Nck adaptor expression in mouse brain. A, domain structure of PAK and Nck proteins involved in the interaction. The N-terminal part of PAK proteins contains an SH3-binding domain located between amino acids 10 and 20. This region interacts with the second SH3 domain of Nck adaptors. The amino acid (aa) numbering of PAK corresponds to those of the human PAK3 sequence. B, amino acid alignment of the N-terminal region of PAK proteins of group I encoded by mouse and human genes. The amino acid sequence of the SH3-binding domain contains a conserved proline-rich motif PPXPP and two critical residues Pro-12 and Ser-20 (numbers refer to PAK3) for Nck interaction are indicated with black and white arrows, respectively. The sequence of the P₁₂ peptide used in the following sections, which inhibits Nck2 interaction, is underlined. C, sequence alignment of the N-terminal domains of primate, mouse, Xenopus, and fish illustrates the phylogenic conservation of the amino extremity of the PAK3 proteins. D, mRNA quantification by quantitative-PCR shows more Nck2 than Nck1 expression in brain. Total RNAs (2 μg) from mouse embryonic E18 brain (E18) and adult brain (AB) were subjected to quantitative RT-PCR. Comparison with Student's t test: **, p < 0.01, n = 4. Error bars indicate the S.E. E, characterization of the Nck sera. The two Nck isoform-specific sera were tested for their ability to specifically recognize their targets. Lysates of COS-7 cells transfected either with HA-Nck1 or HA-Nck2 plasmids were controlled for Nck expression by HA-Western blotting (WB) on total cell lysate (last panel), and HA-precipitated (IP HA) before being analyzed by Western blotting, using a polyclonal HA or the pan-Nck monoclonal antibodies (1st and 2nd panels), or immune Nck1 or Nck2 sera (3rd and 4th panels). Ability for immunoprecipitation of the two new sera was tested from similarly transfected cells, and precipitated complexes were analyzed by HA Western blotting (5th and 6th panels). F, the two Nck and PAK3 proteins were detected in mouse brain by Western blot analysis. E18 and adult brain lysates (25 μg of proteins) were probed with either the Nck1 and the Nck2 antibodies or the PAK3 antibodies and β-actin antibodies for loading control. Data shown in the D–F are representative of a typical experiment.

Preferential binding of PAK3 with Nck2 in vitro and in vivo. A, stronger interaction between PAK3 and Nck2 compared with Nck1. COS-7 cells were co-transfected with plasmids coding for HA-tagged Nck1 or Nck2 with FLAG-PAK3-WT. Nck proteins were HA-immunoprecipitated (IP(HA)). The co-immunoprecipitated PAK3 protein was revealed by anti-FLAG blotting (upper panel). Equal PAK3 expression levels in transfected cells and equal amount of immunoprecipitated Nck proteins were controlled by Western blotting on TCL (lower panel) and immunoprecipitated proteins (IP-HA, WB HA, middle panel), respectively. B, quantification of the amount of PAK3 precipitated with Nck adaptors from the experiments illustrated in A. Comparison with Student's t test: ***, p < 0.001, n = 4. Error bars indicate the S.E. C, PAK3 co-immunoprecipitates with Nck2 but not with Nck1 from mouse brain. E18 or adult brain lysates (10 mg), analyzed by Western blot in Fig. 1F, were immunoprecipitated with preimmune or immune Nck1 or Nck2 sera. Immunoprecipitated Nck proteins were revealed with the pan-Nck antibodies (WB pan Nck). Associated PAK3 proteins were revealed by Western blotting using PAK3 antibodies (WB PAK3). D, three PAK3 proteins bearing the mental retardation mutation A365E, R419Stop, and R67C co-immunoprecipitate with Nck2 with an apparent normal efficiency. COS-7 cells were co-transfected with plasmids coding for HA-tagged PAK3 mutants with FLAG-Nck2. The co-immunoprecipitated HA Nck2 protein was revealed by anti-FLAG blotting (upper panel). Equal Nck2 expression levels in transfected cells and amount of HA-immunoprecipitated PAK3 proteins were controlled by Western blotting on TCL (lower panel) and immunoprecipitated proteins (IP-HA, WB HA, middle panel). Data shown in the A, C, and D are representative of a typical experiment.

PAK3 binds Nck2 independently of its kinase activity. A, in vitro interaction of different PAK3 kinase mutants with the second SH3 domains of Nck1 and Nck2. WT, kinase-dead (kd), and constitutively active (ca) forms of HA-PAK3 were expressed in COS-7 cells, and lysates were incubated with recombinant GST proteins containing the second SH3 domain of Nck1 (1st panel) or Nck2 (2nd panel). Affinity-purified (AP) PAK3 proteins and COS-7 expressed PAK3 proteins (TCL) were revealed by Western blotting (WB) with HA antibodies. Data shown are representative of a typical experiment from three independent experiments. Amount of GST proteins was controlled by Coomassie Blue staining. Note that the lower electrophoretic migration of the GST-Nck1-(SH3)₂ protein is due to the presence of an HA tag. B, quantification of the amount of different PAK3 proteins precipitated with the second SH3 domain of Nck adaptors from the experiments illustrated in A, n = 3. Error bars indicate the S.E. C and D, Cdc42 activation of PAK3 does not suppress its interaction with Nck1 (C) or Nck2 (D). COS-7 cells were co-transfected with plasmids coding for FLAG-PAK3-WT or FLAG-PAK3-kd (kinase-dead), with either active Val-12 or inactive Asn-17 mutants of GFP-Cdc42 vectors, together with HA-tagged Nck1 (C) or Nck2 (D) plasmids. Nck1 or Nck2 proteins were HA-immunoprecipitated (IP (HA)) and visualized after HA immunoblotting (middle panels). PAK3 proteins in immune complexes (upper panels) and in TCLs were revealed by anti-FLAG immunoblotting. GTPase expression was verified using anti-GFP antibody (data not shown). Note that activation of PAK3 by Cdc42-Val-12 induced an electrophoretic shift of PAK3-WT protein as previously reported (25).

Serine 20 residue of PAK3 is not a regulatory site. A–C, two-dimensional chromatography of PAK3 peptides obtained from recombinant GST-PAK3-(2–49) protein (A) or GST-PAK3-(2–49)-S20E protein (B), which were phosphorylated in vitro by CaMKII, before analysis of the tryptic phosphopeptides by two-dimensional mapping. C, two-dimensional mapping of full-length activated wild-type PAK3 protein. HA-PAK3-transfected COS-7 cell lysate was incubated with recombinant GST-Cdc42-V12 before immunoprecipitation. The precipitate was then subjected to a kinase assay with radioactive ATP. Vertical arrows indicate the electrophoretic direction and horizontal arrows the chromatography migration. Black arrow indicates the serine 20-corresponding peptide, and the arrowheads show an uncharacterized phosphorylated peptide. D, phosphorylation of mutated PAK3-N-terminal proteins. The serine residues of the PAK3-N-terminal proteins were successively mutated and tested in a CaMKII kinase assay, as described above. E, Akt phosphorylation of PAK3-N-terminal and huntingtin as positive control. huntingtin and PAK3 recombinant peptides, visualized with Coomassie Blue staining (lower panel), were tested in an Akt kinase assay (upper panel). F, PKA phosphorylation of PAK3-N-terminal and B-Raf as positive control. B-Raf and PAK3 recombinant peptides were tested in a PKA kinase assay. All data shown are representative of a typical experiment from three independent experiments.

Post-synaptic inhibition of the PAKs/Nck2 interaction by perfusion of the inhibitory P₁₂ peptide induced an increase in evoked EPSC. A, P₁₂ peptide decreased the amount of co-immunoprecipitated PAK3 protein with Nck2 but not with Nck1. COS-7 cells were co-transfected with plasmids coding for FLAG-PAK3-WT and HA-tagged Nck1 (left lanes) or Nck2 (right lanes). Nck proteins were HA-immunoprecipitated (IP(HA)) in the absence or the presence of 1 mm of the P₁₂ or the A₁₂ peptide. Nck proteins are visualized after immunoblotting (middle panel). The co-immunoprecipitated PAK3 protein was revealed by anti-FLAG blotting (upper panel). PAK3 expression levels in TCLs were controlled as indicated (lower panel). B, quantification of amount of precipitated PAK3 protein with Nck adaptors illustrated in A (n = 3). C, pulldown assay of PAK1 or PAK3 proteins with the GST-(SH3)₂ domain of Nck2 in presence of the P₁₂ competing peptide or its A₁₂ control peptide_. Lysates of COS-7 cells transfected with FLAG-PAK1 or PAK3-kinase-dead expression vector were incubated 5 min without or with 250 μm of the P₁₂ peptide or the A₁₂ peptide before the PAK proteins were pulled down with the recombinant GST protein (20 μg). Precipitated proteins (left lanes) and expressed proteins in TCL (right lanes) were revealed by FLAG Western blot (WB). D, schematic representation of simultaneous patch clamp recordings. Pyramidal neurons were targeted in layer 5 of parasagittal slices of juvenile rat primary visual cortex. Two simultaneous whole-cell patch clamp recordings were then performed using two pipettes, one of them containing the P₁₂ or A₁₂ peptide, whereas the second consists of control recording. Extracellular electrical stimulations were applied in layer 2/3, on a vertical axis set at 50–70 μm from the recorded neuron apical dendrite. Evoked EPSCs (eEPSCs) were recorded under voltage clamp at a holding potential of −80 mV. Recordings and analysis involving P₁₂ or A₁₂ peptides were performed under blind conditions. E, characterization of the AMPA-mediated evoked excitatory responses in layer 5 neurons. The aCSF panel represents the typical current response obtained under control conditions after the brief extracellular stimulation (as indicated by the corresponding stimulation artifact). Bath application of NMDA (50 μm AP5) or GABA_A (50 μm picrotoxin) receptor blockers had no impact on the evoked EPSC, whereas AMPA receptor blockade 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX, 20 μm) suppressed the response. Current responses represented in this panel are means of three consecutive individual traces. This experiment was replicated three times in different slices. F, superposition of the control eESPC (aCSF condition, black trace) with the eEPSCs recorded under the three different conditions shown in E (gray traces). G, perfusion of the P₁₂ peptide through the patch clamp pipette increases the amplitude of eEPSCs recorded in layer 5 pyramidal neurons by roughly 40% (black square). In contrast, recordings performed with the noninteracting peptide A₁₂ (open dots) showed unmodified amplitudes during more than 1 h (n = 3 cells per condition, p < 0.001; Mann Whitney test). Note that the control recordings (performed without any peptide in the patch clamp pipette, see E) were stable for all cells included in the data set and are not illustrated in this figure. H, significant representation of eEPSC obtained with P₁₂ peptide-containing recording pipette at two different times, during an early step between 10 and 20 min (upper trace) and during a later phase between 50 and 70 min (lower trace).

Mutation of the proline 12 residue and deletion of the N-terminal part of PAK3 suppress co-immunoprecipitation with Nck2 but not with Nck1. A, amino acid sequences of the N-terminal extremity of mouse PAK3 protein and the two mutated proteins used in this study. B, P12A mutation impairs the affinity precipitation of PAK3 protein with the second SH3 domains of Nck1 and Nck2 fused to GST. C, mutation of the proline 12 in PAK3 does not significantly change the interaction with Nck1. FLAG-PAK3 wild-type (wt), with the mutation P12A (P12A) or deleted for the first 20 amino acids (ΔNter) were co-expressed with HA-Nck1 in COS-7 cells. Nck1 protein was HA-immunoprecipitated and the presence of PAK3 in precipitated complexes (IP(HA)) and in TCLs was analyzed by Western blotting (WB) with FLAG antibodies. The amount of Nck1 precipitated proteins was controlled by anti-HA immunoblotting. D, relative binding of PAK3 proteins to Nck1, quantified from three independent experiments illustrated from C and averaged. E, mutation of the proline 12 in PAK3 abolished the interaction with Nck2. PAK3 proteins were co-expressed with HA-Nck2 in COS-7 cells. Nck2 protein was HA-immunoprecipitated and the presence of PAK3 in precipitated complexes (IP(HA)) and in TCL was analyzed by Western blotting with FLAG antibodies. The amount of Nck2 precipitated proteins was controlled by anti-HA immunoblotting. F, relative binding of PAK3 proteins to Nck2, quantified from four independent experiments illustrated from E. Comparison with Student's t test: ***, p < 0.001, n = 3. Errors bars indicate the S.E.

PAK3 acts on spinogenesis and synaptogenesis independently of its interaction with Nck2. A, P12A mutation did not affect spine maturation. Hippocampal neurons were transfected either with GFP as control or GFP-PAK3-WT or GFP-PAK3-P12A constructs at DIV18 and fixed at DIV21. Confocal images showed mature spines (arrows) and filopodia (arrowheads) at DIV21. Scale bar represents 2 μm. B and C, quantitative analysis of protrusion density or length. No significant difference for filopodia (F) or spines (S) between the three expressed constructs was observed. Data are means ± S.E. of measurements obtained from 8 to 11 cells (length of dendrites analyzed: 50–250 μm/cells; 1030–1800 protrusions analyzed). D, P12A mutation did not affect synaptic density. Hippocampal neurons were transfected either with GFP (1st panels) as control, GFP-PAK3-WT (2nd panels) or GFP-PAK3-P12A (3rd panels) constructs at DIV18, fixed at DIV21, and immunolabeled for PSD95 as postsynaptic marker and vGLUT1 as presynaptic marker. Confocal images showed GFP proteins in green, PSD95 in red, and vGLUT1 in blue. PSD95-positive spines were identified from green and red images (arrowhead), and synapses were identified as PSD95-positive spines with adjacent staining of vGLUT1 (arrow). Scale bar, 5 μm. E, quantitative analysis of synaptic cluster density. No significant difference was observed in PSD95-positive spine (left histogram) or synapse (right histogram) density between the three conditions. Data are means ± S.E. of measurements obtained from 15 to 17 neurons (length of dendrites analyzed: 50–250 μm/cell; 680–820 PSD-95 clusters and 230–330 synapses analyzed).

Expression of PAK3-WT down-regulates mEPSC amplitudes in hippocampal neurons through its interaction with Nck2. A, representative sample traces of 5 s recording of mEPSCs in untransfected or GFP-PAK3-WT transfected neurons. Scale bars, 20 pA and 1 s. B, cumulative distribution plots of the mEPSC amplitude, assembled from the first 100 events from each cell recorded. C, quantification of mean mEPSC amplitude (mean ± S.E.). n = 10–15 cells per condition; ***, p < 0.001, Mann Whitney test. D–F, sample traces, cumulative distribution and quantification of mEPSC amplitude (means ± S.E.) recorded in untransfected neurons and in neurons transfected with GFP-PAK3-P12A construct showed no difference. n = 15 cells per condition.

Model of regulation of synaptic transmission by PAK3. Nck2 regulates glutamate receptor stabilization (center diagram). Overexpression of PAK3 titrates Nck2 and induces a decrease of the Nck2-dependent stabilization of glutamate receptors (left diagram). Conversely, perfusion of an inhibitory peptide inhibits the Nck2-PAK3 complex, releasing Nck2 and allowing a Nck2-dependent stabilization of glutamate receptors (right diagram).