Elevated phosphatidylinositol kinase activity in Rous sarcoma virus-transformed cells. Lack of evidence for enzyme translocation

Biochimica et Biophysica Acta 931 (1987) 165-169

165

Elsevier BBA 12158

Elevated phosphatidylinositoi kinase activity in Rous sarcoma virus-transformed cells. Lack of evidence for e n z y m e translocation

Michael A. Tones a,,, Stuart Kellie h , , , and John N. Hawthorne

a

o Department of Biochemistry, University of Nottingham Medical School, Queen's Medical Centre, Nottingham and b Imperial Cancer Research Fund Laboratories, St. Bartholomew's Hospital, London (U.K.)

(Received 3 July 1987)

Key words: Rous sarcoma virus; Phosphatidylinositol kinase; Phosphorylation; Transformation; Protein kinase; (Rat fibroblast)

Phosphatidylinositol kinase (E.C. 2.7.1.67) activity of rat fibroblasts transformed by Rous sarcoma virus (RSV) was measured and compared with immunoprecipitated protein tyrosine kinase activity associated with pp60 v'src. Both enzyme activities were elevated in the particulate fractions from wild-type RSV-transformed cells and cells transformed by a temperature-sensitive mutant of RSV when grown at the permissive temperature. The presence of the non-ionic detergent Nonidet P-40 in the phosphatidylinositol kinase assays stimulated the soluble and particulate forms of the enzyme to different degrees but did not affect the relative differences between transformed and untransformed cells. Our results indicate that phosphatidylinositol kinase activity is a good correlate of RSV transformation and suggest a functional relationship between pp64)v'src and phosphatidylinositol kinase.

Introduction Several retroviral transforming proteins have intrinsic tyrosine-specific protein kinase activity [1] but intense efforts have so far failed to reveal a physiologically relevant protein substrate. The transforming proteins of avian sarcoma viruses R S V and U R 2 have been reported to phosphorylate PtdIns in vitro to form P t d I n s 4 P [2,3]. It is

* Present address: CIBA-Geigy Pharmaceuticals, Horsham, W. Sussex RH12 4AB, U.K. * * Present address: Department of Biochemistry, Royal College of Surgeons of England, The Hunterian Institute, 35-43 Lincoln's Inn Fields, London WC1A 3PN, U.K. Abbreviations: Ptdlns, phosphatidylinositol; Ptdlns4P, phosphatidylinositol 4-phosphate; RSV, Rous sarcoma virus. Correspondence: J.N. Hawthorne, Department of Biochemistry, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, U.K.

now clear that this lipid kinase activity m a y be associated with, but is not intrinsic to, the transforming proteins themselves [4,5]. It has been proposed that phosphatidylinositol kinase m a y be a substrate for the middle T - / p p 6 0 csrc or pp60 .... c tyrosine kinases, and that their co-precipitation by antibodies specific for middle T or pp60 is due to a catalytic interaction [5]. It is important, therefore to know whether this results in a change in the total phosphatidylinositol kinase activity in the cell or in a relevant cellular c o m p a r t m e n t and h o w this compares with pp60v-src-derived protein tyrosine kinase activity. Phosphatidylinositol kinase is k n o w n to exist in b o t h soluble and m e m b r a n e b o u n d forms [7]. As the substrate is an integral part of the cell membrane, translocation of the soluble enzyme to the m e m b r a n e is a potential regulatory mechanism. Such translocation is k n o w n to be involved in the regulation of several enzymes including protein

0167-4889/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

166 kinase C [8] and phosphatidate phosphohydrolase [9]. We have measured both soluble and particulate forms to determine whether the effects of transformation on phosphatidylinositol kinase may involve translocation between cytosol and membrane. Detergents have profound effects on phosphatidylinositol kinase activity [6,7] and so assay conditions may have dramatic effects on the results obtained. We have assayed phosphatidylinositol kinase in fractions prepared from rat fibroblasts transformed by wild-type RSV and by a temperature-sensitive mutant RSV in the presence and absence of detergent. Materials and Methods

Cell culture. All cell lines were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 50 I U / m l penicillin, 50 / t g / m l streptomycin and 5% new-born calf serum in a humidified atmosphere containing 5% CO 2. F o r experiments, cells were plated out onto 100-mm diameter culture dishes at a density of 2 . 1 0 6 cells/dish. The cells were then cultured for 2 days before homogenisation, fractionation and assay of phosphatidylinositol kinase activity. We have used non-transformed Rat-1 fibroblasts and clones which had been transformed by wild-type Prague Strain A RSV (PrA) and a temperature-sensitive mutant tsLA29 (see Ref. 10). Cells transformed by ts LA29 produce a heat-labile pp60 ..... tyrosine kinase such that when cultured at the permissive temperature (35 o C) they are phenotypically transformed but when cultured at the restrictive temperature (39 ° C) they are phenotypically normal. Unless otherwise stated, cells were grown at 37 ° C. Preparation of cell fractions. Culture dishes containing cells were washed once with 10 ml of ice-cold phosphate-buffered saline (Ca 2+ and Mg 2+ free) and once with 10 ml of homogenisation buffer (0.25 M sucrose, 10 m M Tris-HC1, 3 m M EGTA, p H 7.4). Cells were then scraped from the plates into a total vol. of 7 ml (pooling cells from four to seven dishes) and homogenised using a glass-within-glass Dounce homogeniser (10 strokes by hand). The volume was made up to 10 ml with homogenisation buffer and the homogenised cells were centrifuged at 1000 × g for 10

min at 4 ° C . The supernatant was removed and the pellet was discarded. Aliquots of this low-speed supernatant (designated 'homogenate') were taken for subsequent protein and phosphatidylinositol kinase assays, whilst the remainder was subjected to centrifugation at 100000 × g for 60 min at 4 ° C . The resulting supernatant was designated the 'soluble fraction', and the pellet was resuspended in 0.5 ml homogenisation buffer by rapid suction through a fine pipette tip and designated the 'particulate fraction'. All fractions were stored in aliquots at - 7 0 ° C. Protein was assayed by the method of Lowry [11] using bovine serum albumin as a standard, and phosphatidylinositol kinase was assayed as described below. Assay of phosphatidylinositol kinase. The lipid substrate was prepared by sonicating 2 mg of PtdIns in 1 ml of 200 m M Hepes (pH 7.4) for 15 min followed by the addition of 8 ~1 of Nonidet P-40 (except in the experiments of Fig. 2 without detergent) and vortex mixing. Cell fractions were diluted to 40 t~g p r o t e i n / m l using homogenisation buffer. Assay tubes containing 25 t~l of diluted sample and 15/~1 of PtdIns were preincubated on ice for 20 min. The reaction was started by the addition of 10/~1 of 60 m M MgC12 and 10 ~1 of [~,-32p]ATP (3 C i / m m o l , 1.0 m C i / m l obtained from Amersham) and incubation at 3 0 ° C for 10 min. Incubations were terminated by the addition of 60 /~1 of 1 M HC1 to each tube followed by rapid mixing and transfer to an ice bath. Phospholipids were extracted using 120/zl c h l o r o f o r m / methanol (1:1, v / v ) and the extract was washed three times with 30 /~1 of m e t h a n o l / 1 M HCl (1 : 1, v / v ) . The final lower phase was dried down under a stream of N 2, re-dissolved in 20 /~1 of c h l o r o f o r m / m e t h a n o l (2:1, v / v ) and applied to T L C plates (silica gel H impregnated with 1% potassium oxalate). The plates were run in paperlined tanks containing either c h l o r o f o r m / acetone/methanol/glacial acetic a c i d / H 2 ° (40 : 15 : 13 : 12 : 7, v / v ) or c h l o r o f o r m / m e t h a n o l / 35% N H 3 / H 2 0 (45 : 35 : 2 : 8, v / v ) . [32 P]Ptdlns4P was located by autoradiography and co-migration with added carrier PtdIns4P (I2-stained). Radioactivity was quantitated by liquid scintillation counting of silica gel areas. Protein tyrosine kinase activity. This assay was performed essentially by the method of Collet and

167 E r i k s o n [12]. A f t e r f r a c t i o n a t i o n as d e s c r i b e d above, 200 ~ g o f p r o t e i n f r o m each fraction was solubilised in lysis b u f f e r which h a d the following c o m p o s i t i o n : 0.1 M NaC1, 1 m M E D T A , 10 m M Tris HC1 ( p H 7.2), 1% N o n i d e t P-40. A f t e r rem o v a l of a n y i n s o l u b l e material, 5 /tl of a t u m o u r - b e a r i n g r a b b i t serum was a d d e d (genero u s l y p r o v i d e d b y Dr. P.J. E n r i e t t o ) a n d inc u b a t e d on ice for 1 h. T h e n 50 /~1 of a 10% s u s p e n s i o n of fixed p r o t e i n A - c o n t a i n i n g Staphylococcus aureus was a d d e d a n d the i n c u b a t i o n was c o n t i n u e d for a further 30 min. A f t e r w a s h i n g in lysis b u f f e r the p r e c i p i t a t e s were r e s u s p e n d e d in 40 /zl of k i n a s e buffer (0.15 M NaC1, 5 m M M g C I 2 , 10 m M Tris HC1 ( p H 7.0), 1% N o n i d e t P-40. T h e n 10/~1 10 -7 M [3,-32p]-ATP was a d d e d a n d the r e a c t i o n was allowed to p r o c e e d for 10 m i n b e f o r e b e i n g s t o p p e d b y the a d d i t i o n of wash b u f f e r (0.4 M NaC1, 1 m M E D T A , 10 m M TrisHC1 ( p H 8.1), 0.25% d e o x y c h o l a t e , 1% N o n i d e t P-40). A f t e r washing three times, the r e a c t i o n p r o d u c t s were s e p a r a t e d on a 10% S D S - p o l y a c r y l a m i d e gel, d r i e d a n d a u t o r a d i o g r a p h e d . T h e i n c o r p o r a t i o n of 32p into the I g G heavy chain was q u a n t i t a t e d b y s c a n n i n g the a u t o r a d i o g r a p h using a Joyce-Loebl scanning densitometer.

Results and Discussion A l l fractions a s s a y e d h a d easily d e t e c t a b l e p h o s p h a t i d y l i n o s i t o l k i n a s e activity. T a b l e I shows the e n z y m e activity in h o m o g e n a t e s a n d c r u d e cell fractions. T h e r e was s u b s t a n t i a l v a r i a t i o n b e t w e e n the activities m e a s u r e d in the s a m e cell lines between one e x p e r i m e n t a n d the next. N o r m a l i s a t i o n of activities with respect to the activity in the s a m e fraction from Rat-1 cells in the s a m e e x p e r i m e n t revealed that p a r t i c u l a t e fractions f r o m P r A - t r a n s f o r m e d cells h a d d o u b l e the p h o s p h a t i d y l i n o s i t o l k i n a s e activity f o u n d in the n o n - t r a n s f o r m e d Rat-1 cells. There was also v a r i a t i o n in the activities of the total h o m o g e n a t e a n d soluble fractions: in three out o f four e x p e r i m e n t s , the soluble fraction also s h o w e d slightly elevated activity, b u t d u e to e x p e r i m e n t a l v a r i a t i o n this was not statistically significant, a n d in two o u t of four e x p e r i m e n t s there was n e a r l y 200% increase in total h o m o g e n a t e activity, b u t no difference was f o u n d in the o t h e r experiments. Nevertheless, the p a r t i c u l a t e

TABLE I FIBROBLAST PHOSPHATIDYLINOSITOL KINASE ACTIVITY The percent activity refers to the activity of that fraction as a percentage of the activity in the comparable fraction from Rat-I ceils in the same experiment. Data are expressed as mean_+S.E, of four independent experiments, and statistical significance of PrA vs. Rat-1 was tested using a paired t-test: * P < 0.05. Phosphatidylinositol kinase Rat-I

PrA

nmol/min per mg

%

nmol/min per mg

Homogenate 0.180,+0.039 100 0.310,+0.107 164,+45 Soluble 0.079,+0.028 100 0.108,+0.036 141,+20 Particulate 0.210+0.061 100 0.388-+0.100 199_+30 *

fractions o f t r a n s f o r m e d cells consistently exh i b i t e d an increased p h o s p h a t i d y l i n o s i t o l kinase activity. These findings are in a g r e e m e n t with those of S u g i m o t o a n d E r i k s o n [13] who f o u n d higher p h o s p h a t i d y l i n o s i t o l kinase activity in particulate fractions from R S V - t r a n s f o r m e d avian cells. W h e n grown at their restrictive t e m p e r a t u r e (39 ° C ) , ts L A 2 9 - t r a n s f o r m e d cells have a nont r a n s f o r m e d p h e n o t y p e a n d have p h o s p h a t i -

TABLE II PHOSPHATIDYLINOSITOL KINASE ACTIVITY IN CELLS TRANSFORMED BY THE TEMPERATURE-SENSITIVE MUTANT ts LA29 Phosphatidylinositol kinase activity of ts LA29-transformed cells grown at 35°C and 39 °C. Data are expressed as a percent of Rat-I activity in the same experiment, and in the form of mean,+ S.E. of four independent experiments. Significanoe testing between 35 ° C and 39 o C was carried out using a paired t-test: * P < 0.01. Phosphatidylinositol kinase 35°C nmol/min per mg

39°C %

nmol/min per mg

%

Homogenate 0.166,+0.049 90.6+19 0.118-+0.021 68.5-+ 9.5 Soluble 0.057+0.022 66.6_+13 0.048-+0.019 69.8,+10 Particulate 0.325,+0.077 171 ,+29 0.199+0.036 112 -+26 *

168 d y l i n o s i t o l kinase activity in the p a r t i c u l a t e fraction similar to that of n o n - t r a n s f o r m e d cells (Table II). C u l t u r i n g these cells at 35 o C causes develo p m e n t of a t r a n s f o r m e d p h e n o t y p e a n d a statistically significant ( P < 0.01) increase in p h o s p h a t i d y l i n o s i t o l kinase, to activity similar to that f o u n d in P r A t r a n s f o r m e d cells. C o n t r o l e x p e r i m e n t s showed that when Rat-I cells are c u l t u r e d at 35 ° C or 3 9 ° C there is no effect of culture t e m p e r a t u r e p e r se on p h o s p h a t i d y l i n o s i t o l kinase activity in a n y of the fractions d e r i v e d from these n o n - t r a n s f o r m e d cells (results n o t shown). These results i n d i c a t e that p h o s p h a t i d y l i n o s i t o l kinase activity in the p a r t i c u l a t e fraction is a reliable correlate of phenotypic transformation. W e have c a l c u l a t e d the d i s t r i b u t i o n of the total cellular p h o s p h a t i d y l i n o s i t o l kinase b a s e d on enz y m e activities a n d p r o t e i n c o n t e n t of the fractions. Fig. 1 shows that the P r A t r a n s f o r m e d cells have exactly the s a m e d i s t r i b u t i o n of activity between cytosol a n d m e m b r a n e s as the n o n - t r a n s f o r m e d cells. Thus, t r a n s l o c a t i o n is unlikely to be i m p o r t a n t in the increase in m e m b r a n e - a s s o c i a t e d p h o s p h a t i d y l i n o s i t o l k i n a s e activity o b s e r v e d in R S V t r a n s f o r m a t i o n . T h e r e is n o difference in the d i s t r i b u t i o n of e n z y m e in ts L A 2 9 - t r a n s f o r m e d cells either when grown at the permissive a n d non-restrictive t e m p e r a t u r e s . Interestingly, these cells have a higher p r o p o r t i o n of enzyme activity

]

Soluble

]

Padiculate

in the m e m b r a n e - b o u n d form a n d less in the soluble form than either the Rat-I cells or PrAt r a n s f o r m e d cells. T h e reason for this is u n k n o w n but it is u n r e l a t e d to the state of t r a n s f o r m a t i o n of the cells. W e were interested to k n o w w h e t h e r the presence or a b s e n c e of the n o n - i o n i c d e t e r g e n t N o n idet P-40 c o u l d affect our results, so in one experim e n t we a s s a y e d the fractions b o t h with a n d w i t h o u t N o n i d e t P-40 in the assay. Fig. 2 shows that the d e t e r g e n t stimulates p h o s p h a t i d y l i n o s i t o l k i n a s e activity in whichever fraction it is assayed, a n d that the degree of s t i m u l a t i o n is much greater for the p a r t i c u l a t e enzyme than the soluble enzyme. Nevertheless, the relative differences between different cell types are preserved, i n d i c a t i n g that detergents are not required in the assay to observe the effects of t r a n s f o r m a t i o n . W h e n we a s s a y e d the pp60 ..... -associated p r o tein tyrosine kinase activity of the same fractions as were assayed for p h o s p h a t i d y l i n o s i t o l kinase, we f o u n d d r a m a t i c effects of t r a n s f o r m a t i o n . Rat-1 cells a n d L A 2 9 cells grown at a n o n - p e r m i s s i v e t e m p e r a t u r e b o t h h a d very low activity in b o t h the p a r t i c u l a t e a n d soluble fractions ( T a b l e III). PrAt r a n s f o r m e d cells a n d LA29 cells grown at the permissive t e m p e r a t u r e showed elevation greater t h a n 15-fold of tyrosine kinase activity in the p a r t i c u l a t e fraction b u t little change in the soluble fraction. T h e results are consistent with previous evidence that p p 6 0 ..... in LA29 avian cells is t e m p e r a t u r e - l a b i l e with regard to localisation in the p l a s m a m e m b r a n e a n d p r o t e i n kinase activity [14].

100-

>,

80

TABLE

iiiiiiii 31:1:1:

L~

'* 40

iiiiii iiiiii

20

iiiiiii

::::::::::::: RAT-I

Ill

TYROSINE KINASE ACTIVITY OF pp60 v-src IN CELLS TRANSFORMED BY WILD TYPE AND ts ROLLS SARCOMA VIRUS ili!i!i~

C .....

Kinase activity in immunoprecipitates is expressed in arbitrary units derived from densitometric scanning of autoradiographs of SDS-polyacrylamide gels as described. These results are the means of two independent experiments,

~i~ii~i PrA

35o LA

39o - 29

Fig. 1. Subcellular distribution of phosphatidylinositol kinase activity. The histograms represent means__S.E, of four separate experiments.

Tyrosine kinase (arbitrary units) Soluble Particulate

Rat-1

PrA

LA29 (35 °)

138 1248

481 737 19535 15010

LA29(39 ° C) 910 1540

169 PI KINASEOF CONTROLAND RSV-TRANSFORMEDRAT FIBROBLASTS HOMOGENATES

0.5 -

0.2

SOLUBLE

MEMBRANES

1.0 -

NONIDET P40

0.8-

0.4

0.15

r-7

0.2%

~ 0%

--I

A C

2 0.3

0.6

ca.

E -....

o0.2 (a_ ct.

0. I

o-

FL

0.4

~

CON

RSV

0.05

ts-RSV

350

c

0.2

CON RSV

39o

ts-RSV

350

39o

o

--

CON

7

RSV

ts-RSV

350

39o

Fig. 2. Phosphatidylinositol (PI) kinase activity in fractions from non-transformed and RSV-transformed rat fibroblasts assayed in the presence (open bars) and absence (hatched bars) of 0.2% Nonidet P-40. Data are from a single experiment, each assay was performed in duplicate and the mean is presented. The 35 o C and 39 o C columns refer to the temperature-sensitive mutant LA29.

We have shown a close relationship between phosphatidylinositol kinase, protein tyrosine kinase activity and the transformation state. The relative increase in activity of phosphatidylinositol kinase in particulate fractions from transformed cells is modest (99%) compared to the increase in protein tyrosine kinase (greater than 15-fold). From this it may be argued that if phosphatidylinositol kinase was acting as a substrate for pp60 .... c kinase, the effect of phosphorylation is only slight. However, it is possible that a small but functionally important pool of phosphatidylinositol kinase is switched on by pp60 ..... in transformation and plays some role in the development of the transformed phenotype. Acknowledgement

We are grateful to the Wellcome Trust for partial support of this work.

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