Jorrrnul r~l'R.'rrr~oi~~rc~,,li.vrr!. Raven Press. Ltd.. New York !ct 1992 International Society for Neurochernistry
Depolarization-Dependent Tyrosine Phosphorylation in Rat Brain Synaptosomes Sarah Woodrow, Nankie Bissoon, and James W. Gurd Department 01' Biochmnistr~,Scurboroiigh Catpiis. University of Toronto. W%rl Hill, Onturio, Canada
Abstract: Synaptosomes from rat forebrain were analyzed for the presence of phosphotyrosine-containing proteins by immunoblotting with antiphosphotyrosine antibodies. Using this technique, 10- 1 1 phosphotyrosine-containing proteins were detected. Depolarization of synaptosomes by transfer to a high (4 1 mM) Kt medium resulted in increases in the phosphotyrosine content of several synaptosomal proteins, the most pronounced increase being associated with a membrane protein of M, 117,000 (ptpll7). Additional proteins exhibiting depolarization-dependent increases in phosphotyrosine content had molecular weights of 39,000, 104,000. 135,000. and 160,000. The depolarization-dependent increase in the phosphotyrosine content of ptpl17 was apparent within 30 s of the onset of depolarization, reached a maximum between 3 and 5 min, and then
decreased to near control values by 30 min. The increase in tyrosine phosphorylation of ptpl17 was dependent on the concentration of K+ in the depolarizing medium and was maximal with [K+] in excess of 50 mM. It was also calcium dependent and did not occur in the absence of extracellular calcium. The addition of veratridine to the incubation medium also resulted in an increase in the tyrosine phosphorylation of ptp 1 17. The results suggest that the phosphorylation of synaptic proteins on tyrosine residues may be involved in the regulation or modulation of synaptic activity. Key Words: Synaptosome-Depolarization-Tyrosine phosphorylation-Tyrosine kinase. Woodrow S. e t al. Depolarization-dependent tyrosine phosphorylation in rat brain synaptosomes. J. Nezirochem. 59, 857-862 ( 1 992).
Depolarization of the nerve terminal initiates a cascade of events that ultimately leads to the calcium-dependent release of neurotransmitters (Katz and Miladi, 1969: Blaustein, 1975). Depolarization is rapidly followed by changes in the phosphorylation state of a number of synaptic proteins (Kreuger et al., 1977: Dunkley and Robinson, 1986). These depolarizationdependent changes in protein phosphorylation are calcium dependent (Kreuger et al., 1977: Dunkley and Robinson, 1986) and have been attributed to alterations in the activities of Ca'+/calmodulin-dependent protein kinases (Nichols et al., 1987: Wang et al., 1988), protein kinase C (Nichols et al., 1987: Wang et al., 1988), and calcium-dependent protein phosphatases (Robinson et al., 1987) as well as to increased activity of adenylate cyclase (Brostrom et al., 1975; Cheung et al., 1975) resulting in enhanced phosphorylation by cyclic AMP-dependent protein kinase. In addition to serine and threonine protein kinases, the adult brain contains relatively high levels of protein tyrosine kinase activity (Cotton and Brugge,
1983; Dasgupta et al., 1984). A number of tyrosine kinases, including c-yes (Sudol et al., 1988), c-fvn (Veillette and Bolen, 1989), elk, flk (Letwin et al., 1988), trkB (Klein et al.. 1989), c-src, and its neuronspecific homologue c-src' (Levy et al., 1984; Sorge et al., 1984: Brugge et al., 1985; Neer and Lok, 1985; Maness et al., 1988), have been identified in nervous tissue. Tyrosine kinases, including pp60c-s'c, are present at the synapse and tyrosine phosphorylation of endogenous proteins occurs in synaptic vesicles, synaptic membranes, and postsynaptic densities, and in the synaptoplasm (Gurd and Bissoon, 1985; Ellis et al., 1988; Hirano et al., 1988; Pang et al., 1988). Tyrosine phosphorylation has been demonstrated to modulate the functional properties of the nicotinic acetylcholine receptor (Hopfield et al., 1988) and has recently been implicated in the development of long-term potentiation in hippocampal slices (O'Dell et al., 1991). In spite of these observations, the function of synaptic protein tyrosine kinases is not known and a direct relationship between the tyrosine phos-
Received September 6. 199 I : revised manuscript received January 3 1, 1992: accepted February 24. 1992. Address correspondence and reprint requests to Dr. J. W. Gurd at Department of Biochemistry. Scarborough Campus, University ofToronto. West Hill. Ontario MIC IA4, Canada.
.Ibbruviu/ion i r s d MAP kinase. microtubule-associated protein
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phorylation of synaptic proteins and synaptic activity has not been demonstrated. To address these questions, we have analyzed the effects of K+-induced depolarization on tyrosine phosphorylation in rat brain synaptosomes and now report that depolarization results in an increase in the phosphotyrosine content of a number of synaptosomal proteins, the most prominent change being associated with a membrane protein with a molecular weight of 1 17,000 (ptpll7).
MATERIALS AND METHODS Synaptosomes were prepared from the forebrains of 4-5week old rats as described by Dunkley et al. (1988). For depolarization experiments. synaptosomes ( 150-200 pg) were incubated for 45 rnin at 37°C in a modified Krebs buffer containing 143 mMNa+, 4.7 mM K'. 1.2 m M Mg2+, 0. I mM Ca2+, 126 m M Cl-, 24.9 mM NaHC0,-, and 10 mM glucose, and were then transferred to the same buffer containing 4 1 m M K'. The Na+ concentration in the depolarization buffer was decreased appropriately to compensate for the elevated potassium levels. Control samples were treated in the same way except that they were transferred to Krebs buffer containing the normal concentration of Na'. Reactions were stopped by the addition of an equal volume of 2% sodium dodecyl sulfate containing 8 M urea and 2% P-mercaptoethanol (stop solution) and heating at 100°C for 5 min. For experiments in which the effects of calcium were investigated, Ca" was omitted from and 1 mA4 EGTA was added to the preincubation and depolarization buffers. In some experiments, 1 mA4 EGTA was added to the preincubation buffer 5 rnin before depolarization in the absence of Ca2+. Depolarization with veratridine was achieved by transferring synaptosomes that had been preincubated for 45 min to medium that contained 0.05 mM veratridine. To determine the distribution of phosphotyrosine-containing proteins between the synaptosome particulate and soluble fractions, control and depolarized synaptosomes were diluted in 10 volumes of 5 mM Tris, pH 7.6, containing 0. I mMsodium orthovanadate, 0.1 mMphenylmethylsulfonyl fluoride, and 5 pg/ml each of leupeptin, antipain, and aprotinin. Synaptosomes were allowed to stand at 4°C for 15 rnin and then subjected to two cycles of freezing and thawing before centrifugation at 100,000 g,, for 30 min. The supernatant was removed and freeze-dried, and both the soluble and the particulate fractions were solubilized for gel electrophoresis and immunoblotting. In all cases, the protein load was the same for each lane ofthe gel within individual experiments. Between different experiments, the protein load per lane varied between 100 and 150 pg. For the detection of phosphotyrosine-containing proteins, samples were separated on 8% (wt/vol) polyacrylamide gels using the buffer system of Laemmli ( 1970) and electrophoretically transferred to nitrocellulose sheets as described (Cudmore and Curd, 1991). Protein blots were reacted with monoclonal antiphosphotyrosine antibody (Upstate Biotechnology, Inc.. Lake Placid. NY. U.S.A.) as described (Cudmore and Curd, 199 1 ), and immunoreactive proteins were detected by chemiluminescence using the enhanced chemiluminescence reagent kit (Amersham) according to the manufacturer's instructions. Control immunoblotting experiments were performed by adding 2 mA4 phosphotyrosine, phosphoserine. or phosphothreonine to the antibody solution before incubation with the protein blot. J . Nmrochon.. l'd 59. No. 3, 1 Y Y 2
Phosphoamino acid analysis of ptp 1 I7 from depolarized synaptosomes was performed by prelabeling synaptosomes for 45 rnin in the presence of [32P]P04(4 mCi/ml) and then depolarizing for 5 rnin with 4 I mMK+. After electrophoresis, proteins were blotted to Immobilon (Millipore, Inc.). ptpl17, located on the blot by immunodetection with antiphosphotyrosine antibodies as above, was excised from the blot, hydrolyzed with 6 M HCI for 1 h at I IO"C, and phosphoamino acids separated by a two-dimensional procedure as described (Ellis et al.. 1988). In all cases, the results presented are typical of those obtained in at least three, and generally four or five, independent experiments with separate synaptosome preparations.
RESULTS Freshly prepared synaptosomes contained 10- 1 1 proteins that reacted on western blots with antiphosphotyrosine antibodies indicating the presence of phosphotyrosine residues (Fig. 1). With the exception of a decrease in the tyrosine phosphorylation of a protein of molecular weight 104,000, there was little or no change in the tyrosine phosphorylation of synaptosoma1 proteins after a 45-min incubation in modified Krebs buffer (Fig. 1). Additional experiments indicated that the decrease in the phosphotyrosine content of the 104-kDa protein occurred within the first 15 rnin of the incubation. These results demonstrate that the phosphorylation of synaptosomal proteins on tyrosine was in a steady state after the incubation, a necessary precondition for studies of the effect of depolarization on tyrosine phosphorylation. The specificity of the immunoblotting procedure was confirmed by its inhibition by phosphotyrosine, but not by phosphoserine or phosphothreonine (Fig. 2). Depolarization-induced changes in the phosphorylation of synaptosomal proteins has been well documented (Kreuger et al., 1977; Dunkley and Robinson, 1986; Nichols et al., 1987; Wang et al., 1988). The effect of depolarization on tyrosine phosphorylation was assessed by incubating synaptosomes for 45 rnin in Krebs buffer and then transfemng them to a medium containing a high concentration of Kt (41
200FIG. 1. The identification of phosphotyrosine-containingproteins in rat brain synaptosomes. Freshly prepared synaptosomes (0 rnin) or synaptosornes that had been incubated for 45 min in modified Krebs buffer were subjected to gel electrophoresis and phosphotyrosine-containing proteins detected by immunoblottingwith antiphosphotyrosine antibodies. Numbers to the left indicate the position of molecular weight standards.
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TYROSINE PHOSPHOR YLA TION IN SYNAPTOSOMES FIG. 2. Specificity of the immunoblotting reaction with antiphosphotyrosine antibodies. Freshly prepared synaptosomes were solubilized and proteins separated by gel electrophoresis and blotted to nitrocellulose. Blots were probed with antiphosphotyrosine antibody without any additions (lane A) or in the presence of 2 mM phosphoserine (lane B), phosphothreonine (lane C). or phosphotyrosine (lane D), and immunoreactive proteins detected as in Materials and Methods.
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a), a procedure that results in membrane depolarization, changes in protein phosphorylation, and the release of neurotransmitter (Blaustein, 1975; Dunkley and Robinson, 1986). The results obtained with two separate synaptosomal preparations in which the effects of depolarization on the phosphotyrosine content of synaptosomal proteins were determined are presented in Fig. 3A and B. Depolarization resulted in a pronounced increase in the phosphotyrosine content of a protein of molecular weight 117,000 (ptp 1 17). The depolarization-dependent increase in the phosphotyrosine content of ptp 1 17 was consistently observed in 12 experiments with nine separate synaptosome preparations. Depolarization also resulted in an increase in the phosphotyrosine content of a protein of molecular weight 39,000 (ptp39, Fig. 3). This protein was not always well resolved from the major phosphotyrosinylated protein of M, 37,500 and frequently appeared simply as a broadening of the latter protein band (compare Fig. 3A and B). A
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FIG. 3. The effect of depolarization on tyrosine phosphorylation in synaptosomes. Synaptosomeswere preincubatedfor 45 min in modified Krebs buffer and then transferred to control (C) or high K', depolarizing (D) buffer for 3 min. Phosphotyrosine-containing proteins were identified by immunoblotting with antiphosphotyrosine antibodies. Molecular mass standards (in kDa) are indicated on the left and apply to the columns under A. A and B represent results obtained with two separate synaptosome preparations. Large arrow, p t p l l 7 ; arrowhead, ptp39; small arrows, proteins of 104 kDa, 135 kDa, and 160 kDa.
FIG. 4. Identificationof phosphoaminoacids in p t p l l 7 . Synaptosomes were prelabeled for 45 min with [32P]P0, and then depolarized with high K+ for 5 min. p t p l l 7 was isolated and analyzed for the presence of 32P-labeledphosphoaminoacids as described in Materials and Methods. A Ninhydrin stain of standards. B: Autoradiogram of phosphoamino acids.
Other proteins that exhibited minor increases in tyrosine phosphorylation after depolarization included those with molecular weights of 104,000, 135,000, and 160,000 (Fig. 3). The phosphorylation of ptp 1 17 on tyrosine residues was confirmed by analyzing the phosphoamino acid content of ptpll7 isolated from depolarized synaptosomes that had been prelabeled with [32P]P0,. Although phosphoserine and phosphothreonine were the major [32P]phosphoamino acids present in ptp 1 1 7, low levels of phosphotyrosine were also detected (Fig. 4). The depolarization-dependent increase in tyrosine phosphorylation of ptp 1 1 7 was detectable within 30 s after transfer to the high K+ medium, increased to a maximum between 3 and 5 rnin after the onset of depolarization, and then decreased to near control values by 30 rnin (Fig. 5). The tyrosine phosphorylation of proteins of M, 39,000, 104,000, 135,000, and 160,000 followed time courses generally similar to that of ptp 1 1 7. Depolarization of synaptosomes with high [K+]results in an increase in the uptake of Ca2+(Blaustein, 1975) and previous studies have established that the depolarization-dependent changes in synaptosomal protein phosphorylation are Ca2+ dependent (Kreuger et al., 1977: Dunkley and Robinson, 1986; Nichols et al., 1987; Wang et al., 1988). The effect of Ca+ on the tyrosine phosphorylation of synaptosomal proteins was determined by preincubating synaptosomes for 45 rnin in the absence of Ca2+.Depolarization of these synaptosomes in high K+, Ca2+-free buffer failed to elicit an increase in the tyrosine phosphorylation of ptp 1 17 (Fig. 6A, lanes 3 and 4) or of other synaptosomal proteins. Incubation of synaptosomes for 40 rnin in the presence of Ca2+followed by the addition of 1 mM EGTA for 5 rnin did not alter the pattern of phosphotyrosine-containing proteins compared with samples that had been incubated for the full 45 rnin in the absence of Ca2+(compare lanes 3 and 5), indicating that Ca2+alone did not influence basal phosphotyrosine levels. Under the latter condiJ Neurochon.. I ol. 55. No. 3. 1552
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Under these conditions, the maximum increase in tyrosine phosphorylation occurred between 1 and 3 rnin after the start of depolarization. The distribution of phosphotyrosine-containing proteins between the soluble and particulate synaptosome compartments was ascertained by lysing control and depolarized synaptosomes and separating soluble and membrane fractions by centrifugation. The majority of proteins containing phosphotyrosine, including ptpll7, were located in the particulate fraction (Fig. 8). The depolarization-dependent increase in tyrosine phosphorylation of ptp 1 17 was confined to the particulate fraction, whereas that of ptp39 occurred in the soluble fraction.
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FIG. 5. The effect of depolarization time on the tyrosine phosphorylationof synaptosome proteins. Synaptosomeswere depolarized for the indicated period of time in high K+ medium and phosphotyrosine proteins identified by immunoblotting with antiphosphotyrosine antibodies. The arrow and arrowhead indicate the positions of p t p l l 7 and ptp39, respectively. Small arrows indicate the positions of proteins of M, 104K, 135K, and 160K.
DISCUSSION In the present study, we have demonstrated that depolarization of synaptosomes results in the enhanced tyrosine phosphorylation of several proteins. Tyrosine kinases have generally been associated with events involved in the control of cellular growth and differentiation (Hunter and Cooper, 1985) and the high levels of tyrosine kinases in the developing brain (Fults et al., 1985;Weistler and Walter, 1988)are consistent with such a role for tyrosine phosphorylation in the nervous system. The fully differentiated brain continues, however, to exhibit relatively high tyrosine kinase activities (Cotton and Brugge, 1983; Dasgupta et al., 1984) and the functional significance of this activity remains to be determined. We, and others (Gurd and Bissoon, 1985; Ellis et al., 1988; Hirano et al., 1988; Pang et al., 1988), have described the presence of tyrosine kinase activity and phosphotyrosinecontaining proteins in synaptic structures and have suggested that tyrosine kinases may be involved in the regulation of some aspect of synaptic function. The results of the present study are clearly consistent with such a role for tyrosine phosphorylation at the synapse and identify ptp 1 17 as a prominent substrate for depolarization-dependent synaptic tyrosine kinase activity. The Ca2+dependency of the tyrosine phosphorylation of ptp 1 17 further suggests that this reaction may
tions, depolarization in the absence of Ca2+also failed to elicit an increase in the phosphotyrosine content of ptpll7 (Fig. 6A, lanes 5 and 6). These results demonstrate that the increase in phosphotyrosine content of ptp 1 17 after depolarization in the presence of high K+ is Ca2+ dependent. Interestingly, small increases in the tyrosine phosphorylation of proteins of M, 104,000 and 135,000 were observed in the absence of Ca2+,suggesting that a Ca'+-independent alteration in tyrosine phosphorylation may also occur after depolarization. The tyrosine phosphorylation of ptpl17 was also influenced by the concentration of K+ in the depolarizing medium. Fifty percent of the maximum increase in tyrosine phosphorylation of ptpl I7 occurred with K+ concentrations of between 10 and 20 m M and maximum stimulation was apparent with concentrations in excess of 50 m M K+ (Fig. 6B). Veratridine depolarizcs synaptosomes by increasing Na+ permeability (Blaustein and Goldring, 1975), causing an increase in calcium uptake and neurotransmitter release (Blaustein, 1975). Veratridine-induced depolarization also resulted in a pronounced increase in the phosphotyrosine content of ptpl17 (Fig. 7).
FIG. 6. The effects of calcium (A! and K+ (5)on the tyrosine phosphorylation of synaptosomal proteins. A: Synaptosomes were preincubated for 45 min in the presence (lanes 1,2,5, and 6) or absence(lanes 3 and 4) of calcium. EGTA (1 mM) was added after 40 rnin to the samples displayed in lanes 5 and 6. After the preincubation, samples were transferred to control (lanes 1, 3, and 5) or high Kt (lanes 2, 4, and 6) medium in the presence or absence of calcium as indicated for 3 rnin and then analyzed for phosphotyrosine by immunoblotting. B: Synaptosomes were depolarized for 3 min in media containing the indicated increase in [K+] over control media and analyzed for phosphotyrosine by immunoblotting. The arrow in A and B indicates the position of p t p l l 7 .
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TYROSINE PHOSPHOR YLA TION IN SYNAPTOSOMES be a component of the Ca2+-mediated cascade of events involved in the release of neurotransmitters. Although increased tyrosine phosphorylation of ptpll7 could be detected as early as 30 s after the onset of depolarization, the time course of ptpll7 phosphorylation was generally slowerthan depolarization-induced changes in total protein phosphorylation, which may reach maximum levels within 10 s (Kreuger et al., 1977). Tyrosine phosphorylation may therefore be involved in later aspects of the release cycle, such as neurotransmitter regeneration or the translocation and realignment of vesicles in preparation for subsequent depolarizations. The identity of the phosphotyrosine-containing proteins now described are currently not known. Proteins of molecular weight similar to ptp 1 17, including vinculin (Igarashi et al., 1990) and a glycoprotein of M, 1 15,000 (Cheng and Sahyoun, 1990), have been identified as substrates for tyrosine kinase in growth cones and to a lesser extent in synaptosomes, but the relationship of either of these to ptpll7 remains to be determined. ptp39 is of molecular mass similar to a 40-kDa protein associated with microtubule-associated protein 2 kinase (MAP kinase) activity. The phosphotyrosine content of MAP kinase increased after electroconvulsive treatment of rat brain (Stratton et al., 1991 ) and N-methyl-D-aspartate stimulated the tyrosine phosphorylation of a 39-kDa protein, identified as highly related or identical to MAP kinase, in cultured hippocampal cells (Bading and Greenberg, 1991). The tyrosine phosphorylation of a 42-kDa protein with associated MAP kinase activity in chromaffin cells was stimulated by a variety of secretagogues, including high Kf (Ely et al., 1990). The synaptic vesicle-associated protein synaptophysin, M, 38,000, has also been reported to be phosphorylated on tyrosine residues (Barnekow et al., 1990).Synaptophysin is, however, a transmembrane protein (Johnston et al., 1989) and unlikely, therefore, to correspond to ptp39, which occurs primarily in the soluble fraction.
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FIG. 7. The effect of veratridine on tyrosine phosphorylation by rat brain synaptosomes. Synaptosorneswere depolarized for the indicated periods in the presence of 0.05 mM veratridine before analysis for phosphotyrosine by irnrnunoblotting.The arrow indicates the position of p t p l l 7 .
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FIG. 8. The distributionof synaptosomal phosphotyrosine-containing proteins. Control synaptosomes (A) or synaptosornes that had been depolarized for 3 rnin in the presence of high K+ (B)were lysed and soluble (S) and particulate (P) fractions separated by centrifugation as in Materials and Methods. Phosphotyrosine-containing proteins were identified by immunoblotting.The arrow and arrowhead indicate the positions of p t p l l 7 and ptp39, respectively.
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pp6OC-""(Hirano et al., 1988; Cudmore and Gurd, 1991) and c-yes (Sudol et al., 1988) have been identified as synaptic components and are potential candidates for mediating the tyrosine phosphorylation events now described. The former enzyme has been shown to phosphorylate synaptophysin (Barnekow et al., 1990) as well as growth cone-associated tubulin (Matten et al., 1990). To understand the role of tyrosine phosphorylation in synaptic function, it is now clearly important to determine the identity and function of individual synaptic phosphotyrosine-containing proteins and the nature of the regulation of the tyrosine kinase(s) responsible for their phosphorylation. We are currently directing our efforts toward this end. Acknowledgment: The present work was supported by a grant to J.W.G. from the National Science and Engineering Research Council. We thank Dr. John Rostas for helpful discussion during the early part of this work.
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Kreuger B. K.. Forn J., and Greengard P. ( 1977) Depolarization-induced phosphorylation of specific proteins. mediated by calcium influx. in rat brain synaptosomes. J. Biol. Chem. 252, 2764-2773. Laemmli U. K. ( 1970) Cleavage ofstructural proteinsduringassembly of the head of bacteriophage T4. NmiriJ 227, 680-685. Letwin K., Yee S. P.. and Pawson 7. ( I 988) Novel protein tyrosine kinase cDNAs related to.fps[/?s and c y h cloned using antiphosphotyrosine antibodies. Oncugeni. 3, 62 1-627. Levy B. T., Sorge L. K., Meymandi A., and Maness P. F. (1984) pp60"" is in chick and human embryonic tissues. Dev. Bid. 104,9- 17. Maness P. F., Aubry M., Shores C. G.. Frame L.. and Pfenninger K. H. (1988) c-src gene product in developing rat brain is enriched in growth cone membranes. Proc. Natl. .4t.ud. Sci. USA 85,352-360. Matten W. T., Aubry M.. West J.. and Maness P. F. ( I 990) Tubulin is phosphorylated at tyrosine by pp60'~"" in nerve growth cone membranes. J. Cell. Biol. 111, 1959-1970. Neer E. J. and Lok J. M. (1985) Partial purification and characterization of pp60'""-related tyrosine kinase from bovine brain. Proc. Null. Acud. Sci. USA 82, 6025-6029. Nichols R. A.. Haycock J. W.. Wang J. K. T., and Greengard P. ( 1987) Phorbol ester enhancement of neurotransmitter release from rat brain synaptosomes. J. Nc.i(rochem.48,6 15-62 I . ODell T. J., Kandel E. R.. and Grant S. G . N. (1991) Long-term potentiation in the hippocampus is blocked by tyrosine kinase inhibitors. Nutiire 353, 558-560. Pang D. T., Wang J. K. T., Valtorta F., Benfanati F., and Greengard P. ( 1988) Protein tyrosine phosphorylation in synaptic vesicles. Pruc. Null. Acud. Sci. LISA 85, 762-766. Robinson P. J., Hauptschein R., Lovenberg W., and Dunkley P. R.( 1987) Dephosphorylation of synaptosomal proteins P96 and PI 39 is regulated by both depolarization and calcium, but not by a rise in cytosolic calcium alone. J. Neuruchem. 48, 187-1 95. Sorge L. K., Levy R. T., and Maness P. F. ( 1984) pp60'-'" is developmentally regulated in neuronal retina. Cell 36, 249-257. Stratton K. R., Worley P. F.. Litz J. S., Parsons S. J., Huganir R. L., and Baraban J. M. ( I 99 I ) Electroconvulsive treatment induces a rapid and transient increase in tyrosine phosphorylation of a 40-kilodalton protein associated with microtubule-associated protein 2 kinase activity. J. Neirruchem. 56, 147-152. Sudol M., Alvarez-Buylla A.. and Hanafusa H. (1988) Differential cellular srcproteins in the cerebelexpression of cellular~~esand lum. Oncogene Res. 2, 345-355. Veillette A. and Bolen J. B. (1989) Src-related protein tyrosine kinases. in Oncogenes (Benz C. and Liu E.. eds), pp. 121-142. Kluwer Academic Publishers, Nonvell, Massachusetts. Wang J. K. T., Walaas S. I., and Greengard P. ( 1 988) Protein phosphorylation in nerve terminals: comparison ofcalcium/calmodulin-dependent and calcium/diacylglycerol-dependentsystems. J. Niwusci. 8, 28 1-288. Weistler 0. D. and Walter G. (1988) Developmental expression of two forms of pp60'-'" in mouse brain. Mol. Cell. Bid. 8, 502504.
Depolarization-Dependent Tyrosine Phosphorylation in Rat Brain Synaptosomes Sarah Woodrow, Nankie Bissoon, and James W. Gurd Department 01' Biochmnistr~,Scurboroiigh Catpiis. University of Toronto. W%rl Hill, Onturio, Canada
Abstract: Synaptosomes from rat forebrain were analyzed for the presence of phosphotyrosine-containing proteins by immunoblotting with antiphosphotyrosine antibodies. Using this technique, 10- 1 1 phosphotyrosine-containing proteins were detected. Depolarization of synaptosomes by transfer to a high (4 1 mM) Kt medium resulted in increases in the phosphotyrosine content of several synaptosomal proteins, the most pronounced increase being associated with a membrane protein of M, 117,000 (ptpll7). Additional proteins exhibiting depolarization-dependent increases in phosphotyrosine content had molecular weights of 39,000, 104,000. 135,000. and 160,000. The depolarization-dependent increase in the phosphotyrosine content of ptpl17 was apparent within 30 s of the onset of depolarization, reached a maximum between 3 and 5 min, and then
decreased to near control values by 30 min. The increase in tyrosine phosphorylation of ptpl17 was dependent on the concentration of K+ in the depolarizing medium and was maximal with [K+] in excess of 50 mM. It was also calcium dependent and did not occur in the absence of extracellular calcium. The addition of veratridine to the incubation medium also resulted in an increase in the tyrosine phosphorylation of ptp 1 17. The results suggest that the phosphorylation of synaptic proteins on tyrosine residues may be involved in the regulation or modulation of synaptic activity. Key Words: Synaptosome-Depolarization-Tyrosine phosphorylation-Tyrosine kinase. Woodrow S. e t al. Depolarization-dependent tyrosine phosphorylation in rat brain synaptosomes. J. Nezirochem. 59, 857-862 ( 1 992).
Depolarization of the nerve terminal initiates a cascade of events that ultimately leads to the calcium-dependent release of neurotransmitters (Katz and Miladi, 1969: Blaustein, 1975). Depolarization is rapidly followed by changes in the phosphorylation state of a number of synaptic proteins (Kreuger et al., 1977: Dunkley and Robinson, 1986). These depolarizationdependent changes in protein phosphorylation are calcium dependent (Kreuger et al., 1977: Dunkley and Robinson, 1986) and have been attributed to alterations in the activities of Ca'+/calmodulin-dependent protein kinases (Nichols et al., 1987: Wang et al., 1988), protein kinase C (Nichols et al., 1987: Wang et al., 1988), and calcium-dependent protein phosphatases (Robinson et al., 1987) as well as to increased activity of adenylate cyclase (Brostrom et al., 1975; Cheung et al., 1975) resulting in enhanced phosphorylation by cyclic AMP-dependent protein kinase. In addition to serine and threonine protein kinases, the adult brain contains relatively high levels of protein tyrosine kinase activity (Cotton and Brugge,
1983; Dasgupta et al., 1984). A number of tyrosine kinases, including c-yes (Sudol et al., 1988), c-fvn (Veillette and Bolen, 1989), elk, flk (Letwin et al., 1988), trkB (Klein et al.. 1989), c-src, and its neuronspecific homologue c-src' (Levy et al., 1984; Sorge et al., 1984: Brugge et al., 1985; Neer and Lok, 1985; Maness et al., 1988), have been identified in nervous tissue. Tyrosine kinases, including pp60c-s'c, are present at the synapse and tyrosine phosphorylation of endogenous proteins occurs in synaptic vesicles, synaptic membranes, and postsynaptic densities, and in the synaptoplasm (Gurd and Bissoon, 1985; Ellis et al., 1988; Hirano et al., 1988; Pang et al., 1988). Tyrosine phosphorylation has been demonstrated to modulate the functional properties of the nicotinic acetylcholine receptor (Hopfield et al., 1988) and has recently been implicated in the development of long-term potentiation in hippocampal slices (O'Dell et al., 1991). In spite of these observations, the function of synaptic protein tyrosine kinases is not known and a direct relationship between the tyrosine phos-
Received September 6. 199 I : revised manuscript received January 3 1, 1992: accepted February 24. 1992. Address correspondence and reprint requests to Dr. J. W. Gurd at Department of Biochemistry. Scarborough Campus, University ofToronto. West Hill. Ontario MIC IA4, Canada.
.Ibbruviu/ion i r s d MAP kinase. microtubule-associated protein
7 kinase.
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phorylation of synaptic proteins and synaptic activity has not been demonstrated. To address these questions, we have analyzed the effects of K+-induced depolarization on tyrosine phosphorylation in rat brain synaptosomes and now report that depolarization results in an increase in the phosphotyrosine content of a number of synaptosomal proteins, the most prominent change being associated with a membrane protein with a molecular weight of 1 17,000 (ptpll7).
MATERIALS AND METHODS Synaptosomes were prepared from the forebrains of 4-5week old rats as described by Dunkley et al. (1988). For depolarization experiments. synaptosomes ( 150-200 pg) were incubated for 45 rnin at 37°C in a modified Krebs buffer containing 143 mMNa+, 4.7 mM K'. 1.2 m M Mg2+, 0. I mM Ca2+, 126 m M Cl-, 24.9 mM NaHC0,-, and 10 mM glucose, and were then transferred to the same buffer containing 4 1 m M K'. The Na+ concentration in the depolarization buffer was decreased appropriately to compensate for the elevated potassium levels. Control samples were treated in the same way except that they were transferred to Krebs buffer containing the normal concentration of Na'. Reactions were stopped by the addition of an equal volume of 2% sodium dodecyl sulfate containing 8 M urea and 2% P-mercaptoethanol (stop solution) and heating at 100°C for 5 min. For experiments in which the effects of calcium were investigated, Ca" was omitted from and 1 mA4 EGTA was added to the preincubation and depolarization buffers. In some experiments, 1 mA4 EGTA was added to the preincubation buffer 5 rnin before depolarization in the absence of Ca2+. Depolarization with veratridine was achieved by transferring synaptosomes that had been preincubated for 45 min to medium that contained 0.05 mM veratridine. To determine the distribution of phosphotyrosine-containing proteins between the synaptosome particulate and soluble fractions, control and depolarized synaptosomes were diluted in 10 volumes of 5 mM Tris, pH 7.6, containing 0. I mMsodium orthovanadate, 0.1 mMphenylmethylsulfonyl fluoride, and 5 pg/ml each of leupeptin, antipain, and aprotinin. Synaptosomes were allowed to stand at 4°C for 15 rnin and then subjected to two cycles of freezing and thawing before centrifugation at 100,000 g,, for 30 min. The supernatant was removed and freeze-dried, and both the soluble and the particulate fractions were solubilized for gel electrophoresis and immunoblotting. In all cases, the protein load was the same for each lane ofthe gel within individual experiments. Between different experiments, the protein load per lane varied between 100 and 150 pg. For the detection of phosphotyrosine-containing proteins, samples were separated on 8% (wt/vol) polyacrylamide gels using the buffer system of Laemmli ( 1970) and electrophoretically transferred to nitrocellulose sheets as described (Cudmore and Curd, 1991). Protein blots were reacted with monoclonal antiphosphotyrosine antibody (Upstate Biotechnology, Inc.. Lake Placid. NY. U.S.A.) as described (Cudmore and Curd, 199 1 ), and immunoreactive proteins were detected by chemiluminescence using the enhanced chemiluminescence reagent kit (Amersham) according to the manufacturer's instructions. Control immunoblotting experiments were performed by adding 2 mA4 phosphotyrosine, phosphoserine. or phosphothreonine to the antibody solution before incubation with the protein blot. J . Nmrochon.. l'd 59. No. 3, 1 Y Y 2
Phosphoamino acid analysis of ptp 1 I7 from depolarized synaptosomes was performed by prelabeling synaptosomes for 45 rnin in the presence of [32P]P04(4 mCi/ml) and then depolarizing for 5 rnin with 4 I mMK+. After electrophoresis, proteins were blotted to Immobilon (Millipore, Inc.). ptpl17, located on the blot by immunodetection with antiphosphotyrosine antibodies as above, was excised from the blot, hydrolyzed with 6 M HCI for 1 h at I IO"C, and phosphoamino acids separated by a two-dimensional procedure as described (Ellis et al.. 1988). In all cases, the results presented are typical of those obtained in at least three, and generally four or five, independent experiments with separate synaptosome preparations.
RESULTS Freshly prepared synaptosomes contained 10- 1 1 proteins that reacted on western blots with antiphosphotyrosine antibodies indicating the presence of phosphotyrosine residues (Fig. 1). With the exception of a decrease in the tyrosine phosphorylation of a protein of molecular weight 104,000, there was little or no change in the tyrosine phosphorylation of synaptosoma1 proteins after a 45-min incubation in modified Krebs buffer (Fig. 1). Additional experiments indicated that the decrease in the phosphotyrosine content of the 104-kDa protein occurred within the first 15 rnin of the incubation. These results demonstrate that the phosphorylation of synaptosomal proteins on tyrosine was in a steady state after the incubation, a necessary precondition for studies of the effect of depolarization on tyrosine phosphorylation. The specificity of the immunoblotting procedure was confirmed by its inhibition by phosphotyrosine, but not by phosphoserine or phosphothreonine (Fig. 2). Depolarization-induced changes in the phosphorylation of synaptosomal proteins has been well documented (Kreuger et al., 1977; Dunkley and Robinson, 1986; Nichols et al., 1987; Wang et al., 1988). The effect of depolarization on tyrosine phosphorylation was assessed by incubating synaptosomes for 45 rnin in Krebs buffer and then transfemng them to a medium containing a high concentration of Kt (41
200FIG. 1. The identification of phosphotyrosine-containingproteins in rat brain synaptosomes. Freshly prepared synaptosomes (0 rnin) or synaptosornes that had been incubated for 45 min in modified Krebs buffer were subjected to gel electrophoresis and phosphotyrosine-containing proteins detected by immunoblottingwith antiphosphotyrosine antibodies. Numbers to the left indicate the position of molecular weight standards.
11684 5848.5-
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TYROSINE PHOSPHOR YLA TION IN SYNAPTOSOMES FIG. 2. Specificity of the immunoblotting reaction with antiphosphotyrosine antibodies. Freshly prepared synaptosomes were solubilized and proteins separated by gel electrophoresis and blotted to nitrocellulose. Blots were probed with antiphosphotyrosine antibody without any additions (lane A) or in the presence of 2 mM phosphoserine (lane B), phosphothreonine (lane C). or phosphotyrosine (lane D), and immunoreactive proteins detected as in Materials and Methods.
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a), a procedure that results in membrane depolarization, changes in protein phosphorylation, and the release of neurotransmitter (Blaustein, 1975; Dunkley and Robinson, 1986). The results obtained with two separate synaptosomal preparations in which the effects of depolarization on the phosphotyrosine content of synaptosomal proteins were determined are presented in Fig. 3A and B. Depolarization resulted in a pronounced increase in the phosphotyrosine content of a protein of molecular weight 117,000 (ptp 1 17). The depolarization-dependent increase in the phosphotyrosine content of ptp 1 17 was consistently observed in 12 experiments with nine separate synaptosome preparations. Depolarization also resulted in an increase in the phosphotyrosine content of a protein of molecular weight 39,000 (ptp39, Fig. 3). This protein was not always well resolved from the major phosphotyrosinylated protein of M, 37,500 and frequently appeared simply as a broadening of the latter protein band (compare Fig. 3A and B). A
B
iao116a45 a-
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FIG. 3. The effect of depolarization on tyrosine phosphorylation in synaptosomes. Synaptosomeswere preincubatedfor 45 min in modified Krebs buffer and then transferred to control (C) or high K', depolarizing (D) buffer for 3 min. Phosphotyrosine-containing proteins were identified by immunoblotting with antiphosphotyrosine antibodies. Molecular mass standards (in kDa) are indicated on the left and apply to the columns under A. A and B represent results obtained with two separate synaptosome preparations. Large arrow, p t p l l 7 ; arrowhead, ptp39; small arrows, proteins of 104 kDa, 135 kDa, and 160 kDa.
FIG. 4. Identificationof phosphoaminoacids in p t p l l 7 . Synaptosomes were prelabeled for 45 min with [32P]P0, and then depolarized with high K+ for 5 min. p t p l l 7 was isolated and analyzed for the presence of 32P-labeledphosphoaminoacids as described in Materials and Methods. A Ninhydrin stain of standards. B: Autoradiogram of phosphoamino acids.
Other proteins that exhibited minor increases in tyrosine phosphorylation after depolarization included those with molecular weights of 104,000, 135,000, and 160,000 (Fig. 3). The phosphorylation of ptp 1 17 on tyrosine residues was confirmed by analyzing the phosphoamino acid content of ptpll7 isolated from depolarized synaptosomes that had been prelabeled with [32P]P0,. Although phosphoserine and phosphothreonine were the major [32P]phosphoamino acids present in ptp 1 1 7, low levels of phosphotyrosine were also detected (Fig. 4). The depolarization-dependent increase in tyrosine phosphorylation of ptp 1 1 7 was detectable within 30 s after transfer to the high K+ medium, increased to a maximum between 3 and 5 rnin after the onset of depolarization, and then decreased to near control values by 30 rnin (Fig. 5). The tyrosine phosphorylation of proteins of M, 39,000, 104,000, 135,000, and 160,000 followed time courses generally similar to that of ptp 1 1 7. Depolarization of synaptosomes with high [K+]results in an increase in the uptake of Ca2+(Blaustein, 1975) and previous studies have established that the depolarization-dependent changes in synaptosomal protein phosphorylation are Ca2+ dependent (Kreuger et al., 1977: Dunkley and Robinson, 1986; Nichols et al., 1987; Wang et al., 1988). The effect of Ca+ on the tyrosine phosphorylation of synaptosomal proteins was determined by preincubating synaptosomes for 45 rnin in the absence of Ca2+.Depolarization of these synaptosomes in high K+, Ca2+-free buffer failed to elicit an increase in the tyrosine phosphorylation of ptp 1 17 (Fig. 6A, lanes 3 and 4) or of other synaptosomal proteins. Incubation of synaptosomes for 40 rnin in the presence of Ca2+followed by the addition of 1 mM EGTA for 5 rnin did not alter the pattern of phosphotyrosine-containing proteins compared with samples that had been incubated for the full 45 rnin in the absence of Ca2+(compare lanes 3 and 5), indicating that Ca2+alone did not influence basal phosphotyrosine levels. Under the latter condiJ Neurochon.. I ol. 55. No. 3. 1552
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Under these conditions, the maximum increase in tyrosine phosphorylation occurred between 1 and 3 rnin after the start of depolarization. The distribution of phosphotyrosine-containing proteins between the soluble and particulate synaptosome compartments was ascertained by lysing control and depolarized synaptosomes and separating soluble and membrane fractions by centrifugation. The majority of proteins containing phosphotyrosine, including ptpll7, were located in the particulate fraction (Fig. 8). The depolarization-dependent increase in tyrosine phosphorylation of ptp 1 17 was confined to the particulate fraction, whereas that of ptp39 occurred in the soluble fraction.
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FIG. 5. The effect of depolarization time on the tyrosine phosphorylationof synaptosome proteins. Synaptosomeswere depolarized for the indicated period of time in high K+ medium and phosphotyrosine proteins identified by immunoblotting with antiphosphotyrosine antibodies. The arrow and arrowhead indicate the positions of p t p l l 7 and ptp39, respectively. Small arrows indicate the positions of proteins of M, 104K, 135K, and 160K.
DISCUSSION In the present study, we have demonstrated that depolarization of synaptosomes results in the enhanced tyrosine phosphorylation of several proteins. Tyrosine kinases have generally been associated with events involved in the control of cellular growth and differentiation (Hunter and Cooper, 1985) and the high levels of tyrosine kinases in the developing brain (Fults et al., 1985;Weistler and Walter, 1988)are consistent with such a role for tyrosine phosphorylation in the nervous system. The fully differentiated brain continues, however, to exhibit relatively high tyrosine kinase activities (Cotton and Brugge, 1983; Dasgupta et al., 1984) and the functional significance of this activity remains to be determined. We, and others (Gurd and Bissoon, 1985; Ellis et al., 1988; Hirano et al., 1988; Pang et al., 1988), have described the presence of tyrosine kinase activity and phosphotyrosinecontaining proteins in synaptic structures and have suggested that tyrosine kinases may be involved in the regulation of some aspect of synaptic function. The results of the present study are clearly consistent with such a role for tyrosine phosphorylation at the synapse and identify ptp 1 17 as a prominent substrate for depolarization-dependent synaptic tyrosine kinase activity. The Ca2+dependency of the tyrosine phosphorylation of ptp 1 17 further suggests that this reaction may
tions, depolarization in the absence of Ca2+also failed to elicit an increase in the phosphotyrosine content of ptpll7 (Fig. 6A, lanes 5 and 6). These results demonstrate that the increase in phosphotyrosine content of ptp 1 17 after depolarization in the presence of high K+ is Ca2+ dependent. Interestingly, small increases in the tyrosine phosphorylation of proteins of M, 104,000 and 135,000 were observed in the absence of Ca2+,suggesting that a Ca'+-independent alteration in tyrosine phosphorylation may also occur after depolarization. The tyrosine phosphorylation of ptpl17 was also influenced by the concentration of K+ in the depolarizing medium. Fifty percent of the maximum increase in tyrosine phosphorylation of ptpl I7 occurred with K+ concentrations of between 10 and 20 m M and maximum stimulation was apparent with concentrations in excess of 50 m M K+ (Fig. 6B). Veratridine depolarizcs synaptosomes by increasing Na+ permeability (Blaustein and Goldring, 1975), causing an increase in calcium uptake and neurotransmitter release (Blaustein, 1975). Veratridine-induced depolarization also resulted in a pronounced increase in the phosphotyrosine content of ptpl17 (Fig. 7).
FIG. 6. The effects of calcium (A! and K+ (5)on the tyrosine phosphorylation of synaptosomal proteins. A: Synaptosomes were preincubated for 45 min in the presence (lanes 1,2,5, and 6) or absence(lanes 3 and 4) of calcium. EGTA (1 mM) was added after 40 rnin to the samples displayed in lanes 5 and 6. After the preincubation, samples were transferred to control (lanes 1, 3, and 5) or high Kt (lanes 2, 4, and 6) medium in the presence or absence of calcium as indicated for 3 rnin and then analyzed for phosphotyrosine by immunoblotting. B: Synaptosomes were depolarized for 3 min in media containing the indicated increase in [K+] over control media and analyzed for phosphotyrosine by immunoblotting. The arrow in A and B indicates the position of p t p l l 7 .
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TYROSINE PHOSPHOR YLA TION IN SYNAPTOSOMES be a component of the Ca2+-mediated cascade of events involved in the release of neurotransmitters. Although increased tyrosine phosphorylation of ptpll7 could be detected as early as 30 s after the onset of depolarization, the time course of ptpll7 phosphorylation was generally slowerthan depolarization-induced changes in total protein phosphorylation, which may reach maximum levels within 10 s (Kreuger et al., 1977). Tyrosine phosphorylation may therefore be involved in later aspects of the release cycle, such as neurotransmitter regeneration or the translocation and realignment of vesicles in preparation for subsequent depolarizations. The identity of the phosphotyrosine-containing proteins now described are currently not known. Proteins of molecular weight similar to ptp 1 17, including vinculin (Igarashi et al., 1990) and a glycoprotein of M, 1 15,000 (Cheng and Sahyoun, 1990), have been identified as substrates for tyrosine kinase in growth cones and to a lesser extent in synaptosomes, but the relationship of either of these to ptpll7 remains to be determined. ptp39 is of molecular mass similar to a 40-kDa protein associated with microtubule-associated protein 2 kinase (MAP kinase) activity. The phosphotyrosine content of MAP kinase increased after electroconvulsive treatment of rat brain (Stratton et al., 1991 ) and N-methyl-D-aspartate stimulated the tyrosine phosphorylation of a 39-kDa protein, identified as highly related or identical to MAP kinase, in cultured hippocampal cells (Bading and Greenberg, 1991). The tyrosine phosphorylation of a 42-kDa protein with associated MAP kinase activity in chromaffin cells was stimulated by a variety of secretagogues, including high Kf (Ely et al., 1990). The synaptic vesicle-associated protein synaptophysin, M, 38,000, has also been reported to be phosphorylated on tyrosine residues (Barnekow et al., 1990).Synaptophysin is, however, a transmembrane protein (Johnston et al., 1989) and unlikely, therefore, to correspond to ptp39, which occurs primarily in the soluble fraction.
0.25-.5 1 3 Time (rnin.1
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FIG. 7. The effect of veratridine on tyrosine phosphorylation by rat brain synaptosomes. Synaptosorneswere depolarized for the indicated periods in the presence of 0.05 mM veratridine before analysis for phosphotyrosine by irnrnunoblotting.The arrow indicates the position of p t p l l 7 .
86 1
FIG. 8. The distributionof synaptosomal phosphotyrosine-containing proteins. Control synaptosomes (A) or synaptosornes that had been depolarized for 3 rnin in the presence of high K+ (B)were lysed and soluble (S) and particulate (P) fractions separated by centrifugation as in Materials and Methods. Phosphotyrosine-containing proteins were identified by immunoblotting.The arrow and arrowhead indicate the positions of p t p l l 7 and ptp39, respectively.
k.
S
P
S
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pp6OC-""(Hirano et al., 1988; Cudmore and Gurd, 1991) and c-yes (Sudol et al., 1988) have been identified as synaptic components and are potential candidates for mediating the tyrosine phosphorylation events now described. The former enzyme has been shown to phosphorylate synaptophysin (Barnekow et al., 1990) as well as growth cone-associated tubulin (Matten et al., 1990). To understand the role of tyrosine phosphorylation in synaptic function, it is now clearly important to determine the identity and function of individual synaptic phosphotyrosine-containing proteins and the nature of the regulation of the tyrosine kinase(s) responsible for their phosphorylation. We are currently directing our efforts toward this end. Acknowledgment: The present work was supported by a grant to J.W.G. from the National Science and Engineering Research Council. We thank Dr. John Rostas for helpful discussion during the early part of this work.
REFERENCES Bading H. and Greenberg M. E. ( 1 99 1) Stimulation of protein tyrosine phosphorylation by NMDA receptor activation. Science 253,9 12-9 14. Barnekow A.. Jahn R.,and Schartl M. (1990) Synaptophysin: a substrate for the protein tyrosine kinase pp60""" in synaptic vesicles. Oncogene 5, 10 19- 1024. Blaustein M. P. (1975) Effects of potassium. veratridine and scorpion venom on calcium accumulation and transmitter release by nerve terminals in vitro. J. Phvsiol. 247,6 17-655. Blaustein M. P. and Goldring J. M. (1975) Membrane potentials in pinched-off nerve terminals monitored with a fluorescent probe: evidence that synaptosomes have potassium diffusion potentials. J. Physiol. 247, 589-6 15. Brostrom W.. Huang Y.-C.. Breckenndge B. M., and Wolff D. J. (1975) Identification of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase. Proc. Nutl. ,4cad. Sci. LiS.4 72, 64-68. Brugge J. S.. Cotton P. C.. Queral A. E.. Barret J. N., Nonner D.. and Keana W. ( 1985) Neurons express high levels of a structurally modified. activated form of pp60"'. NU/IW 316,95809586. Cheng N. and Sahyoun N. ( 1 990) Neuronal tyrosine phosphorylation in growth cone membranes. J. Biol. Chern. 265, 24172420. Cheung W. Y . . Bradham L. S.. Lynch T. J.. Lin Y . M.. and Tallant
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