Protein tyrosine phosphorylation: Implications for synaptic function

~ Pergamon

Neurochem. Int. Vol. 31, No. 5, pp. 6354549, 1997 © 1997ElsevierScienceLtd P I h S0197-0186(97)00022-3 Printedin Great Britain.All rights reserved 0197-0186/97$17.00+0.00

I N V I T E D REVIEW

PROTEIN TYROSINE PHOSPHORYLATION: IMPLICATIONS FOR SYNAPTIC F U N C T I O N JAMES W. GURD* Division of Life Sciences, University of Toronto at Scarborough, West Hill, Ontario, Canada MIC 1A4 (Received for publication 21 March 1997)

Abstract--The phosphorylation of proteins on tyrosine residues, initially believed to be primarily involved in cell growth and differentiation, is now recognized as having a critical role in regulating the function of mature cells. The brain exhibits one of the highest levels of tyrosine kinase activity in the adult animal and the synaptic region is particularly rich in tyrosine kinases and tyrosine phosphorylated proteins. Recent studies have described the effects of tyrosine phosphorylation on the activities of a number of proteins which are potentially involved in the regulation of synaptic function. Furthermore, it is becoming apparent that tyrosine phosphorylation is involved in the modification of synaptic activity, such as occurs during depolarization, the induction of long-term potentiation or long-term depression, and ischemia. Changes in the activities of tyrosine kinases and/or protein tyrosine phosphatases which are associated with synaptic structures may result in altered tyrosine phosphorylation of proteins located at the synapse leading to both short-term and long-lasting changes in synaptic and neuronal function. © 1997 Elsevier Science Ltd

tyrosine phosphorylation in the function of mature cells. In particular, postmitotic neurons were found to express high levels of tyrosine kinase activity (Dasgupta et al., 1994; Cotton and Brugge, 1983). In view of the well-documented involvement of protein phosphorylation in a variety of synaptic functions it was perhaps not surprising to find that the synapse is associated with relatively high levels of protein tyrosine kinase activity. Analysis of isolated synaptic fractions, including postsynaptic densities (PSDs), synaptic membranes and synaptic vesicles, demonstrated that several synaptic proteins are phosphorylated on tyrosine residues and further that these synaptic organelles are associated with intrinsic tyrosine kinases (Gurd and Bissoon, 1985; Ellis et al., 1988; Hirano et al., 1988; Pang et al., 1988; Barnekow et al., 1990). While these earlier studies suggested that tyrosine phosphorylation was involved in synaptic function it is only in the last few years that progress in identifying specific consequences of tyrosine phos*Tel.: (416) 287-7410; fax: (416) 287-7642; e-mail: phorylation for synaptic activity has been made. This short commentary will review recent advances dealing gurdC~scar.utoronto.ca. 635

Protein phosphorylation is involved in the regulation of a wide range of cellular functions within the CNS. The synaptic region in particular is rich in protein kinase activity and protein phosphorylation plays a central role in the regulation of synaptic function (Nairn et al., 1985; Dunkley and Robinson, 1986; Greengard et al., 1993). More recently, the phosphorylation of proteins on tyrosine residues has attracted increasing attention as a potential mechanism for the regulation of neuronal and synaptic activity. Initially identified in connection with oncogene products and certain growth factor receptors, tyrosine phosphorylation was considered to be primarily involved in the regulation of cell growth and development (reviewed in Hunter, 1996). It soon became apparent, however, that a number of tissues continue to exhibit high levels of tyrosine phosphorylation in the adult animal, indicating a role for

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with the possible implications of tyrosine phosphorylation for synaptic function.

TYROSINE PHOSPHORYLATION OF LIGAND-GATED ION CHANNELS

NMDA

receptor

The N-methyl-D-aspartate (NMDA) subclass of glutamate receptor plays a critical role in a wide variety of phenomena including long-term potentiation (LTP), excitotoxicity, neuronal development and synaptic plasticity. The N M D A receptor is composed of five major types of subunits; N R I , which exists as eight splice variants, and four types of NR2 subunit (NR2A-D). Whereas NR1 subunits will form fully functional homo-oligomeric channels when expressed in transfected cells NR2 subunits do not form functional channels but rather serve to modulate the properties of heteromeric receptors consisting of NR 1 plus 1 or more NR2 subunits (reviewed in Hollman and Heinneman, 1994). Each of the N M D A receptor subunits contains sites for phosphorylation by different protein kinases, and regulation of N M D A receptor function by protein phosphorylation is well documented (Swope et al., 1992; Roche et al., 1994). Several years ago the major PSD-associated glycoprotein of molecular weight 180 000 (PSD-GP180) was identified as a prominent substrate for tyrosine kinases associated with the postsynaptic apparatus (Gurd, 1985; Gurd and Bissoon, 1985; Kearney and Gurd, 1986). Tyrosine phosphorylated PSD-GP180 was widely distributed in different brain regions and its phosphorylation on tyrosine residues was developmentally regulated (Gurd and Bissoon, 1990; Soulliere et al., 1994). The identification of PSD-GPI80 as the NR2B subunit of the N M D A receptor (Moon et al., 1994) provided an important link between these earlier studies of the tyrosine phosphorylation of PSD-GP180 and more recent observations indicating that tyrosine phosphorylation plays an important role in the regulation of N M D A receptor ion channel function. Both NR2A and NR2B subunits of the N M D A receptor are phosphorylated on tyrosine in situ, with NR2B being the more highly phosphorylated subunit. (Moon et al., 1994; Lau and Huganir, 1995; Menegoz et al., 1995). As is frequently the case for tyrosine phosphorylated proteins, the stoichometry of tyrosine phosphorylation of both NR2A and NR2B is low, in the order of 2-3%, in freshly prepared brain tissue (Lau and Huganir, 1995). Tyrosine phosphorylation of the NRI subunit was not detected either in situ or

following in vitro phosphorylation (Lau and Huganir, 1995). Although individual tyrosine phosphorylation sites on the N M D A receptor have not been identified, the proposed intracellular location of the C-terminal domain of glutamate receptors (Tingley et al., 1993; Hollman et al., 1994) is consistent with phosphorylation occurring in this region. In accord with this location of the phosphorylation sites, tyrosine phosphorylation failed to enhance N M D A channel function in transfected HEK 293 cells co-expressing NR1 and a truncated form of NR2A lacking the Cterminal domain (K~hr and Seeburg, 1996; see below). Several studies have addressed the possible relationship between tyrosine phosphorylation and the activity of the N M D A receptor ion channel. Inhibition of protein tyrosine kinase activity with genistein or lavendustin A reduced both N M D A currents and intracellular Ca 2+ responses to applied N M D A in spinal dorsal horn neurons (Wang and Salter, 1994). Furthermore, intracellular application of pp60 ..... or of sodium orthovanadate, conditions which would be expected to enhance tyrosine phosphorylation, resulted in the potentiation of N M D A currents in spinal dorsal horn neurons and hippocampal cells (Wang and Salter, 1994). These authors also provided evidence that protein tyrosine phosphatases play a role in the run-down of N M D A receptor-mediated currents during whole-cell patch-clamp recording and further reported that the application of exogenous protein tyrosine phosphatase to spinal dorsal horn neurons resulted in a decrease in N M D A single channel activity (Wang et al., 1996a). Analysis of the effect of tyrosine phosphorylation on the channel activities of recombinant heteromeric N M D A receptor-channels transiently expressed in HEK 293 cells confirmed that tyrosine phosphorylation by members of the src tyrosine kinase family (Src and Fyn) can lead to potentiation of glutamate-activated currents (K6hr and Seeburg, 1996). Comparison of the consequences of tyrosine phosphorylation for the channel properties of heteromeric N M D A receptors of different subunit composition indicated that phosphorylation of NR2A, but not of NR2B, NR2C or NR1, was responsible for modulation of channel properties (K6hr and Seeburg, 1996). In general accord with these findings, insulin stimulated tyrosine kinase potentiated N M D A currents in the CA 1 region of hippocampal slices and of cloned receptor subunits expressed in X e n o p u s oocytes (Chen and Leonard, 1996; Liu et al., 1995). In contrast to the results of K6hr and Seeburg (1996), however, insulin-induced potentiation of N M D A currents was observed with all combinations of expressed subunits including ~I/E1, ~I/E2, ~l/E4 as well as with

Invited Review homomeric (1 receptors (Chen and Leonard, 1996). These findings may indicate that ~1 (NR1), c2 (NR2B) and c4 (NR2D) subunits are phosphorylated on tyrosine residues under these conditions, or that insulin initiates a sequence leading to modification of N M D A receptor activity that, although requiring tyrosine kinase activity, may not involve tyrosine phosphorylation of the receptor. For example, the application of platelet-derived growth factor to hippocampal cells resulted in a long-lasting inhibition of N M D A receptor function (Valenzuela et aL, 1996). This inhibition apparently did not result from tyrosine phosphorylation of the N M D A receptor, but rather from a sequence involving the binding of PLC-7 to the activated PDGFR, activation of PLC-7, and the elevation of intracellular Ca 2÷ levels (Valenzuela et al., 1996). The results described above are consistent with a model in which tyrosine phosphorylation of one or more N M D A receptor subunits modulates the properties of the ion channel. However, the addition of exogenous protein tyrosine kinases or inhibitors of protein tyrosine phosphatases may result in levels of tyrosine phosphorylation which exceed those which normally occur in situ, giving rise to unphysiological effects. Moreover, as noted by K r h r and Seeburg (1996) discrepancies between results obtained with native receptors and recombinant receptors indicate the potential difficulties of interpreting results obtained with heterologous systems. In contrast to studies of the nicotinic acetylcholine receptor, in which a clear correlation between receptor tyrosine phosphorylation and channel activity has been demonstrated (Hopfield et al., 1988; see below), changes in tyrosine phosphorylation of the N M D A receptor were not confirmed in any of the above reports, leaving open the possibility that the observed effects on channel function may be indirect, resulting from the tyrosine phosphorylation of protein(s) other than, or in addition to the receptor itself. The apparent lack of an effect of Src or Fyn on the channel properties of binary N R 1/NR2B receptors expressed in transfected cells (Krhr and Seeburg, 1996) is surprising in view of the fact that NR2B is the major tyrosine phosphorylated subunit in vivo (Moon et al., 1994; Lau and Huganir, 1995; Menegoz et al., 1995) and suggests either that NR2B is not phosphorylated in the transfected cells or that tyrosine phosphorylation of NR2B serves a role other than modulation of channel properties. The fact that NR2B is phosphorylated by both Src and Fyn under in vitro conditions (Gurd, unpublished results; Suzuki and Okumura-Noji, 1995) would appear to argue against the former possibility.

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As for other possible roles of tyrosine phosphorylation, we have found that tyrosine phosphorylated NR2B and NR2A bind with the SH2 domains of several proteins involved in signal transduction, including Src, PLC-7, Grb2 and the p85 subunit of PI3-kinase (Gurd et al., 1995, 1996 and unpublished results). It has recently become apparent that the N M D A receptor is one of a number of proteins which are linked together by interacting with members of the PSD-95 protein family (Kornau et al., 1995; Lau et al., 1996; Neithammer et al., 1996; Kim et al., 1996). The binding of SH2-containing proteins such as Src and PLC-v to tyrosine phosphorylated NR2B may constitute an additional method by which proteins involved in signaling in the postsynaptic cell may associate with this multifunctional signaling complex. What are the tyrosine kinases which are responsible for the phosphorylation of the N M D A receptor? Fyn and Src are both associated with the postsynaptic density and both will phosphorylate NR2B and NR2A under in vitro conditions (Cudmore and Gurd, 1991; Suzuki and Okumura-Noji, 1995; and Gurd, unpublished results). Src modulates N M D A receptor function in neurons in culture (Wang and Salter, 1994) and both Src and Fyn potentiated N M D A currents in transfected cells (Krhr and Seeburg, 1996). The increase in tyrosine phosphorylation of the N M D A receptor subunit following the induction of LTP in the dentate gyrus (Rostas et al., 1996; Rosenblum et al., 1996), coupled with the inability to induce LTP in f y n - mice (Grant et al., 1992), is consistent with a role for Fyn in receptor phosphorylation. Preliminary results indicating that NR2A/B subunits bind to the SH2 domains of Src (Gurd et al., 1995) further suggest that Src may be associated with the receptor in the PSD. It is interesting that in addition to impaired LTP, mice with t h e f y n mutation exhibit abnormalities in a variety of behaviors including suckling (Yagi et al., 1993), performance in the Morris water maze (Grant et al., 1992), and "emotionality" (Miyakawa et al., 1994). The relationship of these behavioral changes of the f y n mutant mice to specific deficits in tyrosine phosphorylation of synaptic proteins, including the N M D A receptor, is unknown. Changes in tyrosine phosphorylation of NR2A/B subunits of the N M D A receptor may occur under a variety of conditions including the induction of LTP in the rat dentate gyrus (Rostas et al., 1996; Rosenblum et al., 1996; see below), brief periods of transient global ischemia in the rat hippocampus (Gurd et al., 1995; Takagi et al., 1997; see below), and 6-hydroxydopamine lesioning of nigrostriatal dopaminergic

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neurons in the rat striatum (Menegoz et al., 1995). In the latter case the increase in tyrosine phosphorylation occurred primarily on NR2B and was associated with a small increase in striatal tyrosine kinase activity but no detectable change in protein tyrosine phosphatase activity (Menegoz et al., 1995; Girault et al., 1992). Chronic treatment with neuroleptics and conditioned taste aversion training resulted in increased tyrosine phosphorylation of a 180 000 molecular weight protein in the striatum and rat insular cortex, respectively (Girault et al., 1992; Rosenblum et al., 1995) Although the identity of this protein was not determined, it was enriched in PSDs and bound to Con A (Girault et al., 1992), properties in common with PSD-GP180 and the NR2B subunit of the N M D A receptor. Interestingly, mice with the f y n mutation showed normal acquisition of conditioned taste aversion (Shafe et al., 1996), indicating that Fyn is not responsible for the increase in tyrosine phosphorylation of the 180000 molecular weight protein under these conditions. The mechanisms which result in enhanced tyrosine phosphorylation of the N M D A receptor in the above paradigms are not known but it is perhaps relevant that activation of the N M D A receptor itself may stimulate tyrosine kinase activity. For example, the application of glutamate to hippocampal cells stimulated the Ca2+-dependent, APV-sensitive, tyrosine phosphorylation of a 39 000 molecular weight protein (Bading and Greenberg, 1991). In addition, the increase in tyrosine phosphorylation which follows transient cerebral ischemia (Kindy, 1993; Hu and Weiloch, 1994; Ohtsuki et al., 1996) was blocked by the preadministration of MK801, an N M D A antagonist (Hu and Weiloch, 1994). Similarly, MK801 markedly reduced the increase in tyrosine phosphorylation of MAP kinase which was elicited by seizure activity produced by injections of bicuculline (Gass et al., 1993). These findings suggest the possible operation of a feedback loop in which activation of the N M D A receptor stimulates tyrosine phosphorylation, which may in turn result in phosphorylation and modulation of the N M D A receptor. Although there is a considerable amount of information relating to tyrosine phosphorylation of the N M D A receptor, little is known concerning the effects, if any, of tyrosine phosphorylation on other glutamate receptors. A suggestion that the AMPA receptor subunit gluR1 may be a substrate for tyrosine kinases came from a study demonstrating that coexpression of gluR1 (flop) with v-src in HEK 293 cells resulted in tyrosine phosphorylation of the receptor (Moss et al., 1993). The significance of this observation is, however, unclear since the consequences of

the reaction in terms of receptor function are unknown and phosphorylation of the native receptor on tyrosine has not yet been described. Nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (AChR) present at the neuromuscular junction is a pentameric, ligand-gated ion channel consisting of four subunits in the arrangement a2fly6. Each subunit contains four membrane-spanning domains with the a-helices of the M2 transmembrane domain of each subunit lining the cation pore. Regulation of the AChR by phosphorylation on serine and threonine is well documented (reviewed in Huganir and Miles, 1989; Swope et al., 1992). The nicotinic acetyl choline receptor present in the postsynaptic membranes of the electric organ of Torpedo caliJbrnica (Huganir et al., 1984) and in vertebrate skeletal muscle (Smith et al., 1987) is also phosphorylated on tyrosine residues, tyrosine phosphorylation occurring on the fl, ), and 6 subunits (Huganir et al., 1984). Peptide sequence analysis identified the tyrosine phosphorylation sites as Tyr-355 (fl), Tyr 364 (7) and Tyr-372 (6) in the Torpedo AChR (Wagner et al., 1991). Each of these sites occurs in the major cytoplasmic domain between transmembrane segments M3 and M4. Tyrosine phosphorylation of the neuronal AChR has not been demonstrated, although it contains consensus sequences for tyrosine kinases (Swope et al., 1992), A direct relationship between the extent of tyrosine phosphorylation and function of the AChR ion channel was demonstrated by Hopfield et al. (1988). The level of tyrosine phosphorylation of the acetylcholine receptor from Torpedo californica was manipulated by in vitro phosphorylation and dephosphorylation reactions and channel kinetics determined following incorporation of the receptor into lipid vesicles. A 45-fold increase in receptor phosphorylation, from 0.6 to 2.7 mol phosphate per mol receptor, was paralleled by a 7-fold increase in the fast rate of receptor desensitization (Tfast decreasing from 15.3s to 2.2s). A smaller increase in the rate of the slow phase of desensitization also accompanied the increase in tyrosine phosphorylation but other channel properties, including single-channel conductance and apparent mean channel open-time, were unaffected (Hopfield et al., 1988). The tyrosine kinase which is responsible for phosphorylation, and modulation, of the acetylcholine receptor in situ has yet to be identified. One possibility is the receptor-like tyrosine kinase MuSK (Valenzuela et al., 1995a). Clustering of the AChR which is induced by agrin or which occurs during formation of

Invited Review the neuromuscular junction requires the activation of one or more protein tyrosine kinases and involves phosphorylation of the AChR on tyrosine residues (Qu et al., 1990; Wallace et al., 1991; Wallace, 1994; Qu and Huganir, 1994; Ferns et al., 1996). MuSK is essential for the formation of the neuromuscular junction (DeChiara et al., 1996) and appears to serve as the agrin receptor (Glass et al., 1996). Furthermore, when co-expressed with rabsyn in QF-18 cells MuSK phosphorylated the fl subunit of the AChR on Tyr390, which is homologous to the phosphorylation site in the Torpedo receptor (Gillespie et al., 1996). While the evidence is compelling that MuSK is involved in tyrosine phosphorylation of the AChR during formation of the neuromuscular junction and AChR clustering, its role in modulation of receptor function at mature synapses remains unknown. Swope and Huganir (1993) reported that the Src-related tyrosine kinases Fyn and Fyk will phosphorylate the fl, 7 and subunits of the AChR under in vitro conditions. Both of these enzymes associate with the AChR through interaction of their SH2 domains with (P)Tyr residues on the receptor and both were associated with the receptor in the Torpedo postsynaptic membrane (Swope and Huganir, 1994). Whether or not they are responsible for phosphorylation of the receptor in situ is, however, unknown. Protein tyrosine phosphatases, until recently considered to play a largely passive role in regulating levels of tyrosine phosphorylation, are now believed to be dynamic participants in this process (Shenolikar, 1994; Sun and Tonks, 1994). Regulation of protein function by tyrosine phosphorylation implies the presence of protein tyrosine phosphatases which are able to reverse the phosphorylation reaction. The activity of protein tyrosine phosphatases able to act upon the AChR was indicated by the observation that pervanadate, an inhibitor of protein tyrosine phosphatase activity, induced an increase in receptor tyrosine phosphorylation in cultures of chick myotubes (Wallace, 1995). Tanowitz et al. (1996) reported that denervation of rat skeletal muscle was accompanied by a 2fold increase in the activity of an unidentified protein tyrosine phosphatase, a change which may be related to the denervation-induced decrease in tyrosine phosphorylation of the AChR (Qu et al., 1990), although this was not directly demonstrated. Mei and Huganir (1991) isolated a protein tyrosine phosphatase that acts upon the nicotinic acetylcholine receptor from the Torpedo electric organ but the role of this enzyme in regulating receptor tyrosine phosphorylation levels in situ was not examined. In addition to a role in modulating the AChR ion

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channel, tyrosine phosphorylation also participates in the formation and, possibly, the maintenance of the neuromuscular junction. The aggregation of AChRs into clusters during formation of the neuromuscular junction requires tyrosine phosphorylation (Wallace, 1992; Peng et al., 1993) and, as noted above, is critically dependent upon the activity of the receptor-like tyrosine kinase MuSK (Gillespie et al., 1996; DeChiara et al., 1996; summarized in Wallace, 1996). A role for tyrosine phosphorylation in the retention of AChR clusters was suggested by the finding that inhibition of tyrosine kinase activity resulted in an increase in the rate of dispersal of agrin-induced AChR clusters following the removal of agrin from cultured chick and rat myotubes (Wallace, 1994; Ferns et aL, 1996). The requirement for continued tyrosine phosphorylation occurred at a time when the AChR was no longer phosphorylated on tyrosine residues, indicating that other, yet to be identified, tyrosine phosphorylated proteins were involved in maintaining receptor clusters (Ferns et al., 1996). In apparent contrast to these results, denervation of the rat diaphragm was followed by a slow dephosphorylation of tyrosine residues on the AChR so that by 4 weeks after denervation tyrosine phosphate could not be detected at most synapses (Qu et al., 1990). In spite of the decrease in tyrosine phosphorylation, however, the concentration of the AChR at the endplate remained stable for at least 6 weeks following denervation, indicating that in this situation tyrosine phosphorylation was not required for the continued localization of the AChR at the neuromuscular junction (Qu et al., 1990). GABAA receptors GABA^ receptors constitute the major sites of fast synaptic inhibition in the brain. The GABAA receptor is assembled from four classes of subunits, each of which contains several members: ct (1-6), fl (1-3), y (1-3) and 6 (1) (reviewed in Macdonald and Olsen, 1994). GABAA receptors are regulated by protein phosphorylation by several protein kinases including PKC, PKA, PKG and CAM-kinase II (Macdonald and Olsen, 1994). Modulation of GABAA receptors by tyrosine phosphorylation was recently reported by Moss et al. (1995), who found that co-transfection of A293 cells with ctl, fll and y2L GABAA receptor subunits and v-Src resulted in the tyrosine phosphorylation of the y2L, and to a lesser extent of ill, subunits. Site-specific mutational analysis identified the sites of phosphorylation on ),2L as tyrosines 365 and 367 which are located on the major intracellular domain (Moss et al., 1995). Inhibition of tyrosine phosphorylation in transfected cells with genistein

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resulted in a decrease in the tyrosine phosphorylation of ~,2L and a concomitant decrease in the amplitude of the G A B A response. The application of genistein or vanadate to superior cervical ganglion neurons in culture resulted in a decrease or increase, respectively, in the amplitude of the G A B A response, indicating that native receptors are also regulated by tyrosine phosphorylation (Moss et al., 1995). Enhanced tyrosine phosphorylation resulted in an increase in both the mean open time and the probability of channel opening in patches of superior cervical ganglion neurons (Moss et al., 1995). In general accord with these findings, inhibition of tyrosine kinase activity inhibited C1- currents in X e n o p u s oocytes expressing ~lfll or cdfl172L GABAA receptors (Valenzuela et al., 1995b). These authors also reported that inhibition of tyrosine kinase activity decreased the uptake of CI in cortical brain microsacs, indicating that the native receptor was phosphorylated on tyrosine by endogenous tyrosine kinases. This suggestion is supported by the finding that an unidentified tyrosine kinase activity co-purified with the GABAA receptor from bovine cerebral cortex and that this enzyme appeared to phosphorylate the ~2, but interestingly not ~2 or flj, GABAA receptor subunit in the same preparation (Bureau and Laschet, 1995). Additional evidence that tyrosine phosphorylation may be involved in the regulation of GABAergic synaptic transmission was obtained by Tanaka et al. (1996), who reported that the application of basic fibroblast growth factor to hippocampal cells in culture decreased the amplitude of spontaneous Ca 2+ oscillations. This effect was blocked by suramin, indicating that it required binding of the growth factor to the b F G F receptor, and it was prevented by bicuculline, suggesting that b F G F was affecting GABAergic neurotransmission. A direct demonstration of the participation of the receptor tyrosine kinase activity in these events was not, however, reported.

MODULATION OF OTHER ION CHANNELS BY TYROSINE PHOSPHORYLAT1ON

Potassium channels

Regulation of ion channels by the phosphorylation of serine and threonine residues has been well documented (reviewed in Levitan, 1994). More recently it has become evident that tyrosine phosphorylation is also involved in the modulation of the function of several types of ion channels including both potassium and calcium channels. Direct evidence for a role of tyrosine phosphorylation in the regulation of a pot-

assium channel was provided by Huang et al. (1993). These authors demonstrated that activation of the G protein-coupled m I muscarinic acetylcholine receptor expressed in X e n o p u s oocytes or in H E K 293 cells led to suppression of Kvl.2 potassium channels which were co-expressed in the same cells. Current suppression involved a pathway which included activation of phospholipase C and enhanced tyrosine phosphorylation of the potassium channel. Substitution of Tyr132 with phenylalanine reduced the effect of carbachol on ion channel activity and also decreased the carbachoMnduced increase in tyrosine phosphorylation of the ion channel, indicating that phosphorylation ofTyr132 was in part responsible for suppression of ion channel activity. The participation of Ca 2+ ions in the carbachol-induced reduction of potassium currents was suggested by the finding that the degree of channel suppression was reduced in the presence of EGTA. Although the identity of the tyrosine kinase responsible for these effects was not known it is interesting that Lev et al. (1995) reported that the Kvl.2 potassium channel is phosphorylated by the non-receptor tyrosine kinase, PYK2, and further that phosphorylation by PYK2 was accompanied by a suppression of channel currents (Lev et al., 1995). The tyrosine phosphorylation and activity of PYK2 were enhanced in response to elevations in intracellular Ca -,+ and by activation o f P K C (Lev et al., 1995), in general accord with the findings of Huang et al. (1993). The Kvl.3 potassium channel was also found to be suppressed by tyrosine phosphorylation (Holmes et al., 1996a). Co-transfection of H E K 293 cells with the Kvl.3 potassium channel and either a cytoplasmic tyrosine kinase (v-src) or a receptor tyrosine kinase (the E G F receptor) resulted in enhanced tyrosine phosphorylation of the channel protein (8-fold with v-Src and approximately 4-fold with the E G F receptor) as compared to basal phosphorylation levels in cells which were not co-transfected with a tyrosine kinase (Holmes et al., 1996a). These authors further reported that pervanadate treatment of H E K 293 cells transfected with Kvl.3 D N A resulted in an increase in tyrosine phosphorylation of the channel protein and a decrease in Kvl.3 currents, indicating that the channel may be regulated by endogenous tyrosine kinases. Mutation of Tyr449 of the Kvl.3 channel to phenylalanine eliminated much of the tyrosine phosphorylation of the channel as well as the pervanadateinduced suppression of current (Holmes et al., 1996a). A relationship between the human Kv 1.5 potassium channel and tyrosine phosphorylation has also been reported by Holmes et al. (1996b), who found that the potassium channel expressed in HEK 293 cells was

Invited Review phosphorylated on tyrosine residues in cells coexpressing v-Src. Suppression of Kvl.5 channel currents in cell-attached membrane patches was correlated with the presence of v-Src, but phosphorylation of the channel was not directly demonstrated in this situation. An important question relating to the potential control of ion channels by tyrosine phosphorylation concerns the targeting of kinases to the channel protein. As noted above, the N M D A receptor and the A C h R may both interact with members of the Src-family of tyrosine kinases through their SH2 domains. Holmes et al. (1996b) reported that the human Kvl.5 potassium channel was associated with Src in both transfected H E K 293 cells and human myocardium, and further that the channel protein bound to the SH3 domain of Src expressed as a GST-fusion protein, suggesting this as one possible mechanism by which the channel may associate with tyrosine kinases in situ (Holmes et al., 1996b). Endogenous protein tyrosine phosphatase activity present in a cell-free patch-clamp preparation of Aplysia bag cell neurons appeared to be responsible for an increase in the open state probability of a voltagegated cation channel, causing switching from the bursting to the non-bursting mode, and indicating that tyrosine phosphorylation of the channel, or of a closely associated protein, may be involved in regulation of the channel (Wilson and Kaczmarek, 1993). Interestingly, the PTPase which produced these effects appeared to be activated by protein kinase A (Wilson and Kaczmarek, 1993). Thus application of the catalytic subunit of P K A to cell-free patches mimicked the effects of exogenous protein tyrosine phosphatase by a mechanism which was blocked by vanadate, but not by serine/threonine phosphatase. Calcium channels

The potential regulation of a voltage-operated Ca 2÷ channels in vascular smooth muscle cells from rabbit ear artery by tyrosine phosphorylation was suggested in a report by Wijetunge e t al. (1992) in which channel activity was inhibited by the tyrosine kinase inhibitors genistein and tryphostin 1 and tryphostin 23 but not by daidzein, an inactive homologue of genistein. A role for tyrosine phosphorylation in the regulation of L-type calcium channels was indicated by the finding that increases in [Ca2+]i in pituitary GH3 cells elicited by the specific L-type Ca 2+ activator Bay K 8644, or by high K+-induced depolarization, were reduced by genistein, herbimycin A and lavendustin, but not by daidzein, and enhanced by inhibition of protein tyrosine phosphatase activity with vanadate (Cataldi et

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al., 1996). Tyrosine kinase inhibitors also produced a

decrease in voltage-dependent Ba 2+ currents through Ca 2+ channels with characteristics of the L-type (Cataldi et al., 1996). In a study of L-type calcium currents in rat ventricular cells, Yokoshiki et al. (1996) reported that the channel was inhibited by genistein. However, these authors observed similar effects with daidzein, raising the possibility that the inhibitors might have acted directly on the channel rather than via a tyrosine kinase and highlighting at least one of the potential pitfalls associated with the use of kinase inhibitors. In none of the above studies was the tyrosine kinase responsible for the changes in Ca 2÷ channel function identified nor was tyrosine phosphorylation of the calcium channel protein directly demonstrated, and it remains to be seen whether the observed effects are due to phosphorylation of the channel itself or a more indirect action of tyrosine kinases. Regulation of the intracellular concentration of calcium levels may also occur via G protein-coupled receptors acting through phospholipase C to generate the intracellular messengers diacylglycerol and inositol 1,4,5 trisphosphate (IP3); IP3 subsequently triggering the release of Ca 2+ from intracellular stores by activation of the IP3 receptor (see, for example, Berstein et al., 1992; Wang and Augustine, 1995). A possible role for tyrosine phosphorylation in the regulation of calcium release from intracellular stores by this pathway was recently suggested by Jayaraman et al. (1996). These authors reported that stimulation of the T-cell receptor resulted in the association of Fyn with, and tyrosine phosphorylation of, the IP 3 receptor. In addition, the IP3 receptor from brain microsomes was phosphorylated by exogenously added Src or Fyn. The addition of Fyn to cerebellar microsomes fused to planar lipid bilayers, or to the purified IP3 receptor, increased the open probability of the calcium release channel relative to controls which had not been treated with Fyn. Interestingly, activation of the channel by Fyn required that IP 3 be added prior to the kinase, indicating that the channel had to be in the open state in order for Fyn to exert its modulatory effect.

TYROSINE PHOSPHORYLATION AND SYNAPTIC

ACTIVITY Short-term modulation o f synaptic activity by neurotrophins

The neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),

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neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT4/5), were initially described in relation to their ability to promote the survival and differentiation of neurons during development (reviewed in Heumann, 1994; Glass and Yancopoulos, 1993; Dechant et al., 1994). Neurotrophins bind to specific receptor tyrosine kinases that are members of the trk family of protooncogenes; N G F binding to TrkA, BDNF and NT4/5 to TrkB, and NT-3 to TrkC and with lower affinity to TrkA and TrkB. Neurotrophin binding results in trk receptor aggregation and autophosphorylation initiating signaling cascades leading to the appropriate cellular response (Glass and Yancopoulos, 1993; Kaplan and Stephens, 1994; Heumann, 1994; Dechant et al., 1994). In addition to their long-term effects, a role for the neurotrophins in neuronal plasticity has recently been suggested by several observations including: the regulation of neurotrophin synthesis by neuronal activity; the release of N G F in an activity-dependent manner; and the enhancement of the release of neurotransmitters by neurotrophins (reviewed in Lindholm et al., 1994; Lo, 1995; Thoenen, 1995; Berninger and Poo, 1996). In the latter instance NT-3 and BDNF, but not N G F , markedly potentiated both spontaneous and impulse-evoked synaptic activity in Xenopus developing neuromuscular synapses in culture by a mechanism involving an increase in the probability of the release of acetylcholine (Lohof et al., 1993). This effect was apparently dependent upon tyrosine kinase activity of the receptor, being blocked by application of the trk tyrosine kinase inhibitor K-252a. Stoop and Poo (1995) reported that the application of ciliary neurotrophic factor (CNTF) resulted in the rapid potentiation of both spontaneous and impulseevoked transmitter release from developing neuromuscular synapses in Xenopus cell cultures. C N T F was more effective when applied to the cell soma than to the nerve terminal and the effect was abolished if the terminal was isolated from the cell body, indicating that transmitter secretion was under somatic control. Although the mechanism of C N T F action was not determined, the rapidity of the effect suggested that it involved the modification of pre-existing proteins in the cell soma. C N T F may induce tyrosine phosphorylation of the Jak-Tyk family of tyrosine kinases (Stahl et al., 1994) and the activation of PKC (Kalberg et aL, 1993), either or both of which may have accounted for the synaptic potentiation. Neurons of the CNS also exhibit neurotrophininduced increases in synaptic activity (Lel3mann et al., 1994; Knipper et al., 1994a, 1994b; Kang and Schuman, 1995; Kim et al., 1994; Levine et al., 1995).

In accord with the initial findings of Lohof et al. (1993), the potentiation of activity observed in these studies was presynaptic in origin, presumably reflecting an increase in the probability of transmitter release, and was blocked by K-252a, indicating a requirement for the tyrosine kinase activity of the trk receptor (Kim et al., 1994; Kang and Schuman, 1995; Levine et al., 1995). N G F and BNDF enhanced both the spontaneous and depolarization-evoked release of acetylcholine from hippocampal synaptosomes (Knipper et al., 1994a) and N G F stimulated the spontaneous and depolarization-evoked release of glutamate from the same preparation (Knipper et al., 1994b), consistent with a presynaptic action of neurotrophins. The stimulatory effects of N G F on neurotransmitter release from synaptosomes was, in both cases, partially blocked by K-252a, suggesting the possible involvement of a Trk receptor, although this was not established. K-252a-sensitive tyrosine kinase activity appeared to be partially responsible for the up-regulation of acetylcholine release from the motor nerve endings in the diaphragms of rats treated chronically with ~-bungarotoxin (Plomp and Molenaar. 1996). Although the kinase involved in this response was not identified, inhibition of the increase in acetylcholine release by K-252b, a membrane-impermeable inhibitor of similar specificity to K-252a, was consistent with the participation of a neurotrophin receptor. A role for calcium in the neurotrophininduced enhancement of synaptic activity is indicated by the findings that BDNF and NT-3 are able to induce transient increases in [Ca2+]~ in rat hippocampal neurons (Berninger et al., 1993) and that synaptic potentiation evoked by BDNF at Xenopus neuromuscular junctions was accompanied by an increase in presynaptic calcium levels (Stoop and Poo, 1996). A postsynaptic as well as a presynaptic action of BDNF was indicated by an increase in both amplitude and frequency of EPSCs following the application of BDNF to hippocampal neurons, an effect which was blocked by the injection of K-252a into the postsynaptic cell (Levine et al., 1995). In general accord with a postsynaptic action of BDNF isolated postsynaptic densities are associated with functionally active trkB receptors which undergo autophosphorylation in the presence of BDNF (Wu et al., 1997). Depolarization alters the tyrosine phosphorylation o f synaptie proteins

Depolarization of nerve terminals initiates a cascade of events which leads ultimately to the calcium-

Invited Review dependent release of neurotransmitter. Depolarization-dependent changes in the activities of protein kinases and phosphatases and in the phosphorylation state of a number of synaptic proteins has been well documented (Dunkley and Robinson, 1986). More recently, a relationship between depolarization and tyrosine phosphorylation has become apparent (Woodrow et al., 1992; Siciliano et al., 1994; Barrie et al., 1996). Transient increases in the tyrosine phosphorylation of several synaptosomal proteins occurred following depolarization of rat forebrain synaptosomes with high K ÷, veratridine or 4amino pyridine (Woodrow et al., 1992; Barrie et al., 1996). Generally similar results have been obtained with hippocampal slices, neurons in culture and PC12h cells (Siciliano et al., 1994; Okumura et al., 1994). In all cases the increase in tyrosine phosphorylation induced by depolarization was Ca 2÷ dependent. Calcium may regulate the activities of both tyrosine kinases (Lev et al., 1995; Allen et al., 1996; Zhao et al., 1992) and protein-tyrosine phosphatases (Singh, 1990; Ostergaard and Trowbridge, 1991; Mei and Huganir, 1991), either or both of which may be involved in the depolarization-induced changes in tyrosine phosphorylation. Tyrosine kinase activity was increased in synaptosomes following K÷-induced depolarization, and inhibition of tyrosine kinases with genistein decreased the K÷-stimulated, Ca2÷-depen dent release of glutamate in the same preparation (Mullany et al., 1996). An apparently specific inhibitory effect of EGF, but not of insulin or IGF-1, on the Ca2+-dependent component of glutamate release from synaptosomes induced by 4-amino pyridine or high K ÷ was reported by Barrie et al. (1996). In these studies EGF caused a moderate decrease in the depolarization-evoked increase in intracellular Ca 2÷ but did not alter the plasma membrane potential. The authors suggest that the effect of EGF may have been mediated via modulation of voltage-dependent calcium channels, a mechanism which would be in general accord with the inhibitory effects of tyrosine phosphorylation on Ca 2+ channels reported by others (Wijetunge et al., 1992; Cataldi et al., 1996). However, apart from an increase in the tyrosine phosphorylation of the EGF receptor itself, EGF-induced changes in tyrosine phosphorylation were not detected. Tyrosine phosphorylation, long-term potentiation and long-term depression

The induction of LTP, a form of synaptic plasticity involving the long-lasting modification of synaptic behavior (Bliss and Collingridge, 1993), requires tyrosine kinase activity (O'Dell et al., 1991; Abe and Saito,

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1993). An apparently specific requirement for Fyn tyrosine kinase was suggested by gene targeting experiments in which mice with mutations o f f y n , but not of src, yes or abl, exhibited impaired induction of LTP, in spite of a compensatory increase in the activity of Src kinase in t h e f y n mutants (Grant et al., 1992; Grant et al., 1995). T h e f y n mutant mice also displayed decreased tyrosine phosphorylation and activity of the focal adhesion tyrosine kinase (FAK), suggesting that F A K might also be required for the induction of LTP (Grant et al., 1995). Consistent with this suggestion, the tyrosine phosphorylation and activation of FAK is regulated by Ca 2÷ and PKC (Hamawy et al., 1993; Vuori and Rhuoslahti, 1993), both of which are required for the induction of LTP (Bliss and Collingridge, 1993). It is also interesting in this context that activation of neurotransmitter and neuropeptide receptors stimulates the tyrosine phosphorylation of F A K (Gutkind and Robbins, 1992; Zachary et al., 1992). LTP in the dentate gyrus was accompanied by a long-lasting increase in the tyrosine phosphorylation of the NR2B subunit of the N M D A receptor (Rostas et al., 1996; Rosenblum et al., 1996). The increase in tyrosine phosphorylation of NR2B was not detected until 5-15 rain after induction, well after the population EPSP had achieved a stable level of potentiation (Rostas et al., 1996) indicating that, at least under the conditions of these studies, the phosphorylation of NR2B was not involved in the inductive events. The requirement for tyrosine kinase activity for the induction of LTP indicates that other substrates may be phosphorylated on tyrosine during the inductive phase and that phosphorylation of NR2B is involved in the long-term maintenance of LTP. A requirement for tyrosine kinase activity for the induction of long-term depression (LTD) in the cerebellum has also been suggested (Boxall et al., 1996). Using cerebellar slices these authors found that LTD induced by pairing parallel fiber stimulation with postsynaptic Ca 2÷ spiking was blocked by lavendustin A and herbimycin A, two structurally distinct tyrosine kinase inhibitors. The effect of lavendustin A was observed when the inhibitor was included in the recording electrode, suggesting that the inhibitory effect was exerted postsynaptically. Evidence for an interaction between tyrosine kinase activity, PKC and the induction of LTD was also obtained. Inclusion of (-)-indolactam V, a PKC-selective activator, in the recording electrode caused a run-down of the parallel fiber mediated-EPSP that occluded pairing-induced LTD. Preincubation of the cerebellar slices with herbimycin A, to inhibit tyrosine kinase activity, pre-

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vented the depression evoked by ( - ) - i n d o l a c t a m V, indicating that PKC-mediated depression required the activity of tyrosine kinase(s) in the postsynaptic cell (Boxall et al., 1996). Although the mechanism responsible for these effects was not described the authors raise the possibility that increases in [Ca2+]i resulting from stimulation ofmGluR1 at parallel fiber synapses may activate one or more tyrosine kinases which are required for the induction of LTD. The substrates for tyrosine phosphorylation during the induction of LTD were not identified but an intriguing possibility is that the A M P A receptor may be phosphorylated. Decreased sensitivity of A M P A receptors has been implicated in LTD (Ito and Karachot, 1990) and the GluRl subunit of the A M P A receptor is phosphorylated on tyrosine when co-expressed with v-Src in transfected cells (Moss et al., 1993). Whether or not the A M P A receptor is phosphorylated on tyrosine during LTD, and what the functional consequences of this might be, remain topics for future research. Tyrosine phosphorylation and ischemia

Brief periods of global ischemia set in sequence a series of metabolic changes including loss of energy charge and ion homeostasis, membrane depolarization, increases in extracellular glutamate concentrations and an influx of Ca 2+ ions (Choi and Rothman, 1990). Although most of these processes are reversible following the resumption of circulation they initiate long-lasting changes which ultimately may lead to neuronal cell death in sensitive brain regions (Choi and Rothman, 1990; Nitatori et al., 1995). Transient global ischemia results in a marked increase in the tyrosine phosphorylation of a number of proteins in the hippocampus (Kindy, 1993; Hu and Weiloch, 1994), including the postsynaptic glycoprotein PSD-GP180 (Gurd et al., 1995). We have more recently found that both NR2A and NR2B subunits in the hippocampus exhibit large and sustained increases in tyrosine phosphorylation following a 15 rain global ischemic insult (Takagi et al., 1997). In addition, preliminary results have indicated that this change in tyrosine phosphorylation is accompanied by an increase in the ability of the N M D A receptor subunits to bind to the SH2 domains of several signaling proteins, including pp60 src (Gurd et al., 1995; and unpublished observations), pp60 ..... tyrosine kinase activity was enhanced in the hippocampus following transient ischemia in gerbils (Ohtsuki et al., 1996), suggesting that activation of Src may account for the enhanced tyrosine phosphorylation of the N M D A receptor under these conditions. In apparent contrast to the above findings, brief

exposure of hippocampal slices to hypoxia and glucose deprivation resulted in a highly selective and long-lasting decrease in the tyrosine phosphorylation of the glycoprotein PSD-GP180 (Au and Gurd, 1995). The sustained decrease in tyrosine phosphorylation of PSD-GP 180 was dependent upon the presence of Ca 2+ ions, a slow rephosphorylation to control levels occurring if calcium was excluded from the incubation medium. Although PSD-GP180 was not unambiguously identified as the N M D A receptor subunit NR2B in this study, Wang et al. (1996b) subsequently reported that exposure of hippocampal slices to hypoxic conditions caused a reduction in the tyrosine phosphorylation ofNR2A/B subunits which had been immunoprecipitated with subunit-specific antibodies. These authors also found that src-related tyrosine kinase activity was reduced following the hypoxic episode, whereas protein tyrosine phosphatase activity was unchanged. These results, together with those of Au and Gurd (1995), suggest that, at least under in vitro conditions, the influx of Ca 2+ ions during hypoxia leads to a decrease in the activity of src-family tyrosine kinases which result in a change in the tyrosine phosphorylation of the N M D A receptor. The reasons for the different consequences of hypoxia under in vivo and in vitro conditions are unclear but may reflect the presence of factors which could affect tyrosine kinase activity following ischemia in situ, for example neurotrophins (Lindvall et al., 1994), but which may have a reduced effect following hypoxia in hippocampal slices. CONCLUSIONS The evidence described above, and summarized in Table 1, clearly implicates tyrosine phosphorylation in the regulation of a wide range of functions which impact on the biochemical and physiological properties of the synapse. While much of the available information relates to the effects of tyrosine phosphorylation on the activity of ion channels it also seems probable that activity-induced changes in the tyrosine phosphorylation of synaptic proteins will be found to exert more long-lasting effects through the activation of signaling pathways leading to altered gene expression. Indeed, the implied involvement of tyrosine phosphorylation in such diverse phenomena as LTP, ischemia, taste aversion learning, and performance in the Morris water maze indicates that the phosphorylation of synaptic proteins on tyrosine plays an important, and perhaps central, role in both the long- and short-term modulation ofsynaptic function. It is now important to identify the specific tyro-

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Table 1. Tyrosine phosphorylation and the synapse AChR NMDAR GABAAR Potassium channels Calcium channels Depolarization Neurotrophins

LTP LTD lschemia

Short-term effects Tyrosine phosphorylation of Torpedo AChR increases rate of receptor desensitization Tyrosine phosphorylafion involved in AChR clustering at the neuromuscular junction NR2A and NR2B subunits are phosphorylated on tyrosine. Tyrosine phosphorylation potentiates NMDA channel. Activation of NMDA receptor stimulates tyrosine phosphorylation Tyrosine phosphorylation enhances channel currents Kv1.2, Kv1.3 and Kv1.5 potassium channels are suppressed by tyrosine phosphorylation Inhibition of tyrosine kinase reduces activity of voltage-operated Ca:+ channels and L-type Ca 2÷ channels Tyrosine phosphorylation activates IP3 receptor High K +-induced depolarization increases tyrosine phosphorylation of several synaptic proteins Potentiate activity of neuromuscular and central synapses. Acting through Trk receptors stimulate release of neurotransmitter Long-term effects Tyrosine kinase activity required for induction of LTP. NR2B tyrosine phosphorylation increased following induction of LTP Tyrosine kinase activity required for induction of LTD in cerebellar slices General increase in tyrosine phosphorylation in sensitive brain regions following transient cerebral ischemia. Marked increase in NR2A and NR2B tyrosine phosphorylation

For references see text. sine kinases a n d protein tyrosine p h o s p h a t a s e s which are involved in the tyrosine p h o s p h o r y l a t i o n o f individual synaptic proteins, the m e c h a n i s m s by which the activities o f these enzymes are regulated in response to various synaptic stimuli, a n d the precise molecular links between tyrosine p h o s p h o r y l a t i o n , altered functionality of the synapse, a n d long-lasting synapticinduced changes in n e u r o n a l behavior. The present review represents a s n a p s h o t o f the current state o f a n extremely rapidly developing area o f neuroscience. L o n g overlooked because o f its q u a n titatively m i n o r aspect, the i m p o r t a n c e o f tyrosine p h o s p h o r y l a t i o n as a regulator o f m a t u r e n e u r o n a l a n d synaptic function n o w seems clear. A slow trickle of papers o n tyrosine p h o s p h o r y l a t i o n in the nervous system d u r i n g the late 1980s a n d early 1990s has steadily increased over the past few years. (A r o u g h indication o f the g r o w t h in this area is given by the n u m b e r o f listings u n d e r tyrosine p h o s p h o r y l a t i o n in the Society for Neuroscience a n n u a l meeting abstracts; in 1988 one a b s t r a c t was indexed u n d e r this heading, this increased to 32 in 1993 a n d 53 in 1996, n o t taking into a c c o u n t the n u m e r o u s papers dealing with n e u r o t r o p h i n s a n d o t h e r related topics.) G i v e n this g r o w t h it seems safe to predict t h a t m a n y o f the current u n a n s w e r e d questions relating to the mechanisms a n d consequences o f the tyrosine phosp h o r y l a t i o n o f synaptic proteins will soon be answered. REFERENCES

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