Research Paper

Activity-dependent dephosphorylation of paxillin contributed to nociceptive plasticity in spinal cord dorsal horn Xin-Tai Wang, Rui Zheng, Zhan-Wei Suo, Yan-Ni Liu, Zi-Yang Zhang, Zheng-An Ma, Ye Xue, Man Xue, Xian Yang, Xiao-Dong Hu*

Abstract The enzymatic activity of protein tyrosine kinase Src is subjected to the regulation by C-terminal Src kinase (CSK) and protein tyrosine phosphatases (PTPs). Aberrant Src activation in the spinal cord dorsal horn is pivotal for the induction and development of nociceptive behavioral sensitization. In this study, we found that paxillin, one of the well-characterized cell adhesion components involved in cell migration and survival, integrated CSK and PTPs’ signaling to regulate Src-dependent nociceptive plasticity. Paxillin localized at excitatory glutamatergic synapses in the spinal dorsal horn of mice, and the phosphorylation of Tyr118 on paxillin was necessary to associate with and target CSK at synapses. After peripheral tissue injury, the enhanced neuronal activity stimulated N-methyl-D-aspartate (NMDA) subtype glutamate receptors, which initiated PTPs’ signaling to catalyze Tyr118 dephosphorylation. The reduced Tyr118 phosphorylation disrupted paxillin interaction with CSK, leading to the dispersal of CSK out of synapses. With the loss of CSK-mediated inhibition, Src activity was persistently increased. The active Src potentiated the synaptic transmission specifically mediated by GluN2B subunit–containing NMDA receptors. The active Src also facilitated the induction of long-term potentiation of C fiber–evoked field potentials and exaggerated painful responses. In complete Freund’s adjuvant–injected mice, viral expression of phosphomimicking paxillin mutant to resume CSK synaptic localization repressed Src hyperactivity. Meanwhile, this phosphomimicking paxillin mutant blunted NMDA receptor–mediated synaptic transmission and alleviated chronic inflammatory pain. These data showed that PTPs-mediated dephosphorylation of paxillin at Tyr118 was involved in the modification of nociceptive plasticity through CSK-Src signaling. Keywords: Paxillin, C-terminal Src kinase, Protein tyrosine phosphatases, Synaptic plasticity, Pain

1. Introduction Peripheral tissue and nerve injuries activate Src in the spinal cord dorsal horn, which catalyzes the tyrosine phosphorylation of a line of substrates to sensitize nociceptive behaviors.10,18,25 There are 2 important regulatory residues on Src: one within its activation loop (Tyr418) and the other at the extreme carboxyl-terminal tail (Tyr529).43 The phosphorylated Tyr529 interacts intramolecularly with Src homology 2 (SH2) domain to maintain Src in a closed and inactive conformation. When Tyr529 is dephosphorylated by protein tyrosine phosphatases (PTPs), this intramolecular interaction is disrupted, allowing the subsequent Tyr418 autophosphorylation and Src activation.43 In several brain regions, the signaling propagation from PTPs to Src plays an important role in the modification of synaptic transmission and plasticity.22,32,50

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article. Department of Molecular Pharmacology, School of Pharmacy, Lanzhou University, Lanzhou, China *Corresponding author. Address: Department of Molecular Pharmacology, School of Pharmacy, Lanzhou University, Lanzhou, Gansu 730000, China. Tel.: 0086-09318620265; fax: 0086-0931-8915696. E-mail address: [email protected] (X.-D. Hu). PAIN 157 (2016) 652–665 © 2015 International Association for the Study of Pain http://dx.doi.org/10.1097/j.pain.0000000000000415

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In addition to PTPs, Src activity is also controlled by C-terminal Src kinase (CSK), the unique protein kinase responsible for Tyr529 phosphorylation.43 C-terminal Src kinase contains an N-terminal Src homology 3 (SH3) domain, an SH2 domain, and a C-terminal kinase domain. Convincing evidence has indicated that the interaction of SH2 domain with phosphotyrosine proteins is a prerequisite step for cytosolic CSK to locate at the plasma membrane and gain access to membrane-associated Src for a full catalytic action.12,40,51,60 The synaptic localization of CSK constitutively inhibits Src activity, suppresses the synaptic responses mediated by N-methyl-D-aspartate (NMDA) subtype glutamate receptors, and limits Src-dependent long-term potentiation (LTP).57 Although several phosphotyrosine proteins have been shown to provide the docking sites for CSK targeting at the plasma membrane,27,47 the identities of the phosphotyrosine proteins responsible for CSK synaptic distribution in neurons remain to be elucidated. Paxillin is a multidomain scaffolding protein that has been extensively studied at cell adhesions.6 Through direct and indirect interaction with a wide range of signaling components and actin/ microtubule cytoskeleton proteins, paxillin integrates the signals from integrins and growth factor receptors to regulate cell migration.3 There are at least 4 tyrosine residues within the N-terminal region of paxillin.3 Tyrosine phosphorylation of paxillin by focal adhesion kinase and Src creates the binding sites for SH2 domain–containing proteins, including CSK.3,47 The recruitment of CSK by tyrosine-phosphorylated paxillin is proposed PAIN®

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as a molecular mechanism for feedback inhibition of Src activity.39 This study characterized the distribution and functions of paxillin in pain-related spinal dorsal horn and revealed an important role of activity-dependent paxillin dephosphorylation in the long-lasting Src activation during chronic inflammatory pain.

2. Materials and methods 2.1. Expression constructs The full-length paxillin cDNA in pEGFP-N3 vector was a gift from Rick Horwitz (Addgene plasmid No. 15233), which was used as a template to generate paxillin (Y118E) and paxillin (Y118F) mutant by polymerase chain reaction. The full-length CSK cDNA in pcDNA3.1/myc-HisA was obtained from Yingrun Biotechnologies Inc (Changsha, China) and used to generate CSK (K222R) mutant. The pcDNA3.1 encoding cDNA for PTP1B was a gift from Anna Huttenlocher (Addgene plasmid No. 35988). The pIRES2EGFP-SHP2 was a gift from Anton Bennett (Addgene plasmid No. 12283) and the pJ3-SHP1 was a gift from Ben Neel (Addgene plasmid No. 8572). The cDNAs encoding SHP2 and SHP1 were polymerase chain reaction subcloned and ligated into pcDNA3.1 vector. All constructs were confirmed by DNA sequencing. 2.2. Transfection HEK293T cells were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum in an incubator with humidified air (5% CO2) at 37˚C. Cells were transiently transfected with mixed pEGFP-N3 and pcDNA3.1/myc-HisA vectors (1:1, total 10 mg) using a standard calcium phosphate method. Cells were harvested 48 hours after transfection. 2.3. Animals and drugs All experimental procedures were performed with the approval by the Animal Care and Use Committee of Lanzhou University. Male adult Sprague–Dawley rats (180-220 g) or Kunming mice (8-12 weeks) were provided by the Experimental Animal Center of Lanzhou University. The animals were acclimatized to the testing environments for at least 3 days before any experiments were conducted. To induce inflammatory pain, complete Freund’s adjuvant (CFA; Sigma-Aldrich, St. Louis, MO) in 10-mL volume was injected into the plantar surfaces of hindpaws. Control mice received an identical volume of saline. For intrathecal injection,23 the mice were held firmly by a pelvic girdle, and a 30-gauge needle attached to a 25-mL microsyringe was inserted between L5 and L6 vertebrae. A sudden advancement of the needle accompanied by a slight flick of the tail was used as the indicator for the proper insertion into the subarachnoid space. The recombinant adenovirus (1010 pfu/mL) expressing green fluorescent protein (GFP)-tagged paxillin, CSK, and their mutants was commercially obtained from Yingrun Biotechnologies Inc and intrathecally given in 10-mL volume. For spinal plasmid delivery,56 the lipofecter liposomal transfection reagent (10 mL; Beyotime Institute of Biotechnology, Jiangsu, China) was gently mixed with the plasmid (10.0 mg/5 mL) and stood at room temperature for 30 minutes. The mixture in a total volume of 15 mL was intrathecally injected slowly over 5 minutes. After injection, the needle remained in situ for 2 minutes before being withdrawn. Bicuculline, D(-)-2-Amino-5-phosphonopentanoic acid (D-APV), sodium orthovanadate (Sigma-Aldrich), and NMDA (TCI, Tokyo, Japan) were dissolved in saline or artificial cerebrospinal fluid (ACSF; in mM: 119.0 NaCl, 2.5 KCl, 2.5

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CaCl2, 1.3 MgCl2, 1.0 NaH2PO4, 26.0 NaHCO3, 11.0 D-glucose, pH 7.4). Src-family protein kinase inhibitor PP2 (Calbiochem, La Jolla, CA) and GluN2B antagonist ifenprodil (Sigma-Aldrich) were dissolved in dimethyl sulfoxide to prepare the stock solutions, which were diluted with saline or ACSF just before use (with the final concentration of dimethyl sulfoxide less than 0.1%). The chemical reagents were intrathecally given in 5-mL volume. All experiments were conducted blindly by experimenters without knowledge of the manipulations that the animals received. 2.4. Subcellular fractionation The mice were deeply anesthetized with sodium pentobarbital (60-90 mg/kg, intraperitoneally), and their spinal cords were quickly removed into ice-cold ACSF (bubbled with 95% O2 1 5% CO2). The dorsal quadrants of L4-L5 spinal segments were dissected out and homogenized in the lysis buffer (10.0 mM Tris·HCl, pH 7.6, 320.0 mM sucrose, 5.0 mM EDTA, proteases and phosphatases inhibitors [10.0 mM NaF, 1.0 mM Na3VO4, 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 1.0 mg/mL each of aprotinin, chymostatin, leupeptin, antipain, and pepstatin]). The homogenates were centrifuged at 1000g for 10 minutes at 4˚C to remove the nuclei and large debris (P1). The supernatant (S1) was further centrifuged at 10, 000g for 15 minutes to yield the crude synaptosomal fraction (P2) and soluble cytoplasmic fraction (S2) that contained microsomal fractions. To isolate postsynaptic density (PSD), forty mice were divided into 4 groups. The P2 fractions from 10 mice in each group were pooled and resuspended in 320 mM sucrose, 1 mM NaHCO3, and 1 mM PMSF (pH 8.0) and fractionated in sucrose gradients (0.85, 1.0, and 1.2 M) by centrifugation at 82,500g for 2 hours. The synaptosomal plasma membrane between 1.0 and 1.2 M sucrose was incubated with 6 mM Tris·HCl, pH 8.0, 160 mM sucrose, 1 mM PMSF, and 0.5% Triton X-100 at 4˚C for 15 minutes, followed by centrifugation at 32,000g for 20 minutes to obtain PSD fraction.4,5,17 To isolate the synaptosomal membrane fraction (P3), the P2 pellet from 1 mouse was incubated for 15 minutes in the lysis buffer containing 0.5% Triton X-100 and then centrifuged at 32,000g for 20 minutes to obtain P3 fraction, which is enriched with postsynaptic density marker PSD-95.48,59 To assay the protein expression and phosphorylation, the mice were randomly divided into different groups and each group contained 6 mice. The dorsal quadrants of L4-L5 spinal segments were homogenized in radioimmunoprecipitation assay (RIPA) buffer (50.0 mM Tris·HCl, pH 8.0, 150.0 mM NaCl, 1.0 mM EDTA, 1.0% NP-40, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, proteases and phosphatases inhibitors). After centrifugation at 14,000g for 10 minutes, the supernatant was harvested and the protein concentration was measured using BCA protein assay kit (Beyotime Institute of Biotechnology). 2.5. Immunoprecipitation and Western blot To assay Src phosphorylation in mice and paxillin/CSK interaction in HEK293T cells,23 the P2 fractions from mice or HEK293T cells were lysed in RIPA buffer for 30 minutes at 4˚C. After centrifugation at 14,000g for 10 minutes, the supernatant was incubated overnight with primary antibody at 4˚C under gentle rotation, followed by incubation with protein A/G agarose beads for 4 hours at 4˚C. The beads were washed with RIPA buffer 3 times and boiled in sodium dodecyl sulfate sample buffer to elute proteins. For coimmunoprecipitation,23 the P2 fraction was extracted in 50.0 mM Tris·HCl, pH 9.0, 1.0% sodium deoxycholate, 10.0 mM EDTA, proteases and phosphatases inhibitors

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at 37˚C for 30 minutes. This extract was mixed with equal volume of 50.0 mM Tris·HCl, pH 7.4, 150.0 mM NaCl, 1.0% Triton X-100, 0.1% sodium dodecyl sulfate, proteases and phosphatases inhibitors and then gently rotated overnight at 4˚C. After centrifugation at 10,000g for 10 minutes, the supernatant was incubated with primary antibody overnight at 4˚C. The immune complex was isolated by the addition of protein A/G agarose beads. The equal amounts of protein samples were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 5% nonfat milk for 30 minutes at room temperature before incubation overnight with appropriate primary antibody at 4˚C. After washing 3 times with phosphatebuffered saline containing Tween-20, the membranes were incubated with horseradish peroxidase–conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Baltimore, PA) for 60 minutes at room temperature. The blots were visualized by enhanced chemiluminescence (Beyotime Institute of Biotechnology). The primary antibodies used in this study included the mouse anti-paxillin and mouse anti-CSK antibody from BD Transduction (Lexington, KY); rabbit anti-CSK antibody from Santa Cruz (Santa Cruz, CA); rabbit anti–pY118-paxillin, rabbit anti–PSD-95, and rabbit anti–pY418-Src antibody from Invitrogen (Camarillo, CA); mouse anti-Src, rabbit anti-GluN2B, rabbit anti-GluN2A, rabbit anti–pY1472-GluN2B, and mouse anti-synaptophysin antibody from Millipore (Temecula, CA); mouse anti-His and rabbit antiPTP1B antibody from Anbo Biotechnology (JiangSu, China); rabbit anti–pY529-Src antibody from BioSource (Camarillo, CA); rabbit anti-GFP antibody from Clontech Laboratories (Mountain View, CA); mouse anti-GluN1 antibody from BD Pharmingen (San Diego, CA); mouse anti–b-actin antibody from Sigma-Aldrich. 2.6. Behavioral tests For behavioral tests, the mice were randomly divided into different groups and each group contained 6 mice. The 50% paw withdrawal thresholds (PWTs) in response to Von Frey filament (Stoelting, Wood Dale, IL) stimulation were measured using the up–down method as previously described.23 In brief, the mice were placed in a cage with a wire mesh floor, and the calibrated monofilaments were applied perpendicularly to the plantar surfaces until the filaments were bent. The pattern of positive and negative withdrawal responses was converted to 50% PWT. To measure the paw withdrawal latency (PWL), the mice were placed on a clear glass plate, and a beam of light was focused on the plantar surfaces of hindpaws to deliver heat stimuli, with a cutoff of 10 seconds. The time between the onset of heat application and paw withdrawal was recorded as PWL values.26 2.7. Immunohistochemistry The mice were deeply anesthetized with sodium pentobarbital. The lumbar spinal cords were quickly removed into ice-cold ACSF, and the transverse slices (800-mm thickness) were cut on a vibratome stage. The slices were incubated at 36˚C to 37˚C with oxygenated ACSF containing bicuculline, D-APV, NMDA, and/or sodium orthovanadate for the indicated periods, fixed in 4% paraformaldehyde for 4 hours and then cryoprotected in 30% sucrose solution overnight.18 The transverse slices (16-mm thickness) were cut from L4-L5 spinal segments on a cryostat. Six transverse slices from 6 mice were pooled in 1 group. After blocking with 10% normal goat serum and 0.1% Triton X-100 in phosphate-buffered saline, the slices were incubated with mouse

anti-NeuN antibody (Millipore) and rabbit anti–pY118-paxillin antibody. After several washes, the slices were incubated with Alexa Fluor 488–conjugated and Cy3-conjugated secondary antibodies for 2 hours before image capture. 2.8. Electrophysiological recordings The mice (4-5 weeks) were deeply anesthetized with sodium pentobarbital, and the lumbar spinal cords were removed quickly into ice-cold sucrose solution (50.0 mM sucrose, 95.0 mM NaCl, 1.8 mM KCl, 0.5 mM CaCl2, 7.0 mM MgSO4, 1.2 mM NaH2PO4, 26.0 mM NaHCO3, 15.0 mM D-glucose, bubbled with 95% O2 1 5% CO2, pH 7.4).15 A transverse slice (600-mm thickness) with an intact L4 or L5 dorsal root was cut on a vibratome stage, transferred to the recording chamber, and perfused (5 mL/min) with oxygenated ACSF at 30 to 32˚C for at least 1 hour before electrophysiological recordings. The excitatory postsynaptic currents (EPSCs) were recorded with an Axon 700B amplifier (Molecular Devices, Palo Alto, CA). The GFP-expressing lamina II neurons were visually identified using an Olympus BX51WIF microscope (Tokyo, Japan) fitted with a 403 water immersion objective under fluorescence and transmitted light illumination.8,14 The glass pipettes (3-5 MV) were filled with the internal solution containing (in mM) 115 cesium methanesulfonate, 20 CsCl, 10 HEPES, 2.5 MgCl2, 4 Na2ATP, 0.4 Na-GTP, 0.6 EGTA, and 10 sodium phosphocreatine (pH, 7.25; osmolarity, 295-300). Electrical stimulation (0.1 Hz, 0.1-millisecond duration, 3-5 mA) was delivered to the attached dorsal root through a suction electrode.8,15 To calculate a-amino3-hydroxy-5-methylisoxazole-4-propionic acid receptor to N-methyl-D-aspartate receptor (AMPAR/NMDAR) ratios (7 neurons from 7 mice per group), the evoked EPSCs were recorded at -70 mV and 140 mV with the perfusate containing picrotoxin (50.0 mM) and strychnine (2.0 mM). The averaged AMPAR peak currents recorded at -70 mV were divided by averaged NMDAR currents that were measured 49 to 51 milliseconds after the stimulus at 140 mV.49 To pharmacologically isolate the NMDAR component of synaptic responses, AMPAR antagonist 6-cyano7-nitroquinoxaline-2,3-dione (10.0 mM) was also added into the perfusate.8,62 The monosynaptic EPSCs were identified on the basis of a constant latency and the absence of conduction failure in response to high-frequency electrical stimulation (20 Hz).8,62 To plot the input–output curves (10 neurons from 10 mice per group), the synaptic responses were elicited at 6 different stimulation intensities (0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 mA).8 For miniature EPSCs (mEPSCs), the 6 neurons from 6 mice per group were held at -70 mV, and the recordings were performed in the external solution containing tetrodotoxin (0.5 mM; Sigma), picrotoxin, and strychnine. The series and input resistances were monitored online throughout each experiment.14 The recordings were abandoned if any resistances changed more than 15%. The current signals were filtered at 2 kHz and sampled at 10 kHz. 2.9. Long-term potentiation induction in vivo Thirty rats were randomly assigned into 5 groups. The animals were deeply anesthetized with urethane (1.5 g/kg, intraperitoneally).13 The trachea was cannulated and the animal breathed spontaneously. A catheter was inserted into the right femoral vein for intravenous infusion of saline at a rate of 0.8 to 1 mL/h, whereas the right femoral artery was cannulated to monitor the mean arterial blood pressure.7 The left sciatic nerve was dissected free for bipolar electrical stimulation with a silver hook electrode. The exposed nerves were covered with warm paraffin

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oil in a pool made of skin flaps. A laminectomy was performed to expose the lumbar enlargement of spinal cord. The rats were placed in a stereotaxic apparatus, and the dura mater was incised longitudinally. An agarose pool was formed around the exposed spinal segments, which were covered with ACSF. The drugs were applied onto the exposed spinal segments after ACSF was carefully removed. Colorectal temperature was kept constant (37˚C-38˚C) by a feedback-controlled heating blanket. A DAM50 extracellular amplifier (WPI, Sarasota, FL) was used to record C fiber–evoked field potentials in spinal dorsal horn as previously described.7,13 In brief, single-square pulses (0.5-millisecond duration, 25 V, 1-minute interval) were delivered to the sciatic nerves as test stimuli, and field potentials were recorded at the depth of 100 to 300 mm from the surface of spinal cord with a glass pipette (2-3 MV) filled with 135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES (pH 7.4).7 After 30 minutes of recording of stable baseline responses, the conditioning low-frequency electrical stimulation (LFS; 0.5-millisecond duration, 60 V, 2 Hz, 2 minutes) was delivered to the sciatic nerves. After conditioning stimulation, the test stimuli were again applied to the sciatic nerves. The signals were filtered using a bandwidth of 0.1 to 1000 Hz and digitized at a sampling rate of 5 kHz with an A/D converter (LAB-TRAX-4/16; WPI). 2.10. Statistical analysis All data were represented as mean 6 SEM. The peak amplitudes of evoked EPSCs were analyzed by Clampfit 8.0 software (Axon Instruments, Foster City, CA), whereas mEPSCs signals were analyzed with mini-analysis software (Synaptosoft, Fort Lee, NJ). The EPSCs data were compared using paired or unpaired Student t test. The area under the curve of C fiber–evoked field potential was determined offline by Labscribe2 software (WPI). The areas of 5 consecutive field potentials were averaged. The mean area under the curves of field potentials before LTP induction served as a baseline control. Long-term potentiation data were analyzed by 2-way analysis of variance (ANOVA). For immunohistochemical analysis, the images were converted to grayscale 8, and the dorsal horn neurons positive for the phosphorylated paxillin were outlined by Image-Pro Plus 6.0 software. The integrated optical density of the outlined area was measured and divided by the outlined area to obtain the mean density. The immunohistochemical data were analyzed by 1-way ANOVA followed by post hoc Tukey honestly significant difference test. For Western blots, the scanned digital images were quantified by NIH ImageJ software. The relative immunoreactive density of each protein was calculated by the ratio of its signal to b-actin signal, whereas the phosphorylation level was determined by the ratio of the protein phosphorylation signal to its nonphosphorylation signal. The Western blot data were analyzed by unpaired Student t test or 1-way ANOVA followed by post hoc Tukey honestly significant difference test. Behavioral data were analyzed by paired Student t test. The criterion for statistical significance was P , 0.05.

3. Results 3.1. Paxillin phosphorylation at Tyr118 was critical for C-terminal Src kinase binding As the first step to reveal the functions of paxillin in the spinal cord dorsal horn, we characterized its subcellular distribution by differential centrifugation of spinal cord extracts. Paxillin was localized at fractions positive for synaptic vesicle marker synaptophysin (Fig. 1A; n 5 4 experiments). Meanwhile, this

Figure 1. Paxillin localized at excitatory glutamatergic synapses and interacted with C-terminal Src kinase (CSK). (A) The homogenate (H), low-speed supernatant (S1), nuclei (P1), microsomal fraction (S2), synaptosomal fraction (P2), synaptosomal plasma membrane (SPM), and postsynaptic density (PSD) were fractionated from spinal dorsal horn of mice and immunoblotted (IB) with antibodies against paxillin (Pax), CSK, Src, PSD-95, and synaptophysin (Syn). n 5 4 experiments. (B and C) Coimmunoprecipitation (Co-IP) was performed with anti-paxillin (B) or anti-CSK antibody (C) from synaptosomal fraction of spinal dorsal horn of mice. The precipitated paxillin and CSK were probed by immunoblotting. n 5 4 experiments in each group.

CSK-binding protein was detectable at PSD, a specialized biochemical apparatus of excitatory synapses that were enriched with membrane-associated guanylate kinases member PSD-95 (Fig. 1A; n 5 4 experiments). A fraction of CSK and Src was also distributed at PSD (Fig. 1A; n 5 4 experiments). Coimmunoprecipitation demonstrated that anti-paxillin antibody pulled down CSK from the synaptosomal fraction of spinal dorsal horn of mice (Fig. 1B; n 5 4 experiments), and anti-CSK antibody also stably precipitated paxillin (Fig. 1C; n 5 4 experiments). The phosphotyrosines on paxillin have been shown to provide the docking sites for several SH2 domain–containing proteins.35,53 In this study, we found that paxillin phosphorylation at Tyr118 was essential for CSK binding. When His-tagged CSK (His-CSK) was cotransfected with GFP-tagged wild-type paxillin (GFP-paxillin [WT]) in HEK293T cells, anti-GFP antibody was able to precipitate His-CSK protein (Fig. 2A; n 5 4 experiments). However, the binding of His-CSK was dramatically disrupted when HEK293T cells were cotransfected with GFP-paxillin (Y118F), a tyrosine-to-phenylalanine mutant that prevents Tyr118 phosphorylation (Fig. 2A; n 5 4 experiments). In contrast to GFP-paxillin (Y118F), the phosphomimicking GFP-paxillin

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Figure 2. The phosphorylated Tyr118 on paxillin was critical for C-terminal Src kinase (CSK) binding. His-tagged wild-type CSK (His-CSK) was cotransfected in HEK293T cells with green fluorescent protein (GFP)-tagged wild-type paxillin (GFP-PAX [WT]), paxillin (Y118E) mutant (GFP-PAX [Y118E]), or paxillin (Y118F) mutant (GFP-PAX [Y118F]). Anti-GFP (A) or anti-His antibody (B) was used to coimmunoprecipitate His (A) or GFP proteins (B) (up panel). The total amounts of His (middle panel) and GFP proteins (down panel) used for coimmunoprecipitation were also probed. Molecular weight markers were indicated on the right of panels. n 5 4 experiments in each group.

(Y118E) mutant, in which Tyr118 was substituted with glutamic acid, coimmunoprecipitated more His-CSK (Fig. 2A; n 5 4 experiments). Reciprocal experiments verified that the content of GFP-paxillin (Y118F) precipitated by anti-His antibody was much less than that of GFP-paxillin (Y118E) (Fig. 2B; n 5 4 experiments). These results suggested that Tyr118 phosphorylation was required for paxillin-CSK complex formation. 3.2. Activity-dependent regulation of paxillin-Tyr118 phosphorylation in the spinal dorsal horn of mice Under resting conditions, paxillin exhibited a noticeable phosphorylation at Tyr118 in dorsal horn neurons (Fig. 3A). To test whether paxillin phosphorylation was subjected to the regulation by neural activity, the spinal cord slices were incubated with GABAA receptor antagonist bicuculline (10 mM) for 60 minutes before double immunostaining with antibodies against phosphorylated Tyr118 and neuronal marker NeuN. The specificity of the antibody against phosphorylated Tyr118 has been demonstrated by antibody-peptide competition experiment and sitedirected mutation experiment.29 As shown in Figures 3A and B, the enhanced neural activity by bicuculline decreased the phosphorylation level of paxillin-Y118 when compared to control slices (P , 0.05, n 5 6 slices from 6 mice per group). Pretreatment with NMDAR antagonist D-APV (100 mM) for 30 minutes blocked the decrease of paxillin-Y118 phosphorylation induced by bicuculline (Figs. 3A and B; P , 0.05 vs bicuculline, n 5 6 slices from 6 mice per group), whereas direct application of NMDA (50 mM) for 10 minutes mimicked the effect of bicuculline by reducing paxillin-Y118 phosphorylation levels (Figs. 3A and B; P , 0.05 vs control medium, n 5 6 slices from 6 mice per group). Previous studies have demonstrated that PTPs can act downstream of NMDAR to mediate spinal sensitization of nociceptive behaviors.50 We found that previous treatment of spinal cord slices with nonspecific PTPs’ inhibitor orthovanadate (200 mM) for 30 minutes abolished the effect of NMDA (Figs. 3A and B; P , 0.05 vs NMDA, n 5 6 slices from 6 mice per group), suggesting that paxillin dephosphorylation was attributed to PTPs’ activation by NMDAR. Neither bicuculline nor NMDA affected the total

protein level of paxillin (Fig. 3C; n 5 6 experiments). To reveal the identities of PTPs possibly responsible for paxillin dephosphorylation in the spinal dorsal horn, we expressed several PTPs’ members in intact mice,28,33,40 finding that nonreceptor tyrosine phosphatase PTP1B and SH2 domain–containing phosphatase SHP2 effectively reduced paxillin phosphorylation (Fig. 3D; P , 0.05 vs control, n 5 6 experiments). SHP1, another SH2 domain–containing tyrosine phosphatase, lacked this effect (Fig. 3D; n 5 6 experiments). 3.3. Paxillin-Tyr118 dephosphorylation sensitized the nociceptive behaviors in intact mice To reveal the functional significance of altered paxillin phosphorylation, we intrathecally injected recombinant adenovirus encoding GFP-paxillin (Y118E) or GFP-paxillin (Y118F) and monitored the possible changes of pain thresholds. Green fluorescent protein alone had no effects on the PWT in response to Von Frey filament stimuli. The PWTs were comparable to the baseline values throughout the experiments (Fig. 4A; n 5 6 mice). The PWLs in response to thermal stimulation were also unaltered by GFP expression (Fig. 4B; n 5 6 mice). In contrast to GFP control, spinal expression of GFP-paxillin (Y118F) elicited a rapid and persistent reduction of PWT and PWL values in intact mice (Figs. 4A and B; P , 0.05 vs baseline values, n 5 6 mice in each group). The reduction of pain thresholds was observed 1 day after viral injection, reached maximum within 3 to 4 days, and lasted for at least 7 days (Figs. 4A and B). Paxillin (Y118E) expression, however, had no significant effects on PWT and PWL values over the entire observation period (Figs. 4A and B; n 5 6 mice in each group). 3.4. Synaptic C-terminal Src kinase localization and Src activity were regulated by paxillin Paxillin dephosphorylation might realize nociceptive modification through CSK signaling. Compared to GFP control, paxillin (Y118F) significantly reduced the immunoreactive density of CSK at synaptosomal membrane fraction (Fig. 5A; P , 0.05 vs GFP,

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Figure 3. Activity-dependent regulation of paxillin phosphorylation at Tyr118 (paxillin-pY118) in spinal dorsal horn of mice. (A and B) The spinal cord slices were incubated with bicuculline (BIC; 10 mM, 60 minutes) or N-methyl-D-aspartate (NMDA) (50 mM, 10 minutes) before double immunofluorescence for paxillin-pY118 (red) and neuronal marker NeuN (green). N-methyl-D-aspartate receptor antagonist D(-)-2-Amino-5-phosphonopentanoic acid (D-APV; 100 mM, 30 minutes) or protein tyrosine phosphatase (PTP) inhibitor orthovanadate (Ortho; 200 mM, 30 minutes) was used to pretreat the slices before BIC or NMDA application (A). The graph showed the percentage changes of paxillin-pY118 levels (B). *P , 0.05 vs control medium, #P , 0.05 vs BIC, &P , 0.05 vs NMDA, n 5 6 slices from 6 mice in each group. (C) Neither BIC nor NMDA treatment affected the total protein levels of paxillin at spinal homogenates. The equal protein loadings were indicated by b-actin signals. n 5 6 experiments. (D) Paxillin-Tyr118 phosphorylation (pY118) was examined at day 3 after spinal expression of SHP1, SHP2, and PTP1B in intact mice. The polyvinylidene difluoride membrane was stripped and reprobed with anti-paxillin antibody (left). The graph summarized the percentage change of paxillin-pY118 levels (right). *P , 0.05 vs control, n 5 6 experiments.

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Figure 4. Paxillin dephosphorylation at Tyr118 evoked pain sensitization in intact mice. The recombinant adenovirus encoding green fluorescent protein (GFP), GFP-paxillin (Y118E), or GFP-paxillin (Y118F) was intrathecally injected (i.t.), and the changes of paw withdrawal thresholds (A) and paw withdrawal latencies (B) were plotted against time. The arrows indicated the time point of intrathecal injection. *P , 0.05 vs baseline values, n 5 6 mice in each group.

n 5 6 experiments), suggesting that Tyr118 phosphorylation was critical for CSK synaptic distribution. No detectable change of CSK content at synaptosomal membrane fraction was observed with paxillin (Y118E) (Fig. 5A; n 5 6 experiments). To test whether the altered CSK synaptic localization modulated Src activity, we examined Src phosphorylation at Tyr418 and Tyr529 at day 3 after viral expression of GFP, paxillin (Y118E), and paxillin (Y118F). As shown in Figure 5B, Src phosphorylation at the inhibitory Tyr529 residue was substantially reduced at synaptosomal fraction of paxillin (Y118F)-expression mice relative to GFP ones (P , 0.05, n 5 6 experiments), whereas Tyr418 autophosphorylation was significantly enhanced by paxillin (Y118F) (P , 0.05 vs GFP, n 5 6 experiments). There was no difference in Tyr529 or Tyr418 phosphorylation between paxillin (Y118E)-expressing and GFPexpressing mice (Fig. 5B; n 5 6 experiments). The active Src contributed to the pain hypersensitivity induced by paxillin (Y118F). Intrathecal application of Src-family protein kinases inhibitor PP2 (1.5 mg)58 greatly attenuated the reduction of PWT (Fig. 5C; n 5 6 mice) and PWL values (Fig. 5D; n 5 6 mice) in paxillin (Y118F)expressing mice. These data suggested that the synaptic loss of CSK, as a result of paxillin dephosphorylation, caused Src-dependent pain sensitization. To directly examine the role of CSK in spinal nociceptive processing, we intrathecally injected recombinant adenovirus encoding wild-type CSK (CSK [WT]) or CSK (K222R), a catalytically inactive mutant in which lysine at residue 222 was substituted with arginine. The dominant negative interference with CSK activity by CSK (K222R) mimicked paxillin (Y118F) by enhancing Src phosphorylation at Tyr418 and decreasing its phosphorylation at Tyr529 (Fig. 5E; P , 0.05 vs GFP, n 5 6 experiments in each group). Meanwhile, a long-lasting pain sensitization was induced by CSK (K222R) in intact mice (Fig. 5F; n 5 6 mice). Viral expression of CSK (WT), however, had no effects on either Src activity (Fig. 5E; n 5 6 experiments) or pain thresholds (Fig. 5F; n 5 6 mice). 3.5. Paxillin-Tyr118 dephosphorylation specifically boosted the synaptic transmission mediated by GluN2B subunit–containing N-methyl-D-aspartate receptor To directly assay the effects of paxillin phosphorylation on nociceptive synaptic transmission, we prepared the spinal cord slices at day 3 after viral injection and recorded the primary afferent–evoked EPSCs on lamina II neurons under whole-cell patch clamp configuration. Compared to GFP control, viral

expression of paxillin (Y118F) significantly reduced the ratios of AMPAR-mediated EPSCs to NMDAR-mediated EPSCs (AMPAR/NMDAR ratios) (Fig. 6A; P , 0.05, n 5 7 neurons from 7 mice in each group). There was no change of AMPAR/NMDAR ratios in paxillin (Y118E)-expressing neurons relative to GFP ones (Fig. 6A; n 5 7 neurons from 7 mice in each group). Our data showed that the frequencies and amplitudes of AMPARmediated mEPSCs displayed no significant difference among GFP-expressing, paxillin (Y118E)-expressing, and paxillin (Y118F)-expressing neurons (Fig. 6B; n 5 6 neurons from 6 mice in each group). Therefore, the reduction of AMPAR/NMDAR ratios by paxillin (Y118F) might reflect a specific potentiation of NMDAR synaptic responses. Inhibition of Src activity by postsynaptic loading of anti-Src antibody (5 mg/mL) through the recording pipettes reversed the reduction of AMPAR/NMDAR ratios in paxillin (Y118F)-expressing neurons (Fig. 6A; n 5 7 neurons from 7 mice), suggesting that the synaptic potentiation by paxillin (Y118F) was attributed to Src activation. Most NMDAR is composed of GluN1 and GluN2B or GluN2A subunits in the spinal cord. We found that extracellular perfusion of GluN2B-selective antagonist ifenprodil (3.0 mM) decreased NMDAR-EPSCs amplitudes in paxillin (Y118F)-expressing neurons (Fig. 6C; P , 0.05 vs baseline, n 5 6 neurons from 6 mice). The synaptic responses in neurons expressing GFP or paxillin (Y118E) were insensitive to ifenprodil (Fig. 6C; n 5 6 neurons from 6 mice in each group). Immunoblotting demonstrated that paxillin (Y118F) significantly increased the phosphorylation of GluN2B at Tyr1472, a key Src phosphorylation site (Fig. 6D; P , 0.05 vs GFP, n 5 6 experiments). A similar increase of GluN2B tyrosine phosphorylation was also observed with CSK (K222R) (Fig. 6D; P , 0.05 vs GFP, n 5 6 experiments). These data suggested that paxillin dephosphorylation exaggerated GluN2B-mediated nociceptive conveyance through CSK signaling. In support of this possibility, both paxillin (Y118F) and CSK (K222R) enhanced the immunoreactive densities of GluN2B and GluN1 subunits at synaptosomal membrane fraction (Fig. 6E; P , 0.05 vs GFP, n 5 6 experiments), whereas GluN2A content underwent no significant change (Fig. 6E; n 5 6 experiments). 3.6. Paxillin dephosphorylation regulated spinal nociceptive plasticity Peripheral noxious stimuli activate nociceptive C fibers and induce the LTP of C fiber–evoked field potentials in spinal dorsal

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Figure 5. Paxillin phosphorylation at Tyr118 regulated C-terminal Src kinase (CSK) synaptic localization and Src activity in spinal dorsal horn of mice. (A) Immunoblotting analysis of CSK contents at synaptosomal membrane fraction (P3) at day 3 after intrathecal injection of adenovirus encoding green fluorescent protein (GFP), GFP-paxillin (Y118E) (PAX [YE]), or GFP-paxillin (Y118F) (PAX [YF]). The equal protein loadings were indicated by b-actin signals. *P , 0.05 vs GFP, n 5 6 experiments. (B) Src phosphorylation at Tyr418 (pY418) or Tyr529 (pY529) was examined at synaptosomal fraction of mice expressing GFP, GFP-paxillin (Y118E), or GFP-paxillin (Y118F). *P , 0.05 vs GFP, n 5 6 experiments. (C and D) Intrathecal injection (i.t.) of Src family protein tyrosine kinases inhibitor PP2 (1.5 mg), but not saline, attenuated the reduction of paw withdrawal thresholds (C) and paw withdrawal latencies (D) induced by intrathecal injection of adenovirus encoding paxillin (Y118F). The upward and downward arrows indicated the time points when virus and drugs were intrathecally injected, respectively. *P , 0.05 vs the pain thresholds before drug injection, n 5 6 mice in each group. (E) Changes of Src-Tyr418 and Src-Tyr529 phosphorylation at day 3 after intrathecal injection of recombinant adenovirus encoding GFP, GFP-tagged wild-type CSK (GFP-CSK [WT]), or GFP-CSK (K222R) (CSK [KR]). *P , 0.05 vs GFP, n 5 6 experiments. (F) Time course of the changes in paw withdrawal thresholds (left) and paw withdrawal latencies (right) after intrathecal injection of adenovirus encoding GFP, GFPCSK (WT), or GFP-CSK (K222R). *P , 0.05 vs baseline values, n 5 6 mice in each group.

horn. This is widely considered as one of cellular mechanisms underlying central sensitization.42 We tested whether paxillin phosphorylation was involved in spinal plasticity by viral expression of GFP, paxillin (Y118E), or paxillin (Y118F) for 3 days before LTP induction. As shown in Figure 7A, the LFS (2 Hz, 60 V, 0.5-millisecond duration, 2 minutes) of sciatic nerves elicited robust LTP in rats expressing GFP.7 When paxillin (Y118E) was expressed, however, the synaptic potentiation was largely attenuated (Fig. 7A; P , 0.05 vs GFP, n 5 6 rats in each group). Compared to GFP control, paxillin (Y118F) significantly increased the magnitudes of LTP (Fig. 7A; P , 0.05, n 5 6 rats in each group), suggesting that paxillin dephosphorylation facilitated the LTP process. The synaptic potentiation in paxillin (Y118F)expressing rats required the activation of GluN2B receptor and Src kinase because spinal treatment with ifenprodil (10 mg)38 or PP2 (3 mg)10 for 30 minutes before LFS delivery blocked the LTP induction (Fig. 7B; n 5 6 rats in each group). 3.7. Intraplantar injection of complete Freund’s adjuvant dephosphorylated paxillin at Tyr118 in spinal dorsal horn Our data showed that peripheral noxious inputs naturally dephosphorylated paxillin in vivo. Compared to saline control, CFA injection significantly decreased Tyr118 phosphorylation in spinal dorsal horn of mice (Fig. 8A; P , 0.05, n 5 6 experiments). Intrathecal application of NMDAR antagonist

D-APV (5 mg)16 or PTP inhibitor orthovanadate (0.4 mg)50 for 30 minutes abolished paxillin-Tyr118 dephosphorylation by CFA (Fig. 8A; P , 0.05 vs CFA, n 5 6 experiments). These results suggested that NMDAR activity stimulated PTPs to dephosphorylate paxillin during inflammatory pain. With paxillin-Tyr118 dephosphorylation, anti-paxillin antibody coimmunoprecipitated less CSK from the synaptosomal fraction of inflamed mice relative to saline-injected control ones (Fig. 8B; n 5 6 experiments). The PTP1B contents precipitated by anti-paxillin antibody from inflamed mice were comparable to those from control mice (Fig. 8B; n 5 6 experiments). Because inhibition of NMDAR and PTPs enhanced paxillin phosphorylation in CFAinjected mice (Fig. 8A), we tested the effects of D-APV and orthovanadate on paxillin-CSK interaction, finding that both drugs restored CSK binding to paxillin (Fig. 8B; n 5 6 experiments). Immunoblotting illustrated that CFA significantly decreased CSK concentration at synaptosomal membrane fraction when compared to saline control (Fig. 8C; P , 0.05, n 5 6 experiments). This synaptic loss of CSK was attributed to paxillin dephosphorylation because viral expression of paxillin (Y118E) in CFA mice resumed CSK content to control level (Fig. 8C; n 5 6 experiments). Complete Freund’s adjuvant had no effects on the total protein expression of either paxillin or CSK (Fig. 8D; n 5 6 experiments). These data suggested that paxillin dephosphorylation and its dissociation with CSK were closely correlated with inflammatory pain.

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Figure 6. Paxillin-Tyr118 phosphorylation regulated GluN2B receptor–mediated excitatory postsynaptic currents (EPSCs) in spinal dorsal horn of mice. (A and B) The evoked EPSCs (A) and miniature EPSCs (mEPSCs) (B) were recorded in neurons expressing green fluorescent protein (GFP), GFP-paxillin (Y118E) (Pax [Y118E]), or GFP-paxillin (Y118F) (Pax [Y118F]). The a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor to N-methyl-D-aspartate receptor (AMPAR/ NMDAR) ratios ([A] n 5 7 neurons from 7 mice in each group) and the frequencies and amplitudes of mEPSCs ([B] n 5 6 neurons from 6 mice in each group) were summarized. Note that postsynaptic loading of anti-Src antibody (5 mg/mL) through the recording pipettes reversed the reduction of AMPAR/NMDAR ratios in neurons expressing GFP-paxillin (Y118F) (A). *P , 0.05 vs GFP, #P , 0.05 vs Pax (Y118F). (C) Effects of ifenprodil on NMDAR-EPSCs in neurons expressing GFP, GFP-paxillin (Y118E), or GFP-paxillin (Y118F). Sample traces were obtained at the time points indicated by 1 and 2. *P , 0.05 vs baseline values, n 5 6 neurons from 6 mice in each group. (D and E) Viral expression of GFP-paxillin (Y118F) or GFP-CSK (K222R) (CSK [KR]) for 3 days increased GluN2B phosphorylation at Tyr1472 (pY1472) (D) and enhanced the protein expressions of GluN2B and GluN1 subunits at synaptosomal membrane fraction (P3) (E). The synaptic content of GluN2A subunit was unaltered by GFP-paxillin (Y118F) or GFP-CSK (K222R) (E). *P , 0.05 vs GFP, n 5 6 experiments in each group.

3.8. Spinal expression of paxillin (Y118E) and GFP-tagged wild-type C-terminal Src kinase alleviated inflammatory pain Our data showed that the synaptic mistargeting of CSK, as a result of paxillin dephosphorylation, contributed to Src activation during inflammatory pain. Viral expression of either paxillin (Y118E) or CSK (WT) repressed Src hyperactivity in

CFA-injected mice (Fig. 9A; P , 0.05 vs CFA, n 5 6 experiments). Previous studies have demonstrated that Srcdependent NMDAR hyperfunction is critical for the initiation and development of inflammatory pain.10,25 In this study, we found that the potentiation of NMDAR-mediated nociceptive synaptic transmission by CFA was also suppressed by paxillin (Y118E) and

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produced a potent analgesia against chronic inflammatory pain (Fig. 9E; n 5 6 mice in each group), whereas paxillin (Y118F) and CSK (K222R) had no effects (Fig. 9E; n 5 6 mice in each group).

4. Discussion

Figure 7. Paxillin phosphorylation at Tyr118-regulated long-term potentiation (LTP) of C fiber–evoked field potentials in spinal dorsal horn of rats. (A) Lowfrequency stimulation (LFS) was delivered onto sciatic nerves to induce LTP at day 3 after intrathecal injection of adenovirus encoding green fluorescent protein (GFP), GFP-paxillin (Y118E), or GFP-paxillin (Y118F) in rats. The original traces were taken at the time points indicated by number 1 and 2 (up). The area under the curve of C fiber–evoked field potential was plotted against time (down). The arrow indicated the time point of LFS delivery. *P , 0.05 vs GFP, n 5 6 rats in each group. (B) Src family protein tyrosine kinases inhibitor PP2 (3 mg) or GluN2B receptor antagonist ifenprodil (10 mg), when applied 30 minutes before LFS, blocked LTP in rats expressing GFP-paxillin (Y118F). n 5 6 rats in each group.

CSK (WT) (Fig. 9B; n 5 10 neurons from 10 mice in each group). Extracellular perfusion of ifenprodil reduced NMDAR-EPSCs amplitudes in GFP-expressing neurons, but not in paxillin (Y118E)-expressing and CSK (WT)-expressing neurons from CFA-injected mice (Fig. 9C; n 5 6 neurons from 6 mice in each group), suggesting that GluN2B receptor was a key target for paxillin (Y118E) and CSK (WT) to suppress the synaptic responses. Western blot confirmed that CFA-induced GluN2B phosphorylation at Tyr1472 was abolished by paxillin (Y118E) and CSK (WT) (Fig. 9D; n 5 6 experiments). Successive monitoring of pain thresholds demonstrated that paxillin (Y118E) and CSK (WT)

C-terminal Src kinase has been shown to exert a tonic inhibition of NMDAR-dependent synaptic transmission and plasticity through negative control of Src activity.57 This study demonstrated that, in addition to its enzymatic activity, the proper synaptic targeting by paxillin was also necessary for CSK to achieve full synaptic inhibition. We found that the phosphorylated Tyr118 on paxillin complexed with and anchored CSK at postsynaptic compartments under resting conditions, which restricted Src from aberrant activation. Once Tyr118 was dephosphorylated, the synaptic concentration of CSK was dramatically reduced, leading to Src-dependent plastic changes in spinal cord dorsal horn (Fig. 10). Our data demonstrated that paxillin phosphorylation at Tyr118 was dynamically regulated by neuronal activity. Activation of NMDAR by bicuculline or NMDA substantially reduced paxillin phosphorylation levels in vitro. N-methyl-D-aspartate receptordependent paxillin dephosphorylation was also observed after intraplantar CFA injection. We investigated the potential role of PTPs in catalyzing paxillin dephosphorylation, finding that nonspecific PTPs’ inhibition totally abolished Tyr118 dephosphorylation induced by either NMDA or CFA injection. These results were consistent with previous reports that PTPs transduce the signaling from NMDAR to regulate spinal nociceptive transmission50 and suggested that paxillin was an important target of NMDAR-PTP pathway. Given that spinal expression of either PTP1B or SHP2 effectively catalyzed Tyr118 dephosphorylation, it was reasonable that more than 1 PTP member might be motivated by NMDAR during chronic pathological pain to ensure the sufficient and persistent paxillin dephosphorylation. Previous studies have investigated in an extensive fashion the role of paxillin-associated CSK in cell migration.46,54 A prevailing view is that focal adhesion kinase and Src, stimulated by integrins or growth factor receptors, promote paxillin tyrosine phosphorylation, which subsequently recruits CSK to the close vicinity of Src and terminates or represses Src signaling in nonneuronal cells.3,40 In this model, paxillin provides a platform for feedback inhibition of Src activity with important implications for cell mobility and survival.3,39 In spinal dorsal horn neurons, endogenous paxillin displayed a high basal phosphorylation at Tyr118, which might tether adequate CSK at synapses to keep Src at the inactive state. At this time, spinal expression of paxillin (Y118E) generated no significant effects on synaptic CSK localization and Src activity. Through NMDAR/PTPs’ signaling, the enhanced neuronal activity dephosphorylated, rather than phosphorylated, paxillin at Tyr118. This dephosphorylation event reduced the binding affinity of paxillin for CSK and dispersed CSK out of synapses. Viral expression of paxillin (Y118E) to drive CSK synaptic redistribution markedly suppressed Src activity in CFAinjected mice, suggesting that PTPs’ activation by NMDAR impaired the scaffolding function of paxillin and thus disturbed the feedback inhibition conferred by CSK (Fig. 10). Spinal Src hyperactivity plays an important role in chronic pathological pain.25,50 However, the means by which Src activity is rapidly stimulated and persistently maintained are yet to be ascertained. It was noteworthy that paxillin-dephosphorylating PTPs, such as PTP1B and SHP2, can also dephosphorylate Src at the inhibitory Tyr529 residue in several cell lines.41 Based on these observations, we proposed that spinal PTPs might regulate Src

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Figure 8. Intraplantar injection of complete Freund’s adjuvant (CFA) reduced paxillin phosphorylation at Tyr118 and disturbed its interaction with C-terminal Src kinase (CSK) in spinal dorsal horn of mice. (A) Tyr118 phosphorylation on paxillin (pY118) was examined at day 3 after CFA injection. N-methyl-D-aspartate receptor antagonist D(-)-2-Amino-5-phosphonopentanoic acid (D-APV; 5 mg) or protein tyrosine phosphatase (PTP) inhibitor orthovanadate (Ortho; 0.4 mg) was intrathecally applied for 30 minutes before immunoblotting analysis. *P , 0.05 vs saline-injected control mice, #P , 0.05 vs CFA-injected mice, n 5 6 experiments. (B) CFA decreased CSK content coimmunoprecipitated (Co-IP) by anti-paxillin antibody from synaptosomal fraction, which could be blocked by D-APV and orthovanadate. The PTP1B contents in paxillin precipitates were also probed. n 5 6 experiments. (C) The adenovirus encoding paxillin (Y118E) (Pax [YE]) was intrathecally given at 2 hours post-CFA, and the CSK contents at synaptosomal membrane fraction (P3) were examined at day 3 post-CFA. Equal protein loadings were indicated by b-actin signals. *P , 0.05 vs saline-injected control mice, #P , 0.05 vs CFA-injected mice, n 5 6 experiments. (D) CFA had no effects on paxillin and CSK protein levels at spinal homogenates. n 5 6 experiments.

through 2 pathways (Fig. 10). First, PTPs directly dephosphorylated Tyr529 to initiate Src signaling after peripheral lesions.50 Second, PTPs dephosphorylated paxillin at Tyr118, which removed CSK-mediated synaptic inhibition and accounted for the long-lasting Src hyperactivity. In agreement with this possibility, intrathecal application of PTPs’ inhibitors suppresses Src and blocks the induction and maintenance of inflammatory pain.50 Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor and NMDAR are the major ionotropic glutamate receptors that mediate nociceptive conveyance from periphery to higher brain regions. Tyrosine phosphorylation has been shown to regulate the synaptic trafficking of AMPAR and play an important role in synaptic plasticity.1,11,61 However, spinal expression of paxillin (Y118F) had no effect on the frequency and amplitudes of AMPAR-mediated mEPSCs, suggesting that

AMPAR might not be a good substrate of the pool of CSK specifically targeted by paxillin. In contrast to AMPAR, a dramatic increase of GluN2B tyrosine phosphorylation was induced by paxillin (Y118F). As previously reported,31,37 Tyr1472 phosphorylation facilitates GluN2B receptor clustering at postsynaptic membrane by blocking clathrin-mediated endocytosis process. Whole-cell patch clamp recordings in lamina II neurons demonstrated that paxillin (Y118F) significantly boosted GluN2B-mediated synaptic transmission, suggesting that paxillin specifically relayed the signaling to postsynaptic GluN2B receptor. After LTP induction, Tyr1472 phosphorylation is significantly enhanced.30 Knockin mutation of Tyr1472 or pharmacological inhibition of GluN2B receptors largely reduces the magnitudes of LTP.2,31 We investigated the possible role of paxillin in nociceptive plasticity by recording LTP of C fiber–evoked field potentials at the depth of

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Figure 9. Paxillin (Y118E) and wild-type CSK (CSK [WT]) alleviated inflammatory pain by inhibiting Src and N-methyl-D-aspartate receptor (NMDAR) hyperactivity in spinal dorsal horn of mice. Recombinant adenovirus encoding green fluorescent protein (GFP), GFP-paxillin (Y118E) (Pax [YE]), or GFP-CSK (WT) was intrathecally administrated (i.t.) at 2 hours after intradermal (i.d.) saline or complete Freund’s adjuvant (CFA) injection. (A) Src phosphorylation at Tyr418 (pY418) or Tyr529 (pY529) was examined at day 3 post-CFA. *P , 0.05 vs saline-injected GFP-expressing mice, #P , 0.05 vs CFA-injected GFP-expressing mice, n 5 6 experiments. (B) NMDAR–excitatory postsynaptic currents (EPSCs) were elicited at 6 different stimulation intensities (left) and the input (stimulation intensity)–output (synaptic response) curves were plotted (right). *P , 0.05 relative to saline-injected GFP-expressing control mice, #P , 0.05 relative to CFAinjected GFP-expressing mice, n 5 10 neurons from 10 mice in each group. (C) Effects of GluN2B-selective antagonist ifenprodil on NMDAR-EPSCs amplitudes in GFP-expressing, GFP-paxillin (Y118E)-expressing, or GFP-CSK (WT)-expressing neurons from CFA mice. The horizontal bar indicated the period of extracellular ifenprodil perfusion. *P , 0.05 vs baseline values, n 5 6 neurons from 6 mice in each group. (D) GFP-paxillin (Y118E) and GFP-CSK (WT) repressed GluN2B phosphorylation at Tyr1472 (pY1472) in CFA-injected mice. *P , 0.05 relative to saline-injected control mice, #P , 0.05 relative to CFA-injected mice, n 5 6 experiments. (E) Viral expression of GFP-paxillin (Y118E) and GFP-CSK (WT), but not GFP-paxillin (Y118F) (Pax [YF]) or GFP-CSK (K222R) (CSK [KR]), reversed the reduction of paw withdrawal thresholds in CFA-injected mice. *P , 0.05 relative to baseline values, n 5 6 mice in each group.

100 to 300 mm from the surface of spinal cord, a distance that has been shown to be within lamina I-II.7,15,24 We provided evidence that CSK-binding protein paxillin engaged in the regulation of spinal LTP. Through activation of Src and GluN2B receptors, paxillin (Y118F) augmented LTP magnitudes in spinal dorsal horn. The amplitudes of LTP were suppressed by paxillin (Y118E), possibly because of its ability to restrict Src phosphorylation of GluN2B receptors. The lamina II neuronal circuitry is complex and composed of distinct functional populations of interneurons. It was likely that both excitatory and inhibitory interneurons were included in our recordings. However, paxillin (Y118F) expression consistently potentiated the glutamatergic inputs onto lamina II neurons, which correlated well with the pain hypersensitivity in vivo. In agreement with these results, previous studies have proposed that the increased glutamatergic transmission of primary afferent

synapses represents a key mechanism underlying central sensitization.20,21,42,44 Manipulations that block glutamatergic transmission, such as pharmacological inhibition of NMDAR, alleviate pathological pain, suggesting that increased glutamatergic inputs are largely nociceptive.36,44,45,52 The primary afferent inputs after noxious stimulation have also been shown to trigger plastic changes at inhibitory neuronal circuits.19,34 Therefore, the identification of molecular determinants of synaptic strength might provide some important clues as to how nociceptive information is processed at the synaptic level. Our data showed that both PWT and PWL values declined after paxillin (Y118F) expression. The reduced PWT values were probably attributed to the synaptic potentiation caused by paxillin (Y118F) at synapses innervated by low-threshold mechanoreceptive fibers, which have been implicated to transfer

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Figure 10. Schematic illustration of the mechanisms by which paxillin regulated synaptic N-methyl-D-aspartate receptor function. The activitydependent dephosphorylation of Tyr118 by protein tyrosine phosphatases (PTPs) disrupted paxillin interaction with C-terminal Src kinase (CSK) and dispersed CSK out of synapses, which removed CSK-mediated inhibition of Src. The active PTPs might also cause a direct dephosphorylation of Src at Tyr529. Thereafter, the active Src catalyzed GluN2B phosphorylation at Tyr1472 to boost GluN2B-mediated nociceptive transmission. Pax, paxillin; p, phosphorylation; Y, tyrosine residue.

mechanical allodynia.9,21,44,55 The facilitatory effects of paxillin (Y118F) on synaptic transmission between high-threshold nociceptive primary afferent fibers and lamina II neurons might lead to the increased responses to thermal stimuli.9,20,21,44,55 In addition to paxillin (Y118F), CSK (K222R) expression also evoked mechanical allodynia and thermal hyperalgesia in intact mice, suggesting that the synaptic targeting of CSK by phosphorylated paxillin exerted a gating role in the sensitization of nociceptive behaviors. In CFA-injected animals, the enhancement of paxillin phosphorylation or CSK synaptic localization dramatically suppressed glutamatergic transmission and meanwhile alleviated inflammatory pain. These results suggest that plastic changes at synapses between primary afferent fibers and lamina II neurons, resulting from the impaired CSK docking property of paxillin, play an important role in spinal sensitization. Therefore, proper CSK synaptic recruitment might have therapeutic potential in the treatment of pathological pain.

Conflict of interest statement The authors have no conflicts of interest to declare. This work was supported by the National Natural Science Foundation of China (No. 31271186). X. Wang and R. Zheng contributed equally to this work. Article history: Received 25 July 2015 Received in revised form 29 October 2015 Accepted 3 November 2015 Available online 11 November 2015

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