Src family kinases: modulators of neurotransmitter receptor function and behavior

Review

Src family kinases: modulators of neurotransmitter receptor function and behavior Hiroshi Ohnishi1, Yoji Murata2, Hideki Okazawa2 and Takashi Matozaki1,2 1

Laboratory of Biosignal Sciences, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-Machi, Maebashi, Gunma 371-8512, Japan 2 Division of Molecular and Cellular Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe 650-0017, Japan

Src family kinases (SFKs) are non-receptor-type protein tyrosine kinases that were originally identified as the products of proto-oncogenes and were subsequently implicated in the regulation of cell proliferation and differentiation in the developing mammalian brain. Recent studies using transgenic mouse models have demonstrated that SFKs that are highly expressed in the adult brain regulate neuronal plasticity and behavior through tyrosine phosphorylation of key substrates such as neurotransmitter receptors. Here, we provide an overview of these recent studies, as well as discussing how modulation of the endocytosis of neurotransmitter receptors by SFKs contributes, in part, to this regulation. Deregulation of SFK-dependent tyrosine phosphorylation of such substrates might underlie certain brain disorders. Introduction The role of Src family kinases (SFKs) in the central nervous system (CNS) was originally thought to be limited to the regulation of the proliferation and differentiation of neuronal cells [1,2]. However, SFKs are also expressed in differentiated, postmitotic neurons, suggesting that these protein tyrosine kinases (PTKs) might also participate in the regulation of functions in the CNS beyond the developmental stage [3–5]. Indeed, genetic ablation of SFKs such as Fyn or Lyn has been shown to result in behavioral abnormalities in adult mice [6,7], suggesting that SFKs function in the regulation of neuronal plasticity and behavior. However, the molecular mechanisms of such regulation, in particular the identity of the relevant SFK substrates, remained largely unclear. Recent studies using genetically modified mouse models that either lack specific SFK substrates or have specific tyrosine phosphorylation sites mutated within substrates, have begun to shed light on these issues. Here, we provide an overview of recent progress in attempts to define the roles of SFKs in the regulation of neuronal function in the adult brain, with particular emphasis on the modulation of neurotransmitter receptors and behavior.

Corresponding authors: Ohnishi, H. ([email protected]); Matozaki, T. ([email protected]).

SFKs: general domain organization and activation mechanism All SFKs share a conserved domain organization (Box 1). Autophosphorylation of a tyrosine residue (Y416, according to the convention of numbering amino acid residues relative to chicken Src) in the activation loop of the kinase domain is thought to increase the PTK activity of SFKs. By contrast, phosphorylation by other kinases, such as C-terminal Src kinase (Csk) or Csk homologous kinase (Chk), of a tyrosine residue (Y527) near the COOH-terminus of SFKs suppresses the kinase activity. Conversely, dephosphorylation of phosphorylated Y416 or Y527 by protein tyrosine phosphatases (PTPs) results in inactivation or activation of SFKs, respectively (Box 2). Although SFKs are, in general, widely expressed, five members of this family, Src, Fyn, Lyn, Yes and Lck, are present at substantial levels in the adult mammalian brain [8,9]. Roles of SFKs in the regulation of synaptic transmission Recent studies have indicated that SFKs regulate neuronal plasticity and behavior through tyrosine phosphorylation of neurotransmitter receptors. These receptors include ionotropic glutamate receptors of the NMDA and AMPA subclasses (NMDARs and AMPARs), which contribute to excitatory transmission, as well as GABA type A receptors (GABAARs), which mediate the majority of fast synaptic inhibition in the adult mammalian brain. SFKs and the regulation of excitatory transmission NMDARs are ligand-gated cation channels that allow the flow of K+, Na+ and Ca2+ in response to the binding of glutamate. They are thought to be tetramers that consist of two GluN1 (formerly known as NR1 [10]) and two other modulatory subunits, comprised of either GluN2A-D (NR2A-D) or GluN3A-B (NR3A-B) subunits [11]. A role for SFKs in the regulation of ligand-gated neurotransmitter receptor function was first indicated by the observation that the intracellular application of Src in hippocampal slices resulted in potentiation of NMDA-induced current [12]. Treatment with an SFK-activating peptide (pYEEI) was also found to increase NMDAR currents in cultured neurons or hippocampal slices [13]. In addition, activation of SFKs was shown to be important for

0166-2236/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tins.2011.09.005 Trends in Neurosciences, December 2011, Vol. 34, No. 12

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Box 1. Structure of SFKs and regulation of their kinase activity The domain structure of SFKs includes a myristoylated NH2-terminal segment, a unique domain, a Src homology (SH) 3 domain, an SH2 domain, a linker region, a tyrosine kinase (SH1) domain, and a Cterminal tail (Figure Ia). Unlike the other domains, the amino acid sequence of the unique domain is not conserved among SFKs. A synthetic peptide corresponding to the sequence of this region of Src selectively inhibits Src activity [13], suggesting that this region plays a role specific to each SFK. The catalytic activity of SFKs is strictly controlled through intramolecular interactions mediated by the SH2 and SH3 domains and through tyrosine phosphorylation of the kinases themselves, particularly at Y416 and Y527 (Figure Ib). An intramolecular interaction between the SH3 domain and the linker

Src (chicken) Unique domain G2

Linker region

SH3 85

1

Kinase Y416 domain

SH2

139 147

245

Myristoylation site

SH

P

519

533

Active

Y416

Y527 Kinase Csk, Chk

Autophosphorylation 3

Activation loop e Kinas in doma

SH2

Y527

Tyrosine phosphorylation sites

Inactive

3

(b)

260

SH

(a)

region suppresses kinase activity. The activation loop that contains Y416 in the kinase domain also suppresses kinase activity by generating a steric barrier that prevents the formation of the active catalytic center [77]. Autophosphorylation at Y416 within the activation loop induces SFK activation by changing the conformation of the loop and allowing formation of the active conformation of the catalytic center. Another intramolecular interaction between the SH2 domain and phosphorylated Y527 near the C-terminus also suppresses kinase activity. This residue is phosphorylated by Csk and Chk. Dephosphorylation of phosphorylated Y527 is thus important for activation of SFKs, with PTPs such as PTPa and Shp2 being implicated in this reaction (see Box 2).

SH2

P Y416 Kinase domain

Y527

Phosphatase PTPα, Shp2 TRENDS in Neurosciences

Figure I. Structural organization of SFKs and a model for their activation. (a) Domain organization of chicken Src. Tyrosine phosphorylation sites (Y416, Y527) and the myristoylated glycine (G2) site (which are conserved among SFKs) are indicated. The residue numbers of amino acids that delineate the various domains are also shown. (b) Model structures for active and inactive states of SFKs. The activation loop in the kinase domain is indicated as a red line.

the induction of long-term potentiation (LTP), a form of synaptic plasticity that is important for learning and memory, in CA1 pyramidal neurons of hippocampal slices [13]. However, it remains unclear whether the SFK-dependent potentiation of NMDAR function and LTP induction is the result of direct tyrosine phosphorylation of NMDARs by SFKs, although the cytoplasmic tails of GluN2A and GluN2B subunits contain potential phosphorylation sites for these enzymes (see below). In addition, the identity of the SFKs that mediate such regulation remains unknown. Given that SFKs participate in the regulation of LTP, they also probably contribute to the regulation of memory formation and learning. Indeed, both LTP in the hippocampus and spatial learning in the Morris water maze, in which behavior is thought to depend on NMDAR function in the dorsal hippocampus [14], are impaired in Fyn-deficient mice [6]. Spatial learning is also associated with increases in both the activity and expression of Src in the hippocampus, as well as with the interaction of Src with NMDARs and other synaptic proteins such as synapsin I and synaptophysin [15]. It is thus likely that SFKs contribute to spatial learning through the modulation of excitatory synaptic transmission involving NMDARs in the hippocampus. 630

Biochemical studies have shown that Src and Fyn phosphorylate tyrosine residues in the cytoplasmic tails of GluN2A and GluN2B [16–19] (Figure 1). GluN2B in the postsynaptic density (PSD) fraction of the brain has been shown to be tyrosine-phosphorylated by endogenous PTKs, whereas PP2, a specific inhibitor of SFKs [20], inhibits such phosphorylation [21]. Tyrosine phosphorylation of GluN2B is also reduced in Fyn-deficient mice [18]. SFKs are, therefore, likely to be important for the tyrosine phosphorylation of NMDARs in the adult brain. Site-directed mutagenesis studies have identified specific tyrosine residues (i.e. Y1292, Y1325 and Y1387 in GluN2A, andY1252, Y1336 and Y1472 in GluN2B) that are phosphorylated by Src or Fyn when the receptor subunits and kinases are heterologously expressed in human embryonic kidney (HEK) cells [16,18,22]. Furthermore, Y1472 in the GluN2B subunit was shown to be a major site of phosphorylation in the brain [19], and it is hyperphosphorylated in CA1 hippocampal slices after tetanic stimulation of Schaffer collateral inputs [18]. Knockin mice in which Y1472 of GluN2B was replaced by a phenylalanine (F), however, did not manifest any defects in hippocampal LTP [19]. Induction of LTP in the CA1

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Box 2. Modulation of SFK function in the brain by PTPs PTPs catalyze dephosphorylation of tyrosine-phosphorylated proteins and thus are important modulators of SFK function, including in the nervous system. A member of the striatal enriched tyrosine phosphatase (STEP) family of brain-specific, nonreceptor-type PTPs (reviewed in [78]), STEP61 was found to coprecipitate with the NMDAR GluN1 subunit in cell lysates prepared from the spinal cord, hippocampus [79] and primary cultured striatum neurons [80]. This suggests that STEP61 is a component of the NMDAR complex (Figure I). The intracellular application of a functional blocking antibody to STEP, or of a dominant negative form of STEP, increases synaptic NMDAR-mediated currents [79], suggesting that NMDAR activity is negatively regulated by endogenous STEP. STEP also regulates trafficking of NMDARs by controlling the level of tyrosine phosphorylation of the GluN2B subunit of the receptor [81,82]. STEP dephosphorylates Y420 of Fyn (corresponding to Y416 of chicken Src) and suppresses its kinase activity [83]. Given that Fyn is implicated in tyrosine phosphorylation of GluN2B [18,19], STEP probably modulates NMDAR function by inhibiting the activity of Fyn (reviewed in [78]). However, STEP might also directly dephosphorylate NMDARs, resulting in suppression of NMDAR activity [78]. In contrast to STEP, PTPa, a receptor-type PTP, is thought to activate SFKs through dephosphorylation of the inhibitory Y527 residue [84]. Furthermore, it has been demonstrated that both Src and Fyn exhibit reduced activity in the brain of PTPa-deficient mice, as indicated by their increased phosphorylation at Y527 [85]. PTPa is also a component of the NMDAR complex [86]. Intracellular application of recombinant PTPa potentiated NMDAR-mediated miniature EPSCs (mEPSCs) in cultured rat hippocampal neurons, as well as increasing EPSC amplitude in CA1 pyramidal neurons of rat hippocampal slices [86]. The SFK inhibitor PP2 inhibited the potentiation of NMDARmediated mEPSCs induced by the intracellular application of recombinant PTPa, suggesting that the upregulation of NMDAR function by PTPa is mediated by SFKs. In addition, mice lacking PTPa manifest a defect in the induction of LTP, as well as performance

region of the hippocampus is also normal in another knockin mouse in which the Y1325 site of GluN2A was mutated (Y1325F) [22]. Consistently, the learning ability of these two mutant mice in a spatial memory task was also similar to wild type mice [19,22]. It is possible that phosphorylation

(a)

NMDAR

P

GluN2B

SFK Y416 Y527 P PTPα • Activation of Src and Fyn

• Reduction of synaptic NMDAR currents

• Potentiation of synaptic NMDAR currents • Control of spatial learning

Shp2 • Activation of SFKs

TRENDS in Neurosciences

Figure I. Regulation of SFK activity by PTPs in the adult brain. STEP, PTPa, and Shp2 regulate the activity of SFKs (both positively and negatively) and are, therefore, thought to participate in the SFK-mediated modulation of neuronal function. Phosphorylation of NMDARs by SFKs and dephosphorylation of SFKs (or NMDARs) by PTPs are indicated by blue arrows and red bars, respectively.

of Y1325 of GluN2A and that of Y1472 of GluN2B by SFKs have overlapping functions in the hippocampus and that each, therefore, compensates for the loss of the other in the regulation of hippocampal LTP [19]. Alternatively, direct phosphorylation of either Y1325 of GluN2A or Y1472 of

NMDAR D1R

GluN2A

Y1325

P

P

SFK (Src, Fyn)

SFK (Src, Fyn)

Fear-related learning

P

• Inactivation of Fyn

Y1472

• CaMKII binding • Regulation of endocytosis/ synaptic localization • LTP in amygdala

NMDAR

GluN2B

Y

STEP

(b)

NMDAR GluN1

deficits in spatial memory tasks, as measured by the radial-arm water maze test and Morris water maze [87,88]. PTPa is thus thought to participate in the regulation of synaptic plasticity and learning through activation of SFKs in the adult brain. Shp2, a nonreceptor-type PTP that is abundant in the brain [89,90], is also thought to participate in the activation of SFKs ([91,92] and reviewed in [93]). Shp2 has also been identified as a component of the NMDAR complex [94], suggesting that Shp2 may participate in the SFK-dependent regulation of NMDAR function, although this has not yet been demonstrated.

Ca2+ entry CN

PKA DARPP-32 (T34 - P )

Depression-like behavior TRENDS in Neurosciences

Figure 1. Regulation of NMDAR function by SFKs. The Y1325 residue of GluN2A and Y1472 of GluN2B subunits of the NMDAR are major tyrosine phosphorylation sites for SFKs, in particular Src or Fyn [16,18,19,22]. (a) Tyrosine phosphorylation of Y1472 of GluN2B is important for CaMKII binding to NMDARs [19], regulation of endocytosis [24] and proper synaptic localization of the receptors [19], LTP induction in the amygdala [19], and amygdala-dependent fear learning [19]. (b) Tyrosine phosphorylation of Y1325 of GluN2A is important for upregulation of the channel activity of NMDARs [22]. Influx of Ca2+ through NMDARs activates calcineurin (CN), which dephosphorylates DARPP32 at T34. This residue of DARPP-32 is phosphorylated by PKA as a result of D1 dopamine receptor (D1R) stimulation. Given that phosphorylation of DARPP-32 at T34 is important for regulation of depression-like behavior [32], the antidepressive phenotype of GluN2A(Y1325F) knockin mice [22] is probably attributable, at least in part, to increased phosphorylation of DARPP-32 at this residue. Positive or negative regulation is indicated by blue arrows and red bars, respectively.

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Review GluN2B by SFKs may not contribute to the regulation of hippocampal LTP and memory formation. In contrast to the lack of a defect in hippocampal LTP, LTP in the lateral nucleus of the amygdala was markedly impaired in GluN2B(Y1472F) mice [19]. A sequence motif that includes Y1472 (i.e. Y1472EKL) corresponds well to a classical tyrosine binding motif for the m2 subunit of the adaptor protein 2 complex (AP2) [23], which associates with clathrin-coated endocytic vesicles. Phosphorylation of Y1472 by Fyn has been shown to inhibit the binding of AP2 and suppress clathrin-mediated endocytosis of NMDARs ([23] and reviewed in [24,25]). Furthermore, the Y1472F mutation has been shown to suppress interaction of the mutant motif with AP2 [26]. Consistently, the Y1472F mutant GluN2B was abnormally localized at perisynaptic regions, where NMDARs would normally be endocytosed [19]. In addition, the mutant form of GluN2B failed to bind Ca2+- and calmodulin-dependent protein kinase II (CaMKII) [19], a key enzyme in the induction of NMDARdependent LTP [27]. The Y1472 residue of GluN2B is thus important for the proper synaptic localization of NMDARs as well as for the binding of CaMKII, and it thereby contributes to the induction of LTP in the amygdala. Consistent with the impairment of LTP in the amygdala, the GluN2B(Y1472F) knockin mice exhibited impaired fearrelated learning in a fear conditioning test [19], a behavior that is dependent on amygdala function [28]. Fear-related learning in the contextual fear conditioning test is also impaired in Fyn-deficient mice [29]. Given that Y1472 of GluN2B is phosphorylated by Fyn [18], SFK-mediated phosphorylation of this residue is likely to be important for amygdala-dependent fear learning (Figure 1a), although it cannot be ruled out that other substrates of Fyn also contribute to this form of learning. In contrast to the GluN2B(Y1472F) mutant mice, GluN2A(Y1325F) knockin mice show normal behavior in the fear conditioning test [19,22]. Instead, SFK-dependent potentiation of NMDA-induced currents is abolished in medium spiny neurons (MSNs) of the neostriatum in these mice [22]. Intracellular application of recombinant active Src potentiated NMDAR-mediated EPSCs in striatal slices prepared from wild type mice but not in those from mutant mice [22]. This suggests that phosphorylation of Y1325 of GluN2A is required for the Src-induced potentiation of NMDAR current in MSNs. Furthermore, these mutant mice were observed to have reduced immobility in both the forced swim and tail suspension tests, suggesting that the mutant mice exhibit less depressive-like behavior compared to the wild type mice [22]. Indeed, phosphorylation of Y1325 of GluN2A is increased in the striatum of wild type mice during these tests. Phosphorylation of Y416 of SFKs, which reflects SFK activation, is also increased in the striatum of wild type mice during the forced swim test [22]. Taken together, these findings suggest that stress associated with the forced swim or tail suspension tests induces phosphorylation of Y1325 of GluN2A probably through the activation of SFKs, and that such signaling is important for the regulation of depressionrelated behavior (Figure 1b). The molecular mechanisms by which phosphorylation of GluN2A on Y1325 contributes to the regulation of depression-like behavior has not been fully elucidated. However, 632

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phosphorylation of dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) at threonine-34 (Figure 1b) is increased in the striatum of GluN2A(Y1325F) mutant mice compared with wild type mice [22]. Protein kinase A (PKA) activated by D1 dopamine receptors phosphorylates DARPP-32 at T34, whereas calcineurin, a Ca2+and calmodulin-dependent protein phosphatase, dephosphorylates this residue [30,31]. Consistent with the observed increase in the phosphorylation of DARPP-32 on T34, the activity of calcineurin is decreased in MSNs of GluN2A(Y1325F) mutant mice [22]. This reduced activity of calcineurin might be attributable to the defect in SFKmediated potentiation of NMDAR current and the associated reduction in Ca2+ influx in MSNs of the mutant mice. Given that phosphorylation of DARPP-32 at T34 is important for negative regulation of depression-like behavior [32], the antidepressive phenotype of GluN2A(Y1325F) mutant mice is probably due in part to the increased phosphorylation of DARPP-32 on this residue [22]. Forced swim stress also induces an increase in the phosphorylation of Y416 of SFKs and that of Y1472 of GluN2B in the mouse hippocampus [33]. In addition, forced swim stress also induces an increase in the SFK-mediated tyrosine phosphorylation of signal regulatory protein a (SIRPa) in the brain [33]. SIRPa is a transmembrane protein, and tyrosine residues in the cytoplasmic region of SIRPa (Y477 and Y501) are thought to be phosphorylated by SFKs [34]. Tyrosine-phosphorylated SIRPa binds and thereby activates a nonreceptor-type PTP, Src homology 2 domain-containing tyrosine phosphatase 2 (Shp2) [34]. Mice expressing a mutant form of SIRPa that lacks most of the cytoplasmic region exhibit increased depression-like behavior in the forced swim test [33]. Furthermore, SFK-mediated tyrosine phosphorylation of GluN2B is attenuated in the SIRPa mutant mice [33], suggesting that SIRPa positively regulates the phosphorylation of GluN2B, probably through Shp2. In contrast to the tyrosine phosphorylation of GluN2A, modulation of tyrosine phosphorylation of GluN2B by SIRPa may have a suppressive effect on the behavioral immobility in the forced swim test, although further studies assessing synaptic transmission in the SIRPa mutant mice are needed to directly assess this hypothesis. In addition to NMDARs, another subclass of glutamate receptors has been demonstrated to be tyrosine-phosphorylated by SFKs. AMPARs are homo- or hetero-tetramers assembled from four subunits, GluA1 to GluA4 (formerly known as GluR1 to GluR4 [10]) (Figure 2). The GluA2 subunit was found to be tyrosine-phosphorylated in rat hippocampal slices and cultured neurons [35]. The tyrosine phosphorylation of GluA2 is also increased in response to glutamate receptor agonists such as glutamate, AMPA and NMDA in cultured rat cortical neurons, and both the basal and agonist-induced tyrosine phosphorylation of GluA2 are abolished by treatment with PP2 [36], suggesting that SFKs are required for the tyrosine phosphorylation of this subunit. Indeed, the intracellular C-terminal region of GluA2, which contains three tyrosine residues (Y869, Y873, Y876), is phosphorylated by an active form of recombinant Src in vitro [35]. Although it remains unclear whether all three of these tyrosine residues are phosphorylated, Y876 of GluA2 is phosphorylated when this subunit is heterologously

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AMPAR GluA2

Y876 - P

Y876 Endocytosis Y876 BRAG2

SFK (Lyn) BRAG2

Arf6 Endocytosis

Downregulation of BRAG2 binding and endocytosis

Induction of LTD TRENDS in Neurosciences

Figure 2. Regulation of AMPAR endocytosis and function by SFKs. The Y876 residue in the C-terminal region of the GluA2 subunit of AMPARs is phosphorylated by SFKs, in particular by Lyn [36,37]. This residue is important for the interaction of GluA2 with BRAG2, a GEF for Arf6, which promotes the endocytosis of AMPARs. Destabilization of the interaction between GluA2 and BRAG2 by tyrosine phosphorylation of Y876 probably contributes to the suppression of AMPAR endocytosis, and thus, negatively regulates the induction of LTD [40]. In contrast to this model, it has also been proposed that phosphorylation of GluA2 on Y876 promotes the endocytosis of AMPARs [36]. Additional studies are needed to clarify the mechanisms involved in SFK-dependent regulation of AMPARs.

expressed in HEK cells together with SFKs [36]. Lyn may be the PTK responsible for the phosphorylation of AMPARs in vivo, given that it physically associates with these receptors and is rapidly activated in response to AMPAR stimulation in cultured cerebellar neurons [37]. Tyrosine phosphorylation of GluA2 also plays an important modulatory role in the endocytosis of AMPARs [38– 40]. Y876 of GluA2 has been shown to be critical for the interaction between GluA2 and BRAG2, a guanine nucleotide exchange factor (GEF) for the small GTPase ADPribosylation factor 6 (Arf6), which promotes the endocytosis of AMPARs by recruiting the AP2 complex [40] (Figure 2). In addition, application of a synthetic peptide corresponding to the C-terminal region of GluA2 containing the nonphosphorylated forms of the three tyrosine residues increased the GEF activity of BRAG2 for Arf6 in vitro, whereas a peptide containing phosphorylated Y876 did not [40]. The Y876 residue of GluA2 was also dephosphorylated in response to treatment with a metabotropic glutamate receptor (mGluR) agonist that promotes internalization of AMPARs in cultured hippocampal neurons [40]. It is thus likely that phosphorylation of GluA2 on Y876 negatively regulates the endocytosis of AMPARs by preventing the interaction of GluA2 with BRAG2 and the consequent activation of Arf6 (Figure 2). In marked contrast to these observations, PP2 was found to prevent the AMPA- or NMDA-induced internalization of GluA2 in cultured neurons [36]. Moreover, mutation of the Y876 residue of GluA2 (Y876F) resulted in a marked reduction in the extent of the AMPA- or NMDA-induced internalization of receptors containing this subunit [36], suggesting that phosphorylation of Y876 promotes the endocytosis of AMPARs. The C-terminal region of GluA2 adjacent to Y876 is also required for the interaction of GluA2 with the PDZ (PSD-95/Discs-large/ZO-1 homology) domain– containing glutamate receptor interacting proteins, GRIP1 and GRIP2, which promote the synaptic accumulation of AMPARs by preventing receptor endocytosis [41]. Furthermore, Lyn prevented the interaction of GluA2 with GRIP1/2

when these proteins were co-expressed in HEK cells [36]. These results suggest that phosphorylation of the Y876 site within the GluA2 subunit might destabilize the GluA2–GRIP1/2 interaction and thereby promote AMPAR endocytosis [36]. Although the role of GluA2 phosphorylation on Y876 in the regulation of AMPAR endocytosis remains unclear, application of a fusion protein or synthetic peptide containing the nonphosphorylated forms of the three tyrosine residues in the C-terminal region of GluA2 markedly inhibited the induction of long-term depression (LTD) of synaptic transmission by various protocols [35,42–44]. Given that AMPAR endocytosis is important for the induction of LTD [41], tyrosine phosphorylation of GluA2 by SFKs might play a role in LTD induction by regulating the endocytosis of AMPARs (Figure 2). SFKs and the regulation of inhibitory transmission GABAARs are Cl–-selective ligand-gated ion channels. More than 20 distinct GABAAR subunits exist, but the majority of GABAARs in the adult CNS are composed of two a subunits, two b subunits, and one g subunit [45] (Figure 3). The g2 and b1 subunits of GABAARs were first shown to be tyrosinephosphorylated by v-Src, an active mutant of Src, in vitro and in A293 cells [46,47]. The g2 subunit of GABAARs has also been shown to be tyrosine-phosphorylated under basal conditions in cultured cortical neurons and in adult rat brain [48]. Tyrosine phosphorylation of the g2 subunit induced by vanadate, a broad-spectrum inhibitor of PTPs, was found to be markedly reduced in hippocampal slices of Fyn-deficient mice [49], suggesting that SFKs, in particular Fyn, are important for the tyrosine phosphorylation of this GABAAR subunit in the brain. Indeed, Y365 and Y367 in the intracellular domain of the g2 subunit are thought to be major phosphorylation sites for SFKs (reviewed in [50]). Treatment with PP2 markedly reduced the extent of the vanadate-induced tyrosine phosphorylation of the g2 subunit at Y367 in cultured hippocampal neurons [49]. A sequence motif that includes Y367 (i.e. Y367ECL) corresponds well to a 633

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GABAAR β subunit

γ 2 subunit

α subunit

Y365 - P Y367 - P

Y365 Y367 Y365 Y367

μ2

SFK (Fyn) AP2

Clathrin

Endocytosis

Downregulation of AP2 binding and endocytosis

Inhibitory synaptic transmission

Spatial memory formation TRENDS in Neurosciences

Figure 3. Regulation of GABAAR endocytosis and function by SFKs. SFKs, in particular Fyn, phosphorylate Y365 and Y367 of the g2 subunit of GABAARs (reviewed in [50]). These tyrosine residues are important for interaction of the g2 subunit with the m2 subunit of the AP2 complex [50]. The AP2 complex associates with clathrin and promotes endocytosis of GABAARs. Phosphorylation of Y365 and Y367 of the g2 subunit of GABAARs by SFKs is thought to prevent association of the g2 subunit with the AP2 complex, resulting in the inhibition of GABAAR endocytosis [51]. These tyrosine residues have been demonstrated to be important in the modulation of inhibitory synaptic transmission and spatial memory formation [52].

binding motif of AP2 complex (reviewed in [50]). In vitro studies have shown that a peptide containing the Y365GY367ECL sequence of the g2 subunit of GABAARs exhibits robust binding to m2-AP2, whereas a Y365- and Y367-phosphorylated version of this peptide does not [51]. These data suggest that Y365 and/or Y367 residues of the g2 subunit are important for the binding of GABAARs to the AP2 complex and for clathrin-mediated receptor endocytosis, whereas phosphorylation of these residues prevents such events [51] (Figure 3). Mice heterozygous for the g2 (Y365/367F) knockin allele manifest increased cell surface expression of the g2 subunit in the hippocampus [52], with this increase likely to be due in part to a reduced level of endocytosis of GABAARs containing the mutant g2 subunit. In addition, the mean amplitude and frequency of miniature inhibitory postsynaptic currents (mIPSCs) were also increased in CA3 neurons of hippocampal slices prepared from the mutant mice, suggesting that GABAAR function is upregulated as a result of receptor accumulation at the cell surface. Consistent with this deregulation of inhibitory synaptic transmission, the heterozygous mice manifested impaired formation of spatial memory in the object recognition task [52], in which performance is highly dependent on CA3 hippocampal function [53,54]. The Y365 and Y367 residues in the g2 subunit of GABAARs are thus likely to be important for spatial memory formation as a result of their regulation of receptor endocytosis (Figure 3). However, it remains unclear whether phosphorylation of Y365 and Y367 in the g2 subunit by SFKs is important for regulation of GABAAR endocytosis in vivo as well as for spatial memory formation. In addition to the g2 and b1 subunits, the b2 and b3 subunits of GABAARs have also been demonstrated to be tyrosine-phosphorylated in cultured spinal neurons [55]. Tyrosine phosphorylation of b subunits has also been shown 634

to modulate GABAAR function [55], although the role of specific SFKs in such regulation remains uncharacterized. Importance of SFKs in the regulation of responsiveness to alcohol Behavioral responses to drugs of abuse, such as cocaine, morphine and alcohol, are thought to be associated with functional modulation of neurotransmitter receptors and resultant changes in synaptic plasticity (reviewed in [56,57]). Regulation of neurotransmitter receptors by SFKs might thus contribute to drug-evoked plasticity changes and behavioral changes that occur in response to drugs of abuse. Indeed, SFKs are implicated in the central action of alcohol. Fyn-deficient mice manifest an increase in the duration of the ethanol-induced loss of righting reflex (LORR) [58–60], which is used to determine hypnotic sensitivity to ethanol. By contrast, LORR was found to be significantly decreased in mice that overexpress Fyn in the adult forebrain under the control of aCaMKII promoter [61]. Fyn-deficient mice also exhibited impaired acute tolerance to the motor incoordinating effects of ethanol as measured by the use of the stationary dowel [59]. Acute ethanol treatment induces transient reduction of NMDAR-mediated excitatory postsynaptic potentials (EPSPs) in hippocampal slice [58,62]. Striatal-enriched protein tyrosine phosphatase (STEP; Box 2) contributes to such an effect via tyrosine-dephosphorylation of GluN2B [63]. After the transient inhibition of EPSPs, NMDARs exhibit an acute tolerance to the inhibitory effects of ethanol in wild type [62] and heterozygous Fyn knockout mice [58]. Ethanol treatment also decreases NMDAR-mediated EPSCs in rat dorsal striatal slices, whereas after washout of the ethanol, the EPSCs gradually recover and then longterm facilitation (LTF) over the basal level is induced [64]. These phenotypes largely depend on Fyn because

Review Fyn-deficient mice fail to exhibit the acute tolerance to ethanol [58] or the ethanol-induced LTF [64]. The administration of ethanol enhances tyrosine phosphorylation of GluN2B subunit of NMDARs in the hippocampus of control heterozygote but not in Fyn-deficient mice [58]. Ethanolinduced tyrosine phosphorylation of GluN2B at Y1252, as well as activation of Fyn, has also been demonstrated in rat dorsal striatum slice [64]. Thus, Fyn-mediated tyrosine phosphorylation of NMDARs could mediate the acute tolerance of NMDARs to the inhibitory effects of ethanol. By contrast, overexpression of Fyn does not affect the ethanolinduced inhibition of the NMDA current in HEK cells expressing recombinant GluN1/GluN2B-type NMDARs [65]. Contribution of Fyn to the acute tolerance of NMDARs to the ethanol remains to be further explored. The development of addiction involves pathological changes in the cellular and molecular machinery of synaptic plasticity that underlies normal learning and memory [66,67]. Ethanol-induced tyrosine phosphorylation of NMDARs through activation of Fyn might facilitate habitual learning and memory formation that are thought to underlie addictive phenotypes such as compulsive alcohol consumption [64] (Figure 4). However, a definitive role of Fyn in alcohol addiction remains to be determined. Fyn-deficient mice show normal behavior with regard to voluntary ethanol consumption or the rewarding properties of ethanol [60,68], with the exception of one report showing decreased ethanol preference in male, but not female, Fyn-deficient mice [59]. By contrast, microinjection of the SFK inhibitor PP2 into the dorsal striatum of rats markedly reduced alcohol intake compared to vehicle-treated rats, as measured using the operant self-administration test [64,69]. One possible explanation for these findings is that SFKs other than Fyn may compensate the function of Fyn in the regulation of ethanol self-administration and reward in Fyn-deficient mice. Interestingly, increased anxiety-like behavior of wild type mice after withdrawal from chronic alcohol consumption is suppressed in Fyn-overexpressing transgenic mice [70]. GluN2B is hyperphosphorylated in the Fyn-transgenic mice, and a GluN2B-specific antagonist increased anxiety-like behavior of the transgenic mice after alcohol withdrawal [70]. Thus, increased phosphorylation of GluN2B in the Fyn-transgenic mice might contribute to the suppression of alcohol withdrawal-induced increase in anxiety-like behavior. Lyn is another SFK that has been implicated in the central action of alcohol. Alcohol reward is increased in Lyn-deficient mice, whereas overexpression of a constitutively active mutant of Lyn in the ventral tegmental area results in attenuation of such reward [71]. Negative regulation of dopamine release in the mesocorticolimbic system by Lyn was found to underlie this effect [71], suggesting that Lyn may have an important role to play in suppressing the rewarding effects of alcohol. Concluding remarks SFK-mediated tyrosine phosphorylation of neurotransmitter receptors, including NMDARs, AMPARs and GABAARs, as well as modulatory signaling proteins, such as SIRPa has been implicated in the regulation of various brain functions (Figure 4). Phosphorylation-dependent modulation of receptor endocytosis might be a common mechanism underlying

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Substrates

Brain functions

GluN2B pY1472

Fear-related learning

GABAAR γ 2 pY365/367

Spatial memory formation

GluA2 pY876

LTD induction

Disorders

Learning and memory disorder ?

SFKs GluN2A pY1325 SIRPα pY477/501

GluN2B pY?

Regulation of depression-like behavior

Mood disorder ?

Regulation of behavioral response to ethanol

Alcohol addiction ?

TRENDS in Neurosciences

Figure 4. Roles of SFKs and key neurotransmitter substrates and modulatory signaling proteins that are involved in the regulation of adult brain functions. SFKs participate in the regulation of a variety of neuronal functions by catalyzing the tyrosine phosphorylation of specific substrates, including neurotransmitter receptors and proteins that regulate receptor function. Phosphorylation of Y1472 of the GluN2B subunit of NMDARs by SFKs is likely to be important for fear-related learning [19]. Phosphorylation of Y365 and Y367 of the g2 subunit of GABAARs may contribute to spatial memory formation through regulation of GABAAR endocytosis and synaptic inhibition [52]. Phosphorylation of Y876 of GluA2 probably suppresses AMPAR endocytosis and LTD induction [40]. Phosphorylation of Y1325 of the GluN2A subunit of NMDARs, and Y477/501 of SIRPa are important for the regulation of depression-like behavior in mice [22]. SFK-mediated tyrosine phosphorylation of GluN2B might also participate in the regulation of behavioral responses to ethanol [58,60,64,69,70]. Deregulation of SFK-dependent tyrosine phosphorylation of these key neuronal substrates may be involved in neurological disorders, such as those affecting memory or mood, as well as alcohol addiction.

the regulation of neurotransmitter receptor function by SFKs. SFKs also mediate phosphorylation of other ion channels, such as large-conductance Ca2+-activated K+ (BK) channels, voltage-dependent Ca2+ channels, and nicotinic acetylcholine receptors (reviewed in [72,73]). A role for SFKs in the regulation of the endocytosis of these channels warrants further investigation. Genetic mouse models, which lack a specific SFK substrate or relevant tyrosine phosphorylation sites, are powerful tools for investigating molecular mechanisms underlying the regulation of neuronal functions by SFKs in vivo. However, it is sometimes unclear whether results obtained with Y!F knockin mice reflect the importance of tyrosine phosphorylation of the particular SFK substrate. The generation of knockin mice harboring constitutively phosphorylated mutants of SFK substrates would be desirable in this regard (Box 3). Moreover, it is not always easy to directly correlate whether the mutations at the phosphorylation sites are indeed responsible for the observed abnormal behavioral phenotypes in the mutant mice. In addition, unexpected effects of Y!F mutations on development cannot always be excluded. These issues remain to be addressed with the use of conditional knockin mice, in which tyrosine phosphorylation sites within SFK substrates can be regulated in a temporally-defined or tissue-specific manner. The deregulation of SFK-dependent tyrosine phosphorylation of key substrates such as neurotransmitter 635

Review Box 3. Outstanding questions  What are the functional differences among SFKs in terms of phosphorylation of key substrates such as neurotransmitter receptors in the brain?  What are the phenotypes of knockin mice harboring constitutively tyrosine-phosphorylated mutants of SFK substrates?  What are the molecular mechanisms for activation of SFKs in the adult brain? Is there any common upstream mechanism for such activation?  Does deregulation of SFK activity directly contribute to the susceptibility, and/or pathogenesis, of neurological disorders?  Is it possible to design new drugs to specifically modulate SFK activity in the nervous system, and hence, regulate the tyrosine phosphorylation of neural substrates of SFKs?

receptors might be one of the underlying mechanisms leading to neurological disorders such as dementia or depression (Figure 4). In addition, it is interesting to note that a genetic association has been found between a single nucleotide polymorphism in the 50 untranslated region of the human Fyn gene and increased alcohol consumption [74]. Association of this polymorphism with a high risk to develop alcohol dependence has also been reported [75]. However, the functional consequences of this polymorphism were not demonstrated. More recently, suppression of Src-mediated enhancement of NMDAR function by excessive neuregulin-1–ErbB4 signaling was proposed to potentially be one mechanism that may be involved in underlying cognitive deficits associated with schizophrenia [76]. More effort will be required, however, to provide a complete understanding of the physiological and pathological roles of SFKs in adult brain function. Acknowledgments The work in the authors’ laboratories was supported by a Grant-in-Aid for Scientific Research on Priority Areas Cancer, a Grant-in-Aid for Scientific Research (B), a Grant-in-Aid for Scientific Research (C), and a Global Center of Excellence Program grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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