Volume 26, number 1

MOLECULAR • C E L L ~ BIOCHEMISTRY

July 15, 1979

PURIFICATION A N D PROPERTIES OF cAMP INDEPENDENT GLYCOGEN SYNTHASE KINASE A N D PHOSVITIN KINASE FROM HUMAN LEUKOCYTES Henning JUHL Department of Medicine, Marselisborg Hospital, DK-8000 ~rhus C, Denmark (Received March 2, 1979)

Summary cAMP independent glycogen synthase kinase and phosvitin kinase activity was purified from the 180 000 x g supernatant of human polymorphonuclear leukocytes by ammonium sulphate precipitation and phosphocellulose chromatography. The cAMP independent glycogen synthase kinase eluted from the phosphocellulose at 0.54 M NaC1 (peak A) separate from the major phosvitin kinase eluting at 0.68 MNaC1 (peak B). The kinase activity of both peaks tended to form aggregates, but in the presence of 0.6 MNaC1, the peak B enzyme had ~ 250 000, 7.2S and the peak A enzyme Mr 38 000, 3.8S. The ratio between synthase kinase and phosvitin kinase activity in peak A was 1 : 3.2 and in peak B 1 : 31.4. In addition the kinase activities differed with respect to sensitivity to temperature, ionic strength and CaC12. It is suggested that the peak A enzyme represents the cAMP independent glycogen synthase kinase of leukocytes, whereas the peak B enzyme is a phosvitin kinase, which is insignificantly contaminated with some synthase kinase (peak A) and contains a separate, second synthase kinase. Synthase kinase had K ~ p 4.2/ZM for muscle glycogen synthease I and K ~ v 45/~M for ATP. GTP was a poor substrate. The activity was not influenced by cyclic nucleotides, Ca 2+, or Abbreviations : cAMP, adenosine cyclic 3' : 5'monophosphate; DTT, dithiothreitol; EGTA, ethylene glycol-bis-(/3-amino-ethylether)-N,N'-tetraaceticacid; PMSF, phenylmethylsulfonylfluoride;PKI, protein kinase inhibitor; RI, ratio of independence for glycogen synthase; SDS, sodium dodecyl sulphate.

glucose-6-P. Synthase I from muscle and leukocytes was phosphorylated to a ratio of independence of less than 0.05.

Introduction Glycogen synthase I contains multiple phosphorylation sites per tetrameric subunit 1"2. It is of current interest to determine, which enzymes are involved in the phosphorylation reactions. It has been shown that cAMP dependent protein kinase phosphorylates purified muscle synthase I to the (kinetically characterized) D-form 3"4 and in the preceding paper 5, we have shown that the purified catalytic subunit from human leukocytes may phosphorylate synthase I from human leukocytes and rabbit muscle to a ratio of independence (RI) of less than 0.07. Recently, cAMP independent glycogen synthase kinases have been described to occur in renal medulla6, renal cortex 7, several rat tissues s and rabbit skeletal muscle9-11. These enzymes were found to incorporate either one 9A~ or four phosphates per subunit of synthase I ~°. In intact leukocytes an intermediately phosphorylated and kinetically characterized R-form of synthase has been found active in glycogen synthesis~2"13. Under the influence of a-agonists a R to D conversion has been found to occur in the absence of a simultaneous increase in the cAMP level of the cells and it was thought possible that a cAMP independent synthase kinase was responsible for the phosphorylationTM. The present paper describes the purification from human polymorphonuclear leukocytes of a

Dr. W. Junk b.v. Publishers - The Hague, The Netherlands

19

cAMP independent glycogen synthase kinase separate from the major phosvitin kinase.

Experimental Procedures

Materials The sources of the materials used are described in the preceding article 5.

Buffer solutions The following buffers were used repeatedly. Buffer A is 50 rnM glycerol-2-phosphate (pH 7.0), 0.1 rnM EDTA and i mM DTT. Buffer B is buffer A with i n ~ benzamidine, and 0.1 rnM PMSF. Buffer C is 5 mM Tris-HCI (pH 7.5), 1 mM EDTA, 1 mM benzamidine, 0.1 rnM PMSF and 1 naM DTT. Buffer D is buffer C, but with 50 rnM Tris-HC1.

Purification of cAMP independent phosvitin and synthase kinase The 180 000 × g supernatant from 4 or 8 g human polymorphonuclear leukocytes was precipitated with 50% ammonium sulphate and the precipitate dialyzed (buffer C), incubated with cAMP, and chromatographed on DEAEcellulose equilibrated in buffer C, as described in the preceding paper 5. The column was eluted with a continuous gradient of Tris-HC1 (pH 7.5) and the fractions with phosvitin kinase activity pooled, dialyzed against buffer D and loaded on a column (1.5 × 15 cm) of phosphocellulose equilibrated in the same buffer. The column was washed with buffer D until the absorbance of the effluent at 280 nm fell to zero. The breakthrough contained contaminating cAMP dependent histone kinase, whereas phosvitin kinase and synthase kinase activities were completely retained by phosphocellulose. A linear gradient of NaC1 (0-1 M) in buffer D (total volume 500 ml) eluted cAMP independent kinase activity in separate peaks, A and B. The kinase activity of both peaks could be traced with phosvitin as well as synthase I as substrate. Fractions of 6 ml were collected from each of the two peaks, pooled, concentrated by vacuum dialysis to 1-1.5 ml and dialyzed against buffer A. Human serum albumin was added to a final concentration of 1 mg/ml and quantities of 0.1 ml were lyophilized and stored at - 1 5 °C. cAMP independent phosvitin and synthase kinase activities in both peaks were stable for months under these conditions. 20

It was found that an equally efficient purification and separation of peaks A and B could be obtained by omitting the DEAE-cellulose chromatography and loading the phosphocellulose column directly with the resuspended (buffer D) and gelfiltered (Sephadex G-25) ammonium sulphate precipitate. All later experiments were performed with enzymes prepared in this way. It has finally been observed that even the ammonium sulphate precipitation step may be omitted and the 180 000 x g supernatant dialysed against buffer D and loaded directly on the phosphocellulose columns. The 180 000 x g centrffugation separated phosvitin kinase activity in a soluble fraction (40-50%) and a fraction (50-60%) that sedimented with the glycogen pellet. The kinase activity of the glycogen pellet was traced only with phosvitin as substrate. Resuspension of the glycogen pellet and a second centrifugation at 180 000 x g solubilized a further 10% of phosvitin kinase activity. Phosvitin kinase activity associated with the 180 000 x g glycogen pellet was resuspended in buffer D, dialysed against this buffer and loaded directly on a phosphocellulose column equilibrated in buffer D. Glycogen particles and glycogen synthase together with a minor amount of phosvitin kinase activity and all cAMP dependent histone kinase activity were eluted in the break-through upon eluting the column with buffer D. Most of the phosvitin kinase activity was retained on the column and eluted by a linear gradient of NaC1 at precisely the same positions as was found for the soluble fraction.

Assay of cAMP-independent phosvitin kinase The standard reaction mixture contained in a final volume of 0.1 ml : 20 naM glycerol-2phosphate, pH 7.0, 0.4 mM EDTA, 0.2 mM EGTA, 3 mg/ml phosvitin, 0.15 mg/ml PKI, 0.2 n ~ DTT, 1.0 nau [~ - 32p] ATP (2-6 × 107 cpm//zmol), and 10 rnM Mg-acetate. The mixture was incubated at 30 ° and the reaction started by addition of the enzyme sample (20 ~1) and run for 30 min. 75/zl was spotted on 2 × 2 cm filter paper and dropped into icecold 10% trichloro-acetic acid as described by REmlAr,rN et al. 15. A reaction blank in the absence of phosvitin enabled correction to be made for incorporation of 32p into the enzyme sample. For the crude extract, the reaction

velocity was linear with time and enzyme concentration for at least 60 min when less than 0.005 mol 32P/mol phosvitin (Mr 36 000) had reacted. For peak A and peak B enzymes the limits were 120 min and 0.05 mol 32p/molphosvitin. The assays were carried out within these limits. When casein was the substrate, the same reaction mixture was used, but with 6 mg/ml of casein, cAMP independent phosvitin kinase activity with glycogen synthase I as substrate was assayed essentially as described by N ~ l o et al. 9. The incubation contained 25 mM glycerol2-phosphate, (pH 7.0), 0.6 mM EDTA, 0.15 mM EGTA, 0.2 mM D T r , 4 mg/ml crude rabbit protein kinase inhibitor (180 U/mg), 1.0 mM [3'-- a2p] ATP (2-3 x 109 cpm//~mol), 10 mM Mgacetate, and 0.5 mg/ml rabbit muscle glycogen synthase I in a total volume of 0.1 ml. 20/~1 enzyme samples were added. The reaction was initiated by adding the ATP-Mg-acetate solution and run for 3 and 6 min at 30 °C. When the dilute enzyme samples from the D E A E cellulose and phosphocellulose columns were assayed, the assay period was extended to 15 rain. The reaction was terminated as for the phosvitin kinase assay. In addition to correction for 32p incorporated into the kinase preparation, correction was also made for phosphorylation of the muscle synthase I preparation by endogenous protein kinases. The reaction velocity was linear with time and enzyme concentration up to 0.1 tool 32P/mol glycogen synthase subunit had reacted. In single experiments (Table 2), human serum albumin, rabbit muscle phosphorylase b, and histone IIA were substituted for phosvitin in the phosvitin kinase assay. Rabbit muscle synthase I was used as substrate throughout, except for the experiment shown in Figure 7B, where synthase I from leukocytes was used. One unit of phosvitin, casein and glycogen synthase kinase activity is defined as the amount of enzyme that will catalyse the transfer of 1 nmole of phosphate to the substrate per min at 30 ° C.

Other methods Preparation of glycogen synthase I, assay of cAMP dependent and independent histone kinases, glycogen synthase I and D, and protein, as well as sucrose density gradient centrifugation,

gelfiltration and SDS-polyacrylamid gel electrophoresis are described in the preceding article 5. Results

Separation of phosvitin kinases by chromatography DEAE-ceUulose chromatography separated protein kinase activity in three peaks (I-III) of cAMP dependent and independent histone kinase activity and a broad peak having cAMP independent phosvitin kinase activity (see Fig. 1 of the preceding articleS). The peaks of cAMP dependent histone kinase activity could be completely separated from phosvitin kinase activity when the load to the DEAE-cellulose column was preincubated with cAMP (not shown). This did not change the elution position of phosvitin kinase activity, nor did omission of the ammonium sulphate precipitation step. The cAMP independent casein kinase activity eluted as a broad peak at the same position as the phosvitin kinase activity. No casein kinase activity was present in the fractions corresponding to the elution position of the cAMP independent histone kinase activity (see Fig. 1 of the preceding articleS). The cAMP independent protein kinase activity against synthase I as substrate was eluted just in front of, but not separated from the broad peak of phosvitin kinase activity. When, however, these fractions of cAMP independent protein kinase activity were pooled and rechromatographed on phosphocellulose, or when the redissolved ammonium sulphate precipitate was applied directly to the column omitting the DEAE-cellulose step, the activity separated completely in two peaks, A and B, eluting at 0.54 ~ and 0.68 M Nacl. respectively (not shown). The purification of cAMP independent phosvitin kinase activity can be followed in Table 1. The peak A activity was purified 1012 fold and the peak B activity 6012 fold. It is noted, that a 50% decrease in specific activity took place upon concentration and dialysis. Peak A and B were not homogenous and showed multiple bands on SDS-polyacrylamide gel electrophoresis. They did not contain any glycogen synthase or phosphorylase activity. 21

Table 1 Purification table of phosvitin kinase Protein

Volume

Activity

Spec.

activity

Purification

mg

ml

U

U/mg p r o t e i n

fold

Yield

18o ooo x g supernatant

1633

33.8

25.9

o.o158

1

ioo

5o%

lo2

56,7

26.5

o.259

16

lo2

o.28

1.4

4.5

16

lo12

17

(56) x)

(8.2)

(29)

1.4

lo.2

95

6o12

39

(48)

(2o.1)

(187)

(NH4)2SO 4

Peak

A

Phosphocellulose chromatography Peak

B

Phosphocellulose

o.lo7

chromatography

x) Values in b r a c k e t s columns

were

were

obtained

concentrated

and

before

the

eluates

from

the p h o s p h o c e l l u l o s e

dialysed.

Sucrose density gradient centrifugation and gel[iltration of peak A and B enzymes

s e d i m e n t e d to the b o t t o m of the tube. A d d i n g 0 . 6 M NaC1 to the gradient changed the s e d i m e n t a t i o n profile drastically. P e a k A n o w s e d i m e n t e d as a sharp p e a k at 3 . 8 S and p e a k B at 7 . 2 S (not shown). O b v i o u s l y , the e n z y m e s f o r m large aggregates at l o w ionic strength.

P e a k A and p e a k B e n z y m e s w e r e s u b j e c t e d to s u c r o s e density gradient centrifugation and w e r e b o t h f o u n d to s e d i m e n t at 16.4S as a broad p e a k (Fig. 1). S o m e of the activity e v e n A

B

I 6¢

5 u 4c ,

Purification and properties of cAMP independent glycogen synthase kinase and phosvitin kinase from human leukocytes.

Volume 26, number 1 MOLECULAR • C E L L ~ BIOCHEMISTRY July 15, 1979 PURIFICATION A N D PROPERTIES OF cAMP INDEPENDENT GLYCOGEN SYNTHASE KINASE A N...
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