Depolarization-Dependent Tyrosine Phosphorylation in Rat Brain Synaptosomes

Synaptosomes from rat forebrain were analyzed for the presence of phosphotyrosine-containing proteins by immunoblotting with antiphosphotyrosine antibodies. Using this technique, 10-11 phosphotyrosine-containing proteins were detected. Depolarization of synaptosomes by transfer to a high (41 mM) K+ medium resulted in increases in the phosphotyrosine content of several synaptosomal proteins, the most pronounced increase being associated with a membrane protein of M(r) 117,000 (ptp117). 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 ptp117 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 ptp117 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 ptp117. The results suggest that the phosphorylation of synaptic proteins on tyrosine residues may be involved in the regulation or modulation of synaptic activity.     

Peroxynitrite Induces Tyrosine Nitration and Modulates Tyrosine Phosphorylation of Synaptic Proteins

Peroxynitrite, the product of the radical-radical reaction between nitric oxide and superoxide anion, is a potent oxidant involved in tissue damage in neurodegenerative disorders. We investigated the modifications induced by peroxynitrite in tyrosine residues of proteins from synaptosomes. Peroxynitrite treatment (> or =50 microM) induced tyrosine nitration and increased tyrosine phosphorylation. Synaptophysin was identified as one of the major nitrated proteins and pp60src kinase as one of the major phosphorylated substrates. Further fractionation of synaptosomes revealed nitrated synaptophysin in the synaptic vesicles, whereas phosphorylated pp60src was enriched in the postsynaptic density fraction. Tyrosine phosphorylation was increased by treatment with 50-500 microM peroxynitrite and decreased by higher concentrations, suggesting a possible activation/inactivation of kinases. Immunocomplex kinase assay proved that peroxynitrite treatment of synaptosomes modulated the pp60src autophosphorylation activity. The addition of bicarbonate (CO2 1.3 mM) produced a moderate enhancing effect on some nitrated proteins but significantly protected the activity of pp60src against peroxynitrite-mediated inhibition so that at 1 mM peroxynitrite, the kinase was still more active than in untreated synaptosomes. The phosphotyrosine phosphatase activity of synaptosomes was inhibited by peroxynitrite (> or =50 microM) but significantly protected by CO2. Thus, the increase of phosphorylation cannot be attributed to peroxynitrite-mediated inhibition of phosphatases. We suggest that peroxynitrite may regulate the posttranslational modification of tyrosine residues in pre- and postsynaptic proteins. Identification of the major protein targets gives insight into the pathways possibly involved in neuronal degeneration associated with peroxynitrite overproduction.     

In Vivo Phosphorylation of the Postsynaptic Density Glycoprotein gp180

Rats received intraventricular injections of [32P]PO4 and were killed after 30 min for the preparation of postsynaptic densities (PSDs). Gel electrophoretic analysis identified a number of PSD proteins that incorporated 32P under these conditions. Major proteins that were labelled with 32P had Mr of 185,000, 165,000, 140,000, 92,000, and 51,000. Of these p185, p165, and p140 were also labelled when PSDs were incubated with [gamma-32P]ATP in vitro. In contrast p92 and p51 were relatively poorly labelled under in vitro conditions. Analysis of glycoproteins isolated by chromatography on concanavalin A (Con A)-agarose demonstrated that greater than 70-80% of the 32P present in the glycoproteins eluted from Con A-agarose with alpha-methyl-D-mannopyranoside (Con A+ glycoproteins) was associated with the PSD specific glycoprotein gp180 following both in vivo and in vitro labelling. Phosphopeptide maps and phosphoamino acid analysis of gp180 indicated that similar sites were labelled in vitro and in vivo. Analysis of the subcellular distribution of glycoproteins that incorporated 32P during in vivo labelling demonstrated that gp180 was highly concentrated in PSDs, in accord with the previously suggested exclusive association of this glycoprotein with postsynaptic structures.     

Tyrosine Phosphorylation of Microtubule-Associated Protein Kinase After Transient Ischemia in the Gerbil Brain

The tyrosine phosphorylation of microtubule-associated protein (MAP) kinase was examined in the gerbil brain after transient ischemia and reperfusion. Phosphorylation of MAP kinase was maximal within 1 min of reperfusion following 5 min of ischemia and returned to control levels as early as 5 min postischemia. The greatest increase in MAP kinase phosphorylation was detected in the hippocampus, with minor increases in other ischemic regions of the brain. Several tyrosine-phosphorylated proteins were detected in the gerbil hippocampus; however, the ischemia and reperfusion injury only increased tyrosine phosphorylation of MAP kinase. The increase in tyrosine phosphorylation was prevented by the N-methyl-D-aspartate (NMDA) receptor blocker (+)-MK-801, whereas a non-NMDA receptor blocker, 6-cyano-7-nitroquinoxaline-2,3-dione, was ineffective. Pretreatment of gerbils with calcium channel blockers also prevented the tyrosine phosphorylation of MAP kinase in the ischemic brain. Altogether, these results imply an involvement of glutamate receptors and calcium during the tyrosine phosphorylation of MAP kinase. Tyrosine phosphorylation was also prevented when ischemia and reperfusion were conducted under hypothermic conditions, which protect against neurodegenerative damage. These findings implicate a role for MAP kinase in neuronal damage resulting from ischemia and reperfusion.     

Tyrosine Phosphorylation in a Model of Ischemia Using the Rat Hippocampal Slice: Specific, Long-Term Decrease in the Tyrosine Phosphorylation of the Postsynaptic Glycoprotein PSD-GP180

Abstract: The effects of the exposure of hippocampal slices to brief periods of ischemic-like conditions on the tyrosine phosphorylation of proteins and glycoproteins were investigated. Freshly prepared hippocampal slices contained a range of tyrosine-phosphorylated proteins and two prominent tyrosine-phosphorylated glycoproteins of apparent M_r 110,000 (GP110) and 180,000, which we have previously shown to correspond to the postsynaptic density (PSD)-associated glycoprotein PSD-GP180. When hippocampal slices were incubated in oxygenated Krebs-Ringer buffer containing 10 mM glucose (KRB), there was a transient increase in the tyrosine phosphorylation of a protein of M_r 42,000 (p42) and a pronounced increase in the tyrosine phosphorylation of GP110. After these initial changes, the tyrosine phosphorylation of all proteins remained constant for at least 60 min. In vitro “ischemia” was achieved by transferring slices that had been preincubated for 60 min in KRB to KRB that had been equilibrated with N₂ instead of O₂ and that did not contain glucose. Tyrosine-phosphorylated GP110 and PSD-GP180 could no longer be detected after 10 min of exposure of the slices to ischemic-like conditions. GP110 was rapidly rephosphorylated on tyrosine after transfer of slices back to oxygenated, glucose-containing buffer. In contrast, short periods of ischemia (5 or 10 min) resulted in the long-term loss of phosphotyrosine [Tyr(P)]-PSD-GP180 so that it was not detected even after 60 min of reincubation in oxygenated KRB. The sustained decrease in tyrosine phosphorylation of PSD-GP180 after ischemia was Ca²⁺ dependent, the levels of Tyr(P)-PSD-GP180 slowly increasing to preischemic values if Ca²⁺ was omitted from the incubation media. Reoxygenation of ischemic slices also resulted in the Ca²⁺-dependent, transient tyrosine phosphorylation of p42. The major PSD-associated, tyrosine-phosphorylated glycoprotein of molecular mass 180 kDa has recently been identified as the NR2B subunit of the NMDA receptor. The results suggest that changes in tyrosine phosphorylation after an ischemic insult may modulate the NMDA receptor or signal transduction pathways in the postsynaptic cell and are consistent with a role for tyrosine phosphorylation in the sequence of events leading to neuronal cell damage and death.     

Phosphorylation of Tyrosine Hydroxylase by Cyclic GMP-Dependent Protein Kinase

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated and activated by purified cyclic GMP-dependent protein kinase as well as by cyclic AMP-dependent protein kinase catalytic subunit. The extent of activation was correlated with the degree of phosphate incorporated into the enzyme. Comparable stoichiometric ratios (0.6 mol phosphate/mol tyrosine hydroxylase subunit) were obtained at maximal concentrations of either cyclic AMP-dependent or cyclic GMP-dependent protein kinases. The enzymes appeared to mediate the phosphorylation of the same residue based on the observation that incorporation was not increased when both enzymes were present. The major tryptic phosphopeptide obtained from tyrosine hydroxylase phosphorylated by each protein kinase exhibited an identical retention time following HPLC. The purified phosphopeptides also exhibited identical isoelectric points. These data provide support for the notion that the protein kinases are phosphorylating the same residue of tyrosine hydroxylase.     

Tyrosine and Serine Phosphorylation of Dystrophin and the 58-kDa Protein in the Postsynaptic Membrane of Torpedo Electric Organ

Abstract: Dystrophin associates with a 58-kDa and an 87-kDa protein in the postsynaptic membrane of the Torpedo electric organ. We have previously shown that the 87-kDa protein is a major phosphotyrosine-containing protein in these membranes. Immunoprecipitation of the 87-kDa protein from phosphorylated postsynaptic membranes results in coimmunoprecipitation of additional phosphoproteins. These phosphoproteins are identified as dystrophin and the 58-kDa protein. Monoclonal antibodies to dystrophin and the 58-kDa protein immunoprecipitate phosphorylated forms of these proteins from postsynaptic membranes phosphorylated in vitro. Phosphoamino acid analysis reveals that dystrophin and the 58-kDa protein are phosphorylated on serine and tyrosine residues. In addition, both dystrophin and the 58-kDa protein are shown to be phosphorylated on tyrosine residues in vivo. These results suggest that the synaptic function of dystrophin and its associated proteins, the 58-kDa and 87-kDa proteins, may be modulated by tyrosine and serine protein Phosphorylation.     

Nitric Oxide Regulates Cyclic GMP-Dependent Protein Kinase Phosphorylation in Rat Brain

Abstract: Nitric oxide (NO) acts via soluble guanylyl cyclase to increase cyclic GMP (cGMP), which can regulate various targets including protein kinases. Western blotting showed that type II cGMP-dependent protein kinase (cGK II) is widely expressed in various brain regions, especially in the thalamus. In thalamic extracts, the phosphorylation of several proteins, including cGK II, was increased by exogenous NO or cGMP. In vivo pretreatment with a NO synthase inhibitor reduced the phosphorylation of cGK II, and this could be reversed by exogenous NO or cGMP. Conversely, brainstem electrical stimulation, which enhances thalamic NO release, caused a NO synthase-dependent increase in the phosphorylation of thalamic cGK II. These results indicate that endogenous NO regulates cGMP-dependent protein phosphorylation in the thalamus. The activation of cGKII by NO may play a role in thalamic mechanisms underlying arousal.     

Phosphorylation of Synaptic Proteins in Chick Forebrain: Changes with Development and Passive Avoidance Training

We have used synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) to study protein phosphorylation at the synapse in the developing chick forebrain and in 1-day-old chick forebrain following training on a passive avoidance task. Endogenous phosphorylation patterns in SPMs and PSDs prepared by extraction with n-octylglucoside isolated from chick forebrain were investigated by labelling with [32P]ATP. The phosphoprotein components of the SPM and PSD fractions were separated using sodium dodecyl sulphate gradient polyacrylamide gel electrophoresis. Autoradiography and densitometry of the Coomassie Blue protein staining pattern revealed phosphate incorporation into several SPM components including those of molecular mass 52, 37, and 29 kilodaltons (kDa). Bands of similar molecular mass were not phosphorylated in PSD fractions. This difference in phosphorylation between SPMs and PSDs was not due to the detergent n-octylglucoside. In a developmental study in which SPM and PSD fractions were prepared from 1-day-old, 14-day-old, and 21-day-old chickens, the phosphorylation patterns of SPMs were similar throughout, but striking differences occurred in PSDs, both in the level of phosphorylation and in the components phosphorylated. A time-course study was carried out in which phosphorylation of SPMs and PSDs from 1-day-old chicks trained on a passive avoidance task was compared with patterns from control chicks trained on a water-coated bead and untrained chicks. In SPMs prepared from forebrains removed 10 mins following training, a consistent but nonsignificant decrease (-21%) in phosphorylation of a 52 kDa band occurred in chicks with passive avoidance training compared with water-trained and untrained chicks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a Single Protein Kinase C-Mediated Phosphorylation Site in Rat Brain Protein B-50

The neuronal protein B-50 may be involved in diverse functions including neural development, axonal regeneration, neural plasticity, and synaptic transmission. The rat B-50 sequence contains 226 amino acids which include 14 Ser and 14 Thr residues, all putative sites for phosphorylation by calcium/phospholipid-dependent protein kinase C (PKC). Phosphorylation of the protein appears to be a major factor in its biochemical and possibly its physiological activity. Therefore, we investigated rat B-50 phosphorylation and identified a single phosphorylated site at Ser41. Phosphoamino acid analysis eliminated the 14 Thr residues because only [32P]Ser was detected in an acid hydrolysate of [32P]B-50. Staphylococcus aureus protease peptide mapping produced a variety of radiolabelled [32P]B-50 products, none of which had the same molecular weights or HPLC retention times as several previously characterized fragments. Indirect confirmation of the results was provided by differential phosphorylation of major and minor forms of B-60 that have their N-termini at, or C-terminal to, the Ser41 residue and are the major products of specific B-50 proteolysis. Only those forms of B-60 that contained the Ser41 residue incorporated phosphate label. The results are discussed with reference to the substrate requirements for B-50 phosphorylation by PKC and the proposed structure of the B-50 calmodulin binding domain.     

Activation and Multiple-Site Phosphorylation of Tyrosine Hydroxylase in Perfused Rat Adrenal Glands

Tryptic digestion of tyrosine hydroxylase (TH) isolated from rat adrenal glands labeled with 32Pi produced five phosphopeptides. Based on the correspondence of these phosphopeptides with those identified in TH from rat pheochromocytoma cells, four phosphorylation sites (Ser8, Ser19, Ser31, and Ser40) were inferred. Field stimulation of the splanchnic nerves at either 1 or 10 Hz (300 pulses) increased 32P incorporation into TH. At 10 Hz, the phosphorylation of Ser19 and Ser40 was increased, whereas at 1 Hz, Ser19, Ser31, and Ser40 phosphorylation was increased. Stimulation at either 1 or 10 Hz also increased the catalytic activity of TH, as measured in vitro (pH 7.2) at either 30 or 300 microM tetrahydrobiopterin. Nicotine (3 microM, 3 min) increased Ser19 phosphorylation, vasoactive intestinal polypeptide (10 microM, 3 min) increased Ser40 phosphorylation, and muscarine (100 microM, 3 min) increased TH phosphorylation primarily at Ser19 and Ser31. Vasoactive intestinal polypeptide, but not nicotine or muscarine, mimicked the effects of field stimulation on TH activity. Thus, the regulation of rat adrenal medullary TH phosphorylation by nerve impulses is mediated by multiple first and second messenger systems, as previously shown for catecholamine secretion. However, different sets of second messengers are involved in the two processes. The action of vasoactive intestinal polypeptide as a secretagogue involves the mobilization of intracellular calcium, whereas its effects on TH phosphorylation are mediated by cyclic AMP. This latter effect of vasoactive intestinal polypeptide and the consequent increase in Ser40 phosphorylation appear to be responsible for the rapid activation of TH by splanchnic nerve stimulation.     

Changes in the Tyrosine Phosphorylation of Mitogen-Activated Protein Kinase in the Rat Hippocampus During and Following Severe Hypoglycemia

Abstract: The changes in the levels of tyrosine-phosphorylated proteins in the cytosolic fraction of the rat hippocampus subjected to severe hypoglycemia were analyzed. A marked increase in tyrosine phosphorylation of a 43-kDa protein was observed at 30 min of isoelectric EEG and 30 min and 1 h of recovery. Immunostaining of the same blot with antibody against mitogen-activated protein (MAP) kinase demonstrated a double band of ∼42 and 43 kDa. The increased tyrosine phosphorylation of MAP kinase during hypoglycemic coma and the early recovery period suggests that MAP kinase may be involved in neuronal degeneration and repair.     

Activation and Tyrosine Phosphorylation of 44-kDa Mitogen-Activated Protein Kinase (MAPK) Induced by Electroconvulsive Shock in Rat Hippocampus

Abstract: Electroconvulsive shock (ECS) has been reported to induce the phosphorylation and activation of 42-kDa, but not 44-kDa, mitogen-activated protein kinase (MAPK) in rat hippocampus. We studied the activation and tyrosine phosphorylation of MAPKs in rat brain after ECS. We observed the increase of the activities of both 42- and 44-kDa MAPKs in rat hippocampus after ECS. The activities reached peak at 2 min and returned to basal levels by 15 min after ECS. We also observed the increased phsophorylation on the tyrosine residue of 42-kDa MAPK in rat hippocampus after ECS, but not on that of 44-kDa MAPK. However, when we examined the immunoprecipitated 44-kDa MAPK, we could demonstrate that the tyrosine phosphorylation of 44-kDa MAPK at 2 min after ECS was markedly increased, in accordance with the increase of kinase activity. These results indicate that ECS induces the transient activation and tyrosine phosphorylation of 44-kDa MAPK, as well as 42-kDa MAPK, in rat hippocampus, although the amount of tyrosine phosphorylation is far less and the kinase activity is lower in 44-kDa MAPK than in 42-kDa MAPK.     

Fibroblast Growth Factors Stimulate Protein Tyrosine Phosphorylation and Mitogen-Activated Protein Kinase Activity in Primary Cultures of Hippocampal Neurons

Abstract: Fibroblast growth factors (FGFs) are not only mitogens, but they also promote the differentiation of various cell types. For instance, basic FGF (bFGF) provides a critical trophic support for hippocampal neurons in culture. To elicit their biological effects, FGFs interact with high-affinity receptors that are transmembrane proteins with a cytoplasmic portion containing a tyrosine kinase activity. The tyrosine phosphorylation pattern was examined in primary cultures of hippocampal neurons derived from rat embryos. In these cultures grown for 3 days in the absence of serum, the addition of bFGF causes a rapid increase of tyrosine phosphorylation for various proteins with an optimal level after 5 min of bFGF exposure. Concomitantly, bFGF activates mitogen-activated protein kinase (MAP kinase) activity measured with a selective MAP kinase peptide. The activity increased rapidly after the addition of bFGF and remained elevated even when cultures were treated for 1 h with bFGF. Both acidic and basic FGF were able to enhance protein tyrosine phosphorylation and MAP kinase activity, whereas nerve growth factor and epidermal growth factor did not elicit any of these responses. These data indicate that some of the transduction signals (i.e., tyrosine phosphorylation and activation of MAP kinase) that have been described for the proliferative effect of FGFs are also involved when FGFs act as trophic factors for postmitotic neurons in culture.     

Immunohistochemical Localization of Ca2+/Calmodulin-Dependent Protein Kinase II in Rat Brain and Various Tissues

Polyclonal antibodies against Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) of rat brain were prepared by immunizing rabbits and then purified by antigen-affinity column. The antibodies which recognized both subunits of the enzyme with Mrs 49K and 60K were used for the study on the distribution of CaM kinase II in formalin-fixed, paraffin-embedded tissues. In the brain, a light-microscopic study demonstrated strong immunoreactivity in neuronal somata and dendrites and weak immunoreactivity in nuclei. The densely stained regions included cerebral cortex, hippocampal formation, striatum, substantia nigra, and cerebellar cortex. In substantia nigra, neurites were stained, but not neuronal somata. Electron microscopy revealed that the immunoreactive product was highly concentrated at the postsynaptic densities. In addition to neurons, weak immunoreactivity was also demonstrated in glial cells, such as astrocytes and ependymal cells of ventricles and epithelial cells of choroid plexus. In other tissues, strong immunoreactivity was observed in the islet of pancreas and moderate immunoreactivity in skeletal muscle and kidney tubules. Immunoreactivity was demonstrated in all of the tissues tested. The results suggest that CaM kinase II is widely distributed in the tissues.     

Phosphorylation of the solubilized insulin receptor by the gene product of the Rous sarcoma virus, pp60src

Both the insulin receptor and the gene product of the Rous sarcoma virus, pp60src, are protein kinases which phosphorylate themselves and other proteins on tyrosine residues. Addition of the solubilized insulin receptor to purified pp60src increased the phosphorylation of the beta-subunit of the insulin receptor. Phosphorylation of the insulin receptor by pp60src occurred both in the absence and presence of insulin but did not alter the insulin dose response for autophosphorylation of the receptor. Increasing concentrations of pp60src increased the phosphorylation of the receptor and at high concentrations equaled the maximal effect produced by insulin. Our observations suggest a possible mechanism by which the metabolically regulated insulin receptor tyrosine kinase could be altered by other tyrosine kinases such as that associated with pp60src. Further studies will be required to determine if the insulin receptor is phosphorylated by pp60src in Rous sarcoma virus-infected cells.     

LA－90细胞在温度转化过程中蛋白质酪氨酸磷酸化作用研究

ＬＡ－９０细胞在温度转化过程中蛋白质酪氨酸磷酸化作用研究夏英,高漫,颜卉君,吴国利（北京师范大学生物系生物化学及分子生物学研究室,１００８７５）关键词酪氨酸蛋白激酶;磷酸酪氨酸蛋白磷酸酶;细胞转化ｉｓ－ＲＳＶＬＡ－９０细胞是ＲＳＶ转染的小鼠３Ｔ３细胞...     

Protein Tyrosine Kinase Activity and Its Endogenous Substrates in Rat Brain: A Subcellular and Regional Survey

The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.     

Synaptic Protein Tyrosine Kinase: Partial Characterization and Identification of Endogenous Substrates

The subcellular distribution of protein tyrosine kinase in rat forebrain was determined using [Val5]-angiotensin II as exogenous substrate. Enzyme activity was present in each of the fractions analyzed and was enriched in synaptic membranes (SMs) and the synaptosomal soluble fraction (2.2- and 2.5-fold over the homogenate, respectively). SMs also phosphorylated polyglutamyltyrosine (pGT; molar ratio of 4:1), the Vmax for angiotensin and pGT phosphorylation being 26.3 +/- 1.6 and 142 +/- 4 pmol/min/mg, respectively. Extraction of SMs with several different detergents resulted in enhanced enzyme activity and the solubilization of 33-37% of the angiotensin and 43-70% of the pGT-phosphorylating activity. Isolated postsynaptic densities (PSDs) contained tyrosine kinase and phosphorylated angiotensin and pGT. The Vmax values for angiotensin and pGT phosphorylation by PSDs were 17 +/- 5 and 23 +/- 1 pmol/min/mg, respectively. Six putative endogenous substrates for SM tyrosine kinase, with molecular weights of 205K, 180K, 76K, 60K, 50K, and 45K, were identified. Each of these proteins, except p76, was phosphorylated in the detergent-insoluble residue obtained following the extraction of SMs with Triton X-100 as well as in PSDs, indicating that the postsynaptic apparatus is an active site of tyrosine phosphorylation. The phosphorylation of p76 was localized to the Triton X-100 extract and also occurred in the synaptosomal soluble fraction. The results indicate that tyrosine kinase and its substrates are located in both pre- and postsynaptic compartments and suggest a role for this enzyme in synaptic function.