Journal of Nrurorhrmisrr). 1976 Vol. 26, pp. 573-577. Pergamon Press. Printed in Great Britain
PHOSPHORYLATION OF ENDOGENOUS PROTEINS IN MYELIN OF RAT BRAIN E. MIYAMOTO The Research Division and Clinical Laboratory, Nakamiya Mental Hospital, Hirakata, Osaka, Japan (Receiaed 12 June 1975. Accepted 20 August 1975)
Abstract-The phosphorylation of endogenous proteins occurring in the myelin of rat brain was examined using the method of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Two myelin basic proteins and at least five more proteins were phosphorylated after incubation of myelin fraction in the presence of ATP Mg2+.The apparent molecular weights of the proteins other than the myelin basic proteins were 120,000, 76,000, 60,000, 41,000 and 38,000, respectively. The proteins of mol wt 60,OOO. 41,000 and 38,000 were extracted by treatment with hydrochloric acid, whereas those of mol wt 120,000 and 76,000 were insoluble in hydrochloric acid and chloroform-methanol. Folch-Lees proteolipid protein was not found to be phosphorylated under the conditions studied. The endogenous phosphorylation of the proteins was not stimulated by adenosine 3',5'-monophosphate.
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AFTER the discovery of the adenosine 3',5'-monophos-
The present study describes experiments demonstratphate (cyclic AMP)dependent protein kinase in skele- ing the Occurrence of at least seven endogenous protal muscle (WALSHe f al., 1968), liver (LANGAN, 1968) teins in myelin of rat brain, which are phosphorylated and brain (MIYAMOTO et al., 1969), it was proposed after incubation with ATP + Mg2+. that the diverse actions of cyclic AMP in various tissues are mediated through the activation of the cyclic MATERIALS AND METHODS AMP-dependent protein kinase (Kuo & GREENGARD, Materials. Cyclic AMP was obtained from Kohjin Co. 1969; GREENGARD & Kuo, 1970). The myelin of brain and spinal cord contains a Cytochrome c, myoglobin, chymotrypsinogen A, ovalbubasic protein which is known as an experimental min, bovine serum albumin and ?-globulin were purchased (carrier-free) allergic encephalomyelitogenic protein, since it in- from Schwarz-Mann. Orth~[~~P]phosphate was obtained from Japan Radioisotope Association. duces the disease in animals when injected with Phosphorylation of myelin fraction and extraction of Freund's complete adjuvant (LAATSCH et al., 1962). various fractions from phosphorylated myelin fraction. The The purified myelin basic protein has been found to myelin fraction was prepared from the cerebrum of male serve as a g o d substrate for the cyclic AMP-depen- SpragueDawley rat according to the method of UYEMURA dent protein kinases iq cytosol fractions (CARNEGIEet al. (1972) with a slight modification (MIYAMOTO & KAKet al., 1973, 1974; MIYAMOTO et al., 1974; MIYAMOTO WCHI, 1974). A typical experiment for the phosphorylation & KAKIUCHI,1974). The incubation of the myelin of the myelin fraction was carried out as follows. A myelin fraction in the presence of ATP + Mgz+ resulted in fractiop containing 46Opg of protein was incubated at the phosphorylation of the protein (CARNEGE et al., 30°C fob 2 or 40 min in a mixture consisting of 200 pmol 1974; MIYAMOTO et al., 1974; MNAMOTO & KAKIU- of sodium acetate buffer, pH 6.0, 20.0nmol of [Y-~'P]ATP (4 to 10 x lo6 c.p.m.), 1.2 pnol of ethylene glycol bis-(/?CHI, 1974; STECK& APPEL,1974). Furthermore, the aminoethy1ether)-N.N'-tetra-acetic and and 40p o l of protein has been shown by injection of orthophos- magnesium acetate with or without 10mwNaF in a total phate into ventricle of rat brain to be phosphorylated volume of 4.0 ml. After incubation, the solution was kept in tiiw (MIYAMOTO et al., 1974; MIYAMOTO & KAKILJ- at 0°C and then centrifuged at 22,0009 for 15 min at 0°C. CHI,1974; STECK& APPEL,1974) and to occur ori- The conditions, in which the samples were kept at 0°C et al., and centrifuged at 0°C for 15 min after incubation, caused ginally in a phosphorylated form (MIYAMOTU 1974; MIYAMOTO & KAKNCHI,1974). The enzyme no change in the amount of phosphate incorporated into systems of protein kinase (MIYAMOTO, 1975) and of the myelin fraction in both 2-min and 40-min incubations. phosphoprotein phosphatase (MIYAMOTO & KAKIU- The precipitate was suspended in 5 ml of a mixture of ether CHI,1975) in myelin of rat brain, which are respon- and ethanol (3:2, v/v). The suspension was centrifuged at 22,0009 for 15min. The supernatant fluid was decanted sible for the phosphorylation and dephosphorylation into a test tube. The extraction of phosphorylated myeli of myelin basic protein, have also been characterized. fraction with the mixture of ether and ethanol was perAbbreviations used: Cyclic AMP, adenosine 3',5'-monophosphate; SDS, sodium dodecyl sulphate; TCA, trichloroacetic acid; EE, ether-ethanol (2:3, v/v); CM, chloroform-methanol (2: 1, v/v).
formed three times. The supernatant fluids were combined, dried in a vacuum desiccator and designated as ether and ethanol (EE) extract. The precipitate obtained after the treatment with the mixture of ether and ethanol, which was designated as partially delipidated myelin fraction, was
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5 74
lamide gel electrophoresis, the gels were stained, destained and then cut into 2-mm slices. Radioactivity was mainly found in slices which corresponded to the bands of the myelin basic proteins. The value reached about 62% of the total radioactivity observed in the gel. Insoluble material which remained at the origin of the gel contained about 24% of the radioactivity. The autoradiogram of the gels showed the main location of the radioactivity to be in the region of the myelin basic protein bands (Fig. 1). Cyclic AMP stimulated neither the incorporation of radioactivity into two myelin basic proteins nor any special protein. The amino acid residues into which phosphate was incorporated were examined in phosphorylated myelin fraction incubated for 2 min without cyclic AMP Sodium dodecyl sulphate (SDS) polyacrylamide gel elecaccording to the method of MIYAMOTO & KAKIUCHI trophoresis. SDS polyacrylamide gel electrophoresis was (1974). After correction for hydrolysis of phosphoserperformed according to the method of WEBER& OSBORN (1969) using 10% acrylamide. Gels were stained in a solu- ine and phosphothreonine, 74, 11 and 15% of radioaction containing 0.025% Coomassie Brilliant Blue, 10% is& tivity were found to be associated with phosphoserine, propyl alcohol and 10% acetic acid for 14 h and destained phosphothreonine and other amino acids or polypepby diffusion in a solution of 5% methanol and 7% acetic tides, respectively, with recovery of about 95% comacid. Radioactivity in the gel was located by two methods. pared to the radioactivity of the myZlin fraction deterIn one method, the gel was stained, destained and then mined directly as an insoluble precipitate with tricut into 2-mm slices. Each slice of the gel was crushed chloroacetic acid (TCAJ. with a spatula. Radioactivity of each sample was deterStaining pattern of proteins in the fractions extracted mined by a liquid scintillation spectrometer of Packard model 3310. In the other method, the gel, after being from phosphorylated myelin paction on SDS polyacrystained and destained, was placed on a wet filter paper lamide gel electrophoresis and the autoradiography of and wrapped in Saran wrap. Radioactivity was located by the gels. EE extract, HCI extract, C M extract and autoradiography with Fuji KX medical X-ray film. Mc- insoluble residue which were obtained from the phoslecular weights were estimated by calibrating each electro- phorylated myelin fraction contained 3, 50, 8 and 5%, phoretic analysis with six protein standards containing respectively, of the total radioactivity incorporated cytochrome c (12,400), myoglobin (17,800), chymotryp into myelin fraction which was determined as an insinogen A (25,000),ovalbumin (45,000), bovine serum albumin (67,000) and y-globulin (160,000). Relative mobilities soluble precipitate with TCA. These fractions were of the various proteins were determined using the mig- subjected to SDS polyacrylamide gel electrophoresis; the pattern is shown in Fig. 2. The EE extract conration of cytochrome c as reference. Other methods. [Y-~'P]ATPwas prepared by the method tained almost no stain for a protein band. From the of POST & SEN (1967). Protein was measured by the extraction procedures, HCI extract, CM extract and et al. (1951), with bovine serum albumin insoluble residue were expected to contain myelin method of LOWRY as the protein standard. basic proteins, Folch-Lees proteolipid protein (FOLCH-PI& LEES,1951) and Wolfgram proteolipid RESULTS protein (WOLFGRAM,1966), respectively. The HCI Staining pattern of the proteins in myelin of rat brain extract included two myelin basic proteins and some on SDS polyacrylamide gel electrophoresis and the proteins of larger molecular weights. When a smaller autoradiography of the gels. The myelin fraction pre- amount of proteins of HCI extract was subjected to pared from rat cerebrum was incubated at 30°C for the electrophoresis, two protein bands, the molecular 2min in the presence of [y-32P]ATP M g Z f . After weights of which are 38,000 and 41,000, respectively, incubation, the reaction mixture was centrifuged at were clearly observed on the gel (not shown). These 22,OOOg for 15 min. The myelin fraction precipitated proteins were not seen on the gel of the electrowas treated 3 times with a mixture of ether and eth- phoresis for partially delipidated myelin fraction (Fig. anol (3:2, v/v). The staining pattern of proteins in 1). The main protein band in C M extract was found a partially delipidated myelin fraction on SDS polyaG to be Folch-Lees proteolipid protein which is also rylamide gel electrophoresis is illustrated in Fig. 1. shown in Fig. 1. Two more protein bands which The two myelin basic proteins and Folch-Lees pro- migrated more slowly than the proteolipid protein teolipid protein (FOLCH-PI& LEES, 1951) were the were observed on the gel of the electrophoresis for major myelin proteins which migrated as three pro- C M extract. The insoluble residue showed fine protein bands. Many other components were also visible. tein bands which were also observed on the gel for The electrophoretic pattern was very similar to the partially delipidated myelin fraction. samples incubated in the presence and absence of The autoradiography for the gel of each fraction 1 pM-cyclic AMP. After completion of SDS polyacry- is shown in Fig. 2. The radioactivity was found
suspended in 0.5 ml of 0.2 M-HCl and stirred at 4°C for 14h. The suspension was centrifuged at 2 2 , W g for 15min. The supernatant was decanted into a test tube. The extraction procedure with 0.2 M-HCl was repeated twice more. The supernatant fluids were combined, dried fn a Vacuum desigcator over sodium hydroxide and designated as HCI extract. The precipitate was suspended in 3 ml of a mixture of chloroform and methanol (2: 1, v/V). m e suspension was centrifuged at l W g for 10min. The precipitate was suspended in 3 ml of the mixture of chloroform and methanol, and centrifuged in the same way as described above. The supernatant fluids were combined, dried in a vacuum desiccator and designated chloroformmethanol (CM) extract. The precipitate obtained after the treatment with the mixture of chloroform and methanol was dried in a vacuum desiccator and designated insoluble residue.
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FIG. I . SDS polyacrylamide gel electrophoresis of proteins in partially delipidated myelin fraction after phosphorylation, and autoradiography of the gels. Myelin fraction containing 230 pg of protein was incubated at 30°C for 2 min in the absence (A) and presence (B) of 1 pM-cyclic AMP under the standard conditions as described in the text. After incubation, the phosphorylated myelin fraction was treated three times with 3 ml of a mixture of ether and ethanol (3:2, v/v) and then subjected to SDS polyacrylamide gel electrophoresis which was carried out at 5 mA per tube for 1 h, 20 min. Protein migrated from top to bottom. Left, protein staining pattern. PL, Folch-Lees proteolipid protein; SBP, small myelin basic protein ; LBP, large myelin basic protein. Right, autoradiogram, made as described in the text. The gels were placed in close contact with film for 3 days and the film was developed.
FIG.2. SDS polyacrylamide gel electrophoresis of proteins in the fractions obtained from phosphorylated myelin fraction and autoradiography of the gels. Myelin fraction containing 460 pg of protein was incubated at 30°C for 2 min in the absence (A-D) and presence (E-H) of 1 pM-cyclic AMP under the standard conditions as described in the text. After incubation, EE extract containing 20 pg of protein, HCI extract containing 144 pg of protein, CM extract containing 80 pg of protein and insoluble residue containing 28 pg of protein were obtained from phosphorylated myelin fraction as described in the text, respectively. The total amount of EE extract (A,E), HCI extract (B,F), CM extract (C,G) and insoluble residue @,H) were subjected to SDS polyacrylamide gel electrophoresis which was carried out a t 5 mA per tube for 1 h, 20 min. Protein migrated from top to bottom. Left, protein staining pattern. Pr. 1, protein 1; Pr. 2, protein 2; Pr. 3, protein 3 ; SBP, small myelin basic protein; LBP, large myelin basic protein. Right, autoradiogram, made as described in the text. The gels were placed in close contact with film for 3 days and the film was developed. NC-574
FIG.3. The autoradiography of the gels for the samples which were incubated for longer times. The myelin fraction containing 230 pg of protein was incubated at 30°C for 40 min with 10 nmol of (y-32P)ATP (1.2 x lo7 c.p.m.) in the presence of 1 0 mM-NaF under the standard conditions as described in the text. Partially delipidated myelin fraction (A,C) and HCI extract (B,D,E) were obtained from 230 pg and 460 pg of myelin fraction as protein, respectively. In A and B, SDS disc gel electrophoresis was performed as described in the text. In C and D, 8 M-urea was added to the gels with 0.1 % SDS in the use of 10% acrylamide, to which the samples containing 0 . 5 % 2-mercaptoethanol, 1.5 % SDS and 4 M-urea, were applied. In E, disc gel electrophoresis was carried out according to the method of REISFELD et nl. (1962) in the use of 15 % acrylamide. Protein migrated from top to bottom. In A to D, top was cathode and bottom anode. In E, the electrode was revcrsed. The autoradiography was performed as described in the text. The gels were placed in close contact with film for one day and the film was developed. Pr. 1, protein 1; Pr. 4, protein 4 ; Pr. 5 , protein 5 . FIG.4. Autoradiography of gels. CM extract (A) and insoluble residue (B) were obtained from 460 pg of phosphorylated myelin fraction as protein. Incubation conditions were as described in the legend to Fig. 3. The total amounts of CM extract and insoluble residue were subjected to SDS disc gel electrophoresis which was carried out at 5 mA per tube for 1 h, 30 min. Protein migrated from top to bottom. The autoradiography was performed as described in the text. The gels were placed in close contact with film for 1 day and the film was developed.
Phosphorylation of proteins in myelin of rat brain mainly in the protein bands of the HCl extract. In the gel of the HCI extract, two myelin basic proteins and three proteins of larger molecular weights were founds to be phosphorylated (Fig. 2). The apparent molecular weights of the phosphorylated proteins other than myelin basic proteins were found to be 60,000 (protein l), 41,000 (protein 2) and 38,000 (protein 3), respectively, using 6 protein standards. The direct measurement of radioactivity in the gel slices showed that protein 1, protein 2 3 and two myelin basic proteins contained about 5, 20 and 69% of the total radioactivity observed in the gel of the HCl extract, respectively. Cyclic AMP did not stimulate the phosphorylation of these five proteins under the conditions used. The total radioactivity in the gel of the HCl extract and the incorporation of radioactivity into each protein band were similar between the samples incubated in the presence and absence of CyClic AMP. A fine protein band (mol wt 21,000) was observed on the gel from electrophoresis of the HCI extract which migrated more slowly than large myelin basic proteins. It was not clear, from the detection methods of radioactivity used, whether or not this protein is phosphorylated. The gels from electrophoresis of the CM extract and insoluble residue contained radioactivity, the location of which corresponded to the origin and myelin basic protein bands, but not to those of proteins in CM extract and in insoluble residue (Fig. 2). The radioactivity observed at the origin of the gels for CM extract and insoluble residue may account for most of the material that stayed at the origin of the gel for partially delipidated myelin fraction. The direct measurement of radioactivity in the gel slices revealed that the total radioactivity observed at the position of myelin basic proteins in the gels for CM extract and insoluble residue was found to constitute about 3 and 1%of that observed in the gel for HCl extract, respectively. The radioactivity derived from myelin basic proteins is probably contaminating the CM extract and insoluble residue. According to extraction procedures used, the percentage of radioactivity located at the position of myelin basic protein bands on the gels for CM extract and insoluble residue fluctuated in the range of each value k0.5 to 1.0%. From the results of five similar experiments, this radioactivity included in the gels was not found to be stimulated by cyclic AMP. It was, therefore, concluded that proteins soluble in chlorofom-methanol after treatment with hydrochloric acid, and insoluble in hydrochloric acid and chloroform-methanol are not phosphorylated under the conditions used. The endogenous phosphorylation of proteins in myeZinpaction by incubation for longer times. The myelin fraction was incubated for 40 min with radioactive ATP of higher specific activity in the presence of 10mM-NaF. NaF had no effect on the labelling pattern of the myelin proteins or incorporation of phosphate into myelin fraction incubated for 2 min (MIYA-
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MoTo, 1975). In contrast, the amount of phosphate incorporated into myelin fraction which was incubated for 40min increased 6 to 7 times more in the Presence Of 10mwNaF than into that incubated for 2min Or for Mmin in the absence of NaF at the fixed concentration of ATP (MIYAMOTO, 1975). Partially delipidated myelin fraction, HCl extract, CM extract and insoluble residue which were obtained from phosphorylated myelin fractions contained 62, 42, 5 and 5”/,, respectively, of total radioactivity incorporated into the myelin fraction, which was determined as an insoluble precipitate with TCA. These fractions were subjected to disc gel electrophoresis, The partially delipidated myelin fraction and HCI extract were electrophoresed on the gels in three different conditions, i.e. SDS, SDS plus 8M-urea and pH 4.3. After disc gel electrophoresis, staining and destaining of proteins in the gels, the autoradiography was performed (Fig. 3). The phosphorylation of protein 1 was more visible on the gels for partially delipidated myelin fraction than for HCl extract by incubation for longer times. Little incorporation of phosphate into proteins 2 and 3 were observed on the gels for the partially delipidated myelin fraction. The phosphorylation of two more proteins which migrated more slowly than protein 1 was observed, and designated as proteins 4 and 5. The molecular weights of proteins 4 and 5 were determined to be 120,000 and 76,000, respectively. Myelin basic proteins and proteins 1, 4 and 5 contained 49: 6, 4 and 5%, respectively, of total radioactivity incorporated into the partially delipidated myelin fraction. On the electrophoresis gel performed according to the method of REISFELDet al. (1962), incorporation of phosphate was only observed into the bands of myelin basic proteins (Fig. 3E). In separate experiments, incorporation of phosphate into two fine protein bands which migrated more slowly than myelin basic proteins was observed on the gels for HCl extract, performed at pH 4.3. These proteins may correspond to proteins 1: 2 or 3. The electrophoretic pattern of proteins was identical on the gels containing SDS and SDS plus 8 M-urea. The autoradiograms of the electrophoresis gels performed by the two methods were also essentially similar to each other (Fig. 3B and D). Therefore, it seems unlikely that proteins 1 , 2 and 3 are aggregate forms of large and small myelin basic proteins. However, the possibility is not yet excluded, that the phosphorylated higher molecular weight proteins, such as proteins 1 to 5, are formed from myelin basic proteins during incubation or fractionation, since a polymer formed from monomeric peptides of myelin basic protein appears under certain conditions on column chromatography on gel filtration (MARTENSON et al., 1975), and a polymer of myelin basic protein exists with a monomer even on SDS polyacrylamide gel & DEIBLER, 1975). The electrophoresis (MARTENSON autoradiogram of the gels for insoluble residue showed incorporation of phosphate into proteins 1,
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4 and 5 (Fig. 4B). In view of the extraction procedures teins in myelin may be due to the fact that Cyclic performed, the possibility cannot be excluded that a AMP can not reach the regulatory site of the enzyme protein of mol wt 60,000 observed on the gels for in myelin. Another reason may be that the regulatory partially delipidated myelin fraction and insoluble subunit, an inhibitory component for the enzymic aG residue is not identical with protein 1 observed on tivity, cannot be removed from the protein kinase for the integration of enzyme molecule into myelin strue the gel for HCl extract. The amino acid residues into which phosphate was ture, since evidence has been presented that the incorporated were examined in the phosphorylated mechanism by which cyclic AMP activates Cyclic myelin fraction incubated for 40 min without cyclic AMP-dependent protein kinase involves dissociation AMP in the presence of 10mM-NaF according to the of the holoenzyme into a regulatory subunit and catamethod of MIYAMOTO & KAKIUCHI (1974). After cor- lytic subunit (GILL& GARREN,1970; TAOet al., 1970; et al., 1971; REIMANN rection for hydrolysis of phosphoserine and phos- KIJMONet al., 1970; ERLICHMAN et al., 1971, 1973). It may be phothreonine, 85, 6 and 9% of radioactivity were et al., 1971; MIYAMOTO found to be associated with phosphoserine, phos- possible that the phosphorylation of the proteins is phothreonine and other amino acids or polypeptides, dependent on cyclic AMP under the appropriate conrespectively, with recovery of about 70%, compared ditions, as the activity of solubilized protein kinase to radioactivity determined directly as insoluble pre- from myelin fraction is stimulated by cyclic AMP (MIYAMOTO, 1975), and the phosphorylation of puricipitate with TCA. fied myelin basic protein is dependent on cyclic AMP DISCUSSION by incubation with cytosol enzymes (CARNEGIE et al., The results described in this communication indi- 1973; MJYAMOTO et al., 1974; MIYAMOTO & KAKILIcate that at least seven proteins which can be phos- CHI, 1974). phorylated occur in the myelin of rat brain, two of The protein kinase modulator which acts primarily which are large and small myelin basic proteins, and on the catalytic site of protein kinase had no effect the other proteins of mol wt 120,000, 76,000, 60,000, on the endogenous phosphorylation of the myelin 41,000 and 38,000, respectively. The proteins of mol fraction, whereas it inhibited the activity of the solubiwt 120,000 and 76,000 were insoluble in hydrochloric lized enzyme from the myelin fraction (MIYAMOTO, acid and chloroform-methanol. In view of the mo- 1975). The addition of histone or exogenous protein lecular weights (WIGGINSet at., 1974), these proteins kinase to the endogenous phosphorylation system may not be identical with Wolfgram proteolipid pro- resulted in a slight increase in the incorporation of tein. The phosphorylation of Folch-Lees proteolipid phosphate into myelin fraction without NaF (MIYAprotein was not observed under the conditions stud- MOT0 & KAKIUCHI, 1974). However, this increase was ied. The phosphorylation of proteins 1 to 5 was not much smaller than that caused by the addition of shown in the experiments of CARNEGIE et at. (1974), 1OmM-NaF to the system and, therefore, was not our previous report (MIYAMOTO & KAKILICHI, 1974) thought to reflect the real effect of histone or the exoor STECK& APPEL (1974). This is thought to be for genous protein kinase (MIYAMOTO, 1975). Thus, it was the following reasons. (a) We performed disc gel elec- found that exogenous substrate added to the endotrophoresis for HC1 extract according to the method genous phosphorylation system of myelin did not of REIsFELDet al. (1962). As shown in Fig. 3, the phos- serve as substrate for the myelin-associated protein phorylation of proteins 1, 2 and 3 which were kinase (MIYAMOTO, 1975). Furthermore, exogenous extracted by treatment with hydrochloric acid were protein kinase added to the endogenous phosphorylanot easily Seen under the conditions of the electro- tion system could not utilize the endogenous subphoresis. Proteins 4 and 5 were not extracted by treat- strate in the myelin fraction (MIYAMOTO, 1975). These ment with hydrochloric acid. (b) Incorporation of results indicate that the catalytic site of enzyme and phosphate into proteins 1 to 5 was much smaller than substrate protein in myelin may be integrated into that into large and small myelin basic proteins in the membrane structure, and that the exogenous subterms of rate and amount. The phosphorylation of strate or exogenous protein kinase is, therefore, not these proteins was observed only by incubation of accessible to their appropriate sites. It suggests that the myelin fraction for longer times using radioactive the in vitro endogenous phosphorylation of myelin ATP of high specific activity and fairly large amounts fraction described in this study may be based on the of myelin fraction as protein. spatial orientation of enzyme and substrate in vivo; The endogenous phosphorylation of the proteins random orientation of both may cause the non-speciwas not stimulated by cyclic AMP under the condi- fic phosphorylation of alternative sites. tions used. It has been reported that the exogenous It has been reported that a protein of mol wt 50,000 cyclic AMP was only partially accessible to cyclic which can be phosphorylated, is found in prepAMP-binding site in myelin and that the regulatory arations from cytoplasm of mammalian brain (UEDA subunit of cyclic AMP-dependent protein kinase in et al., 1973), ductus deferens, uterus and small intesmyelin may be integrated into its membrane structure tine (CASNELLIE & GREENGARD, 1974) and membrane (MIYAMOTO, 1975). The inabilify of cyclic AMP to fractions of toad bladder (DELORENZOet al., 1 9 9 , stimulate the phosphorylation of the endogenous pro- ductus deferens, uterus and small intestine (CASNELLIE
Phosphorylation of proteins in myelin of rat brain
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& GREENGARD, 1974). This protein is thought to be LOMY 0. H., ROSEBROUGH N . J., FARRA. L. & RANDALL the regulatory subunit of a cycic AMP-dependent R. J. (1951) J . biol. Chem. 193, 265-275. protein kinase in the case of brain cytoplasm (MAENO MAENOH., REYESP. L., UEDAT., RUDOLPHS. A. & GREENGARD P. (1974) Archs Biochem. Biophys. 164, 551-559. et al., 1974). In view of the fact that both cyclic AMPR. E. & DEIBLER G. E. (1975) J . Neurocheni. dependent protein kinase and cyclic AMP-binding MARTENSON 24, 79-88. protein are present in myelin of rat brain (MIYAMOTO, MARTENSON R. E., DEIBLERG . E. & KRAMER A. J. (1975) 1975), one of the proteins in myelin, which can be J . Neurochem. 24, 173-182. phosphorylated, may be identical with those found MIYAMOTO E. (1975) J . Neurochem. 24, 503-512. in preparations from the cytoplasm and the mem- MIYAMOTOE. & KAKIUCHIS. (1974) J . biol. Chem. 249, brane fractions described above. However, a protein 2769-2777. of the same molecular weight was not found among MIYAMOTO E. & KAKIUCHI S. (1975) Biochim. biophys. Acta those phosphorylated in myelin. Further study will 384, 458-465. MIYAMOTO E., KAKKJCIII S. & KAKJMOTO Y. (1974) Nuture, be needed to clarify this problem. L m d . 249, 151-152. MIYAMOTO E., K ~ J J. O F. & GREENGARD P. (1969) Science, N.Y 165, 63-65. MIYAMOTO E., PETZOLD G. L., HARRISJ. S. & GREENGARD P. (1971) Biochem. biophys. Res. Commun. 44, 305-312. REFERENCES MIYAMOTO E., PETZOLDG. L., Kuo J. I;. & GREENGARD CARNEGIEP. R., KEMPB. E., DUNKLEY P. R. & MURRAY P. (1973) J . biol. Chem. 248, 179-189. POSTR. L. & SEN A. K. (1967) in Methods in Enzymology A. W. (1973) Biochem. J . 135, 569-572. R. W. & PULLMAN M. E., eds.) Vol. X, pp. CARNEGIE P. R., DUNKLEY P. R., KEMPB. E. & MURRAY (ESTABROOK 773-776. Academic Press, New York. A. W. (1974) Nature, Lond. 249, 147-150. C. O., CORBINJ. D., KING C. CASNELLIE J. E. & GREENGARD P. (1974) Proc. natn. Acad. REIMANNE. M., BROSTROM A. C KREBSE. G. (1971) Biochem. biophys. Res. Cornmun. Sci. U.S.A. 71, 1891-1895. 42, 187-194. DELORENZOR. J., WALTONK. G., CURRAN P. F. & R., LEWISU. & WILLIAMS D. (1962) Nafure, Lond. GREENCARD P. (1973) Proc. natn. Acad. Sci. U.S.A. 70, REISFELD 195, 281-283. 88&884. ERLICHMAN J., HIRSCHA. H. & ROSEN0. M. (1971) Proc. STECKA. J. & APPEL S. H. (1974) J . bid. Chern. 249, 541&5420. natn. Acad. Sci. U.S.A. 68, 731-735. FOLCH-PI J. & LEESM. (1951) J . bid. Chem. 191, 807-817. TAOM., SALASM. L. & LIPMANNF. (1970) Proc. natn. Acud. Sci. U.S.A. 67, 408414. GILL G. N. & GARREN L. D. (1970) Biochem. biophys. Re.?. UEDAT., MAENOH. & GREENGARD P. (1973) J . biol. Chem. Commun. 39, 335-343. 248, 8295--8305. GREENGARD P. & K u o J. F. (1970) in Role of Cyclic A M P K., TOBARIC., HIRANOS. & TSUKADA Y. (1972) in Cell Function (GREENGARD P. & COSTAE., eds.) pp. UYEMURA J . Neurochem. 19, 2607-2614. 287-306. Raven Press, New York. J. P. & KRERSE. G. (1968) J . bid. KUMONA,, YAMAMURA H. & NISHIZUKA Y. (1970) Biochem. WALSHD. A,, PERKINS Chem. 243, 3763-3765. biophys. Res. Commun. 41, 129&1297. Kuo J. F. & GREENGARD P. (1969) Proc. natn. Acad. Sci. WEBER K. & OSBORNM. (1969) J . biol. Chem. 244, 440-4 12. U.S.A. 64, 1349-1355. LAATSCHR. H., KIES M. W., GORDON S. & ALVORDE. WIGGINSR. C., JOFFES., DAVIDSOND. & DEI. VALLE u. (1974) J . Neurochem. 22, 171-175. C. (1962) J . exp. Med. 115, 777-788. WOLFGRAM F. (1966) J . Neurochem. 13, 461-470. LANGAN T. A. (1968) Science, N.Y 162, 579-581.
Acknowledgemenl-The author wishes to thank Mr. A. NISHIDAof this laboratory for his help with the autoradiography and photography.
PHOSPHORYLATION OF ENDOGENOUS PROTEINS IN MYELIN OF RAT BRAIN E. MIYAMOTO The Research Division and Clinical Laboratory, Nakamiya Mental Hospital, Hirakata, Osaka, Japan (Receiaed 12 June 1975. Accepted 20 August 1975)
Abstract-The phosphorylation of endogenous proteins occurring in the myelin of rat brain was examined using the method of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Two myelin basic proteins and at least five more proteins were phosphorylated after incubation of myelin fraction in the presence of ATP Mg2+.The apparent molecular weights of the proteins other than the myelin basic proteins were 120,000, 76,000, 60,000, 41,000 and 38,000, respectively. The proteins of mol wt 60,OOO. 41,000 and 38,000 were extracted by treatment with hydrochloric acid, whereas those of mol wt 120,000 and 76,000 were insoluble in hydrochloric acid and chloroform-methanol. Folch-Lees proteolipid protein was not found to be phosphorylated under the conditions studied. The endogenous phosphorylation of the proteins was not stimulated by adenosine 3',5'-monophosphate.
+
AFTER the discovery of the adenosine 3',5'-monophos-
The present study describes experiments demonstratphate (cyclic AMP)dependent protein kinase in skele- ing the Occurrence of at least seven endogenous protal muscle (WALSHe f al., 1968), liver (LANGAN, 1968) teins in myelin of rat brain, which are phosphorylated and brain (MIYAMOTO et al., 1969), it was proposed after incubation with ATP + Mg2+. that the diverse actions of cyclic AMP in various tissues are mediated through the activation of the cyclic MATERIALS AND METHODS AMP-dependent protein kinase (Kuo & GREENGARD, Materials. Cyclic AMP was obtained from Kohjin Co. 1969; GREENGARD & Kuo, 1970). The myelin of brain and spinal cord contains a Cytochrome c, myoglobin, chymotrypsinogen A, ovalbubasic protein which is known as an experimental min, bovine serum albumin and ?-globulin were purchased (carrier-free) allergic encephalomyelitogenic protein, since it in- from Schwarz-Mann. Orth~[~~P]phosphate was obtained from Japan Radioisotope Association. duces the disease in animals when injected with Phosphorylation of myelin fraction and extraction of Freund's complete adjuvant (LAATSCH et al., 1962). various fractions from phosphorylated myelin fraction. The The purified myelin basic protein has been found to myelin fraction was prepared from the cerebrum of male serve as a g o d substrate for the cyclic AMP-depen- SpragueDawley rat according to the method of UYEMURA dent protein kinases iq cytosol fractions (CARNEGIEet al. (1972) with a slight modification (MIYAMOTO & KAKet al., 1973, 1974; MIYAMOTO et al., 1974; MIYAMOTO WCHI, 1974). A typical experiment for the phosphorylation & KAKIUCHI,1974). The incubation of the myelin of the myelin fraction was carried out as follows. A myelin fraction in the presence of ATP + Mgz+ resulted in fractiop containing 46Opg of protein was incubated at the phosphorylation of the protein (CARNEGE et al., 30°C fob 2 or 40 min in a mixture consisting of 200 pmol 1974; MIYAMOTO et al., 1974; MNAMOTO & KAKIU- of sodium acetate buffer, pH 6.0, 20.0nmol of [Y-~'P]ATP (4 to 10 x lo6 c.p.m.), 1.2 pnol of ethylene glycol bis-(/?CHI, 1974; STECK& APPEL,1974). Furthermore, the aminoethy1ether)-N.N'-tetra-acetic and and 40p o l of protein has been shown by injection of orthophos- magnesium acetate with or without 10mwNaF in a total phate into ventricle of rat brain to be phosphorylated volume of 4.0 ml. After incubation, the solution was kept in tiiw (MIYAMOTO et al., 1974; MIYAMOTO & KAKILJ- at 0°C and then centrifuged at 22,0009 for 15 min at 0°C. CHI,1974; STECK& APPEL,1974) and to occur ori- The conditions, in which the samples were kept at 0°C et al., and centrifuged at 0°C for 15 min after incubation, caused ginally in a phosphorylated form (MIYAMOTU 1974; MIYAMOTO & KAKNCHI,1974). The enzyme no change in the amount of phosphate incorporated into systems of protein kinase (MIYAMOTO, 1975) and of the myelin fraction in both 2-min and 40-min incubations. phosphoprotein phosphatase (MIYAMOTO & KAKIU- The precipitate was suspended in 5 ml of a mixture of ether CHI,1975) in myelin of rat brain, which are respon- and ethanol (3:2, v/v). The suspension was centrifuged at 22,0009 for 15min. The supernatant fluid was decanted sible for the phosphorylation and dephosphorylation into a test tube. The extraction of phosphorylated myeli of myelin basic protein, have also been characterized. fraction with the mixture of ether and ethanol was perAbbreviations used: Cyclic AMP, adenosine 3',5'-monophosphate; SDS, sodium dodecyl sulphate; TCA, trichloroacetic acid; EE, ether-ethanol (2:3, v/v); CM, chloroform-methanol (2: 1, v/v).
formed three times. The supernatant fluids were combined, dried in a vacuum desiccator and designated as ether and ethanol (EE) extract. The precipitate obtained after the treatment with the mixture of ether and ethanol, which was designated as partially delipidated myelin fraction, was
573
E. MIYAMOTO
5 74
lamide gel electrophoresis, the gels were stained, destained and then cut into 2-mm slices. Radioactivity was mainly found in slices which corresponded to the bands of the myelin basic proteins. The value reached about 62% of the total radioactivity observed in the gel. Insoluble material which remained at the origin of the gel contained about 24% of the radioactivity. The autoradiogram of the gels showed the main location of the radioactivity to be in the region of the myelin basic protein bands (Fig. 1). Cyclic AMP stimulated neither the incorporation of radioactivity into two myelin basic proteins nor any special protein. The amino acid residues into which phosphate was incorporated were examined in phosphorylated myelin fraction incubated for 2 min without cyclic AMP Sodium dodecyl sulphate (SDS) polyacrylamide gel elecaccording to the method of MIYAMOTO & KAKIUCHI trophoresis. SDS polyacrylamide gel electrophoresis was (1974). After correction for hydrolysis of phosphoserperformed according to the method of WEBER& OSBORN (1969) using 10% acrylamide. Gels were stained in a solu- ine and phosphothreonine, 74, 11 and 15% of radioaction containing 0.025% Coomassie Brilliant Blue, 10% is& tivity were found to be associated with phosphoserine, propyl alcohol and 10% acetic acid for 14 h and destained phosphothreonine and other amino acids or polypepby diffusion in a solution of 5% methanol and 7% acetic tides, respectively, with recovery of about 95% comacid. Radioactivity in the gel was located by two methods. pared to the radioactivity of the myZlin fraction deterIn one method, the gel was stained, destained and then mined directly as an insoluble precipitate with tricut into 2-mm slices. Each slice of the gel was crushed chloroacetic acid (TCAJ. with a spatula. Radioactivity of each sample was deterStaining pattern of proteins in the fractions extracted mined by a liquid scintillation spectrometer of Packard model 3310. In the other method, the gel, after being from phosphorylated myelin paction on SDS polyacrystained and destained, was placed on a wet filter paper lamide gel electrophoresis and the autoradiography of and wrapped in Saran wrap. Radioactivity was located by the gels. EE extract, HCI extract, C M extract and autoradiography with Fuji KX medical X-ray film. Mc- insoluble residue which were obtained from the phoslecular weights were estimated by calibrating each electro- phorylated myelin fraction contained 3, 50, 8 and 5%, phoretic analysis with six protein standards containing respectively, of the total radioactivity incorporated cytochrome c (12,400), myoglobin (17,800), chymotryp into myelin fraction which was determined as an insinogen A (25,000),ovalbumin (45,000), bovine serum albumin (67,000) and y-globulin (160,000). Relative mobilities soluble precipitate with TCA. These fractions were of the various proteins were determined using the mig- subjected to SDS polyacrylamide gel electrophoresis; the pattern is shown in Fig. 2. The EE extract conration of cytochrome c as reference. Other methods. [Y-~'P]ATPwas prepared by the method tained almost no stain for a protein band. From the of POST & SEN (1967). Protein was measured by the extraction procedures, HCI extract, CM extract and et al. (1951), with bovine serum albumin insoluble residue were expected to contain myelin method of LOWRY as the protein standard. basic proteins, Folch-Lees proteolipid protein (FOLCH-PI& LEES,1951) and Wolfgram proteolipid RESULTS protein (WOLFGRAM,1966), respectively. The HCI Staining pattern of the proteins in myelin of rat brain extract included two myelin basic proteins and some on SDS polyacrylamide gel electrophoresis and the proteins of larger molecular weights. When a smaller autoradiography of the gels. The myelin fraction pre- amount of proteins of HCI extract was subjected to pared from rat cerebrum was incubated at 30°C for the electrophoresis, two protein bands, the molecular 2min in the presence of [y-32P]ATP M g Z f . After weights of which are 38,000 and 41,000, respectively, incubation, the reaction mixture was centrifuged at were clearly observed on the gel (not shown). These 22,OOOg for 15 min. The myelin fraction precipitated proteins were not seen on the gel of the electrowas treated 3 times with a mixture of ether and eth- phoresis for partially delipidated myelin fraction (Fig. anol (3:2, v/v). The staining pattern of proteins in 1). The main protein band in C M extract was found a partially delipidated myelin fraction on SDS polyaG to be Folch-Lees proteolipid protein which is also rylamide gel electrophoresis is illustrated in Fig. 1. shown in Fig. 1. Two more protein bands which The two myelin basic proteins and Folch-Lees pro- migrated more slowly than the proteolipid protein teolipid protein (FOLCH-PI& LEES, 1951) were the were observed on the gel of the electrophoresis for major myelin proteins which migrated as three pro- C M extract. The insoluble residue showed fine protein bands. Many other components were also visible. tein bands which were also observed on the gel for The electrophoretic pattern was very similar to the partially delipidated myelin fraction. samples incubated in the presence and absence of The autoradiography for the gel of each fraction 1 pM-cyclic AMP. After completion of SDS polyacry- is shown in Fig. 2. The radioactivity was found
suspended in 0.5 ml of 0.2 M-HCl and stirred at 4°C for 14h. The suspension was centrifuged at 2 2 , W g for 15min. The supernatant was decanted into a test tube. The extraction procedure with 0.2 M-HCl was repeated twice more. The supernatant fluids were combined, dried fn a Vacuum desigcator over sodium hydroxide and designated as HCI extract. The precipitate was suspended in 3 ml of a mixture of chloroform and methanol (2: 1, v/V). m e suspension was centrifuged at l W g for 10min. The precipitate was suspended in 3 ml of the mixture of chloroform and methanol, and centrifuged in the same way as described above. The supernatant fluids were combined, dried in a vacuum desiccator and designated chloroformmethanol (CM) extract. The precipitate obtained after the treatment with the mixture of chloroform and methanol was dried in a vacuum desiccator and designated insoluble residue.
+
FIG. I . SDS polyacrylamide gel electrophoresis of proteins in partially delipidated myelin fraction after phosphorylation, and autoradiography of the gels. Myelin fraction containing 230 pg of protein was incubated at 30°C for 2 min in the absence (A) and presence (B) of 1 pM-cyclic AMP under the standard conditions as described in the text. After incubation, the phosphorylated myelin fraction was treated three times with 3 ml of a mixture of ether and ethanol (3:2, v/v) and then subjected to SDS polyacrylamide gel electrophoresis which was carried out at 5 mA per tube for 1 h, 20 min. Protein migrated from top to bottom. Left, protein staining pattern. PL, Folch-Lees proteolipid protein; SBP, small myelin basic protein ; LBP, large myelin basic protein. Right, autoradiogram, made as described in the text. The gels were placed in close contact with film for 3 days and the film was developed.
FIG.2. SDS polyacrylamide gel electrophoresis of proteins in the fractions obtained from phosphorylated myelin fraction and autoradiography of the gels. Myelin fraction containing 460 pg of protein was incubated at 30°C for 2 min in the absence (A-D) and presence (E-H) of 1 pM-cyclic AMP under the standard conditions as described in the text. After incubation, EE extract containing 20 pg of protein, HCI extract containing 144 pg of protein, CM extract containing 80 pg of protein and insoluble residue containing 28 pg of protein were obtained from phosphorylated myelin fraction as described in the text, respectively. The total amount of EE extract (A,E), HCI extract (B,F), CM extract (C,G) and insoluble residue @,H) were subjected to SDS polyacrylamide gel electrophoresis which was carried out a t 5 mA per tube for 1 h, 20 min. Protein migrated from top to bottom. Left, protein staining pattern. Pr. 1, protein 1; Pr. 2, protein 2; Pr. 3, protein 3 ; SBP, small myelin basic protein; LBP, large myelin basic protein. Right, autoradiogram, made as described in the text. The gels were placed in close contact with film for 3 days and the film was developed. NC-574
FIG.3. The autoradiography of the gels for the samples which were incubated for longer times. The myelin fraction containing 230 pg of protein was incubated at 30°C for 40 min with 10 nmol of (y-32P)ATP (1.2 x lo7 c.p.m.) in the presence of 1 0 mM-NaF under the standard conditions as described in the text. Partially delipidated myelin fraction (A,C) and HCI extract (B,D,E) were obtained from 230 pg and 460 pg of myelin fraction as protein, respectively. In A and B, SDS disc gel electrophoresis was performed as described in the text. In C and D, 8 M-urea was added to the gels with 0.1 % SDS in the use of 10% acrylamide, to which the samples containing 0 . 5 % 2-mercaptoethanol, 1.5 % SDS and 4 M-urea, were applied. In E, disc gel electrophoresis was carried out according to the method of REISFELD et nl. (1962) in the use of 15 % acrylamide. Protein migrated from top to bottom. In A to D, top was cathode and bottom anode. In E, the electrode was revcrsed. The autoradiography was performed as described in the text. The gels were placed in close contact with film for one day and the film was developed. Pr. 1, protein 1; Pr. 4, protein 4 ; Pr. 5 , protein 5 . FIG.4. Autoradiography of gels. CM extract (A) and insoluble residue (B) were obtained from 460 pg of phosphorylated myelin fraction as protein. Incubation conditions were as described in the legend to Fig. 3. The total amounts of CM extract and insoluble residue were subjected to SDS disc gel electrophoresis which was carried out at 5 mA per tube for 1 h, 30 min. Protein migrated from top to bottom. The autoradiography was performed as described in the text. The gels were placed in close contact with film for 1 day and the film was developed.
Phosphorylation of proteins in myelin of rat brain mainly in the protein bands of the HCl extract. In the gel of the HCI extract, two myelin basic proteins and three proteins of larger molecular weights were founds to be phosphorylated (Fig. 2). The apparent molecular weights of the phosphorylated proteins other than myelin basic proteins were found to be 60,000 (protein l), 41,000 (protein 2) and 38,000 (protein 3), respectively, using 6 protein standards. The direct measurement of radioactivity in the gel slices showed that protein 1, protein 2 3 and two myelin basic proteins contained about 5, 20 and 69% of the total radioactivity observed in the gel of the HCl extract, respectively. Cyclic AMP did not stimulate the phosphorylation of these five proteins under the conditions used. The total radioactivity in the gel of the HCl extract and the incorporation of radioactivity into each protein band were similar between the samples incubated in the presence and absence of CyClic AMP. A fine protein band (mol wt 21,000) was observed on the gel from electrophoresis of the HCI extract which migrated more slowly than large myelin basic proteins. It was not clear, from the detection methods of radioactivity used, whether or not this protein is phosphorylated. The gels from electrophoresis of the CM extract and insoluble residue contained radioactivity, the location of which corresponded to the origin and myelin basic protein bands, but not to those of proteins in CM extract and in insoluble residue (Fig. 2). The radioactivity observed at the origin of the gels for CM extract and insoluble residue may account for most of the material that stayed at the origin of the gel for partially delipidated myelin fraction. The direct measurement of radioactivity in the gel slices revealed that the total radioactivity observed at the position of myelin basic proteins in the gels for CM extract and insoluble residue was found to constitute about 3 and 1%of that observed in the gel for HCl extract, respectively. The radioactivity derived from myelin basic proteins is probably contaminating the CM extract and insoluble residue. According to extraction procedures used, the percentage of radioactivity located at the position of myelin basic protein bands on the gels for CM extract and insoluble residue fluctuated in the range of each value k0.5 to 1.0%. From the results of five similar experiments, this radioactivity included in the gels was not found to be stimulated by cyclic AMP. It was, therefore, concluded that proteins soluble in chlorofom-methanol after treatment with hydrochloric acid, and insoluble in hydrochloric acid and chloroform-methanol are not phosphorylated under the conditions used. The endogenous phosphorylation of proteins in myeZinpaction by incubation for longer times. The myelin fraction was incubated for 40 min with radioactive ATP of higher specific activity in the presence of 10mM-NaF. NaF had no effect on the labelling pattern of the myelin proteins or incorporation of phosphate into myelin fraction incubated for 2 min (MIYA-
+
\
V. 26 3-
J
575
MoTo, 1975). In contrast, the amount of phosphate incorporated into myelin fraction which was incubated for 40min increased 6 to 7 times more in the Presence Of 10mwNaF than into that incubated for 2min Or for Mmin in the absence of NaF at the fixed concentration of ATP (MIYAMOTO, 1975). Partially delipidated myelin fraction, HCl extract, CM extract and insoluble residue which were obtained from phosphorylated myelin fractions contained 62, 42, 5 and 5”/,, respectively, of total radioactivity incorporated into the myelin fraction, which was determined as an insoluble precipitate with TCA. These fractions were subjected to disc gel electrophoresis, The partially delipidated myelin fraction and HCI extract were electrophoresed on the gels in three different conditions, i.e. SDS, SDS plus 8M-urea and pH 4.3. After disc gel electrophoresis, staining and destaining of proteins in the gels, the autoradiography was performed (Fig. 3). The phosphorylation of protein 1 was more visible on the gels for partially delipidated myelin fraction than for HCl extract by incubation for longer times. Little incorporation of phosphate into proteins 2 and 3 were observed on the gels for the partially delipidated myelin fraction. The phosphorylation of two more proteins which migrated more slowly than protein 1 was observed, and designated as proteins 4 and 5. The molecular weights of proteins 4 and 5 were determined to be 120,000 and 76,000, respectively. Myelin basic proteins and proteins 1, 4 and 5 contained 49: 6, 4 and 5%, respectively, of total radioactivity incorporated into the partially delipidated myelin fraction. On the electrophoresis gel performed according to the method of REISFELDet al. (1962), incorporation of phosphate was only observed into the bands of myelin basic proteins (Fig. 3E). In separate experiments, incorporation of phosphate into two fine protein bands which migrated more slowly than myelin basic proteins was observed on the gels for HCl extract, performed at pH 4.3. These proteins may correspond to proteins 1: 2 or 3. The electrophoretic pattern of proteins was identical on the gels containing SDS and SDS plus 8 M-urea. The autoradiograms of the electrophoresis gels performed by the two methods were also essentially similar to each other (Fig. 3B and D). Therefore, it seems unlikely that proteins 1 , 2 and 3 are aggregate forms of large and small myelin basic proteins. However, the possibility is not yet excluded, that the phosphorylated higher molecular weight proteins, such as proteins 1 to 5, are formed from myelin basic proteins during incubation or fractionation, since a polymer formed from monomeric peptides of myelin basic protein appears under certain conditions on column chromatography on gel filtration (MARTENSON et al., 1975), and a polymer of myelin basic protein exists with a monomer even on SDS polyacrylamide gel & DEIBLER, 1975). The electrophoresis (MARTENSON autoradiogram of the gels for insoluble residue showed incorporation of phosphate into proteins 1,
516
E. MWAMOTO
4 and 5 (Fig. 4B). In view of the extraction procedures teins in myelin may be due to the fact that Cyclic performed, the possibility cannot be excluded that a AMP can not reach the regulatory site of the enzyme protein of mol wt 60,000 observed on the gels for in myelin. Another reason may be that the regulatory partially delipidated myelin fraction and insoluble subunit, an inhibitory component for the enzymic aG residue is not identical with protein 1 observed on tivity, cannot be removed from the protein kinase for the integration of enzyme molecule into myelin strue the gel for HCl extract. The amino acid residues into which phosphate was ture, since evidence has been presented that the incorporated were examined in the phosphorylated mechanism by which cyclic AMP activates Cyclic myelin fraction incubated for 40 min without cyclic AMP-dependent protein kinase involves dissociation AMP in the presence of 10mM-NaF according to the of the holoenzyme into a regulatory subunit and catamethod of MIYAMOTO & KAKIUCHI (1974). After cor- lytic subunit (GILL& GARREN,1970; TAOet al., 1970; et al., 1971; REIMANN rection for hydrolysis of phosphoserine and phos- KIJMONet al., 1970; ERLICHMAN et al., 1971, 1973). It may be phothreonine, 85, 6 and 9% of radioactivity were et al., 1971; MIYAMOTO found to be associated with phosphoserine, phos- possible that the phosphorylation of the proteins is phothreonine and other amino acids or polypeptides, dependent on cyclic AMP under the appropriate conrespectively, with recovery of about 70%, compared ditions, as the activity of solubilized protein kinase to radioactivity determined directly as insoluble pre- from myelin fraction is stimulated by cyclic AMP (MIYAMOTO, 1975), and the phosphorylation of puricipitate with TCA. fied myelin basic protein is dependent on cyclic AMP DISCUSSION by incubation with cytosol enzymes (CARNEGIE et al., The results described in this communication indi- 1973; MJYAMOTO et al., 1974; MIYAMOTO & KAKILIcate that at least seven proteins which can be phos- CHI, 1974). phorylated occur in the myelin of rat brain, two of The protein kinase modulator which acts primarily which are large and small myelin basic proteins, and on the catalytic site of protein kinase had no effect the other proteins of mol wt 120,000, 76,000, 60,000, on the endogenous phosphorylation of the myelin 41,000 and 38,000, respectively. The proteins of mol fraction, whereas it inhibited the activity of the solubiwt 120,000 and 76,000 were insoluble in hydrochloric lized enzyme from the myelin fraction (MIYAMOTO, acid and chloroform-methanol. In view of the mo- 1975). The addition of histone or exogenous protein lecular weights (WIGGINSet at., 1974), these proteins kinase to the endogenous phosphorylation system may not be identical with Wolfgram proteolipid pro- resulted in a slight increase in the incorporation of tein. The phosphorylation of Folch-Lees proteolipid phosphate into myelin fraction without NaF (MIYAprotein was not observed under the conditions stud- MOT0 & KAKIUCHI, 1974). However, this increase was ied. The phosphorylation of proteins 1 to 5 was not much smaller than that caused by the addition of shown in the experiments of CARNEGIE et at. (1974), 1OmM-NaF to the system and, therefore, was not our previous report (MIYAMOTO & KAKILICHI, 1974) thought to reflect the real effect of histone or the exoor STECK& APPEL (1974). This is thought to be for genous protein kinase (MIYAMOTO, 1975). Thus, it was the following reasons. (a) We performed disc gel elec- found that exogenous substrate added to the endotrophoresis for HC1 extract according to the method genous phosphorylation system of myelin did not of REIsFELDet al. (1962). As shown in Fig. 3, the phos- serve as substrate for the myelin-associated protein phorylation of proteins 1, 2 and 3 which were kinase (MIYAMOTO, 1975). Furthermore, exogenous extracted by treatment with hydrochloric acid were protein kinase added to the endogenous phosphorylanot easily Seen under the conditions of the electro- tion system could not utilize the endogenous subphoresis. Proteins 4 and 5 were not extracted by treat- strate in the myelin fraction (MIYAMOTO, 1975). These ment with hydrochloric acid. (b) Incorporation of results indicate that the catalytic site of enzyme and phosphate into proteins 1 to 5 was much smaller than substrate protein in myelin may be integrated into that into large and small myelin basic proteins in the membrane structure, and that the exogenous subterms of rate and amount. The phosphorylation of strate or exogenous protein kinase is, therefore, not these proteins was observed only by incubation of accessible to their appropriate sites. It suggests that the myelin fraction for longer times using radioactive the in vitro endogenous phosphorylation of myelin ATP of high specific activity and fairly large amounts fraction described in this study may be based on the of myelin fraction as protein. spatial orientation of enzyme and substrate in vivo; The endogenous phosphorylation of the proteins random orientation of both may cause the non-speciwas not stimulated by cyclic AMP under the condi- fic phosphorylation of alternative sites. tions used. It has been reported that the exogenous It has been reported that a protein of mol wt 50,000 cyclic AMP was only partially accessible to cyclic which can be phosphorylated, is found in prepAMP-binding site in myelin and that the regulatory arations from cytoplasm of mammalian brain (UEDA subunit of cyclic AMP-dependent protein kinase in et al., 1973), ductus deferens, uterus and small intesmyelin may be integrated into its membrane structure tine (CASNELLIE & GREENGARD, 1974) and membrane (MIYAMOTO, 1975). The inabilify of cyclic AMP to fractions of toad bladder (DELORENZOet al., 1 9 9 , stimulate the phosphorylation of the endogenous pro- ductus deferens, uterus and small intestine (CASNELLIE
Phosphorylation of proteins in myelin of rat brain
577
& GREENGARD, 1974). This protein is thought to be LOMY 0. H., ROSEBROUGH N . J., FARRA. L. & RANDALL the regulatory subunit of a cycic AMP-dependent R. J. (1951) J . biol. Chem. 193, 265-275. protein kinase in the case of brain cytoplasm (MAENO MAENOH., REYESP. L., UEDAT., RUDOLPHS. A. & GREENGARD P. (1974) Archs Biochem. Biophys. 164, 551-559. et al., 1974). In view of the fact that both cyclic AMPR. E. & DEIBLER G. E. (1975) J . Neurocheni. dependent protein kinase and cyclic AMP-binding MARTENSON 24, 79-88. protein are present in myelin of rat brain (MIYAMOTO, MARTENSON R. E., DEIBLERG . E. & KRAMER A. J. (1975) 1975), one of the proteins in myelin, which can be J . Neurochem. 24, 173-182. phosphorylated, may be identical with those found MIYAMOTO E. (1975) J . Neurochem. 24, 503-512. in preparations from the cytoplasm and the mem- MIYAMOTOE. & KAKIUCHIS. (1974) J . biol. Chem. 249, brane fractions described above. However, a protein 2769-2777. of the same molecular weight was not found among MIYAMOTO E. & KAKIUCHI S. (1975) Biochim. biophys. Acta those phosphorylated in myelin. Further study will 384, 458-465. MIYAMOTO E., KAKKJCIII S. & KAKJMOTO Y. (1974) Nuture, be needed to clarify this problem. L m d . 249, 151-152. MIYAMOTO E., K ~ J J. O F. & GREENGARD P. (1969) Science, N.Y 165, 63-65. MIYAMOTO E., PETZOLD G. L., HARRISJ. S. & GREENGARD P. (1971) Biochem. biophys. Res. Commun. 44, 305-312. REFERENCES MIYAMOTO E., PETZOLDG. L., Kuo J. I;. & GREENGARD CARNEGIEP. R., KEMPB. E., DUNKLEY P. R. & MURRAY P. (1973) J . biol. Chem. 248, 179-189. POSTR. L. & SEN A. K. (1967) in Methods in Enzymology A. W. (1973) Biochem. J . 135, 569-572. R. W. & PULLMAN M. E., eds.) Vol. X, pp. CARNEGIE P. R., DUNKLEY P. R., KEMPB. E. & MURRAY (ESTABROOK 773-776. Academic Press, New York. A. W. (1974) Nature, Lond. 249, 147-150. C. O., CORBINJ. D., KING C. CASNELLIE J. E. & GREENGARD P. (1974) Proc. natn. Acad. REIMANNE. M., BROSTROM A. C KREBSE. G. (1971) Biochem. biophys. Res. Cornmun. Sci. U.S.A. 71, 1891-1895. 42, 187-194. DELORENZOR. J., WALTONK. G., CURRAN P. F. & R., LEWISU. & WILLIAMS D. (1962) Nafure, Lond. GREENCARD P. (1973) Proc. natn. Acad. Sci. U.S.A. 70, REISFELD 195, 281-283. 88&884. ERLICHMAN J., HIRSCHA. H. & ROSEN0. M. (1971) Proc. STECKA. J. & APPEL S. H. (1974) J . bid. Chern. 249, 541&5420. natn. Acad. Sci. U.S.A. 68, 731-735. FOLCH-PI J. & LEESM. (1951) J . bid. Chem. 191, 807-817. TAOM., SALASM. L. & LIPMANNF. (1970) Proc. natn. Acud. Sci. U.S.A. 67, 408414. GILL G. N. & GARREN L. D. (1970) Biochem. biophys. Re.?. UEDAT., MAENOH. & GREENGARD P. (1973) J . biol. Chem. Commun. 39, 335-343. 248, 8295--8305. GREENGARD P. & K u o J. F. (1970) in Role of Cyclic A M P K., TOBARIC., HIRANOS. & TSUKADA Y. (1972) in Cell Function (GREENGARD P. & COSTAE., eds.) pp. UYEMURA J . Neurochem. 19, 2607-2614. 287-306. Raven Press, New York. J. P. & KRERSE. G. (1968) J . bid. KUMONA,, YAMAMURA H. & NISHIZUKA Y. (1970) Biochem. WALSHD. A,, PERKINS Chem. 243, 3763-3765. biophys. Res. Commun. 41, 129&1297. Kuo J. F. & GREENGARD P. (1969) Proc. natn. Acad. Sci. WEBER K. & OSBORNM. (1969) J . biol. Chem. 244, 440-4 12. U.S.A. 64, 1349-1355. LAATSCHR. H., KIES M. W., GORDON S. & ALVORDE. WIGGINSR. C., JOFFES., DAVIDSOND. & DEI. VALLE u. (1974) J . Neurochem. 22, 171-175. C. (1962) J . exp. Med. 115, 777-788. WOLFGRAM F. (1966) J . Neurochem. 13, 461-470. LANGAN T. A. (1968) Science, N.Y 162, 579-581.
Acknowledgemenl-The author wishes to thank Mr. A. NISHIDAof this laboratory for his help with the autoradiography and photography.
