The Shank Family of Postsynaptic Density Proteins Interacts with and Promotes Synaptic Accumulation of the {beta}PIX Guanine Nucleotide Exchange Factor for Rac1 and Cdc42*

Eunhye Park {ddagger}, Moonseok Na {ddagger}, Jeonghoon Choi {ddagger}, Seho Kim {ddagger}, Jae-Ran Lee {ddagger}, Jiyoung Yoon {ddagger}, Dongeun Park §, Morgan Sheng ¶ || and Eunjoon Kim {ddagger} **

From the {ddagger} Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea, § School of Biological Sciences, Seoul National University, Seoul 151-742, Korea, Picower Center for Learning and Memory, RIKEN-MIT Neuroscience Research Center and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received for publication, January 30, 2003 , and in revised form, March 5, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Shank/ProSAP family of multidomain proteins is known to play an important role in organizing synaptic multiprotein complexes. Here we report a novel interaction between Shank and {beta}PIX, a guanine nucleotide exchange factor for the Rac1 and Cdc42 small GTPases. This interaction is mediated by the PDZ domain of Shank and the C-terminal leucine zipper domain and the PDZ domain-binding motif at the extreme C terminus of {beta}PIX. Shank colocalizes with {beta}PIX at excitatory synaptic sites in cultured neurons. In brain, Shank forms a complex with {beta}PIX and {beta}PIX-associated signaling molecules including p21-associated kinase (PAK), an effector kinase of Rac1/Cdc42. Importantly, overexpression of Shank in cultured neurons promotes synaptic accumulation of {beta}PIX and PAK. Considering the involvement of Rac1 and PAK in spine dynamics, these results suggest that Shank recruits {beta}PIX and PAK to spines for the regulation of postsynaptic structure.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic spines are actin-rich morphological specializations in neurons that mediate most excitatory synaptic transmission (1, 2, 3). The postsynaptic density (PSD)1 is a microscopic structure within dendritic spines that is associated with the postsynaptic membrane and contains a variety of scaffolding and signaling proteins (4, 5).

The Shank/ProSAP/SSTRIP family of multidomain proteins (Shank1, Shank2, and Shank3) plays important roles in organizing the PSD (6, 7). Shank is a relatively large protein (~200 kDa) and contains various protein interaction domains including, from the N terminus, ankyrin repeats, an SH3 domain, a PDZ domain, a long (>1000 aa residues) proline-rich region and a SAM domain. The ankyrin repeats interact with {alpha}-fodrin, an actin-regulating protein, and Sharpin, a protein implicated in Shank multimerization (8, 9). The Shank PDZ domain interacts with the GKAP/SAPAP family of synaptic scaffold proteins and various membrane proteins including the calcium-independent receptor for latrotoxin, somatostatin receptors, and metabotropic glutamate receptors (10, 11, 12, 13, 14, 15, 16). The long proline-rich region of Shank associates with IRSp53 (an insulin receptor tyrosine kinase substrate protein), Homer (an immediate early gene product that binds the group I metabotropic receptors and inositol 1,4,5-trisphosphate receptors), dynamin (a GTPase that regulates endocytosis), and cortactin (a regulator of the cortical actin cytoskeleton) (16, 17, 18, 19, 20). The C-terminal SAM domain mediates multimerization of Shank proteins (10). There are several splice variants of Shank with alternative translational start and stop codons, suggesting that the Shank protein interactions are regulated by alternative splicing (11, 12, 21, 22).

Functionally, Shank is involved in the morphogenesis of dendritic spines (3, 23). Overexpression of Shank proteins promotes the maturation of spines in cultured neurons (24). The enhanced spine maturation by Shank requires the interaction of Shank with Homer, a protein that binds to metabotropic glutamate receptors and inositol 1,4,5-trisphosphate receptors (16). In addition, expression of dominant-negative Shank constructs decreases spine density, suggesting that Shank is involved in spine formation or maintenance.

PIX/Cool is a family ({alpha}PIX and {beta}PIX) of guanine nucleotide exchange factors for the Rac1 and Cdc42 small GTPases (25, 26, 27). PIX binds p21-activated kinase (PAK), a family of Rac/Cdc42-activated serine/threonine kinases (28), and promotes functional coupling of Rac1/Cdc42 and PAK (25). PIX also interacts with GIT/Cat/PKL/p95-APP, a family of multidomain signaling integrators with GTPase-activating protein activity for ADP ribosylation factor small GTPases, and regulates the dynamics of focal adhesion complexes (29). The function of PIX in neurons was suggested recently (30) by a genetic study on dPIX, a Drosophila homolog of PIX (31). Deletion of the dpix gene leads to defects in the structure of the neuromuscular junction and decreased synaptic levels of proteins including PAK, the PDZ domain-containing protein Dlg, and the glutamate receptor subunit GluRIIA (30). This suggests that PIX is an important organizer at the neuromuscular junction, but it remains unknown whether PIX plays a role in central synapses and, if so, how PIX regulates synaptic organization.

Here we report a novel interaction of Shank with {beta}PIX and show that Shank promotes the synaptic localization of {beta}PIX and {beta}PIX-associated PAK. In light of the fact that Rac/Cdc42 and PAK regulate the actin cytoskeleton (28) and that dendritic spines are actin-rich structures (2), our results suggest that Shank recruits {beta}PIX and {beta}PIX-associated proteins to spines and regulates postsynaptic structure.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Two-hybrid Assay—Yeast two-hybrid assay was performed using the L40 yeast strain as described previously (32). Full-length (aa 1–646) {beta}PIX were amplified by PCR from mouse {beta}PIX-a splice variant (27) and subcloned into the EcoRI-SalI site of pBHA (a vector containing LexA DNA-binding domain). Small cDNA fragments of {beta}PIX (aa 640–646 wild-type and point mutants) were generated by annealing oligonucleotides and subcloning them into the EcoRI-BamHI site of pBHA. The following PDZ domains were subcloned into pGAD10 (a prey vector; Clontech): Shank2 PDZ (aa 30–137; BamHI), Shank3 PDZ (aa 121–328; BamHI-EcoRI), SAP97 PDZ1–2 (aa 224–404; BamHI-EcoRI), GRIP2 PDZ1 (aa 41–136; BamHI-EcoRI), GRIP2 PDZ2 (aa 148–245; BamHI-EcoRI), and GRIP2 PDZ3 (aa 247–339; BamHI-EcoRI). The other pGAD10 PDZ constructs have been described previously (32, 33, 34).

Antibodies—Polyclonal {beta}PIX antibodies were generated by immunizing rabbits (1254) and guinea pigs (1257) with H6-{beta}PIX (aa 294–646) as immunogen. Specific {beta}PIX antibodies were affinity-purified after immobilizing the protein to a polyvinylidene difluoride membrane. The following antibodies have been described: EGFP (1167) (35), PSD-95 (SM55) (35), Shank (3856, pan-Shank) (10), Shank (1123, pan-Shank) (9), and GIT1 (du139) (36). The following antibodies were obtained from commercial sources: HA rabbit polyclonal (Santa Cruz Biotechnology), FLAG M2 monoclonal (Sigma), synaptophysin SVP38 (Sigma), MAP2 (Sigma), phospho-neurofilament H N52 (Sigma), PAK N-20 (Santa Cruz Biotechnology), vinculin hVIN-1 (Sigma), and p130Cas (Transduction Laboratories).

Expression Constructs—To generate various EGFP-tagged {beta}PIX constructs, the following regions of {beta}PIX were PCR amplified and subcloned into pEGFP-C1 (Clontech) using the indicated enzyme sites: full-length (aa 1–646; SalI-KpnI), SH3 (aa 1–70; EcoRI-BamHI), DH (aa 89–286; EcoRI-KpnI), PH (aa 280–417; EcoRI-BamHI), PXXP (aa 396–502; EcoRI-BamHI), GBD (aa 486–566; EcoRI-BamHI), LZ (aa 575–642; EcoRI-BamHI), {Delta}SH3 (aa 61–646; EcoRI-KpnI), {Delta}DH (aa 1–646 {Delta}100–279; SalI-KpnI-BamHI), {Delta}PH (aa 1–646 {Delta}287–400; SalI-KpnI-KpnI), {Delta}PXXP (aa 1–646 {Delta}407–494; SalI-KpnI-KpnI), {Delta}GBD (aa 1–646 {Delta}496–555; SalI-KpnI-KpnI), {Delta}LZ (aa 1–646 {Delta}587–639; EcoRI-KpnI-KpnI), {Delta}ETNL (aa 1–642; EcoRI-KpnI), and {Delta}(LZ-ETNL) (aa 1–586; EcoRI-KpnI). EGFP-tagged full-length {alpha}PIX (aa 1–728) was subcloned into the EcoRI site of pEFGP-C1. The last seven aa of {beta}PIX (aa 640–646) and GKAP (aa 660–666) were generated by annealing oligonucleotides and subcloning them into the KpnI-BamHI site of pEGFP-C1. The following constructs have been described: FLAG-tagged full-length GIT1 (36), HA-tagged full-length Shank2 and Shank3 (21), and HA-tagged full-length and deletion variants of Shank1B (24).

GST Pull Down Assay—For pull down, the last seven aa of {beta}PIX (wild-type and L646A mutant) were generated by annealing oligonucleotides and subcloning them into the BamHI-EcoRI site of pGEX4T-1. The PDZ domain of Shank1 (aa 584–690) was subcloned into the BamHI site of pGEX4T-1. For pull down, HEK293T cells were transfected with various {beta}PIX constructs, GIT1, GKAP last seven aa, and Shank2. Two days after transfection, HEK293T cells were harvested and extracted by incubating with phosphate-buffered saline containing 1% Triton X-100 (for cells transfected with various {beta}PIX and GIT1) or with radioimmune precipitation assay buffer (pH 7.5, 50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS; for cells transfected with GKAP last seven aa and Shank2) at 4 °C for 30 min. After centrifugation, the supernatant was incubated with 5 µg of GST fusion proteins, or GST alone, for 30 min at room temperature, followed by precipitation with glutathione-Sepharose 4B resin. The precipitates were analyzed by immunoblotting with antibodies against HA (0.4 µg/ml), EGFP (1167, 1:1000), and FLAG (1 µg/ml).

Preparation of PSD and Subcellular Fractions—Subcellular fractionation of rat brain was performed as described previously (37). Adult (~6 weeks) rat brains were homogenized in ice-cold homogenization buffer (0.32 M sucrose, 4 mM HEPES, pH 7.3) supplemented with protease inhibitors. After centrifuging the homogenates twice at 900 and 1000 x g for 10 min, the supernatant was centrifuged at 12000 x g for 15 min. The supernatant was saved as the cytosolic fraction (S2). The pellet was resuspended in homogenization buffer and centrifuged at 13000 x g for 15 min to obtain the crude synaptosomal fraction (pellet, P2). PSD fractions were purified as described previously (38).

Coimmunoprecipitation Assay in Heterologous Cells and in Rat Brain—Transfected HEK293T cells were extracted with binding buffer (phosphate-buffered saline containing 1% Triton X-100 or radioimmune precipitation assay buffer) and incubated with HA-agarose (Sigma) or antibodies against EGFP (1167; 4 µg/ml) at 4 °C for 90 min, followed by precipitation with protein A-Sepharose (Amersham Biosciences). For in vivo coimmunoprecipitation, the crude synaptosomal fraction of adult rat brain was solubilized with DOC buffer (50 mM Tris-HCl, 1% sodium deoxycholate, pH 9.0), dialyzed against binding/dialysis buffer (50 mM Tris-HCl, 0.1% Triton X-100, pH 7.4), and centrifuged. The supernatant was incubated with {beta}PIX (1254; 7 µg/ml) antibody, Shank (3856; 10 µg/ml) antibody, or rabbit IgG (10 µg/ml; negative control) for 2 h and then with protein A-Sepharose for 2 h. The precipitates were analyzed by immunoblotting with antibodies against EGFP (1167; 1:1000), HA (0.4 µg/ml), {beta}PIX (1254; 0.2 µg/ml or 1257; 1 µg/ml), Shank (3856; 1:2000), GIT1 (du139; 1:2000), PAK (1 µg/ml), vinculin (1:1000), and p130Cas (1:1000).

Immunohistochemistry on Rat Brain Sections—Adult rats were perfused with 4% paraformaldehyde, and brain sections (50 µm) were cut using a vibratome. Brain sections were permeabilized by incubation in phosphate-buffered saline containing 50% ethanol at room temperature for 30 min. For immunofluorescence staining, brain sections were incubated with {beta}PIX (1254; 1 µg/ml) antibodies overnight at room temperature, followed by Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) for 2 h at room temperature. Images were captured using a LSM510 confocal laser scanning microscope (Zeiss).

Primary Neuron Culture, Transfection, and Immunocytochemistry— Cultured hippocampal neurons were prepared from embryonic (E18) rat brain as described (39). Disassociated neurons were placed in neurobasal medium supplemented with B27, 0.5 mM L-glutamine, 12.5 µM glutamate, and penicillin-streptomycin (Invitrogen) for 3 h and grown in fresh medium without glutamate. Low density cultures were used for colocalization studies. At 21 days in vitro (DIV), hippocampal neurons were fixed and permeabilized with precooled methanol at -20 °C for 15 min and incubated with primary antibodies against {beta}PIX (1254; 1 µg/ml), Shank (1123; 1:150), synaptophysin (1:200), MAP2 (1:500), neurofilament-H (1:500), and PAK (10 µg/ml), followed by Cy3- or fluorescein isothiocyanate-conjugated secondary antibodies. Neurons were transfected at DIV 19 using a mammalian transfection kit (Invitrogen) and stained at DIV 21 using the same method used for colocalization experiments.

Image Acquisition and Analysis—Images were analyzed blind using MetaMorph image analysis software (Universal Imaging). The parameter settings were kept constant for all scans. Transfected neurons were chosen randomly for quantitation from immunostained coverslips from two to three independent experiments. Synaptic areas were defined as discrete Shank-positive regions. For each neuron studied, the synaptic targeting of {beta}PIX was determined by measuring the average fluorescence intensity of {beta}PIX in 10 individual synaptic areas per neuron. Statistical significance was determined by Student's t test. N numbers refer to the number of neurons quantified.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Interaction between Shank and {beta}PIX in Vitro—We reported recently (40) that {beta}PIX is enriched in the PSD and associates with the GIT-liprin-{alpha}-GRIP complex that is involved in the regulation of synaptic targeting of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors. We noticed that the C terminus of {beta}PIX ends with Asp-Glu-Thr-Asn-Leu (DETNL), a sequence that fits to the class I PDZ-binding consensus (41) and closely mimics that of GKAP/SAPAP (QTRL), a family of proteins that interact with Shank (42, 43, 44). When tested for binding to various PDZ domains in a yeast two-hybrid assay, the C terminus of {beta}PIX interacted with the PDZ domains from the Shank family proteins (Shank1, Shank2, and Shank3) but not with those from other proteins including PSD-95, SAP97, S-SCAM, GRIP1, and GRIP2/ABP (Fig. 1A). A point mutation of the {beta}PIX C terminus in which the last aa, Leu-646, of {beta}PIX was converted into Ala (DETNA) but not a conserved mutation (DQTNL) eliminated the Shank-{beta}PIX interaction (Fig. 1B). Intriguingly mutation of the Asp residue at the -4 position to Asn (NETNL and NQTNL) eliminated the Shank-{beta}PIX interaction (Fig. 1B), suggesting that the residues upstream of the known PDZ-binding consensus sites (in general the last three residues) are also important for PDZ recognition in the Shank-{beta}PIX interaction.



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FIG. 1.
Characterization of the interaction between Shank and {beta}PIX in vitro. A, specific interaction of {beta}PIX with Shank PDZ domains in the yeast two-hybrid assay. The last seven aa residues of {beta}PIX in pBHA were tested for binding to PDZ domains from various PDZ-domaincontaining proteins in pGAD10. HIS3 activity was as follows: +++, >60%; ++, 30–60%; +, 10–30%; -, no significant growth. {beta}-Galactosidase was as follows: +++, <45 min; ++, 45–90 min; +, 90–240 min; -, no significant {beta}-galactosidase activity. B, interaction of the C terminus of {beta}PIX with Shank. Variants of the {beta}PIX C terminus (wild-type and point mutants of the backbone of the last seven aa residues) in pBHA were tested against Shank1 PDZ in pGAD10 in a yeast two-hybrid assay. PXXP, proline-rich region. Small numbers refer to aa residues at the boundaries of domains. The mutated residues are underlined in bold. C, pull down of Shank by GST fusion proteins of {beta}PIX (last seven residues, wild-type, and point mutant). Lysates of HEK293T cells transfected with HA-Shank2 were pulled down by indicated {beta}PIX GST fusion proteins and characterized by immunoblotting with HA antibodies. D and E, coimmunoprecipitation between Shank and {beta}PIX in heterologous cells. HEK293T cell lysates transfected with full-length HA-Shanks (Shk) + EGFP-{beta}PIX, EGFP-{beta}PIX alone, or HA-Shank2 alone were immunoprecipitated with HA or EGFP antibodies and characterized by immunoblotting with HA and EGFP antibodies. Transf, transfection; IP, immunoprecipitation. F, absence of detectable interaction between Shank and {alpha}PIX in coimmunoprecipitation assay.

 

In GST pull down assay, a GST fusion protein containing the last seven residues of {beta}PIX (GST-{beta}PIX last seven aa) but not GST alone pulled down Shank2 expressed in HEK293T cells (Fig. 1C). However, a GST-{beta}PIX last seven aa mutant in which the last residue Leu was changed into Ala (GST-{beta}PIX last seven aa L646A) did not pull down Shank2. These results are consistent with the yeast two-hybrid results and indicate that the Shank-{beta}PIX interaction is mediated by the canonical PDZ-peptide interaction.

In HEK293T cell lysates doubly transfected with HA-tagged Shanks (HA-Shank1, HA-Shank2, or HA-Shank3) and EGFP-tagged {beta}PIX (EGFP-{beta}PIX), HA antibodies immunoprecipitated HA-Shanks and coprecipitated {beta}PIX (Fig. 1D). HA antibodies did not bring down singly expressed {beta}PIX. Conversely, incubation of the cell lysates doubly transfected with HA-Shank2 + EGFP-{beta}PIX with EGFP antibodies brought down {beta}PIX and coprecipitated HA-Shank (Fig. 1E). These results indicate that full-length Shank and {beta}PIX form a coimmunoprecipitable complex in heterologous cells.

The PIX family contains two members, {alpha}PIX and {beta}PIX, that share similar domain structure (25). Like {beta}PIX, {alpha}PIX contains an LZ domain that shares 75% aa sequence identity with the {beta}PIX LZ domain, although {alpha}PIX does not have a PDZ-binding motif at its C terminus. However, {alpha}PIX did not coimmunoprecipitate with Shank1 (Fig. 1F), suggesting that Shank specifically interacts with {beta}PIX but not with {alpha}PIX.

The LZ Domain and the C-terminal PDZ-binding Motif of {beta}PIX Mediate the Interaction with the PDZ Domain of Shank—Although the results mentioned above clearly suggest that the C-terminal PDZ-binding motif of {beta}PIX interacts with the PDZ domain of Shank, it is possible that other regions of Shank and {beta}PIX may mediate the interaction. We first tested this possibility by pulling down various deletion variants of {beta}PIX (depicted in Fig. 2A) with the GST fusion protein containing the PDZ domain of Shank1 (GST-Shank1 PDZ) (Fig. 2B). GST-Shank1 PDZ pulled down the last seven aa residues of {beta}PIX (EGFP-{beta}PIX last seven aa), as expected. Intriguingly, GST-Shank1 PDZ also pulled down EGFP-{beta}PIX containing the LZ domain with a strong coiled-coil property (EGFP-{beta}PIX LZ), suggesting that the LZ domain of {beta}PIX also binds to the PDZ domain of Shank. In contrast, none of the other domains of {beta}PIX (SH3, DH, PH, PXXP, and GBD) was pulled down by GST-Shank1 PDZ. Consistently, {beta}PIX deletions lacking the LZ domain (EGFP-{beta}PIX {Delta}LZ) and the last four residues (EGFP-{beta}PIX {Delta}ETNL) showed a significantly reduced and undetectable pull down, respectively, by GST-Shank1 PDZ. As expected, EGFP-{beta}PIX lacking the region from the LZ domain to the C terminus (EGFP-{beta}PIX {Delta}(LZ-ETNL)) was not pulled down by the Shank PDZ. In contrast, deletion of the other domains of {beta}PIX ({Delta}SH3, {Delta}DH, {Delta}PH, {Delta}PXXP, and {Delta}GBD) did not affect the pull down of {beta}PIX by the Shank PDZ. These results indicate that the LZ domain and the C-terminal PDZ-binding motif of {beta}PIX mediate its interaction with Shank.



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FIG. 2.
The LZ domain and the C-terminal-binding motif of {beta}PIX mediate the interaction with Shank in a pull down assay. A, diagram depicting deletion variants of {beta}PIX and summary of their interaction with Shank in pull down (Fig. 2) and coimmunoprecipitation (Fig. 3) assays. CoIP, coimmunoprecipitation. B, pull down of {beta}PIX (individual domains and deletion variants) by Shank1 PDZ. Lysates of HEK293T cells transfected with indicated EGFP-{beta}PIX variants were pulled down by GST-Shank1 PDZ, and the precipitates were characterized by immunoblotting with EGFP antibodies.

 



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FIG. 3.
The LZ domain and the C-terminal PDZ-binding motif of {beta}PIX mediate the interaction with Shank in a coimmunoprecipitation assay. Lysates of HEK293T cells doubly transfected with HA-Shank1 + EGFP-{beta}PIX individual domains (A), HA-Shank1 + EGFP-{beta}PIX deletions variants (B) or singly transfected with EGFP-{beta}PIX variants were immunoprecipitated by HA-agarose and characterized by immunoblotting with HA and EGFP antibodies. Input, 2%.

 
In coimmunoprecipitation assays, EGFP-{beta}PIX LZ or EGFP-{beta}PIX last seven aa but not other domains of {beta}PIX formed a complex with HA-Shank1 full-length in transiently transfected HEK293T cells (Fig. 3A). Consistently, EGFP-{beta}PIX {Delta}LZ and EGFP-{beta}PIX {Delta}ETNL showed reduced coimmunoprecipitation with Shank (Fig. 3B), and EGFP-{beta}PIX {Delta}(LZ-ETNL) completely lost its Shank binding. In contrast, deletion of the other domains in {beta}PIX ({Delta}SH3, {Delta}DH, {Delta}PH, {Delta}PXXP, and {Delta}GBD) did not affect the coimmunoprecipitation of {beta}PIX with Shank. The small but significant coimmunoprecipitation observed in EGFP-{beta}PIX {Delta}ETNL (Fig. 3B) contrasts with the complete loss of its interaction with the Shank PDZ in the pull down assay (Fig. 2B). This could be because of the fact that the Shank constructs used in the two assays were different; the PDZ domain and the full-length were used in the pull down and coimmunoprecipitation assays, respectively. Indeed, EGFP-{beta}PIX {Delta}ETNL failed to coimmunoprecipitate with HA-Shank1 PDZ (data not shown), although further details remain to be determined. Taken together, these results, summarized in Fig. 2A, further confirm that the LZ domain and the C-terminal PDZ-binding motif of {beta}PIX mediate its interaction with Shank.

Conversely, in HEK293T cells cotransfected with EGFP-{beta}PIX full-length and HA-Shank1B deletion variants (depicted in Fig. 4A), all of the HA-Shank deletion variants containing the PDZ domain coimmunoprecipitated with {beta}PIX (Fig. 4B). In contrast, Shank deletions lacking the PDZ domain did not show any detectable coimmunoprecipitation with {beta}PIX. These results, summarized in Fig. 4A, suggest that the PDZ domain of Shank is the major determinant of {beta}PIX binding.



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FIG. 4.
The PDZ domain of Shank mediates the interaction with {beta}PIX. A, diagram depicting deletion variants of Shank1B, a splice variant of Shank1 lacking the C-terminal SAM domain (24), and summary of their interaction with {beta}PIX in coimmunoprecipitation assays. Ank, ankyrin repeats; PDZ, PSD-95/Dlg/ZO-1 domain; Pro, proline-rich region. PDZPDZ indicates a tandem construct. B, coimmunoprecipitation of {beta}PIX with deletion variants of Shank1B. Lysates of HEK293T cells transfected with EGFP-{beta}PIX full-length + HA-Shank1B deletion variants, or EGFP-{beta}PIX full-length alone, were immunoprecipitated with HA-agarose and characterized by immunoblotting with HA and EGFP antibodies. Input, 2%.

 

Spatiotemporal Expression of {beta}PIX and Its Association with Shank in Rat Brain—To study {beta}PIX in vivo, in particular its spatiotemporal expression and the association with Shank, we generated polyclonal antibodies against {beta}PIX (rabbit 1254 and guinea pig 1257) using a H6 fusion protein containing the second half (aa 294–646) of {beta}PIX as immunogen. The 1254 {beta}PIX antibody specifically recognized {beta}PIX but not {alpha}PIX in immunoblot analysis (Fig. 5A). Similar results were obtained for the 1257 {beta}PIX antibody (data not shown). In rat brain, the {beta}PIX (1254) antibody recognized four major bands (66–105 kDa; see Fig. 5B), which may represent splice variants of {beta}PIX (45, 46). In support of this, one of the brain {beta}PIX bands matched the size of the {beta}PIX-a splice variant expressed in heterologous cells (Fig. 5B).



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FIG. 5.
Spatiotemporal expression pattern of {beta}PIX and its association with Shank in rat brain. A, specific recognition of {beta}PIX by the 1254 {beta}PIX antibody. HEK293T cells transfected with EGFP-{alpha}PIX or EGFP-{beta}PIX were characterized by immunoblotting with {beta}PIX (1254) and EGFP (for normalization) antibodies. The numbers (1 and 0.1) indicate relative amounts of proteins loaded. B, {beta}PIX proteins expressed in brain. The crude synaptosomal (P2) and cytosolic (S2) fractions of adult rat brain were immunoblotted with {beta}PIX (1254) antibodies. The asterisks indicate {beta}PIX bands expressed in brain, one of which matches the size of the {beta}PIX-a splice variant expressed in HEK293T (293T) cells. Un, untransfected HEK293T cells. C, expression of {beta}PIX in various brain regions. Total homogenates of adult rat brain regions were immunoblotted with {beta}PIX (1254) and PSD-95 (Sm55) antibodies. Ctx, cortex; Cb, cerebellum; Hc, hippocampus; R, other regions of the brain. D, expression of {beta}PIX during postnatal brain development. Total homogenates of rat brain at various stages of postnatal development were immunoblotted with {beta}PIX (1254), PSD-95, and Shank (3856) antibodies. P1, postnatal day 1; Ad, adult (6 weeks). E, lack of PSD enrichment in PAK. PSD fractions extracted with Triton X-100 once (PSD I), twice (PSD II), or with Triton X-100 and a strong detergent Sarcosyl (PSD III) were immunoblotted with PAK, {beta}PIX (1254), and PSD-95 antibodies. F and G, coimmunoprecipitation of Shank with {beta}PIX and {beta}PIX-associated proteins in brain. Detergent lysates of the crude synaptosomal fraction of adult rat brain were immunoprecipitated with {beta}PIX (1254), Shank (3856), or rabbit IgG (IgG) and characterized by immunoblotting with the indicated antibodies.

 

Expression of {beta}PIX proteins was detected in various brain regions including the cortex, cerebellum, and hippocampus (Fig. 5C). During the postnatal development of rat brain, expression levels of {beta}PIX reached a peak around postnatal day 7 and then gradually decreased to adult levels (Fig. 5D). This contrasts with the steady increase in expression levels of PSD-95 and Shank during the first 3 weeks of postnatal development (Fig. 5D). In contrast to the reported enrichment of {beta}PIX and {beta}PIX-binding GIT1 in the PSD (40), PAK, another {beta}PIX-binding protein, was not enriched in the PSD although a significant portion of PAK was detected in the crude synaptosomal fraction as was {beta}PIX (Fig. 5E), suggesting that PAK is not a core component of the PSD.

We next determined whether Shank and {beta}PIX form a complex in brain. Incubation of extracts of the crude synaptosomal fraction of adult rat brain with {beta}PIX antibodies brought down {beta}PIX and coprecipitated Shank and {beta}PIX-associated GIT1 and PAK (Fig. 5F). Irrelevant proteins such as vinculin and p130Cas were not coimmunoprecipitated with {beta}PIX. Conversely, Shank antibodies pulled down Shank and coprecipitated {beta}PIX, GIT1, and PAK but not vinculin and p130Cas (Fig. 5G). These results suggest that Shank forms a complex with {beta}PIX and {beta}PIX-associated proteins in brain.

Shank and {beta}PIX Colocalize at Synaptic Sites in Cultured Neurons—Shank proteins are mainly localized to synaptic sites in cultured neurons (10, 11). However, little is known about the subcellular distribution pattern of {beta}PIX. Using the {beta}PIX (1254) antibody, we determined the subcellular distribution of {beta}PIX in cultured hippocampal neurons (DIV 21) (Fig. 6). Immunofluorescence signals of {beta}PIX colocalized with both MAP2-positive dendrites and MAP2-negative axons (arrow; see Fig. 6A). Consistently, {beta}PIX colocalized with neurofilament-H-positive axons (Fig. 6B, arrow), suggesting that {beta}PIX distributes to both dendrites and axons. At higher magnifications, {beta}PIX immunoreactivity was mainly detected in punctate structures (Fig. 6C1). Some of the punctate {beta}PIX-positive structures colocalized with synaptophysin, a marker for the presynaptic nerve terminal, but a significant portion of {beta}PIX structures did not (Fig. 6C), suggesting that {beta}PIX proteins are widely distributed to both synaptic and non-synaptic sites. Some {beta}PIX signals colocalized with Shank (Fig. 6D), suggesting that {beta}PIX is localized to excitatory synaptic sites.



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FIG. 6.
Subcellular localization of {beta}PIX and its colocalization with Shank in cultured neurons. Cultured hippocampal neurons (DIV 21) were stained by double immunofluorescence staining for {beta}PIX (1254) (red) and MAP2, neurofilament-H (NF-H), synaptophysin (Syn-Phy), or Shank (1123) (green). Arrows in A and B indicate axons. Merged images of A1 + A2, B1 + B2, and C1 + C2 are shown in A3, B3, and C3, respectively. Scale bar, 30 µm.

 

Distribution of {beta}PIX in Brain Regions—In rat brain slices, immunofluorescence signals of {beta}PIX were widely detected in various regions of rat brain including the cortex (Fig. 7A), hippocampus (Fig. 7B), and cerebellum (Fig. 7C). At higher magnifications, strong {beta}PIX signals were observed in hippocampal pyramidal neurons (Fig. 7, D and E, examples from CA1 and CA3 regions of hippocampus, respectively) and cerebellar Purkinje cells (Fig. 7F). Preincubation of {beta}PIX antibodies with immunogen eliminated the signal (data not shown). Double immunofluorescence staining for {beta}PIX and glial fibrillary acidic protein, a marker for glial cells, showed no colocalization between the two proteins at least in cortex and hippocampus (data not shown), suggesting that {beta}PIX is mainly expressed in neurons. Taken together, these results suggest that {beta}PIX is widely expressed in brain regions.



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FIG. 7.
Distribution of {beta}PIX in brain regions. Adult rat brain slices were incubated with {beta}PIX (1254) antibodies, followed by Cy3-conjugated secondary antibodies. A, cortex; B, hippocampus; C, cerebellum; D, CA1 region of hippocampus; E, CA3 region of hippocampus; F, cerebellum at a higher magnification. The scale bar was as follows: A, 100 µm; B and C, 400 µm; D and E, 50 µm; F, 100 µm.

 

Overexpression of Shank in Cultured Neurons Promotes Synaptic Accumulation of {beta}PIX and PAK—The majority of Shank proteins distribute to synaptic sites (10, 11), whereas a significant portion of {beta}PIX staining is detected at extrasynaptic sites (Fig. 6, C and D). Biochemically, Shank proteins are mainly detected in the crude synaptosomal fraction of rat brain (9), whereas {beta}PIX distributes to both synaptosomal and cytosolic fractions (Fig. 5E) (40). These results suggest the hypothesis that Shank may recruit {beta}PIX to spines. To this end, we tested the effect of Shank overexpression on the subcellular localization of endogenous {beta}PIX in cultured neurons (Fig. 8). Overexpression of Shank1B in cultured hippocampal neurons markedly increased the colocalization of endogenous {beta}PIX with Shank (Fig. 8A), in contrast to the partial synaptic localization of {beta}PIX in untransfected neurons (Fig. 6, C and D). Quantitative analysis indicated that the immunofluorescence staining intensity of {beta}PIX at synapses (as defined by the average fluorescence intensity of {beta}PIX in synaptic area) was significantly increased in Shank-overexpressing neurons (181.2 ± 25.7, n = 30 cells; *, p < 0.0001; see Fig. 8D), compared with untransfected neurons (111.1 ± 41.6, n = 30; see Fig. 8D). These results, considering a previous report that overexpressed Shank proteins are mostly targeted to postsynaptic spines (24), suggest that Shank promotes accumulation of {beta}PIX in dendritic spines. In addition, synaptic labeling of PAK was also significantly increased by Shank overexpression (190.3 ± 18.3, n = 25; *, p < 0.0001; see Fig. 8, B and E), relative to untransfected neurons (136.0 ± 28.7, n = 25; see Fig. 8, C and E). Taken together, these results suggest that Shank promotes recruitment of {beta}PIX and PAK to spines.



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FIG. 8.
Overexpression of Shank in cultured neurons promotes accumulation of {beta}PIX and PAK in dendritic spines. A and B, cultured hippocampal neurons (DIV 19) were transfected with HAShank1B (exo-Shank), and changes in the subcellular distribution of endogenous {beta}PIX and endogenous PAK were monitored by double immunofluorescence staining for {beta}PIX (A1, red; 1254, rabbit polyclonal), PAK (B1, red; rabbit polyclonal), and Shank (A2 and B2, green; 1123, guinea pig polyclonal) at DIV 21. Neurons expressing exogenous Shank could be easily identified by their higher immunofluorescence relative to neighboring untransfected neurons. C, limited colocalization of endogenous PAK and Shank. Cultured hippocampal neurons (DIV 21) were doubly labeled for PAK (C1, red) and Shank (C2, green). Scale bar, 30 µm. D and E, quantitation of changes in spine accumulation of {beta}PIX (D) and PAK (E) induced by Shank overexpression. Immunofluorescence staining intensities of {beta}PIX and PAK at synapses (as defined by the average fluorescence intensity of {beta}PIX and PAK in synaptic area) in untransfected (Un) and transfected (Shank) neurons were compared. Histograms show mean ± S.D. *, p < 0.0001. AU, arbitrary unit.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A Novel Mode of Protein-Protein Interactions in the Shank PDZ Domain—Our results indicate that both the LZ domain and the C-terminal PDZ-binding motif of {beta}PIX are involved in the interaction with the PDZ domain of Shank. We recently determined (47) the crystal structure of the Shank PDZ in a complex with a peptide mimicking the C-terminal PDZ-binding motif of GKAP/SAPAP.2 The structure of the Shank PDZ-peptide complex indicates an association of the peptide with the peptide-binding groove, a well known region in the PDZ domain for peptide binding (41). Assuming that a similar binding mode also applies to the interaction between the Shank PDZ and the {beta}PIX C terminus, the LZ domain of {beta}PIX is likely to bind to a region of the Shank PDZ outside the groove. The participation of "non-groove" surfaces of the PDZ domain in protein interactions is not uncommon. The PDZ7 of GRIP1 uses a novel hydrophobic surface distinct from the groove to interact with GRASP-1 (48), a neuronal Ras guanine nucleotide exchange factor (49). Similarly, a novel surface of the PDZ6 of GRIP1 outside the groove mediates self-dimerization (50). This mode of parallel binding, simultaneous binding of the LZ and the extreme C terminus to the PDZ, may ensure a stronger interaction between Shank and {beta}PIX and/or provide additional sites for regulation of the interaction.

The LZ domain of {beta}PIX is known to mediate homo- and heterodimerization (46, 51). It remains to be determined whether the dimerization affects the Shank-{beta}PIX interaction or vice versa. However, if these two interactions occur independently, the Shank-{beta}PIX interaction may function as a mechanism to bring additional {beta}PIX and {beta}PIX-binding proteins (PAK, Rac1/Cdc42, and GIT) into the vicinity of Shank. Alternatively, {beta}PIX dimers may further stabilize Shank multimers that are known to be formed by the C-terminal SAM domain (10). This hypothesis is reminiscent of the proposed functions of Homer that, through self-multimerization, links Shank to inositol 1,4,5-trisphosphate receptors and metabotropic glutamate receptors (10, 16, 24).

Functions of the Interaction between Shank and {beta}PIX—Over-expression of Shank in cultured neurons promotes synaptic accumulation of {beta}PIX (Fig. 8, A and D), suggesting that Shank recruits {beta}PIX to spines. This model is supported by the immunohistochemical and biochemical results that Shank mainly distributes to synaptic sites (9, 10, 11, 16, 22) whereas {beta}PIX is partially synaptic (see Fig. 5E and Fig. 6, C and D) (40). This is also consistent with the hypothesis that Shank, through local translation of dendritic Shank mRNAs, may act as a scaffold recruiting various synaptic proteins to spines (6, 13).

In addition to {beta}PIX, synaptic accumulation of PAK is also increased by Shank overexpression (Fig. 8, B and E). The enhanced synaptic accumulation of PAK appears to occur through its interaction with {beta}PIX. In support of this, {beta}PIX is enriched in the PSD (Fig. 5E) (40), whereas PAK is not enriched in the PSD although a significant fraction of it is present in the crude synaptosomal fraction (Fig. 5E). Critically, a Drosophila genetic study demonstrated that mutations in the dpix gene lead to a complete loss of synaptic localization of dPAK (30). These results are also consistent with the results from non-neuronal cells that {beta}PIX binding is required for localization of PAK to focal complexes (25).

How might Shank promote synaptic accumulation of {beta}PIX? It may occur through the direct or indirect interaction between Shank and {beta}PIX, which are not mutually exclusive. Previously, Sala et al. (24) have shown that the Shank-induced synaptic accumulation of Homer is eliminated by mutations that disrupt the Shank-Homer interaction. However, we could not take a similar approach, because the PDZ domain of Shank, the region to which {beta}PIX binds, has been shown to be critical for synaptic targeting of Shank (24), making it impossible to distinguish between direct and indirect mechanisms. Nevertheless, it has been shown in non-neuronal cells that the LZ domain of {beta}PIX is critical for targeting of {beta}PIX to the cell periphery and inducing membrane ruffles and microvillus-like structures (46, 51). These results and our finding that the {beta}PIX LZ binds to the Shank PDZ support the first hypothesis of direct recruitment, although further details remain to be elucidated.

Neuronal Functions of {beta}PIX—We demonstrated that the expression levels of {beta}PIX reaches a peak around postnatal day 7 and then decreases gradually to adult levels (Fig. 5D). Because dendritic spines are in general poorly developed during early postnatal stages (1–2 weeks), the high levels of {beta}PIX around the first week suggest that {beta}PIX may have roles in developing neurons. {beta}PIX activates Rac1 and Cdc42 (25), small GTPases known to regulate various aspects of neuronal morphogenesis including neurite initiation, growth, guidance, branching, polarity, and synapse formation (52). Thus {beta}PIX expressed at early developmental stages may have a role associated with the Rac/Cdc42-dependent regulation of neuronal morphogenesis.

We observed steady, although reduced, levels of {beta}PIX expression in the later stages of postnatal development (Fig. 5D), suggesting that {beta}PIX also has functions in mature neurons. In mature neurons, {beta}PIX is localized to excitatory synaptic sites (Fig. 6D), enriched in the PSD (Fig. 5E) (40), and redistributed, along with PAK, to synaptic sites by Shank (Fig. 8), suggesting that {beta}PIX may regulate functions associated with dendritic spines. In dendritic spines, {beta}PIX may induce local activation of Rac1/Cdc42 and PAK, molecules known to regulate spine morphogenesis. Constitutively active Rac1 leads to the development of supernumerary spines of very small sizes in cerebellar Purkinje neurons of transgenic mice (53) and generation of filopodia- and lamellipodia-like structures in neurons of rat hippocampal and cortical slices (54, 55). In contrast, dominant-negative Rac1 leads to a progressive elimination of dendritic spines in hippocampal slices (55). Consistently, Kalirin-7, a brain-specific guanine nucleotide exchange factor for Rac1 enriched in the PSD (56), interacts with various PDZ-containing proteins including PSD-95 (57) and, by upstream stimulation of Eph receptors, increases the number and size of spine-like structures in transfected neurons in a Rac1- and PAK-dependent manner (57, 58, 59). PAK is known to regulate the actin cytoskeleton (28), a major determinant of the shape, stability, and plasticity of dendritic spines (2, 60, 61, 62). In Drosophila, dPAK is a key mediator of the dPIX-dependent regulation of postsynaptic structure and protein targeting (30). Taken together, our data, along with previous results, suggest that Shank may regulate spine dynamics through synaptic accumulation of {beta}PIX and local activation of the Rac1-PAK signaling pathway. It has been reported (24) that overexpression of Shank in cultured neurons promotes spine maturation while not affecting spine density and that overexpression of dominant-negative constructs of Shank reduces spine density. Considering the association of Shank with {beta}PIX, a possible interpretation of these results is that overexpressed dominant-negative Shank proteins may inhibit synaptic targeting of endogenous Shank that is required for spine recruitment of {beta}PIX and formation/maintenance of dendritic spines.

In conclusion, we have demonstrated that Shank associates with {beta}PIX and recruits {beta}PIX and PAK to synaptic sites. These molecular mechanisms may contribute to Shank-dependent organization of the PSD and to the regulation of dendritic spine dynamics. We are currently investigating the functions of {beta}PIX and {beta}PIX-associated proteins in the morphogenesis of dendritic spines.


    FOOTNOTES
 
* This work was supported in part by grants from the Korean Ministry of Science and Technology, the Korea Science and Engineering Foundation, and the Korea Research Foundation (to E. K.) and by National Research Laboratory Program M1-0203-00-0072 (to D. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Investigator of the Howard Hughes Medical Institute. Back

** To whom correspondence should be addressed. Tel.: 42-869-2633; Fax: 42-869-2610; E-mail: kime{at}mail.kaist.ac.kr.

1 The abbreviations used are: PSD, postsynaptic density; SH3, Src homology 3; SAM, sterile alpha motif; aa, amino acid(s); PAK, p21-associated kinase; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; PH, pleckstrin homology; DH, Dbl homology; LZ, leucine zipper; GST, glutathione S-transferase; HEK, human embryonic kidney; MAP, mitogen-activated protein; DIV, days in vitro; GBD, GIT-binding domain. Back

2 S. Park, E. Kim, and S. Eom, unpublished data. Back


    ACKNOWLEDGMENTS
 
We are grateful to Dr. Carlo Sala at CNR Institute of Neuroscience for the gift of Shank deletion constructs.



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