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Phosphorylation of surface proteins of HeLa cells using an exogenous protein kinase and [γ-32P]ATP
Biochimica et Biophysica dcta, 322 (1973) 337-351 ~) Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - P r i n t e d in The N e t h e r l a n d s
BBA
36520
P H O S P H O R Y L A T I O N OF SURFACE P R O T E I N S OF H E L A CELLS USING AN EXOGENOUS P R O T E I N KINASE AND [y-z~PIATP
V O L K E R K I N Z E L * AND G E R A L D C. M U E L L E R * *
McArdle Laboratory for Cancer Research, University of Wisconsin Medical Center, Madison, Wisc. 53706 (U.S.A.) (Received March I9th, 1973)
SUMMARY
The enzymatic phosphorylation of surface proteins of living cells with the aid of a protein kinase isolated from rat skeletal muscle and [s*p~ATP has been described. Utilizing monolayer cultures of HeLa cells the degree of phosphorylation was dependent on the amount of the protein kinase used, the labeled E32P]ATP and the incubation time. The phosphorylation was stimulated to a limited degree by cyclic AMP. After a Io-min labeling period, 86-95% of the label was removed with a brief treatment of 0.05% trypsin. Partial characterization of the labeled surface components revealed an instability of the label to hot NaOH, but good stability in hydroxylamine. The label was not removed by lipid extraction. Using a high resolution gel electrophoresis system it was shown that the label is distributed among at least 15 bands. The mildness of the procedure was verified by the continued growth of the cells.
INTRODUCTION
Recent studies suggest that cell membranes may play a dominant role in the control of replication and differentiation of cells. In addition, they may be important targets in the malignant transformation of cells by oncogenic viruses. To explore their role in these cellular processes our laboratory is investigating effects of selective labeling and chemical modification of surface proteins on their metabolic fate and the behavior of the treated cells. A recent review by Hoelzel-Wallach 1 and papers by Hubbard and Cohn ~ and Rifkin et al. ~ summarize related studies in this area. The present report describes a method for the phosphorylation of surface membrane proteins with radioactive phosphorus using an exogenous protein kinase (EC 2.7.1.37) from rat skeletal muscle and E~-~PIATP. Preliminary evidence relating to the stability and metabolic fate of surface proteins from HeLa cells is reported. * P r e s e n t address: G e r m a n Cancer Research Center ( D K F Z ) , K i r s c h n e r s t r a s s e 6, 69 Heidelberg, G e r m a n y . ** To w h o m r e p r i n t r e q u e s t s should be sent.
338
v . K I N Z E L , G. C. M U E L L E R
The data document that the phosphorylation of cell membranes provides a physiologically mild means for the selective labeling of surface proteins for studies of their metabolic fate and influence on the function of the cell. MATERIALS AND METHODS
The following chemicals were purchased as indicated: [y-a2Pladenosine 5'-triphosphate (La2P]ATP) (11-17. 7 Ci/mmole) from New England Nuclear, L8-aH]adenosine 5'-triphosphate from Amersham/Searle (E3HJATP, I I Ci/mmole), adenosine 3',5'-monophosphate (cyclic AMP) from Sigma, disodium adenosine 5'-triphosphate (Na2ATP) from P-L-Biochemicals, Inc., Milwaukee, calf thymus histone (B grade) from Calbiochem, theophylline (anhydrous) from Schwarz/Mann, sodium dodecylsulfate from Pierce Chemical Co.; the other chemicals for the polyacrylamide gel electrophoresis were obtained from BioRad Laboratories, DEAE-cellulose was purchased from Sigma (o.85 mequiv/g, medium mesh), bovine albumin (Fraction V) from Armour Pharmaceutical Corp., hydroxylamine was prepared according to Hokin et al. 4. Scintisol was obtained from Isolab Incorporated, Akron, Ohio. Trypsin was purchased from Nutritional Biochemicals Corp. Trichloroacetic acid, EDTA, and other chemicals were of analytical grade. H e L a $3 cells were cultured in suspension as described earlier 5. For the described experiments the cells were centrifuged, resuspended in Eagle's HeLa medium (BEHM) containing I O ~ bovine serum and transferred to 4 - I 5 - c m plastic petri dishes (Falcon). Subsequent incubations were carried out in a CO 2 incubator (air-CO2, 95:5, v/v) for at least 12 h prior to phosphorylation experiments. For most of the studies described 4-cm dishes were used with 2 ml medium. The cell counting was done with a Coulter Counter (model B). The protein kinase used in the experiments was prepared according to Kuo and Greengard 6 and Miyamoto et al. 7 from skeletal muscle of 3 4 -month-old female Sprague-Dawley rats. In most experiments the enzyme was purified through the DEAE-cellulose colunm step; however, in certain cases the (NH4)2SO 4 fractionated enzyme was used. The D E A E eluates were dialyzed against o.I M potassium phosphate buffer (pH 7, with 2 mM EDTA). In certain cases the enzyme was further concentrated by filtration with an Amicon Diaflo ultrafilter, PM-Io which retains molecular weights larger than IO ooo. This procedure resulted occasionally in a slight loss of enzyme activity. The enzyme solution was usually concentrated about five times based on the protein content and the enzyme solution stored at --22 °C without the addition of any stabilizing factors. A decrease in specific activity to about 5o~,~ within three months was noted for certain preparations. Protein kinase activity was assayed according to the procedure of Miyamoto et al. 7 except the ATP level was reduced to 25 pmoles per assay tube, which contained 5' IO-6 M cyclic AMP and 4o/~g calf thymus histones as a receptor. One unit of enzyme activity corresponds to an amount of enzyme which transfers i pmole of ~2p from [7-aePIATP to receptor protein in 5 min at 3o °C. This unit is approx. 4o times larger than that defined in the work of Miyamoto ct al. 7. Enzymatic phosphorylation of HeLa cells in 4-cm dishes (or 6-cm dishes) The medium was removed by suction, the cells were washed two times (washing procedure A) with 2 ml (or 4 ml in 6-cm dishes) of a prewarmed solution containing
PHOSPHORYLATION
OF CELL SURFACE PROTEINS
339
Tris-acetic acid buffer (0.03 M,pH 7-5 at 23 °C), NaC1 (o.15 M) and magnesium acetate (0.02 M) (Solution A). The cells were then preincubated at 37 °C for 5 min with the prewarmed incubation mixture adapted from Agren and Ronquist s and Miyamoto et al. 7. Tris-acetic acid buffer (0.03 M, pH 7.5), NaC1 (o.15 M), magnesium acetate (0.02 M), N a F (o.oi M), theophylline (4 raM) ; 4- cyclic AMP (approx. 5" IO GM) and lOO/~1 of protein kinase solution as indicated in the individual experiments per o.8-ml incubation mixture per dish (respectively, 250 #I enzyme per 2 ml per 6-cm dish). Since the enzyme solution was prepared in o.I M potassium phosphate, the final phosphate concentration of inorganic phosphate in the incubation mixture was 12.5 raM. The incubation mixture was freshly prepared each time. The reaction was started by the addition of IV-32PIATP (amounts given in the individual experiments). The incubation was carried out at 37 °C in the C02 incubator. To stop the reaction the incubation mixture was removed by suction and the dishes washed two times with 2 ml (4 ml in 6-cm dishes) of ice cold washing solution B (i.e. Solution A containing IO 3 M unlabeled Na2ATP) and then fixed with I ml (2 ml in 6-cm dishes) of ice cold 5 % trichloroacetic acid. The cultures were allowed to stand at least I h at o °C prior to washing again with i ml (2 ml in 6-cm dishes) trichloroacetic acid. The cells were removed using a rubber policeman and transferred to centrifuge tubes. Following centrifugation for IO rain at 2000 rev./min in an International PR-2 centrifuge at o °C in a No. 269 head, the residue from one dish was dissolved in 0.3 ml (0.6 ml in 6-cm dishes) ice cold o.I M NaOH and immediately reprecipitated b y the addition of I ml (2 ml in 6-cm dishes) 5 % trichloroacetic acid. This sequence was performed two times in order to get rid of contaminating ATP 9. The final precipitates were dissolved in 0.5 M NaOH and 4oo-#1 aliquots were counted in Scintisol using a scintillation counter. Protein determinations were carried out on 5O-lOO-/,1 allquots. The phosphorylation of larger batches of cells was done in a slightly different way. After two washings with 25 ml Solution A the I5-cm dishes were preincubated with the incubation mixture for 5 rain at 37 °C on a rocker platform (18 rev./min; Belco) and the reaction started by the addition of labeled ATP. A total volume of 6 ml of incubation mixture per I5-cm dish was used. The amounts of enzyme and A T P used per dish are given in the legends for the particular experiments. To stop the reaction the dishes were rinsed two times with 25 ml wash solution B and the cells used for the indicated purposes (i.e. membrane preparations, etc.). The release of 3 2 p i from Iz2PIATP during the phosphorylation of cells was determined according to the procedure of Martin and Doty TM as modified by Lindberg and Ernster n. 200 #1 from the organic phase was counted in IO ml Scintisol. The protein measurement was done by the method of Lowry et al. TM with bovine serum albumin as the standard. The radioactivity measurements were made in liquid scintillation spectrometers (a Packard Tricarb with automatic external standardization (efficiency about 45%) or a computerized Nuclear Chicago Isocap 300 (efficiency about 80%)). Membranes were prepared according to the ZnCI~ method of Warren and Glick TM as adapted in our laboratory for H e L a cells in monolayer cultures. Cells from two large dishes (3°. lO 6 cells/I5-cm dish) were washed two times with saline A and removed from the surface by 25 ml of I mM E D T A in saline within 20 rain and transferred to centrifuge tubes. After two washings in saline A (to remove the EDTA) the
340
v . K I N Z E L , G. C. M U E L L E R
cells were resuspended in I mM ZnC12 and were processed as described by Warren and Glick 13. Usually five strokes in a 7-ml Dounce, with pestle B, were sufficient to disrupt about 95 % of the cells. The purification of the released surface membranes was done in two ways. The membranes were sedimented through a discontinuous 5-ml sucrose gradient of 55, 50, 45, 40, and 35% sucrose in 3o-ml Cortex tubes using a Sorval RC-2 refrigerator centrifuge (HB- 4 rotor) at 15oo × g for 30 min. This procedure which is essentially the same as described by Warren and Glick 13 yielded membranes at the 40/45 and 45/50% sucrose interface. The membranes were re-sedimented according to the original procedure ~3. The second membrane isolation procedure employed was essentially the twophase polymer method given by Brunette and Till 14. The only modification which was introduced was the reduction of the speed in the "high" speed centrifugations from 8500 to 4000 rev./min (Sorvall RC-2, HB- 4 rotor). With remixing and recentrifuging the supernatants (i.e. the two-phase system plus membranes) five times high yields of well purified membranes were obtained; these appeared very clean when examined under phase contrast microscopy. The polyacrylamide gel electrophoresis followed essentially the method given by LaemmlP s. The autoradiography of longitudinally sliced gels was done with minor modifications according to the method of Fairbanks et al. 16. The scanning of gels (Coomassie blue stain: 57 ° n m ) and autoradiography (600 nm) was done in a Gilford Spectrophotometer with a linear transport apparatus. RESULTS
Properties of protein kinase from rat skeletal muscle The present studies were carried out using several preparations of protein kinase prepared according to the methods of Kuo and Greengard 6 and Miyamoto et al/. As revealed in Table I, the typical protein kinase preparation phosphorylates a receptor protein, histone, using [7-zPIATP as a substrate. Preparations of intermediate purity are characteristically stimulated by the addition of cyclic AMP, indicating the TABLE
I
PROPERTIES
OF THE
PROTEIN
KINASE
FROM
RAT SKELETAL
MUSCLE
P r o t e i n k i n a s e (a D E A E c o l u m n - p u r i f i e d f r a c t i o n ) w a s a s s a y e d a s d e s c r i b e d u n d e r M a t e r i a l a n d M e t h o d s : 28 p m o l e s [ y - 3 z P ] A T P ( 3 . 8 9 . lO 4 d p m / p m o l e ) w e r e u s e d p e r t u b e . T h e t e s t b a c k g r o u n d m e a s u r e d w i t h o u t h i s t o n e , w i t h o u t e n z y m e a n d c y c l i c A M P w a s 1.65 p m o l e s / t u b e w h i c h w a s subtracted from the values given in the table. Each value represents the mean of two determinations.
Enzyme
Cyclic A MP
Histone
pmoles s~p transferred to recovered protein
+ + +
+ -+
+ + --
--
+
+
--+
-+
+ ---
9.6o ± o.09 3 . 9 6 zk 0.3 0.64 ~ 0.o5 o.41 ± o.oz 0.26 ~ 0.07 0.07 ± 0.03 0.28 ± 0.06
341
PHOSPHORYLATION OF CELL SURFACE PROTEINS TABLE II DEPENDENCE
OF THE CELL PHOSPHORYLATION
REACTION
ON C Y C L I C A M P
lOs cells per 4-cm dish were phosphorylated as described under Materials and Methods in the presence or absence of cyclic AMP (5" lO-8 M) using two different protein kinase preparations, (23.4 units/dish) and Lp-8~P]ATP (23 ° pmoles/dish, 2.58. lO4 dpm/pmole). Incubation time was io min. As indicated the phopshokinase from the (NH,)2SO 4 fractionation was used directly or further purified by chromatography on DEAE. Background phosphorylation was measured in two dishes without enzyme with or without the addition of cyclic AMP yielding lO2 :£ 5" lO-3 pmoles a2P/mg cell protein and 97 ~: i . lO-8 pmoles 3*P/mg cell protein, respectively. These values were subtracted from the enzyme-mediated values in the table. Each value represents the mean of two cultures.
pmoles 3*P/rng cell protein × zo 3
(NH~)zSO 4 Concentrated D E A E eluate
Without cyclic A M P
With cyclic A M P
lO4 -- 6
857 4- 74
386 ± 95
685 ± 31
presence of the usual regulatory unit. Purification of the protein kinase activity by chromatography
on D E A E
columns serves to largely remove this regulatory unit
(Table II).
T h e e n z y m e p r e p a r a t i o n s w h i c h w e r e u s e d w e r e r e l a t i v e l y free o f e n d o genous acceptor proteins.
Characteristics of the cell phosphorylation reaction T h e t i m e c o u r s e o f t h e t y p i c a l p h o s p h o r y l a t i o n r e a c t i o n is p r e s e n t e d in Fig. I. U s i n g IO u n i t s o f p r o t e i n k i n a s e p e r 1.4" l o 6 cells in a 4 - c m p e t r i d i s h i t is e v i d e n t t h a t t h e r e a c t i o n p r o c e e d s m o r e r a p i d l y d u r i n g t h e first 5 m i n w h e r e u p o n it c o n t i n u e s
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Fig. I. Kinetics of cell phosphorylation without (O---O) and with 5" lO-6 M cyclic AMP ( O - - O ) . 1. 4. Io e cells per 4-cm dish were phosphorylated as described under Materials and Methods; the reaction was terminated after the indicated times. The reaction medium contained: protein kinase (IO units/dish) and [V-32P]ATP (200 pmoles/dish, 1.74.1o 4 dpm/pmole). Each point represents the mean of two cultures.
342
v.
KINZEL,
G. C. M U E L L E R
a t an a p p r o x i m a t e l y linear r a t e over the n e x t 4o min. The a d d i t i o n of cyclic A M P has its m a j o r effect d u r i n g the first 5 min. The effect of v a r y i n g the c o n c e n t r a t i o n of the e n z y m e on t h e cell p h o s p h o r y l a t i o n r e a c t i o n is shown in Fig. 2. I n this case all m e a s u r e m e n t s were m a d e in the presence of cyclic AMP. I t is a p p a r e n t t h a t the degree of p h o s p h o r y l a t i o n with the higher level of e n z y m e is r o u g h l y p r o p o r t i o n a l to t h a t e x p e c t e d from the lower level of a c t i v i t y . I t is also e v i d e n t in these e x p e r i m e n t s t h a t t h e e n z y m e m e d i a t e s the t r a n s f e r of the 7-32P label from A T P r a t h e r t h a n f a c i l i t a t i n g the u p t a k e of the entire nucleotide molecule b y the cell as m e a s u r e d with I3HiATP. The h e a t d e n a t u r e d enzyme (heated to 8o °C for i o min) did not effect the endogenous use of these nucleotides for these two p a t h w a y s b y the cells. W i t h the e x p e r i m e n t a l conditions e m p l o y e d c o - p r e c i p i t a t i o n of A T P as i n d i c a t e d b y the values o b t a i n e d with [3HIATP was not a problem. Z ~A 2.2-
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Fig. 2. Kinetics of cell phosphorylation with different enzyme levels using [7-32P]ATP (1~--O) and [3H]ATP ( ( ) - - - Q ) . io 6 cells in 4-cm dishes were phosphorylated as described under Materials and Methods and the reaction terminated after the indicated times. Protein kinase was varied from o, 5.8 or 23. 4 units/dish as indicated using 23o pmoles DJ-32P]ATP (2.58. lO4 dpm/pmole) or 23o pmoles E3HIATP (2.42. lO4 dpm/pmole) per dish. Each point represents the mean of t w o cultures. W h e n a s s a y e d in the presence of cyclic AMP, e n z y m e p r e p a r a t i o n s at two levels of purification ((NH4)2SO 4 fractions v s D E A E eluate) e x h i b i t e d c o m p a r a b l e degrees of p h o s p h o r y l a t i o n of cell surfaces. A s s a y e d in the absence of cyclic A M P the higher level of i n h i b i t o r in the (NH4)2SO 4 f r a c t i o n a t e d e n z y m e is e v i d e n c e d b y the low level of p h o s p h o r y l a t i o n o b t a i n e d (Table II). Since the D E A E - p u r i f i e d p r o t e i n kinase was e m p l o y e d in the m a j o r i t y of the e x p e r i m e n t s , the d e p e n d e n c y on cyclic A M P was minimized. The degree of cell p h o s p h o r y l a t i o n was clearly d e p e n d e n t on the level of A T P used in the p h o s p h o r y l a t i o n procedure. S a t u r a t i o n of t h e s y s t e m was n o t o b t a i n e d even a t a level of lO ~ M A T P in the presence of or absence of cyclic A M P (Fig. 3). I n c r e a s i n g t h e cell n u m b e r per p e t r i dish from o.6 to 2.6. lO 6 cells increased the t o t a l a m o u n t of p h o s p h o r y l a t i o n per dish in a n e a r l y p r o p o r t i o n a l m a n n e r (Fig. 4). F o r future m e t a b o l i c a n d b e h a v i o r a l studies, it was n e c e s s a r y to assess w h e t h e r
P H O S P H O R Y L A T I O N OF CELL S U R F A C E P R O T E I N S
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343
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ATP C O N C E N T R A T I O N Fig. 3. E f f e c t o f i n c r e a s i n g A T P c o n c e n t r a t i o n s w i t h o u t ( 0 - - 0 ) a n d w i t h 5" IO-S M c y c l i c A M P ( ( 2 ) - - © ) . 6 ' 1 o 5 ceils p e r 4 - c m d i s h w e r e p h o s p h o r y l a t e d as d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s . P r o t e i n k i n a s e ( i o u n i t s / d i s h ) a n d t h e i n d i c a t e d l e v e l s o f [~-82P]ATP w e r e e m p l o y e d . [ ~ - 8 2 P ] A T P w a s d i l u t e d 20 t i m e s b y c o l d N % A T P t o a s p e c i f i c a c t i v i t y : 97 ° d p m / p m o l e . I n c u b a tion time was io min. Each point represents the mean of two cultures.
the phosphorylated cells survived and replicated. As shown in Fig. 5 the phosphorylation of cells on Day I after planting had little or no effect on the subsequent growth when compared to control cultures. Similarly, phase contrast microscopy failed to reveal any major morphological alteration in the treated cultures.
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1224 2.525 CELL NUMBER PER DISH X 30"s 0585
F i g . 4. T h e e f f e c t o f v a r y i n g cell n u m b e r s o n t h e l e v e l o f p h o s p h o r y l a t i o n . G i v e n a r e t h e s p e c i f i c a c t i v i t y ( Q - - O ) a n d t h e t o t a l a c t i v i t y p e r c u l t u r e ((2) . . . . ©). C u l t u r e s c o n t a i n i n g o.58, 1.2 o r 2. 5. lO 6 cells p e r 4 - c m d i s h w e r e p h o s p h o r y l a t e d a s d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s u s i n g p r o t e i n k i n a s e ( i o u n i t s ] d i s h ) a n d [ y - s s P ] A T P (i i o p m o l e s / d i s h , 2.o 5 - lO 4 d p m / p m o l e ) . I n c u b a t i o n t i m e w a s i o rain. E a c h p o i n t r e p r e s e n t s t h e m e a n o f t w o c u l t u r e s . T h e d e t e r m i n a t i o n s o f t h e cell numbers were made on two additional dishes which were not phosphorylated, but were washed with Solution A in a similar manner.
Localization and partial characterization of the phosphorylated surface components Data supporting the conclusion that the phosphorylated components are surface membrane proteins are presented in Fig. 6. When cells, which had been phosphoryl-
344
v. KINZEL, G. C. MUELLER
,x
ro --12"
z
12I.I-
0
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01-
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T I M E (DAYS)
2 5i
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Fig. 5. Effect of cell surface phosphorylation on cell viability. Cells were plated on day zero in 4-cm dishes. On day I (~) the cells were phosphorylated as described under Materials and Methods. In this case all solutions were sterilized using Millipore filters (pore size 0.45 pro). Four different cell treatments were employed: × , ATP plus enzyme; O, ATP only; + , enzyme only; A, buffer control. Protein kinase (io units/dish) and unlabelled Na2ATP (I 12 pmoles/dish) were used with a io-min incubation. After the usual phosphorylation procedure the cultures were further incubated in BEHM. Each symbol represents the mean of two dishes. To avoid complexity only one line has been drawn through the points. Fig. 6. Removal of phosphorylated surface components by trypsin. The phosphorylated cells were treated without (O--Q) or with ( Q - - O ) trypsin for the indicated times. 8. Io ~ cells per 4-cm dish were phosphorylated as described under Materials and Methods using protein kinase (23.4 units/dish) and {y-3zP~ATP (23 ° pmoles/dish, 1.33. io a dpm/pmole) ; incubation time was IO rain. After rinsing the cultures with wash solution B the controls were further incubated with 2 ml of prewarmed saline A. The other cultures received 2 ml of 0.05% trypsin solution in saline A which was prewarmed to 37 °C just prior to addition. After the indicated times the control cultures were rinsed two times with 2 ml ice cold saline, fixed in 2 ml 5% trichloroacetic acid and processed as described in Materials and Methods. The so called zero values of the trypsin group were processed in the same way. In the other cases the trypsinized cells were susepnded with a pasteur pipette prior to transferring to centrifuge tubes containing 2 ml of ice cold saline and centrifuged (iooo rev./min, 5 min International refrigerated centrifuge). The cell sediment was washed twice with i-ml aliquots of ice cold saline, fixed with 5 O//otrichloroacetic acid and processed like the control culture. Each value represents the mean of two cultures. a t e d for IO m i n , are e x p o s e d t o a l o w l e v e l o f t r y p s i n ( o . o 5 % t r y p s i n ) , 8 6 - 9 5 % o f t h e l a b e l e d m a t e r i a l is r e l e a s e d q u i c k l y as a s o l u b l e f o r m . T o e x c l u d e t h e p o s s i b i l i t y t h a t a n e x t r a c e l l u l a r c o m p o n e n t o f t h e p h o s p h o r y l a t i o n m e d i a w a s a d s o r b e d on t h e s u r f a c e o f t h e cells, a n e x p e r i m e n t w a s c a r r i e d o u t in w h i c h cells w e r e e x p o s e d t o s u c h m e d i u m a f t e r t h e p h o s p h o r y l a t i o n s t e p . A s d e m o n s t r a t e d in Fig. 7, t h e r e w a s no adsorption of pre-phosphorylated components from such a reaction mixture even t h o u g h t h e cells w e r e e x p o s e d u n d e r c o n d i t i o n s i d e n t i c a l t o t h e u s u a l p h o s p h o r y l a t i o n reaction procedure. S o m e s t a b i l i t y c h a r a c t e r i s t i c s o f t h e p h o s p h o r y l a t e d c o m p o n e n t s o f t h e cell s u r f a c e are p r e s e n t e d in T a b l e I I I . I n a g r e e m e n t w i t h t h e k n o w n p r o p e r t i e s o f p h o s p h o p r o t e i n s , t h e 32p in t h e cell r e s i d u e w a s r e a s o n a b l y s t a b l e t o a b r i e f e x p o s u r e t o c o l d I.O M N a O H ; in c o n t r a s t , e x t r a c t i o n w i t h h o t I.O M N a O H r e m o v e d t h e label. E x t r a c t i o n w i t h c h l o r o f o r m - m e t h a n o l (2 :I, v / v ) a n d t r e a t m e n t w i t h h y d r o x y l a m i n e (0.8 M) f a i l e d t o e x t r a c t t h e l a b e l t h e r e b y s u g g e s t i n g t h a t t h e p h o s p h o r u s w a s n o t
345
P H O S P H O R Y L A T I O N OF C E L L S U R F A C E P R O T E I N S
0.8z 0.7-
0.6-
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0.5-
n~" ~_ 0.4-
w
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0.2-
0.I-
;
,b
TIME (MINUTES)
Fig. 7. Phosphorylation of cells versus incubation of cells with a pre-phosphorylated incubation mixture. A first group of cultures were phosphorytated as described in Materials and Methods for different periods of time indicated ( 0 - - 0 ) . Protein kinase: 3 ° units/dish; E32P~ATP, 23 ° pmoles/dish, 7.76. lO 3 dpm/pmole, 8- lO5 cells/4-cm dish. The second group got a phosphorylated incubation mixture for the times indicated (@--(2)). The pre-phosphorylated incubation mixture was obtained in the following way: preincubation of 4.2-ml incubation mixture with 7 × 3 ° units of protein kinase but without labeled ATP for 5 min at 37 °C in a shaker water b a t h (15o rev./min) followed by the addition of 7 × 23 ° pmoles [32p]ATP (0. 7 ml), 7.76. IO3 dpm/pmole and incubation for further io min at 37 °C. To stop a possible phosphorylation 0. 7 ml unlabeled ATP (8. IO 3 M Na2ATP ) was added to a final concentration of lO-3 M giving a total volume of 5.6 ml and also resulting in the final concentrations of the other compounds in the mixture. This phosphorylated incubation mixture was distributed to the second group of cultures (0.8 ml/culture) which were further incubated for the intervals indicated. The stop procedure was carried out as described above. Each point represents the mean of two dishes. associated with phospholipid or some labile phosphate trichloroacetic acid released 4 6% of the label.
ester. Extraction
with hot
Hydrolysis of extracellular A T P by cultured cells While the above experiments have been carried out in the presence of large amounts of extracellular inorganic phosphorus in the attempt to minimize the backg r o u n d i n c o r p o r a t i o n o f i n o r g a n i c p h o s p h o r u s l a b e l i n t o t h e cells, w e w e r e s t i l l interested to know how much inorganic phosphorus was released from [V-3zpIATP u n d e r t h e c o n d i t i o n s e m p l o y e d t o p h o s p h o r y l a t e t h e cell s u r f a c e s . A s o b s e r v e d b y ~ g r e n et al. 17 u s i n g d i f f e r e n t t i s s u e c u l t u r e cells, H e L a cells also h a v e a c o n s i d e r a b l e l e v e l o f A T P a s e a c t i v i t y o n t h e i r cell s u r f a c e (Fig. 8). T h e r e l e a s e o f i n o r g a n i c p h o s p h o r u s i n t h e cell s y s t e m w a s i n c r e a s e d a p p r o x . 35 % o v e r a 4 o - m i n a s s a y p e r i o d b y the addition of the protein kinase preparation.
Stability of the phosphorylated membrane components in living cells A s d e m o n s t r a t e d i n Fig. 5, t h e p h o s p h o r y l a t e d cells r e m a i n v i a b l e a n d c o n t i n u e to grow in the culture dishes. Accordingly, it was of interest to determine whether or n o t t h e l a b e l e d s u r f a c e m e m b r a n e c o m p o n e n t s w e r e t u r n e d o v e r i n t h e cells d u r i n g
346
v. KINZEL, G. C. MUELLER
TABLE III PARTIAL
CHARACTERIZATION
OF THE
PHOSPHORYLATED
CELL
SURFACE
COMPONENTS
6o. lO6 ceils in two i5-cm dishes were phosphorylated as described under Materials and Methods using protein kinase (45 ° units/dish) and ET-3~P]ATP (13.15 nmoles/dish, 1.o6.1o 4 dpm/pmole) over an incubation time of io rain. After the washing procedure B the cells were fixed with 2o ml ice cold 5% trichloroacetic acid for i h, removed from the petri dishes using a rubber policeman and centrifuged. The cell precipitate was dissolved in 6 ml of ice cold o.1 M NaOH and immediately reprecipitated by adding 20 ml 5% trichloroacetic acid. This precipitate was again dissolved with 16 ml o.i M NaOH and distributed in i-ml portions to centrifuge tubes. The solubilized protein was then immediately reprecipitated by 3 ml 5 % trichloroacetic acid per tube. The precipitate for hot and cold trichloroacetic acid treatment was suspended in I ml lO% trichloroacetic acid. In this case, the controls were kept in the cold while the test samples were heated for 15 min in a boiling water bath. After cooling the precipitates were washed once with 2 nil 5% trichloroacetic acid and dissolved in o. 5 M NaOH for assay of radioactivity and protein content. [n the case of the hot and cold NaOH treatments the cell precipitates were resolubilized with I ml I M NaOH. The control tubes (cold NaOH) were kept in an ice bath while the test samples were heated in a boiling water bath for 15 min. The cooled tubes were reprecipitated with 6 ml 4o% trichloroacetic acid and diluted with 4° ml water prior to centrifugation. The sediment was dissolved in o. 5 M NaOH for assay of radioactivity and protein content. Hydroxylamine treatment was done according to Hokin et al. 4. The precipitate was once washed with 4 ml water and suspended in either 4 ml o.8 M hydroxylamine or with 4 ml o.64 M NaC1 for the control and incubated for 15 min at 37 °C. The reaction was terminated by adding i mi ice cold 4o°/, trichloroacetic acid. The sediment was dissolved in o. 5 M NaOH for assay of radioactivity and protein content. For the lipid extraction the sediment was washed once with 4 ml water and suspended two times in 3 ml chloroform methanol (2 :i, v/v); each time the protein was reprecipitated by 2 ml 4o% trichloroacetic acid. The final precipitate was dissolved in 88% formic acid and an aliquot counted in Io ml Scintisol. Each value represents the mean of two tubes.
Trealment
pmoles 3~p/mg cell protein
Starting material Hot trichloroacetic acid (io%) Cold trichloroacetic acid (io%) Hot NaOH (I M) Cold NaOH (2 M) Hydroxylamine (0.8 M) NaC1 (o.6 4 1V[) Chloroform-methanol (2 :i, v/v)
2.3548 1.282 2.3753 o.1334 1.5949 1.9596 2.4372 2.296
m ± ± ± ± ~ ± ±
0.0254 0.0095 o.o8i o.o155 o.1349 0.0058 0.o435 o.1757
t h e s u b s e q u e n t g r o w t h i n t e r v a l . F o r t h i s p u r p o s e , cells w e r e l a b e l e d for 5- a n d I o - m i n i n t e r v a l s , w a s h e d w i t h c o l d A T P ( S o l u t i o n B) t o r e m o v e t h e [ y - a ~ P ] A T P a n d p h o s p h o r y l a t i o n m e d i u m , a n d i n c u b a t e d i n f r e s h B E H M m e d i u m (Fig. 9). W h e r e a s t h e r e w a s a s i g n i f i c a n t loss o f l a b e l f r o m t h e cells d u r i n g t h e first 3 5 - 4 ° m i n ( 2 o % loss f r o m cells p h o s p h o r y l a t e d 5 m i n a n d 4 o % loss f r o m cells p h o s p h o r y l a t e d IO m i n ) t h e r e w a s v e r y l i t t l e loss o v e r a s u b s e q u e n t I 5 o - m i n g r o w t h i n t e r v a l . W h e t h e r or n o t t h e r e w a s r e l o c a t i o n o f t h e c o m p o n e n t s f r o m t h e cell s u r f a c e t o o t h e r s i t e s in t h e cell or m e t a b o l i c a l t e r a t i o n s in t h e c a r r i e r m o l e c u l e s is n o t k n o w n a t t h i s t i m e a n d is a s u b j e c t o f f u t u r e i n v e s t i g a t i o n .
S t u d i e s on isolated m e m b r a n e s For further analysis of the phosphorylated surface proteins, plasma membranes w e r e p r e p a r e d in c e r t a i n e x p e r i m e n t s e s s e n t i a l l y a c c o r d i n g t o t h e ZnCI~ m e t h o d o f W a r r e n a n d Glick13; t h e s e w e r e t h e n p u r i f i e d e i t h e r b y s e d i m e n t a t i o n in s u c r o s e d e n s i t y g r a d i e n t s as t h e y d e s c r i b e d or r e f i n e d in t h e t w o - p h a s e p o l y m e r s y s t e m o f B r u n e t t e a n d T i l P 4. T h e p o l y m e r s y s t e m g a v e m i c r o s c o p i c a l l y c l e a n m e m b r a n e s in
PHOSPHORYLATION OF CELL SURFACE PROTEINS
347
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Fig. 8. Kinetics of [7-31P]ATP hydrolysis u n d e r p h o s p h o r y l a t i o n conditions, lO 8 cells per 4-cm dish were exposed to [y-32P]ATP (230 pmoles/dish, 1.75. io4 d p m / p m o l e ) in the absence ( © - - O ) or presence of p r o t e i n kinase ( O - - I ) (23.4 units/dish). At the indicated times the a m o u n t a~Pi which was released was d e t e r m i n e d as described u n d e r Materials and Methods. E a c h point r e p r e s e n t s the m e a n of two cultures. The 3~Pi level in 23 ° pmoles ~y-8*PJATP was 14.8 pmoles. Fig. 9. The metabolic stability of the p h o s p h o r y l a t e d surface m e m b r a n e c o m p o n e n t s . 8. lO s cells per 4-cm dish were p h o s p h o r y l a t e d as described u n d e r Materials and Methods for the indicated times using p r o t e i n kinase (3 ° units/dish) and [7-32p]ATP (230 pmoles/dish, 7.76. lO 3 d p m / p m o l e ) . The o-, 5- and I o - m i n values were o b t a i n e d in the usual way. F o r the chase e x p e r i m e n t s the cult u r e s were washed two times w i t h 2 ml p r e w a r m e d w a s h solution B and i n c u b a t e d f u r t h e r in B E H M culture m e d i u m (2 ml/dish). At the indicated times r e p r e s e n t a t i v e cultures were analyzed for p h o s p h o r y l a t e d surface c o m p o n e n t s as described u n d e r MateIials and Methods. E a c h p o i n t represents the m e a n of t w o cultures.
substantially higher yields than the sucrose gradient procedure; membranes from the latter also appeared to be contaminated with particulates. However, in comparing the specific activities of the two preparations relative to whole cells (Table IV) the membranes from the polymer system had a specific activity of only one-half of the whole cells. In contrast, the sucrose gradient prepared membranes exhibited a specific activity of 2.3 times that of whole cells. In view of the trypsin experiments cited earlier, it would appear that the polymer system may have displaced the labeled TABLE IV THE SPECIFIC ACTIVITY OF MEMBRANES ISOLATED FROM PHOSPHORYLATED CELLS Cell m e m b r a n e s were isolated according to the ZnC12 procedure of W a r r e n a n d Glick ]3 and either s e p a r a t e d b y sucrose gradient centrifugation or the t w o - p h a s e p o l y m e r s y s t e m of B r u n e t t e and Till 14 and c o m p a r e d w i t h total cell protein.
pmoles 32Piing protein × IO a Cells 199 2638 535 326
Membranes~cells
Separation procedure
2.28 2.45 0.63 0.5
sucrose sucrose two-phase system two-phase system
Membranes ± ! ± •
II 5° 4 4°
455 614o 334 165
~: ± ! ±
14 15o 2 3
348
v. KINZEL, G. C. MUELLER
m e m b r a n e c o m p o n e n t s f r o m t h e cells. I n a c c o r d w i t h t h i s c o n c e p t w a s t h e o b s e r v a t i o n t h a t t h e d i r e c t e x p o s u r e o f Z n C 1 2 - t r e a t e d w h o l e cells t o t h e p o l y m e r s y s t e m r e s u l t e d in a 2 5 % d r o p in specific a c t i v i t y f o r t h e w h o l e cells. A c c o r d i n g l y , b o t h p r o c e d u r e s a p p e a r t o p r e s e n t s o m e i n a d e q u a c i e s for m e m b r a n e i s o l a t i o n f r o m H e L a cells; t h e y also p o i n t t o t h e i n s t a b i l i t y o f t h e s t r u c t u r e s , e v e n a f t e r t h e ZnC12 t r e a t m e n t .
Electr@horesis of prolein.from labeled cells T o g a i n s o m e i n s i g h t i n t o t h e r a n g e o f s u r f a c e p r o t e i n s p h o s p h o r y l a t e d in t h e s e
E
I 2
T
I 2
Fig. IO. l~esolution of 3~P-labeled surface components. Residue proteins from phosphorylated cells which had been released from the petri dishes with EDTA (El, E2) or trypsin (TI, T2) were electrophoresed directly (El, TI) in sodium dodecyl sulfate gels or after NaOH-trichloroacetic acid extraction (E2, T2) and the distribution of proteins and ~2p determined. For this purpose, 4" Io6 cells per 6-cm dish were phosphorylated using protein kinase (75 units/dish) and [7-82P]ATP (3.o9 nmoles/dish; 1.87-1o 4 dpm/pmole). The phosphorylation time was io rnin. After the washing procedure B the cells were removed from the dishes either b y I mM EDTA (in saline A) within 2o rain or by o.o59/o trypsin (in saline A) within io min. The cells were washed twice in saline A and either directly solubilized in sample buffer (containing in ioo ml: io ml glycerol, 5 ml 2-mercaptoethanol, 3-4 g sodium dodecylsulfate and 2.27 g Tris base, adjusted to p H 8.8) ~for gel electrophoresis to about 2oo g protein/Ioo 1 or plecipitated with trichloroacetic acid] or solubilized after NaOH-trichloroacetic acid procedure as described in Materials and Methods. ioo-/d aliquots containing 2oo #g protein were electrophoresed on io-cm gels (6 m m diameter) containing IO.5~o acrylamide and o.42 ~o methylene bisacrylamide using a stacking gel on top (2 mA/gel). The gels were fixed, stained with Coomassie blue, scanned and sliced longitudinally for the autoradiography. The autoradiography was done using Kodak medical X-ray film R P Royal X - O m a t (exposure time 16 days).
349
PHOSPHORYLATION OF CELL SURFACE PROTEINS
experiments, labeled HeLa cells and their extracted residues were solubilized in sodium dodecylsulfate and subjected to electrophoresis in the stacking sodium dodecylsulfate-gel system described under Materials and Methods. The cell residues were prepared by extraction with NaOH and trichloroacetic acid as under Materials and Methods to remove 32P-labeled nucleotide and small molecular weight contaminants. Fig. IO presents the autoradiagraphs and the photographs of Coomassie bluestained gels after electrophoresis of unextracted and NaOH-trichloroacetic acid residues from control or trypsin-treated cells. A scan of the stained gel and the autoradiograph from the NaOH-trichloroacetic acid extracted residue of nontrypsinized cells is also presented (Fig. II). It is evident from this study that the Coomassiebluescan
C) 570nm 0.8 ~ ~
(~
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Autoradiographyscan
t
Fi
j
~_
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i
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25.7 IFI 0.5 Mobility
L
0.6
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0.7
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Fig. I I. A scan of the distribution of proteins and y-32P-labeled cell surface c o m p o n e n t s in the e l e c t r o p h e r o g r a m of the residues of p h o s p h o r y l a t e d cells. The Coomassie blue-stained gel E2 (Fig. io) and the corresponding a u t o r a d i o g r a p h were scanned as described u n d e r Materials and Methods. D a t a are expressed in relative units.
radioactive components are concentrated in at least 15 bands which are readily removed from the cell by Io-min treatment with o.o5% trypsin; this treatment has little effect, however, on the overall distribution of the proteins as determined from the stained gels. The observation that a significant portion of the asp migrates faster than the stainable proteins is unexplained. This entity appears to be unsusceptible to the trypsin treatment but is partially removed in NaOH-trichloroacetic acid extraction procedure. DISCUSSION
In this study the practicality of using a phosphoprotein kinase and [7-3~PIATP
35 °
V. KINZEL, G. C. MUELLER
to label the surface proteins of living cells was explored. The degree of phosphorylation was found to be dependent on the amount of enzyme, the level of FT-a2PIATP and duration of the labeling period. Cyclic AMP stimulated the process somewhat with the enzyme preparations employed. Under optimum conditions at least 15 bands of proteins, as measured by size fractionation in gel electrophoresis, are phosphorylated. Their location to the surface was largely shown by the ready releasability of the isotopically marked component from the intact cells with mild trypsinization. Since at least 15 bands of protein are labeled in the phosphorylation step it would appear that the protein kinase is not highly specific (a result also shown by Miyamoto et al. 7 for protein kinase from brain) and that the cell surface has a sizeable number of potential acceptor proteins in an exposed state. The phosphorylation of these proteins, as well as the procedure itself, however, appear to be relatively innoccuous since the cells continued to grow and replicate; during this interval the labeled components are reduced in amount. To our knowledge this is the first example of using phosphoprotein kinase as a reagent to label living cells; although it has been established that cells incubated with E),-32PIATP only become labeled 18 and yield asP-labeled serine and 32P-labeled threonine on hydrolysis of the proteins 19. The likelihood that this labeling is mediated by an endogenous phosphoprotein kinase is supported by the observation that cyclic AMP stimulated the labeling 2°. Whether the phosphorylations with exogenous ATP - - w i t h or without exogenous phosphoprotein kinase--can be related to the endogenous phosphorylation of membrane proteins with stimulation of certain cell fractions21, 22 remains to be explored. It is of further interest to assess whether selective treatments of the cells with hormones, nutrients, ions, and inhibitors expose different surface units for phosphorylation from without. One complication of the procedure involves the presence of surface ATPase 17. In addition to the destruction of the valuable substrate, the released inorganic phosphate, which is released by the enzyme has to be controlled; including 12.5 mM phosphate in the reaction system provides an ample extracellular pool for trapping this ion. Using more active phosphoprotein kinase preparations, higher levels of ATP, and shorter incubation intervals can be expected to further improve the selectivity of the phosphorylation procedure as a means of labeling the surface components of living cells. ACKNOWLEDGEMENTS
This investigation was supported b y U.S. Public Health Service Grants ToICA-5oo2 and CA-o7175. V.K. was holder of a fellowship from the Deutscher Akademischer Austauschdienst (DAAD). G.C.M. is a recipient of a research career award, U.S. Public Health Service. We wish to express our appreciation for m a n y helpful suggestions and discussions to Drs V. P. Wray, G. W. Wray, and W. LeStourgeon and Ms Mary LeMahieu for her assistance in the preparation of this manuscript. REFERENCES I Hoelzel-Wallach, D. F. (1972) Biochim. Biophys. Acta 265, 61-83 2 H u b b a r d , A. L. and Cohn, Z. A. (1972 ) J. Cell Biol. 55, 390-405
PHOSPHORYLATION OF CELL SURFACE PROTEINS
351
3 Rifkin, D. B., Compans, R. W. and Reich, E. (1972) J. Biol. Chem. 247, 6432-6437 4 Hokin, L. E., Sastry, P. S., Galsworthy, P. R. and Yoda, A. (1965) Proc. Natl. Acad. Sei. U.S. 54, 177-184 5 Mueller, G. C., Kajiwara, K., Stubblefield, E. and Rueckert, R. R. (1962) Cancer Res. 22, lO84 i o9 ° 6 Kuo, J. F. and Greengard, P. (1969) Proc. Natl. Acad. Sci. U.S. 64 , 1349-1355 7 Miyamoto, E., IZuo, J. F. and Greengard, P. (1969) J. Biol. Chem. 244, 6395-6402 8 2kgren, G. and Ronquist, G. (1969) Acta Physiol. Scand. 75, 124-128 9 Greenaway, P. J. (1972) Biochem. Biophvs. Res. Commun. 47, 639-644 IO Martin, J. B. and Doty, D. M. (1949) Anal. Chem. 21, 965-969 i i Lindberg, O. and Ernster, L. (1956) Methods Biochem. Anal. 3, 1-22 12 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193,265275 13 Warren, L. and Glick, M. C. (1969) in Fundamental Techniques in Virology (Habel, K. and Salzman, N. P., eds), pp. 66-71, Acadenlic Press, New York 14 Brunette, D. M. and Till, J. E. (1971) J. Membrane Biol. 5, 215-224 15 Laemmli, U. K. (197 o) Nature 227, 680-685 16 Fairbanks Jr, G., Levinthal, C. and Reeder, R. H. (1965) Biochem. Biophys. Res. Commun. 20, 393-399 17 Agren, G., Ponten, J., Ronquist, G. and Westermark, B. (1971) J. Cell. Physiol. 78, 171-176 18 ~_gren, G. and Ronquist, G. (197 o) Acta Physiol. Scand. 79, I25 -I28 19 Ronquist, G. and ~gren, G. (197 o) Acta Chem. Scand. 24, 728-73 ° 20 Duffy, M. J. and Schwarz, V. (1972 ) Biochem. J. 126, I2p 21 Majumder, G. C. and Turkington, R. W. (1972 ) J. Biol. Chem. 247, 7207 7217 22 Greengard, P., McAfree, D. A. and Kebabian, J. w . (1972) in Advances in Cyclic Nucleolide Research (Greengard, P. and Robinson, G. A., eds), Vol. I, pp. 337-356, Raven Press, New York
BBA
36520
P H O S P H O R Y L A T I O N OF SURFACE P R O T E I N S OF H E L A CELLS USING AN EXOGENOUS P R O T E I N KINASE AND [y-z~PIATP
V O L K E R K I N Z E L * AND G E R A L D C. M U E L L E R * *
McArdle Laboratory for Cancer Research, University of Wisconsin Medical Center, Madison, Wisc. 53706 (U.S.A.) (Received March I9th, 1973)
SUMMARY
The enzymatic phosphorylation of surface proteins of living cells with the aid of a protein kinase isolated from rat skeletal muscle and [s*p~ATP has been described. Utilizing monolayer cultures of HeLa cells the degree of phosphorylation was dependent on the amount of the protein kinase used, the labeled E32P]ATP and the incubation time. The phosphorylation was stimulated to a limited degree by cyclic AMP. After a Io-min labeling period, 86-95% of the label was removed with a brief treatment of 0.05% trypsin. Partial characterization of the labeled surface components revealed an instability of the label to hot NaOH, but good stability in hydroxylamine. The label was not removed by lipid extraction. Using a high resolution gel electrophoresis system it was shown that the label is distributed among at least 15 bands. The mildness of the procedure was verified by the continued growth of the cells.
INTRODUCTION
Recent studies suggest that cell membranes may play a dominant role in the control of replication and differentiation of cells. In addition, they may be important targets in the malignant transformation of cells by oncogenic viruses. To explore their role in these cellular processes our laboratory is investigating effects of selective labeling and chemical modification of surface proteins on their metabolic fate and the behavior of the treated cells. A recent review by Hoelzel-Wallach 1 and papers by Hubbard and Cohn ~ and Rifkin et al. ~ summarize related studies in this area. The present report describes a method for the phosphorylation of surface membrane proteins with radioactive phosphorus using an exogenous protein kinase (EC 2.7.1.37) from rat skeletal muscle and E~-~PIATP. Preliminary evidence relating to the stability and metabolic fate of surface proteins from HeLa cells is reported. * P r e s e n t address: G e r m a n Cancer Research Center ( D K F Z ) , K i r s c h n e r s t r a s s e 6, 69 Heidelberg, G e r m a n y . ** To w h o m r e p r i n t r e q u e s t s should be sent.
338
v . K I N Z E L , G. C. M U E L L E R
The data document that the phosphorylation of cell membranes provides a physiologically mild means for the selective labeling of surface proteins for studies of their metabolic fate and influence on the function of the cell. MATERIALS AND METHODS
The following chemicals were purchased as indicated: [y-a2Pladenosine 5'-triphosphate (La2P]ATP) (11-17. 7 Ci/mmole) from New England Nuclear, L8-aH]adenosine 5'-triphosphate from Amersham/Searle (E3HJATP, I I Ci/mmole), adenosine 3',5'-monophosphate (cyclic AMP) from Sigma, disodium adenosine 5'-triphosphate (Na2ATP) from P-L-Biochemicals, Inc., Milwaukee, calf thymus histone (B grade) from Calbiochem, theophylline (anhydrous) from Schwarz/Mann, sodium dodecylsulfate from Pierce Chemical Co.; the other chemicals for the polyacrylamide gel electrophoresis were obtained from BioRad Laboratories, DEAE-cellulose was purchased from Sigma (o.85 mequiv/g, medium mesh), bovine albumin (Fraction V) from Armour Pharmaceutical Corp., hydroxylamine was prepared according to Hokin et al. 4. Scintisol was obtained from Isolab Incorporated, Akron, Ohio. Trypsin was purchased from Nutritional Biochemicals Corp. Trichloroacetic acid, EDTA, and other chemicals were of analytical grade. H e L a $3 cells were cultured in suspension as described earlier 5. For the described experiments the cells were centrifuged, resuspended in Eagle's HeLa medium (BEHM) containing I O ~ bovine serum and transferred to 4 - I 5 - c m plastic petri dishes (Falcon). Subsequent incubations were carried out in a CO 2 incubator (air-CO2, 95:5, v/v) for at least 12 h prior to phosphorylation experiments. For most of the studies described 4-cm dishes were used with 2 ml medium. The cell counting was done with a Coulter Counter (model B). The protein kinase used in the experiments was prepared according to Kuo and Greengard 6 and Miyamoto et al. 7 from skeletal muscle of 3 4 -month-old female Sprague-Dawley rats. In most experiments the enzyme was purified through the DEAE-cellulose colunm step; however, in certain cases the (NH4)2SO 4 fractionated enzyme was used. The D E A E eluates were dialyzed against o.I M potassium phosphate buffer (pH 7, with 2 mM EDTA). In certain cases the enzyme was further concentrated by filtration with an Amicon Diaflo ultrafilter, PM-Io which retains molecular weights larger than IO ooo. This procedure resulted occasionally in a slight loss of enzyme activity. The enzyme solution was usually concentrated about five times based on the protein content and the enzyme solution stored at --22 °C without the addition of any stabilizing factors. A decrease in specific activity to about 5o~,~ within three months was noted for certain preparations. Protein kinase activity was assayed according to the procedure of Miyamoto et al. 7 except the ATP level was reduced to 25 pmoles per assay tube, which contained 5' IO-6 M cyclic AMP and 4o/~g calf thymus histones as a receptor. One unit of enzyme activity corresponds to an amount of enzyme which transfers i pmole of ~2p from [7-aePIATP to receptor protein in 5 min at 3o °C. This unit is approx. 4o times larger than that defined in the work of Miyamoto ct al. 7. Enzymatic phosphorylation of HeLa cells in 4-cm dishes (or 6-cm dishes) The medium was removed by suction, the cells were washed two times (washing procedure A) with 2 ml (or 4 ml in 6-cm dishes) of a prewarmed solution containing
PHOSPHORYLATION
OF CELL SURFACE PROTEINS
339
Tris-acetic acid buffer (0.03 M,pH 7-5 at 23 °C), NaC1 (o.15 M) and magnesium acetate (0.02 M) (Solution A). The cells were then preincubated at 37 °C for 5 min with the prewarmed incubation mixture adapted from Agren and Ronquist s and Miyamoto et al. 7. Tris-acetic acid buffer (0.03 M, pH 7.5), NaC1 (o.15 M), magnesium acetate (0.02 M), N a F (o.oi M), theophylline (4 raM) ; 4- cyclic AMP (approx. 5" IO GM) and lOO/~1 of protein kinase solution as indicated in the individual experiments per o.8-ml incubation mixture per dish (respectively, 250 #I enzyme per 2 ml per 6-cm dish). Since the enzyme solution was prepared in o.I M potassium phosphate, the final phosphate concentration of inorganic phosphate in the incubation mixture was 12.5 raM. The incubation mixture was freshly prepared each time. The reaction was started by the addition of IV-32PIATP (amounts given in the individual experiments). The incubation was carried out at 37 °C in the C02 incubator. To stop the reaction the incubation mixture was removed by suction and the dishes washed two times with 2 ml (4 ml in 6-cm dishes) of ice cold washing solution B (i.e. Solution A containing IO 3 M unlabeled Na2ATP) and then fixed with I ml (2 ml in 6-cm dishes) of ice cold 5 % trichloroacetic acid. The cultures were allowed to stand at least I h at o °C prior to washing again with i ml (2 ml in 6-cm dishes) trichloroacetic acid. The cells were removed using a rubber policeman and transferred to centrifuge tubes. Following centrifugation for IO rain at 2000 rev./min in an International PR-2 centrifuge at o °C in a No. 269 head, the residue from one dish was dissolved in 0.3 ml (0.6 ml in 6-cm dishes) ice cold o.I M NaOH and immediately reprecipitated b y the addition of I ml (2 ml in 6-cm dishes) 5 % trichloroacetic acid. This sequence was performed two times in order to get rid of contaminating ATP 9. The final precipitates were dissolved in 0.5 M NaOH and 4oo-#1 aliquots were counted in Scintisol using a scintillation counter. Protein determinations were carried out on 5O-lOO-/,1 allquots. The phosphorylation of larger batches of cells was done in a slightly different way. After two washings with 25 ml Solution A the I5-cm dishes were preincubated with the incubation mixture for 5 rain at 37 °C on a rocker platform (18 rev./min; Belco) and the reaction started by the addition of labeled ATP. A total volume of 6 ml of incubation mixture per I5-cm dish was used. The amounts of enzyme and A T P used per dish are given in the legends for the particular experiments. To stop the reaction the dishes were rinsed two times with 25 ml wash solution B and the cells used for the indicated purposes (i.e. membrane preparations, etc.). The release of 3 2 p i from Iz2PIATP during the phosphorylation of cells was determined according to the procedure of Martin and Doty TM as modified by Lindberg and Ernster n. 200 #1 from the organic phase was counted in IO ml Scintisol. The protein measurement was done by the method of Lowry et al. TM with bovine serum albumin as the standard. The radioactivity measurements were made in liquid scintillation spectrometers (a Packard Tricarb with automatic external standardization (efficiency about 45%) or a computerized Nuclear Chicago Isocap 300 (efficiency about 80%)). Membranes were prepared according to the ZnCI~ method of Warren and Glick TM as adapted in our laboratory for H e L a cells in monolayer cultures. Cells from two large dishes (3°. lO 6 cells/I5-cm dish) were washed two times with saline A and removed from the surface by 25 ml of I mM E D T A in saline within 20 rain and transferred to centrifuge tubes. After two washings in saline A (to remove the EDTA) the
340
v . K I N Z E L , G. C. M U E L L E R
cells were resuspended in I mM ZnC12 and were processed as described by Warren and Glick 13. Usually five strokes in a 7-ml Dounce, with pestle B, were sufficient to disrupt about 95 % of the cells. The purification of the released surface membranes was done in two ways. The membranes were sedimented through a discontinuous 5-ml sucrose gradient of 55, 50, 45, 40, and 35% sucrose in 3o-ml Cortex tubes using a Sorval RC-2 refrigerator centrifuge (HB- 4 rotor) at 15oo × g for 30 min. This procedure which is essentially the same as described by Warren and Glick 13 yielded membranes at the 40/45 and 45/50% sucrose interface. The membranes were re-sedimented according to the original procedure ~3. The second membrane isolation procedure employed was essentially the twophase polymer method given by Brunette and Till 14. The only modification which was introduced was the reduction of the speed in the "high" speed centrifugations from 8500 to 4000 rev./min (Sorvall RC-2, HB- 4 rotor). With remixing and recentrifuging the supernatants (i.e. the two-phase system plus membranes) five times high yields of well purified membranes were obtained; these appeared very clean when examined under phase contrast microscopy. The polyacrylamide gel electrophoresis followed essentially the method given by LaemmlP s. The autoradiography of longitudinally sliced gels was done with minor modifications according to the method of Fairbanks et al. 16. The scanning of gels (Coomassie blue stain: 57 ° n m ) and autoradiography (600 nm) was done in a Gilford Spectrophotometer with a linear transport apparatus. RESULTS
Properties of protein kinase from rat skeletal muscle The present studies were carried out using several preparations of protein kinase prepared according to the methods of Kuo and Greengard 6 and Miyamoto et al/. As revealed in Table I, the typical protein kinase preparation phosphorylates a receptor protein, histone, using [7-zPIATP as a substrate. Preparations of intermediate purity are characteristically stimulated by the addition of cyclic AMP, indicating the TABLE
I
PROPERTIES
OF THE
PROTEIN
KINASE
FROM
RAT SKELETAL
MUSCLE
P r o t e i n k i n a s e (a D E A E c o l u m n - p u r i f i e d f r a c t i o n ) w a s a s s a y e d a s d e s c r i b e d u n d e r M a t e r i a l a n d M e t h o d s : 28 p m o l e s [ y - 3 z P ] A T P ( 3 . 8 9 . lO 4 d p m / p m o l e ) w e r e u s e d p e r t u b e . T h e t e s t b a c k g r o u n d m e a s u r e d w i t h o u t h i s t o n e , w i t h o u t e n z y m e a n d c y c l i c A M P w a s 1.65 p m o l e s / t u b e w h i c h w a s subtracted from the values given in the table. Each value represents the mean of two determinations.
Enzyme
Cyclic A MP
Histone
pmoles s~p transferred to recovered protein
+ + +
+ -+
+ + --
--
+
+
--+
-+
+ ---
9.6o ± o.09 3 . 9 6 zk 0.3 0.64 ~ 0.o5 o.41 ± o.oz 0.26 ~ 0.07 0.07 ± 0.03 0.28 ± 0.06
341
PHOSPHORYLATION OF CELL SURFACE PROTEINS TABLE II DEPENDENCE
OF THE CELL PHOSPHORYLATION
REACTION
ON C Y C L I C A M P
lOs cells per 4-cm dish were phosphorylated as described under Materials and Methods in the presence or absence of cyclic AMP (5" lO-8 M) using two different protein kinase preparations, (23.4 units/dish) and Lp-8~P]ATP (23 ° pmoles/dish, 2.58. lO4 dpm/pmole). Incubation time was io min. As indicated the phopshokinase from the (NH,)2SO 4 fractionation was used directly or further purified by chromatography on DEAE. Background phosphorylation was measured in two dishes without enzyme with or without the addition of cyclic AMP yielding lO2 :£ 5" lO-3 pmoles a2P/mg cell protein and 97 ~: i . lO-8 pmoles 3*P/mg cell protein, respectively. These values were subtracted from the enzyme-mediated values in the table. Each value represents the mean of two cultures.
pmoles 3*P/rng cell protein × zo 3
(NH~)zSO 4 Concentrated D E A E eluate
Without cyclic A M P
With cyclic A M P
lO4 -- 6
857 4- 74
386 ± 95
685 ± 31
presence of the usual regulatory unit. Purification of the protein kinase activity by chromatography
on D E A E
columns serves to largely remove this regulatory unit
(Table II).
T h e e n z y m e p r e p a r a t i o n s w h i c h w e r e u s e d w e r e r e l a t i v e l y free o f e n d o genous acceptor proteins.
Characteristics of the cell phosphorylation reaction T h e t i m e c o u r s e o f t h e t y p i c a l p h o s p h o r y l a t i o n r e a c t i o n is p r e s e n t e d in Fig. I. U s i n g IO u n i t s o f p r o t e i n k i n a s e p e r 1.4" l o 6 cells in a 4 - c m p e t r i d i s h i t is e v i d e n t t h a t t h e r e a c t i o n p r o c e e d s m o r e r a p i d l y d u r i n g t h e first 5 m i n w h e r e u p o n it c o n t i n u e s
zO.$ hi I--" 0n " (:L d --I LI.I
0.2t~
E n." LU 0.. ithout
O.
cAMP
m OI-
w J 0
; 6
2b
go
T~ME IMtNUTESl
Fig. I. Kinetics of cell phosphorylation without (O---O) and with 5" lO-6 M cyclic AMP ( O - - O ) . 1. 4. Io e cells per 4-cm dish were phosphorylated as described under Materials and Methods; the reaction was terminated after the indicated times. The reaction medium contained: protein kinase (IO units/dish) and [V-32P]ATP (200 pmoles/dish, 1.74.1o 4 dpm/pmole). Each point represents the mean of two cultures.
342
v.
KINZEL,
G. C. M U E L L E R
a t an a p p r o x i m a t e l y linear r a t e over the n e x t 4o min. The a d d i t i o n of cyclic A M P has its m a j o r effect d u r i n g the first 5 min. The effect of v a r y i n g the c o n c e n t r a t i o n of the e n z y m e on t h e cell p h o s p h o r y l a t i o n r e a c t i o n is shown in Fig. 2. I n this case all m e a s u r e m e n t s were m a d e in the presence of cyclic AMP. I t is a p p a r e n t t h a t the degree of p h o s p h o r y l a t i o n with the higher level of e n z y m e is r o u g h l y p r o p o r t i o n a l to t h a t e x p e c t e d from the lower level of a c t i v i t y . I t is also e v i d e n t in these e x p e r i m e n t s t h a t t h e e n z y m e m e d i a t e s the t r a n s f e r of the 7-32P label from A T P r a t h e r t h a n f a c i l i t a t i n g the u p t a k e of the entire nucleotide molecule b y the cell as m e a s u r e d with I3HiATP. The h e a t d e n a t u r e d enzyme (heated to 8o °C for i o min) did not effect the endogenous use of these nucleotides for these two p a t h w a y s b y the cells. W i t h the e x p e r i m e n t a l conditions e m p l o y e d c o - p r e c i p i t a t i o n of A T P as i n d i c a t e d b y the values o b t a i n e d with [3HIATP was not a problem. Z ~A 2.2-
~2.0.J .-I 1.8W '0{31 1.6-
: ~ ~'32p-ATP °--'° 3 H ~
j ~ 234 ~
F L4n-. wn 1.21.0(3-
03 O
o.6-
23,4
0.4/ "/'
o20
0
/ ~ i
20 TIME (MINUTES)
o5"8
i
40
Fig. 2. Kinetics of cell phosphorylation with different enzyme levels using [7-32P]ATP (1~--O) and [3H]ATP ( ( ) - - - Q ) . io 6 cells in 4-cm dishes were phosphorylated as described under Materials and Methods and the reaction terminated after the indicated times. Protein kinase was varied from o, 5.8 or 23. 4 units/dish as indicated using 23o pmoles DJ-32P]ATP (2.58. lO4 dpm/pmole) or 23o pmoles E3HIATP (2.42. lO4 dpm/pmole) per dish. Each point represents the mean of t w o cultures. W h e n a s s a y e d in the presence of cyclic AMP, e n z y m e p r e p a r a t i o n s at two levels of purification ((NH4)2SO 4 fractions v s D E A E eluate) e x h i b i t e d c o m p a r a b l e degrees of p h o s p h o r y l a t i o n of cell surfaces. A s s a y e d in the absence of cyclic A M P the higher level of i n h i b i t o r in the (NH4)2SO 4 f r a c t i o n a t e d e n z y m e is e v i d e n c e d b y the low level of p h o s p h o r y l a t i o n o b t a i n e d (Table II). Since the D E A E - p u r i f i e d p r o t e i n kinase was e m p l o y e d in the m a j o r i t y of the e x p e r i m e n t s , the d e p e n d e n c y on cyclic A M P was minimized. The degree of cell p h o s p h o r y l a t i o n was clearly d e p e n d e n t on the level of A T P used in the p h o s p h o r y l a t i o n procedure. S a t u r a t i o n of t h e s y s t e m was n o t o b t a i n e d even a t a level of lO ~ M A T P in the presence of or absence of cyclic A M P (Fig. 3). I n c r e a s i n g t h e cell n u m b e r per p e t r i dish from o.6 to 2.6. lO 6 cells increased the t o t a l a m o u n t of p h o s p h o r y l a t i o n per dish in a n e a r l y p r o p o r t i o n a l m a n n e r (Fig. 4). F o r future m e t a b o l i c a n d b e h a v i o r a l studies, it was n e c e s s a r y to assess w h e t h e r
P H O S P H O R Y L A T I O N OF CELL S U R F A C E P R O T E I N S
o'6t '°1
343
/
withcAMP/
m z t2!
~-"
/
(LI--
omto
~.j
8
1,//
2 ~,...~,, ~/
,
=66 5xt07
,--
Id~ a
5xp~
l~?
ATP C O N C E N T R A T I O N Fig. 3. E f f e c t o f i n c r e a s i n g A T P c o n c e n t r a t i o n s w i t h o u t ( 0 - - 0 ) a n d w i t h 5" IO-S M c y c l i c A M P ( ( 2 ) - - © ) . 6 ' 1 o 5 ceils p e r 4 - c m d i s h w e r e p h o s p h o r y l a t e d as d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s . P r o t e i n k i n a s e ( i o u n i t s / d i s h ) a n d t h e i n d i c a t e d l e v e l s o f [~-82P]ATP w e r e e m p l o y e d . [ ~ - 8 2 P ] A T P w a s d i l u t e d 20 t i m e s b y c o l d N % A T P t o a s p e c i f i c a c t i v i t y : 97 ° d p m / p m o l e . I n c u b a tion time was io min. Each point represents the mean of two cultures.
the phosphorylated cells survived and replicated. As shown in Fig. 5 the phosphorylation of cells on Day I after planting had little or no effect on the subsequent growth when compared to control cultures. Similarly, phase contrast microscopy failed to reveal any major morphological alteration in the treated cultures.
I
6o I
L~
d LU /
~05.
/
/
/
/
/
~0.2-
/ ~E
/
/
/
/
/
/ 50 ,~ o x 4o
30 o~
-20 wm L_ -10 ¢x
¢ i
i
1224 2.525 CELL NUMBER PER DISH X 30"s 0585
F i g . 4. T h e e f f e c t o f v a r y i n g cell n u m b e r s o n t h e l e v e l o f p h o s p h o r y l a t i o n . G i v e n a r e t h e s p e c i f i c a c t i v i t y ( Q - - O ) a n d t h e t o t a l a c t i v i t y p e r c u l t u r e ((2) . . . . ©). C u l t u r e s c o n t a i n i n g o.58, 1.2 o r 2. 5. lO 6 cells p e r 4 - c m d i s h w e r e p h o s p h o r y l a t e d a s d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s u s i n g p r o t e i n k i n a s e ( i o u n i t s ] d i s h ) a n d [ y - s s P ] A T P (i i o p m o l e s / d i s h , 2.o 5 - lO 4 d p m / p m o l e ) . I n c u b a t i o n t i m e w a s i o rain. E a c h p o i n t r e p r e s e n t s t h e m e a n o f t w o c u l t u r e s . T h e d e t e r m i n a t i o n s o f t h e cell numbers were made on two additional dishes which were not phosphorylated, but were washed with Solution A in a similar manner.
Localization and partial characterization of the phosphorylated surface components Data supporting the conclusion that the phosphorylated components are surface membrane proteins are presented in Fig. 6. When cells, which had been phosphoryl-
344
v. KINZEL, G. C. MUELLER
,x
ro --12"
z
12I.I-
0
x "I" 03
w n,w8 n
/
n," LU 113 :3 Z4 J J I~J ¢0
L0-
0.9without trypsin
08~0.7r~ 0.6~" 0.5-
Q4IE
0.30.2-
with ltrypsin
01-
o
i
0
T I M E (DAYS)
2 5i
5' TIME (MINUTES)
12
Fig. 5. Effect of cell surface phosphorylation on cell viability. Cells were plated on day zero in 4-cm dishes. On day I (~) the cells were phosphorylated as described under Materials and Methods. In this case all solutions were sterilized using Millipore filters (pore size 0.45 pro). Four different cell treatments were employed: × , ATP plus enzyme; O, ATP only; + , enzyme only; A, buffer control. Protein kinase (io units/dish) and unlabelled Na2ATP (I 12 pmoles/dish) were used with a io-min incubation. After the usual phosphorylation procedure the cultures were further incubated in BEHM. Each symbol represents the mean of two dishes. To avoid complexity only one line has been drawn through the points. Fig. 6. Removal of phosphorylated surface components by trypsin. The phosphorylated cells were treated without (O--Q) or with ( Q - - O ) trypsin for the indicated times. 8. Io ~ cells per 4-cm dish were phosphorylated as described under Materials and Methods using protein kinase (23.4 units/dish) and {y-3zP~ATP (23 ° pmoles/dish, 1.33. io a dpm/pmole) ; incubation time was IO rain. After rinsing the cultures with wash solution B the controls were further incubated with 2 ml of prewarmed saline A. The other cultures received 2 ml of 0.05% trypsin solution in saline A which was prewarmed to 37 °C just prior to addition. After the indicated times the control cultures were rinsed two times with 2 ml ice cold saline, fixed in 2 ml 5% trichloroacetic acid and processed as described in Materials and Methods. The so called zero values of the trypsin group were processed in the same way. In the other cases the trypsinized cells were susepnded with a pasteur pipette prior to transferring to centrifuge tubes containing 2 ml of ice cold saline and centrifuged (iooo rev./min, 5 min International refrigerated centrifuge). The cell sediment was washed twice with i-ml aliquots of ice cold saline, fixed with 5 O//otrichloroacetic acid and processed like the control culture. Each value represents the mean of two cultures. a t e d for IO m i n , are e x p o s e d t o a l o w l e v e l o f t r y p s i n ( o . o 5 % t r y p s i n ) , 8 6 - 9 5 % o f t h e l a b e l e d m a t e r i a l is r e l e a s e d q u i c k l y as a s o l u b l e f o r m . T o e x c l u d e t h e p o s s i b i l i t y t h a t a n e x t r a c e l l u l a r c o m p o n e n t o f t h e p h o s p h o r y l a t i o n m e d i a w a s a d s o r b e d on t h e s u r f a c e o f t h e cells, a n e x p e r i m e n t w a s c a r r i e d o u t in w h i c h cells w e r e e x p o s e d t o s u c h m e d i u m a f t e r t h e p h o s p h o r y l a t i o n s t e p . A s d e m o n s t r a t e d in Fig. 7, t h e r e w a s no adsorption of pre-phosphorylated components from such a reaction mixture even t h o u g h t h e cells w e r e e x p o s e d u n d e r c o n d i t i o n s i d e n t i c a l t o t h e u s u a l p h o s p h o r y l a t i o n reaction procedure. S o m e s t a b i l i t y c h a r a c t e r i s t i c s o f t h e p h o s p h o r y l a t e d c o m p o n e n t s o f t h e cell s u r f a c e are p r e s e n t e d in T a b l e I I I . I n a g r e e m e n t w i t h t h e k n o w n p r o p e r t i e s o f p h o s p h o p r o t e i n s , t h e 32p in t h e cell r e s i d u e w a s r e a s o n a b l y s t a b l e t o a b r i e f e x p o s u r e t o c o l d I.O M N a O H ; in c o n t r a s t , e x t r a c t i o n w i t h h o t I.O M N a O H r e m o v e d t h e label. E x t r a c t i o n w i t h c h l o r o f o r m - m e t h a n o l (2 :I, v / v ) a n d t r e a t m e n t w i t h h y d r o x y l a m i n e (0.8 M) f a i l e d t o e x t r a c t t h e l a b e l t h e r e b y s u g g e s t i n g t h a t t h e p h o s p h o r u s w a s n o t
345
P H O S P H O R Y L A T I O N OF C E L L S U R F A C E P R O T E I N S
0.8z 0.7-
0.6-
~
0.5-
n~" ~_ 0.4-
w
~
0.2-
0.I-
;
,b
TIME (MINUTES)
Fig. 7. Phosphorylation of cells versus incubation of cells with a pre-phosphorylated incubation mixture. A first group of cultures were phosphorytated as described in Materials and Methods for different periods of time indicated ( 0 - - 0 ) . Protein kinase: 3 ° units/dish; E32P~ATP, 23 ° pmoles/dish, 7.76. lO 3 dpm/pmole, 8- lO5 cells/4-cm dish. The second group got a phosphorylated incubation mixture for the times indicated (@--(2)). The pre-phosphorylated incubation mixture was obtained in the following way: preincubation of 4.2-ml incubation mixture with 7 × 3 ° units of protein kinase but without labeled ATP for 5 min at 37 °C in a shaker water b a t h (15o rev./min) followed by the addition of 7 × 23 ° pmoles [32p]ATP (0. 7 ml), 7.76. IO3 dpm/pmole and incubation for further io min at 37 °C. To stop a possible phosphorylation 0. 7 ml unlabeled ATP (8. IO 3 M Na2ATP ) was added to a final concentration of lO-3 M giving a total volume of 5.6 ml and also resulting in the final concentrations of the other compounds in the mixture. This phosphorylated incubation mixture was distributed to the second group of cultures (0.8 ml/culture) which were further incubated for the intervals indicated. The stop procedure was carried out as described above. Each point represents the mean of two dishes. associated with phospholipid or some labile phosphate trichloroacetic acid released 4 6% of the label.
ester. Extraction
with hot
Hydrolysis of extracellular A T P by cultured cells While the above experiments have been carried out in the presence of large amounts of extracellular inorganic phosphorus in the attempt to minimize the backg r o u n d i n c o r p o r a t i o n o f i n o r g a n i c p h o s p h o r u s l a b e l i n t o t h e cells, w e w e r e s t i l l interested to know how much inorganic phosphorus was released from [V-3zpIATP u n d e r t h e c o n d i t i o n s e m p l o y e d t o p h o s p h o r y l a t e t h e cell s u r f a c e s . A s o b s e r v e d b y ~ g r e n et al. 17 u s i n g d i f f e r e n t t i s s u e c u l t u r e cells, H e L a cells also h a v e a c o n s i d e r a b l e l e v e l o f A T P a s e a c t i v i t y o n t h e i r cell s u r f a c e (Fig. 8). T h e r e l e a s e o f i n o r g a n i c p h o s p h o r u s i n t h e cell s y s t e m w a s i n c r e a s e d a p p r o x . 35 % o v e r a 4 o - m i n a s s a y p e r i o d b y the addition of the protein kinase preparation.
Stability of the phosphorylated membrane components in living cells A s d e m o n s t r a t e d i n Fig. 5, t h e p h o s p h o r y l a t e d cells r e m a i n v i a b l e a n d c o n t i n u e to grow in the culture dishes. Accordingly, it was of interest to determine whether or n o t t h e l a b e l e d s u r f a c e m e m b r a n e c o m p o n e n t s w e r e t u r n e d o v e r i n t h e cells d u r i n g
346
v. KINZEL, G. C. MUELLER
TABLE III PARTIAL
CHARACTERIZATION
OF THE
PHOSPHORYLATED
CELL
SURFACE
COMPONENTS
6o. lO6 ceils in two i5-cm dishes were phosphorylated as described under Materials and Methods using protein kinase (45 ° units/dish) and ET-3~P]ATP (13.15 nmoles/dish, 1.o6.1o 4 dpm/pmole) over an incubation time of io rain. After the washing procedure B the cells were fixed with 2o ml ice cold 5% trichloroacetic acid for i h, removed from the petri dishes using a rubber policeman and centrifuged. The cell precipitate was dissolved in 6 ml of ice cold o.1 M NaOH and immediately reprecipitated by adding 20 ml 5% trichloroacetic acid. This precipitate was again dissolved with 16 ml o.i M NaOH and distributed in i-ml portions to centrifuge tubes. The solubilized protein was then immediately reprecipitated by 3 ml 5 % trichloroacetic acid per tube. The precipitate for hot and cold trichloroacetic acid treatment was suspended in I ml lO% trichloroacetic acid. In this case, the controls were kept in the cold while the test samples were heated for 15 min in a boiling water bath. After cooling the precipitates were washed once with 2 nil 5% trichloroacetic acid and dissolved in o. 5 M NaOH for assay of radioactivity and protein content. [n the case of the hot and cold NaOH treatments the cell precipitates were resolubilized with I ml I M NaOH. The control tubes (cold NaOH) were kept in an ice bath while the test samples were heated in a boiling water bath for 15 min. The cooled tubes were reprecipitated with 6 ml 4o% trichloroacetic acid and diluted with 4° ml water prior to centrifugation. The sediment was dissolved in o. 5 M NaOH for assay of radioactivity and protein content. Hydroxylamine treatment was done according to Hokin et al. 4. The precipitate was once washed with 4 ml water and suspended in either 4 ml o.8 M hydroxylamine or with 4 ml o.64 M NaC1 for the control and incubated for 15 min at 37 °C. The reaction was terminated by adding i mi ice cold 4o°/, trichloroacetic acid. The sediment was dissolved in o. 5 M NaOH for assay of radioactivity and protein content. For the lipid extraction the sediment was washed once with 4 ml water and suspended two times in 3 ml chloroform methanol (2 :i, v/v); each time the protein was reprecipitated by 2 ml 4o% trichloroacetic acid. The final precipitate was dissolved in 88% formic acid and an aliquot counted in Io ml Scintisol. Each value represents the mean of two tubes.
Trealment
pmoles 3~p/mg cell protein
Starting material Hot trichloroacetic acid (io%) Cold trichloroacetic acid (io%) Hot NaOH (I M) Cold NaOH (2 M) Hydroxylamine (0.8 M) NaC1 (o.6 4 1V[) Chloroform-methanol (2 :i, v/v)
2.3548 1.282 2.3753 o.1334 1.5949 1.9596 2.4372 2.296
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t h e s u b s e q u e n t g r o w t h i n t e r v a l . F o r t h i s p u r p o s e , cells w e r e l a b e l e d for 5- a n d I o - m i n i n t e r v a l s , w a s h e d w i t h c o l d A T P ( S o l u t i o n B) t o r e m o v e t h e [ y - a ~ P ] A T P a n d p h o s p h o r y l a t i o n m e d i u m , a n d i n c u b a t e d i n f r e s h B E H M m e d i u m (Fig. 9). W h e r e a s t h e r e w a s a s i g n i f i c a n t loss o f l a b e l f r o m t h e cells d u r i n g t h e first 3 5 - 4 ° m i n ( 2 o % loss f r o m cells p h o s p h o r y l a t e d 5 m i n a n d 4 o % loss f r o m cells p h o s p h o r y l a t e d IO m i n ) t h e r e w a s v e r y l i t t l e loss o v e r a s u b s e q u e n t I 5 o - m i n g r o w t h i n t e r v a l . W h e t h e r or n o t t h e r e w a s r e l o c a t i o n o f t h e c o m p o n e n t s f r o m t h e cell s u r f a c e t o o t h e r s i t e s in t h e cell or m e t a b o l i c a l t e r a t i o n s in t h e c a r r i e r m o l e c u l e s is n o t k n o w n a t t h i s t i m e a n d is a s u b j e c t o f f u t u r e i n v e s t i g a t i o n .
S t u d i e s on isolated m e m b r a n e s For further analysis of the phosphorylated surface proteins, plasma membranes w e r e p r e p a r e d in c e r t a i n e x p e r i m e n t s e s s e n t i a l l y a c c o r d i n g t o t h e ZnCI~ m e t h o d o f W a r r e n a n d Glick13; t h e s e w e r e t h e n p u r i f i e d e i t h e r b y s e d i m e n t a t i o n in s u c r o s e d e n s i t y g r a d i e n t s as t h e y d e s c r i b e d or r e f i n e d in t h e t w o - p h a s e p o l y m e r s y s t e m o f B r u n e t t e a n d T i l P 4. T h e p o l y m e r s y s t e m g a v e m i c r o s c o p i c a l l y c l e a n m e m b r a n e s in
PHOSPHORYLATION OF CELL SURFACE PROTEINS
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Fig. 8. Kinetics of [7-31P]ATP hydrolysis u n d e r p h o s p h o r y l a t i o n conditions, lO 8 cells per 4-cm dish were exposed to [y-32P]ATP (230 pmoles/dish, 1.75. io4 d p m / p m o l e ) in the absence ( © - - O ) or presence of p r o t e i n kinase ( O - - I ) (23.4 units/dish). At the indicated times the a m o u n t a~Pi which was released was d e t e r m i n e d as described u n d e r Materials and Methods. E a c h point r e p r e s e n t s the m e a n of two cultures. The 3~Pi level in 23 ° pmoles ~y-8*PJATP was 14.8 pmoles. Fig. 9. The metabolic stability of the p h o s p h o r y l a t e d surface m e m b r a n e c o m p o n e n t s . 8. lO s cells per 4-cm dish were p h o s p h o r y l a t e d as described u n d e r Materials and Methods for the indicated times using p r o t e i n kinase (3 ° units/dish) and [7-32p]ATP (230 pmoles/dish, 7.76. lO 3 d p m / p m o l e ) . The o-, 5- and I o - m i n values were o b t a i n e d in the usual way. F o r the chase e x p e r i m e n t s the cult u r e s were washed two times w i t h 2 ml p r e w a r m e d w a s h solution B and i n c u b a t e d f u r t h e r in B E H M culture m e d i u m (2 ml/dish). At the indicated times r e p r e s e n t a t i v e cultures were analyzed for p h o s p h o r y l a t e d surface c o m p o n e n t s as described u n d e r MateIials and Methods. E a c h p o i n t represents the m e a n of t w o cultures.
substantially higher yields than the sucrose gradient procedure; membranes from the latter also appeared to be contaminated with particulates. However, in comparing the specific activities of the two preparations relative to whole cells (Table IV) the membranes from the polymer system had a specific activity of only one-half of the whole cells. In contrast, the sucrose gradient prepared membranes exhibited a specific activity of 2.3 times that of whole cells. In view of the trypsin experiments cited earlier, it would appear that the polymer system may have displaced the labeled TABLE IV THE SPECIFIC ACTIVITY OF MEMBRANES ISOLATED FROM PHOSPHORYLATED CELLS Cell m e m b r a n e s were isolated according to the ZnC12 procedure of W a r r e n a n d Glick ]3 and either s e p a r a t e d b y sucrose gradient centrifugation or the t w o - p h a s e p o l y m e r s y s t e m of B r u n e t t e and Till 14 and c o m p a r e d w i t h total cell protein.
pmoles 32Piing protein × IO a Cells 199 2638 535 326
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348
v. KINZEL, G. C. MUELLER
m e m b r a n e c o m p o n e n t s f r o m t h e cells. I n a c c o r d w i t h t h i s c o n c e p t w a s t h e o b s e r v a t i o n t h a t t h e d i r e c t e x p o s u r e o f Z n C 1 2 - t r e a t e d w h o l e cells t o t h e p o l y m e r s y s t e m r e s u l t e d in a 2 5 % d r o p in specific a c t i v i t y f o r t h e w h o l e cells. A c c o r d i n g l y , b o t h p r o c e d u r e s a p p e a r t o p r e s e n t s o m e i n a d e q u a c i e s for m e m b r a n e i s o l a t i o n f r o m H e L a cells; t h e y also p o i n t t o t h e i n s t a b i l i t y o f t h e s t r u c t u r e s , e v e n a f t e r t h e ZnC12 t r e a t m e n t .
Electr@horesis of prolein.from labeled cells T o g a i n s o m e i n s i g h t i n t o t h e r a n g e o f s u r f a c e p r o t e i n s p h o s p h o r y l a t e d in t h e s e
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Fig. IO. l~esolution of 3~P-labeled surface components. Residue proteins from phosphorylated cells which had been released from the petri dishes with EDTA (El, E2) or trypsin (TI, T2) were electrophoresed directly (El, TI) in sodium dodecyl sulfate gels or after NaOH-trichloroacetic acid extraction (E2, T2) and the distribution of proteins and ~2p determined. For this purpose, 4" Io6 cells per 6-cm dish were phosphorylated using protein kinase (75 units/dish) and [7-82P]ATP (3.o9 nmoles/dish; 1.87-1o 4 dpm/pmole). The phosphorylation time was io rnin. After the washing procedure B the cells were removed from the dishes either b y I mM EDTA (in saline A) within 2o rain or by o.o59/o trypsin (in saline A) within io min. The cells were washed twice in saline A and either directly solubilized in sample buffer (containing in ioo ml: io ml glycerol, 5 ml 2-mercaptoethanol, 3-4 g sodium dodecylsulfate and 2.27 g Tris base, adjusted to p H 8.8) ~for gel electrophoresis to about 2oo g protein/Ioo 1 or plecipitated with trichloroacetic acid] or solubilized after NaOH-trichloroacetic acid procedure as described in Materials and Methods. ioo-/d aliquots containing 2oo #g protein were electrophoresed on io-cm gels (6 m m diameter) containing IO.5~o acrylamide and o.42 ~o methylene bisacrylamide using a stacking gel on top (2 mA/gel). The gels were fixed, stained with Coomassie blue, scanned and sliced longitudinally for the autoradiography. The autoradiography was done using Kodak medical X-ray film R P Royal X - O m a t (exposure time 16 days).
349
PHOSPHORYLATION OF CELL SURFACE PROTEINS
experiments, labeled HeLa cells and their extracted residues were solubilized in sodium dodecylsulfate and subjected to electrophoresis in the stacking sodium dodecylsulfate-gel system described under Materials and Methods. The cell residues were prepared by extraction with NaOH and trichloroacetic acid as under Materials and Methods to remove 32P-labeled nucleotide and small molecular weight contaminants. Fig. IO presents the autoradiagraphs and the photographs of Coomassie bluestained gels after electrophoresis of unextracted and NaOH-trichloroacetic acid residues from control or trypsin-treated cells. A scan of the stained gel and the autoradiograph from the NaOH-trichloroacetic acid extracted residue of nontrypsinized cells is also presented (Fig. II). It is evident from this study that the Coomassiebluescan
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radioactive components are concentrated in at least 15 bands which are readily removed from the cell by Io-min treatment with o.o5% trypsin; this treatment has little effect, however, on the overall distribution of the proteins as determined from the stained gels. The observation that a significant portion of the asp migrates faster than the stainable proteins is unexplained. This entity appears to be unsusceptible to the trypsin treatment but is partially removed in NaOH-trichloroacetic acid extraction procedure. DISCUSSION
In this study the practicality of using a phosphoprotein kinase and [7-3~PIATP
35 °
V. KINZEL, G. C. MUELLER
to label the surface proteins of living cells was explored. The degree of phosphorylation was found to be dependent on the amount of enzyme, the level of FT-a2PIATP and duration of the labeling period. Cyclic AMP stimulated the process somewhat with the enzyme preparations employed. Under optimum conditions at least 15 bands of proteins, as measured by size fractionation in gel electrophoresis, are phosphorylated. Their location to the surface was largely shown by the ready releasability of the isotopically marked component from the intact cells with mild trypsinization. Since at least 15 bands of protein are labeled in the phosphorylation step it would appear that the protein kinase is not highly specific (a result also shown by Miyamoto et al. 7 for protein kinase from brain) and that the cell surface has a sizeable number of potential acceptor proteins in an exposed state. The phosphorylation of these proteins, as well as the procedure itself, however, appear to be relatively innoccuous since the cells continued to grow and replicate; during this interval the labeled components are reduced in amount. To our knowledge this is the first example of using phosphoprotein kinase as a reagent to label living cells; although it has been established that cells incubated with E),-32PIATP only become labeled 18 and yield asP-labeled serine and 32P-labeled threonine on hydrolysis of the proteins 19. The likelihood that this labeling is mediated by an endogenous phosphoprotein kinase is supported by the observation that cyclic AMP stimulated the labeling 2°. Whether the phosphorylations with exogenous ATP - - w i t h or without exogenous phosphoprotein kinase--can be related to the endogenous phosphorylation of membrane proteins with stimulation of certain cell fractions21, 22 remains to be explored. It is of further interest to assess whether selective treatments of the cells with hormones, nutrients, ions, and inhibitors expose different surface units for phosphorylation from without. One complication of the procedure involves the presence of surface ATPase 17. In addition to the destruction of the valuable substrate, the released inorganic phosphate, which is released by the enzyme has to be controlled; including 12.5 mM phosphate in the reaction system provides an ample extracellular pool for trapping this ion. Using more active phosphoprotein kinase preparations, higher levels of ATP, and shorter incubation intervals can be expected to further improve the selectivity of the phosphorylation procedure as a means of labeling the surface components of living cells. ACKNOWLEDGEMENTS
This investigation was supported b y U.S. Public Health Service Grants ToICA-5oo2 and CA-o7175. V.K. was holder of a fellowship from the Deutscher Akademischer Austauschdienst (DAAD). G.C.M. is a recipient of a research career award, U.S. Public Health Service. We wish to express our appreciation for m a n y helpful suggestions and discussions to Drs V. P. Wray, G. W. Wray, and W. LeStourgeon and Ms Mary LeMahieu for her assistance in the preparation of this manuscript. REFERENCES I Hoelzel-Wallach, D. F. (1972) Biochim. Biophys. Acta 265, 61-83 2 H u b b a r d , A. L. and Cohn, Z. A. (1972 ) J. Cell Biol. 55, 390-405
PHOSPHORYLATION OF CELL SURFACE PROTEINS
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3 Rifkin, D. B., Compans, R. W. and Reich, E. (1972) J. Biol. Chem. 247, 6432-6437 4 Hokin, L. E., Sastry, P. S., Galsworthy, P. R. and Yoda, A. (1965) Proc. Natl. Acad. Sei. U.S. 54, 177-184 5 Mueller, G. C., Kajiwara, K., Stubblefield, E. and Rueckert, R. R. (1962) Cancer Res. 22, lO84 i o9 ° 6 Kuo, J. F. and Greengard, P. (1969) Proc. Natl. Acad. Sci. U.S. 64 , 1349-1355 7 Miyamoto, E., IZuo, J. F. and Greengard, P. (1969) J. Biol. Chem. 244, 6395-6402 8 2kgren, G. and Ronquist, G. (1969) Acta Physiol. Scand. 75, 124-128 9 Greenaway, P. J. (1972) Biochem. Biophvs. Res. Commun. 47, 639-644 IO Martin, J. B. and Doty, D. M. (1949) Anal. Chem. 21, 965-969 i i Lindberg, O. and Ernster, L. (1956) Methods Biochem. Anal. 3, 1-22 12 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193,265275 13 Warren, L. and Glick, M. C. (1969) in Fundamental Techniques in Virology (Habel, K. and Salzman, N. P., eds), pp. 66-71, Acadenlic Press, New York 14 Brunette, D. M. and Till, J. E. (1971) J. Membrane Biol. 5, 215-224 15 Laemmli, U. K. (197 o) Nature 227, 680-685 16 Fairbanks Jr, G., Levinthal, C. and Reeder, R. H. (1965) Biochem. Biophys. Res. Commun. 20, 393-399 17 Agren, G., Ponten, J., Ronquist, G. and Westermark, B. (1971) J. Cell. Physiol. 78, 171-176 18 ~_gren, G. and Ronquist, G. (197 o) Acta Physiol. Scand. 79, I25 -I28 19 Ronquist, G. and ~gren, G. (197 o) Acta Chem. Scand. 24, 728-73 ° 20 Duffy, M. J. and Schwarz, V. (1972 ) Biochem. J. 126, I2p 21 Majumder, G. C. and Turkington, R. W. (1972 ) J. Biol. Chem. 247, 7207 7217 22 Greengard, P., McAfree, D. A. and Kebabian, J. w . (1972) in Advances in Cyclic Nucleolide Research (Greengard, P. and Robinson, G. A., eds), Vol. I, pp. 337-356, Raven Press, New York