181

Biochimica et Biophysica Acta, 5 8 4 ( 1 9 7 9 ) 1 8 1 - - 1 9 5 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 28851

ADENOSINE 3',5'-MONOPHOSPHATE-DEPENDENT PROTEIN KINASE(S) IN DIPLOID AND SV40 TRANSFORMED HUMAN FIBROBLASTS

K E I T H P. R A Y *, T H O M A S B. M I L L E R , Jr. a n d R E G I N A L D W. B U T C H E R **

Department of Biochemistry, University of Massachusetts Medical School, Worcester, MA 01605 (U.S.A.) (Received July llth, 1978) (Revised m a n u s c r i p t received O c t o b e r 23rd, 1 9 7 8 )

Key words: Cyclic AMP;Protein kinase; Transformation; (SV40, Human fibroblast)

Summary Cyclic AMP-dependent protein kinases (EC 2.7.1.37; ATP:protein phosphotransferase) in the human diploid fibroblast WI-38 and an SV40-transformant WI-38-VA13-2RA (VA13) have been compared on the basis of their concentrations in cells, isoenzyme composition and susceptibility to hormonal activation. In high population density cultures, total soluble cyclic AMP-dependent kinase activities measured with histone were essentially the same in WI-38 and VA13. Two soluble protein kinase forms separated by chromatography on DEAEcellulose were present in both cell lines. The concentration of cyclic AMP required for half-maximal activation of both enzyme forms was 10--30 nM. Overall kinase stimulation was greater for the Peak I enzymes. Kinase activation induced in the presence of 0.5 M KCI was more rapid and complete for the Peak I enzymes. Under conditions which elevated the concentration of cyclic AMP in WI-38 and VA13 cells the activities of the soluble histone kinases were increased. Incubation of the cells with either of 5.7 ~M prostaglandin E1 Or 1 pM isopropylnorepinephrine induced complete activation of the cyclic AMP-dependent histone kinases within 5 min and maintained the effect for 20 min. When intraceUular cyclic AMP levels were raised by prostaglandin El, activation of glycogen phosphorylase (assayed-AMP) suggested that this enzyme cascade involving cyclic AMP-dependent protein kinase(s) was intact and responsive in both cell lines.

* Present address: School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG0 U.K. ** To whom reprint requests should b e addressed.

182

Introduction The human diploid lung fibroblast strain, WI-38 derived by H a y r i c k [ 1] and a clone derived by SV40 transformation of WI-38 cells, WI-38-VA13-2RA (VA13) have been partially characterized in terms of their ability to accumulate cyclic AMP in response to various hormones or other agents [2]. The t w o cell lines differ quite markedly in their cyclic AMP responses and differences in the enzymes controlling cyclic AMP metabolism have been noted [2]. Cyclic AMP-dependent protein kinases are now recognized as important intracellular receptors for cyclic AMP [3,4] mediating the control of many differentiated functions in response to various extracellular stimuli [5,6]. It has also been suggested that intrinsic mechanisms controlling cell growth involve cyclic AMP |7,8] and that this regulation may be affected by a cell cycle specific expression of cyclic AMP-dependent protein kinases [9]. Although not studied in human diploid fibroblast cell lines, protein kinases have been studied in other cultured cells [9--18]. Several reports have compared the kinase of transformed cells with those of the normal cell lines or tissues [ 11,14--16]. Some of these investigations have revealed either a defect or difference in the properties of kinases in transformed cell lines [11,15,16] while others have failed to show any differences [ 14,18]. In this report we have examined the cyclic AMP-dependent protein kinases in WI-38, a density-inhibited fibroblast, and a transformant, VA13, which lacks density-dependent inhibition of growth. Kinase activities in these two cell lines have been compared on the basis of (i) their enzyme content, (ii) their activation by cyclic AMP and inhibition b y protein kinase inhibitor and (iii) their responsiveness to activation following hormonal treatment of cells. Some preliminary investigations of the protein kinases in other cultured fibroblast cell lines are also described. Materials and Methods Materials. WI-38 cells were obtained from Dr. L. Hayflick and used at population doubling levels o f 25--48. SV40-transformed WI-38 cells (WI-38-VA132RA, referred to here as VA13) were from Dr. A n t h o n y Girardi of the Wistar Institute. IMR-90, a human diploid lung fibroblast, was obtained from the Institute for Medical Research and WI-26-VA4, an SV40-transformed derivative of WI-26 cells, was from the American Type Culture Collection. The mouse fibroblast cell lines 3T3 and SV-3T3 were obtained from Dr. Harvey Ozer of the Worcester Foundation for Experimental Biology. Prostaglandin E1 was provided by Dr. John Pike of the Upjohn Company. Isopropyl-norepinephrine was obtained from K & K Laboratories. Mixed histones, Type II from calf thymus, and rabbit liver glycogen, Type III, were from Sigma Chemical Company. Vitamin-free casein was treated according to the m e t h o d o f Reimann et al. [19]. Radiochemicals were from New England Nuclear, Boston. Amberlite Monobed ion-exchange resin was a gift from the R o h m and Haas Company. Other biochemicals and enzymes were obtained from Boehringer Mannheim. Cell culture methods. Cells were propagated from frozen stock in Eagle's minimal essential medium or Dulbeccos medium (3T3 and S V - 3 T 3 ) s u p -

183 plemented with penicillin-streptomycin (100 units each per ml), and either 5% (VA13-2RA, WI26-VA4 and SV-3T3) or 10% (WI-38, IMR-90 and 3T3) fetal bovine serum (Grand Island Biological Co., Grand Island, NY). Nutrient medium was renewed every 3 or 4 days and the cells were maintained at 37°C in a humidified 95% air/5% CO2 atmosphere. Cells were seeded into 100-mm plastic Petri dishes 3 to 4 days prior to experimentation to obtain confluent cell cultures. In this study the term confluent indicates that cells were grown to cover the entire growth surface. Incubation procedure for intact cell studies. For intact cell experiments growth medium was replaced with serum-free medium containing 20 mM Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid} buffer, pH 7.4, instead of bicarbonate, and the cells were allowed to equilibrate in a humidified air atmosphere at 37°C. After a 30 min preincubation period, hormones or effector molecules were added according to the experiment. Duplicate dishes were prepared for each experimental point unless stated otherwise. Incubations were terminated by the rapid removal of medium using a suction device. Cell sheets were rinsed twice with ice cold extraction buffer comprising 150 mM potassium chloride/5 mM potassium phosphate/2 mM EDTA, pH 6.8 (Buffer A). After the removal of excess buffer the cells were frozen by floating the dishes on liquid nitrogen. The dishes were stored at --20°C for up to 2 h until extracts could be prepared for assay. Homogenization for intact cell studies. Frozen cells were allowed to thaw at 2°C for 5 min, then 1 ml of Buffer A was added to each dish and the cells were scraped. Homogenization-sonication was carried out at 2°C using a Willems Polytron for 15 s at 2/3 full speed. The homogenate was centrifuged at 12 000 × g for 15 min at 2°C, after which the supernatant and pellet were separated. The supernatant extracts were placed on ice and assayed for kinase activity immediately. Extraction o f protein kinases for broken cell studies. Soluble and particulate cell fractions required for the determination of kinase activities i n c r u d e cell extracts were prepared in Buffer B, comprising 5 mM potassium phosphate/2 mM EDTA, pH 6.8, at 2°C. After removal of the growth medium, cells (attached to dishes) were rinsed four times with 5 ml of the buffer and the dishes were placed on ice. The cells from three dishes were removed by scraping with a rubber policeman in a total volume of 1 ml of the extraction Buffer B. The suspension was homogenized in a small Dounce homogenizer using 10 strokes of a tight-fitting pestle. Supernatant and pellet fractions were separated by centrifugation at 12 000 × g for 15 min at 2°C. The pellet fraction was resuspended directly in the original extraction volume of Buffer B by five strokes in the Dounce homogenizer. Protein kinase assay. Protein kir;ase activity was determined essentially as described by Corbin and Reimann [20] using 20-~1 aliquots of the enzyme preparation. Each sample was assayed in duplicate with or without histone in the presence or absence of added cyclic AMP. 20 gl of crude cell extract or enzyme fraction were added to tubes containing 50 pl of 17 mM potassium phosphate, pH 6.8, 500 ~g of Type IIa calf thymus histone or casein, 0.3 mM [~,-32P]ATP (approx. 35 cpm/pmol), 6 mM magnesium acetate and 2 pM cyclic AMP. After 10 min at 30°C, 50 pl of each reaction mixture were withdrawn

184 and spotted onto a strip of Whatman ET31 filter paper (1 × 2 cm) which was then dropped into ice-cold 0.6 M trichloroacetic acid to terminate the reaction. The filter papers were washed three times more with fresh 0.6 M trichloroacetic acid, then rinsed twice with ethanol and dried. 32p incorporation into acidinsoluble material b o u n d to the papers was measured by liquid scintillation spectrometry. Kinase fractions were diluted with the appropriate extraction buffers to ensure that linear reaction rates were obtained during the assay. Preliminary studies indicated that for incubations containing 10--40 pg of soluble extract protein or 5--20 t~g of particulate cell extract protein, 3~p incorporation measured in the presence or absence of histone was linear for up to 20 min. Unless indicated otherwise, protein kinase activity is defined as the amount o f enzyme required to catalyze the transfer of 1 pmol of phosphate from [7-32p]ATP to histone (or casein) and/or endogenous substrate in 1 min. The terminology 'histone kinase' implies that histone was used as the substrate and not that the kinase was necessarily specific for histone. A preparation of an inhibitor of cyclic AMP-dependent kinase was isolated from bovine skeletal muscle according to the method of Schlender and Reimann [21] in a typical incubation with 15 pg of soluble protein kinase extract per assay tube, 2 pg of the inhibitor preparation was required to cause maximal inhibition of the cyclic AMP stimulated 32p incorporation measured with the histone substrate. Glycogen phosphorylase, extraction and assay. All studies were performed using cells which had been preincubated with fresh growth medium for 1 h. The preparation of cells, experimental incubation procedure and enzyme extraction method were otherwise similar to those described above for intact cell studies of protein kinase, but the extraction buffer comprised 50 mM ~-(N-morpholino)-ethanesulfonic acid (MES)/50 mM K F / 6 0 mM 2-mercaptoethanol, pH 6.1, at 0--4°C. Phosphorylase activities in cell supernatant extracts were determined by measuring the incorporation of radioactivity from D-[U14C]glucose 1-phosphate into glycogen according to the m e t h o d of Gilboe et al. [22]. The rabbit liver glycogen substrate was treated with an Amberlite M o n o b e d ion exchange resin to remove any contaminating nucleotides. DEAE-cellulose chromatography. Soluble cell extracts prepared in 5 mM Tris-HC1 buffer, pH 7.2, containing 2 mM EDTA (for m e t h o d see broken cell studies) were subjected to chromatography on columns (0.7 × 8 cm) of DEAEcellulose (DE-52) which had been pre-equilibrated with the same buffer at 4°C. Following application of the sample containing 3--20 mg of protein, each column was washed with approx. 20 ml o f the Tris buffer, then eluted with a linear gradient of NaC1 to a final concentration of 0.5 M NaC1 in Tris buffer. An LKB ultrograd gradient mixer was used to deliver a total gradient volume o f approx. 40 ml with a flow rate of 20 ml/h. Preparation of [7-32p]ATP. Adenosine 5'-triphosphate labelled in the T-position with a2p was prepared to a high specific radioactivity by the m e t h o d of Glynn and Chappell [23] and purified with charcoal as described by Walsh et al. [24]. Protein measurements. Determinations of protein were made according to the m e t h o d of Lowry et al. [25] with bovine serum albumin as the standard.

185 Results Kinase activities in crude cellular extracts Protein kinase activities present in WI-38 and VA13 were first measured in soluble and particulate extracts prepared from cell homogenates. Values for the 32p incorporation into trichloroacetic acid-insoluble material obtained with these crude enzyme fractions are summarized in Table I. Histone kinase activities have been expressed allowing for the endogenous phosphorylation measured in the absence of histone. Cyclic AMP-dependent histone kinases were found to be concentrated in the soluble fractions o f both cell lines and the total histone kinase activities measured in the presence of cyclic AMP (2 ~M) were very similar. When cyclic AMP was omitted, histone addition caused an apparent inhibition of phosphorylation in soluble extracts from VA13 cells, to a level below that measured with the endogenous substrates alone. The negative values which appear in Table I indicate where this phenomenon occurred. The phosphorylation of endogenous substrates was both more rapid and extensive in soluble extracts from VA13 and might suggest differences in either the availability or the character of the substrates for phosphorylation in the transformed cell line. The histone kinase activities of particulate cell extracts were much lower than those found in the soluble fractions, but showed similar properties. However, no a t t e m p t was made to wash the particulate preparations, and contamination by soluble histone kinase was not determined. Endogenous kinase activities present in the particulate extracts were not affected by cyclic AMP and similar values were obtained in both WI-38 and VA13. The soluble kinase activities measured for confluent cultures o f several other cell lines are presented in Table II. Histone kinase activity was markedly cyclic AMP-dependent in each case. Total histone kinase activities for the density

TABLE I PROTEIN KINASE ACTIVITY IN SOLUBLE AND PARTICULATE

E X T R A C T S O F WI-38 A N D V A 1 3

C r u d e k i n a s e f r a c t i o n s w e r e p r e p a r e d f r o m c o n f l u e n t c u l t u r e s o f WI-38 a n d V A 1 3 . E a c h cell s h e e t w a s r i n s e d , t h e n s c r a p e d a n d h o m o g e n i z e d in 5 m M p o t a s s i u m p h o s p h a t e p H 6 . 8 / 2 m M E D T A b u f f e r . F o l l o w i n g c e n t r i f u g a t i o n a t 12 0 0 0 X g f o r 15 m i n t h e s u p e r n a t a n t e x t r a c t w a s r e m o v e d and t h e p e l l e t resusp e n d e d in e x t r a c t i o n b u f f e r . K i n a s e a c t i v i t i e s , w e r e m e a s u r e d in t h e p r e s e n c e o r a b s e n c e o f a d d i t i o n a l c y c l i c A M P (2 ~ M ) c A M P u s i n g a m i x e d h i s t o n e f r a c t i o n as t h e s u b s t r a t e . D e t a i l s o f t h e assay p r o c e d u r e are g i v e n u n d e r M e t h o d s . K i n a s e a c t i v i t i e s are g i v e n as t h e p m o l o f 3 2 p i n c o r p o r a t e d p e r r a i n p e r m g o f e n z y m e e x t r a c t p r o t e i n . A c t i v i t i e s f o r h i s t o n e k i n a s e h a v e b e e n c o r r e c t e d to allow f o r t h e e n d o g e n o u s p h o s p h o r y l a t i o n m e a s u r e d with the crude extract alone. Values represent the m e a n s of a n u m b e r (N) of s e p a r a t e e x p e r i m e n t a l d e t e r m i n a t i o n s m e a s u r e d in d u p l i c a t e , + t h e s t a n d a r d d e v a t i o n o f t h e m e a n . Cell f r a c t i o n

Cell t y p e

Percentage cell protein

Kinase activity (pmol/min per mg)

Endogenous p r o t e i n

Histone

--cAMP

--cAMP

+cAMP

Soluble

WI-38 (8) V A 1 3 (8)

63 58

78 ± 201 ±

37 64

Particulate

WI-38 (6) V A 1 3 (6)

37 42

3 3 7 -+ 1 3 0 370 ± 102

i00 + 243 +

31 77

359 + 147 401 + 100

105 + ---46 +

+cAMP 63 42

65 + 40 --14 + 110

475 + 487 +

70 79

1 3 0 + 58 84 ± 121

186 T A B L E II P R O T E I N K I N A S E A C T I V I T Y IN S O L U B L E A N D P A R T I C U L A T E ITED AND SV40-TRANSFORMED FIBROBLASTS

EXTRACTS

OF DENSITY

INHIB-

E x t r a c t s w e r e p r e p a r e d f r o m c o n f l u e n t cells and a s s a y e d for their kinase c o n t e n t as d e s c r i b e d u n d e r M e t h o d s in t h e p r e s e n c e o r a b s e n c e o f a d d i t i o n a l c y c l i c A M P ( c A M P ) (2 t i M ) u s i n g a m i x e d h i s t o n e f r a c t i o n as t h e s u b s t r a t e . O t h e r d e t a i l s are given in t h e l e g e n d t o T a b l e I.

Cell f r a c t i o n

Cell t y p e

Percentage cell p r o t e i n

IMR-90 (3) VA-4 3T3(3) 3 V 3 T 3 (3)

Soluble

63 57 56 55

Kinase activity (pmol/min

per mg)

Endogenous protein

Histone

~cAMP

+cAMP

--cAMP

44 186 95 139

76 239 125 194

~ ÷ ÷ ~

19 52 12 19

± t ± ±

17 77 15 36

82 --30 9 9

± 36 t 27 t 12 + 1

+cAMP 580 373 342 393

* 152 ~ 58 + 13 ± 102

inhibited cells were similar to those measured for the SV-40 transformed celt lines and values were comparable to those measured for WI-38 and VA13. Phosphorylation of soluble endogenous substrates was greater for extracts from the transformed cells. Particulate kinase activities were similar to those described for the WI-38 and V A 1 3 cell lines (data not shown). The susceptibility o f soluble protein kinase activities present in WI-38 and V A 1 3 cells to inhibition by the protein kinase modulator was tested using a preparation of this inhibitor from bovine skeletal muscle. The data presented in Table III show that with histone as the substrate basal (-- cyclic AMP) phosphorylation was reduced in the presence of the inhibitor. Part of the basal histone kinase activity measured for both cell lines might, therefore, have been due to activity of the free catalytic subunit o f cyclic AMP-dependent protein kinase. Total (+ cyclic AMP) histone kinase activities were inhibited considerably, but these activities remained higher than the basal inhibited levels, even though inhibition was essentially maximal under the conditions used. The patTABLE III EFFECTS OF AN INHIBITOR KINASE ACTIVITIES

OF CYCLIC

AMP-DEPENDENT

PROTEIN

KINASES

ON SOLUBLE

S o l u b l e e x t r a c t s w e r e p r e p a r e d f r o m c o n f l u e n t W I - 3 8 a n d V A 1 3 c u l t u r e s as d e s c r i b e d u n d e r M e t h o d s . Ext r a c t s w e r e d i l u t e d for t h e assay o f kinase a c t i v i t y t o p r o v i d e 1 0 - - 1 5 /zg e x t r a c t p r o t e i n p e r i n c u b a t i o n . 5 p g of the inhibitor preparation per incubation was included where indicated. This inhibitor c o n c e n t r a t i o n w a s s u f f i c i e n t t o e n s u r e m a x i m a l i n h i b i t i o n o f h i s t o n e ldnase a c t i v i t y m e a s u r e d in t h e p r e s e n c e o f c y c l i c A M P (2 p M ) . T h e average v a l u e s o f kinase a c t i v i t i e s m e a s u r e d in t w o s e p a r a t e e x p e r i m e n t s are given, in pmol/min per mg. Cell type

Inhibitot

Endogenous protein

Histone

Casein

-~cAMP

+cAMP

~AMP

+cAMP

--cAMP

+cAMP

WI-38

-+

83 135

141 130

137 95

374 168

448 435

537 477

VA-13

-+

234 321

296 328

--39 --109

302 --27

505 499

582 484

187

tern of inhibition obtained with an alternative substrate casein was quite different. Casein kinase activity was largely independent of cyclic AMP. The inhibitor preparation had no effect on basal casein kinase activities but prevented the small increase in total kinase activities measured in the presence o f cyclic AMP. Therefore, only a minor c o m p o n e n t o f the total kinase activities towards casein, appeared to be related to cyclic AMP-dependent kinase activities in these fibroblasts. Effects of the inhibitor on endogenous phosphorylation appear to be comparable to those obtained with casein. The high values for endogenous phosphorylation in the presence of the inhibitor may be explained by the incorporation of 32p into material present in the inhibitor preparation. DEAE-cellu lose ch rom a tography o f solu ble p ro tein k inases

Two forms of cyclic AMP-dependent protein kinase with activity towards histone were detected in soluble extracts from both WI-38 and VA13 cells (Fig. 1). Peak I protein kinases were eluted by salt concentrations of 0.06 to 0.1 M NaC1 while the Peak II enzyme forms eluted between 0.15 and 0.25 M NaCl. In addition, an intermediate peak or shoulder of kinase activity was noted as a minor c o m p o n e n t in a number of experiments. On the basis of activ-

Ioo

tO0

W/-38

80

-c

80

60

0.5

< < .J

40

40

2o

2.0

c~

(b < cc (b cL oc (D

Io

I0

~J

8

8

--

C~ I

I

I

I

I

I

4

8

12

16

20

INCUBATION PERIOD+ PGE (MIN) 1 Fig. 6. E f f e c t o f p r o s t a g l a n d i n E l ( P G E 1) o n t h e a c t i v i t y o f g l y c o g e n p h o s p h o r y l a s e i n W I - 3 8 a n d V A 1 3 . Cells w e r e p r e p a r e d for i n c u b a t i o n w i t h 5 . 7 p M p r o s t a g l a n d i n E 1 u s i n g t h e p r o c e d u r e d e s c r i b e d i n t h e l e g e n d t o T a b l e I V . I n c u b a t i o n s w e r e t e r m i n a t e d at t h e t i m e s i n d i c a t e d a n d p h o s P h o r y l a s e a c t i v i t i e s in t h e cell s u p e r n a t a n t e x t r a c t s ( 1 2 0 0 0 X g for 1 5 rain) w e r e m e a s u r e d . T h e p h o s P h o r y l a s e a c t i v i t y ratios (activi t y m e a s u r e d w i t h o u t A M P / a c t i v i t y a s s a y e d in t h e p r e s e n c e o f 3 m M A M P ) w e r e d e t e r m i n e d for W I - 3 8 ( e ) a n d V A 1 3 (A) u s i n g t w o d i s h e s o f cells at e a c h t i m e p o i n t . T h e average v a l u e s w e r e p l o t t e d against t h e t i m e o f i n c u b a t i o n w i t h p r o s t a g l a n d i n E 1.

193 the b form of the enzyme present in skeletal muscle. The total phosphorylase measured in VA13 cell extracts in the presence of AMP was at least double that observed for WI-38. Incubation of WI-38 and VA13 cells for 10 min with prostaglandin E1 (5.7 pM) led to activation of the phosphorylase. Values for these phosphorylase activities were 3--4 times higher than the controls (assayed without AMP) but did not reach the total phosphorylase values (assayed in the presence of AMP). The increased total phosphorylase measured in hormonally treated WI-38 cells suggests that AMP may also affect the activated form of the enzyme. However, the total phosphorylase in VA13 cells did not change following activation. Time course for the prostaglandin E~ mediated activation of phosphorylase present in WI-38 and VA13 cells are shown in Fig. 6. The activity of phosphorylase at each time point has been expressed as the ratio of the activity measured in the presence of AMP. The rapid activation of phosphorylase in both cell lines was complete within 5 min and persisted over a 20-min period in the presence of the prostaglandin. Discussion

A number of recent studies (Refs. 7 and 27 and reviews in Refs. 8 and 28) have suggested that cyclic AMP might be important in controlling the densitydependent inhibition of growth observed in confluent fibroblast cultures, and that a defect in the metabolism of this nucleotide may be responsible for the abnormal growth characteristics of transformed cells [29,30]. Our own studies [31], however, have indicated that cyclic AMP concentrations in prostaglandin E~-treated VA13 fibroblasts could be maintained significantly above control values for prolonged periods with no apparent inhibition of cell growth. A possible explanation for this result was that the cyclic AMP-dependent protein kinase(s) controlling cell growth might be either defective or unable to function normally in the transformed cell. On the basis of evidence presented in this paper we have been unable to detect significant differences between the cyclic AMP-dependent protein kinase of the confluent, density inhibited fibroblast WI-38 and the transformant, VA13. The possibility remains, however, that differences between specific cyclic AMP-dependent protein kinases involved with growth regulation may have been missed using the analytical methods adopted for this study. Total soluble cyclic AMP-dependent kinase activities measured with histone as the substrate were very similar for confluent cultures of both cell lines, and the enzyme forms detected during growth and after the cells had reached confluency were comparable. Comparison of basal cyclic AMP-dependent kinase activities was complicated by the fact that histone could inhibit the phosphorylation of endogenous substrates present in soluble VA13 extracts. Data obtained using the inhibitor of cyclic AMP-dependent protein kinase suggest that catalytic activity derived from cyclic AMP-dependent enzymes may contribute part but not all of the basal kinase activity present in these cells. The major enzyme forms resolved by ion-exchange chromatography appear to correspond to Type I and II cyclic AMP-dependent protein kinases which have been characterized in other mammalian tissues [26]. Hormonal treatments of intact WI-38 and VA13 cells caused essentially complete activation of the

194

cyclic AMP-dependent histone kinases, indicating that both the Peak I and II enzymes were susceptible to activation when the intracellular concentrations of cyclic AMP were elevated. Gharrett et al. [16] have reported that only Type II protein kinase was present in two density-inhibited 3T3 cell lines, whereas both Type I and Type II kinases were found in SV40-transformed 3T3 cells. On the basis of this evidence they have proposed that an altered kinase composition resulted from the viral transformation of these cells. In contrast we have identified two kinase forms in soluble extracts prepared from confluent, density-inhibited and SV40transformed 3T3 cell lines currently used in this laboratory. Thus it would appear that the different isoenzyme compositions may not necessarily be related to viral transformation per se but rather may reflect the characteristics of the particular clones of cells used. An interesting difference between the WI-38 and VA13 cells was the a m o u n t of 32p incorporated into endogenous substrates present in soluble cell fractions. As 32p incorporation into particulate material did not vary significantly between the two, it would appear that the transformed cells maintain a higher concentration of soluble substrates rather than an altered cellular distribution compared to the normal cell lines. However, while differences in substrate composition cannot be discounted, an equally logical alternative would be that the basal phosphorylation state of these substrates was lower in the transformed cell lines, thus allowing a greater incorporation of 32p to occur. Identification of the kinase(s) responsible for the phosphorylation of endogenous substrates and their relationship to the cyclic AMP-dependent enzymes requires a more critical investigation of their properties. However, the cyclic AMP-independent nature of these reactions may indicate that enzymes other than the cyclic AMP-dependent histone kinases are involved. Protein kinase activity which was not affected by an inhibitor of cyclic AMP dependent kinase has been demonstrated in both cell lines (Table II). The use of casein rather than histone as the enzyme substrate appears to be more appropriate for the detection of such activities. Gray et al. [32] have reported a protein kinase with activity towards endogenous substrates present in HeLa cells which was inhibited by histone. A similar inhibitory effect of histone noted in this study was apparently limited to the phosphorylation of endogenous substrates present in transformed fibroblasts. A possible explanation for this effect could be that some soluble endogenous substrates are histones released from the nuclear material during homogenization, and inhibition of the kinase results from similarities between these native substrates and one or more components of the mixed histone material used for the assay. The complex nature of the histone mixture clearly makes such results difficult to interpret. Preliminary studies of glycogen phosphorylase present in WI-38 and VA13 indicate that the enzyme has properties similar to those of skeletal muscle phosphorylase. The prostaglandin mediated activation of phosphorylase was consistent with the currently accepted model for cyclic AMP-dependent activation of glycogenolysis and suggests that this enzyme cascade system involving the cyclic AMP-dependent protein kinases is intact and responsive in both cell lines. Glycogen breakdown in these fibroblasts may also be regulated by a p o t e n t allosteric activation of glycogen phosphorylase involving AMP. The data

195 shown in Table IV suggest that effects of this metabolite may be more significant in the transformed cells, where the total phosphorylase activity expressed in the presence of AMP far exceeded that due to the prostaglandin E1 activation alone and was twice that measured in the normal cells. These differences might be explained if only part of the total phosphorylase present in the V A 1 3 cells were available for prostaglandin E i-mediated activation. Alternatively, this enzyme could be more susceptible to activation by AMP than the phosphorylase forms present in WI-38 cells. It is not clear at present whether these differences relate to cellular transformation or simply indicate an altered metabolic requirement in the V A 1 3 cells. Acknowledgements We wish to thank Agneta Brown for her excellent technical assistance. This work was supported by Research Grant AM 13904 from the National Institutes of Health, United States Public Health Service. References 1 2 3 4 5

6 7 8 9 10 11 12 19 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

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