Characterization of a protein kinase-phosphoprotein system in free cytoplasmic ribonucleoprotein particles of plasma cell tumours

549

Biochimica et Biophysica Acta, 454 ( 1 9 7 6 ) 5 4 9 - - 5 5 7 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 9 8 7 7 4

CHARACTERIZATION OF A PROTEIN KINASE-PHOSPHOPROTEIN SYSTEM IN FREE CYTOPLASMIC RIBONUCLEOPROTEIN PARTICLES OF PLASMA CELL TUMOURS

J.M. EGLY, M. S C H M I T T and J. K E M P F Unit~ de Recherche sur la Biochimie de la Cellule Canc$reuse de I'INSERM, and Centre de Neurochimie du CNRS, 11 Rue Humann, 67085 Strasbourg Cedex (France)

(Received May 5th, 1976)

Summary A protein kinase, associated with free cytoplasmic ribonucleoprotein particles (free dRNP) has been purified from mouse plasma cell turnouts. This protein kinase is able to phosphorylate in vitro endogenous protein from free dRNP. Some characteristics of this protein kinase have been studied. This protein kinase behaves as being cyclic AMP independent. The properties of this protein kinase were compared with other protein kinases: soluble, ribosome-bound, and nuclear protein kinases. Although there are minor differences it is very similar to a ribosome-associated protein kinase from the plasma cell tumours.

Introduction Recent evidence [1--5] indicates that several proteins of eukaryotic ribosomal subunits are phosphorylated in vitro and in vivo with some protein kinase which can be cyclic AMP dependent or independent. Some protein kinases are tightly bound to intact ribosome and cannot be extracted with buffers of moderate ionic strength (<0.5 M KCI) [6]. On the other hand, previous studies have demonstrated that a phosphoprotein fraction is also associated with dRNA in free cytoplasmic ribonucleoprotein particles (dRNP) [7,8] and a phosphorylated nucleoprotein complex, containing dRNA, has also been shown in the 0.5 M KC1 wash of purified plasmocytoma ribosomes uncontaminated with free dRNP [9]. More recently, the presence of phosphoprotein bound HnRNA in nuclear particles has been detected [10]. The occurrp-~e of phosphorylated proteins in these different phases of celluAbbreviations: dRNA, DNA-like RNA; dRNP, dRNA protein particles; HnRNP, heterogeneous nuclear RNA-protein particles; I-InRNA, heterogeneous nuclear RNA.

550 lar information transfer could play a role in the regulation of gene expression in mammalian cells. Phosphorylation has for a long time been suspected to play a regulative role in the case of chromatin-associated proteins [ 11 ]. Different considerations have prompted a search for the existence of the enzyme responsible of this phosphorylation in the different ribonucleoprotein particles. In the present study, we describe and characterize the isolation of a protein kinase which is tightly bound to free cytoplasmic dRNP from mouse plasma cell tumours. Material and Methods RPCs plasma cell tumours transplanted on Balb/c mice were used. RNA was labelled by intraperitoneal injection of 1 mCi of tritiated uridine 2 h before killing. The tumours were homogenized using a glass homogenizer with a teflon pestle in 3 vol. of ice-cold 20 mM triethanolamine-HC1 buffer pH 7.6 containing 150 mM KC1, 4 mM magnesium acetate, 6 mM 2-mercaptoethanol and 1.1 M sucrose. Preparation o f the 0.5 M KCl dRNP wash. Ribonucleoprotein particles from a post mitochondrial supernatant were fractionated on 3H20/sucrose gradient [12]. After centrifugation for 20 h at 200 000 × g, the heavy 3H:O/sucrose layer was removed and used as the free dRNP fraction. This fraction was then dialysed 12 h against a Tris • HC1 buffer pH 7.6 (30 mM Tris/5 mM magnesium acetate/150 mM KC1/250 mM sucrose/6 mM 2-mercaptoethanol). The free dRNP particles (200 A260n~ units) were brought to 0.5 M KC1 and stirred in the cold for 30 min; the suspension was then centrifuged at 250,000 X g for 2 h and the upper two thirds of the supernatants were removed and dialysed 12 h against a Tris • HC1 buffer pH 7.6 (10 mM Tris/50 mM KC1/0.2 mM EDTA/ 6 mM 2-mercaptoethanol). Enzyme activity determinations. Protein kinase activity was determined by the incorporation of 32p from [7-3:p]ATP (NEN). The incubation mixture contained, in a total volume of 0.1 ml, 3.5 pmoles Tris • HC1 pH 7.6; 0,5 pmol MgCl:; 10 pmol KC1; 0.1 #mol dithiothreitol; 15 nmol [7-32p]ATP (specific activity 50 000 cpm/nmol); 50 pl of the column fractions and 10--30 pg of casein as substrate. Samples were incubated for 30 min at 37°C and adsorbed on 1 × 2 cm Whatmann filter paper which was suspended in 10% trichloracetic acid at 4 ° C. The filter was heated in 5% trichloracetic acid at 95°C for 10 min. washed in ethanol, ethanol/diethyl ether (v/v) and diethyl ether, and then counted in omnifluor toluene scintillation fluid in a spectrometer. Intertechnique ABAC SL40. The results are expressed in nmoles of 32p incorporated per mg of protein, calculated on the basis of the specific activity of the added [~/-32p]ATP. In all assays, endogenous phosphorylation of the enzyme was measured by omitting substrate protein from the reaction mixture. This value was substracted from the values measured in the presence of substrate. Protein determinations. Protein was estimated by the m e t h o d of Z a m e n h o f et al. [ 13] using bovine serum albumin as standard. Polyacrylamide gel electrophoresis. Electrophoresis on polyacrylamide gels was performed according to Davis [14]. The separating gel was 8% in poly-

551

acrylamide. The pH of the gel was 8.3. The pH of 0.05 M Tris/0.1 M glycine buffer solution was 8.3 For the estimation of molecular weight, proteins were analysed by SDSpolyacrylamide gel electrophoresis under the same conditions as described previously [15]. Reference proteins were catalase, bovine serum albumin and phosphorylase a obtained from Sigma Chemical Company. Results

Phosphorylation in vitro of "free dRNP" proteins In the absence of exogenous phosphorylable protein substrate, the free d R N P fraction was able to catalyse the in vitro incorporation of radioactive phosphate from [ 7 ) 2 P ] A T P into an insoluble form (Table I). The incorporation of 7 -32PO~- into proteins was verified by its insolubility in h o t 5% trichloracetic acid, its sensitivity to pronase and its resistance to ribonuclease. It was shown that intact d R N P contained an endogenous substrate for the protein kinase and that most of the endogenous phosphorylated substrates remained in the washed d R N P after 0.5 M KC1 treatment.

Isolation of the protein kinase from plasmocytoma free dRNP Characterisation of protein kinase activity. After treatment of the free dRNP with 0 . 5 M KC1, the protein kinase activity of the different fractions was tested. Most of the protein kinase activity (87%) (Table I) was localized in the 0.5 M KC1 dRNP wash. Little activity was found in washed dRNP particles. The sum of the protein kinase activity of the 0.5 M KC1 " d R N P wash" and of the washed d R N P was lower than the protein kinase activity of the untreated dRNP. This may be due to a greater stability of the enzyme in the untreated d R N P where it is held in a more active conformation. Purification of the "free dRNP" protein kinase. To isolate the protein kinase and to characterize it further, the 0.5 M KC1 " d R N P wash" was applied to a DEAE-cellulose column as is shown in Fig. 1. The protein kinase activity was eluted at a b o u t 0.15 M KC1. The fraction containing the protein kinase activity

TABLE I DETECTION OF PROTEIN KINASE ACTIVITY AND ENDOGENOUS PHOSPHOPROTEINS T h e free d R N P particles w e r e w a s h e d w i t h 0 . 5 M KCL c e n t r i f u g e d a n d resuspended in the i n c u b a t i o n m e d i u m ; t h e 0.5 M KC1 d R N P wash a n d w a s h e d d R N P w e r e tested for protein k i n a s e a c t i v i t y using casein as e x o g e n o u s s u b s t r a t e . T h e e n d o g e n o u s p h o s p h o p r o t e i n s w e r e d e t e c t e d u n d e r the same c o n d i t i o n s but, w i t h o u t e x o g e n o u s s u b s t r a t e . T h e values are the a v e r a g e o f t r i p l i c a t e assays.

Endogenous P h o s p h o p r o t e i n s

P r o t e i n kinase activity

(nmol/30 rnin/mg protein)

F r e e d R N P particles 0.5 M KC1 d R N P w a s h Washed d R N P

32 p i n c o r p o r a t e d nmol/30 rain/rag e n z y m a t i c protein

% Enzyme activity

4,10 3.60 0,21

100 87 5

15.20 6.72 9,13

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Fig. 1. D E A E - c e l l u l o s e c h r o m a t o g r a p h y o f 0 . 5 M KCI d R N P w a s h . A b o u t 1 0 m g o f 0 . 5 M K C l w a s h p r o t e i n w a s a p p l i e d on a 1 X 1 5 c m c o l u m n o f D E A E - c e l l u l o s e e q u i l i b r a t e d w i t h a Tris • HC1 b u f f e r p H 7.6 ( 1 0 m M T r i s / 5 0 m M KC1/0.2 m M E D T A / 6 m M 2 * m e r c a p t o e t h a n o l ) a n d c h r o m a t o g r a p h e d u s i n g a KCl g r a d i e n t (m-I ) . T h e e l u t e d p r o t e i n s w e r e m e a s u r e d b y a b s o r b a n c e at 2 8 0 n m (A A). F r a c t i o n s o f 1 m l w e r e c o l l e c t e d a n d t h e p r o t e i n k i n a s e a c t i v i t y w a s d e t e r m i n e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s w i t h c a s e i n as s u b s t r a t e ( e ~'), T h e t u b e s c o n t a i n i n g e n z y m e a c t i v i t y w e r e p o o l e d , d i a l y s e d a g a i n s t a Tris • HCI b u f f e r p H 7.9 c o n t a i n i n g 10 m M Tris, 50 m M KCI, 0 . 2 m M E D T A a n d 6 m M 2 - m e r c a p t o e t h a n o l . T h e m i x t u r e w a s c o n c e n t r a t e d in a D i a f l o cell t h r o u g h a XM 1 0 0 A m e m b r a n e u n t i l t h e v o l u m e r e a c h e d 5 ml. o o, 3 H l a b e l l e d R N A i n s o l u b l e in c o l d t r i c h l o r o a c e t i c acid.

was free of nucleic acids which were eluted from the column only at a concentration of 0.5 M KCI. For further purification, the fraction containing the peak of protein kinase activity was collected, dialysed and applied to a phosphocellulose column (Fig. 2); the protein kinase activity was eluted at about 0.55 M KC1. After elution the tubes contain':ng enzyme activity were pooled and dialysed against a Tris • HC1 buffer pH 7.6 containing a KC1 concentration of 50 raM. The mixture was then concentrated in a Diaflo cell through a XM 100 A membrane until the protein concentration reached 0.5 mg/ml. At this stage, the purified enzyme may be stored frozen at --70°C for at least several weeks without loss of activity. The specific activity of the purified enzyme did n o t increase, compared with the activity of the enzyme in the native free dRNP fraction. This could be due to a deactivation of the enzyme during the different steps of the purification. Polyacrylamide gel electrophoresis: molecular weight. The purified enzyme moved as a major band on polyacrylamide gel electrophoresis, with two very minor bands. The absorbance at the top of the gel could be due to some aggregation (Fig. 3a). When enzyme activity was directly tested in the gel, the recovered activity corresponded to a single band (Fig. 3b). When electrophoresis was carried out at +4°C in the presence of 0.1% sodium dodecyl sulphate the

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enzyme migrated only as a single band, but no activity could be detected. From calibrated gels with proteins of known molecular weight, the molecular weight of the protein kinase was estimated to be 66 000 (+2%) (Fig. 4). In a gel filtration experiment, the protein kinase elutes from a Sephadex A + B+ ~" o 'o ~=

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Fig. 4. D e t e r m i n a t i o n o f the m o l e c u l a r w e i g h t o f free d R N P p r o t e i n kinase b y d o d e c y l s u l p h a t e - p o l y a c r y l a m i d e gels. T h e m o l e c u l a r w e i g h t o f the r e f e r e n c e p r o t e i n s w e r e (1) p h o s p h o r y l a s e a m o n o m e r 94 0 0 0 ; (2) s e r u m a l b u m i n , 68 0 0 0 ; (3) catalase m o n o m e r 58 0 0 0 ; (4) t r y p s i n e 23 000. F = f r o n t .

G200 column as a single highly symmetrical peak. Its molecular weight was estimated to be a b o u t 64 000 from the calibration curve obtained with different size markers (results n o t shown). Comparing this value with that obtained above in SDS-polyacrylamide gels (tool. wt. 66 000) we conclude that this protein kinase is probably a m o n o m e r .

Enzymatic characteristics of the protein kinase Optimal conditions for protein kinase activity were determined. All reactions were carried o u t at 37°C for 30 rain. Under these conditions, p h o s p h o r y l a t i o n was linear with the a m o u n t o f protein used. An optimal pH near 7.5 in the presence o f 5 to 10 mM Mg 2+ was observed. The activity is markedly Mg 2÷ dependent. When p h o s p h o r y l a t i o n of casein was catalysed by free dRNP protein kinase, an apparent Michaelis constant Km can be calculated for this substrate, we f o u n d it to be 3.98 • 10 -9 M. The m a x i m u m velocity of this kinase was 32.7 nmol o f z 2p i n c o r p o r a t e d / 3 0 min by 1 mg of enzyme. Thus it appears that there may be a high level of specificity of " f r e e d R N P " bound protein kinase for casein. Effect of cyclic AMP. When we tested the effect of various concentrations of cAMP on the p h o s p h o r y l a t i o n of the different substrates by the free dRNP associated protein kinase, no significant stimulation was detected. Compared

555 TABLE II SUBSTRATE SPECIFICITY OF FREE dRNP PROTEIN KINASE COMPARED TO SOLUBLE PROTEIN KINASE T h e a s s a y s were performed under standard assay conditions u s i n g 15 /~g p r o t e i n k i n a s e either soluble ( f r o m S i g m a C h e m . C o m p . ) [ 1 6 ] o r f r e e d R N P a n d 30 ~g of acceptor protein. All v a l u e s w e r e corrected b y substracting endogenous 7 -32 p incorporation.

Substrate

Cyclic A M P i0 ~mol

nmol 32p/30 rain/rag protein kinase Free d R N P

Casein Histone H 1 H i s t o n e s H 2 a , H 2 b , H 3, H4 Serum albumin Protein fraction of free d R N P ( B I ) [15]

PK l

Soluble PK 2

+ -+ --

4.00 4.10 0.05 0.03

1.52 0.20 4.55 3.05

+ --

0 0

+ ---

0.03 0 2.20

17 11

0 0 0.94

with a soluble cyclic AMP dependent protein kinase [ 16] the protein-kinase of the free d R N P seems to be cyclic AMP independent (Table II). It was verified that the absence of stimulation b y cyclic AMP was independent of the incubation medium as well as the addition of different inhibitors of the phosphodiesterase such as theophylline or papaverine. Substrate specificity. The substrate specificity of the free dRNP protein kinase PKt was compared with that of a soluble cytoplasmic cyclic AMP-dependent protein kinase PK2 [16]. Each protein kinase was tested with different proteins as substrate such as casein, histone and serum albumin. The results are shown in Table II. It can be seen that under standard assay conditions, the casein was the preferred substrate for the enzyme PK1 which did n o t phosphorylate histone (0.05 nmol 32P/30 min/mg). PK2 had a very high substrate specificity with chicken erythrocyte histones (arginine rich histone H4 was a better phosphate acceptor than histone Hi ) and casein was phosphorylated to a limited extent under these reaction conditions. It was also shown that the free dRNP protein kinase has a much higher specificity for a protein fraction of free dRNP (analogous to the fraction which is called BI and described elsewhere [15]) than the soluble protein kinase PK2. If we compared the properties of this free d R N P protein kinase with the classification established by Traugh et al. [17], this enzyme is of type III, without any stimulation b y cyclic AMP and any specificity for the different histones. Discussion

The isolation procedure yields a preparation of a highly purified protein kinase of molecular weight 6 6 000. This protein kinase is able to phosphorylate in vitro some proteins from the free dRNP particles. The majority of the phosphorylable proteins remained in the washed dRNP after 0.5 M KC1 treatment

556 of the free d R N P particles. This suggests that the phosphorylated proteins are more tightly b o u n d to the d R N A than some non-phosphorylated species. A similar p h e n o m e n o n has been found in the H n R N P from the HeLa cell nucleus [10]. The former results have shown that the purified protein kinase is not stimulated by cyclic AMP. Moreover, cyclic AMP-dependent protein kinases can be cyclic AMP-independent under certain conditions [ 18]. Thus it is important to distinguish true cyclic AMP-independent kinases from either modified forms of the protein kinase holo-enzyme (R-C) or its catalytic subunit (C). In our case, the dissociation of the holo-enzyme by KC1 treatment during the purification of the protein kinase could be suggested as shown by Lemay et al. [18]. It was also shown that when free d R N P were not treated with 0.5 M KC1, they did not bind cyclic [3H]AMP which had no effect on the increase of the protein kinase activity. Therefore it is probable that our free d R N P protein kinase is a cyclic AMP-independent protein kinase. It should also be emphasized that cyclic AMP-dependent protein phosphorylation appears to include only a fraction of the total amount of protein phosphorylation which occurs in tissues [19]. In an earlier study [9] protein kinase activity was identified in the plasmoc y t o m a ribosome KC1 wash. The similarity of this plasmocytoma-ribosome associated protein kinase and the free d R N P protein kinase is emphasized both are cyclic AMP-independent and phosphorylate essentially casein as exogenous substrate under the same experimental conditions (pH optimum = 7.6; Mg ~÷= 6 raM). They have similar molecular weights: free d R N P protein kinase 66 000, ribosome-bound protein kinase 60 000. They phosphorylate also under the same conditions a protein of the free d R N P [15] (Protein I 60, mol. wt. 60 000 which has no kinase activity b y itself) (unpublished results). The identification of other proteins phosphorylated by the free DRNP protein kinase are under investigation. Different possible roles for the protein kinase may exist. The investigations of Spohr et al. [20] demonstrate that newly synthesized m R N A appears in the cytoplasm first free from ribosome b u t associated with proteins in the form of ribonucleoprotein complexes. The free dRNPs could be a precursor which is able to bind to ribosome to give functional polysomes. In our case, it was shown that the free d R N P protein kinase is present with some of its substrates in the free d R N P particles. It could also be possible that the phosphorylated substrates of this enzyme could regulate RNA-protein or protein-protein interactions and so would be able to play a regulating role during the transport and/or the attachment of the m R N A to the ribosome in protein synthesis. In fact, from various studies on the protein kinase substrate system in differentiated cell types, the picture which is emerging is one of a highly compartmentalized system in which enzymes for phosphorylation and dephosphorylation are found in close association with the phosphorylation substrates and are involved in some specific regulatory roles [21]. It is of interest that a variety of protein kinase activities have been found in the cell nuclei. Most of these kinases phosphorylate nuclear proteins by a cyclic nucleotide independent mechanism [22,23]. The protein kinase associated with the HnRNP, and extracted by 0.5 M KC1 phosphorylated preferentially casein as exogenous substrate. Their relationship to cytoplasmic kinases is

557 unknown, b u t the analogies mentioned above, between the different RNP associated protein kinases suggest that the protein kinase-phosphoprotein system could play a role in the translocation of the d R N A from the nucleus to the cytoplasm.

Acknowledgements This investigation was supported by a grant (No. 74 703 61) from the D~l~gation G6n~rale ~ la Recherche Scientifique et Technique. We thank Prof. P. Mandel and Dr. C. Quirin-Stricker for continuous interest in this work. Thanks are also due to Dr. M. Gross-Bellard for her gift of various histone preparations and valuable discussions, and to Prof. L. Austin for his help in the preparation of this manuscript. We are indebted to Mrs. N. Pfleger and Miss C. Flesh for their advice and skilful technical assistance. J.M.E. is an Attach~ de Recherche I'INSERM.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

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