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Protein kinase(s) in bovine brain coated vesicles
238
Biochimica et Biophvsica Acta,
799 (1984) 238 245 Elsevier
BBA 21772
P R O T E I N KINASE(S) IN BOVINE BRAIN COATED VESICLES ALAIN PAULOIN a JACQUES LOEB b and PIERRE JOLLieSa,. Laboratoire des Protbines (E. R. 102 CNRS, U- 116 I N S E R M ) Universitb de Paris II, 45 rue des Saints - Pbres, F - 752 70 Paris Cbdex 06 and h Laboratoire de Biochimie des Rbgulations, I.R.S.C., B.P. No. 9, F-94800 Villejuif (France)
(Received February 6th, 1984)
Key words: Protein kinase; Phosphorylation; (Bovine brain coated vesicle)
Purified bovine brain coated vesicles contain protein kinase activity which phosphorylates 165, 54 and 50 kDa protein substrates. These phosphorylations do not seem to be induced by a unique protein kinase: indeed, the three substrates present different localizations in coated vesicles, the phosphorylation sites are either serine or threonine residues and vanadate and ATP[¥S] have different effects on 32p incorporation in the substrates. Comparison of the coated vesicle protein and phosphorylation patterns from different tissues and animal origins shows that only the 50 kDa protein phosphorylation is always observed, compared to the great diversity in other minor phosphorylations which are observed or not in the various coated vesicles. The possible presence of a 50 kDa phosphoprotein phosphatase is also discussed. It is suggested that the 50 kDa protein with its connected specific kinase and phosphatase seems to constitute a regulatory system present in coated vesicles.
Introduction The coated pit region of plasma membrane is the site of localization of receptor-bound ligands prior to internalization [1]. It has been assumed that the transfer of ligands is carried out by the intermediary of coated vesicles pinched off from coated pits [1-4]. The latter as well as coated vesicles have also been implicated in membrane recycling [5], intracellular protein translocation [6,7] and exocytosis of newly synthesized proteins [8]. Biochemical studies involving the bristle coat have been greatly facilitated by the purification of coated vesicles, which have a membrane core enclosed by a polyhedral protein lattice [9,10]. The * To whom correspondenceshould be addressed. Abbreviations: Mes, 2-(N-morpholino)ethanesulfonic acid; EGTA, ethyleneglycolbis(fl-aminoethylether)-N,N'-tetraacetic acid; PMSF, phenylmethylsulfonylfluoride; SDS, sodium dodecyl sulfate; ATP['tS], adenosine-5'-O-(3-thiotriphosphate). 0304-4165/84/$03.00 © 1984 ElsevierSciencePublishers B.V.
latter is composed predominantly of clathrin, a 180 kDa protein associated with 36 and 33 kDa polypeptides [11]. Under native conditions, the basic assembly unit is a molecular complex of 630 kDa composed of three clathrin molecules with three polypeptides of 36 and 33 kDa [12-14]. Additional minor proteins of 110-100 kDa and 55-50 kDa have also been reported [15]. We have previously described the presence of a cyclic nucleotides and Ca2+-independent protein kinase activity in bovine brain coated vesicles [16], which has been corroborated by others [17,18]. This protein kinase catalyses specifically the transfer of the y-phosphate of ATP to the 50 kDa protein of coated vesicles. The phosphorylation of other proteins has also been described [17-19]. Protein phosphorylation is an important mechanism by which cellular metabolism or functions are regulated [20]. On a molecular level, phosphorylation of proteins is known to alter their conformations
239 and thereby modulates their activities [21]. The different expressions of coated vesicle protein kinase(s) imply a fine regulation of coated vesicles functions. The phosphorylation conditions of the different protein substrates in bovine brain coated vesicles are compared in the present paper. Our results show: (i) differences in phosphorylation conditions of each substrate; (ii) the constant presence of 50 kDa protein phosphorylation in coated vesicles from different tissues and species; (iii) the association of the 50 kDa protein kinase with the clathrin-light chain complex; (iv) a phosphatase activity specific for the 50 kDa phosphoprotein. The possible existence in coated vesicles of multiple protein kinases and the origin of each protein substrate are discussed. Materials and Methods
Materials Both pig and bovine brains were obtained from the Centre National de Recherches Zootechniques (Jouy-en-Josas, France). Wistar rats were obtained from IRSC( Villejuif, France). [7-32p]ATP with a specific activity of 60 C i / m m o l and Aquasol-2 were from New England Nuclear. ATP, egg phosvitin, urea, Mes, EGTA, MgC12, 2-mercaptoethanol and PMSF were obtained from Sigma. Sucrose and Tris were obtained from Merck. All components used for sodium dodecyl sulfate polyacrylamide gels were from Bio-Rad. Adenosine-5'O-(3-thiotriphosphate) was purchased from Boehringer (Mannheim), sodium orthovanadate from Ventron and 2H20 from CEA (Saclay, France). Coated vesicles from adrenal gland were a gift from Dr. A. Alfsen.
Purification of coated vesicles All procedures were carried out at 4°C. Brains were cleansed of meninges and the white matter removed by dissection. The grey matter was homogenized in 1 vol. of Mes buffer: 0.1 M Mes, pH 6.5, 0.1 mM EGTA, 2 mM MgCI 2, 1 mM 2mercaptoethanol, 0.2% NaN 3, 50/xg. ml-1 PMSF. The homogenate was centrifuged at 20 000 × g for 30 min in a Sorvall GSA rotor. The resulting pellet was subjected twice to the same procedure. All the supernatants were pooled and centrifuged at 100000 x g for 1 h in a Beckman 60 Ti rotor. The
soluble fraction was removed and the pelleted material was resuspended in Mes buffer. Coated vesicles were purified by two successive sucrose gradients [16,22]. The highly enriched upper 5% sucrose solution of the second gradient was used for the final purification using a 1 H 2 0 / 2 H 2 0 8% sucrose step gradient as described [23]. Rat liver coated vesicles were prepared from ten rats killed by decapitation. Livers were removed, washed with distilled water and homogenized in the Mes buffer. The coated vesicles were purified according to the method of Nandi et al. [23].
Phosphorylation assays The standard assay involved incubation of 100 #g of coated vesicles at 22 ° C with 1 #Ci (70 nmol. m1-1) of [y-32p]ATP in 12 mM Tris-HCl (pH 7.5), 32 mM KCI, 2 mM MgC12, 2.3 mM 2-mercaptoethanol in a total volume of 38 #1. Various quantities of unlabelled ATP were added (0.01-10 mM) for kinetic studies. For temperature dependence determinations the coated vesicles were preincubated for 2 min in a water bath (Thermomix 1410 Braun) at the selected temperatures prior to labelling. In each case the reaction, started by the rapid addition of labelled ATP, was carried out for 10 s at 22 ° C and was stopped immediately by addition of 60/xl SDS gel sample buffer (4% SDS, 3 % 2-mercaptoethanol, 10% glycerol and 0.1% Bromophenol blue in 80 mM Tris-HC1, pH 6.8) and boiling for 2 min at 100 o C. The polypeptides composition was analyzed by SDS-polyacrylamide gel (5-10%) electrophoresis according to Laemmli [24]. The following proteins were run in parallel as molecular weight markers: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa) and carbonic anhydrase (30 kDa). Clathrin (180 kDa) was used as internal molecular weight marker. The electrophoresis was carried out at 30 mA for 2 h at constant current until the Bromophenol blue as tracking dye was eluted. The protein bands were visualized by staining with 0.25% Coomassie blue and destaining in a solution of 5% acetic acid/25% ethanol. Autoradiography was performed by exposing the stained gel to a X-Omat AR Kodak film. 32p incorporation into 50, 54 and 165 kDa bands was quantitated by excising these bands from the dried gel and counting in 2.5 ml Aquasol-2
240
Phosphoamino acid analysis Coated-vesicle sample (300 ~g) was phosphorylated for 20 min at 22°C under standard conditions as described above, and analyzed by preparative gel electrophoresis. At the end of the migration, the gel was freezed and autoradiography was performed. The polyacrylamide radioactive gels corresponding to 50, 54 and 165 kDa bands on the processed film were excised, homogenized in 10 ml SDS-electrophoresis buffer [23] and eluted under stirring for 48 h at 20 o C. The homogenates were centrifuged at 20000 x g in a Sorvall SS-34 rotor. The supernatants were removed, filtered on 0.22 # m Millex-GS (Millipore) and extensively dialyzed against water. A 200 /~1 fraction of each sample was kept for SDS-polyacrylamide gel electrophoresis and control autoradiography; the remaining part of the samples was lyophilized. The dried extracts were dispersed in 200 ffl 6 M HC1. The tubes were sealed under vacuum and heated at l l 0 ° C for 1.5 h. HC1 was removed and the samples were dissolved in 25 t~l electrophoresis buffer, p H 3.5 (glacial acetic a c i d / p y r i d i n e / water, 10 : 1 : 89, v/v). 5-/~1 aliquots were mixed with 2 #1 of a solution containing 5 m g / m l of phosphoserine, phosphothreonine and phosphotyrosine, spotted on Whatman 3 MM chromatography paper and electrophoresed for 50 min at 60 V / c m . Phosphoamino acids were detected with ninhydrin and radioactive spots by autoradiography.
filtered on Sephadex G-50 (25 x 0.8 cm) equilibrated with the Mes buffer. Protein fractions contained in the void volume were pooled (1.66 ffCi 32p-labelled coated vesicles/ml) and incubated at 37 o C. Aliquots (200/tl) were removed between 0 and 5 h. The reaction was stopped by freezing; after lyophilization and addition of 60/~1 SDS-gel sample buffer, the aliquots were analyzed by SDS-polyacrylamide gel electrophoresis and the 32p incorporation into the 50, 54, 165 kDa bands was quantitated. Results
Phosphorylation of coated vesicles The protein gel patterns of the bovine brain coated vesicles after Coomassie blue staining and autoradiography are shown in Fig. 1. Several proteins associated with clathrin and the light chains were regularly observed: a thin band of high molecular weight (about 300 kDa), a group of seven bands from 105 to 136 kDa with one major band of 105 kDa, the protein doublet of 57 and 54
Preparation of clathrin-stripped vesicles Clathrin-stripped vesicles which constituted inner cores were prepared by incubating coated vesicles for 2 h at 2 0 ° C with 3 M urea in Mes buffer. They were pelleted for 1 h at 100000 × g in a Beckman 60 Ti rotor and resuspended in the Mes buffer. The supernatant contained most of the clathrin-light chains complex [17,25]. Both the supernatant (S) and resuspended pellet (P) were dialyzed overnight at 4 ° C against the Mes buffer prior to phosphorylation assays. In order to determine the kinase activity, 10 #g egg phosvitin were added in the two fractions prior to labelling.
Phosphatase activity assay Coated vesicles (400 #g) were phosphorylated for 10 min at 22°C under standard conditions. In order to eliminate ATP, the reaction mixture was
Fig. 1. Comparison of the protein gel patterns after Coomassie blue staining (A) and autoradiography (B) of bovine brain coated vesicles.
241
kDa associated to the 50 kDa protein and some other very discrete bands (part A). On the autoradiography, three proteins were phosphorylated: they have molecular weights of 165, 54 and 50 kDa. A slight phosphorylation corresponding to a very high molecular weight substance (greater than 900 kDa) was also detected (part B).
tion into the 50 kDa protein and the phosvitin area was quantitated. The ratios corresponding to cpm in the supematant versus cpm in the pellet were the same for the two substrate systems, 3.4 and 3.3 for the 50 kDa protein and the phosvitin area, respectively. It is noteworthy that the 54 kDa phosphorylation was very small and was only de-
Phosphorylations in both outer coat and inner vesicle structures of coated vesicles The protein gel patterns after Coomassie blue staining and autoradiography of the outer coats and the inner vesicles which correspond to the supernatant (S) and the pellet (P), respectively (see preparation of clathrin-stripped vesicles), are shown in Fig. 2. Nearly all the 300 kDa band and the clathrin light chains complex were solubilized in the supernatant and all the 57-54 kDa doublet remained associated with the pellet. The urea treatment removed approx. 30% of both the 105-136 kDa protein group and the 50 kDa band which appeared in the supematant; the majority of these constituents remained associated with the pellet (part A). On the autoradiography, the 165 kDa radioactive band was exclusively associated with the inner vesicle, when the 50 kDa radioactive band was detected in the outer coat fraction (part B). Phosvitin as exogenous substrate [17] was added to the inner vesicle and the outer coat fractions. As shown in part C, the phosvitin kinase activity was detected in both fractions, but it was more pronounced in the outer coat. 32P incorpora-
s
+
-
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Fig. 2. Comparison of the protein get patterns after Coomassie blue staining (A) and autoradiography (B) of the supematant (S) and the pellet (P) of urea-treated coated vesicles. Phosphorylation of phosvitin as exogenous substrate (C).
Fig. 3. Identification of phosphoaminoacids in the 50 kDa protein (lane A), mixture of 54 and 50 kDa proteins (lane B) and 165 kDa protein (lane C). Control autoradiogram of SDSpolyacrylamide gel electrophoresis of each purified substrate is shown in the upper part.
242
tected in the supernatant, in spite of the presence of both the phosvitin kinase activity and nearly all the 54-57 kDa doublet in the pellet fraction.
kDa proteins occurred at 44°C. An upper temperature optimum of 46°C for the 165 kDa compound phosphorylation was regularly observed.
Phosphoamino acid analysis The phosphorylated amino acid in each purified coated vesicle phosphoprotein was identified by paper electrophoresis after acid hydrolysis (Fig. 3). Phosphothreonine was the only radioactive amino acid detected in the 50 kDa protein. The two other substrates (54 and 165 kDa fractions) were phosphorylated on serine residues. No phosphorylation of tyrosine was observed.
Effects of adenosine 5'-O-(3-thiotriphosphate) and vanadate (VOJ +) on phosphorylation in coated vesicles
Time-course phosphorylation in coated vesicles The phosphorylation at 22 ° C with a 0.08 mM ATP concentration occurred differently for the three coated vesicle substrates. The 165 and 54 kDa compound phosphorylations were linear for at least 3 min, whereas the 50 kDa phosphorylation was linear only for 30 s, before reaching a plateau in 10 min (Fig. 4). Temperature effect on the phosphorylation of the coated vesicles substrates After 2 min of temperature equilibration, coated vesicles were labelled during 10 s with [32p]ATP under standard conditions. Phosphorylation of each substrate is shown in Fig. 5. Temperature increase induced a considerable enhancement of the 50 kDa protein phosphorylation rate compared to the 54 and 165 kDa protein phosphorylations. Optimum phosphorylation of the 54 and 50
The effect of 10 mM vanadate and the substitution of ATP[yP] by ATP[yS] on the 32p incorporation into coated vesicles are shown in Fig. 6 A and B, respectively. If both vanadate and ATP[yS] enhanced the 3Zp incorporation into the 50 kDa protein, neither of them modified the 32p incorporation into the 165 kDa protein substrate. In the case of the 54 kDa protein, vanadate strongly inhibited its phosphorylation, whereas ATP[yS] had no effect.
Dephosphorylation of the coated vesicle phosphoproteins T h e 32p-coated vesicles d e v o i d of A T P were i n c u b a t e d at 37 o C for various time intervals. The evolution of the r a d i o a c t i v i t y of the 50, 54 a n d 165 k D a p r o t e i n s are shown in Fig. 7. O n l y a decrease o f the 50 k D a p r o t e i n p h o s p h o r y l a t i o n a p p e a r e d with time, whereas the labelling of b o t h the 54 a n d 165 k D a b a n d s r e m a i n e d constant.
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Fig. 4. Time-course of phosphorylation of coated vesicle proteins. 165 kDa (zx A), 54 kDa (O O) and 50 kDa (A A) protein, cv, coated vesicles.
0
10
2b
3b
4b
~0
"C
Fig. 5. Optimum temperature determination for coated-vesicle phosphorylation substrates: 165 kDa (A A), 54 kDa (© ©) and 50 kDa (A A) protein, cv, coated vesicles.
243
A
B
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Fig. 6. Effect of 10 mM vanadate (A) and ATP[yS] (B) on the phosphorylation of 165 kDa protein (A zx), 54 kDa protein ( O O) and 50 kDa protein (A A). Control experiments are shown by full lines ( ); vanadate added or ATP[yS] instead of ATP[y P] are represented by dashed lines ( . . . . . . ).
Phosphorylation of coated vesicles from different origins
¢9
Autoradiography of the protein gel patterns of the coated vesicles purified from bovine brain, pig brain, rat liver and bovine adrenal gland is shown in Fig. 8: the 50 kDa labelled band is the only common band and the most labelled in the four
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Time (h) Fig. 7. Dephosphorylation of the coated vesicle phosQ) and phoproteins: 165 kDa (z~ zx), 54 kDa (O 50 kDa ( A ~ A ) phosphoprotein.
Fig. 8, Phosphorylation of coated vesicles from different origins, Comparison of the protein gel patterns after Coomassie blue staining (A) and autoradiography (B) of the coated vesicles from rat liver, bovine brain, bovine adrenal gland and pig brain.
244 coated vesicles species. The other phosphorylated compounds are characteristic for each kind of coated vesicles. Discussion
Protein phosphorylation is now recognized to be the major general mechanism by which intracellular events in mammalian tissues are controlled by external physiological stimuli. The part played by ATP in coated vesicle functions becomes thus quite important. ATP is implicated in protein phosphorylation [16-18], in a proton pump [26,27] and in releasing of clathrin from coated vesicles [28], and might also be involved in clathrin recruitement and coated vesicle movements into the cell [29]. In a preceding paper [16] we used an [),-32p]ATP with a weak specific activity of 2 Ci/mM. Only the 50 kDa band was clearly phosphorylated, whereas the phosphorylation of other bands was so slight that they were considered as back ground. The use of ATP of higher specific activity (60 Ci/mM) conducted regularly to the phosphorylation of three bovine brain coated vesicle proteins of 165, 54 and 50 kDa. The slight phosphorylation of the approx. 900 kDa band might correspond to coated vesicles not dissociated by SDS. Only the 50 kDA protein is really coated-vesicle specific [16,18], in contrast to the 54 kDa protein recently identified as fl-tubulin [18,30,31]. The 165 kDa compound is only detected after 32p-labelling. Its presence in trace amounts raises the question of its coated-vesicle constitutivity as it might be an internalized compound. This hypothesis is supported by: (i) the exclusive association of the 165 kDa radiolabelled band with the inner core vesicles; (ii) the higher temperature optimum for the 165 kDa protein phosphorylation compared to that of the 54 and 50 kDa proteins - this might be explained by a protective effect of the vesicles against thermic denaturation of the kinase-165 kDa substrate system. Presence of multiple protein kinases in coated vesicles is shown by different ways. (i) The disappearance of the radiolabelled 54 kDa band in both the supernatant and the pellet of urea-stripped vesicles might indicate the presence of a distinct 54 kDa protein kinase inactivated by urea. (ii) The
time-courses of the phosphorylations were different. (iii) The effects of vanadate known to act on phosphorylation mechanisms: it inhibited a variety of enzymes such as (Na++ K+)-ATPase [32[, alkaline phosphatase [33], acid phosphatase [34], membrane phosphotyrosyl phosphatase [35], phosphofructokinase [36] and adenylate kinase [37]. In contrast, adenylate cyclase was stimulated [38]. Vanadate had an opposite effect on 54 and 50 kDa protein phosphorylations, which were inhibited and enhanced, respectively. No effect on the 105 kDa protein phosphorylation was detected. (iv) Using a thiophosphate analogue of ATP it was shown that coated vesicle kinases were capable to transfer ~,-thiophosphoryl groups from ATP[yS] to their respective substrates. Substitution of phosphoATP by thioATP did not modify the initial velocity of 32p incorporation into the 165 and 54 kDa proteins, indicating that ATP[vP], ATP[vS] and [3,32p]ATP can be indistinctly used. On the other hand, the 50 kDa compound phosphorylation was enhanced by ATP~,S. Thiophosphoproteins are known to be dephosphorylated slowly by phosphatases [39]. The unlabelled thiophosphoproteins formed in coated vesicles might block the specific phosphatase pool which became inactive to dephosphorylate the 32p-labelled 50 kDa protein. The possible presence of a 50 kDa phosphoprotein phosphatase can be suggested by the decrease with time of the 32p labelling of the 50 kDa protein, whereas the radioactivity of the two other proteins remained constant. However, the loss of radioactivity is relatively slow. The 50 kDa phosphatase might require an activator, but at the present stage a proteolytic activity cannot be excluded. Comparative phosphorylation of several species of coated vesicles shows that the labelled 50 kDa protein is the only one which is present in every case and which is the most phosphorylated protein. Other minor labelled proteins are characteristic of the coated-vesicle origin. Thus, it was recently demonstrated that tubulin is a molecular component of coated vesicles [30,31], This must be restricted to the coated vesicles from brain and nervous tissue, where the tubulin is known to be tightly associated with membrane, compared to both the adrenal gland and liver coated vesicles, where tubulin is present only in trace amount. Phosphorylation pattern of coated vesicle minor
245 proteins might reflect the m e m b r a n o u s n a t u r e from which coated vesicles p i n c h e d off. I n conclusion, the p h o s p h o r y l a t i o n c o n d i t i o n s of the 165, 54 a n d 50 k D a c o m p o n e n t s of b o v i n e b r a i n coated vesicle proteins differ greatly. All these differences raise the p r o b l e m of multiple substrate-specific p r o t e i n kinases. O n l y the 50 k D a p r o t e i n - k i n a s e system seems to play an i m p o r t a n t role in the coated-vesicle working mechanisms: (i) b y its high enzymatic activity; (ii) b y the possible existence of a 50 k D a p h o s p h o p r o t e i n phosphatase; (iii) because it is n o t restricted to one type of coated vesicle, b u t seems to be present in the same way as clathrin. At present, the physiological f u n c t i o n of these p h o s p h o r y l a t i o n s r e m a i n s unclear, b u t the selective i n t e r n a l i z a t i o n of ligands, the choice of intracellular targets or the repeated m e m b r a n e fusion a n d fission m u s t require regulations characterized b y some p h o s p h o r y l a t i o n steps. Acknowledgements This research was supported by the I.N.S.E.R.M. ( U n i t 6 U-116), the C.N.R.S. (E.R. 102) a n d the F o n d a t i o n p o u r la Recherche M r d i cale Franqaise. We t h a n k Dr. Fevre (C.N.R.Z.) for p r o v i d i n g the b o v i n e b r a i n s a n d Mrs. C. Creuzet (I.R.S.C.) for her technical assistance, as well as Dr. I. Bernier for her helpful c o m m e n t s o n the manuscript.
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Biochimica et Biophvsica Acta,
799 (1984) 238 245 Elsevier
BBA 21772
P R O T E I N KINASE(S) IN BOVINE BRAIN COATED VESICLES ALAIN PAULOIN a JACQUES LOEB b and PIERRE JOLLieSa,. Laboratoire des Protbines (E. R. 102 CNRS, U- 116 I N S E R M ) Universitb de Paris II, 45 rue des Saints - Pbres, F - 752 70 Paris Cbdex 06 and h Laboratoire de Biochimie des Rbgulations, I.R.S.C., B.P. No. 9, F-94800 Villejuif (France)
(Received February 6th, 1984)
Key words: Protein kinase; Phosphorylation; (Bovine brain coated vesicle)
Purified bovine brain coated vesicles contain protein kinase activity which phosphorylates 165, 54 and 50 kDa protein substrates. These phosphorylations do not seem to be induced by a unique protein kinase: indeed, the three substrates present different localizations in coated vesicles, the phosphorylation sites are either serine or threonine residues and vanadate and ATP[¥S] have different effects on 32p incorporation in the substrates. Comparison of the coated vesicle protein and phosphorylation patterns from different tissues and animal origins shows that only the 50 kDa protein phosphorylation is always observed, compared to the great diversity in other minor phosphorylations which are observed or not in the various coated vesicles. The possible presence of a 50 kDa phosphoprotein phosphatase is also discussed. It is suggested that the 50 kDa protein with its connected specific kinase and phosphatase seems to constitute a regulatory system present in coated vesicles.
Introduction The coated pit region of plasma membrane is the site of localization of receptor-bound ligands prior to internalization [1]. It has been assumed that the transfer of ligands is carried out by the intermediary of coated vesicles pinched off from coated pits [1-4]. The latter as well as coated vesicles have also been implicated in membrane recycling [5], intracellular protein translocation [6,7] and exocytosis of newly synthesized proteins [8]. Biochemical studies involving the bristle coat have been greatly facilitated by the purification of coated vesicles, which have a membrane core enclosed by a polyhedral protein lattice [9,10]. The * To whom correspondenceshould be addressed. Abbreviations: Mes, 2-(N-morpholino)ethanesulfonic acid; EGTA, ethyleneglycolbis(fl-aminoethylether)-N,N'-tetraacetic acid; PMSF, phenylmethylsulfonylfluoride; SDS, sodium dodecyl sulfate; ATP['tS], adenosine-5'-O-(3-thiotriphosphate). 0304-4165/84/$03.00 © 1984 ElsevierSciencePublishers B.V.
latter is composed predominantly of clathrin, a 180 kDa protein associated with 36 and 33 kDa polypeptides [11]. Under native conditions, the basic assembly unit is a molecular complex of 630 kDa composed of three clathrin molecules with three polypeptides of 36 and 33 kDa [12-14]. Additional minor proteins of 110-100 kDa and 55-50 kDa have also been reported [15]. We have previously described the presence of a cyclic nucleotides and Ca2+-independent protein kinase activity in bovine brain coated vesicles [16], which has been corroborated by others [17,18]. This protein kinase catalyses specifically the transfer of the y-phosphate of ATP to the 50 kDa protein of coated vesicles. The phosphorylation of other proteins has also been described [17-19]. Protein phosphorylation is an important mechanism by which cellular metabolism or functions are regulated [20]. On a molecular level, phosphorylation of proteins is known to alter their conformations
239 and thereby modulates their activities [21]. The different expressions of coated vesicle protein kinase(s) imply a fine regulation of coated vesicles functions. The phosphorylation conditions of the different protein substrates in bovine brain coated vesicles are compared in the present paper. Our results show: (i) differences in phosphorylation conditions of each substrate; (ii) the constant presence of 50 kDa protein phosphorylation in coated vesicles from different tissues and species; (iii) the association of the 50 kDa protein kinase with the clathrin-light chain complex; (iv) a phosphatase activity specific for the 50 kDa phosphoprotein. The possible existence in coated vesicles of multiple protein kinases and the origin of each protein substrate are discussed. Materials and Methods
Materials Both pig and bovine brains were obtained from the Centre National de Recherches Zootechniques (Jouy-en-Josas, France). Wistar rats were obtained from IRSC( Villejuif, France). [7-32p]ATP with a specific activity of 60 C i / m m o l and Aquasol-2 were from New England Nuclear. ATP, egg phosvitin, urea, Mes, EGTA, MgC12, 2-mercaptoethanol and PMSF were obtained from Sigma. Sucrose and Tris were obtained from Merck. All components used for sodium dodecyl sulfate polyacrylamide gels were from Bio-Rad. Adenosine-5'O-(3-thiotriphosphate) was purchased from Boehringer (Mannheim), sodium orthovanadate from Ventron and 2H20 from CEA (Saclay, France). Coated vesicles from adrenal gland were a gift from Dr. A. Alfsen.
Purification of coated vesicles All procedures were carried out at 4°C. Brains were cleansed of meninges and the white matter removed by dissection. The grey matter was homogenized in 1 vol. of Mes buffer: 0.1 M Mes, pH 6.5, 0.1 mM EGTA, 2 mM MgCI 2, 1 mM 2mercaptoethanol, 0.2% NaN 3, 50/xg. ml-1 PMSF. The homogenate was centrifuged at 20 000 × g for 30 min in a Sorvall GSA rotor. The resulting pellet was subjected twice to the same procedure. All the supernatants were pooled and centrifuged at 100000 x g for 1 h in a Beckman 60 Ti rotor. The
soluble fraction was removed and the pelleted material was resuspended in Mes buffer. Coated vesicles were purified by two successive sucrose gradients [16,22]. The highly enriched upper 5% sucrose solution of the second gradient was used for the final purification using a 1 H 2 0 / 2 H 2 0 8% sucrose step gradient as described [23]. Rat liver coated vesicles were prepared from ten rats killed by decapitation. Livers were removed, washed with distilled water and homogenized in the Mes buffer. The coated vesicles were purified according to the method of Nandi et al. [23].
Phosphorylation assays The standard assay involved incubation of 100 #g of coated vesicles at 22 ° C with 1 #Ci (70 nmol. m1-1) of [y-32p]ATP in 12 mM Tris-HCl (pH 7.5), 32 mM KCI, 2 mM MgC12, 2.3 mM 2-mercaptoethanol in a total volume of 38 #1. Various quantities of unlabelled ATP were added (0.01-10 mM) for kinetic studies. For temperature dependence determinations the coated vesicles were preincubated for 2 min in a water bath (Thermomix 1410 Braun) at the selected temperatures prior to labelling. In each case the reaction, started by the rapid addition of labelled ATP, was carried out for 10 s at 22 ° C and was stopped immediately by addition of 60/xl SDS gel sample buffer (4% SDS, 3 % 2-mercaptoethanol, 10% glycerol and 0.1% Bromophenol blue in 80 mM Tris-HC1, pH 6.8) and boiling for 2 min at 100 o C. The polypeptides composition was analyzed by SDS-polyacrylamide gel (5-10%) electrophoresis according to Laemmli [24]. The following proteins were run in parallel as molecular weight markers: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa) and carbonic anhydrase (30 kDa). Clathrin (180 kDa) was used as internal molecular weight marker. The electrophoresis was carried out at 30 mA for 2 h at constant current until the Bromophenol blue as tracking dye was eluted. The protein bands were visualized by staining with 0.25% Coomassie blue and destaining in a solution of 5% acetic acid/25% ethanol. Autoradiography was performed by exposing the stained gel to a X-Omat AR Kodak film. 32p incorporation into 50, 54 and 165 kDa bands was quantitated by excising these bands from the dried gel and counting in 2.5 ml Aquasol-2
240
Phosphoamino acid analysis Coated-vesicle sample (300 ~g) was phosphorylated for 20 min at 22°C under standard conditions as described above, and analyzed by preparative gel electrophoresis. At the end of the migration, the gel was freezed and autoradiography was performed. The polyacrylamide radioactive gels corresponding to 50, 54 and 165 kDa bands on the processed film were excised, homogenized in 10 ml SDS-electrophoresis buffer [23] and eluted under stirring for 48 h at 20 o C. The homogenates were centrifuged at 20000 x g in a Sorvall SS-34 rotor. The supernatants were removed, filtered on 0.22 # m Millex-GS (Millipore) and extensively dialyzed against water. A 200 /~1 fraction of each sample was kept for SDS-polyacrylamide gel electrophoresis and control autoradiography; the remaining part of the samples was lyophilized. The dried extracts were dispersed in 200 ffl 6 M HC1. The tubes were sealed under vacuum and heated at l l 0 ° C for 1.5 h. HC1 was removed and the samples were dissolved in 25 t~l electrophoresis buffer, p H 3.5 (glacial acetic a c i d / p y r i d i n e / water, 10 : 1 : 89, v/v). 5-/~1 aliquots were mixed with 2 #1 of a solution containing 5 m g / m l of phosphoserine, phosphothreonine and phosphotyrosine, spotted on Whatman 3 MM chromatography paper and electrophoresed for 50 min at 60 V / c m . Phosphoamino acids were detected with ninhydrin and radioactive spots by autoradiography.
filtered on Sephadex G-50 (25 x 0.8 cm) equilibrated with the Mes buffer. Protein fractions contained in the void volume were pooled (1.66 ffCi 32p-labelled coated vesicles/ml) and incubated at 37 o C. Aliquots (200/tl) were removed between 0 and 5 h. The reaction was stopped by freezing; after lyophilization and addition of 60/~1 SDS-gel sample buffer, the aliquots were analyzed by SDS-polyacrylamide gel electrophoresis and the 32p incorporation into the 50, 54, 165 kDa bands was quantitated. Results
Phosphorylation of coated vesicles The protein gel patterns of the bovine brain coated vesicles after Coomassie blue staining and autoradiography are shown in Fig. 1. Several proteins associated with clathrin and the light chains were regularly observed: a thin band of high molecular weight (about 300 kDa), a group of seven bands from 105 to 136 kDa with one major band of 105 kDa, the protein doublet of 57 and 54
Preparation of clathrin-stripped vesicles Clathrin-stripped vesicles which constituted inner cores were prepared by incubating coated vesicles for 2 h at 2 0 ° C with 3 M urea in Mes buffer. They were pelleted for 1 h at 100000 × g in a Beckman 60 Ti rotor and resuspended in the Mes buffer. The supernatant contained most of the clathrin-light chains complex [17,25]. Both the supernatant (S) and resuspended pellet (P) were dialyzed overnight at 4 ° C against the Mes buffer prior to phosphorylation assays. In order to determine the kinase activity, 10 #g egg phosvitin were added in the two fractions prior to labelling.
Phosphatase activity assay Coated vesicles (400 #g) were phosphorylated for 10 min at 22°C under standard conditions. In order to eliminate ATP, the reaction mixture was
Fig. 1. Comparison of the protein gel patterns after Coomassie blue staining (A) and autoradiography (B) of bovine brain coated vesicles.
241
kDa associated to the 50 kDa protein and some other very discrete bands (part A). On the autoradiography, three proteins were phosphorylated: they have molecular weights of 165, 54 and 50 kDa. A slight phosphorylation corresponding to a very high molecular weight substance (greater than 900 kDa) was also detected (part B).
tion into the 50 kDa protein and the phosvitin area was quantitated. The ratios corresponding to cpm in the supematant versus cpm in the pellet were the same for the two substrate systems, 3.4 and 3.3 for the 50 kDa protein and the phosvitin area, respectively. It is noteworthy that the 54 kDa phosphorylation was very small and was only de-
Phosphorylations in both outer coat and inner vesicle structures of coated vesicles The protein gel patterns after Coomassie blue staining and autoradiography of the outer coats and the inner vesicles which correspond to the supernatant (S) and the pellet (P), respectively (see preparation of clathrin-stripped vesicles), are shown in Fig. 2. Nearly all the 300 kDa band and the clathrin light chains complex were solubilized in the supernatant and all the 57-54 kDa doublet remained associated with the pellet. The urea treatment removed approx. 30% of both the 105-136 kDa protein group and the 50 kDa band which appeared in the supematant; the majority of these constituents remained associated with the pellet (part A). On the autoradiography, the 165 kDa radioactive band was exclusively associated with the inner vesicle, when the 50 kDa radioactive band was detected in the outer coat fraction (part B). Phosvitin as exogenous substrate [17] was added to the inner vesicle and the outer coat fractions. As shown in part C, the phosvitin kinase activity was detected in both fractions, but it was more pronounced in the outer coat. 32P incorpora-
s
+
-
165
f 54 w- 50
-
kDA
kDA kDA
+
Orthophosphote
+
P-serine
+
P-threonine
+
P-tyrosine
P
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a
C
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Fig. 2. Comparison of the protein get patterns after Coomassie blue staining (A) and autoradiography (B) of the supematant (S) and the pellet (P) of urea-treated coated vesicles. Phosphorylation of phosvitin as exogenous substrate (C).
Fig. 3. Identification of phosphoaminoacids in the 50 kDa protein (lane A), mixture of 54 and 50 kDa proteins (lane B) and 165 kDa protein (lane C). Control autoradiogram of SDSpolyacrylamide gel electrophoresis of each purified substrate is shown in the upper part.
242
tected in the supernatant, in spite of the presence of both the phosvitin kinase activity and nearly all the 54-57 kDa doublet in the pellet fraction.
kDa proteins occurred at 44°C. An upper temperature optimum of 46°C for the 165 kDa compound phosphorylation was regularly observed.
Phosphoamino acid analysis The phosphorylated amino acid in each purified coated vesicle phosphoprotein was identified by paper electrophoresis after acid hydrolysis (Fig. 3). Phosphothreonine was the only radioactive amino acid detected in the 50 kDa protein. The two other substrates (54 and 165 kDa fractions) were phosphorylated on serine residues. No phosphorylation of tyrosine was observed.
Effects of adenosine 5'-O-(3-thiotriphosphate) and vanadate (VOJ +) on phosphorylation in coated vesicles
Time-course phosphorylation in coated vesicles The phosphorylation at 22 ° C with a 0.08 mM ATP concentration occurred differently for the three coated vesicle substrates. The 165 and 54 kDa compound phosphorylations were linear for at least 3 min, whereas the 50 kDa phosphorylation was linear only for 30 s, before reaching a plateau in 10 min (Fig. 4). Temperature effect on the phosphorylation of the coated vesicles substrates After 2 min of temperature equilibration, coated vesicles were labelled during 10 s with [32p]ATP under standard conditions. Phosphorylation of each substrate is shown in Fig. 5. Temperature increase induced a considerable enhancement of the 50 kDa protein phosphorylation rate compared to the 54 and 165 kDa protein phosphorylations. Optimum phosphorylation of the 54 and 50
The effect of 10 mM vanadate and the substitution of ATP[yP] by ATP[yS] on the 32p incorporation into coated vesicles are shown in Fig. 6 A and B, respectively. If both vanadate and ATP[yS] enhanced the 3Zp incorporation into the 50 kDa protein, neither of them modified the 32p incorporation into the 165 kDa protein substrate. In the case of the 54 kDa protein, vanadate strongly inhibited its phosphorylation, whereas ATP[yS] had no effect.
Dephosphorylation of the coated vesicle phosphoproteins T h e 32p-coated vesicles d e v o i d of A T P were i n c u b a t e d at 37 o C for various time intervals. The evolution of the r a d i o a c t i v i t y of the 50, 54 a n d 165 k D a p r o t e i n s are shown in Fig. 7. O n l y a decrease o f the 50 k D a p r o t e i n p h o s p h o r y l a t i o n a p p e a r e d with time, whereas the labelling of b o t h the 54 a n d 165 k D a b a n d s r e m a i n e d constant.
44*
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15
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g 8
~
f
/
i
0
_o o fi,..
>
5,
-c o. ii. I 10
[ 20
I 3O
Time (min)
Fig. 4. Time-course of phosphorylation of coated vesicle proteins. 165 kDa (zx A), 54 kDa (O O) and 50 kDa (A A) protein, cv, coated vesicles.
0
10
2b
3b
4b
~0
"C
Fig. 5. Optimum temperature determination for coated-vesicle phosphorylation substrates: 165 kDa (A A), 54 kDa (© ©) and 50 kDa (A A) protein, cv, coated vesicles.
243
A
B
.A ~ A I ,,/A
A
>
7.
/ tF/
o~
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~, 3. ~
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o
ATP (mM)
oi~ o:2 ATRP or ATP, S (mM]
Fig. 6. Effect of 10 mM vanadate (A) and ATP[yS] (B) on the phosphorylation of 165 kDa protein (A zx), 54 kDa protein ( O O) and 50 kDa protein (A A). Control experiments are shown by full lines ( ); vanadate added or ATP[yS] instead of ATP[y P] are represented by dashed lines ( . . . . . . ).
Phosphorylation of coated vesicles from different origins
¢9
Autoradiography of the protein gel patterns of the coated vesicles purified from bovine brain, pig brain, rat liver and bovine adrenal gland is shown in Fig. 8: the 50 kDa labelled band is the only common band and the most labelled in the four
' O 60.
O 40,
4=
3! - ~ - ~ - - ~ 8 2. 1 i k--~---~---~--~ 0
0
~,
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i
i
i
i
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1
2
3
4
5
Time (h) Fig. 7. Dephosphorylation of the coated vesicle phosQ) and phoproteins: 165 kDa (z~ zx), 54 kDa (O 50 kDa ( A ~ A ) phosphoprotein.
Fig. 8, Phosphorylation of coated vesicles from different origins, Comparison of the protein gel patterns after Coomassie blue staining (A) and autoradiography (B) of the coated vesicles from rat liver, bovine brain, bovine adrenal gland and pig brain.
244 coated vesicles species. The other phosphorylated compounds are characteristic for each kind of coated vesicles. Discussion
Protein phosphorylation is now recognized to be the major general mechanism by which intracellular events in mammalian tissues are controlled by external physiological stimuli. The part played by ATP in coated vesicle functions becomes thus quite important. ATP is implicated in protein phosphorylation [16-18], in a proton pump [26,27] and in releasing of clathrin from coated vesicles [28], and might also be involved in clathrin recruitement and coated vesicle movements into the cell [29]. In a preceding paper [16] we used an [),-32p]ATP with a weak specific activity of 2 Ci/mM. Only the 50 kDa band was clearly phosphorylated, whereas the phosphorylation of other bands was so slight that they were considered as back ground. The use of ATP of higher specific activity (60 Ci/mM) conducted regularly to the phosphorylation of three bovine brain coated vesicle proteins of 165, 54 and 50 kDa. The slight phosphorylation of the approx. 900 kDa band might correspond to coated vesicles not dissociated by SDS. Only the 50 kDA protein is really coated-vesicle specific [16,18], in contrast to the 54 kDa protein recently identified as fl-tubulin [18,30,31]. The 165 kDa compound is only detected after 32p-labelling. Its presence in trace amounts raises the question of its coated-vesicle constitutivity as it might be an internalized compound. This hypothesis is supported by: (i) the exclusive association of the 165 kDa radiolabelled band with the inner core vesicles; (ii) the higher temperature optimum for the 165 kDa protein phosphorylation compared to that of the 54 and 50 kDa proteins - this might be explained by a protective effect of the vesicles against thermic denaturation of the kinase-165 kDa substrate system. Presence of multiple protein kinases in coated vesicles is shown by different ways. (i) The disappearance of the radiolabelled 54 kDa band in both the supernatant and the pellet of urea-stripped vesicles might indicate the presence of a distinct 54 kDa protein kinase inactivated by urea. (ii) The
time-courses of the phosphorylations were different. (iii) The effects of vanadate known to act on phosphorylation mechanisms: it inhibited a variety of enzymes such as (Na++ K+)-ATPase [32[, alkaline phosphatase [33], acid phosphatase [34], membrane phosphotyrosyl phosphatase [35], phosphofructokinase [36] and adenylate kinase [37]. In contrast, adenylate cyclase was stimulated [38]. Vanadate had an opposite effect on 54 and 50 kDa protein phosphorylations, which were inhibited and enhanced, respectively. No effect on the 105 kDa protein phosphorylation was detected. (iv) Using a thiophosphate analogue of ATP it was shown that coated vesicle kinases were capable to transfer ~,-thiophosphoryl groups from ATP[yS] to their respective substrates. Substitution of phosphoATP by thioATP did not modify the initial velocity of 32p incorporation into the 165 and 54 kDa proteins, indicating that ATP[vP], ATP[vS] and [3,32p]ATP can be indistinctly used. On the other hand, the 50 kDa compound phosphorylation was enhanced by ATP~,S. Thiophosphoproteins are known to be dephosphorylated slowly by phosphatases [39]. The unlabelled thiophosphoproteins formed in coated vesicles might block the specific phosphatase pool which became inactive to dephosphorylate the 32p-labelled 50 kDa protein. The possible presence of a 50 kDa phosphoprotein phosphatase can be suggested by the decrease with time of the 32p labelling of the 50 kDa protein, whereas the radioactivity of the two other proteins remained constant. However, the loss of radioactivity is relatively slow. The 50 kDa phosphatase might require an activator, but at the present stage a proteolytic activity cannot be excluded. Comparative phosphorylation of several species of coated vesicles shows that the labelled 50 kDa protein is the only one which is present in every case and which is the most phosphorylated protein. Other minor labelled proteins are characteristic of the coated-vesicle origin. Thus, it was recently demonstrated that tubulin is a molecular component of coated vesicles [30,31], This must be restricted to the coated vesicles from brain and nervous tissue, where the tubulin is known to be tightly associated with membrane, compared to both the adrenal gland and liver coated vesicles, where tubulin is present only in trace amount. Phosphorylation pattern of coated vesicle minor
245 proteins might reflect the m e m b r a n o u s n a t u r e from which coated vesicles p i n c h e d off. I n conclusion, the p h o s p h o r y l a t i o n c o n d i t i o n s of the 165, 54 a n d 50 k D a c o m p o n e n t s of b o v i n e b r a i n coated vesicle proteins differ greatly. All these differences raise the p r o b l e m of multiple substrate-specific p r o t e i n kinases. O n l y the 50 k D a p r o t e i n - k i n a s e system seems to play an i m p o r t a n t role in the coated-vesicle working mechanisms: (i) b y its high enzymatic activity; (ii) b y the possible existence of a 50 k D a p h o s p h o p r o t e i n phosphatase; (iii) because it is n o t restricted to one type of coated vesicle, b u t seems to be present in the same way as clathrin. At present, the physiological f u n c t i o n of these p h o s p h o r y l a t i o n s r e m a i n s unclear, b u t the selective i n t e r n a l i z a t i o n of ligands, the choice of intracellular targets or the repeated m e m b r a n e fusion a n d fission m u s t require regulations characterized b y some p h o s p h o r y l a t i o n steps. Acknowledgements This research was supported by the I.N.S.E.R.M. ( U n i t 6 U-116), the C.N.R.S. (E.R. 102) a n d the F o n d a t i o n p o u r la Recherche M r d i cale Franqaise. We t h a n k Dr. Fevre (C.N.R.Z.) for p r o v i d i n g the b o v i n e b r a i n s a n d Mrs. C. Creuzet (I.R.S.C.) for her technical assistance, as well as Dr. I. Bernier for her helpful c o m m e n t s o n the manuscript.
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