• knockout mice;
  • NMDA receptor;
  • proline-rich tyrosine kinase 2;
  • protein tyrosine phosphatase alpha;
  • src family kinases;
  • synaptosome


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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.

Abbreviations used

bovine serum albumin


Ca2+-dependent tyrosine kinase


Child & Family Research Institute


cell adhesion kinase β


focal adhesion kinase


human embryonic kidney


long-term potentiation


NMDA receptor


nonidet P-40


phenylmethylsulfonyl fluoride


postsynaptic density-95


protein tyrosine kinase


protein tyrosine phosphatase


polyvinylidene fluoride


proline-rich tyrosine kinase 2


radioimmunoprecipitation assay


sodium dodecyl sulfate


SDS − polyacrylamide gel electrophoresis


src family kinase



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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Reagents and antibodies

Anti-NR2A, -NR2B and -NR2A/B antibodies were obtained from Chemicon (Temecula, CA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-NR1 antibodies were purchased from Pharmingen (Mississauga, ON, Canada). Anti-phosphotyrosine clone 4G10 antibodies were from Upstate Biotechnology (Lake Placid, NY, USA). Anti-v-src antibodies were from Oncogene (San Diego, CA, USA). Antibodies to yes, Pyk2 and PSD-95 were obtained from BD Transduction Laboratories (Mississauga, ON, Canada). Anti-lyn and -lck antibodies were from Santa Cruz Biotechnology. Anti-fyn antibodies were obtained from BD Transduction Laboratories and Santa Cruz Biotechnology. Antibodies to the transferrin receptor were from Zymed Laboratories, Inc. (South San Francisco, CA, USA). SFK phosphorylation status was investigated using phosphosite-specific antibodies raised to the C-terminal phosphorylation site (pTyr527) and autophosphorylation site (pTyr416) of src (BioSource International, Camarillo, CA, USA). Anti-phospho-Pyk2 (Tyr402) antibody was from Cell Signaling Technology (Beverly, MA, USA). Other reagents were from Sigma (St Louis, MO, USA).


Mice deficient for PTPα were generated as described (Ponniah et al. 1999) and maintained as an advanced intercross line (129SvEv × Black Swiss, 50 : 50 mixed background). Animal care and use followed the guidelines of the Canadian Council on Animal Care and the University of British Columbia.

Preparation of crude synaptosomal P2 fraction

Brain fractionation was carried out essentially as described by Huttner et al. (1983) and Lin et al. (1998). Frozen whole mouse brains were homogenized in ice-cold homogenizing buffer [320 mm sucrose, 10 mm Tris (Sigma) pH 7.4, 1 mm NaHCO3, 1 mm MgCl2, 1 mm Na3VO4, 20 mm NaF, 1 mm phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL aprotinin and 5 µm leupeptin] using a Dounce homogenizer. The homogenates were centrifuged at 1000g for 10 min at 4°C to remove nuclei and large debris. Supernatants were collected and further centrifuged at 10 000g for 15 min at 4°C. The resulting pellets (crude synaptosomal fraction, P2) were kept at −80°C for later studies.

Preparation of Triton X-100 soluble and insoluble fractions

Crude synaptosomal fractions were lysed in Triton X-100 buffer (20 mm Tris pH 7.5, 150 mm NaCl, 2 mm EDTA, 1 mm NaVO4, 20 mm NaF, 1 mm PMSF, 10 µg/mL aprotinin, 5 µm leupeptin and 1% Triton X-100 (Sigma)) on ice for 1 h. The lysates were centrifuged at 16 000g for 15 min. The resulting supernatants and pellets were designated as Triton X-100 soluble and insoluble fractions, respectively. Supernatants were assayed for protein concentration using Biorad reagent. Pellets were dissolved in 1% sodium dodecyl sulfate (SDS), boiled for 3 min, diluted 10-fold with Triton X-100 buffer, and centrifuged to obtain clear homogenates.

Cell culture and transient transfection

HEK293T cells were obtained from American Type Culture Collection (CRL-1573) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and streptomycin (100 µg/mL)/penicillin (100 units/mL). Cells (60–70% confluency, 10 cm dish) were transfected with combinations of the following plasmids: 5 µg of pRK5-NR2A or pRK5-NR2B, 2 µg of pGW1-EGFP-PSD-95, 5 µg of pXJ41neo-fyn and 1 µg of pXJ41neo-PTPα (Bhandari et al. 1998); or 2 µg of pcDNA3-myc-Pyk2, 2 µg of pXJ41neo-fyn and 0.5 µg of pXJ41neo-PTPα, using a standard calcium transfection method. Cells were harvested for analysis 30 h post-transfection.

Preparation of cell lysates

Cells were washed with cold phosphate-buffered saline and then lysed in modified radioimmunoprecipitation assay (RIPA) buffer (20 mm Tris pH 7.5, 150 mm NaCl, 2 mm EDTA, 1 mm Na3VO4, 20 mm NaF, 1 mm PMSF, 10 µg/mL aprotinin, 5 µm leupeptin, 1% Triton X-100, 0.5% NaDOC and 0.1% SDS). In experiments to determine the association of Pyk2 and fyn, the Triton X-100, SDS, and NaDOC were replaced with 1% Nonidet P-40 (NP-40). Cell homogenates were incubated on ice for 30 min and then centrifuged at 16 000g for 15 min. The resulting supernatants were collected and assayed for protein concentration using Biorad reagent.

Immunoprecipitation and immunoblotting

Immunoprecipitation was performed by incubating the pre-cleared protein homogenates with the indicated antibody on a rotator at 4°C for 2 h to overnight. Protein G Plus Protein A Agarose (Santa Cruz Biotechnology) was added for another 2 h at 4°C and the immunocomplexes were washed three times with lysis buffer and eluted with SDS sample buffer at 100°C for 5 min. Immunoprecipitated proteins were resolved by SDS − polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 2% bovine serum albumin (BSA) or 5% skim milk, subjected to anti-phosphotyrosine immunoblotting if appropriate, and then stripped and probed for other proteins of interest. To examine the expression of synaptosomal proteins, 10–20 µg of protein were loaded on SDS–PAGE.

Kinase assay

Fyn immunoprecipitates were prepared from lysates of transfected HEK293 cells using anti-fyn antibody (FYN 3-G, Santa Cruz Biotechnology). These were used in in vitro kinase assays as described (Ponniah et al. 1999), and immunoblotted with anti-fyn antibody (BD Transduction Laboratories).


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Localization of PTPα, SFKs, Pyk2 and NMDAR subunits in Triton X-100 solubilized synaptosomes

The NMDAR and multiple associated signaling proteins are highly enriched in detergent-insoluble post-synaptic densities (Moon et al. 1994; Husi et al. 2000). As an initial step in determining the functional effects of PTPα upon NMDAR tyrosine phosphorylation, we investigated the physical localization of PTPα, NMDAR subunits and non-receptor tyrosine kinases that can regulate NMDAR phosphorylation, in detergent-fractionated synaptosomes. We also determined whether ablation of PTPα affected the detergent solubility or developmental expression of synaptosomal NMDAR subunits and non-receptor tyrosine kinases.

Crude synaptosomes (P2 fraction) were prepared from whole brains of WT and PTPα–/– mice and solubilized in 1% Triton X-100 (Fig. 1a). PTPα and the NR1, NR2A and NR2B subunits were detectable in both Triton X-100-soluble and -insoluble fractions prepared from animals at post-natal day 7 (P7) to day 60 (P60) ages. PTPα was expressed in the Triton X-100-soluble and -insoluble fractions at all ages. Increased expression of NR2A, NR2B, NR1 and PSD-95 in the Triton X-100-insoluble fractions was observed post-P7, consistent with other reports (Sheng et al. 1994; Petralia et al. 2005). Four (fyn, src, yes and lyn) of five SFKs that are reported to be expressed in the CNS (Thomas and Brugge 1997) were present in Triton X-100-soluble and -insoluble fractions of the synaptosomal preparations. The fifth CNS SFK, lck, was not reliably detected in these fractions, although, as described below, it could be immunoprecipitated from Triton X-100-insoluble synaptosomal fractions. Another tyrosine kinase, Pyk2, was present in both detergent-soluble and -insoluble fractions, with increasing expression detected in the latter fractions with age as reported (Menegon et al. 1999). We did not observe differences in NMDAR subunit, SFK or Pyk2 expression levels or distribution between WT and PTPα–/– mice of any age examined.


Figure 1.  Developmental expression of PTPα, NMDAR, PSD-95 and tyrosine kinases in detergent-fractionated crude synaptosomes of WT and PTPα-null mice. (a) Triton X-100-soluble and -insoluble fractions prepared from synaptosomes of WT (+/+) and PTPα–/– (–/–) mice at post-natal days 7, 14, 30 and 60 (P7–P60) were immunoprobed for developmental expression of the indicated proteins. The relative protein expressions within either the detergent-soluble or -insoluble samples can be compared, but not between detergent-soluble and -insoluble fractions because of unequal sample and loading amounts. (b) The relative protein expression between Triton X-100-soluble (S) and -insoluble (I) fractions was determined in P60 samples after equal sample fractionation and loading. Tr-R denotes transferrin receptor. The migration positions of molecular mass markers (kDa) are shown.

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The protein expression shown in Fig. 1(a) does not reflect the relative protein amounts in Triton X-100-soluble versus -insoluble fractions, as these two types of fractions were analyzed in amounts that enabled protein visualization rather than equivalency. To examine the relative expression levels in these fractions, P60 synaptosome preparations were fractionated into equal volumes of detergent-soluble and -insoluble material and equal volumes of these fractions assessed by immunoprobing (Fig. 1b). Comparatively low amounts of PTPα were present in the insoluble fraction (estimated at ∼ 2–5% of the total synaptosomal PTPα). Consistent with previous reports (Blahos and Wenthold 1996; Perez and Bredt 1998), the majorities of NR2A, NR2B, NR1 and PSD-95 were localized in the insoluble fraction in mature animals. More fyn and yes were found to be Triton X-100-insoluble, whereas more src, lyn and Pyk2 were localized in the soluble fraction.

Altered protein tyrosine phosphorylation in PTPα–/– synaptosomal fractions

The tyrosine phosphorylation status of proteins in crude synaptosomal fractions of WT and PTPα-deficient mouse brain was compared. The absence of PTPα resulted in reduced phosphorylation of Triton X-100-insoluble proteins that migrated at ∼ 180 and ∼ 120 kDa, with the former detectable from P14 onwards, and the latter from P30 onwards (Fig. 2, top right-hand panel). In contrast, in the same detergent-insoluble fractions the absence of PTPα also resulted in the enhanced tyrosine phosphorylation, readily observed from P14 onwards, of a protein(s) that migrated at ∼ 60 kDa (Fig. 2, top right-hand panel), about the same size as SFKs. Enhanced phosphorylation of a ∼ 60 kDa protein(s) was also detectable in Triton X-100-soluble fractions (Fig. 2, top left-hand panel), although no other PTPα-dependent changes in tyrosine phosphorylation were observed in these fractions.


Figure 2.  Phosphotyrosyl proteins and SFK tyrosine phosphorylation status in detergent-fractionated crude synaptosomes of WT and PTPα-null mice. Triton X-100-soluble and -insoluble fractions prepared from synaptosomes of WT (+/+) and PTPα–/– (–/–) mice at post-natal days 7, 14, 30 and 60 (P7–P60) were immunoprobed for protein tyrosine phosphorylation using anti-phosphotyrosine antibody (upper panels). The arrows indicate differences detected between WT and PTPα–/– samples. These fractions were also probed with phosphosite-specific antibodies to the autophosphorylation site phosphotyrosine residue of SFKs (anti-Src pTyr416) and to the inhibitory C-terminal tail region phosphotyrosine residue of SFKs (anti-Src pTyr527) (middle panels), and with anti-actin antibody (bottom panels). The migration positions of molecular mass markers (kDa) are shown on the left.

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Previous studies have demonstrated that brain src/fyn kinase activities in PTPα–/– mice are reduced to about half of those of WT animals (Ponniah et al. 1999; Su et al. 1999). This is concomitant with elevated phosphorylation of the regulatory C-terminal tyrosine residue of src (Tyr527) and fyn. Full SFK activation requires autophosphorylation at another tyrosine residue (Tyr416 in src) in the activation loop region (Brown and Cooper 1996; Xu et al. 1999). However, the phosphorylation status of brain src/fyn at this site in PTPα–/– brain has not been reported. Phosphospecific antibodies towards src phosphoTyr527 and phosphoTyr416 were used to determine which tyrosine residue of SFKs was hyperphosphorylated in the synaptosomal fractions. These antibodies recognize equivalent phosphotyrosyl residues in other SFKs such as fyn and yes (Reinehr et al. 2004). Both antibodies recognized a protein(s) that co-migrated with the antiphosphotyrosine-reactive ∼ 60 kDa protein(s). Phosphorylation at Tyr416 (or equivalent) did not differ in the Triton X-100-soluble and -insoluble fractions between WT and PTPα–/– animals at any age (Fig. 2, middle panels). However, phosphorylation at Tyr527 (or its equivalent tyrosine residue) was enhanced in the detergent-soluble and -insoluble fractions of PTPα–/– animals at the same ages as was the enhanced phosphorylation of the ∼ 60 kDa band(s) (Fig. 2, bottom panels). This indicates that the phosphotyrosyl 60 kDa band is likely to represent one or more SFKs, and that enhanced phosphorylation of one or more SFKs at the C-terminal tyrosine site is observed in the absence of PTPα. Equivalent actin amounts were present in the paired WT and PTPα–/– samples (Fig. 2), indicating that the observed changes in protein tyrosine phosphorylation were not due to unequal sample loading.

SFK hyperphosphorylation in PTPα–/– synaptosomes

To identify which SFKs exhibited enhanced C-terminal tyrosine phosphorylation in PTPα–/– synaptosomes, individual SFKs were immunoprecipitated and probed for phosphotyrosine. Fyn, src and yes displayed significantly increased tyrosine phosphorylation in Triton X-100-soluble (2.3-, 1.6- and 2.5-fold increases, respectively) and -insoluble (1.9-, 1.9- and 2.6-fold increases, respectively) fractions from PTPα–/– samples compared with WT samples (Figs 3a and c, and 4a). In accord with enhanced overall SFK C-terminal tyrosine phosphorylation detected in the synaptosomal fractions, this was confirmed to be due to enhanced phosphorylation at the C-terminal regulatory residue, as probing src or fyn immunoprecipitates with anti-src phosphoTyr416 antibody demonstrated no significant differences in activation loop tyrosine phosphorylation of these kinases in the PTPα–/– samples relative to that of the kinases from WT samples (Figs 3b and d). Although lck was not detected by probing the fractions with anti-lck antibody, low amounts of lck could be immunoprecipitated from Triton X-100-insoluble fractions, and the phosphotyrosine content of lck from PTPα–/– samples was 2-fold higher than that of lck from WT samples (Fig. 4b). In contrast to the altered tyrosine phosphorylation of fyn, src and yes, the lack of PTPα did not affect lyn tyrosine phosphorylation in either the Triton X-100-soluble or -insoluble fractions (Fig. 4c). Thus four SFKs (fyn, src, yes and lck) were hyperphosphorylated in PTPα–/–synaptosomal fractions, and the tyrosine phosphorylation status of a fifth SFK, lyn, remained unaltered upon PTPα ablation.


Figure 3.  Altered tyrosine phosphorylation of the synaptosomal SFKs src and fyn in mice lacking PTPα. Src and fyn were individually immunoprecipitated from Triton X-100-soluble (S) and -insoluble (I) fractions prepared from synaptosomes of WT (+/+) and PTPα–/– (–/–) mice at post-natal day 60. Fyn (a and b) and src (c and d) immunoprecipitates were probed for phosphotyrosine content (a and c, upper panels) or with a phosphospecific antibody that recognizes src phosphoTyr416 or the equivalent autophosphorylation site in fyn (b and d, upper panels), and with antibody to the specific kinase that was immunoprecipitated (lower panels). The bar charts to the right show the amount of phosphotyrosine per amount of immunoprecipitated kinase (± SD), as determined from densitometric scanning of the results of several independent experiments (n is shown in brackets above each chart). The gray bars represent WT (+/+) samples and are set at 1 unit, and the black bars represent PTPα–/– (–/–) samples with the units expressed relative to those of the WT samples. The asterisk denotes a significant difference (p ≤ 0.002) between WT and PTPα–/– samples.

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Figure 4.  Tyrosine phosphorylation of the synaptosomal SFKs yes, lck and lyn in mice lacking PTPα. Yes, lck and lyn were individually immunoprecipitated from Triton X-100-soluble (S) and -insoluble (I) fractions prepared from synaptosomes of WT (+/+) and PTPα–/– (–/–) mice at post-natal day 60. Yes (a), lck (b) and lyn (c) immunoprecipitates were probed for phosphotyrosine content (upper panels) and with antibody to the specific kinase that was immunoprecipitated (lower panels). The bar charts to the right show the amount of phosphotyrosine per amount of immunoprecipitated kinase (± SD), as determined from densitometric scanning (n = 3). The gray bars represent WT (+/+) samples and are set at 1 unit, and the black bars represent PTPα–/– (–/–) samples with the units expressed relative to those of the WT samples. The asterisks denote significant differences (*p ≤ 0.003; **p ≤ 0.01) between WT and PTPα–/– samples.

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Reduced NR2A and NR2B tyrosine phosphorylation in PTPα–/– synaptosomes

The NMDAR NR2A and NR2B subunits are SFK substrates (Tezuka et al. 1999; Cheung and Gurd 2001; Nakazawa et al. 2001; Yang and Leonard 2001). The ∼ 180 kDa phosphotyrosyl protein(s) detected in Triton X-100-insoluble synaptosomal fractions co-migrated with the NMDAR NR2A and NR2B subunits (data not shown). Furthermore, the reduced phosphorylation of the ∼180 kDa protein(s) that was apparent in PTPα–/– Triton X-100-insoluble fractions correlates with the reduced SFK activity expected upon the observed increased C-terminal tyrosine phosphorylation of SFKs. To ascertain if the tyrosine phosphorylation of these subunits was indeed altered by the ablation of PTPα, NR2A and NR2B were immunoprecipitated from the P14, P30 and P60 fractions and probed with anti-phosphotyrosine antibody. NR2A tyrosine phosphorylation was significantly reduced in all PTPα–/– samples compared with WT samples (Figs 5a and b). Similar reduced levels of NR2B tyrosine phosphorylation were detected in PTPα–/– samples (Figs 5c and d).


Figure 5.  Reduced tyrosine phosphorylation of NR2A and NR2B in synaptosomal fractions of PTPα–/– mice. (a) NR2A and (c) NR2B were immunoprecipitated from Triton X-100-insoluble fractions of WT (+/+) and PTPα–/– (–/–) mice at post-natal days 14, 30 and 60 (P14–P60), and probed for phosphotyrosine (top panels) and for NR2A or NR2B as shown (bottom panels). The results of several such experiments were quantified by densitometry and are shown in the bar charts in (b) (NR2A) and (d) (NR2B). The bars represent the amount of phosphotyrosine per amount of NR2A or NR2B (± SD). This ratio was set at 100% for WT samples (gray bars) at each age, and the ratio from PTPα–/– samples (black bars) is expressed as a relative percentage. The number of paired samples analyzed is shown in brackets above the bars. Asterisks depict significant differences between the WT and PTPα–/– samples (*p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0005).

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Pyk2 autophosphorylation is reduced in synaptosomes lacking PTPα

Pyk2 is a component of the NMDAR complex, and can function upstream of src to up-regulate NMDAR function (Husi et al. 2000; Huang et al. 2001; Liu et al. 2001; Seabold et al. 2003). The autophosphorylation of Pyk2 at Tyr402, and src or fyn binding to this site, correlates with Pyk2 activation (Dikic et al. 1996; Sieg et al. 1998; Li et al. 1999; Huang et al. 2001). We found that Pyk2 Tyr402 phosphorylation was reduced in Triton X-100-soluble synaptosomal fractions prepared from P7–P60 PTPα–/– mice (Fig. 6a). Quantification of phospho-Tyr402 of immunoprecipitated Pyk2 from adult (P60) mouse synaptosomes demonstrated that ablation of PTPα resulted in about a 40% reduction in phosphorylation (Fig. 6b). The low level of Pyk2 in Triton X-100-insoluble fractions prevented us from accurately determining the Tyr402 phosphorylation status of Pyk2 in these fractions. To determine if increased PTPα activity promoted Pyk2 activation, HEK293 cells were transfected with Pyk2 alone, with fyn, or with fyn and PTPα. Co-expressed PTPα and/or fyn did not increase the phosphorylation of transfected Pyk2 at Tyr402, perhaps because of endogenous cellular factors or conditions promoting maximal or near-maximal autophosphorylation of Pyk2 at this site (Fig. 6c). However, co-transfected fyn was detected in association with Pyk2, and the co-expression of PTPα further enhanced the association of fyn with Pyk2 by about 2-fold (Figs 6c and d). The above results indicate that PTPα may positively regulate properties of Pyk2, such as Tyr402 autophosphorylation and SFK binding, that are associated with Pyk2 activation.


Figure 6.  Pyk2 autophosphorylation and association with fyn are altered by ablated or increased PTPα expression. Pyk2 was immunoprecipitated from Triton X-100-soluble fractions of synaptosomes prepared from WT (+/+) and PTPα–/– (–/–) mice at post-natal days 7, 14, 30 and 60 (P7–P60). (a) The immunoprecipitates were probed for phosphoTyr402 (top panel) and for Pyk2 amount (bottom panel). (b) The results of six such immunoprecipitations from paired P60 samples were quantified by densitometry. The bars represent the amount of autophosphorylation per amount of Pyk2 (± SD). This ratio is expressed as 100% for the WT samples and the ratio from PTPα–/– samples is expressed as a relative percentage. (c) HEK293 cells were transiently co-transfected with plasmids expressing Pyk2, fyn and/or PTPα as shown. Pyk2 immunoprecipitates were probed for Pyk2 phosphorylation at Tyr402, for Pyk2 amount, and for associated fyn (the top three panels). Cell lysates were also probed for fyn and PTPα amounts (the bottom two panels). (d) The amount of fyn associated with Pyk2 in the absence or presence of co-expressed PTPα in experiments as in (c) (n = 4) was quantified by densitometry. The bars represent the amount of fyn per unit of Pyk2 in arbitrary units (± SD). Asterisks in (b) and (d) depict significant differences between samples with or without PTPα (*p ≤ 0.002; **p ≤ 0.0004). IP, immunoprecipitation; IB, immunoblotting.

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PTPα enhances fyn-mediated phosphorylation of NR2A in HEK293 cells

The ability of PTPα to regulate SFK-mediated NMDAR phosphorylation has never been directly demonstrated. The HEK293 cell line lacking endogenous NMDAR has been shown in many studies to be a useful system in which to reconstitute NMDAR phosphorylation and signaling pathways (Tezuka et al. 1999; Nakazawa et al. 2001; Yang and Leonard 2001). We therefore transiently transfected these cells with NR2A or NR2B, fyn and PTPα, and examined the tyrosine phosphorylation of immunoprecipitated NR2A/2B. Virtually no tyrosine phosphorylation of NR2A was detected in cells expressing NR2A alone or with PTPα(Fig. 7a). The co-expression of fyn with NR2A resulted in NR2A phosphorylation, and this was further enhanced by the expression of PTPα (Fig. 7a). Similar results were obtained when cells were transfected with NR2B, fyn and PTPα (Fig. 7d). PTPα activated fyn (Fig. 7c), and promoted a fyn-dependent 2.6- to 2.8-fold increase in NR2A/2B tyrosine phosphorylation over that catalyzed by fyn alone (Figs 7b and e). Consistent with the finding of a previous study (Tezuka et al. 1999), the scaffolding protein PSD-95 enhanced fyn-mediated phosphorylation of NR2B (Fig. 7f). The expression of PSD-95 in our transfected cells increased fyn-mediated NR2B tyrosine phosphorylation by about 6-fold (6.0 ± 0.30). The co-expression of PTPα with NR2B, fyn and PSD-95 further increased NR2B phosphorylation (Fig. 7f) (to 13.0 ± 3.67-fold over fyn alone). Thus PTPα effects a 2–3-fold enhancement of fyn-mediated NR2B phosphorylation whether in the presence or absence of PSD-95.


Figure 7.  PTPα enhances fyn-mediated NR2A and NR2B tyrosine phosphorylation. HEK293 cells were transiently co-transfected with plasmids expressing NR2A [(a)-(c)] or NR2B [ (d)- (f)], fyn, PTPα and/or PSD-95 as shown. (a) , (d), (f) NR2A or NR2B was immunoprecipitated from cell lysates and analyzed for phosphotyrosine content (top panels), NR2A or NR2B amount (second panels), fyn or PSD-95 expression (third panels), and PTPα expression (bottom panels). (b) , (e) The results of three independent experiments as in (a) and (d) were quantified by densitometric scanning. The bars represent NR2A or NR2B tyrosine phosphorylation per amount of NR2A or NR2B (arbitrary units ± SD) in cells co-expressing fyn with or without PTPα. (c) Fyn was immunoprecipitated from lysates of HEK293 cells co-expressing NR2A and with or without co-expressed PTPα, and assayed for kinase activity in an in vitro assay. Fyn autophosphorylation and phosphorylation of the exogenous fyn substrate enolase are shown in the top panel. Portions of the immunoprecipitates were probed for fyn amount (bottom panel).

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Lynn Raymond and Alaa El-Husseini (University of British Columbia) for their respective gifts of NR2 subunit and PSD-95 expression plasmids, and Michael Schaller (University of North Carolina) and Michael Gold (University of British Columbia) for the gift of Pyk2 expression plasmid. HTL was the recipient of a Child & Family Research Institute (CFRI) Studentship. LM was the recipient of a Bertram Hoffmeister Fellowship from the CFRI. CJP holds a CFRI Investigatorship Award. This work was supported by a grant from the Canadian Institutes of Health Research (CJP).


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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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