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. Author manuscript; available in PMC: 2018 Apr 25.
Published in final edited form as: Sheng Li Xue Bao. 2017 Oct 25;69(5):657–665.

Local substrates of non-receptor tyrosine kinases at synaptic sites in neurons

Li-Min Mao 1, Ryan Geosling 2, Brian Penman 2, John Q Wang 1,2,*
PMCID: PMC5672811  NIHMSID: NIHMS916616  PMID: 29063113

Abstract

Several non-receptor tyrosine kinase (nRTK) members are expressed in neurons of mammalian brains. Among these neuron-enriched nRTKs, two Src family kinase members (Src and Fyn) are particularly abundant at synaptic sites and have been most extensively studied for their roles in the regulation of synaptic activity and plasticity. Increasing evidence shows that the synaptic subpool of nRTKs interacts with a number of local substrates, including glutamate receptors (both ionotropic and metabotropic glutamate receptors), postsynaptic scaffold proteins, presynaptic proteins, and synapse-enriched enzymes. By phosphorylating specific tyrosine residues in the intracellular domains of these synaptic proteins either constitutively or in an activity-dependent manner, nRTKs regulate these substrates in trafficking, surface expression, and function. Given the high sensitivity of nRTKs to changing synaptic input, nRTKs are considered to act as a critical regulator in the determination of the strength and efficacy of synaptic transmission.

Keywords: N-methyl-D-aspartic acid, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, metabotropic glutamate receptor, postsynaptic density protein 95, Src, Fyn, ERK, Cdk5, synaptophysin, synapsin I

Tyrosine kinases catalyze the transfer of a phosphate group from ATP to a tyrosine residue of a protein, i.e., phosphorylation. The first subgroup of tyrosine kinases are receptor tyrosine kinases which switch on or off other enzymes in a cell through phosphorylation. The second subgroup of tyrosine kinases are non-receptor tyrosine kinases (nRTK) which are cytoplasmic and phosphorylate a number of substrates to regulate a variety of cellular functions [1]. While nRTKs phosphorylate their targets, nRTKs themselves are regulated by a phosphorylation-dependent mechanism. With few exceptions, phosphorylation of a tyrosine site in the activation loop of nRTKs enhances enzymatic activity [1]. This phosphorylation in the activation loop is induced by an autophosphorylation mechanism or by a different nRTK. Notably, this phosphorylation step is sensitive to changing cellular input. Thus, it regulates cellular responses to stimulation and is linked to the pathogenesis of various diseases.

At present, thirty-two nRTK members have been identified in mammalian cells. These nRTKs are classified into several subfamilies [1]. Notably, a number of nRTKs are highly expressed in the brain. These nRTKs include five (Src, Fyn, Yes, Lyn, and Lck) out of nine members of the Src family kinase (SFK), two members (Fak and Pyk2) of the focal adhesion kinase (Fak) family kinase, and a member (c-Abl) of the Abl family kinase [2–4]. Among these brain-enriched nRTKs, Src and Fyn are particularly abundant at synaptic sites. They are therefore conceived to target local synaptic substrates to regulate the strength and efficacy of synaptic transmission [2].

Indeed, a number of synaptic proteins have been demonstrated to be direct chemical substrates of nRTKs. These substrates include glutamate receptors and other proteins. Among them, glutamate receptors have been most thoroughly investigated and characterized. Two classes of glutamate receptors are expressed at synaptic sites in the central nervous system: ionotropic glutamate receptors (iGluR) and metabotropic glutamate receptors (mGluR). The former is a ligand-gated ion channel, while the latter is a family of G protein-coupled receptors (GPCR) [5,6]. Two classes of glutamate receptors carry out different functions. While iGluRs mediate fast synaptic transmission, mGluRs modulate a variety of cellular and synaptic activities [5,6]. Both iGluRs and mGluRs are subjected to the regulation by phosphorylation and are in fact substrates of nRTKs. As a result, nRTKs can phosphorylate glutamate receptors at specific amino acid sites located in the intracellular domain such as the C-terminal (CT) tail. By changing the constitutive phosphorylation of glutamate receptors or by mediating activity-dependent phosphorylation induced by changing synaptic input, nRTKs modulate the biochemistry, biophysics, and physiology of glutamate receptors [7–10]. This review thus summarizes the relationship between nRTKs and their substrates within the synaptic microdomain. From accumulated evidence, it is clear that nRTKs interact directly or indirectly with synaptic substrates and act as a set of key kinases controlling the number and function of modified proteins.

1 Phosphorylation of glutamate receptors by nRTKs

1.1 iGluRs

There are three classes of iGluRs: α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPAR), N-methyl-D-aspartate receptors (NMDAR), and kainate receptors [5].

NMDARs form functional channels by assembling GluN1 (formerly known as NR1) with GluN2 subunits, mainly GluN2A (NR2A) and GluN2B (NR2B) [5]. AMPARs are assembled by four subunits (GluA1–4, previously named GluR1–4). All NMDAR and AMPAR subunits have an intracellular CT domain which has been found to accommodate dynamic protein-protein interactions and tyrosine phosphorylation [7–10]. GluN2A CT and GluN2B CT are large and are phosphorylated by nRTKs (Src and/or Fyn) at multiple tyrosine sites [11–14]. Like NMDARs, AMPARs are also tyrosine-phosphorylated. The SFK members, including Src, Fyn and Lyn, phosphorylated GluA2 at Y876, while the Fak family of nRTKs (Fak and Pyk2) did not [15].

The Src/Fyn-mediated phosphorylation of NMDARs controls subcellular or subsynaptic distributions of the receptors. For instance, Fyn by phosphorylating GluN2B determined expression of GluN2B-containing NMDARs via a mechanism involving the calpain-dependent cleavage of GluN2B [16]. GluN2B phosphorylation at Y1472 and Y1336 sites selectively enriched GluN2B/NMDAR abundance in synaptic versus extrasynaptic compartments, respectively [17]. Functionally, Src phosphorylated GluN2A Y1325, and thereby potentiated the efficacy of NMDARs [13]. Src and Fyn tyrosine-phosphorylated GluN2A and GluN2B to potentiate NMDAR currents [18]. Like NMDARs, AMPARs are regulated in expression and function by tyrosine phosphorylation. Tyrosine phosphorylation at Y876 of GluA2 disrupted the association of GluA2 with glutamate receptor interacting proteins 1 and 2 (GRIP1/2). Since GRIP1/2 stabilize surface expression of AMPARs, the disruption of their associations with GluA2 accelerated endocytosis of GluA2, leading to reduction of the abundance of surface-expressed AMPARs [15].

1.2 mGluRs

Compared to iGluRs which are extensively investigated for tyrosine phosphorylation, mGluRs are less studied for their regulation by nRTKs. There are currently eight mGluR subtypes (mGluR1–8), which are grouped into three functional groups (group I, II, and III). At present, group I mGluRs (mGluR1/5) have been analyzed for their relationship with nRTKs. mGluR1/5 are GPCRs [6], and stimulation of mGluR1/5 activates Gαq-coupled phospholipase Cβ1 and thereby hydrolyzes phosphoinositide into inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG then trigger Ca2+ release from internal Ca2+ stores and activate protein kinase C (PKC), respectively, to regulate cellular and synaptic activities. As typical membrane-bound GPCRs, mGluR1/5 have multiple intracellular domains. Intracellular CT tails are particularly large and harbor most protein-protein interactions so far discovered [19, 20]. Moreover, CT regions are the only intracellular domain containing tyrosine residues and thus have the potential for tyrosine phosphorylation.

Tyrosine phosphorylation signals were detected in mGluR5 in an early study [21]. In mGluR5 proteins immunopurified from the rat brain, robust phosphotyrosine signals were seen. Moreover, the level of tyrosine phosphorylation of mGluR5 was enhanced by a protein phosphatase inhibitor. Thus, mGluR5 undergoes tyrosine phosphorylation which is controlled by an active phosphorylation and dephosphorylation cycle. Since NMDA enhanced mGluR5 tyrosine phosphorylation [21], the tyrosine phosphorylation may act as an activity-dependent step linking NMDARs to mGluR5, although accurate phosphorylation sites in mGluR5 and the specific nRTK member(s) responsible for catalyzing tyrosine phosphorylation are currently unclear. Functional relevance of tyrosine phosphorylation of mGluR5 is also unknown. Given the finding that inhibition of tyrosine phosphorylation of mGluR5 did not affect the mGluR5-assoicated phosphoinositide hydrolysis [21], tyrosine phosphorylation of mGluR5 is less likely involved in the regulation of the conventional mGluR5 signaling transduction.

Another group I mGluR subtype (mGluR1) is also regulated by tyrosine phosphorylation. A recent study observed that recombinant Fyn proteins directly bound to mGluR1a at a consensus binding motif located in mGluR1a CT in vitro [22]. Similarly, endogenous Fyn formed complexes with mGluR1a in rat cerebellar neurons in vivo. The mGluR1a-associated Fyn was constitutively active and phosphorylated mGluR1a at a conserved tyrosine residue in the CT region. In cerebellar neurons and transfected HEK293T cells, Fyn facilitated surface expression of mGluR1a and augmented the efficacy of mGluR1a in triggering its postreceptor signaling. Thus, mGluR1a is a novel substrate of Fyn at synaptic sites. By binding to and phosphorylating mGluR1a, Fyn regulates surface expression and signaling of the receptors.

SFKs are known to be sensitive to group I mGluR input. Evidence shows that pharmacological stimulation of group I mGluRs with the selective agonist DHPG consistently activated Src and/or Fyn in cortical and cerebellar neurons [23, 24], although precise signaling pathways linking group I mGluRs to SFKs are incompletely investigated. Thus, it is possible that SFKs participate in the formation of an activity-dependent signaling loop which exerts the positive feedback regulation of mGluR signaling. That is, activation of group I mGluRs activates SFKs which in turns augment the conventional group I mGluR signaling.

2 Phosphorylation of synaptic scaffold proteins by nRTKs

2.1 Postsynaptic density protein 95 (PSD-95)

PSD-95, also known as synapse-associated protein 90 (SAP-90), is a member of the membrane-associated guanylate kinase (MAGUK) family. It serves as a multimeric scaffold protein for clustering receptors, ion channels, and other signaling proteins at postsynaptic sites and thereby plays a pivotal role in stabilizing synaptic proteins and forming synaptic plasticity. As the best studied member of MAGUK family, PSD-95 was found to be associated with nRTKs. An early study showed that PSD-95 promoted Fyn-mediated tyrosine phosphorylation of GluN2A [25]. This may be due to a role of PSD-95 in facilitating formation of multiple protein complexes within the PSD microdomain. PSD-95 was associated with both GluN2A and Fyn at different regions, which mediated complex formation of Fyn with GluN2A. Additionally, PSD-95 is associated with other SFK members, such as Src, Yes, and Lyn. These results support the role of PSD-95 as a critical scaffold protein tethering SFKs to glutamate receptors and likely other synaptic substrates at synaptic sites.

PSD-95 is also a major target for tyrosine phosphorylation by SFKs. The direct evidence that PSD-95 is tyrosine-phosphorylated by SFKs has been provided. For instance, PSD-95 was phosphorylated by Src/Fyn in vitro [26]. In transfected COS7 cells, Src/Fyn interacted with PSD-95 and phosphorylated PSD-95 primarily at a tyrosine site (Y523). Overexpression of a phosphorylation-deficient mutant (Y523F) in cultured hippocampal neurons abolished the facilitating effect of PSD-95 on the glutamate- or NMDA-stimulated currents. In contrast, a phosphomimetic mutant (Y523D) induced GluN2A tyrosine phosphorylation [27]. Thus, by phosphorylating PSD-95, SFKs positively regulate NMDAR function. In addition to Src and Fyn, Pyk2 bound to PSD-95 [27]. Overexpression of PSD-95 in PC6-3 cells induced autophosphorylation of Pyk2, the first step in Pyk2 activation [28]. A decrease in PSD-95 Y523 phosphorylation reduced the interaction of Pyk2 with PSD-95 and impaired the stimulating effect of PSD-95 on Pyk2 [27]. Based on these findings, a synaptic model involving the physical and functional integration of multiple proteins is proposed. Src phosphorylates PSD-95 to integrate Pyk2 into PSD-95 complexes. Both Src and Pyk2 then tyrosine-phosphorylate GluN2A to upregulate GluN2A/NMDARs and synaptic transmission. In addition to PSD-95, Fak and Pyk2 interacted with SAP90/PSD-95-associated protein-3 (SAPAP3) [4]. This interaction may contribute to anchoring Fak and Pyk2 in the PSD region and processing the Fak-mediated regulation of the synaptic function [29].

c-Abl is a member of the Abl family kinase of nRTKs and is expressed in mouse brain synapses. Increasing evidence supports that c-Abl is implicated in synaptic transmission and plasticity. Indeed, cAbl associated with PSD-95 and phosphorylated PSD-95 at Y533 [30]. Chemical or genetic inhibition of c-Abl reduced tyrosine phosphorylation of PSD-95, resulting in reduction of PSD-95 clustering and synapses in hippocampal neurons. Mutation of Y533 produced similar results. Thus, c-Abl modulates formation of synapses by tyrosine-phosphorylating and clustering PSD-95.

2.2 PSD-93

In addition to PSD-95, PSD-93 also known as disks large homolog 2 (DLG2) or channel-associated protein of synapse-110 (chapsyn-110) is another substrate of Fyn. As a member of the MAGUK family, PSD-93 interacts with a large number of synaptic proteins to support synaptic networks and participate in synaptic transmission. Indeed, PSD-93 interacted with Fyn in the cerebral cortex as detected by coimmunoprecipitation [31]. In PSD-93 knockout mice, the expression level of Fyn but not other SFK members (Src, Yes, and Lyn) was reduced in synaptosomal membranes. Recombinant PSD-93 was phosphorylated at Y384 by Fyn in vitro [32]. In Fyn knockout mice, tyrosine phosphorylation of PSD-93 was reduced in brain tissue. Functionally, PSD-93 deletion blocked the SFK-mediated tyrosine phosphorylation of GluN2A and GluN2B in cultured cortical neurons. These findings collectively indicate that PSD-93 is a substrate of Fyn and plays a role in the Fyn-mediated tyrosine phosphorylation and regulation of NMDARs in synaptic locations.

2.3 SPIN90

SPIN90 (SH3 protein interacting with Nck, 90 kDa) is a binding protein of PSD-95 and Shank and plays a role in constructing spine structures and synaptic network. Recent evidence shows that SPIN90 was tyrosine-phosphorylated by Src in vitro and in neurons [33]. Tyrosine phosphorylation of SPIN90 targeted the protein to dendritic spines in cultured hippocampal neurons, while a SPIN90 phospho-deficient mutant failed to do so. In contrast, overexpression of a phospho-mimicking mutant of SPIN90 enlarged spine heads and enhanced postsynaptic function in hippocampal neurons. Thus, SPIN90 functions to modulate synaptic activity via a SFK-dependent mechanism.

3 Phosphorylation of synapse-enriched enzymes by nRTKs

3.1 Mitogen-activated protein kinases (MAPK)

MAPKs are serine/threonine kinases that are expressed in postmitotic neurons of mammalian brains [34]. Among three subclasses of MAPKs, extracellular signal-regulated kinases (ERKs) have been most thoroughly investigated and characterized in neural activity. ERKs usually reside in the cytoplasmic compartment. Upon activation, ERKs translocate into the nucleus and activate a set of transcription factors to regulate gene expression and to transcriptionally regulate cellular activities and responses [35]. In addition, ERKs are notably distributed in neuronal peripheries, i.e., postsynaptic dendritic spines [36–38]. This subpool of ERKs is sensitive to changing synaptic input [39, 40] and is believed to regulate local substrates to control synaptic transmission and plasticity. ERKs are characterized to be regulated by nRTKs. In various heterologous cells, Src, Fyn, Fak and Pyk2 are responsive to GPCR signals. Inhibition of responses of these nRTKs to GPCRs by either a pharmacological inhibitor or dominant negative mutants blocked activation of ERKs [41, 42], indicating the role of nRTKs in linking GPCRs to ERKs. Consistent with this, the endocannabinoid-stimulated ERK in hippocampal slices was lost in Fyn knockout mice [43]. NMDA-induced ERK activation in cultured striatal neurons was blocked by the SFK inhibitor PP2 [44]. The SFK inhibitor SU6656 also reduced the transient global ischemia-induced ERK phosphorylation in the hippocampus [45]. ERKs are activated by the MAPK kinase (MEK) which is activated by Raf-1. Src is known to activate Raf-1 by phosphorylating Raf-1 at Y340 and Y341 [46, 47]. Thus, within the Ras-Raf-MAPK-ERK pathway, Src affects Raf-1 to regulate ERK activity. Alternatively, Src could directly phosphorylate protein phosphatase 2A (PP2A) at Y307 to inactivate PP2A [48, 49]. Since PP2A is an ERK phosphatase that dephosphorylates and thereby inactivates ERK, Src may inactivate PP2A to activate ERK [50]. Given that Src and ERKs are both located at synaptic sites, Src may regulate local ERKs to modulate synaptic transmission.

3.2 Cyclin-dependent kinases (Cdk)

Cdks are a family of proline-directed serine/threonine kinases that are expressed in proliferating cells and thus regulate cell cycle-associated events. Unlike most Cdk members, Cdk5 is abundant in postmitotic neurons, while it is also expressed in a number of tissues, and has no functional role in the cell cycle. Instead, Cdk5’s role is restricted to the nervous system, including synaptic activity [51]. Cdk5 is activated by binding to the activator, p35 (Cdk5R1) or p39 (Cdk5R2). Early work found that Fyn and c-Abl or receptor tyrosine kinases stimulated Cdk5 by phosphorylating Cdk5 at Y15 [52–54]. Such inducible Y15 phosphorylation leads to structural changes, including neurite and spine reaction and dendrite outgrowth. A recent study reveals that active Fyn increases p35 protein levels but not Cdk5 Y15 phosphorylation in neurons [55]. This indicates that Fyn may activate Cdk5 by increasing the amount of p35 proteins rather than the phosphorylation of Cdk5 at Y15.

4 Phosphorylation of presynaptic proteins by nRTKs

4.1 Synaptophysin

Synaptophysin is one of the most abundant proteins on synaptic vesicle membranes in the presynaptic nerve terminals. The CT tails of synaptophysin are characterized by the existence of multiple tyrosine residues, indicating that synaptophysin might be a target of tyrosine kinases. In fact, recombinant synaptophysin proteins were tyrosine phosphorylated in vitro by Src and Fyn [56–58]. Similarly, native synaptophysin proteins immunoprecipitated from animal brain homogenates were tyrosine phosphorylated [59, 60]. Tyrosine phosphorylation of synaptophysin may not play a significant role in neurotransmitter release, but evidence clearly shows that tyrosine phosphorylation of synaptophysin is critical for synaptic plasticity [61]. The level of tyrosine phosphorylation of synaptophysin was increased in parallel with glutamate release from hippocampal slices undergoing a typical form of synaptic plasticity, i.e., long-term potentiation (LTP) [60]. Inhibition of tyrosine kinases prevented an increase in tyrosine phosphorylation of synaptophysin and glutamate release as well as LTP. Moreover, in the rat hippocampus, presynaptic Src activity was increased after a memory training, which was accompanied by an increase in the Src-synaptophysin interaction [62]. Thus, Src may be related to an increase in tyrosine phosphorylation of synaptophysin, which contributes to the development of synaptic plasticity. Consistent with this, an early study has found that phosphorylation of synaptophysin at tyrosine 273 within its C-terminus is critical for the binding of synaptophysin to Src and for the activation of Src [63].

4.2 Synapsin I

Synapsin I is another synaptic vesicle-associated protein which is involved in the regulation of neurotransmitter release. Onofri et al. found that the Src homology 3 (SH3) domain of Src bound to a proline-rich domain D of synapsin I [64]. The interaction of the two proteins resulted in a substantial increase in Src tyrosine kinase activity. In contrast, removal of synapsin I from purified synaptic vehicles induced a decrease in Src kinase activity. These results suggest that Src and synapsin I may form a functional complex implicated in the regulation of synaptogenesis and transmitter release. In support of this, synapsin I was found to be phosphorylated at tyrosine 301 by Src [65]. This phosphorylation was sensitive to depolarization. Functionally, Src-mediated phosphorylation of synapsin I enhanced the binding of synapsin I to synaptic vesicles and actin and the formation of synapsin I dimers, all of which are critical for synaptic vesicle clustering.

5 Conclusion

nRTKs are among protein kinases that are enriched at synaptic sites. Abundant expression of nRTKs enables them to actively regulate a large number of local substrates. Indeed, a set of synaptic substrates have been identified, which include synaptic receptors, scaffold and structural proteins, enzymes, and presynaptic proteins (Fig. 1). By directly phosphorylating these substrates at specific tyrosine residues, nRTKs regulate subsynaptic distributions and trafficking of modified substrates. More importantly, nRTKs thus play a significant functional role in shaping structural changes of synapses and in regulating strength and efficacy of synaptic transmission. While previous studies over decades have significantly advanced our knowledge on nRTKs and their synaptic protein substrates, more studies are needed to deepen our understanding of this topic. First, previous studies have focused on Src and Fyn. In addition to Src and Fyn, several other nRTK members are expressed in the central nervous system and are present at synaptic sites, such as Yes, Lyn, Lck, Fak, Pyk2, etc., but they are less studied. Thus, more studies targeting these members are needed as these kinases are deemed to be active in the regulation of local substrates and synaptic transmission. Second, synaptic structures accommodate a large number of proteins at both pre-and postsynaptic sites. In addition to synaptic substrates that have been identified, including the substrates mentioned above as well as GABAA receptors and cholinergic receptors reviewed previously [8], nRTKs are believed to interact with additional substrates within the synaptic microdomain. Thus, future studies can be designed to find new substrates of nRTKs and roles of a new substrate in linking nRTK activity to a specific function. Finally, different nRTK members are thought to work in concert to regulate discrete substrates and to maintain synaptic homeostasis. Substantial crosstalk among different nRTK-associated signaling pathways is believed to exist at multiple levels. Moreover, nRTKs may integrate with serine/threonine kinases to coordinately phosphorylate and regulate the same set of substrates. These types of crosstalk involving synaptic nRTKs need to be investigated in the future.

Fig. 1.

Fig. 1

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A schematic diagram illustrating the regulation of glutamate receptors by Src family kinases. Src or Fyn directly phosphorylates AMPA receptor GluA1 subunits, NMDA receptor GluN2A and GluN2B subunits, and mGluR1a. The Src/Fyn-mediated phosphorylation occurs at specific tyrosine residues located in the intracellular C-terminal tails of these receptors. Src family kinases also phosphorylate postsynaptic density protein 95 (PSD-95) that interacts with and stabilizes GluN2A and GluN2B subunits. By phosphorylating these tyrosine sites, Src and Fyn regulate subsynaptic and surface distributions and function of modified receptors.

Acknowledgments

Research from the corresponding author’s laboratory was supported by NIH (No. R01 DA010355 and R01 MH061469).

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