Characterization of two distinct protein kinase activities bound to membrane-free rat liver polysomes

Characterization of two distinct protein kinase activities bound to membrane-free rat liver polysomes

BIOCHIMIE, 1978, 60, 813-816. Characterization of two distinct protein kinase activities bound to membrane-free rat liver polysomes. Y. CF,N A T I E ...

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BIOCHIMIE, 1978, 60, 813-816.

Characterization of two distinct protein kinase activities bound to membrane-free rat liver polysomes. Y. CF,N A T I E M P 0 ~ and A. J. COZZONE *<>. A. G ~ N O T * ~ and J. P. REBOUD * *

(5-6-1978).

" Laboratoire de Biologie Mol~culaire, "" Laboratoire de Biochimie M~dicale, Uniuersit~ de L y o n , ~3, Bd du Onze Nouembre, 69621 Villeurbanne.

Mots cl~s: rat liver, phosphorylation, polysomes, associated kinases.

Introduction. P r o t e i n kinases h a v e been f o u n d associated w i t h r i b o s o m e s in v a r i o u s e u k a r y o t i c systems inc l u d i n g n a m e l y rat l i v e r [1, 2, 31. Most of these enzymes are a c t i v a t e d by c y c l i c a d e n o s i n e 3' : 5'm o n o p h o s p h a t e and catalyze the t r a n s f e r of the t e r m i n a l p h o s p h a t e of A ~ P to s e r i n e a n d t h r e o n i ne r e s i d u e s of specific r i b o s o m a l p r o t e i n s [4, 5, 6]. A large p r o p o r t i o n of the b o u n d k i n a s e a c t i v i t y has been r e p o r t e d to be e x t r a c t e d f r o m r i b o s o m e s by w a s h i n g w i t h 0.5 M-1.0 M p o t a s s i u m c h l o r i d e or by other p r o c e d u r e s that p r o m o t e d i s s o c i a t i o n of 80,S m o n o s o m e s into t h e i r subunits [7, 8, 9, 10, 11]. This p r o p e r t y has led several authors to ask w h e t h e r p r o t e i n kinases are integral r i b o s o m a l p r o t e i n s or, instead, are cytosolic enzymes adventitiously attached to r i b o s o m e s d u r i n g cell fract i o n a t i o n p r o c e d u r e s , t h e r e b y a c c o u n t i n g for the r i b o s o m e - a s s o c i a t e d kinase activity [3]. Most e x p e r i m e n t s h a v e been c a r r i e d out on the total r i b o s o m a l m a t e r i a l of the cell without, in par_ t i t u l a r , any d i s c r i m i n a t i o n b e t w e e n the e n z y m e a c t i v i t y of r i b o s o m e s w h i c h are not engaged actively in p r o t e i n synthesis and that of r i b o s o m e s inv o l v e d in f u n c t i o n a l polysomes. Such d i s c r i m i n a tion m a y be of interest if one c o n s i d e r s the regulatory role possibly p l a y e d by p h o s p h o r y l a t i o n in the i n i t i a t i o n of p o l y p e p t i d e c h a i n synthesis [12, 13, 14, 151. A study w a s t h e r e f o r e p r e v i o u s l y u n d e r taken, in the p a r t i c u l a r case of rat liver, to c h e c k the p r e s e n c e of any kinase activity specifically associated w i t h p o l y s o m e s [16]. M e m b r a n e - f r e e p o l y s o m e s w e r e selectively e x a m i n e d in o r d e r to a v o i d any possible i n t e r f e r e n c e due to c o n t a m i nant p r o t e i n kinases b o u n d to cellular m e m b r a n e s .

To whom all correspondence should be addressed.

S i n c e t h e y w e r e s h o w n to h a r b o r i n d e e d an enzyme f r a c t i o n able to catalyze p r o t e i n p h o s p h o r y 1,ation, an attempt w a s made, in t h e p r e s e n t w o r k , to investigate f u r t h e r the nature of this p a r t i c u l a r kinase activity. The e x p e r i m e n t a l m a t e r i a l hereafter r e p o r t e d i n d i c a t e s m a i n l y that at least t w o dist i n c t t y p e s of p r o t e i n kinases w i t h different substrate specificities are b o u n d to polysomes. These t w o enzymes ~vere m o r e o v e r d i f f e r e n t i a t e d on the basis of t h e i r ,activity d e p e n d e n c e on c y c l i c AMP, d i a m i d e and salt c o n c e n t r a t i o n ,

Materials and Methods. Preparation of membrane-free polysomes. The method used for preparing polysomes is a modification [17] of that previously described [18]. Briefly, Sprague-Dawley male rats weighing 200-2~2,0 g were fasted for 24. h and sacrificed by deeapitation. Livers were pooled and homogenized in 2 volumes of 0.25 M sucrose in TKM buffer (50 mM triethanolaminc-HC1 pH 7.4, 25 mM KCA, 5 mM MgCI~). The post-mitoehondrial supernatant was prepared by centrifuging the homogenate for 10 mn at 10000 g. It was then centrifuged for 24 h at 303 000 g in a 50.2 Ti Beckman rotor (Palo A,lto, California, U.S.A.) through three successive layers of sucrose (1.35 M/1.6 M/2.0 M~ sucrose). Membrane-free polysomes were thus obtained as a pellet while polysomes bound to cellular membranes remained at the 1.6 1~/2.~) M sucrose interface and were discarded. In vitro phosphorylation assay. Polysomes (1 A~o unit) were suspended in 250 ~1 of an incubation mixture similar to that already described [19]. This mixture contained 30 mM Tris-HC1 pH 7.4, 0.3 M ethylene glycol-bis-(amino-2 ethyl ether)N,N'-tetraeetic acid, 3.5 mM theophyllin, 20 mM NaF, 12 mM MgCI_~. Histone II-A from calf thymus or (t-casein from milk (Sigma Chemical Co., Saint Louis, ~o., U.S.A.) were added to the final concentration of 1 mg/ml each. The kinase activity of polysomes was

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Y. Cenatiempo and colt.

e s t i m a t e d f r o m the a m o u n t of o r t h o p h o s p h a t e incorporated into p r o t e i n a t the expense of ATP. F o r this, radioactive [~_32p~ ATP (10{)-250 c p m / p m o l , Radiochemical A m e r s h a m Centre, England) was added, at zero time, to the final c o n c e n t r a t i o n of 40~ ~M. I n c u b a tion was carried out at 30°C. Aliquots (5,0 ~l) were w i t h d r a w n periodically t h e n spotted on W h a t m a n 3 M1V~p a p e r discs as described [20] and t h e i r radioactivity was counted. The i n i t i a l slope of the kinetic curves t h u s o b t a i n e d w a s d e t e r m i n e d a n d kinase activities were expressed in e n z y m a t i c u n i t s defined as the n u m b e r of picomoles of o r t h o p h o s p h a t e incorporated per m i n u t e . I n some e x p e r i m e n t s , i n c u b a t i o n was carried o u t in t h e presence of 10 ~M cyclic AMP added to the m e d i u m . I n others, i n c u b a t i o n was also p e r f o r m e d in the presence of either d i a m i d e (5 or 20, raM) or p o t a s s i u m chloride (100, 200 or 280 raM) i n a d d i t i o n to cyclic AI~P as indicated i n Results. Diamide (azodicarboxylic acid-bis-dimethylamide), and cyclic adenosine 3':5'm o n o p h o s p h a t e were o b t a i n e d f r o m Sigma Chemical Co.

Salt treatment of polysomes. The e x t e n t of s o l u b i l i z a t i o n of k i n a s e activities by salts was e s t i m a t e d b y first s u s p e n d i n g 13,0, A~o u n i t s of purified polysomes in 2.4 m l of TKM buffer. The KC1 c o n c e n t r a t i o n was t h e n e i t h e r m a i n t a i n e d at 25 mlVf or raised to various h i g h e r values (see Results) a n d the suspension was i n c u b a t e d for 1 h at 37°C. T h e n polysomes were layered onto 2.8 ml of 30 per cent sucrose in a buffer c o n t a i n i n g 5'0~ mM triethanolamine-HC1 pH 7.4, 5 mM MgCI.o a n d a KC1 c o n c e n t r a t i o n equal to t h a t used d u r i n g i n c u b a t i o n , a n d they were centrifuged in a SW 60 B e c k m a n r o t o r at 430 00~0 g f o r 3~h at 4°C. The polysome-free s u p e r n a t a n t was collected, t h e n dialyzed overnight a g a i n s t TKM~~buffer a n d finally quickly frozen a n d stored at - - 4 0 ° C for f u r t h e r kinase activity assays. Salt-treated polysomes o b t a i n e d as a pellet were r e s u s p e n d e d a n d centrifuged once more u n d e r the same conditions as those above described. The final pellet was suspended i n TKM buffer t h e n divided into aliquots p r i o r to freezing a n d storage at - - 4 0 ° C . The r e s i d u a l k i n a s e a c t i v i t y of polysomes t h u s t r e a t e d was assayed as a l r e a d y indicated.

Results. I n a f i r s t set of e x p e r i m e n t s , t h e a ~ i v i t y of t h e previously described [16] p o l y s o m e - a s s o c i a t e d k i n a s e w a s a n a l y z e d t h r o u g h its a b i l i t y t o c a t a l y z e in vitro t h e p h o s p h o r y l a t i o n of t w o d i f f e r e n t p r o teins : casein and histone. For this, purified polys o m e s w e r e i n c u b a t e d i n t h e p r e s e n c e of e i t h e r p r o t e i n a n d t h e a m o u n t of r a d i o a c t i v e p h o s p h a t e i n c o r p o r a t e d , i n e a c h case, at t h e e x p e n s e of E~/-a~P~ AT:P w a s m e a s u r e d . As s h o w n i n t a b l e I, both casein and histone are phosphorylated to a significant extent under these conditions. S i n c e s u c h r e s u l t s m a y s u g g e s t a priori t h a t t w o s e p a r a t e t y p e s of p r o t e i n k i n a s e s w i t h d i s t i n c t

BIOCHIMIE, 1978,

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substrate specificities could be simultaneously b o u n d to p o l y s ~ m e s , f u r t h e r e x p e r i m e n t s w e r e t h e n p e r f o r m e d b y i n v e s t i g a t i n g t h e in vitro act i o n of t h r e e d i f f e r e n t e f f e c t o r s o n e n z y m e a c t i v i t i e s : .cyclic AMP, d ~ a m i d e a n d salts. All r e s u l t s a r e e x p r e s s e d i n t a b l e I.

TABLE I. In vitro activity of protein kinases

associated with polysomes.

cyclic AMP (~tM)

Kiaase activity (units)

system

Assay

KCI (raM)

diamide (mM)

histone

casein

--

--

--

0.89

0.80

10 10 10 10 10 10

--

--

4.71

0.80

5 20 ----

0.30 0.29 2.52 2.21 1 73

0.78 0.74 2.25 3 69 2.43

--100 200 280

Histone kinase a n d casein kinase activities were m e a s u r e d in the p h o s p h o r y l a t i o n assay m e d i u m deseribed in the Materials a n d Methods section. In most experiments, t h i s m e d i u m was s u p p l e m e n t e d w i t h effeetors (cyclic AMP', .KC1 a n d diamide) using various c o m b i n a t i o n s as indicated. In each case, t o t a l e n z y m a tic u n i t s per A~o u n i t of polysomes are given. All expressed values were d e t e r m i n e d a f t e r s n b s t r a c t i n g the p r o t e i n kinase activity responsible for a u t o p h o s p h o r y l a t i o n of polysomes, as m e a s u r e d in the absence of h i s t o n e a n d casein. - - , effector absent.

F i r s t l y , it a p p e a r s t h a t a d d i t i o n of 10 ~M c y c l i c A M P to t h e i n c u b a t i o n m i x t u r e i n d u c e s a f i v e f o l d i n c r e a s e i n t h e p h o s p h o r y l a t i o n d e g r e e of h i s t o n e w h e r e a s n o s t i m u l a t i o n is o b t a i n e d w i t h c a s e i n . S e c o n d l y , t h e a d d i t i o n of d i a m i d e , a t h i o l - o x i d i zing agent which inhibits various eukaryotic prot e i n k i n a s e s [21, 22, 23], r e s u l t s i n a s e v e r e i n h i b i t i o n i n t h e t r a n s f e r of p h o s p h a t e to h i s t o n e e v e n w h e n a d d e d at a c o n c e n t r a t i o n as l o w as 5 m M i n p r e s e n c e of 10 ~M c y c l i c AMP. I n c o n t r a s t , n o s i g n i f i c a n t v a r i a t i o n is f o u n d i n t h e e a s e of c a s e i n . No s e n s i b l e d i f f e r e n c e i n d i a m i d e e f f e c t s is o b s e r v e d i n e i t h e r c a s e w h e n its c o n c e n t r a t i o n is i n c r e a s e d t o 20 mM. F i n a l l y , b y a n a l y z i n g t h e i n f l u e n c e of t h r e e d i f f e r e n t c o n c e n t r a t i o n s of p o t a s s i u m c h l o r i d e ,(from 100 m M to 280 raM) it a p p e a r s t h a t , i n all i n s t a n c e s , t h e a d d i t i o n of s a l t p r o m o t e s a n e x t e n s i v e a c t i v a t i o n of c a s e i n p h o s p h o r y l a t i o n a n d a c o n c o m i t a n t i n h i b i t i o n of h i s t o n e p h o s p h o r y l a t i o n i n p r e s e n c e of c y c l i c A M P as p r e v i o u s l y described for other sinfilar eukaryotic enzymes [24]. T h e r e f o r e it s e e m s c l e a r t h a t c y c l i c AMP,

P r o t e i n k i n a s e s b o u n d to rat l i v e r p o l y s o m e s .

d i a m i d e a n d KC1 have separately quite different effects on the p h o s p h o r y l a t i o n of historic and casein as catalyzed by polysome-associated kinase activity. Since the results based on the study of enzymatic properties all c o n c u r in i n d i c a t i n g that membrane-free polysomes h a r b o r at least two distinct kinase activilies, it seemed of interest to achieve f u r t h e r differentiation and c h a r a c t e r i z a t i o n of such activities by other criteria. For this purpose, the tightness of kinase bind:rig to polysomes was studied u p o n i n c u b a t i o n ot particles in the presence of i n c r e a s i n g c o n c e n t r a t i o n s of potassium chloride v a r y i n g from 25 mM to 1.5 M. I n each case, the n a t u r e a n d the a m o u n t of soluble activity released from polysomes were determined. E n z y m e s were assayed u n d e r optimal c o n d i t i o n s : in presence of 0.2 M KC1 for casein kinase and 10 ~M cyclic AMP for histone kinase. The results s h o w n in figure 1 clearly i n d i c a t e that histone kinase activity is already m a x i m a l l y removed from polysomes after i n c u b a t i o n i n a salt concent r a t i o n as low as 25 mM. I n contrast, m u c h h i g h e r salt c o n c e n t r a t i o n s are r e q u i r e d to solubilize the casein k i n a s e activity since m a x i m a l release can be a t t a i n e d only b y t r e a t i n g polysomes w i t h KC1 no l o w e r t h a n 0.5 M. It thus exists a clear-cut difference i n the b i n d i n g strength of the two enzymes to polysomes, the h i s t o n e kinase b e i n g m u c h more loosely attached t h a n the casein kinase. I n order to evaluate the extent of solubilization of each enzyme by salts, bolh casein kinase and ~oo

"d ~

15o

~

1oo 0

0 0

~ o

5o

O~

12

oa

O.4 KCI

concentration

14 ( M )

FIG. 1. - - Effects of salt treatment on the release of protein kinases from pol!tsomes. Polysomes (13'0, A~o units) were treated by various inereasing KC1 concentrations as described in the Materials and Methods section. Total histone kinase (O) and casein kinase (0) activities thus solubilized were measured, in each case, after overnight dialysis against TK~ buffer. Total casein kinase activity remaining in salt-treated polysomes (11) was also determined after substraeting the protein kinase activity responsible for autophosphorylation of polysomes.

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histone kinase activities were then measured in polysomes after t r e a t m e n t by KC1 c o n c e n t r a t i o n s i n c r e a s i n g from 25 mM to 1.5 M. On the one h a n d , the casein kinase activity r e m a i n i n g in polysomes was s h o w n to decrease progressively as a f u n c t i o n of salt c o n c e n t r a t i o n and reach about 10 per cent of its i n i t i a l value after i n c u b a t i o n in 0.25 M KC1 (fig. 1). It was equal to zero only after t r e a t m e n t by a least 0.5 M KC1 w h i c h is therefore the m i n i real salt c o n c e n t r a t i o n r e q u i r e d for complete solubilization of this enzyme activity. On the other h a n d , no residual histone kinase activity could be detected i n polysomes even after t r e a t m e n t by a KC1 c o n c e n t r a t i o n as low as 25 raM. These data are thus in good agreement w i t h the above mentioned results c o n c e r n i n g the different b i n d i n g strength of the two enzymes to polysomes. Moreover, they b r i n g the a d d i t i o n a l i n f o r m a t i o n that, u n d e r our assay conditions, the plateau values reported in figure 1 for released kinase activities correspond, in each case, to the complete solubilization of the total enzyme activity i n i t i a l l y present i n salt-untreated polysomes.

Discussion.

Rat liver polysomes h a r b o r two d i s t i n c t h i s t o n e kinase a n d casein kinase activities w h i c h can be classified respectively in group I and group III E25] by c o n s i d e r i n g t h e i r differential b e h a v i o u r in the p r e s e n c e of cyclic AMP, d i a m i d e a n d potassium chloride. It c a n n o t be concluded, however, w h e t h e r these two enzymes are actually the only kinases b o u n d to polysomes. One can envisage i n d e e d that other types of kinases m a y be attached to the particles as well. To check this possibility, the polysome-associated activity should be f u r t h e r analyzed w i t h respect to its specificity for other substrates, e.g. prot a m i n e or p h o s v i t i n , its d e p e n d e n c y on various enzyme activators or i n h i b i t o r s a n d its capacity to utilize p r e c u r s o r s other t h a n ATP as phosphoryl donors. The existence of distinct kinase activities b o u n d to ribosomes, as described here in the p a r t i c u l a r case of rat h e p a t i c polysomes, has been already r e p o r t e d i n other eukaryoti.c systems i n o l u d i n g namely horse t h y r o i d glands E26J, yeast E27], mouse p l a s m a cell t u m o r s [28], b o v i n e corpus luteum [29] a n d r a b b i t reticulocytes [30]. However their i n d i v i d u a l physiological role is not clearly u n d e r stood yet. It has been s h o w n that some p r o t e i n kinases p r e f e r e n t i a l l y p h o s p h o r y l a t e acidic pro-

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Y. Cenatiempo

teins like casein and phosvitin while other kinases are, instead, rather specific for basic proteins like h i s t o n e a n d p r o t a m i n e [30]. If o n e a s s u m e s t h a t the two different kinase activities found in rat p o l y s o m e s a r e r e s p o n s i b l e , at l e a s t i n p a r t , f o r t h e e n d o g e n o u s p h o s p h o r y l ~ t i , o n of r i b o s o m a l p r o t e i n s , it b e c o m e s t h e n c o n c e i v a b l e t h a t t h e y m a y a c t o n d i f f e r e n t g r o u p s of p r o t e i n s : c a s e i n k i n a s e , for instance, would catalyze mainly the phosphor y l a t i o n of a c i d i c r i b o s o m a l p r o t e i n s ~ v h e r e a s h i s tone kinase would selectively phosphorylate basic ribosomal proteins. Preliminary experiments perf o r m e d i n o u r l a b o r a t o r y a,c t u a l l y f a v o r t h i s h y p o thesis.

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