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Mice lacking protein tyrosine phosphatase alpha (PTPα) exhibited defects in NMDA receptor (NMDAR)-associated processes such as learning and memory, hippocampal neuron migration, and CA1 hippocampal long-term potentiation (LTP). In vivo molecular effectors linking PTPα and the NMDAR have not been reported. Thus the involvement of PTPα as an upstream regulator of NMDAR tyrosine phosphorylation was investigated in synaptosomes of wild-type and PTPα-null mice. Tyrosine phosphorylation of the NMDAR NR2A and NR2B subunits was reduced upon PTPα ablation, indicating a positive effect of this phosphatase on NMDAR phosphorylation via intermediate molecules. The NMDAR is a substrate of src family tyrosine kinases, and reduced activity of src, fyn, yes and lck, but not lyn, was apparent in the absence of PTPα. In addition, autophosphorylation of proline-rich tyrosine kinase 2 (Pyk2), a tyrosine kinase linked to NMDAR signaling, was also reduced in PTPα-deficient synaptosomes. Altered protein tyrosine phosphorylation was not accompanied by altered expression of the NMDAR or the above tyrosine kinases at any stage of PTPα-null mouse development examined. In a human embryonic kidney (HEK) 293 cell expression system, PTPα enhanced fyn-mediated NR2A and NR2B tyrosine phosphorylation by several-fold. Together, these findings provide evidence that aberrant NMDAR-associated functions in PTPα-null mice are due to impaired NMDAR tyrosine phosphorylation resulting from the reduced activity of probably more than one of the src family kinases src, fyn, yes and lck. Defective NMDAR activity in these mice may also be linked to the loss of PTPα as an upstream regulator of Pyk2.
The N-methyl-d-aspartate receptor (NMDAR), a ligand-gated ion channel, is an important regulator of synaptic plasticity, brain development and excitotoxicity in the central nervous system (Nakazawa et al. 2004; Waxman and Lynch 2005). The receptor itself is a complex composed of NR1 and NR2 (types A–D) subunits (Cull-Candy et al. 2001). The NMDAR is associated with numerous other proteins, including signaling enzymes, scaffolding, cytoskeletal and adaptor proteins, and cell adhesion molecules (Husi et al. 2000; Sheng and Pak 2000). Reversible tyrosine phosphorylation of the NMDAR modulates channel function, receptor trafficking, and NMDAR multiprotein complex composition and associated downstream signaling pathways (Salter and Kalia 2004). Several protein tyrosine kinases (PTKs) [such as src, fyn, lyn and proline-rich tyrosine kinase 2 (Pyk2)] and protein tyrosine phosphatases (PTPs) (such as SHP-2, PTPMEG, MKP2 and STEP61) are components of the NMDAR complex (Lin et al. 1999; Tezuka et al. 1999; Hironaka et al. 2000; Husi et al. 2000; Pelkey et al. 2002; Kalia and Salter 2003). In particular, the src family kinases (SFKs) src and fyn phosphorylate several tyrosine residues in the cytoplasmic tails of the NR2A and NR2B subunits, and this is associated with NMDAR activation (Wang and Salter 1994; Kohr and Seeburg 1996; Yu et al. 1997; Tezuka et al. 1999; Cheung and Gurd 2001; Nakazawa et al. 2001; Yang and Leonard 2001), and with regulated synaptic localization and surface expression of the receptor (Grosshans et al. 2002; Thornton et al. 2003; Prybylowski et al. 2005; Suvarna et al. 2005). Conversely, STEP61 inhibits NMDAR function by opposing SFK-mediated NMDAR activation (Pelkey et al. 2002).
Receptor protein tyrosine phosphatase alpha (PTPα) is a ubiquitously expressed transmembrane protein that is enriched in brain (Kaplan et al. 1990; Krueger et al. 1990; Matthews et al. 1990; Sap et al. 1990). PTPα is a positive physiological regulator of the tyrosine kinases src and fyn. In brains of PTPα-null mice, these kinases exhibit enhanced tyrosine phosphorylation of their regulatory C-terminal tyrosine residues and concomitantly reduced kinase activity (Ponniah et al. 1999; Su et al. 1999). These mice also display defects in processes linked to NMDAR function such as learning and memory, hippocampal neuron migration, and CA1 hippocampal long-term potentiation (LTP) (Petrone et al. 2003; Skelton et al. 2003). Evidence of physical interactions between PTPα, src and fyn and the NMDAR comes from studies demonstrating that PTPα associates, as do fyn and src, with the NMDAR through the postsynaptic density-95 (PSD-95) scaffolding protein intermediate (Lei et al. 2002). The introduction of PTPα into NR1/NR2A-expressing PTPα–/– fibroblasts enhances NMDAR-mediated currents in a manner dependent on functional SFKs, while antibody inhibition of PTPα in hippocampal neurons reduces NMDAR-mediated currents (Lei et al. 2002). These lines of evidence suggest that PTPα plays a positive role in mediating NMDAR function, most likely via SFKs.
The SFKs src, fyn, yes, lyn and lck are expressed in the central nervous system (Thomas and Brugge 1997). To investigate the physiological regulation of specific SFK function by PTPα, particularly with respect to SFK-catalyzed NMDAR tyrosine phosphorylation, we determined SFK and NMDAR tyrosine phosphorylation status in synaptosomal fractions of wild-type (WT) and PTPα-deficient mice. We also examined the phosphorylation status of Pyk2, a tyrosine kinase that can act upstream of src to regulate NMDAR function (Huang et al. 2001). Our results demonstrate reduced tyrosine phosphorylation of Pyk2, enhanced inhibitory tyrosine phosphorylation of four SFKs, and reduced phosphorylation of NR2A and NR2B in PTPα-deficient synaptosomes, consistent with PTPα acting as a positive physiological regulator of SFK-mediated NMDAR tyrosine phosphorylation. In support of this, we show that heterologous expression of PTPα with fyn/NR2A/NR2B in human embryonic kidney (HEK) 293 cells enhances fyn-mediated phosphorylation of NR2A or NR2B.
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We report here that tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDAR is reduced in detergent-resistant synaptosomal fractions from PTPα–/– mice, a persistent effect that is detectable from 2 weeks of age into adulthood. This is consistent with the reduced phosphorylation of NR2B at Tyr1472 reported in hippocampi of adult PTPα–/– mice (Petrone et al. 2003). Tyrosine phosphorylation of NR2 subunits is reduced by about 25–35% in the absence of PTPα but is not abolished. These findings indicate that that PTPα is a physiological upstream activator of NMDAR phosphorylation.
The SFKs src and fyn regulate NMDAR tyrosine phosphorylation (Tezuka et al. 1999; Cheung and Gurd 2001; Nakazawa et al. 2001; Yang and Leonard 2001). These kinases can be regulated by PTPα (Ponniah et al. 1999; Su et al. 1999). Our investigation of SFK activation as determined by kinase tyrosine phosphorylation status reveals that, in synaptosomal fractions from mice lacking PTPα, the SFKs src and fyn, and also yes and lck, exhibit reduced activity as indicated by their enhanced C-terminal tyrosine phosphorylation. The phosphorylation/activation of a fifth synaptosomal SFK, lyn, remained unchanged in the absence of PTPα. This demonstrates that PTPα has a broad range of action towards several neuronal SFKs. The similar structures and regulation of SFK family members suggest it is unlikely that lyn cannot be dephosphorylated by PTPαper se, and thus synaptosomal lyn may be sublocalized such that it is not in proximity to PTPα and/or specific conditions are required to stimulate a functional interaction. Although only src and fyn have been shown to phosphorylate the NMDAR, our results suggest that not only these SFKs but also yes and lck are candidate kinase intermediates in the observed PTPα-dependent regulation of NMDAR tyrosine phosphorylation in mouse synaptosomes. All of these SFKs and PTPα are present in the Triton X-100-insoluble synaptosomal fraction where most of the NMDAR is found. Interestingly, PTPα and the SFKs src, fyn and yes can physically associate with the NMDAR via their interactions with the scaffolding protein PSD-95 (Tezuka et al. 1999; Kalia and Salter 2003). PSD-95 binding could thus promote phosphatase–kinase–NMDAR proximity to enhance NMDAR phosphorylation upon the PTPα-mediated dephosphorylation and activation of these SFKs.
Direct evidence supporting a physiological dephosphorylation–phosphorylation mechanism of signaling linking PTPα, SFKs and the NMDAR was obtained from experiments utilizing heterologous co-expression of these proteins in HEK293 cells. Fyn-catalyzed tyrosine phosphorylation of NR2A and NR2B in HEK293 cells was significantly enhanced in the presence of PTPα. Although PSD-95 has been shown to promote Fyn-induced NR2A phosphorylation (Tezuka et al. 1999), a similar effect on NR2B has not been reported. Introduction of PSD-95 to the cells markedly increased fyn-dependent NR2B phosphorylation in the absence of co-expressed PTPα, and a further increase was observed upon PTPα co-expression. Nevertheless, PTPα effected an approximate 2- to 3-fold increase in NR2B phosphorylation in either the presence or absence of PSD-95. Thus optimal NR2B tyrosine phosphorylation occurred when PTPα and PSD-95 were co-expressed with fyn, and this was an additive rather than a synergistic increase. This indicates that while PSD-95 is important in promoting fyn–NR2B interactions, it is not essential for the PTPα-mediated activation of fyn per se. However, in neuronal cells, the abilities of PTPα and fyn/src to associate with PSD-95 (Tezuka et al. 1999; Lei et al. 2002; Kalia and Salter 2003) may be advantageous in promoting PTPα-catalyzed activation of a population of fyn/src within the NMDAR complex at the synaptic membrane.
The tyrosine kinase Pyk2 (CAKβ/CADTK) acts downstream of G-protein coupled receptors and integrins and upstream of SFKs to mediate NMDAR tyrosine phosphorylation and NMDAR-induced LTP (Huang et al. 2001; Heidinger et al. 2002; Bernard-Trifilo et al. 2005). We found that Pyk2 phosphorylation at its key activation site, Tyr402, was significantly reduced in PTPα–/– synaptosomes. In a converse situation in transfected HEK293 cells, increased PTPα expression promoted an enhanced association of fyn and Pyk2. PTPα-dependent modulation of Pyk2 activation by regulation of Pyk2 autophosphorylation and binding to SFKs has not been previously observed. Pyk2 Y402 phosphorylation occurs by an autocatalytic mechanism (Li et al. 1999). In synaptoneurosomes, the SFK inhibitor PP2 inhibits integrin-stimulated NMDAR tyrosine phosphorylation, but not Pyk2 Tyr402 phosphorylation (Bernard-Trifilo et al. 2005). This and other observations (Huang et al. 2001) place the SFKs downstream rather than upstream of Pyk2. However, Pyk2 tyrosine phosphorylation, including that at Tyr402, is dramatically decreased in hippocampi of mice lacking fyn (Corvol et al. 2005). Pyk2 is closely related to focal adhesion kinase (FAK). In integrin signaling, the PTPα-catalyzed activation of src and fyn promotes FAK autophosphorylation, and FAK association with these SFKs, at a site analogous to Pyk2 Tyr402 (Zeng et al. 2003). PTPα could potentially regulate Pyk2 by a similar mechanism, although this, and the nature of the upstream signals that engage PTPα to lead to Pyk2 activation, requires further investigation. Pyk2 has been implicated in LTP (Huang et al. 2001), and impaired LTP in mice lacking PTPα (Petrone et al. 2003) could involve altered Pyk2 activity.
This study provides evidence that aberrant NMDAR-associated functions reported in PTPα-null mice are due to impaired NMDAR tyrosine phosphorylation. Our results indicate that a key mechanism by which PTPα is involved in neuronal NMDAR-mediated processes such as learning and memory, hippocampal neuron migration, and LTP is through controlling NMDAR tyrosine phosphorylation via its upstream action on the SFKs src, fyn, yes and/or lck. This is the first report of the physiological regulation of yes and lck by PTPα. Since fyn is only one of several neuronal substrates of PTPα, this may contribute to the partial but not complete overlap between PTPα–/– and fyn–/– phenotypes. Impaired NMDAR function and/or reduced SFK activity in these mice result in alterations of downstream effectors such as Pyk2 that may further contribute to defective NMDAR-related processes. Accumulating evidence of the association of PTPα with various cell surface receptors, including F3/contactin, integrins, NCAM and the NMDAR itself (Lei et al. 2002; von Wichert et al. 2003; Zeng et al. 1999; Bodrikov et al. 2005), suggests that PTPα action as an activator of SFKs is regulated by its interactions with ligand-stimulated receptors. Further studies are required to determine which of the multiple neuronal receptors that regulate SFK-catalyzed NMDAR tyrosine phosphorylation and function/localization (Salter and Kalia 2004) may do so via a PTPα-mediated signaling mechanism.