with the anti- AUT-PK 500 IgG revealed an intracytoplasmic localization of the enzyme in the fixed RADC, cell monolayers and in the freshly isolated tumor cells. AUT-PK 500 was not apparent in the nuclei or nucleoli. Studies of viable RADC, cell mem- branes were negative, indicative that the localization of AUT- PK 500 was principally intracellular.

Since none of the known substrates were phosphorylated by AUT-PK 500, a search was made to identify its endogenous substrate(s). Our initial studies suggested that the phosphor- ylation of certain rat adrenal cytoplasmic proteins was en- hanced by AUT-PK 500, but the pattern was too complex to identify one specific AUT-PK 500-catalyzed phosphorylated protein. It was, therefore, considered necessary to study the AUT-PK 500-dependent phosphorylation of the proteins re- siding in specialized rat cell components. The immunofluo- rescence results had revealed a cytoplasmic localization of the enzyme, and since the source of AUT-PK 500 was from the rat neoplastic adrenal cortex, the cytoplasmic fractions of rat tissues were selected for these studies. The normal tissue counterpart, t.he rat. adrenal gland, is severely limited in size. Therefore, we elected to examine the AUT-PK 500-dependent phosphorylation of proteins contained in membrane-bound ribosomes of rat liver. AUT-PK 500 catalyzed the phosphor- ylation of M , = 31,000 protein. This phosphorylation was not inhibited by cyclic AMP-dependent protein kinase inhibitor. The anti-AUT-PK 500 IgG inhibited the AUT-PK 500-de- pendent phosphorylation of this protein, while the preimmune rabbit serum IgG was unable to do so. AUT-PK 500-inde- pendent phosphorylation of other membrane-bound ribo- somal proteins was not influenced by either the specific im- mune IgG or the preimmune IgG. These results revealed that the membrane-bound ribosomal protein M , = 31,000 was a specific substrate of AUT-PK 500.

It is known that cyclic AMP-dependent protein kinase has a broad substrate specificity (2 ,3) . It was pertinent, therefore, to evaluate whether the M, = 31,000 membrane-bound ribo- somal protein also served as a substrate of cyclic AMP- dependent protein kinase. Phosphorylation of the protein was markedly increased by the catalytic subunit of cyclic AMP- dependent protein kinase, and this increment was blocked by the heat-stable inhibitor. These results indicated that the M , = 31,000 protein is also a substrate of cyclic AMP-dependent protein kinase. It needs to be determined whether the protein domains of phosphorylation by the two kinases (cyclic AMP- dependent and AUT-PK 500) are distinct or identical.

The studies described in this report indicate that AUT-PK 500 is a novel cyclic nucleotide-independent autophosphory- lating protein kinase. The enzyme appears to uniquely regu-

late the activity of one membrane-bound ribosomal protein. Neither the biological significance of this regulation nor the mechanisms by which the enzyme is regulated is known. It is anticipated that the availability of a sufficient quantity of homogeneous enzyme and of specific antibodies against this enzyme described herein will be useful in achieving these goals.

Acknowledgments-We thank Dr. W. Y. Cheung for the critical review and comments on this investigation, Dr. Gypsy Majumdar for repeating some experiments. We are also grateful to Dr. Marc S. Lewis, National Institutes of Health, for determining the molecular weight of AUT-PK 500 with the use of the photoelectric scanner of the Beckman Model E ultracentrifuge a t 230 nm, and Vera W. Rossi for excellent typing.

1. 2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21. 22.

23.

24.

25.

26. 27.

28.

29.

REFERENCES Greengard, P. (1978) Science (Wash. D. C.) 199, 146-152 Krebs, E. G., and Beavo, J. A. (1979) Annu. Reu. Biochem. 48,

Sharma, R. K. (1982) in Progress in Nucleic Acid Research and Molecular Biology (Cohn, W. E., ed) Vol. 27, pp. 233-288, Academic Press, New York

Cohen, P., Burchell, A., Foulkes, J. G., Cohen, P. T. W., Vana- man, T. C., and Nairn, A. C. (1978) FEBS Lett. 92, 287-293

DePaoli-Roach, A. A., Roach, P. J., and Larner, J . (1979) J. Bid. Chem. 254, 4212-4219

Dabrowska, R., Sherry, J . M. F., Aromatorio, D. K., and Hart- shorne, D. J. (1978) Biochemistry 17, 253-258

Yagi, K., Yazawa, M., Kakiuchi, S., Ohshima, M., and Uenishi, K. (1978) J. Biol. Chem. 253,1338-1340

Hathaway, D. R., and Adelstein, R. S. (1979) Proc. Natl. Acacl. Sci. U. S. A . 76, 1652-1657

Walsh, M. P., Vallet, B., Autric, F., and Demaille, J. G. (1979) J. Biol. Chem. 254, 12136-12144

Adelstein, R. S., and Klee, C. B. (1981) J. Biol. Chem. 256,7501- 7509

Payne, M. E., and Soderling, T. R. (1980) J. Biol. Chem. 255,

Takai, Y., Kishimoto, A,, Iwasa, Y., Kawahara, Y., Mori, T., and

Wise, B. C., Raynor, R. L., and Kuo, J. F. (1982) J. Bid. Chem.

Farrell, P. J., Balkow, K., Hunt, T., Jackson, R. J., and Traschel,

Gross, M., and Rabinovitz, M. (1973) Biochem. Biophys. Res.

Ranu, R. S., and London, I. M. (1976) Proc. Natl. Acaa'. Sci. CJ. S. A. 73,4349-4353

Trachsel, H., Ranu, R. S., and London, I. M. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 3654-3658

Tahara, S. M., Traugh, J . A., Sharp, S. B., Lundak, T. S., Safer, B., and Merrick, W. C. (1978) Proc. Natl. Acad. Sci. U. S. A . 75, 789-793

Levin, D. H., and London, J. M. (1978) Proc. Natl. Acad. Sci.

Lenz, J. R., and Baglioni, C. (1978) J. Biol. Chem. 253, 4219-

Ushiro, H., and Cohen, S. (1980) J. Biol. Chem. 255,8363-8365 Carpenter, G., King, L., Jr., and Cohen, S. (1979) J. Bid. Chem.

Cohen, S., Carpenter, G., and King, L. Jr., (1980) J. Biol. Chem.

Witte, 0. N., Dasgupta, A., and Baltimore, D. (1980) Nature

Eckhart, W., Hutchinson, M. A., and Hunter, T. (1979) Cell 18,

Beemon, K. (1981) Cell 24, 145-153 Hathaway, G. M., and Traugh, J . A. (1979) J. Bid. Chem. 254,

Tuazon, P. T., Bingham, E. W., and Traugh, J . A. (1979) Eur. J.

Tahara, S. M., and Traugh, J. A. (1981) J . Biol. Chem. 256,

923-959

8054-8056

Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695

257,8481-8488

H. (1977) Cell 11, 187-200

Commun. 50,832-838

U. S. A. 75, 1121-1125

4223

254,4884-4891

255, 4834-4842

(Land.) 283,826-831

925-933

762-768

Biochem. 94,497-504

11558-11564

by guest on June 26, 2020http://w

ww

.jbc.org/D

ownloaded from

Adrenocortical Carcinoma Autophosphorylating Protein Kinase 500 5969

30. Tuazon, P. T., Merrick, W. C., and Traugh, J. A. (1980) J. Biol.

31. Kuroda, Y., and Sharma, R. K. (1982) Arch. Biochem. Biophys.

32. Kuroda, Y., Merrick, W. C., and Sharma, R. K. (1982) Arch.

33. Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fischer, E.

34. Ahrens, H., Paul, A. K., Kuroda, Y., and Sharma, R. K. (1982)

35. O'Farrell, P. H. (1975) J. Bid. Chem. 250,4007-4021 36. Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685 37. Moore, R. E., Ahrens, H., and Sharma, R. K. (1980) Cell. Mol.

38. Engbaek, C., Gross, R., Burgess, R. (1976) Mol. Gen. Genet. 143,

39. Orstein, L. (1964) Ann. N. Y. Acad. Sci. 121, 321-349 40. Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121,404-427 41. Ames, G. F. L., and Nikaido, K. (1976) Biochemistry 15, 616-

42. Siegel, L. E., and Monty, K. J. (1966) Biochim. Biophys. Acta.

43. Martin, R. G., and Ames, B. N. (1961) J . Biol. Chem. 236,1372-

44. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 45. Ikehara, Y., and Pitot, H. C. (1973) J. Cell. Biol. 59, 28-44

Chem. 255, 10954-10958

2 17,588-596

Biochem. Biophys. 213, 271-275

H., and Krebs, E. G. (1971) J. Biol. Chem. 246, 1977-1985

Arch. Biochem. Biophys. 251,597-609

Biol. 25,435-443

291-295

623

112,346-362

1379

46. Garvey, J. S., Cremer, N. E., and Sussdorf, D. H. (1977) in Methods in Immunology, A Laboratory Text for Instruction and Research, 3rd Ed, pp. 218-219,328-336,340-346,394-397,522, W. B. Benjamin Inc., New York

47. Schmidt-Ullrich, R., Wallach, D. F .H., andDavis, F. D. G. (1976) J. Nat. Cancer Inst. 57, 1117-1126

48. Williams, C. A., and Chase, M. W. (1971) Methods in Immunol- ogy, Vol. 3, p. 468, Academic Press, New York

49. Roberts, A. N., Smith, K. L., Dowell, B. L., and Hubbard, A. K. (1978) Cancer Res. 38, 3033-3043

50. Snell, K. C., and Stewart, H. L. (1959) J . Nat. Cancer Inst. 22, 1119-1155

51. Sluyterman, L. A. AE, and Elgersma, 0. (1978) J . Chromatog. ~~" ""

150, 17-30 52. Sluyterman, L. A. AE, and Wijdenes, J. (1978) J. Chromatog.

53. Ion Exchange Chromatography, Principles and Methods, Phar- macia Fine Chemicals AB, Box 175, S-75104 Uppsala 1, Sweden

54. Ashby, C. D., and Walsh, D. A. (1972) J. Biol. Chem. 247,6637- 6642

55. Traugh, J. A., DelGrande, R. W., and Tuazon, P. T. (1981) Cold Spring Harbor Conf. Cell Proliferation 8, 999-1012

56. Rosen, 0. M., and Erlichman, J . (1975) J . Biol. Chem. 250,7788- 7794

57. Walton, G. M., and Gill, G. N. (1981) J . Biol. Chem. 256, 1681- 1688

150,31-44

by guest on June 26, 2020http://w

ww

.jbc.org/D

ownloaded from

C Ganguly, A N Roberts, Y Kuroda and R K Sharmaimmunological characterization, and substrate specificity.

autophosphorylating protein kinase 500. Purification, biochemical and Rat adrenocortical carcinoma 494 autophosphorylating protein kinase,

1984, 259:5959-5969.J. Biol. Chem. 

  http://www.jbc.org/content/259/9/5959Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/259/9/5959.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on June 26, 2020http://w

ww

.jbc.org/D

ownloaded from