Protein kinase and its endogenous substrates in coated vesicles

306

Biochimica et Biopl~vsica Acta, 798 (1984) 306 312 [:Asevicr

BBA21724

P R O T E I N KINASE A N D ITS E N D O G E N O U S S U B S T R A T E S IN C O A T E D VESICLES MIHOKO USAMI, AKIRA TAKAHASHI and KEN KADOTA *

Division of Neurochemistry, Psychiatric Research Institute of Tokyo, Kamikitazawa, Setagava- ku, Tokyo 156 (Japan) (Received May 31st, 1983) (Revised manuscript received December 30th, 1983)

Key words: Protein kinase; Phosvitin; (Coated vesicle)

Coated vesicles prepared from bovine brains contained a protein kinase activity which catalyzed the phosphorylation of endogenous structural proteins, M r 150000, 120000, 48000 and 32 000. An endogenous protein, M r 48000 was most strongly phosphorylated by this kinase. This protein kinase also phosphorylated exogenous proteins, phosvitin intensely and casein slightly but not histone or protamine. The enzyme activity was independent of cyclic nucleotides or Ca2+/calmodulin. Mg 2+ stimulated the kinase activity. Some divalent cations were substituted for Mg2+; the potency decreased in the order Mn 2+ , Mg 2+ , Co 2+ , Ca 2+ , Zn 2+. Two separate subfractions, the outer coat and the inner vesicle (core), were prepared from coated vesicles by a urea treatment followed by sucrose density gradient centrifugation and dialysis. The kinase activity was found predominantly in the coat subfraction. Introduction The coated vesicle which possesses a lattice-like network covering an inner vesicle, exists in a variety of eukaryotic cells. It was identified as a structural correlate of specific protein transport by Roth and Porter in 1964 [1], and hexagonal and pentagonal features of its lattice were first described by Kaneseki and Kadota [2]. Thereafter, morphological studies of the coat work have been carried out by m a n y workers [3-5]. Recent studies have shown that the coated vesicle is involved in receptormediated endocytosis [6-8], intracellular transport of macromolecules [9,10], presynaptic membrane recycling [11,12] and secretion [13,14]. Coated vesicles have been purified from various tissues [10,15-17] and characterized biochemically by many investigators [10,18,19]. The vesicle con* To whom correspondence should be addressed. Abbreviations: EGTA, ethylene glycol bis(fl-aminoethyl ether)N,N,N',N'-tetraacetic acid; Mes, 2-(N-morpholino)ethanesulfonic acid. 0304-4165/84/$03.00 © 1984 Elsevier Scientific Publishers B.V.

tained a major protein species of M r 180000, clathrin, which covered the inner vesicle [15]. A treatment of coated vesicles with urea, Tris-HC1 or MgCI 2 resulted in the disruption of the coat structure and the release of coat proteins in soluble form [19,20]. Calmodulin was found to associate with coated vesicles which had been purified in the presence of Ca 2÷ and radiolabeled calmodulin was bound to coated vesicles in a specific manner [21]. Other studies showed that coated vesicles from brains or neurophypophyses sequestered Ca 2+ by an ATP-requiring process [18]. Recent reports have shown the presence of a cyclic nuc|eotides and Ca 2+ independent protein kinase activity in coated vesicles [22-24]. We report here detailed properties of the protein kinase in coated vesicles prepared from whole brain tissues. Materials and Methods

Materials and preparations. [y-32p]ATP was prepared according to the method of Glynn and

307 Chappell [25]. Calmodulin was partially purified following the method of Lin et al. [26]. Phosvitin, casein, histone IIA (calf thymus) and protamine (salmon sperm) and cAMP-dependent protein kinase (bovine heart) were purchased from Sigma. Other reagents were all of analytical grade and were available commercially. Preparation of coated vesicles. Coated vesicles were isolated from fresh bovine brains with slight modifications of the method of Pearse [15]. The purification was carried out at 4°C. Bovine brains were homogenized in 1 vol. of a Mes buffer (pH 6.5) containing 0.1 M Mes, 1 mM EGTA, 0.5 mM MgCI 2 and 0.02% sodium azide. The homogenate was centrifuged at 20 000 × g for 30 min, and the resulting supernatant was centrifuged at 55 000 x g for 60 min. The pellet was suspended in a 50 ml of the Mes buffer containing 10% sucrose. Coated vesicles were precipitated by centrifugation for 60 min at 100 000 x g. The pellet was homogenized in a small volume of the buffer and layered on a top of a continuous 30-50% sucrose density gradient (in the Mes buffer). The gradient was centrifuged for 16 h at 50000 x g. A turbid band at about 35-40% sucrose was collected, diluted 4-fold with the buffer and concentrated by centrifugation at 10000 x g for 60 min. The pellet was resuspended, layered on a top of a continuous 5-30% sucrose gradient and centrifuged at 100000 × g for 60 min. A band at the density of about 14-20% sucrose was collected, pooled and precipitated by centrifugation. The purified coated vesicles were resuspended in a Mg2÷-free Mes buffer.

Preparation of coat and core subfractions from coated vesicles. To dissolve the coat structure a part of the coated vesicle preparation was incubated for 2 h at 4°C in 3 M urea in the Mes buffer following the procedure of Woodward et al. [20]. Then the material was applied onto a layer of 0.6 M sucrose and centrifuged at 180000 x g for 60 min. The dissociated coat was located in the upper (3 M urea) layer and the inner vesicle (core) in the 0.6 M sucrose layer. Each layer was collected separately and dialyzed against a 50 mM Mes buffer for reassembling lattice-like networks of the coat. The subfractions of the reassembled coat and core were centrifuged and resuspended in a small volume of the Mes buffer, respectively. The purity of the coated vesicle preparation and

its subfractions were examined by electron microscopy as described previously [16]. The specimens were negatively stained with 1% aqueous solution of uranyl acetate. For section of materials, a piece of coated vesicle preparation was prefixed with 3% ( v / v ) glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 30 min and then postfixed in 2% OsO 4 in the same buffer for 1 h. The specimens were block-stained with 1% uranyl acetate in a veronal acetate buffer, 50 mM (pH 5.2), dehydrated, and embedded in Epon 812.

Assay of protein kinase activity of coated vesicles. Under a standard assay condition, each preparation of coated vesicles, coat or core (30-100 #g protein) was preincubated in 100/~1 of a solution containing 20 mM imidazole (pH 7.0), 0.13 M KCI, 0.2 mM EGTA with or without 2 mM MgCI 2. In some cases, the reaction mixture was contained 100 /~M cAMP or cGMP. The reaction was initiated by the addition of 2 - 2 5 / t M [y-a2P]ATP (15 /~Ci/nmol). Following incubation at 25°C, the reaction was stopped by the addition of 20/~1 of a treatment buffer containing 4% SDS, 10% 2mercaptoethanol, 20% glycerol and 0.125 M TrisHC1 (pH 6.8).

SDS-polyacrylamide gel electrophoresis, autoradiography, and protein assay. The discontinuous buffer system described by Laemmli [27] was used with 10 and 4% acrylamide for the separating and stacking gels, respectively. After electrophoresis at 30 mA for 4 h, the gels were stained with Coomassie brilliant blue. Molecular weight ( M r) standards included RNA-polymerase (165 000, 155 000 and 39 000), phosphorylase b ( M r 94 000), bovine serum albumin ( M r 68 000) and catalase ( M r 60 000). For autoradiography, the stained and vacuum-dried gels were exposed to fine-grain positive film 7302 (Kodak). The incorporation of 32p into coated vesicle proteins was analyzed with an Aloka Chromatography Scanner (fl-ray scan). Protein concentration was determined by the method of Lowry et al. [28]. Results

Mophological properties and protein components of coated vesicles. The coated vesicle fraction prepared from bovine brain was virtually free of contaminating membrane fragments or synaptic

308

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Fig. 1. SDS-polyacrylamide gel electrophoretic pattern of proteins and autoradiographs of phosphorylated proteins of coated vesicles. Coated vesicles (73 #g of protein) were incubated in 100/~1 of a solution containing 20 mM imidazole (pH 7.0), 0.13 M KCI, 0.2 mM EGTA and 2 #M [y-32p]ATP with or without 2 mM MgCI 2 at 25°C for 10 min. The reaction was stopped by the addition of 20 #1 of a SDS solubilizing solution. The samples were analyzed by SDS-gel electrophoresis according to Laemmli [27] on 4% stacking/10% separating gels. A, Coomassie blue stained gel lane illustrating M, standard (a) and the protein components of coated vesicles (b). B, autoradiograph illustrating the phosphorylation of proteins. Lane 1, without MgCI2; lane 2, with 2 mM MgCI 2.

vesicles. The coated vesicles amounted to approx. 95% of all components in the fraction. The coated vesicle contained one major and five minor proteins with M r 180000, 120000, 52000, 48 000 and two polypeptides in a M r 30 000-40 000 range (Fig. 1A). A predominant protein of the purified coated vesicle was M r 180000, the reported M r of clathrin [15].

Properties of an endogenous phosphorylation system of coated vesicles. The coated vesicle was incubated with [y-32 P]ATP and resulting 32P-labeled phosphoproteins were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiogra-

phy. In the absence of Mg 2+, the phosphorylation of proteins of the coated vesicle was not significant (Fig. 1B). When Mg 2+ was added to the reaction mixture, four proteins, M r 32 000, 48 000, 120 000 and 150000, were endogenously phosphorylated (Fig. 1B). A protein of M r 48000, termed C-48 [24], was strongly phosphorylated while other proteins were only slightly phosphorylated. cAMP or c G M P had no effect on the kinase activity. The phosphorylation of the coated vesicle proteins was not increased in the extent with the addition of cAMP-dependent protein kinase and cAMP. Ca 2+ and calmodulin were without effect on the phosphorylation of the coated vesicle proteins. A high concentration of E G T A (2 mM) was added to the reaction mixture to test its effect on protein kinase activity in the coated vesicle, since the coated vesicle fraction has been reported to contain calmodulin [21]. The protein kinase activity was not affected by this treatment. Trifluoperazine, a calmodulin blocking agent, was ineffective on the kinase activity. Divalent cations, especially Mg 2÷, markedly stimulated the protein kinase activity (Fig. 2, Table I). Table I shows the effect of various divalent cations on the extent of the phosphorylation of C-48 in comparison with that of Mg 2+. Mn 2+

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Fig. 2. Effects of various divalent cations on protein kinase activity in coated vesicles. Experimental conditions were as described in Fig. 1, except that each of divalent cations (2 raM) was added to the reaction miture in place of Mg 2+. The concentrations of coated vesicles and [y-32p]ATP was 70 t~g protein/100/~l and 10/tM, respectively. Lane, 1, none; lane 2, MgC12; lane 3, MnCl2; lane 4, CoCl2; lane 5, ZnCl2; lane 6, CaCI2; Lane 7, FeC12; lane 8, CuC12.

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Fig. 3. Phosphorylation of exogenous proteins by protein kinase in coated vesicles. Coated vesicles (73 /~g of protein) were incubated for pbosphorylation reaction as described in Fig. 1. Phosvitin (20#g), casein (20/~g), histone (20 #g) or protamine (20 /~g) and 10 # M [y-32p]ATP were added in the reaction mixture. Lane 1 was without addition of MgCI 2. lanes 2 - 6 were with 2 m M MgC12. lanes 3 - 6 in the presence of phosvitin (lane 3), casein (lane 4), histone, (lane 5, and protamine (lane 6), respectively.

could be substituted for Mg 2+. Co 2+, Ca 2+ and Zn 2+ stimulated the phosphorylation of the C-48 protein while Fe 2÷ and Cu 2÷ did not. The order of the stimulating potency was Mn 2÷ > Mg 2+ > Co 2+ > Ca 2 + > Zn 2+. Mn 2+ especially intensified the phosphorylation of M r 120000 protein of the coated vesicle.

Phosphorylation of exogenous proteins with the protein kinase in coated vesicles. The substrate specificity of the protein kinase was studied using

TABLE I EFFECTS O F D I V A L E N T C A T I O N S ON T H E PHOSP H O R Y L A T I O N O F C-48 P R O T E I N O F C O A T E D VESICLES Phosphorylation of the coated vesicle and SDS-polyacrylamide gel electrophoresis were carried out as described in Fig. 2. The incorporation of 32Pi into C-48 protein of the coated vesicle was measured with an Aloka Chromatography Scanner. Divalent cations

(%)

None Mg 2 + M n 2+ Ca 2 + Co 2 + Zn 2 + Fe 2+ Cu 2+

6.1 + 2.6 100.0 105.8 5:4.7 28.7 + 7.3 73.9 + 3.5 17.9 5:7.6 0 0

exogenous protein substrates, phosvitin, casein, histone IIA and protamine. The autoradiographs in Fig. 3 show that the protein kinase in coated vesicles phosphorylates phosvitin and casein. Phosvitin was especially a good substrate for this kinase. In addition phosvitin stimulated the phosphorylation of component proteins of the coated vesicle. Histone and protamine were not phosphorylated at all. However, these proteins inhibited or stimulated the incorporation of the radioactive phosphate into some endogenous acceptor proteins. These basic proteins reduced considerably the extent of phosphorylation of C-48 while they increased that of M r 32 000 protein.

Fig. 4. Electron micrographs of the coat and the core subfractions of the coated vesicle, a, urea-treated coated vesicle: negatively stained (bar = 0.1/~m); b, c, coat subfraction: b, negatively stained (bar = 0.1/~m); c, thin section (bar = 0.5 #m); d, core subfraction; negatively stained (bar = 0.1/~m).

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Separate localization of protein kinase in coat subfraction and endogenous substrate in core subfraction. The coated vesicle was divided into two substructural components, the coat and the core, by a treatment with urea. The coat structure of the coated vesicle was dissociated after the incubation in 3 M urea. There was no lattice of the coat (Fig. 4a). Polygonal lattice structures were reassembled after the dialysis of the subfraction obtained from the urea layer. This coat subfraction contained many empty coat structures and a few coated vesicles (Fig. 4b and c). The reassembled coat had similar morphologies to those of the coat found in the untreated coated vesicles. Fig. 4d shows an inner vesicle (core) subfraction obtained after the dialysis of the 0.6 M sucrase layer. This subfraction contained vesicles without the outer coat structure, and amorphorous materials, possibly broken coat structure of the coated vesicle (Fig. 4d). The coat subfraction was rich in clathrin ( M r 180000) whereas the core one, two proteins with M r of 52000 and 48000 (C-48) (Fig. 5A). The electrophoretic pattern of proteins of the coat subfraction was apparently similar to that shown by Woodward et al. [20]. In order to determine whether the protein kinase activity of the coated vesicle associated with the coat or the core structure, we examined the protein

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! Fig. 5. Pbospborylation of endogenous and exogenous proteins by coat or core subfraction. Coat and core subfractions prepared from coated vesicles were incubated in a phosphorylation medium, subjected to electrophoresis and autoradiography as described in Fig. 3. A, Coomassie blue stained gel lane. lane 1, coat fraction; lane 2, core fraction; B and C, autoradiographs illustrating the phosphorylation of the proteins of the coat (35 #g of protein) and the core (33 #g of protein). Lane 2, phosvitin (phos); lane 3, casein (cas); lane 4, histone (his); lane 5, protamine (pro).

phosphorylation in each fraction. An exogenous substrate, phosvitin, was strongly phosphorylated with the coat subfraction but slightly with the core subfraction (Fig. 5B and C). No significant endogenous phosphorylation was found in the core subfraction although this subfraction contained an abundance of the endogenous substrate protein, C-48. These results suggest that the protein kinase closely associates with the coat structure of coated vesicles while the substrate protein, C-48, with the inner vesicular structure. Discussion

The coated vesicle isolated from bovine brain contained an endogenous protein kinase activity as well as its substrate proteins. The endogenous substrates were proteins with M r 32000, 48000 (C-48), 120000 and 150000 by SDS-polyacrylamide gel electrophoresis. The C-48 was the most suitable substrate of these four proteins and was recovered mainly in the inner vesicle (core) subfraction of the coated vesicle. A major element of the coat protein, clathrin, [15] was not phosphorylated. This protein kinase has been suggested to associate closely with the coat structure of the coated vesicle (Figs. 4 and 5). The kinase activity in the coated vesicle dose not seem to be crosscontamination by phosvitin kinase in plasma membrane or cytosol in brain. The reasons for that are: (1) our coated vesicle fraction is nearly pure, (2) phosvitin kinase activity in plasma membrane prepared from bovine brain is about 10-fold lower in specific activity than that of the protein kinase in the coated vesicle (unpublished data), (3) during the dissociation-reassembly-precipitation steps for the purification of the coat lattice, a large part of the contaminated proteins may be removed from the coat subfraction. In brain, many protein kinases have been reported to exist in cytosol [29-32,34], plasma membrane [31,32], and synaptic vesicles [33]. Kennedy and Greengard [32] have suggested that the brain tissue contains at least four distinct calmodulindependent kinases and two types of cAMP dependent protein kinases. Burke and DeLorenzo have demonstrated that Ca2+/calmodulin-dependent protein kinases are present in synaptic vesicles [33] and cytosol [34]. The kinase in the coated vesicle

311

fraction differs from these protein kinases, in that its activity is independent of cyclic nucleotides or Ca z ÷/calmodulin. This kinase appears to be different from the free catalytic subunit of cAMP-dependent protein kinase, since it did not catalyze the phosphorylation of histone [35]. Furthermore, the addition of cAMP-dependent protein kinase and cAMP did not increase the phosphorylation of C-48, an endogenous substrate protein of protein kinase in the coated vesicle. Some workers have reported the existence of cyclic nucleotides or Ca2÷/calmodulin-independent protein kinases in brain [29-31], skeletal muscle [36,37], liver [38] or heart [39], which catalyze the phosphorylation of phosvitin and casein. From these preceding findings and the present reasults, it appears that the protein kinase in the coated vesicle shares properties with the casein kinase shown in skeletal muscle [36,37] and brain [29]. It remains to be determined which amino acid residues of its substrates are phosphorylated by the present protein kinase in the coated vesicle. Protein phosphorylation is one of the most important control mechanisms for regulating the activities of various enzymes [37,38] and muscle constituents [36]. The coated vesicle was known to mediate the endocytotic uptake of some macromolecules in tissues and cells in culture [6-8]. Recently some workers observed the phosphorylation of hormone receptors which are reported to be internalized by coated endocytosis [40,41]. At present, functional roles of the protein phosphorylation system in the coated vesicle are yet unclear. One possibility is that the phosphorylation of coated vesicle proteins may be involved in the receptor-mediated endocytosis. When the ligands for this type of endocytosis associate with the receptor components on plasma membrane, the phosphorylation system in the coated vesicle might be stimulated to affect the coated endocytotic processes as depicted by Goldstein et al. [6].

Acknowledgment The authers are grateful to Dr. T. Kadota, Department of Anatomy, School of Medicine, Chiba University, for her support in electron microscopy.

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