Purification and partial characterization of the catalytic subunit of cAMP-dependent protein kinases from reticulocytes

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 197, No. 2, October 15, pp. 618-629, 1979

Purification and Partial CAMP-Dependent NIKODEM

Characterization of the Catalytic Subunit Protein Kinases from Reticulocytes

GRANKOWSKI, Clayton

GISELA

KRAMER,

AND BOYD

of

HARDESTY’

Foundation Biochemical Institute, Department of Chemistry, The University of Texas, Austin, Texas 78712 Received May 22, 1979; revised July 9, 1979

The catalytic subunit of cyclic AMP-dependent protein kinases from rabbit reticulocytes has been purified to near homogeneity. It has a molecular weight of 43,000 as judged from gel filtration and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and appears to be similar in physical properties and substrate specificity to the comparable enzyme isolated from muscle or liver. The enzyme phosphorylates histones, a protein of 40 S ribosomal subunits from reticulocytes and from Atiemia salina, and the low molecular weight heat-stable phosphatase inhibitor-l (G. A. Nimmo and P. Cohen, 1978, Eur. J. Biochem. 87,341-351). No evidence has been obtained for a direct or indirect role of this enzyme in the regulation of protein synthesis.

Cyclic AMP-dependent protein kinases have been extensively studied, particularly in relation to their role in glycogen metabolism in liver and muscle. It is clearly established, that these kinases phosphorylate and thereby participate with their cognate phosphatases in the regulation of a number of enzymes involved in carbohydrate and lipid metabolism (1, 2). They appear to be the principle, if not the sole mediator of the cAMP2 response in animals. Two types of CAMP-dependent protein kinases have been reported (3). The enzymes have different regulatory subunits, but have a common catalytic subunit and therefore apparently similar substrate specificity (4). Both are inhibited by a heat-stable low molecular weight protein (5). Traugh and Traut (6) described several different protein kinases in rabbit reticulo1 To whom correspondence should be sent. * Abbreviations used: HCR, hemin-controlled repressor; BME, /3-mercaptoethanol; SDS, sodium dodecyl sulfate; CAMP, cyclic AMP; DTE, dithioerythritol; GMP-P(CH,)P, guanosine 5’-(P,y-methylene)triphosphate; eIF-2, eukaryotic initiation factor 2; HS, heat&able protein; HL, heat-labile protein; solution A, 20 mM Tris-HCl (pH 7.5), 10 mM BME. 0003-9861/79/120618-12$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

618

cytes. They chromatographed postribosomal supernatant on DEAE-cellulose and phosphocellulose, then detected two CAMPdependent and one CAMP-independent protein kinase using casein and histones as substrate in the presence or absence of CAMP. The role, if any, of these CAMPdependent kinases in the regulation of protein synthesis is not clear. In reticulocytes, protein synthesis is regulated by at least two apparently separate and distinct protein kinases that phosphorylate the smallest subunit of the peptide initiation factor eIF-2 (7-10). One of the enzymes is activated in the absence of heme and is called the hemin-controlled repressor or HCR (11). This same kinase may be activated in the presence of heme by a cascade sequence of reactions that involves first a heat-stable protein, HS, isolated from the postribosomal supernatant of reticulocytes. HS may be activated by either pressure (12) or heat (13). In turn with ATP, this protein activates a heat-labile protein, HL, of yet unknown function. The eIF-2 kinase of the HCR system appears to be a 95,000 to 100,000 M, peptide that is activated by phosphorylation (14, 15). A protein of about

RETICULOCYTE

CAMP-DEPENDENT

90,000 M, is involved in this activation (16). The second eIF-2 kinase is indirectly activated by low concentrations of doublestranded RNA in lysates of reticulocytes (7) or certain interferon-treated cells (17- 19). Both of these eIF-2 kinase systems have been found by several laboratories to involve CAMP-independent protein kinases. However, Ochoa and his co-workers (20) obtained data that were interpreted to reflect an effect of CAMP on the eIF-2 kinase of the HCR system. They presented a particularly intriguing model in which the CAMP-dependent protein kinase of rabbit reticulocytes phosphorylated and thereby activated this eIF-2 kinase. Evidence was presented indicating that the reticulocyte CAMP-dependent enzyme was activated in the absence of heme (21). However, this interpretation has been questioned (22-24). Here, we described a procedure for purification to apparent homogeneity of the catalytic subunit of the CAMP-dependent protein kinases from reticulocytes. The enzyme was partially characterized with respect to its physical properties and substrate specificity. It is shown that this enzyme from reticulocytes will phosphorylate a low-molecular weight, heat-stable phosphatase inhibitor from muscle that is known to be activated by a CAMP-dependent protein kinase (25, 26). The possibility that CAMP might function in regulation of protein synthesis by a mechanism involving this inhibitor and its cognate phosphatase is discussed. EXPERIMENTAL

PROCEDURES

Materials Histones IIA, phosphocellulose, CNBr-activated Sepharose 4B, and GMP-P(CH,)P were purchased from Sigma Chemical Company (St. Louis, MO.). Hydroxylapatite was obtained from Clarkson Chemical Company (Williamsport, Pa.), and Sephadex G-75 from Pharmacia Fine Chemicals (Uppsala, Sweden). Microgranular DE-52 cellulose was from Whatman Biochemical, Ltd. (Kent, England). Hexokinase was bought from Boehringer-Mannheim, Germany. [Y-~*P]ATP and carrier-free [3ZP]phosphate were purchased from ICN Life Science Group (Irvine, Calif.). [y32P]ATP was also prepared by minor modification (27) of the procedure described by Glynn and Chappell

PROTEIN

KINASE

PURIFICATION

619

(28). X-ray no-screen film was from Eastman Kodak Company.

Buffer Solutions Solution A, 20 mM Tris-HCl (pH 7.5), 10 mM BME. Solution B, 75 mM K,HPO,/KH,PO, (pH 7.51, 5 mM BME, 1 mM EDTA. Solution C, 350 mM K,HPO,/KH,PO, (pH 7.5), 5 mM BME, 1 mM EDTA. Solution D, 20 mM Tris-HCl (pH 7.5), 5 mM BME, 1 mM EDTA.

Preparation of Substrates for Assay of Protein Kinase Reticulocyte 40 and 60 S ribosomal subunits were prepared as previously described (29). The procedure for the preparation of the ribosomal subunits from Artemia salina has also been reported (30). Briefly, KCl-washed reticulocytes and A. salina ribosomes were dissociated in solutions containing 0.5 and 0.7 M KCl, respectively. Then the subunits were separated by centrifugation through a linear sucrose gradient (15 to 30% w/v) in a Ti-14 zonal rotor (Beckman Instruments, Palo Alto, Calif.). The protein phosphatase inhibitor 1 from rabbit skeletal muscle was prepared as described by Nimmo and Cohen (26) from the heat-stable fraction of the tissue extract. It was purified up to the Sephadex G-100 step.

Preparation of Reticulocyte Postribosomal Supernatant The postribosomal supernatant fraction was prepared from rabbit reticulocytes by the procedure described previously (12), then stored at -80°C until used.

Assays for Protein Kinases The activity of CAMP-dependent protein kinases or their catalytic subunit was assayed in two different ways. (a) Qualitative determination and autoradiography. Assay

by gel electrophoresis

reaction mixtures contained in a total volume of 30 ~1: 20 mM Tris-HCl (pH 7.5), 5 mM MgCl,, 2.5 mM dithiothreitol, 0.5 mM [y-“2P]ATP (100-1000 Ci/mol) plus substrate, enzyme and additional components in the amounts indicated in the text and figure legends. Usually, 20 pg of reticulocyte 40 S ribosomal subunits containing a 32,000 M, protein capable of being phosphorylated by the enzyme, was used as substrate. Reaction mixtures were incubated for 10 min at 37°C and placed on ice. Thirty microliters of “electrophoresis sample buffer”(50mMTris-HCI, pH6.8; IOOmMDTE, 2% SDS, 10% glycerol, and 0.008% bromophenol blue) were added immediately. Then the samples were heated at 95°C for 5 min and loaded on a 15%

620

GRANKOWSKI,

KRAMER.

SDS-polyacrylamide gel prepared as described (31, 32). Electrophoresis and autoradiography were performed as detailed previously (8). (b) Quantitative determination using histones as substrate. Reaction mixtures as described above under (a) were increased to a final volume of 100 ~1 containing 16 pg of histones as substrate. After incubation for 20 min at 37”C, 200 pg of bovine serum albumin in 10 ~1 H,O and then 2 ml of 5% trichloroacetic acid were added. The mixtures were placed on ice for 5 min, then the samples were filtered through glass-fibers filters (Schleicher and Schiill, No. 29), washed twice with 5 ml each of 5% trichloroacetic acid, dried, and counted by liquid scintillation. Saturating amounts of highly purified or crude enzyme preparations gave about 1750 pmol of [3ZP]phosphate incorporation into histones under these assay conditions. Blank reactions were run without histones. For most samples containing CAMP-dependent protein kinase these values were less than 2% of those observed in the presence of histones. These blank values were subtracted from the total for estimation of CAMPdependent kinase activity. A concentration curve of the enzyme fraction versus [3ZP]phosphate incorporated was prepared for quantitative estimation of each enzyme fraction. The enzymatic activity per unit volume of the enzyme fraction was estimated from at least three points falling in the linear region of this curve. Fractions containing homogeneous catalytic subunit give 53 pmol of phosphate/rig of enzyme protein in this assay system. A unit of enzyme is defined as that amount of enzyme necessary to transfer 1 nmol of [32P]PO; from [y-32P]ATP into histones in 20 min.

Preparation of the Protein Inhibitor of CAMP-dependent Protein Kinuses The inhibitor was prepared from rabbit hind leg muscle by minor modifications of the procedures described originally by Walsh et al. (5) and altered by Cohenet al. (33). The muscle tissue was homogenized with 2.5 vol of 4 mM EDTA, centrifuged (Sorvall, rotor GSA; 10 min at 9500 rpm), and the supernatant adjusted to pH 6.1 with 1 M acetic acid. After 10 min on ice, centrifugation was repeated for 30 min. The resulting supernatant was raised to pH ‘7.0 with 6 M NH,OH, then heated for 20 min in a boiling water bath, cooled on ice, and filtered. The inhibitor fraction was precipitated by the addition of 50% trichloroacetic acid to give a final concentration of 15%. Precipitated protein was collected by centrifugation, then resuspended and dialyzed against 10 mM Tris-HCl (pH 7.5). About 8 mg of the inhibitor fraction was loaded on a 0.9 x 55-cm column containing Sephadex G-75 that was equilibrated in 10 mM Tris-HCl (pH 7.5). Fractions were assayed for activity that would inhibit phosphorylation of histones by a crude

AND HARDESTY

fraction of reticulocyte CAMP-dependent protein kinase in the presence of 2 pM CAMP as described above.

Preparation

of Histone -Sepharose .GB

Sepharose 4B bearing covalently linked histones was prepared according to the procedure recommended by Pharmacia (34). Cyanogen bromide-activated Sepharose 4B, 2 g, was washed with 400 ml of 1 mM HCl and then with 500 ml of coupling solution (0.1 M NaHCO,, adjusted to pH 8.3, 0.5 M NaCl). The beads of the Sepharose 4B were suspended in 15-20 ml of the same solution to which 70 mg of histones was added. The coupling reaction was carried out at 4°C for 18 h. The resulting suspension was washed with an additonal 200 ml of the coupling solution, then unreacted active groups remaining on the Sepharose were blocked by the addition of 40 ml 0.1 M Tris-HCl, pH 8.0. This reaction was carried out for 2 h at room temperature. Finally, the product was washed successively with 200 ml 0.1 M sodium acetate buffer (pH 4.0) containing 0.5 M NaCl, 400 ml of coupling solution, and solution D containing 120 mM KCl.

Protein Determination Protein concentrations were determined routiriely by the method of Warburg and Christian (35) and checked occasionally with the procedure involving amidoschwarz staining as described by Schaffner and Weissman (36).

Analysis of Proteins by Isoelectric Focusing The purified enzyme preparation (5 pg of protein) was subjected to isoelectric focusing on 5% polyacrylamide gels (“Ampholine PAGplates,” pH 3.5-9.5, purchased from LKB, Rockville, Md.). Electrode solutions were 1 M H,PO, and 1 M NaOH, respectively. Samples of rabbit hemoglobin and bovine serum albumin (10 Hg of protein each) were included in the same experiment. The proteins were run at -2°C for 5 h with limits of 15 W or 1000 V (Model 494 power supply, Instrumentation Specialties Co., Lincoln, Nebr.). At the end of the run, a 0.5-cm-wide strip of the gel was cut into l-cm pieces that were submerged into 1 ml of 50 mM KCl. After at least 2 h, pH for each slice was determined. Proteins were fixed in 15% trichloroacetic acid, washed in 10% acetic acid plus 45% methanol, then stained, destained, and dried as described for SDS-polyacrylamide gels (8).

Binding of [35S]Methionyl-tRNAf to Reticulocyte 40 S Ribosomal Subunits The preparation of components for the formation of the 40 S initiation complex has been described

RETICULOCYTE

CAMP-DEPENDENT

PROTEIN

Catalytic dependent

FIG. 1. Purification procedure from rabbit reticulocytes.

KINASE

subunit Protein

of tile Kinare

cA”P-

for the catalytic subunit of CAMP-dependent

elsewhere (37). The assay conditions were originally described by Gupta et al. (38) and modified by Spremulli et al. (39). To measure protein kinasemediated inhibition, a three-step assay was performed as described previously (29). In step 1, during 5 min at 37”C, 0.1 A,,, unit of 40 S ribosomal subunits, 2 ~g of eIF-2, a protein kinase fraction as detailed in the figure legend, and 0.4 mM ATP were incubated under conditions described for the protein kinase assay. The preincubation was terminated by addition of 0.35 unit hexokinase and glucose to give a final concentration of 1 mM in a final volume of 50 ~1. Incubation was continued for 3 min at 37°C. Finally, in step 3 the preincubated components were mixed with a reaction mixture containing, in a final volume of 100 ~1: 20 mM Tris-HCl, pH 7.5; 4 mM MgCI,; 85 mM KCl; 2.5 mM DTE; 0.2 mM GMP-P(CH,)P; 75 pM spermine; 25 wg of FII (cf. 37); and 8 pmol of [35S]methionyl-tRNAf (1.5 to 2 Ci/mmol). Incubation was continued at 37°C for 5 min, then at 0°C for 5 min. Reaction mixtures were diluted with ice-cold buffer containing 20 mM Tris-HCl, pH 7.5; 4 mM MgC&; and 85 mM KCI. The samples were filtered slowly through nitrocellulose filters (0.45~pm pore size, type HAWG, Millipore Corp., Beford, Mass.), washed with the same buffer, dried, and counted as described above. A blank value (usually about 5 to 10%) for [35S]methionyltRNAr bound to Millipore filters in the absence of 40 S particles served as a control. This value was subtracted to calculate binding to 40 S ribosomal subunits.

621

PURIFICATION

protein

kinases

RESULTS

Purification of the Catalytic CAMP-Dependent Protein Rabbit Reticulocytes

Subunit of Kinases from

The catalytic subunit of CAMP-dependent protein kinases from the postribosomal supernatant of rabbit reticulocytes was purified to an extent that it approached apparent homogeneity by the fractionation steps given in Fig. 1. This procedure includes batch elution from DEAE-cellulose and then hydroxylapatite, separation of the catalytic and regulatory subunits of CAMP-dependent protein kinases by chromatography on DEAE-cellulose in the presence of CAMP, then consecutive chromatography of the catalytic subunit on columns of phosphocellulose, histone-Sepharose, and Sephadex G-75. Activity for CAMPdependent protein kinase or its catalytic subunit was followed qualitatively by its ability to phosphorylate a 32,000 M, protein of reticulocyte 40 S ribosomal subunits. This analysis was done by gel electrophoresis and autoradiography as described under Experimental Procedures. Quantita-

622

GRANKOWSKI,

KRAMER, TABLE

AND

HARDESTY

I

PURIFICATION OF THE CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN KINASE Step of purification DEAE-cellulose 250 mM KC1 fraction’ Ammonium sulfate fractionation (O-50%) Hydroxylapatite 350 mM potassium phosphate fraction’ DEAE-cellulose 50 mM KC1 fraction Phosphocellulose gradient fraction Histone-Sepharose 4B 200 mM KC1 fraction Sephadex G-75

Total proteina (md

Total activity (units)*

Specific activity (units/mg)

3,324

1,452,‘708

437

1

1,901

1,271,824

669

1.5

87

961

1,005,762

1,047

2.4

69

27

607,745

22,509

51.5

42

10.2

274,676

26,929

61.5

19

3.5 1.1

155,378 58,300

44,394 53,000

Purification (fold)

101 121

Yield (a) 100

11 4

d Four liters of postribosomal supernatant containing 145 g of protein was applied to the first DE-52 column. The CAMP-dependent kinase activity cannot be determined reliably in the postribosomal supernatant. b One unit is defined as that amount of enzyme necessary to transfer 1 nmol of [32P]PO; from [@*P]ATP into histones in 20 min. c In steps 1 to 3 protein kinase activity was assayed in the presence of 2 x lo-’ M cycIic AMP.

tive determination of enzymatic activity was measured by incorporation of [32P]phosphate from [Y-~~P]ATP into histones. All fractionation procedures were carried out at 0-4°C. Four liters of postribosomal supernatant prepared as described previously (12) was used for a typical preparation. A quantitative estimation of the purification obtained at each step of the isolation procedure is presented in Table I. DEAE-cellulose chromatography and ammonium sulfate fractionation. A 7.5 x IlO-cm column was filled with washed microgranular DE-52, then flushed with 7.5 liters of solution A containing 20 mM KCl. Postribosomal supernatant (4 liters containing about 145 g of protein) was applied to the column which was then washed with solution A plus 20 mM KC1 until the absorbance at 280 nm of the eluate was below 0.5. Most of the hemoglobin present in postribosomal supernatant is removed from the column by this procedure. Adsorbed protein was eluted with solution A containing 250 mM KCl. Typically 2.2 liters containing about 3.5 g of protein was collected in this fraction. The enzyme was precipitated by

the slow addition of finely ground crystalline ammonium sulfate to give 50% saturation. The pH was maintained at pH 7.5 by the dropwise addition of 1 N NH,OH added as required along with the ammonium sulfate. Precipitated protein containing about 87% of enzymatic activity eluted from the DE-52 column was collected by centrifugation (10,OOOg for 20 min), resuspended in a minimum volume of solution B, then dialyzed against two Z-liter volumes of solution B for a total of 18 h. Hydroxylapatite chromatography. About 1.9 g of protein from the previous step was applied to a 2.5 X 40.0~cm column of hydroxylapatite that had been washed overnight with about 3 liters of solution B. A chromatographic loading density of about 10 mg of protein/ml of hydroxylapatite was found to be necessary to obtain satisfactory resolution and recovery of activity under the conditions used. Nonadsorbed protein was removed from the column by extensive washing with solution B, then the enzyme was eluted with solution C. Fractions with an absorbance at 280 nm greater than 0.1 were pooled (total volume of about 500 ml>,

RETICULOCYTE

CAMP-DEPENDENT

FIG. 2. Chromatography on phosphocellulose. About 2’7 mg of protein from the second DE-52 chromatography was chromatographed on phosphocellulose as described in detail in the text. The column was washed with the application buffer, then, where indicated by the arrow, a linear gradient running from 50 to 400 mM KC1 was started. Protein kinase activity (A) was measured by formation of 32P-labeled histones as described under Experimental Procedures; (O), absorbance at 280 nm; (- - -) KC1 concentration,

concentrated to about 30 ml with an Amicon ultrafiltration system equipped with a PM-10 membrane, then dialyzed overnight against two changes of 1 liter each of solution D plus 50 mM KC1 and 20 PM CAMP. Second DEAE-cellulose chromatography. A 2 x 4’7~cm column was loaded with washed, microgranular DE-52 cellulose, then equilibrated overnight by flushing with about 800 ml of solution D containing 50 mM KC1 and 20 PM CAMP. Approximately 1g of protein from the previous step was applied to the column which was then eluted with additional solution D containing 50 mM KC1 and 20 pM CAMP. The regulatory and catalytic subunit of the CAMP-dependent protein kinases are dissociated and the latter is not retained on the column under these conditions (40). Fractions containing nonadsorbed protein (about 147 ml) were combined and then concentrated to about 10 ml by ultrafiltration as described above. The resulting fraction, designated F1, contains nearly all of the histone kinase activity that can be recovered from the column and is used for further purification of the enzyme as described below. Protein adsorbed to the DEAE-cellulose under these conditions was eluted in two steps, first with solution D plus 100 mM KCl,

PROTEIN

KINASE

PURIFICATION

623

then solution D plus 300 InM KCl. These fractions are designated Fz and F,, respectively. Fraction F2 contains trace amounts of CAMP-dependent kinase. Fraction F, is free of measurable amounts of this enzyme, but is a rich source of several CAMPindependent protein kinases that will be described elsewhere. Phosphocellulose chromatography. A 1.0 x 7.0~cm column was filled with washed phosphocellulose, then flushed with 500 ml of solution D containing 50 InM KCl. About 2’7 mg of protein from the previous step (Fraction Fl) was applied. Nonadsorbed protein was removed by extensive washing with the application buffer. The enzyme was eluted with a 120-ml linear gradient of 50 to 400 InM KC1 in solution D. Fractions of 2 ml were collected and assayed for protein kinase activity measured by [32P]phosphate incorporation into histones. The elution profile of protein kinase activity from this column is shown in Fig. 2. Fractions 30 to 50 were pooled and concentrated by ultrafiltration as described above, then dialyzed overnight against 2 x 500 ml of solution D containing 120 mM KCl. Typically lo-12 mg of protein containing 250,000300,000 units of enzyme was recovered in this fraction. Histone-Sepharose chromatography. Protein from the previous step (lo-12 mg) was applied to a column of histone-Sepharose (1 x 5 cm) that had been equilibrated previously with solution D containing 120 mM KCI, then the column was washed with about 50 ml of the same solution. The catalytic subunit of CAMP-dependent protein kinases was eluted from this column with solution D containing 200 mM KCl. Fractions containing protein were combined and concentrated by dialysis against solution D plus 100 mM KC1 and 50% glycerol. Sephadex G-75 chromuto~phy. The concentrated sample from the previous step (about 3.5 mg of protein in 0.9 ml buffer) was loaded on a Sephadex G-75 column (1 x 50 cm) equilibrated with solution D plus 100 MM KCl. The column was developed at a flow rate of about 1 ml/h and l-ml fractions were collected. The elution profile of absorbance at 230 nm and of protein kinase activity is given in Fig. 3. Fractions 19 to

624

GRANKOWSKI,

KRAMER.

22 were combined and concentrated by dialysis against solution D containing 50% glycerol. The enzyme preparation was stored in small aliquots at -80°C. General remarks concerning the puti$cation procedure. As described in the previous sections and as shown in Table I, about 1.1 mg of the isolated catalytic subunit of CAMP-dependent protein kinases was obtained from about 4 liters of reticulocyte postribosomal supernatant containing approximately 145 g of protein. A large portion of this protein is hemoglobin, most of which is separated from the enzyme in the first chromatographic step. It should be noted that the calculated purification factors presented in Table I are not based on the postribosomal supernatant, but rather on the fraction obtained from the next step of purification, the first chromatography on DEAE-cellulose. CAMP-dependent protein kinase activity, as judged by an increase in phosphorylation of histones in the presence of CAMP, is not detected in fractions other than those obtained from the first three steps of purification. Rechromatography of the enzyme on DEAE-cellulose in the presence of CAMP results in a particularly large purification. Under the conditions used, CAMP causes dissociation of the CAMPdependent protein kinase into its regulatory and catalytic subunit. The latter is not adsorbed to DEAE-cellulose even with relatively low salt concentrations, 50 InM KC1 (40). Thus the catalytic subunit is effectively separated from not only the regulatory subunit but many other proteins that are eluted at 250 InM KC1 during the first chromatography on DEAE-cellulose but remain bound at the lower salt concentration used in this second chromatography on this medium. The peptide distribution pattern from SDS-gel electrophoresis of the pooled enzyme fractions obtained from the five final fractionation steps is shown in Fig. 4. All steps were found to be required to give the catalytic subunit at a stage of purity approaching homogeneity. A trace amount of a minor contaminant was seen occasionally in some preparations when they were analyzed in relatively large amounts by SDSgel electrophoresis. This peptide is visible in track 5 of Fig. 4 at a point corresponding to a molecular weight of about 26,000.

AND HARDESTY

FRACTION

NUMBER

FIG. 3. Gel filtration on Sephadex G-75. About 3.5 mg of protein from the histone-Sepharose fraction was applied to a column containing Sephadex G-75. The column was developed as detailed in the text. Absorbance at 280 nm (0) and formation of 3ZP-labeled histones (A) were determined. In a separate run, the void volume (q,) and the position at which soybean trypsin inhibitor eluted (M, = 21,300, indicated by T. I.), were measured.

A relatively large loss of enzymatic activity occurs during chromatography on phosphocellulose with relatively little increase in specific activity of the enzyme. However, omission of this step resulted in contamination of the protein kinase obtained at the final step of purification as indicated by the appearance of several additional bands following SDS-gel eleetrophoresis. Characterization Subunit

of the Purified

Catalytic

The catalytic subunit of CAMP-dependent protein kinases purified as described in the previous sections appears to be a single protein with an apparent molecular weight of 43,000 as judged from the elution pattern of the Sephadex G-75 column and from analysis by polyacrylamide gel electrophoresis in the presence of SDS (Fig. 4, track 5). When subjected to isoelectric focusing, the isoelectric point of this protein was found to be about pH 8.0 (Fig. 5). A minor component was observed to focus near pH 7.0. This does not appear to be the trace contamination visible in track 5 of Fig. 4 in that it was observed in approximately the same proportion to the major band in several prepara-

RETICULOCYTE

CAMP-DEPENDENT

+a

+b +C

+d

I2345 FIG. 4. Analysis by SDS-polyacrylamide gel electrophoresis of fractions containing the catalytic subunit of CAMP-dependent protein kinase. Aliquots from the last five steps of the purification procedure were analyzed for their peptide pattern. The experimental details are given under Experimental Procedures. Track 1 = 25 pg of protein after chromatography on hydroxylapatite; track 2 = 20 pg of protein after the second chromatography on DE-52; track 3 = 15 pg protein of the phosphocellulose fraction; track 4 = 10 pg protein of the histone-Sepharose fraction; track 5 = 10 pg of protein obtained after gel filtration on Sephadex G-75. Molecular weight markers are indicated by arrows: a = bovine serum albumin (68,000 MJ, b = fumarase (subunit; 48,500 M,), c = glyceraldehyde Y-phosphate dehydrogenase (subunit; 37,000 M,), d = trypsin inhibitor (21,300 M,).

PROTEIN

KINASE

PURIFICATION

isolated enzyme were determined using [Y-~~P]ATP and different substrates. The results are shown in Fig. 6. Besides histones (track 4), a 32,00OM, protein of the reticulocyte 40 S ribosomal subunit is strongly phosphorylated (track 6). The ribosomal subunits or the other protein fractions used as substrates do not contain endogenous protein kinase activity (tracks 2, 5, 7, 9, and 11). Phosphorylation is also observed with a protein of the Artemia saEina 40 S ribosomal subunits (track lo), although this protein is somewhat smaller (about 30,000 M,) than the protein of reticulocyte ribosomes that is phosphorylated under the same conditions. This result may reflect homology between these two ribosomal proteins. Only slight phosphorylation is seen with some proteins of the 60 S ribosomal subunits, especially with those derived from A. salina (Fig. 6, track 12). Also shown in Fig. 6 (track 3) is the phosphorylation of the phosphatase inhibitor-l that has been partially purified from rabbit skeletal muscle as described under Experimental Procedures. This inhibitor is involved in the regulation of the multifunctional phosphatase that dephosphorylates several enzymes of the glycogen metabolism. The phosphatase inhibitor-l is active only after it has been phosphorylated by a CAMP-dependent protein kinase (25, 26). Phosphorylation of substrate proteins by

Hb

tions containing different amounts of the contaminating peptide. It is likely that the band observed at about pH 7.0 following isoelectric focusing reflects another form of the reticulocyte kinase. Multiple forms have been detected by isoelectric focusing of the catalytic subunit in bovine heart (41) and rabbit muscle (42). Enzymatic activity and specificity of the

625

FIG. 5. Isoelectric

EGA

focusing of the enzyme fraction. Isoelectric focusing was performed on LKB 5% polyacrylamide gels as described under Experimental Procedures. About 5 c(g of the enzyme fraction after the last step of purification was applied. A strip of the gel was taken for pH determination as detailed under Experimental Procedures. Marker proteins were rabbit hemoglobin and bovine serum albumin. Their isoelectric points are indicated by arrows as Hb and BSA, respectively.

626

GRANKOWSKI,

KRAMER,

AND HARDESTY

FIG. 6. Substrate specificity of the catalytic subunit of reticulocyte CAMP-dependent protein kinase. Different substrates were incubated with 1 or 1.5 gg of the catalytic subunit, where indicated, and 0.15 mM [@*P]ATP (100 Cilmol) as detailed under Experimental Procedures. Proteins were separated on 15% SDS-polyacrylamide gels and analyzed for phosphorylation by autoradiography. (A) Stained gel; (B) radioautograph of the same gel. Tracks 2,5,7,9, and 11 no enzyme; track 1 = enzyme alone; tracks 2 + 3 = 9 pg muscle phosphatase inhibitor-l; track 4 = 2 wg histones; tracks 5 + 6 = 20 pg of reticulocyte 40 S ribosomal subunits; tracks ‘7 + 8 = 40 pg of reticulocyte 60 S ribosomal subunits; tracks 9 + 10 = 20 pg of A. salina 40 S ribosomal subunits; tracks 11 + 12 = 40 pg of A. saliva 60 S ribosomal subunits. Molecular markers are the same as in Fig. 4.

CAMP-dependent protein kinases (in the presence of CAMP) or by their catalytic subunit is greatly reduced by the low molecular weight protein inhibitor specific for these kinases, as described by Walsh et al. (5). The effect of this inhibitor, isolated from rabbit muscle as described under Experimental Procedures, on the activity of the reticulocyte catalytic subunit was tested with the results given in Fig. 7. Phosphorylation of either the 32,000 M, protein of reticulocyte 40 S ribosomal subunits or of histones was inhibited in the presence of this heatstable protein (tracks 2 and 4, respectively). It may be of physiological significance that this inhibitor protein could barely be detected in reticulocytes when the isolation procedure described above was used (unpublished results). Previous results (23) indicated that the catalytic subunit of the reticulocyte CAMPdependent protein kinase does not catalyze phosphorylation of the (Ysubunit (38,000 M,) of the peptide initiation factor eIF-2, or a 95,000 to 100,000 M, peptide that appears to function as a protein kinase for this eIF-2 subunit (14). Neither CAMP nor the catalytic subunit had a detectable effect on protein synthesis in lysates of rabbit reticulocytes. The data presented in Fig. 8 indicate that the catalytic subunit has no effect on the

FIG. 7. Inhibitor of the catalytic subunit of reticulocyte CAMP-dependent protein kinase. The enzyme (0.1 pg of protein) was incubated with histones and 40 S ribosomal subunits, respectively, in the absence or presence of the low molecular weight, heat-stable inhibitor (5) and [y-z2P]ATP. Then radioactive proteins were analyzed as described under Experimental Procedures. A, Stained gel; B, radioautograph. Tracks 1 + 2 = 20 pg of reticulocyte 40 S ribosomal subunits; tracks 3 + 4 = 5 clg histones. Tracks 2 + 4 also contain 7 pg of the inhibitor fraction prepared as described under Experimental Procedures.

RETICULOCYTE

CAMP-DEPENDENT

-0

D

0

-o-catalytic

I

IO PROTEIN,

KINASE

PURIFICATION

627

DISCUSSION

I

I

PROTEIN

tubunlt

I

20 ,,Q

FIG. 8. No effect of the catalytic subunit of CAMPdependent protein kinases on binding of [“S]methionyltRNA, to 40 S ribosomal subunits. The assay was performed as described under Experimental Procedures. Increasing amounts of the protein kinase fractions were added in step one as indicated on the abscissa. (0) Catalytic subunit of CAMP-dependent protein kinase after the second DE-52 chromatography (Fraction 1 in Fig. 1); (A) An HCR fraction obtained from the same chromatography (Fraction 3 in Fig. 1).

eIF-Z-dependent binding of methionyltRNAr to 40 S ribosomal subunits. Both eIF-2 and reticulocyte 40 S ribosomal subunits were incubated with ATP and either the catalytic subunit of CAMP-dependent protein kinases or a fraction containing the HCR eIF-2 kinase (fraction F, from the second DEAE-chromatography, cf. Fig. 1). Residual ATP was eliminated in the reaction mixture by incubation with glucose and hexokinase, then the activity of the preincubated eIF-2 plus 40 S ribosomal subunits to support binding of [35S]methionyl-tRNAf to the 40 S subunits was determined. In contrast to HCR that inactivates the components for this reaction, the phosphorylation of the 40 S ribosomal subunits by the CAMP-dependent protein kinase does not affect their ability to support the methionyltRNAr binding reaction. These results are consistent with our earlier conclusion that CAMP is not directly involved in the regulation of protein synthesis (23).

The catalytic subunit of CAMP-dependent protein kinases from rabbit reticulocytes has been purified to a degree that approaches homogeneity. A molecular weight of 43,000 was determined by gel electrophoresis in the presence of SDS. This is similar in molecular weight to 41,300 and 39,000 reported for the same type of catalytic subunit isolated from rabbit skeletal muscle (42) and from bovine heart (41). Bechtel et al. (42) commented on the similarity if not identity of the catalytic subunit isolated from these two different sources. Two isozymes were detected by isoelectric focusing of the catalytic subunit from skeletal muscle (42) and three from bovine heart (41). Data have been presented (41,42) indicating that the catalytic subunits contained about 2 mol phosphate/m01 enzyme. We have not determined the phosphate content of the reticulocyte enzyme, however, it appears to consist of two distinct isozymes that differ in isoelectric points. We did not detect [3zP]phosphate incorporation into the 43,000 M, peptide following incubation of the purified catalytic subunit with [32P]ATP. This might indicate that the reticulocyte enzyme is extensively phosphorylated at the time of isolation or that it does not undergo autophosphorylation. In any event the catalytic subunit from reticulocytes appears to be similar, if not identical to the enzyme subunit from heart and muscle. A number of proteins can function in vitro as substrates for the catalytic subunit from reticulocytes, however these reactions may be of no physiological significance. Histones are an effective substrate for the reticulocyte enzyme even though these anucleated cells may not contain histones. A number of proteins in crude enzyme fractions from reticulocytes including a protein of the 40 S ribosomal subunit are phosphorylated in vitro by the enzyme. The 40 S subunit protein has an apparent molecular weight of 32,000 and may be S6 by the new nomenclature suggested for mammalian ribosomal subunits (43). This appears to be the same ribosomal protein reported by Traugh and Porter (44) to be phosphorylated by fractions from reticulocytes that contained CAMP-dependent protein kinases or their

628

GRANKOWSKI,

KRAMER,

catalytic subunit. No evidence was found for a change in biological function that could be attributed to this phosphorylation. In agreement with these earlier data, the results of Fig. 8 demonstrate that reticulocyte 40 S ribosomal subunits phosphorylated by the reticulocyte enzyme are as active in methionyl-tRNAr binding as untreated subunits. Thus, the physiological significance of this phosphorylation is obscure. Recently we have presented evidence interpreted to indicate that the catalytic subunit of the CAMP-dependent kinase did not activate an eIF-2 kinase or cause an inhibition of protein synthesis in reticulocyte lysates (23). The data presented here support this interpretation. We believe these data considered with those published earlier justify the conclusion that eIF-Ba-phosphorylation in reticulocytes is not regulated by CAMP through the CAMP-dependent kinase. However, even though no effect on protein synthesis was detected in these experiments, the possibility remained that CAMP might function to regulate protein synthesis through an effect on eIF-2 phosphatase. In this regard, it is of potential significance that a 25,000 M,, heat-stable protein from muscle has been demonstrated to block the multifunctional phosphatase that functions with several enzymes of glycogen metabolism (26). This inhibitor protein is activated by phosphorylation with the CAMP-dependent kinase. Such an inhibitor functioning with the eIF-2 phosphatase might lead to a CAMP-dependent increase in phosphorylation of eIF-2 and thereby cause an inhibition of protein synthesis only in the presence of low levels of eIF-2 kinase activity. Such an effect might have been missed in our earlier experiments. The inhibitor protein from muscle is phosphorylated by the catalytic subunit of the reticulocyte enzyme (Fig. 6). However, no similar protein could be detected in reticulocytes (unpublished results). Furthermore, the phosphorylated inhibitor protein from muscle has been found not to inhibit the reticulocyte eIF-2 phosphatase (unpublished results). These results support our earlier conclusion. It appears unlikely that protein synthesis in reticulocytes or their lysates is controlled directly by CAMP.

AND

HARDESTY ACKNOWLEDGMENTS

The authors wish to thank M. Hardesty, M. Rodgers, K. Pham, and D. Lehmusvirta for their excellent technical assistance, F. Hoffmann for his preparation of the photographs for figures, and M. A. Henderson for preparation of the typescript. This grant is supported in part by Grants CA-1660805 and CA-09182-04, awarded by the National Cancer Institute, DHEW, to Dr. B. Hardesty.

REFERENCES 1. NIMMO, H. G., AND COHEN, P. (1977) Advan. Cyclic Nucleotide Res. 8, 145-266. 2. BLOCH, K., AND VANCE, D. (1977) Annu. Rev. Biochem. 46, 263-298. 3. RUBIN, C. S., AND ROSEN, 0. M. (1975) Annu. Rev. Biochem. 44, 831-887. 4. SHENOLIKAR, S., AND COHEN, P. (1978) FEBS Lett. 86, 92-98. 5. WALSH, D. A., ASHBY, C. D., GONZALES, C., CALKINS, D., FISCHER, E., AND KREBS, E. (1971) J. Biol. Chem. 246, 1977-1985. 6. TRAUGH, J. A., AND TRAUT, R. R. (1974) J. Biol. Chem. 249, 1207-1212. 7. FARRELL,, P. J., BALKOW, K., HUNT, T., JACKSON, R. J., AND TRACHSEL, H. (1977) Cell 11, 187200. J. M., AND HARDESTY, 8. KRAMER, G., CIMADEVILLA, B. (1976) Proc. Nat. Acad. Sci. USA 73, 30783082. D. H., RANU, R. S., ERNST, V., AND 9. LEVIN, LONDON, I. M. (1976) Proc. Nat. Acad. Sci. USA 73, 3112-3116. 10. PETRYSHYN, R., TRACHSEL, H., AND LONDON, I. M. (1979) Proc. Nut. Acad. Sci USA 76, 1575-1579. 11. GROSS, M., AND RABINOVITZ, M. (1972) Biochim. Biophys. Acta 287, 340-352. A. B., AND HARDESTY, B. (1978) 12. HENDERSON, Biochem. Biophys. Res. Commun. 83,715-723. A. B., MILLER, A., AND HARDESTY, 13. HENDERSON, B. (1979) Proc. Nat. Acad. Sci. USA 76, 2605-2609. 14. TRACHSEL, H., RANU, R. S., AND LONDON, I. M. (1978) Proc. Nat. Acad. Sci. USA 75, 36543658. M., AND MENDELEWSKI, J. (1978) Biochim. 15. Gross, Biophys. Acta 520, 650-663. 16. WALLIS, M. H., KRAMER, C., AND HARDESTY, B. (1979) Fed. Proc. 38, 781. A., FEDERMAN, P., SHULMAN, L., 17. ZILBERSTEIN, AND REVEL, M. (1976)FEBSLett. 68,119-U& 18. ROBERTS, W. K., HOVANESSIAN, A., BROWN, R. E., CLEMENS, M., AND KERR, I. (1976) Nature (London) 264, 477-480. 19. LEBLEU, B., SEN, G. C., SHAILA, S., CARBRER, B.,

RETICULOCYTE

CAMP-DEPENDENT

AND LENGYEL, P. (1976) Proc. Nat. Acad. Sci. USA 73, 3107-3111. 20. DATTA, A., DEHARO, C., SIERRA, J. M., AND OCHOA, S. (197’7) Proc. Nat. Acad. Sci. USA 74, 1463-1467. 21. DA’ITA, A., DEHARO, C., AND OCHOA, S. (1978) Proc. Nat. Acad. Sci. USA 75, 1148-1152. 22. GRANKOWSKI, N., AND HARDESTY, B. (1979) Fed. Proc. 38, 329. 23. GRANKOWSKI, N., KRAMER, G., AND HARDESTY, B. (1979) J. Biol. Chenz. 254, 3145-3147. 24. ERNST, V., AND LEVIN, D. (1979) Fed. Proc. 38, 781. 25. HUANG, F. L., AND GLINSMANN, W. H. (1976) Eur. J. Biochem. 70, 419-426. 26. NIMMO, G. A., AND COHEN, P. (1978) Eur. J. Biothem. 87, 341-351. 27. KEENAN, R. W., ZISHKA, M. K., ANDNISHIMURA, J. S. (1972) Anal. Biochem. 47. 601-608. 28. GLYNN, I. M., AND CHAPPELL, J. B. (1964) Biothem. J. 90, 147-149. 29. KRAMER, G., HENDERSON, A. B., PINPHANICHAKARN, P., WALLIS, M. H., AND HARDESTY, B. (1977) Proc. Nat. Acad. Sci. USA 74, 14451449. 30. KRAMER, G., ODOM, 0. W., AND HARDESTY, B. (1979) in Methods in Enzymology (Moldave, K., and Grossman, L., eds.), Vol. 60, pp. 555-566. Academic Press, New York. 31. LAEMMLI, U. K. (1970) Nature (London) 227, 680-685.

PROTEIN

KINASE

PURIFICATION

629

32. ANDERSON, C. W., BAUNI, P. R., AND GESTELAND, R. F. (19’73) J. Viral. 12, 241-252. 33. COHEN, P., NIMMO, G., AND ANTONIW, J. (1977) Biochem. J. 162, 435-444. 34. AFFINITY CHROMATOGRAPHY (1974) Pharmacia Fine Chemicals, Uppsala, Sweden. 35. WARBURG, O., AND CHRISTIAN, W. (1942) Biothem. 2. 310, 384-421. 36. SCHAFFNER, W., AND WEISSMANN, C. (1973) Anal. Biochem. 56, 502-514. 37. ODOM, 0. W., KRAMER, G., HENDERSON, A. B., PINPHANICHAKARN, P., AND HARDESTY, B. (1978) J. Biol. Chem. 253, 1807-1816. 38. GUPTA, N. K., CHATTERJEE, B., CHEN, Y., MAJUMDAR, A. (1975) J. Biol. Chem. 250, 853-862. 39. SPREMULLI, L., WALTHALL, B., LAX, S., AND RAVEL, J. (1977) Arch. Biochem. Biophys. 178, 565-575. 40. RUBIN, C. S., ERLICHMAN, J., AND ROSEN, 0. M. (1972) J. Biol. Chem. 247, 36-44. 41. PETERS, K., DEMAILLE, J., AND FISCHER, E. (1977) Biochemistry 16, 5691-5697. 42. BECHTEL, P., BEAVO, J., AND KREBS, E. (1977) J. Biol. Chem. 252, 2691-2697. 43. MCCONKEY, E. H., BIELKA, H., GORDON, J., LASTIK, S. M., LIN, A., OGATA, K., REBOUD, J. P., TRAUGH, J. A., TRAUT, R. R., WARNER, J. R., WELFLE, H., AND WOOL, I. G. (1979) Mol. Gen. Genet. 169, l-6. 44. TRAUGH, J. A., AND PORTER, G. G. (19’76) Biochemistry 15, 610-616.