From the
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.
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ABSTRACT |
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INTRODUCTION |
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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
-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 (PIX and
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 PIX and show that Shank promotes the synaptic localization of
PIX and
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
PIX and
PIX-associated proteins to spines and regulates postsynaptic structure.
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EXPERIMENTAL PROCEDURES |
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AntibodiesPolyclonal PIX antibodies were generated by immunizing rabbits (1254) and guinea pigs (1257) with H6-
PIX (aa 294646) as immunogen. Specific
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 ConstructsTo generate various EGFP-tagged PIX constructs, the following regions of
PIX were PCR amplified and subcloned into pEGFP-C1 (Clontech) using the indicated enzyme sites: full-length (aa 1646; SalI-KpnI), SH3 (aa 170; EcoRI-BamHI), DH (aa 89286; EcoRI-KpnI), PH (aa 280417; EcoRI-BamHI), PXXP (aa 396502; EcoRI-BamHI), GBD (aa 486566; EcoRI-BamHI), LZ (aa 575642; EcoRI-BamHI),
SH3 (aa 61646; EcoRI-KpnI),
DH (aa 1646
100279; SalI-KpnI-BamHI),
PH (aa 1646
287400; SalI-KpnI-KpnI),
PXXP (aa 1646
407494; SalI-KpnI-KpnI),
GBD (aa 1646
496555; SalI-KpnI-KpnI),
LZ (aa 1646
587639; EcoRI-KpnI-KpnI),
ETNL (aa 1642; EcoRI-KpnI), and
(LZ-ETNL) (aa 1586; EcoRI-KpnI). EGFP-tagged full-length
PIX (aa 1728) was subcloned into the EcoRI site of pEFGP-C1. The last seven aa of
PIX (aa 640646) and GKAP (aa 660666) 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 AssayFor pull down, the last seven aa of 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 584690) was subcloned into the BamHI site of pGEX4T-1. For pull down, HEK293T cells were transfected with various
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
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 FractionsSubcellular 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 BrainTransfected 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 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),
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 SectionsAdult 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 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 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 AnalysisImages 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 PIX was determined by measuring the average fluorescence intensity of
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.
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RESULTS |
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In GST pull down assay, a GST fusion protein containing the last seven residues of PIX (GST-
PIX last seven aa) but not GST alone pulled down Shank2 expressed in HEK293T cells (Fig. 1C). However, a GST-
PIX last seven aa mutant in which the last residue Leu was changed into Ala (GST-
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-
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 PIX (EGFP-
PIX), HA antibodies immunoprecipitated HA-Shanks and coprecipitated
PIX (Fig. 1D). HA antibodies did not bring down singly expressed
PIX. Conversely, incubation of the cell lysates doubly transfected with HA-Shank2 + EGFP-
PIX with EGFP antibodies brought down
PIX and coprecipitated HA-Shank (Fig. 1E). These results indicate that full-length Shank and
PIX form a coimmunoprecipitable complex in heterologous cells.
The PIX family contains two members, PIX and
PIX, that share similar domain structure (25). Like
PIX,
PIX contains an LZ domain that shares 75% aa sequence identity with the
PIX LZ domain, although
PIX does not have a PDZ-binding motif at its C terminus. However,
PIX did not coimmunoprecipitate with Shank1 (Fig. 1F), suggesting that Shank specifically interacts with
PIX but not with
PIX.
The LZ Domain and the C-terminal PDZ-binding Motif of PIX Mediate the Interaction with the PDZ Domain of ShankAlthough the results mentioned above clearly suggest that the C-terminal PDZ-binding motif of
PIX interacts with the PDZ domain of Shank, it is possible that other regions of Shank and
PIX may mediate the interaction. We first tested this possibility by pulling down various deletion variants of
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
PIX (EGFP-
PIX last seven aa), as expected. Intriguingly, GST-Shank1 PDZ also pulled down EGFP-
PIX containing the LZ domain with a strong coiled-coil property (EGFP-
PIX LZ), suggesting that the LZ domain of
PIX also binds to the PDZ domain of Shank. In contrast, none of the other domains of
PIX (SH3, DH, PH, PXXP, and GBD) was pulled down by GST-Shank1 PDZ. Consistently,
PIX deletions lacking the LZ domain (EGFP-
PIX
LZ) and the last four residues (EGFP-
PIX
ETNL) showed a significantly reduced and undetectable pull down, respectively, by GST-Shank1 PDZ. As expected, EGFP-
PIX lacking the region from the LZ domain to the C terminus (EGFP-
PIX
(LZ-ETNL)) was not pulled down by the Shank PDZ. In contrast, deletion of the other domains of
PIX (
SH3,
DH,
PH,
PXXP, and
GBD) did not affect the pull down of
PIX by the Shank PDZ. These results indicate that the LZ domain and the C-terminal PDZ-binding motif of
PIX mediate its interaction with Shank.
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Conversely, in HEK293T cells cotransfected with EGFP-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
PIX (Fig. 4B). In contrast, Shank deletions lacking the PDZ domain did not show any detectable coimmunoprecipitation with
PIX. These results, summarized in Fig. 4A, suggest that the PDZ domain of Shank is the major determinant of
PIX binding.
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Spatiotemporal Expression of PIX and Its Association with Shank in Rat BrainTo study
PIX in vivo, in particular its spatiotemporal expression and the association with Shank, we generated polyclonal antibodies against
PIX (rabbit 1254 and guinea pig 1257) using a H6 fusion protein containing the second half (aa 294646) of
PIX as immunogen. The 1254
PIX antibody specifically recognized
PIX but not
PIX in immunoblot analysis (Fig. 5A). Similar results were obtained for the 1257
PIX antibody (data not shown). In rat brain, the
PIX (1254) antibody recognized four major bands (66105 kDa; see Fig. 5B), which may represent splice variants of
PIX (45, 46). In support of this, one of the brain
PIX bands matched the size of the
PIX-a splice variant expressed in heterologous cells (Fig. 5B).
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Expression of 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
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
PIX and
PIX-binding GIT1 in the PSD (40), PAK, another
PIX-binding protein, was not enriched in the PSD although a significant portion of PAK was detected in the crude synaptosomal fraction as was
PIX (Fig. 5E), suggesting that PAK is not a core component of the PSD.
We next determined whether Shank and PIX form a complex in brain. Incubation of extracts of the crude synaptosomal fraction of adult rat brain with
PIX antibodies brought down
PIX and coprecipitated Shank and
PIX-associated GIT1 and PAK (Fig. 5F). Irrelevant proteins such as vinculin and p130Cas were not coimmunoprecipitated with
PIX. Conversely, Shank antibodies pulled down Shank and coprecipitated
PIX, GIT1, and PAK but not vinculin and p130Cas (Fig. 5G). These results suggest that Shank forms a complex with
PIX and
PIX-associated proteins in brain.
Shank and PIX Colocalize at Synaptic Sites in Cultured NeuronsShank proteins are mainly localized to synaptic sites in cultured neurons (10, 11). However, little is known about the subcellular distribution pattern of
PIX. Using the
PIX (1254) antibody, we determined the subcellular distribution of
PIX in cultured hippocampal neurons (DIV 21) (Fig. 6). Immunofluorescence signals of
PIX colocalized with both MAP2-positive dendrites and MAP2-negative axons (arrow; see Fig. 6A). Consistently,
PIX colocalized with neurofilament-H-positive axons (Fig. 6B, arrow), suggesting that
PIX distributes to both dendrites and axons. At higher magnifications,
PIX immunoreactivity was mainly detected in punctate structures (Fig. 6C1). Some of the punctate
PIX-positive structures colocalized with synaptophysin, a marker for the presynaptic nerve terminal, but a significant portion of
PIX structures did not (Fig. 6C), suggesting that
PIX proteins are widely distributed to both synaptic and non-synaptic sites. Some
PIX signals colocalized with Shank (Fig. 6D), suggesting that
PIX is localized to excitatory synaptic sites.
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Distribution of PIX in Brain RegionsIn rat brain slices, immunofluorescence signals of
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
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
PIX antibodies with immunogen eliminated the signal (data not shown). Double immunofluorescence staining for
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
PIX is mainly expressed in neurons. Taken together, these results suggest that
PIX is widely expressed in brain regions.
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Overexpression of Shank in Cultured Neurons Promotes Synaptic Accumulation of PIX and PAKThe majority of Shank proteins distribute to synaptic sites (10, 11), whereas a significant portion of
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
PIX distributes to both synaptosomal and cytosolic fractions (Fig. 5E) (40). These results suggest the hypothesis that Shank may recruit
PIX to spines. To this end, we tested the effect of Shank overexpression on the subcellular localization of endogenous
PIX in cultured neurons (Fig. 8). Overexpression of Shank1B in cultured hippocampal neurons markedly increased the colocalization of endogenous
PIX with Shank (Fig. 8A), in contrast to the partial synaptic localization of
PIX in untransfected neurons (Fig. 6, C and D). Quantitative analysis indicated that the immunofluorescence staining intensity of
PIX at synapses (as defined by the average fluorescence intensity of
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
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
PIX and PAK to spines.
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DISCUSSION |
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The LZ domain of PIX is known to mediate homo- and heterodimerization (46, 51). It remains to be determined whether the dimerization affects the Shank-
PIX interaction or vice versa. However, if these two interactions occur independently, the Shank-
PIX interaction may function as a mechanism to bring additional
PIX and
PIX-binding proteins (PAK, Rac1/Cdc42, and GIT) into the vicinity of Shank. Alternatively,
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 PIXOver-expression of Shank in cultured neurons promotes synaptic accumulation of
PIX (Fig. 8, A and D), suggesting that Shank recruits
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
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 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
PIX. In support of this,
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
PIX binding is required for localization of PAK to focal complexes (25).
How might Shank promote synaptic accumulation of PIX? It may occur through the direct or indirect interaction between Shank and
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
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
PIX is critical for targeting of
PIX to the cell periphery and inducing membrane ruffles and microvillus-like structures (46, 51). These results and our finding that the
PIX LZ binds to the Shank PDZ support the first hypothesis of direct recruitment, although further details remain to be elucidated.
Neuronal Functions of PIXWe demonstrated that the expression levels of
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 (12 weeks), the high levels of
PIX around the first week suggest that
PIX may have roles in developing neurons.
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
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 PIX expression in the later stages of postnatal development (Fig. 5D), suggesting that
PIX also has functions in mature neurons. In mature neurons,
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
PIX may regulate functions associated with dendritic spines. In dendritic spines,
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
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
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
PIX and formation/maintenance of dendritic spines.
In conclusion, we have demonstrated that Shank associates with PIX and recruits
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
PIX and
PIX-associated proteins in the morphogenesis of dendritic spines.
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FOOTNOTES |
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|| Investigator of the Howard Hughes Medical Institute.
** 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.
2 S. Park, E. Kim, and S. Eom, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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