Cyclic Nucleotide-independent Protein Kinase from Rat Liver

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 22, Issue of October 5, pp. 12280-12286 1985 Printed in d.S.A.

Cyclic Nucleotide-independentProtein Kinase from Rat Liver PURIFICATION AND CHARACTERIZATION OF A MULTIFUNCTIONAL PROTEIN KINASE* (Received for publication, March 6, 1985)

Seethala Ramakrishna and William B. Benjamin From the Diabetes Research Laboratory, Department of Physiology and Biophysics, School of Medicine, Health Sciences Center, State University of New York, Stony Brook, New York11 794

A rat liver CAMP-independent protein kinase that multifunctional protein kinase and to detail some of its propphosphorylates peptideb of ATP-citratelyase (Ramak- erties. The accompanying paper (12) describes this protein rishna, S., Pucci, D. L., and Benjamin,W. B. (1983) J. kinase’s effects on glycogen synthase. Biol. Chem. 258, 4950-4956) hasbeenpurified to apparent homogeneity. The molecular weight, deterEXPERIMENTALPROCEDURES minedbypolyacrylamide gel electrophoresisinthe Materials-Pbosphocellulose P-11 was purchased from Whatman, presenceofsodiumdodecyl sulfate, sucrosedensity Blue Sepharose, Polybuffer, Pharmalytes, CNBrl-activated Sephagradient, andby gel filtration, was found to be 36,000. rose, DEAE-Sephadex, CM-Sephadex, and Sephadex G-100 superfine This protein kinase phosphorylates in vitro ATP-cit- from Pharmacia, and silver stainingkit from Bio-Rad. Glycogen rate lyase, acetyl-coA carboxylase, and glycogensyn- synthase and inhibitor-2 were obtained from Dr. T. Soderling, Vanderbilt university. Artemia salina 40 S ribosomal subunits were a gift thase and does not phosphorylate phosphorylase, phosphorylase kinase, histone, phosvitin, and casein. It has from Dr. 0. M. Rosen, Memorial Sloan-Kettering Cancer Center, and from rabbit reticulocytes was a gift from Dr. B. Safer, National F, (activity factor) activity stimulating the ATP.Mg- eIF-2 Heart and Lung Institute, National Institutes of Health. Phosphodependent phosphatase andis therefore named a mul- rylase b (Sigma) was phosphorylated with [y-32P]ATP andphosphotifunctional protein kinase. This kinase differs from rylase kinase (Sigma) by the method of Antoniw et al. (13). Polyglycogen synthase kinase-3 with regard to substrate lysine Sepharose 4B was prepared as described by Yang et al. (14). specificity, kinetic parameters,andphysicochemical TSK columns were from Beckman. HPLC solvents were purchased from Pierce Chemical Co. All other chemicals were analytical grade. properties. Phosphorylation Assay-Protein kinase activity was assayed as described previously (10). The reaction mixture (50 pl) contained 50 mM MES buffer (pH 6.8), 5 mM 2-mercaptoethanol, 8 mM magnesium Insulin acts bothto increase and decrease the phosphoryla- acetate, 0.3 mM EGTA, 0.5 mM EDTA, 0.05 mM [Y-~’P]ATP(100tion of some enzymes of intermediary metabolism (1-4). In 1000 cpm/pmol), 1 mg/ml of bovine serum albumin, 1-3 mg/ml of ATP-citrate lyase, acetyl-coA carboxylase, or glycogen synthase and proteins that are phosphorylated at multiple sites, hormone the appropriateprotein kinase. The reaction was terminated by action may change the phosphorylation of each site independ- adding sample buffer and boiling in water bath for 3 min. Samples ently and in opposite direction (3-7). For example, insulin were loaded on SDS-acrylamide slab gels and electrophoresis was acts on ATP-citrate lyase to increase the phosphorylation of carried out asdescribed (15). When ATP-citrate lyase was substrate, peptide a and todecrease the phosphorylation of peptide b ( 5 , gels weretreated with 1N HC1, 7% acetic acid for 1h to remove acid6). In our studies on ATP-citrate lyase phosphorylation, we labile histidine phosphate (autophosphorylated phosphate). HCl was from the gel with 7% acetic acid, 10% methanol. Gels were isolated a CAMP-independent protein kinase that specifically washed stained with 0.1% Coomassie Blue G in 50% methanol, 7% acetic phosphorylates in vitro aserine and threonine residue on acid. Whenacetyl-coA carboxylase or glycogen synthase was the peptide B (8-lo), most likely the same peptide andsites substrate, the gel was directly stained with Coomassie Blue G and phosphorylated on peptide b in vivo. This kinase, initially destained with 10% methanol, 7% acetic acid, the protein band was named ATP-citrate lyase kinase was subsequently found to cut from the gel, and itsradioactivity was determined (10). A unit of phosphorylate acetyl-coA carboxylase (11)but not to phos- protein kinase is that amount of enzyme which catalyzes the transfer 1.0 pmol of phosphate/min. phorylate histone-HBB, casein, phosphorylase kinase, pyru- of Purification of MultifunctionalProtein Kinase-All procedures vate kinase, phosvitin, or phosphorylase. Recently, it hasbeen were carried out at0-4 “C. At each step of purification, phosphorylanoted that ATP-citrate lyase kinase phosphorylates glycogen tion of ATP-citrate lyase was assayed. Liver (1 kg) from fed 250 g, synthase at sites 2 and 3 (12). Because the phosphate contents body weight, Sprague-Dawley rats was homogenized with 3 volumes of sites 2 and 3 in glycogen synthase and peptide b in ATP- of 50 mM Tris-HC1, (pH 7.5), 0.5 mM EDTA, 15 mM 2-mercaptoethcitrate lyase are decreased by insulin action (2,3,6,8-10,12), anol, 0.5 mM PMSF, 10 mM a-amino caproic acid, 10 mM benzamidine, 10 p~ TLCK, 1 mg/l of leupeptin and pepstatin. The homogwe believe it is important to describe the purification of this enate was centrifuged at 25,000 X g for 20 min and then at140,000X g for 45 min. The supernatant was decanted through glass wool and * This investigation was supported by a grant from the Juvenile then stirred for 3 h with 1 1 of settled phosphocellulose P-11 equiliDiabetes Foundation International, and Grants AM 18905 and AM brated with 50 mM Tris-HC1 (pH 7.51, 15 mM 2-mercaptoethanol, 32150 from the National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health. This workwas The abbreviations used are: CNBr, cyanogen bromide; EGTA, presented at the69th Annual Meeting of the Federation of American ethylene glycol bis(6-aminoethyl ether)-N,N,N’N’-tetraacetic acid; Societies for Experimental Biology, Anaheim, CA, April 21-26, 1985 Mes, 4-morpholineethanesulfonic acid; HPLC, high performance liq(Ramakrishna, S., and Benjamin, W.B. (1985) Fed. Proc. 44, 704 uid chromatography; SDS, sodium dodecyl sulfate; PMSF, phenyl(Abstr. 1713)). The costs of publication of this article were defrayed methylsulfonyl fluoride; TPCK-trypsin, L-1-tosylamido-2-phenylin part by the payment of page charges. This article must therefore ethyl chloromethyl ketone-treated trypsin; eIF-2, eukaryotic initiabe hereby marked “advertisement” in accordance with 18 U.S.C. tion factor-2; l, liter; Hepes, 4-(2-hydroxyethyl)-l-piperazineethaneSection 1734 solelyto indicate this fact. sulfonic acid.

12280

Multifunctional Protein Kinase 0.25 mM PMSF, and 5% glycerol (buffer A). The slurry was filtered on a sintered glass funnel, extensively washed with buffer A (4 l), followed by 0.075 M KC1 in buffer A (8 1) until all the dark browncolored proteins were washed from the ion exchanger. The multifunctional protein kinase elutes from phosphocellulose between 0.230.30 M KC1. With hatch elution, phosphocellulose was stirred overnight with 1.2 1 of 0.35 M KC1 in buffer A, filtered, and washed with 1.2 1 of 0.35 M KC1 in buffer A. The 0.35 M KC1 filtrate and washings were pooled (2.35 1) and solid ammonium sulfate was added to 30% saturation. After standing for 30 min the sample was centrifuged and the precipitate was discarded. Ammonium sulfate was added to the supernatant to 60% saturation. After stirring for 1 h, the suspension was centrifuged at 15,000 X g for 30 min and the precipitate was washed in buffer A containing 60% saturated ammonium sulfate. The precipitate was dissolved in 200 mlof buffer A, and 50% polyethylene glycol (w/v) was added dropwise with stirring to obtain 7% concentration. Thesuspension was centrifuged at 20,000 X g for 15 min and the precipitate was discarded. 50% polyethylene glycol was added to the supernatant to a final concentration of 20% and stirred for 1 h. The suspension was collected by centrifugation, and the pellet was carefully rinsed with cold H,O without mixing. The precipitate was dissolved in 200 ml of20mM MES, 15 mM 2-mercaptoethanol, 0.2 mM PMSF, 1 mM EDTA, 10% glycerol (buffer B) using a Dounce homogenizer. The suspension was centrifuged at 20,000 X g for 10 min and the pellet was discarded. The supernatant was stirred for 1 h with 200 ml of settled DEAE-Sephadex equilibrated in buffer B and filtered on a sintered glass funnel. The resin was washed with another 200 ml of buffer B. The filtrate and washings were pooled and stirred with 200 ml of settled CM-Sephadex equilibrated in buffer B, washed with 500 ml of buffer B, 200 ml of 0.1 M NaCl in buffer B, and eluted with 200 ml of 0.4 M NaCl in buffer B. Ammonium sulfate was added to the 0.4 M NaCl eluate to 60% saturation. After stirring for 1 h, the suspension was centrifuged and the supernatant was discarded. The pellet was dissolved in 3ml of buffer B containing0.2 M NaCl and centrifuged. The clear supernatant fluid was further resolved on Sephadex G-100 (superfine, 2.6 X 100 cm) using buffer B containing 0.2 M NaCl (Fig. l a ) . Fractions with protein kinase activity for ATP-citrate lyase were pooled and concentrated by precipitation with ammonium sulfate to 60% saturation. The pellet was dissolved in 2ml of buffer B containing0.2 M NaCl, clarified by centrifugation, and resolved by HPLC on TSK 300/2000 (75 X 600-mm) columns connected in series. Fractions with most protein kinase activity were pooled and dialyzed overnight against 50 volumes of buffer B. The protein sample was then loaded on a Blue Sepharose (1.5 X 15-cm) column washed with 50 ml of buffer B followed by 50 ml of 0.1 M NaCl in buffer B and eluted with a 200 ml gradient of 0.10 to 0.75 M NaCl in buffer B. Fractions (2.5 ml) were collected and aliquots (0.02 ml) were assayed for protein kinase activity. Fig. l b gives the chromatographic pattern. Peak fractions (25-36)werepooled and dialyzed overnight against buffer B. The dialyzed sample was applied on a second Blue Sepharose (1 X IO-cm) column equilibrated with 0.075 M NaCl in buffer b. The column was washed with 50 ml of same buffer and eluted with 200 ml of a linear gradient of 0.075 to 0.45 M NaCl in buffer B. Fractions with protein kinase activity were pooled and dialyzed against buffer B followed by buffer B containing 50% glycerol. Other Method~-[y-~~P]ATP and [T-~’P]GTP were prepared by the method of Walseth and Johnson (16). Sucrose gradient centrifugation was performed by applying protein kinase fraction (in 0.2 ml) or the marker proteins hemoglobin ( M , = 64,000), ovalbumin ( M , = 43,000), chymotrypsinogen (M, = 25,000), and cytochrome c ( M , = 12,500) on linear gradients of 5 to 20% sucrose and centrifuged in a Beckman SW 40 rotor for 40 h a t 2 “C. The sedimentation coefficient and molecular weight were determined according to Martin and Ames (17).Preparative isoelectric focussing was performed in column (LKB) or flat bed on a Multiphor electrophoresis apparatus (LKB), Chromatofocussing was done on a Polybuffer 11-8 (Pharmacia) matrix equilibrated with 0.025 M triethylamine-HC1, pH l l , and proteins eluted with Pharmalyte-HCl, pH 7. ATP-citrate lyase and acetylCoA carboxylase were purified from rat liver as described previously (18, 19). Catalytic subunit of CAMP-dependent protein kinase was prepared from rabbit skeletal muscle by method 11 of Bechtel et al. (20) with an additional hydroxylapatite chromatographic step. Protein concentration was determined by the method of Lowry et al. (21) or Bradford (22) using bovine serum albumin as standard. ATP. Mgdependent protein phosphatase was prepared and assayed as described elsewhere (14, 23). A unit of phosphatase is that amount of enzyme that catalyzes the release of 1.0 pmol of phosphate/min.

12281

I

a -Il.*

12

0

m

N

a

V

1

I

‘. 0

”

0

20

1

40 60 F R A C T I O N NUMBER

a0

0

FIG. 1. Chromatography of multifunctional protein kinase. a, material from step 5 was applied on a ‘Sephadex G-100 superfine column (2.6 X 100 cm) equilibrated in buffer B containing 0.2 M NaCl, and proteins were eluted with the same buffer. Absorbance at 280 nm (-----) and ATP-citrate lyase phosphorylation ( O ” 0 ) by each fraction were determined. b, dialyzed material from step 7 was applied on to Blue Sepharose column (1.5 X 15 cm) washed with buffer B until absorbance of the eluate (-----) was below 0.01 at 280 nm. The column was washed with 50 ml of 0.1 M NaCl in buffer B and theneluted with a linear gradient of 0.10 to 0.75 M NaCl in buffer B. Conductivity ( X - - - - - X ) and ATP-citrate lyase phosphorylation activity ( 0 4 )of the fractions were determined.

RESULTS

Purification-A multifunctional protein kinase that phosphorylates ATP-citrate lyase specifically at peptide B (5, 8, 9) was purified to near homogeneity from rat liver. A summary of its purification using ATP-citrate lyase as substrate is given in Table I. The enzyme was isolated from the 140,000 X g supernatant fraction using various chromatographic steps, and about 0.5 mg of purified protein kinase was obtained from 1kg of liver. The purified protein corresponds to a purification of 16,000-fold and a yield of 5%. The enzyme is stable for 6 months at -20 “C in 50% glycerol after purification through steps 6 and 7. Enzyme activity was found to be stable for not more than 4 weeks at -20 “C in 50% glycerol after additional purification by affinity chromatography on Blue Sepharose. After the second Blue Sepharose step, protein kinase activity was stable for a much shorter period. Substrate Specificity-This higher purified protein kinase phosphorylates in addition to ATP-citrate lyase (8-lo), acetyl-coA carboxylase ( l l ) , and glycogen synthase (see Fig. 2 of accompanying paper). Summary of the purification of this protein kinase using various protein substrates is given in Table 11. The specific activity of the enzyme increased with each step of purification and the ratio of synthase to lyase phosphorylation was constant from step 6 onwards. However, the specific activity of histone kinase and casein kinase activities changed little in the first few steps of purification and

12282

Multifunctional Protein Kinase

decreased after step 5 with little histone or casein kinase activity remaining after the Blue Sepharose step. Because Pi inhibited this protein kinase, as itdoes glycogen synthase kinase-3, it was determined if this protein kinase also activated ATP. Mg-dependent phosphatase activity (14). Fractions from each step of the purification activated ATP. Mg-dependent protein phosphatase and this stimulatory activity co-purified with protein kinase activity for ATP-citrate activities increased similarly lyase (Table 11).After step 3 both and the phosphatase stimulatory activity and protein kinase activity did not diverge even after two chromatographic purifications on Blue Sepharose. Phosphorylation of ATP-citrate lyase and acetyl-coA carboxylase by the purified multifunctional protein kinase was linear up to 1 h and thereafter plateaued with about 0.55 and 0.6 mol of phosphate incorporated per mol of subunit, respectively, at 3 h (Fig. 2). Details of the glycogen synthase phosphorylation are described elsewhere (12). This protein kinase was originally isolated as an ATPcitrate lyase kinase (8). It was later found to phosphorylate acetyl-coA carboxylase (11)and glycogen synthase (12). With TABLE I Purification of multifunctional protein kinase from 1 kg of rat liver Protein kinase activity was measured with ATP-citrate lyase as substrate (1.2 mg/ml, 11 PM) from each stepas described under "Experimental Procedures."

equal molar concentrations of these substrates, glycogen synthase was phosphorylated to a greater extent than the other substrates (Table 111).Glycogen synthase phosphorylation by the multifunctional proteinkinase was %foldhigher than that of ATP-citrate lyase and about 7-fold that of acetyl-coA carboxylase. This purified protein kinase did not phosphorylate casein, phosphorylase, phosphorylase kinase, pyruvate kinase, casein, phosvitin, and histone H2B (Table 111). Physical Properties-Fractions containing the most highly purified protein kinase (step 9), when analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate and silver staining, gave a single major band at a molecular weight of 36,000 (Fig. 3). Incubation of the purified enzyme with [y32P]ATPand Mg2+ showedautophosphorylation of the M , = 36,000 band (Fig. 3). Note that with [y-32P]azido-ATP (ICN Radiochemicals), only the M , = 36,000 band was labeled (data not given). Fresh preparations containedonly the M, = 36,000 band (lanes 5 and 6, Fig. 3). However, electrophoretic runs of some preparations of the extensively purified protein kinase showed a doublet of protein bands at M, = 32,000 and 36,000 (lane 2) but only M, = 36,000 was autophosphorylated (lane 4 ) . The Mr = 32,000 proteinband could be a proteolytic product as when the purified protein fractions were kept on ice for a few days it was found that in addition to thedoublet additional higher (>120,000) and lower (about 22,000) M, phosphorylated protein bands appeared (not shown in the figure). With increasing storage the relative amounts of these

-z I-

a

nmolf min

nmolf mgl mm

162,000 1,600

697 672

0.004 0.42

670

650

mg

1. Extract 2. Phosphocellulose 3. Polyethylene glycol 4. DEAE-Sephadex 5. CM-Sephadex 6. Sephadex G-100 7. TSK 8. Blue Sepharose-1 9. Blue Sepharose-2

I

0.97

n-fold

?Lo 0.6 -

%

-I

0

1 98

100 96

225

93

3

/ ; - /// ./ 1 9 c w

0.4

-

0.2

459

610

1.33

309

88

232 24.5

522 194

2.25 7.93

523 1,844

75 28

13.7 2.8

129 95

9.44 33.65

2,195 7,826

19 14

34

68.89

16.020

5

0.48

-B

"""

;

&"O""

I

0

40

"~-""""""-""

"""-o"""" 80

0

I

160

120

T I M E lrninutesl

FIG. 2. Time course of phosphorylation of ATP-citrate lyase and acetyl-coA carboxylase by the multifunctional protein kinase. Phosphorylation assays were performed from 6 min to 3 h as described. ATP-citrate lyase (0and e)and acetyl-coA carboxylase (0and W) were either phosphorylated with [Y-~*P]ATP (solid symbols) or [Y-~'P]GTP(open symbols).

TABLEI1 Specific activity of protein kinase with different substrates Fractions from each step were used for phosphorylation of glycogen synthase (1.2 mg/ml), histone H2B (2 mg/ ml), or casein (5 mg/ml). Specific activity of ATP-citrate lyase is from Table I. ATP.Mg-dependent phosphatase activation was assayed by the method of Yang e t al. (14). The Faactivity of the purified enzyme waslow compared to Ref. 23 which may be due to thefact that ATP.Mg-dependent phosphatase preparation contained alarge basal activity. Specific activity Step

ATP-citrate lvase

Glycogen svnthase

0.004 0.42 0.97 1.33 2.25 7.93 9.44 33.65 68.89

0.373 2.61 3.40 4.73 7.97 23.70 27.80 80.63 172.20

Histone H2B

Ratio Casein

Phosphatase

0.51 1.19 1.88 1.90 3.62 3.15 1.48 0.18 0.00

0.0 0.32 1.66 2.45 4.28 14.37 17.32 63.89 106.70

Synthase lvase

Phosphatase lvase

6.21 3.51 3.56 3.54 2.99 2.94 2.40 2.50

0.76 1.71 1.84 1.90 1.81 1.83 1.90 1.55

nmolfmgfmin

1. Extract

2. 3. 4. 5. 6. 7. 8. 9.

Phosphocellulose Polyethylene glycol DEAE-Sephadex CM-Sephadex Sephadex G-100 TSK Blue Sepharose-l Blue Sepharose-2

1.55 7.04 5.07 5.75 9.78 5.27 1.23 0.19 0.00

Kinase

ProteinMultifunctional

12283

TABLE 111 Properties of some cyclic AMP-independent protein kinases This table is a summary of Tables 1-111 as published (31) (the originals should be consulted for the details) and a summary of results using the liver multifunctional protein kinase. Abbreviations: ND, not determined; PrP-1, protein phosphatase-1; GSK-3, GSK-4, GSK-5, glycogen synthase kinases -3 -4, and -5; CK-1, casein kinase -1; these CAMP-independent protein kinase are from rabbit skeletal muscle. Relative activity 0 =