PSD-95 is a negative regulator of the tyrosine kinase Src in the NMDA receptor complex
2006; Springer Nature; Volume: 25; Issue: 20 Linguagem: Inglês
10.1038/sj.emboj.7601342
ISSN1460-2075
AutoresLorraine V. Kalia, Graham M. Pitcher, Kenneth A. Pelkey, Michael W. Salter,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoArticle21 September 2006free access PSD-95 is a negative regulator of the tyrosine kinase Src in the NMDA receptor complex Lorraine V Kalia Lorraine V Kalia Division of Neurology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada Search for more papers by this author Graham M Pitcher Graham M Pitcher Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada Department of Physiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Kenneth A Pelkey Kenneth A Pelkey Laboratory of Cellular and Synaptic Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Michael W Salter Corresponding Author Michael W Salter Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada Department of Physiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Lorraine V Kalia Lorraine V Kalia Division of Neurology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada Search for more papers by this author Graham M Pitcher Graham M Pitcher Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada Department of Physiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Kenneth A Pelkey Kenneth A Pelkey Laboratory of Cellular and Synaptic Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Michael W Salter Corresponding Author Michael W Salter Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada Department of Physiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Author Information Lorraine V Kalia1,2, Graham M Pitcher2,3, Kenneth A Pelkey4 and Michael W Salter 2,3 1Division of Neurology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada 2Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada 3Department of Physiology, University of Toronto, Toronto, Ontario, Canada 4Laboratory of Cellular and Synaptic Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA *Corresponding author. Program in Neurosciences and Mental Health, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Tel.: +1 416 813 6272; Fax: +1 416 813 7921; E-mail: [email protected] The EMBO Journal (2006)25:4971-4982https://doi.org/10.1038/sj.emboj.7601342 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The tyrosine kinase Src upregulates the activity of the N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) and tyrosine phosphorylation of this receptor is critical for induction of NMDAR-dependent plasticity of synaptic transmission. A binding partner for Src within the NMDAR complex is the protein PSD-95. Here we demonstrate an interaction of PSD-95 with Src that does not require the well-characterized domains of PSD-95. Rather, we show binding to Src through a 12-amino-acid sequence in the N-terminal region of PSD-95, a region not previously known to participate in protein–protein interactions. This region interacts directly with the Src SH2 domain. Contrary to typical SH2 domain binding, the PSD-95–Src SH2 domain interaction is phosphotyrosine-independent. Binding of the Src-interacting region of PSD-95 inhibits Src kinase activity and reduces NMDAR phosphorylation. Intracellularly administering a peptide matching the Src SH2 domain-interacting region of PSD-95 depresses NMDAR currents in cultured neurons and inhibits induction of long-term potentiation in hippocampus. Thus, the PSD-95–Src SH2 domain interaction suppresses Src-mediated NMDAR upregulation, a finding that may be of broad importance for synaptic transmission and plasticity. Introduction The non-receptor protein tyrosine kinase Src is expressed widely throughout the central nervous system (CNS) and is present at high levels in neurons of the brain and spinal cord. A major function of Src in the adult CNS is to regulate glutamatergic transmission and synaptic plasticity (Salter and Kalia, 2004). Src acts postsynaptically at glutamatergic synapses to upregulate the activity of the N-methyl-D-aspartate receptor (NMDAR) (Yu et al, 1997), a principal subtype of glutamate receptor crucial for neuronal development, neuroplasticity and excitotoxicity (Dingledine et al, 1999). An increasing body of evidence indicates that Src serves as a point of convergence through which a number of signalling pathways modulate NMDAR activity. These pathways involve G-protein-coupled receptors, Ras, cytokine receptors, receptor protein tyrosine kinases, integrins, insulin and lipoprotein receptors (Salter and Kalia, 2004; Macdonald et al, 2005; Chen et al, 2005; Jones and Leonard, 2005). By upregulating the activity of NMDARs at Schaffer collateral–CA1 synapses within the hippocampus, Src functions as an intermediary in the induction of long-term potentiation (LTP) (Lu et al, 1998), a form of lasting enhancement of synaptic transmission and the predominant cellular model of learning and memory (Bliss and Collingridge, 1993). Tyrosine phosphorylation of the NMDAR subunit protein NR2B, the most prominent tyrosine phosphorylated protein within postsynaptic densities at glutamatergic synapses (Moon et al, 1994), has been found to increase in LTP in CA1 hippocampus (Nakazawa et al, 2001) and dentate gyrus (Rostas et al, 1996). Tyrosine phosphorylation of NR2B subunits has also been shown to increase in several pathological processes in the CNS, including ischemia, seizure and hyperalgesia (Salter and Kalia, 2004). Thus, the tyrosine kinase Src has emerged as a key regulator of NMDAR function and thereby of excitatory synaptic transmission, plasticity and pathophysiology. NMDARs are multiprotein complexes comprised of the core NMDAR subunit proteins, which form the ion permeation pathway (Dingledine et al, 1999), together with a variety of molecular scaffolds and signalling enzymes (Husi et al, 2000). Src has been identified as a component of the NMDAR complex (Yu et al, 1997; Husi et al, 2000). We have discovered two proteins within the complex that interact with Src: ND2 (Gingrich et al, 2004) and PSD-95 (Kalia and Salter, 2003). For ND2, it has been determined that a region within its C-terminal domain interacts with the unique domain of Src and that ND2 functions to anchor Src within the NMDAR complex (Gingrich et al, 2004). By contrast, it is unknown how PSD-95 interacts with Src and moreover, the function of this interaction remains obscure. Therefore, here we sought to characterize the interacting regions in PSD-95 and Src, and to determine the functional consequences of this interaction. Results SH2 domain of Src interacts with the N-terminal region of PSD-95 We explored the region of Src involved in the association with PSD-95 by testing GST fusion proteins of three main regions of Src: the unique domain, SH3 domain and SH2 domain (Brown and Cooper, 1996; Thomas and Brugge, 1997) (Figure 1A). We found that the Src SH2 domain GST fusion protein pulled down PSD-95 from rat forebrain extracts (Figure 1B). In contrast, PSD-95 was not pulled down by the other GST fusion proteins or by GST alone. Thus, PSD-95 interacts with the SH2 domain of Src but not the Src unique domain or Src SH3 domain. Figure 1.PSD-95 interacts with the SH2 domain of Src. (A) Illustration of GST fusion proteins of Src unique domain, Src SH3 domain and Src SH2 domain. (B) Pull-down assays using rat forebrain extracts (600 μg) with GST fusion proteins shown in panel A. Proteins were probed with anti-PSD-95 (upper). Input was 60 μg of protein from brain extracts. GST fusion proteins were stained with Coomassie blue (lower). Molecular weight markers are indicated on left. Results are representative of five experiments. Download figure Download PowerPoint We therefore next determined the region of PSD-95 that interacts with the Src SH2 domain. PSD-95 is the lead member of the PSD-95 family of membrane-associated guanylate kinase (MAGUK) proteins (Cho et al, 1992). The domain structure of PSD-95 includes three PDZ domains, one SH3 domain and a guanylate kinase homology (GuK) domain (Figure 2A), each of which have been found to mediate protein–protein interactions (Kim and Sheng, 2004). To investigate the association of PSD-95 with the Src SH2 domain, we used a series of PSD-95 deletion constructs that were individually expressed in HEK293 cells. Each of the deletion constructs lacked one or more of the known protein–protein interaction modules of PSD-95 (Figure 2A). We found that the Src SH2 domain GST fusion protein pulled down PSD-95 from lysates of HEK293 cells transfected with wild-type PSD-95 (Figure 2B). The Src SH2 domain GST fusion protein also pulled down the PSD-95 deletion proteins lacking the PDZ1 and PDZ2 domains (ΔPDZ1/2), the PDZ3 domain (ΔPDZ3) or the SH3 and GuK domains (ΔSH3/GuK). In contrast, GST alone did not pull down any of the PSD-95 proteins (Figure 2B). Thus, removal of each of the known protein–protein interaction domains of PSD-95 failed to eliminate the interaction with the Src SH2 domain. Figure 2.Src SH2 domain binds directly to the N-terminal region of PSD-95. (A) Illustration of PSD-95 deletion constructs. (B) Pull-down assays were performed using lysates (300 μg) of HEK293 cells transfected with constructs shown in panel A. Proteins that associated with GST alone or GST-Src SH2 were probed with anti-PSD-95. Input was 30 μg of protein from cell lysates. Arrows indicate positions of corresponding PSD-95 proteins. Results are representative of five experiments. (C) GST alone or GST-Src SH2 was incubated with the PSD-95(1–54) peptide (1.0 μM). PSD-95(1–54) that bound was probed with an anti-PSD-95 antibody raised against aa 18–32 of PSD-95. Input was 10% of starting material. (D) Left: densitometric quantification from three experiments, one of which is represented in panel C. Bars correspond to mean (±s.e.m.) normalized to maximum gray value for each experiment. Right: modified ELISAs to measure pull-down of GST-Src SH2 domain by PSD-95(1–54). Bars correspond to mean (±s.e.m.) normalized to maximum absorbance reading for each experiment and are representative of three experiments. Download figure Download PowerPoint The only region common to all of the PSD-95 deletion mutants was amino acids 1–54. Because we had observed previously that ΔSH3/GuK PSD-95 is sufficient to bind to Src (Kalia and Salter, 2003), we used ΔSH3/GuK PSD-95 to determine whether amino acids 1–54 are necessary for binding to the Src SH2 domain. To this end, we made a protein (Δ(1–54)/SH3/GuK) in which these residues were deleted from the ΔSH3/GuK PSD-95 construct. We found that the Δ(1–54)/SH3/GuK PSD-95 protein was not pulled down by GST-Src SH2 (Figure 2B), implying that amino acids 1–54 are required for binding to the Src SH2 domain. We previously observed that the SH3 domain of PSD-95 may also interact with Src (Kalia and Salter, 2003), but here we found that the PSD-95 SH3 domain does not interact with the SH2 domain of Src (data not shown). Thus, the region of amino acids 1–54 of PSD-95, but not the PSD-95 SH3 domain, may mediate binding to the SH2 domain of Src. Therefore, for the remainder of the present study, we focused on this region of PSD-95. To determine whether the region of amino acids 1–54 of PSD-95 interacts directly with the Src SH2 domain, we tested the binding of the Src SH2 domain GST fusion protein and PSD-95(1–54) peptide, which had been cleaved from GST. We found that GST-Src SH2 domain interacted with PSD-95(1–54) in pull-down assays (Figure 2C and D, left) and conversely, that PSD-95(1–54) bound to GST-Src SH2 domain in modified ELISAs (Figure 2D, right). The binding of PSD-95(1–54) was detectable at peptide concentrations ⩾0.1 μM, and the amount of PSD-95(1–54) that was pulled down increased with increasing concentrations of the peptide up to 2.0 μM, the maximum concentration used. The GST-Src SH2 domain interacted with PSD-95(1–54) at all concentrations of the Src SH2 domain tested (0.625–2.5 μM) in modified ELISAs. Taken together, these results imply that PSD-95(1–54) directly interacts with the SH2 domain of Src and that the interaction occurs at low micromolar concentrations of either protein. We next determined whether PSD-95(1–54) was sufficient to interact with Src (Figure 3). We found that the PSD-95(1–54) GST fusion protein pulled down recombinant Src as well as Src from rat forebrain extracts. In contrast, GST alone, GST-PSD-95 PDZ1 or GST-PSD-95 PDZ2 did not pull down Src. To ensure that the purified recombinant PDZ domains were in their native conformations and hence should have interacted with PDZ ligands, we probed the proteins pulled down by the PDZ domain proteins for NR2A/B subunits of the NMDAR, which have been shown to directly bind to the PDZ1 and PDZ2 domains of PSD-95 (Kornau et al, 1995). We found that GST-PSD-95 PDZ1 and GST-PSD-95 PDZ2 pulled down NR2A/B subunits. Conversely, neither GST alone nor GST-PSD-95(1–54) pulled down NR2A/B. From these results, we conclude that the region comprising the first 54 amino acids of PSD-95 is a protein–protein interaction module in PSD-95. This is a region of PSD-95 not previously suspected as mediating interactions with other proteins. Figure 3.N-terminal region of PSD-95 is sufficient for the interaction with Src. (A) Illustration of GST fusion proteins of aa 1–54, PDZ1 domain and PDZ2 domain of PSD-95. (B) Pull-down assays with full-length recombinant Src and GST fusion proteins illustrated in panel A. Src that bound was probed with anti-Src (upper). Pull-down assays from rat forebrain extracts (600 μg) with the GST fusion proteins were performed. Membrane was probed with anti-Src, stripped and then re-probed with anti-NR2A/B (middle). The amount of NR2A/B pulled down by PDZ2 was greater than that by PDZ1 as expected from the reported differences in affinities (Lim et al, 2002). Input was 20% of starting material. GST fusion proteins were stained with Coomassie blue (lower). Molecular weight markers are shown on left. Similar results were seen in each of three experiments. Download figure Download PowerPoint Interaction between the Src SH2 domain and N-terminal region of PSD-95 is phosphotyrosine-independent The SH2 domain of Src and SH2 domains of other signalling molecules typically bind ligands containing phosphorylated tyrosine residues (Songyang et al, 1993). The sequence of amino acids 1–54 of PSD-95 contains three tyrosine residues but none is contained in a consensus phosphorylation sequence according to a peptide library-based searching algorithm (Yaffe et al, 2001). The GST fusion proteins and PSD-95(1–54) peptide used in the experiments described above were expressed in Escherichia coli BL21 cells that do not express protein tyrosine kinases and consequently, were not phosphorylated on tyrosine (confirmed by immunoblotting with anti-phosphotyrosine (anti-pY) antibody; data not shown). It has been reported that Src does not phosphorylate PSD-95 in vitro (Nada et al, 2003) and that PSD-95 lacks tyrosine phosphorylation, as detected by immunoblotting (Tezuka et al, 1999; Nada et al, 2003). To test whether PSD-95 from rat brain extracts used in the present study was tyrosine phosphorylated, we immunoprecipitated PSD-95 and then probed with anti-pY (Figure 4A). Tyrosine phosphorylation of PSD-95 from the extracts was not detected. Thus, we inferred that the pull-down of PSD-95 by GST-Src SH2 as well as the in vitro interaction between the PSD-95(1–54) peptide and Src SH2 domain occurred without tyrosine phosphorylation of the interacting region in PSD-95. Figure 4.Src SH2 domain binding does not require tyrosine phosphorylation of the N-terminal region of PSD-95. (A) Immunoprecipitations from PSD fractions (100 μg) with anti-PSD-95. Immunoprecipitants were sequentially probed with a different anti-PSD-95 antibody (left) and anti-pY (right). A band corresponding to PSD-95 was not detectable with anti-pY even following longer exposures (data not shown). Input was 8 μg of PSD proteins. Molecular weight markers are shown on right. Similar results were observed in three separate experiments. (B) Upper: pull-down assays with GST-PSD-95(1–54) and recombinant Src preincubated with no peptide, EPQYEEIPIA or EPQ(pY)EEIPIA (1 mM). Src that remained bound to GST-PSD-95(1–54) was probed with anti-Src. Middle: pull-down assays from rat forebrain extracts (600 μg) with GST-Src SH2 preincubated with no peptide, EPQYEEIPIA or EPQ(pY)EEIPIA (1 mM). Proteins that remained associated with GST-Src SH2 were probed with anti-PSD-95. Lower: immunoprecipitations with anti-Src from lysates of HEK293 cells (300 μg) cotransfected with PSD-95 and Src, and incubated with no peptide, EPQYEEIPIA or EPQ(pY)EEIPIA (1 mM). Proteins were probed with anti-PSD-95. Similar results were found in each of three experiments. (C) Immunoprecipitations with anti-Src from lysates of HEK293 cells (300 μg) transfected with PSD-95 and no Src (upper), wild-type Src (middle) or R175K mutant Src (lower). Immunoprecipitants were probed with anti-PSD-95. Input was 30 μg of cell lysate proteins. Similar results were found in each of three experiments. (D) Pull-down assays from forebrain extracts (600 μg) with GST alone, GST-Src SH2 or GST-R175K Src SH2. Proteins were sequentially probed with anti-pY (upper) and anti-PSD-95 (lower). Molecular weight markers are indicated on right. Results are representative of three experiments. Download figure Download PowerPoint Nevertheless, it is possible that binding of PSD-95 to the Src SH2 domain might depend upon the same binding surface in the Src SH2 domain as that used to bind phosphotyrosine ligands. This possibility was tested in a series of experiments using a phosphotyrosine peptide, EPQ(pY)EEIPIA. This peptide is a high-affinity ligand for the Src SH2 domain (Songyang et al, 1993) and prevents binding of other ligands to the domain (Gilmer et al, 1994). We performed three experiments with this peptide: in vitro pull-down assays with GST-PSD-95(1–54) and recombinant Src (Figure 4B, upper); pull-down assays with GST-Src SH2 domain and forebrain extracts (Figure 4B, middle); and co-immunoprecipitations using lysates from HEK293 cells transiently cotransfected with Src and PSD-95 (Figure 4B, lower). In all three experiments, we found that including EPQ(pY)EEIPIA eliminated the Src–PSD-95 interaction. But when we included no peptide or the non-phosphorylated peptide, EPQYEEIPIA, which does not bind to the Src SH2 domain, the Src–PSD-95 interaction was not affected. The results from this series of experiments demonstrate that the EPQ(pY)EEIPIA peptide precludes the binding between Src and PSD-95, indicating that the Src SH2 domain cannot simultaneously bind a phosphotyrosine ligand and PSD-95(1–54). Within the binding surface of the Src SH2 domain, the arginine residue at amino-acid position 175 (R175) is involved in an ion-pairing interaction with the phosphate group on tyrosine in the ligand (Waksman et al, 1993). Mutation of this critical arginine residue to lysine (R175K) has been shown to reduce binding of tyrosine phosphorylated peptides to less than 10% of that of wild-type Src SH2 domain (Bibbins et al, 1993). Because binding of PSD-95 to the Src SH2 domain does not require tyrosine phosphorylation of PSD-95, we wondered whether binding might also not require R175 in the SH2 domain. We expressed Src with or without the R175K mutation together with PSD-95 and found that the R175K mutation did not reduce the amount of PSD-95 co-immunoprecipitated with Src (Figure 4C). In addition, we made a GST fusion protein of the R175K Src SH2 domain. We found that the amount of tyrosine phosphorylated proteins pulled down by GST-R175K Src SH2 domain was dramatically less than that pulled down by wild-type Src SH2 domain (Figure 4D, upper), confirming that this mutation had greatly reduced the interaction of the Src SH2 domain with tyrosine phosphorylated proteins. In contrast to the decrease in tyrosine phosphorylated proteins pulled down by GST-R175K Src SH2, the amount of PSD-95 pulled down by this mutant protein was not reduced (Figure 4D, lower). Together these findings indicate that, although PSD-95 is not tyrosine phosphorylated, binding of PSD-95 to the SH2 domain of Src depends upon the binding surface of the Src SH2 domain which is involved in interactions with phosphotyrosine-containing peptides. However, R175 within the Src SH2 domain which mediates binding to phosphotyrosine is not required for the Src SH2 domain–PSD-95 interaction. Deletion of the N-terminal region of PSD-95 enhances Src-mediated tyrosine phosphorylation of NMDAR subunits To begin to investigate the functional consequences of the interaction between the Src SH2 domain and N-terminal region of PSD-95, we determined whether this region of PSD-95 regulates the phosphorylation of the NR2B subunit of the NMDAR, a major target of Src (Cheung and Gurd, 2001). NR2B binds directly to PSD-95 via the PDZ1 or PDZ2 domain of PSD-95 (Kornau et al, 1995). Therefore, we reconstituted the NMDAR–PSD-95 complex and expressed recombinant NR1/NR2B NMDARs together with a constitutively active form of Src (Y527F Src) (Brown and Cooper, 1996) in HEK293 cells. Without coexpressing Y527F Src, tyrosine phosphorylation of NR2B was not detectable (data not shown). To test the effect of the N-terminal region of PSD-95 on Src-mediated tyrosine phosphorylation of NR2B, we compared the level of NR2B tyrosine phosphorylation when wild-type PSD-95 was coexpressed versus when a mutant PSD-95 lacking amino acids 14–54 (Δ(14–54) PSD-95) was coexpressed. In the mutant PSD-95, we retained the first 13 amino acids of PSD-95, as they contain the consensus sequence for palmitoylation required for normal trafficking of PSD-95 to the plasma membrane (El Husseini et al, 2000). We found that the tyrosine phosphorylation level of NR2B was enhanced by 1.9-fold when NMDARs were coexpressed with Δ(14–54) PSD-95 as compared with wild-type PSD-95 (Figure 5A). Tyrosine phosphorylation of NR2B was dependent on Src kinase activity because NR2B phosphorylation was undetectable when a catalytically inactive form of Src (K295R Src) (Kamps and Sefton, 1986) was expressed instead of constitutively active Y527F Src. As with NR2B, we found that Src-mediated tyrosine phosphorylation of NR2A was enhanced by Δ(14–54) PSD-95 as compared with wild-type PSD-95 (Supplementary Figure S1). Figure 5.Deleting the N-terminal region of PSD-95 increases Src-mediated NMDAR tyrosine phosphorylation. (A) Upper: immunoprecipitations with anti-NR2 from lysates of HEK293 cells (300 μg) cotransfected with NR1/2B, Y527F Src or K295R Src, and wild-type (WT) PSD-95 or Δ(14–54) PSD-95. Immunoprecipitated NR2B was sequentially probed with anti-pY and anti-NR2B. Blots shown are representative of three separate experiments. Lower: densitometric quantification from three experiments, one of which is represented above. Band intensity was quantitated as mean gray value and the ratio of pY NR2B to total NR2B was calculated. Bars correspond to mean (±s.e.m.) ratios normalized to ratios obtained for cotransfection of NR1/2B with Y527F Src and wild-type PSD-95. (*P<0.0005, t-test versus Y527F Src and wild-type PSD-95). Inset: representative 5-point standard curve (R2=0.99, P 0.4, t-test versus WT PSD-95). (C) Upper: immunoprecipitations with a pan anti-Src antibody from lysates of HEK293 cells (300 μg) transfected with Y527F Src plus no PSD-95, WT PSD-95 or Δ(14–54) PSD-95. Immunoprecipitated Src was immunoblotted with anti-pY416 Src (upper) and anti-Src (lower). Input was 30 μg of proteins from cells cotransfected with Y527F Src and Δ(14–54) PSD-95. Positions of IgG heavy chains are indicated. Results are representative of three separate experiments. Lower: densitometric analysis from three experiments, one of which is represented above. Bars correspond to mean (±s.e.m.) band intensity, which was quantitated as mean gray value (*P<0.01, t-test versus no PSD-95 or versus Δ(14–54) PSD-95). Download figure Download PowerPoint We found that protein expression of Y527F Src and NR2B in cells expressing wild-type PSD-95 was not significantly different from that in cells expressing Δ(14–54) PSD-95 (Figure 5B). Thus, there were no significant changes in the relative amounts of the kinase or its substrate that could have accounted for the difference in NR2B phosphorylation. Another potential explanation for the lower level of NR2B phosphorylation in cells expressing wild-type PSD-95 might have been that less Y527F Src was associated with the NMDAR complex in these cells. However, we found that the amount of Y527F Src co-immunoprecipitating with anti-NR2B when wild-type PSD-95 was coexpressed was not different from that when Δ(14–54) PSD-95 was coexpressed (Figure 5B). Thus, deleting the 14–54 region of PSD-95 did not alter the association of Y527F Src with the NMDAR complex. To determine whether the 14–54 region of PSD-95 affects the catalytic activity of Src, we expressed in HEK293 cells constitutively active Y527F Src, alone or together with wild-type PSD-95 or Δ(14–54) PSD-95. We reasoned that, if the interaction of Src with the 14–54 region of PSD-95 suppresses Src catalytic activity, the coexpression of wild-type PSD-95 but not Δ(14–54) PSD-95 should suppress Src activity. We assessed the level of Src activation by using an antibody that recognizes an active form of Src in which tyrosine 416 (Y416) of Src is phosphorylated. Y416 is located within the activation loop of the kinase and phosphorylation of this residue is required for full activity of Src. Therefore, the level of phosphorylated Y416 (pY416) is a surrogate marker of Src tyrosine kinase activity (Brown and Cooper, 1996; Thomas and Brugge, 1997). We immunoprecipitated Src from the cell lysates with a pan anti-Src antibody, which recognizes both Y416 and pY416 forms of Src. By probing with an antibody that recognizes pY416, but not Y416, we found that the degree of Src activation was reduced by coexpressing with wild-type PSD-95 versus no PSD-95 (Figure 5C). In contrast, Src activity was not affected by coexpressing Δ(14–54) PSD-95. The total amount of Y527F Src immunoprecipitated was not affected by coexpression of either PSD-95 or Δ(14–54) PSD-95. Thus, these results indicate that PSD-95 suppresses Src activity and that this suppression depends upon the 14–54 region of PSD-95. Furthermore, these results imply that the increase in Src-mediated tyrosine phosphorylation of NR2B by Δ(14–54) PSD-95, compared with wild-type PSD-95, is due to removal of the suppression of Src catalytic activity. PSD-95(43–57) binds directly to the SH2 domain and suppresses the tyrosine kinase activity of Src The above findings imply that there is a stretch of amino acids N-terminal to the first PDZ domain of PSD-95 that depresses the catalytic activity of Src. To map this stretch, we used a partially overlapping series of four 15-mer synthetic peptides. In order that each of these peptides was of the same length, the series collectively spanned amino acids 6–57 of PSD-95 (Figure 6A). The peptides were synthesized without phosphoamino acids. We tested the peptides individually for their ability to disrupt the direct interaction between the PSD-95(1–54) GST fusion protein and recombinant Src in vitro. We observed that when Src was preincubated with the PSD-95(43–57) peptide, the pull-down of Src by GST-PSD-95(1–54) was eliminated (Figure 6B, left). However, preincubating with each of the other three PSD-95 peptides did not reduce the amount of Src that interacted with GST-PSD-95(1–54). The inhibition of the Src–PSD-95(1–54) interaction by the PSD-95(43–57) peptide was concentration-dependent (Figure 6B, right). Moreover, we found that the Src SH2 domain GST fusion protein bound directly to the PSD-95(43–57) peptide (Figure 6C). The amount of GST-Src SH2 that bound to the other PSD-95 peptides was not different from the background binding level in these experiments. Therefore, we concluded that the PSD-95(43–57) peptide directly interacts with the Src SH2 domain. Figure 6.PSD-95(43–57) peptide suppresse
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