JOURNAL OF NEUROCHEMISTRY
| 2008 | 107 | 61–72
doi: 10.1111/j.1471-4159.2008.05585.x
, ,
*Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Japan Department of Anatomy, Fujita Health University School of Medicine, Toyoake, Japan àMolecular Biotechnology Center and §Center for Experimental Research and Medical Studies, University of Turino, Turin, Italy ¶Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Japan
Abstract p140Cap (Cas-associated protein) is an adaptor protein considered to play pivotal roles in cell adhesion, growth and Src tyrosine kinase-related signaling in non-neuronal cells. It is also reported to interact with a pre-synaptic membrane protein, synaptosome-associated protein of 25 kDa, and may participate in neuronal secretion. However, properties and precise functions of p140Cap in neuronal cells are almost unknown. Here we show, using biochemical analyses, that p140Cap is expressed in rat brain in a developmental stagedependent manner, and is relatively abundant in the synaptic plasma membrane fraction in adults. Immunohistochemistry showed localization of p140Cap in the neuropil in rat brain and immunofluorescent analyses detected p140Cap at synapses
of primary cultured rat hippocampal neurons. Electron microscopy further revealed localization at pre- and postsynapses. Screening of p140Cap-binding proteins identified a multidomain adaptor protein, vinexin, whose third Src-homology 3 domain interacts with the C-terminal Pro-rich motif of p140Cap. Immunocomplexes between the two proteins were confirmed in COS7 and rat brain. We also clarified that a presynaptic protein, synaptophysin, interacts with p140Cap. These results suggest that p140Cap is involved in neurotransmitter release, synapse formation/maintenance, and signaling. Keywords: neuron, p140Cap, synapse, synaptophysin, vinexin. J. Neurochem. (2008) 107, 61–72.
p140Cap (Cas-associated protein) is a hydrophilic 140-kDa protein that comprises two predicted coiled-coil domains, two highly charged regions, and two proline-rich domains with multiple Pro-rich (PPXY and PXXP) motifs (Fig. 1a) (Di Stefano et al. 2004). Many proteins binding to the motifs are thought to play important roles in cell morphology, cell–cell contact, cell adhesion, and cell signaling. Based on the characteristic domain structure, p140Cap is thought to function as an adaptor protein. p140Cap was first identified as an interactive partner of p130Cas (Crk-associated substrates), a protein highly tyrosine phosphorylated in fibroblasts transformed with v-Src or v-Crk oncogene [for review, see Defilippi et al. (2006)]. In non-transformed cells, p130Cas is tyrosine phosphorylated upon ligation of integrins and the phosphorylation is thought to be crucial for linkage of the actin cytoskeleton to the extracellular matrix during cell migration, cell invasion, and cell transformation
(Defilippi et al. 2006). Recently, p130Cas was reported to be a key component in the netrin signal transduction pathway and to play an important role in guiding commissural axons in vivo (Liu et al. 2007).
Received June 30, 2008; revised manuscript received July 7, 2008; accepted July 7, 2008. Address correspondence and reprint requests to Dr Koh-ichi Nagata, Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya, Kasugai 4800392, Japan. E-mail:
[email protected] Abbreviations used: Cap, Cas-associated protein; Cas, Crk-associated substrates; DIV, days in vitro; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; N-WASP, neural-Wiskott-Aldrich syndrome protein; SH3, Src-homology 3; SNAP-25, synaptosome-associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive fusion attachment protein receptors.
2008 The Authors Journal Compilation 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 61–72
61
62 | H. Ito et al.
(a)
(b)
Fig. 1 Structure of p140Cap, synaptophysin, and the mutants constructed into expression vectors. Structure of p140Cap (a), synaptophysin (b), and the mutants. Structural domains are abbreviated as follows: Pro, proline-rich region; CC, coiled-coil region; Q, charged sequence region where approximately 35% of amino acids are charged; TM, membrane spanning region; SYP, synaptophysin; Cap, Cas-associated protein.
p140Cap is also tyrosine-phosphorylated upon integrindependent adhesion or epidermal growth factor treatment, indicating a possible cooperative role with p130Cas as a downstream molecule in cell matrix and in growth factor signaling (Di Stefano et al. 2004). In this context, it is of interest that p140Cap has been shown to participate in regulating Src and the downstream signaling, leading to inhibit breast cancer cell spreading, motility, invasion, and growth (Di Stefano et al. 2007). In addition, p140Cap colocalizes with actin stress fibers in endothelial cells, suggesting that in some cell types the protein is involved in actin cytoskeletal organization (Di Stefano et al. 2004). However, precise molecular mechanisms linking p140Cap to integrin- or growth factor-mediated signaling and actin cytoskeleton-related cellular events are almost unknown. In addition to the above functions in non-neuronal cells, p140Cap appears to be involved in yet unidentified cellular events in neurons as this protein was also identified as a synaptosome-associated protein of 25 kDa (SNAP-25)-inter-
acting protein and termed SNIP (Chin et al. 2000). SNAP-25 is a component of soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNAREs), playing a major role in membrane docking of synaptic vesicles during neurotransmitter release (Hong 2005). As subcellular fractionation analyses suggest that p140Cap interacts with the cortical actin cytoskeleton as well as SNAP-25, it appears to serve as a linker protein connecting SNAP-25 to the submembranous cytoskeleton (Chin et al. 2000). In contrast to the case in non-neuronal cells, only fragmentary information is available about the functions and properties of p140Cap in brain tissues. In the present study, we therefore carried out comprehensive analyses to gain insight into the physiologic functions of p140Cap in neuronal cells. We determined expression profiles of p140Cap in rat brain during embryonic and postnatal developmental stages and confirmed localization at synapses by comprehensive analyses. In addition, we identified vinexin, a member of a novel family of adaptor proteins, as an interactive partner. It should be noted that this protein is enriched at synapses in hippocampal neurons (Ito et al. 2007). Furthermore, we show that p140Cap interacts with the pre-synaptic vesicle membrane protein, synaptophysin. The results obtained here suggest that p140Cap is involved in neurotransmitter release by coordinated interaction with synaptophysin as well as SNAP-25 and in the regulation of vinexin-mediated cellular events such as cytoskeletal organization and signaling.
Materials and methods Plasmid construction Expression plasmids harboring green fluorescent protein (GFP)- or Myc-p140Cap and Myc-p140Cap-Pro, lacking the C-terminal Prorich motif, were as described previously (Fig. 1a) (Di Stefano et al. 2004, 2007). Cap-N (aa 1–680), Cap-C2 (aa 677–1217), Cap-Cent (aa 677–866), Cap-CC (aa 677–787), and Cap-C (aa 1023–1217) were made and constructed into various vectors including pGEX-4T3, pYTH9, pEGFP-C2, and pRK5-Myc (Fig. 1a). Rat synaptophysin and the mutants, DN (aa 128–313) and DC (aa 1–247) (Fig. 1b), human neural-Wiskott-Aldrich syndrome protein (N-WASP), and human vinexin were amplified by PCR and inserted into the pRK5 vector. VinexinbDSH3-3 (aa 1–251) lacking the third Src-homology 3 (SH3) domain was also made and constructed into pRK5-Flag vector. For RNAi experiments, two single-stranded DNA oligonucleotides (top strand, 5¢-TGCTGTGATACTTCGGTCTT GGATATGTTTTGGCCACTGACTGACATATCCAACCGAAGTATCA-3¢; bottom strand, 5¢-CCTGTGATACTTCGGTTGGATATGTCAGTCAGTGGCCAAAACATATCCAAGACCGAAGTATCAC-3¢) were designed, annealed, and cloned into the pcDNA6. 2-GW/EmGFP-miR (Invitrogen, Carlsbad, CA, USA). All constructs were verified by DNA sequencing. Antibodies Using glutathione S-transferase (GST)-Cap-C expressed in Escherchia coli as an antigen, a rabbit polyclonal antibody (anti-Cap-C)
2008 The Authors Journal Compilation 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 61–72
p140Cap as part of a pre-synaptic complex | 63
was generated and affinity-purified on a column to which the antigen had been conjugated. Polyclonal anti-vinexin antibody was raised and purified as described (Ito et al. 2007). Monoclonal antisynaptophysin antibody was from Progen (Heidelberg, Germany). Monoclonal anti-Flag M2, polyclonal anti-Flag, and anti-tubulin antibodies were from Sigma-Aldrich (St Louis, MO, USA). Monoclonal anti-hemagglutinin 12CA5, anti-Myc 9E10, anti-GFP, anti-glial fibrillary acidic protein (GFAP), and polyclonal anti-GFP and anti-Myc antibodies were from Santa Cruz Biotech (Santa Cruz, CA, USA). Monoclonal anti-Tau-1 and anti microtubule-associated protein 2 antibodies were obtained from Chemicon (Temecula, CA, USA). Preparation of rat brain extracts and immunoblotting Preparation of rat brain extracts at various developmental stages was carried out as described (Morishita et al. 1999). Whole extracts of various adult rat brain regions were prepared with 50 mM Tris–HCl buffer (pH 7.5) containing 0.1 M NaF, 5 mM EDTA, 10 lg/mL aprotinin, 10 lg/mL leupeptin, and 2% sodium dodecyl sulfate, and subcellular fractionation was carried out (Jones and Matus 1974; Ichtchenko et al. 1995; Hata et al. 1996). Cytosol and membrane fractions of adult rat brain were prepared as described (Sudo et al. 2006). Protein concentrations were determined with a microbicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL, USA). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10% gel unless otherwise indicated) and immunoblotting was performed as detailed earlier (Nagata and Inagaki 2005). Immunohistochemical studies and electron microscopy Immunohistochemistry was performed essentially as reported (Shinohara et al. 1998; Sudo et al. 2007) and images were obtained using a BX61 microscope with an attached DP-70 digital camera (Olympus, Tokyo, Japan). Immunoelectron microscopy with microslicer sections of cerebral cortex was conducted as described previously (Ito et al. 2007). Cell culture, transfection, and immunofluorescence Primary cultured rat hippocampal neurons and astrocytes were obtained as described (Aloisi et al. 1988; Goslin et al. 1998). COS7 and REF52 cells were cultured (Nagata et al. 2004) and transient transfection was carried out with the Lipofectamine method (GibcoBRL, Rockville, MD, USA). Immunofluorescence analysis was conducted as detailed earlier (Nagata et al. 2004) with Alexa Fluor 488- or 568-labeled IgG (Molecular Probes, Eugene, OR, USA) as secondary antibodies. Fluorescent images were obtained using a FLUOVIEW confocal microscope (FV-1000; Olympus). Yeast two-hybrid analyses pYTH9-Cap-C was used as a bait in the two-hybrid screen with a human brain cDNA library fused to pACT2 (BD Biosciences Clontech, San Jose, CA, USA), following the Matchmaker twohybrid system protocol. Subsequent two-hybrid interaction analyses were carried out as described (Nagata et al. 1998). Immunoprecipitation Immunoprecipitation was performed using RIPA buffer containing 150 mM NaCl, 1% Nonidet P-40 (Sigma-Aldrich), 0.5% deoxy-
(a)
(b)
(c)
(d)
Fig. 2 Characterization of anti-Cap-C and detection of p140Cap in the rat brain. (a) Lysates from COS7 cells (30 lg of proteins) expressing Myc-p140Cap or Myc-tag (vector) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoblotting with anti-Cap-C (upper left panel) or the antibody pre-absorbed with the antigen (upper right panel). The blots were re-probed with anti-tubulin antibody as a loading control (lower panels). Molecular size markers are shown on the left. (b) COS7 cells were transfected with pcDNA-Myc-p140Cap, pcDNA6.2-GW/EmGFP-miR-p140Cap, and the vacant vector (Control) in various combinations and cell lysates (30 lg) were immunoblotted with anti-Cap-C (upper panel) or anti-GFP (lower panel). (c) Myc-p140Cap was immunoprecipitated with anti-Cap-C or rabbit IgG (2 lg per assay) from extracts (100 lg) of COS7 cells expressing Myc-p140Cap. Extract (10 lg; Input) and the precipitated materials (20%) were immunoblotted with 9E10. (d) Various adult rat brain regions were dissected and whole extracts were prepared (20 lg) and subjected to immunoblotting with anti-CapC (upper panel) or anti-synaptophysin (lower panel). Cap, Cas-associated protein; SYP, synaptophysin; GFP, green fluorescent protein.
cholic acid, 0.1% SDS, and 50 mM Tris–HCl (pH 8.0) essentially as detailed earlier (Nagata et al. 2003).
Results Detection of p140Cap in neuronal tissues using anti-Cap-C To explore the physiologic significance of p140Cap, we developed a rabbit polyclonal antibody (anti-Cap-C) and confirmed the specificity by immunoblotting using lysates of COS7 cells expressing Myc-p140Cap (Fig. 2a). Pre-incuba-
2008 The Authors Journal Compilation 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 61–72
64 | H. Ito et al.
tion of anti-Cap-C with the antigen eliminated the immunoreactivity of over-expressed as well as endogenous p140Cap (Fig. 2a and data not shown). Immunoreactivity was also markedly reduced when Myc-p140Cap expression was suppressed by co-transfection of pcDNA6.2-GW/EmGFPmiR-p140Cap, which expresses microRNA for RNAi of p140Cap (Fig. 2b). Expression of GFP confirmed efficient transfection of the plasmids (Fig. 2b). Anti-Cap-C was found to immunoprecipitate Myc-p140Cap expressed in COS7 cells (Fig. 2c). When tissue distribution in rat was analyzed, p140Cap was found to be expressed dominantly in brain as described previously (data not shown) (Chin et al. 2000). On dissection of 13 regions for immunoblot analyses using antiCap-C (Fig. 2d, upper panel), relative abundance was noted in the cerebellum and telencephalon, including the hippocampus, neocortex, entorhinal cortex, and visual cortex. As a control experiment, the synaptophysin distribution pattern was evaluated (Fig. 2d, lower panel). It remains to be clarified if the approximately 120 kDa-protein detected in cerebellum is a splicing variant. We next examined protein expression level of p140Cap in rat brain during embryonic and postnatal developmental stages. As shown in Fig. 3a upper panel, p140Cap dramatically increased between embryonic days E14.5 and E18.5, suggesting an involvement of the protein in neuronal development. The developmental process was confirmed by visualizing glial cell differentiation marker, GFAP (Fig. 3a, lower panel). As shown in Fig. 3b, p140Cap was also detected in primary cultured rat hippocampal neurons. We further concluded that p140Cap is also expressed in astrocytes, as astrocytes were found to be contaminants in cultured neuron dishes whereas neurons were not present with cultured astrocytes (Fig. 3c). Biochemical fractionation and immunoblotting showed PSD95 and p140Cap to be enriched in the synaptic plasma membrane fraction of brain homogenates (Fig. 3d).
Fig. 3 Expression profile of p140Cap in rat brain. (a) Extracts (30 lg of proteins) of brain at various developmental stages (E12.5–P32) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoblotting with anti-Cap-C (upper panel) or anti-GFAP (lower panel). (b) Lysates (20 lg) of cultured rat hippocampal neurons and astrocytes were immunoblotted with antiCap-C. (c) The membrane for (b) was then stripped and re-probed with anti-synaptophysin (SYP), anti-GFAP, and anti-tubulin. (d) Aliquots of brain fractions (10 lg) were immunoblotted with anti-Cap-C (upper panel). H, homogenate; P1, nuclear fraction; S1, crude synaptosomal fraction; S2, cytosolic synaptosomal fraction; P2, crude synaptosomal pellet fraction; LP2, synaptosomal vesicle fraction; LP1, synaptosomal membrane fraction; SPM, synaptic plasma membrane fraction. The blot was then re-probed with anti-PSD-95 (middle panel) and anti-SYP (lower panel). GFAP, glial fibrillary acidic protein; Cap, Cas-associated protein; PSD, postsynaptic density protein.
Immunohistochemical analyses of p140Cap in adult rat brain As the p140Cap expression in cultured neurons and astrocytes might be related to artificial culture conditions, the localization profile of p140Cap in brain was determined by immunohistochemical analyses with anti-Cap-C at a dilution of 1 : 2000. The overall distribution of p140Cap in a horizontal paraffin section partially overlapped with that of vinexin at
hippocampus (Fig. 4a and b). In sections of hippocampus, distinct immunoreactivity of p140Cap was observed in the axons and/or dendrites of CA1 (Fig. 4c and d) and CA3 regions (data not shown), with a strong suggestion of enrichment at excitatory synapses. Strong staining was also observed in axons/dendrites in the dentate gyrus (data not shown). In addition, p140Cap was enriched in the neuropil rather than soma in the thalamus, corpus striatum, and
Fig. 4 Immunohistochemical analyses of p140Cap and vinexin in adult rat central nervous tissues. p140Cap (a) or vinexin (b) was stained with horseradish peroxydase/diaminobenzidine (HRP/DAB) in horizontal paraffin sections of the brain. Staining of p140Cap (c and d) or vinexin (f and g) in paraffin sections of hippocampus CA1 region, using HRP/DAB. Double-staining was carried out using HRP/DAB to detect p140Cap (e) or vinexin (h), and 5-bromo-4-chloro-3-indolyl phosphate phosphatase/Nitroblue tetrazolium (BCIP/NBT) was used
to detect synaptophysin (e and h). Staining of synaptophysin was shown using BCIP/NBT (i and j). Cryosections of corpus callosum were immunostained for glial fibrillary acidic protein (GFAP) and for p140Cap (l) and vinexin (m) with BCIP/NBT (k) and HRP/DAB, respectively. Double-staining using HRP/DAB to detect p140Cap (n) or vinexin (o) and BCIP/NBT to detect GFAP (n and o) was carried out. Scale Bars, 50 lm. Cap, Cas-associated protein.
(a)
(c)
(b)
(d)
2008 The Authors Journal Compilation 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 61–72
p140Cap as part of a pre-synaptic complex | 65
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
2008 The Authors Journal Compilation 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 61–72
66 | H. Ito et al.
(i)
(k)
(n)
(j)
(l)
(m)
(o)
Fig. 4 (Continued).
2008 The Authors Journal Compilation 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 61–72
p140Cap as part of a pre-synaptic complex | 67
(a)
(b)
(c)
Fig. 5 Localization of p140Cap in primary cultured rat hippocampal neurons. (a and b) Neurons cultured for 3 days were double-stained for p140Cap with Tau-1 (a) or MAP2 (b). Merged images are also shown. Scale Bars, 50 lm. (c) Neurons cultured for 21 days were double-stained for p140Cap with synaptophysin. Images in the upper panels are demonstrated at higher magnification in the lower panels. Merged images are also shown. Scale Bars, 25 lm (upper panels) and 10 lm (lower panels). Cap, Cas-associated protein; MAP, microtubule associated protein.
cerebral cortex (data not shown). Double-staining with synaptophysin at CA1 region was shown (Fig. 4e). On the other hand, vinexin was enriched at cytoplasm but clearly
detected at the axons and/or dendrites of CA1 region (Fig. 4f and g), and partially co-localized with synaptophysin (Fig. 4h). Synaptophysin per se was stained in a very similar manner to p140Cap at the CA1 region (Fig. 4i and j). It should be noted that p140Cap was hardly detected in astrocytes in paraffin sections of corpus callosum where neurons are hardly seen (data not shown). Instead, we detected the protein in astrocytes using a cryosection with the antibody at a dilution of 1 : 100 (Fig. 4k and l). Under the conditions, vinexin was also detected in astrocytes (Fig. 4m). Double-staining of GFAP with p140Cap or vinexin further demonstrated expression of these two proteins in astrocytes (Fig. 4n and o). Subcellular localization of p140Cap in neurons In neuronal tissues, p140Cap is likely to be involved in synapse network formation and/or maintenance as (i) the protein level dramatically increases from E14.5 to E18.5 (Fig. 3a), (ii) the protein is abundant in biochemically isolated synaptic plasma membranes (Fig. 3d), (iii) strong staining of the neuropil was evident on immunohistochemical analyses (Fig. 4), and (iv) the protein is reported to interact with a SNARE component, SNAP-25 (Chin et al. 2000). We thus examined the subcellular distribution of p140Cap in primary cultured rat hippocampal neurons using confocal microscopy and found distribution throughout the cell body but clearly co-localization with an axonal marker, Tau-1, in 3 days in vitro (DIV) neurons (Fig. 5a). p140Cap was also weakly distributed in dendrites in 3 DIV neurons (Fig. 5b). In 21 DIV neurons, p140Cap was well colocalized with a pre-synaptic marker, synaptophysin (Fig. 5c). Although p140Cap was dominantly distributed in the neuropil on immunohistochemical analyses, it was strongly detected in soma in cultured neurons. We assume that the observed discrepancy may be at least partly attributable to the artificial neuron culture conditions. Further analyses using immuno-electron microscopy demonstrated the localization of p140Cap at pre-synapses
Fig. 6 Immunoperoxidase electron microscopy of p140Cap. The asterisk and arrowhead indicate typical pre-synaptic terminals and post-synaptic density, respectively. Scale Bars, 500 nm. Cap, Cas-associated protein.
2008 The Authors Journal Compilation 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 61–72
68 | H. Ito et al.
(Fig. 6). However, it should be noted that p140Cap was enriched at the pre-synapse and post-synapse in approximately 10% and 80% of synapses analyzed, respectively (data not shown). In the remaining, approximately 10% of the synapses, p140Cap was localized at both pre- and postsynaptic sides (data not shown). Although the localization at pre-synapses is consistent with a previous report of interaction of p140Cap with SNAP-25 (Chin et al. 2000), strong accumulation at post-synapses suggests yet unidentified role of p140Cap there. Interaction of p140Cap with an adaptor protein, vinexin, and synaptophysin Although p140Cap is present at pre-synapses and might regulate neurotransmitter release through interaction with SNAP-25, it could play additional roles at synapses by interacting with yet unidentified proteins. To address this issue, we searched for interactive partners. Using p140Cap-C containing the C-terminally located Pro-rich motif as a bait (Fig. 7a), we performed yeast two-hybrid screening of a human brain cDNA library (Nagata et al. 1998) and identified vinexin, a member of a novel adaptor protein family. The results obtained by two-hybrid screening strongly suggest that p140Cap and vinexin form a complex in cellular context. To test this possibility, immunoprecipitation experiments were performed using the COS7 cell transient expression method. As shown in Fig. 7b, p140Cap was immunoprecipitated with vinexinb. As p140Cap-C contains only one Pro-rich motif (PPPPPRR) which is C-terminally located, we assume that p140Cap binds through the motif to one or more of the SH3 domains of vinexin. We thus asked if p140Cap interacts with a vinexinb mutant, vinexinbDSH3-3, which lacks the third SH3 domain. As shown in Fig. 7b, the association was lost when vinexinbDSH3-3 was used instead of the wild type. To assess the role of the C-terminal Pro-rich motif in p140Cap-vinexin interactions, we examined if Flagvinexinb co-immunoprecipitates a p140Cap mutant lacking the Pro-motif (Myc-p140Cap-Pro) by COS7 cell expression methods. The mutant failed to bind to vinexinb (Fig. 7c). It should be noted that the interaction of p140Cap with vinexin is highly specific, as among 12 Pro-rich motifs in p140Cap, only that located at the C-terminus interacted with the third SH3 domain of vinexin. Immunocomplex formation of p140Cap and vinexinb in COS7 cells strongly suggests their physiologic interaction in brain tissues. We thus tested if endogenous p140Cap and vinexin associate with each other in brain. When endogenous p140Cap was immunoprecipitated from adult rat brain extracts, the protein was found to form complexes with vinexina/c, isoforms dominantly expressed in brain tissues (Fig. 7d). While p140Cap was mainly present in the membrane fraction of rat cerebrum and cerebellum, it was clearly detected in the cytosol where vinexin was dominantly
(a)
(b)
(c)
(d)
(e)
Fig. 7 Identification of vinexin as a p140Cap-binding protein. (a) Structures of p140Cap and vinexin. Structural domains of vinexin are abbreviated as follows: SoHo, sorbin-homology; SH3, Srchomology 3. The region (p140Cap-C; aa, 1023–1217) used as bait in the yeast two-hybrid screening is underlined. (b) Lysates from COS7 cells (100 lg of protein) expressing GFP-p140Cap with Flag-vinexinb or -vinexinbDSH3-3 were immunoprecipitated with M2 (1 lg per assay) and the precipitated materials were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoblotting with a mixture of M2 and anti-GFP. Expression of each protein was confirmed by immunoblotting with M2 and anti-GFP (Exp; 20 lg). (c) Lysates from COS7 cells expressing Myc-p140Cap or -p140Cap-Pro with or without Flag-vinexinb were immunoprecipitated with M2 and analyzed as in (b). (d) Endogenous p140Cap was immunoprecipitated with anti-Cap-C or rabbit IgG (2 lg per assay) from adult rat brain extracts (1000 lg). The precipitated materials (20%) were immunoblotted with anti-Cap-C (upper panel) or antivinexin (lower panel). Extract (Input: 10 lg) was used as a control. (e) Cytosolic (cyto; 20 lg) and membrane (memb; 30 lg) fractions were prepared from adult rat cerebrum and cerebellum as described (Sudo et al. 2007), and subjected to immunoblotting using anti-Cap-C (upper panel) or anti-vinexin (lower panel). Cap, Cas-associated protein; IP, immunoprecipitation; Exp, expression.
distributed (Fig. 7e). Based on these findings, the immunocomplex observed in Fig. 7d is likely to be composed of cytosolic p140Cap and vinexin.
2008 The Authors Journal Compilation 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 61–72
p140Cap as part of a pre-synaptic complex | 69
Physiologic functions of p140Cap and vinexin in neurons are almost unknown. The functional meaning of p140Capvinexin interaction also remains to be clarified. To gain some clue to understand the functional role of the interaction, we asked if subcellular localization of one protein is affected by the other. When expressed in rat embryonic fibroblast REF52 cells, Myc-p140Cap per se was distributed in the perinuclear region while Flag-vinexina was accumulated at focal adhesions (Fig. 8a, upper and middle panels). When the two proteins were co-expressed, vinexina came to co-localize with p140Cap in the perinuclear region, especially in cells where vinexina was highly expressed (Fig. 8a, bottom panels). We next tested if p140Cap affects interaction of (a)
vinexin with other binding partners. Neuronal (N)-WASP has been shown to bind with the third SH3 domain of vinexin (Mitsushima et al. 2006). As shown in Fig. 8b, this N-WASP-vinexin interaction was suppressed when p140Cap was co-expressed under the conditions used. Interestingly, immunocomplexes from rat brain extracts contained a synaptic vesicle membrane protein, synaptophysin (Fig. 9a), known to regulate SNARE core complex formation through recruitment of synaptobrevin. On the other hand, two other SNARE proteins, synaptobrevin and syntaxin 1, were not detected in the immunocomplexes (data not shown). We then confirmed the interaction of Myc-p140Cap with Flag-synaptophysin in COS7 cell expression experiments (Fig. 9b). Synaptophysin possesses four membrane spanning regions and the N- and C-termini face to the cytoplasmic side. The synaptophysin-p140Cap interaction was lost when synaptophysin DN or DC mutant was used instead of the wild type (Fig. 9b), suggesting that both N- and C-terminal flanking regions are required to bind with p140Cap. As for p140Cap, the center region containing coiled-coil domains and a charged sequence region was found to be important to bind with synaptophysin (Fig. 9c). Based on the results in Figs 7c and 9c, p140Cap was supposed to associate with synaptophysin and vinexin via distinct regions. Indeed, p140Cap, synaptophysin, and vinexin formed a ternary complex in COS7 cells (Fig. 9d). p140Cap may thus interact with vinexin, synaptophysin, and SNAP-25 in a complex manner at pre-synapses and thus regulate presynapse functions such as SNARE complex formation and synaptic vesicle fusion to the plasma membrane.
(b)
Discussion
Fig. 8 Effects of p140Cap on vinexin localization and the interaction between vinexin and N-WASP. (a) Myc-p140Cap (top panels) or Flagvinexina (middle panels) was expressed in COS7 cells and doublestained with rhodamine-conjugated phalloidin. Myc-p140Cap and Flag-vinexina were co-expressed and double-stained with M2 and polyclonal anti-Myc antibody (bottom panels). Merged images are also shown. Scale Bar, 20 lm. (b) COS7 Cells were transfected with pcDNA-Myc-p140Cap, pRK5-Flag-vinexin, and -HA-N-WASP in various combinations, and cell lysates (100 lg) were immunoprecipitated with M2. Immunoblotting was carried out with 9E10, 12CA5, and M2 to detect Myc-p140Cap, -Flag-vinexin and -HA-N-WASP, respectively (left panels). Expression of each protein was confirmed by immunoblotting with a mixture of 9E10, 12CA5, and M2 (right panels). Cap, Cas-associated protein; HA, hemagglutinin; N-WASP, neuronalWiskott-Aldrich syndrome protein.
In the present study, use of a newly produced antibody, anti-Cap-C, showed p140Cap to be abundant in the telencephalon of rat brain and enriched in the biochemically separated synaptic plasma membrane fraction, consistent with a previous study (Chin et al. 2000). We also found that p140Cap is expressed in a developmental stagedependent manner in rat brain; the protein was detected at E14.5 and dramatically increased by E18.5. In adult rat brain, p140Cap appeared to be dominantly present in synapses, especially excitatory ones, on immunohistochemical and immunofluorescence analyses. Using a biochemical approach, we confirmed that p140Cap is relatively abundant in the synaptic plasma membrane. Electron microscopy analyses then revealed that p140Cap is located mainly at post-synapses but also at pre-synapses. These results suggest that p140Cap plays roles in the formation and maintenance of synapses during brain developmental processes. p140Cap is implicated in neuronal secretion as it has been reported to interact with a SNARE protein, SNAP-25, in vitro and inhibit Ca2+-dependent exocytosis from PC-12 cells
2008 The Authors Journal Compilation 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 61–72
70 | H. Ito et al.
(a)
(b)
(c)
(d)
Fig. 9 Interaction of p140Cap with a synaptic vesicle protein, synaptophysin (SYP). (a) p140Cap was immunoprecipitated with antiCap-C or rabbit IgG (2 lg) from adult rat brain extracts (1000 lg). The precipitated materials (20%) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (15% gel) followed by immunoblotting with anti-synaptophysin (SYP). Extract (Input: 10 lg) was used as a control. (b) Lysates from COS7 cells expressing Myc-p140Cap, Flag-synaptophysin, -DN, and -DC in various combinations were immunoprecipitated with M2. The precipitated materials (IP; 20%) were analyzed by immunoblotting with a mixture of polyclonal anti-Flag and anti-Myc antibodies. Expression of each protein was confirmed by immunoblotting with M2 and 9E10 (Exp; 20 lg). (c) Lysates from COS7 cells expressing Flag-synaptophysin, GFP-p140Cap, -Cap-N, -Cap-C2, -Cap-Cent, Cap-CC, and Myc-Cap-
C, in various combinations were immunoprecipitated with M2. The precipitated materials (IP; 20%) were analyzed by immunoblotting with a mixture of polyclonal anti-Flag, anti-Myc, and anti-GFP antibodies. Expression of each protein was confirmed by immunoblotting with M2, 9E10, and monoclonal anti-GFP antibodies (Exp; 20 lg). (d) Lysates from COS7 cells expressing Myc-p140Cap, GFP-synaptophysin, and Flag-vinexin in various combinations were immunoprecipitated with M2. The precipitated materials (IP; 20%) were analyzed by immunoblotting with a mixture of polyclonal anti-GFP, anti-Flag, and anti-Myc antibodies. Expression of each protein was confirmed by immunoblotting with monoclonal anti-GFP, M2, and 9E10 (Exp; 20 lg). Cap, Cas-associated protein, GFP, green fluorescent protein; IP, immunoprecipitation; Exp, expression.
(Chin et al. 2000). The SNARE complex is a conserved set of membrane proteins mediating exocytosis of synaptic vesicles and composed of a vesicle membrane protein, synaptobrevin, and two plasma membrane proteins, syntaxin, and SNAP-25. The cyclic assembly and disassembly of SNARE proteins constitutes crucial steps in synaptic vesicle fusion, and assembly is thought to result in tight binding of a vesicle to the plasma membrane. However, less is known about how the complex formation is regulated. We here found that p140Cap interacts with another synaptic vesicle protein, synaptophysin, which is not a component of the SNARE complex. Synaptophysin is reported to control the targeting of synaptobrevin to synaptic vesicles (Pennuto et al. 2003). The binding to synaptobrevin is specific and exclusive: synaptobrevin bound to synaptophysin cannot
enter the SNARE complex, and conversely, synaptobrevin in the SNARE complex cannot interact with synaptophysin. Thus, synaptophysin is most likely to serve as a regulator for the incorporation of synaptobrevin into the SNARE complex. Given our results and the findings reported by Chin et al. (2000), p140Cap may take part in the regulation of neurotransmitter release at two protein (synaptophysin and SNAP-25) levels, although the physiologic significance and the precise regulatory mechanisms remain to be clarified. We here identified vinexin, a member of a multidomain adaptor family, as a binding partner for p140Cap. Vinexin family is composed of vinexin, c-Cbl associated protein/ ponsin, and Arg-binding protein 2 (Kioka et al. 2002). The molecular architecture of this family is highly conserved; they share a N-terminally located sorbin homology domain
2008 The Authors Journal Compilation 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 61–72
p140Cap as part of a pre-synaptic complex | 71
and three SH3 domains toward the C-terminal region (Fig. 7a). Vinexin is transcribed into several alternative forms including vinexina, b, and c (Kioka et al. 1999; Matsuyama et al. 2005). While vinexina and c are larger variants highly expressed in brain tissues (Ito et al. 2007) and have a sorbin homology domain, vinexinb is a short form containing only three SH3 domains. In non-neuronal cells, vinexin is localized at focal adhesions and shown to be involved in growth factor- and integrin-mediated signal transduction, actin cytoskeletal organization, cell spreading, motility, and growth [for review see Kioka et al. (2002)]. We reported earlier that vinexin is co-localized with synaptophysin in matured primary cultured rat hippocampal neurons (Ito et al. 2007). Immunoelectron microscopy analyses also revealed that vinexin is present in pre-synapses and postsynapses of hippocampal neurons (Ito et al. 2007). Based on the earlier and present findings, we concluded that vinexin and p140Cap are co-localized at pre-synapses. It is notable that extracellular signal-regulated kinase-mediated phosphorylation appears to be important for the vinexin functions both in neuronal and cancer cells (Ito et al. 2007; Mizutani et al. 2007). Vinexin is also reported to localize at cell–cell junctions in epithelial cells (Kioka et al. 1999) and the synapse is a specialized variant form of cell–cell junction in neuronal tissues (Govek et al. 2005). Like p140Cap, vinexin is enriched at synapses and is suggested to play as yet unidentified physiologic role(s) there (Ito et al. 2007). Although the physiologic significance of the vinexinp140Cap interaction at synapses is not known, the results obtained here suggest that subcellular localization of these proteins is affected by each other, and that p140Cap alters mode of interaction of vinexin with other proteins. Vinexinp140Cap complex might be involved in the formation and/or maintenance of synapses. In addition to p140Cap, N-WASP, and a membraneassociated guanylate kinase family protein, lp-dlg/ KIAA0583, have been reported to bind to vinexin (Wakabayashi et al. 2003; Mitsushima et al. 2006). N-WASP is a key factor which modulates actin polymerization crucial for synapse formation/maintenance. As p140Cap inhibits N-WASP-vinexin interaction, it might impact on the actin cytoskeletal organization at synapses. As lp-dlg/KIAA0583 is co-localized and interacts with vinexin and b-catenin at sites of cell–cell contact in epithelial cells (Wakabayashi et al. 2003), p140Cap could form multicomplexes with vinexin, lp-dlg/KIAA0583, and b-catenin at synapses. The precise localization of lp-dlg/KIAA0583, which is expressed in human brain, has yet to be determined. We here provided evidence that p140Cap may be involved in synapse functions, such as neurotransmitter release, signaling, and synapse formation/maintenance. Although molecular mechanisms governing these cellular events are enigmatic, it is tempting to speculate that synaptic vesicle fusion regulated by interactions among
p140Cap, vinexin, synaptophysin, and SNAP-25, and synapse formation/maintenance regulated by interactions among p140Cap, vinexin, and lp-dlg/KIAA0583 are controlled in an integrated manner. We also assume that p140Cap participates in neuronal cell developmental processes. Further analyses are warranted to further clarify its functional roles.
Acknowledgements This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Technology, Sports, and Culture of Japan and by grants from the Takeda Foundation and the Aichi Cancer Research Foundation.
References Aloisi F., Agresti C. and Levi G. (1988) Establishment, characterization, and evolution of cultures enriched in type-2 astrocytes. J. Neurosci. Res. 21, 188–198. Chin L. S., Nugent R. D., Raynor M. C., Vavalle J. P. and Li L. (2000) SNIP, a novel SNAP-25-interacting protein implicated in regulated exocytosis. J. Biol. Chem. 275, 1191–1200. Defilippi P., Di Stefano P. and Cabodi S. (2006) p130Cas: a versatile scaffold in signaling networks. Trends Cell Biol. 16, 257–263. Di Stefano P., Cabodi S., Boeri Erba E., Margaria V., Bergatto E., Giuffrida M. G., Silengo L., Tarone G., Turco E. and Defilippi P. (2004) P130Cas-associated protein (p140Cap) as a new tyrosinephosphorylated protein involved in cell spreading. Mol. Biol. Cell 15, 787–800. Di Stefano P., Damiano L., Cabodi S. et al. (2007) p140Cap protein suppresses tumour cell properties, regulating Csk and Src kinase activity. EMBO J. 26, 2843–2855. Goslin K., Asmussen H. and Banker G. (1998) Rat hippocampal neurons in low-density culture, in Culturing Nerve Cells (Banker G. and Goslin K., eds), ed 2, pp. 339–370. MIT, Cambridge, MA. Govek E. E., Newey S. E. and Van Aelst L. (2005) The role of the Rho GTPases in neuronal development. Genes Dev. 19, 1–49. Hata Y., Butz S. and Sudhof T. C. (1996) CASK: a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein kinase domain identified by interaction with neurexins. J. Neurosci. 16, 2488–2494. Hong W. (2005) SNAREs and traffic. Biochim. Biophys. Acta 1744, 493–517. Ichtchenko K., Hata Y., Nguyen T., Ullrich B., Missler M., Moomaw C. and Sudhof T. C. (1995) Neuroligin 1: a splice site-specific ligand for beta-neurexins. Cell 81, 435–443. Ito H., Usuda N., Atsuzawa K. et al. (2007) Phosphorylation by extracellular signal-regulated kinase of a multidomain adaptor protein, vinexin, at synapses. J. Neurochem. 100, 545–554. Jones D. H. and Matus A. I. (1974) Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient centrifugation. Biochim. Biophys. Acta 356, 276–287. Kioka N., Sakata S., Kawauchi T., Amachi T., Akiyama S. K., Okazaki K., Yaen C., Yamada K. M. and Aota S. (1999) Vinexin: a novel vinculin-binding protein with multiple SH3 domains enhances actin cytoskeletal organization. J. Cell Biol. 144, 59–69. Kioka N., Ueda K. and Amachi T. (2002) Vinexin, CAP/ponsin, ArgBP2: a novel adaptor protein family regulating cytoskeletal organization and signal transduction. Cell Struct. Funct. 27, 1–7.
2008 The Authors Journal Compilation 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 61–72
72 | H. Ito et al.
Liu G., Li W., Gao X. et al. (2007) p130CAS is required for netrin signaling and commissural axon guidance. J. Neurosci. 27, 957–968. Matsuyama M., Mizusaki H., Shimono A., Mukai T., Okumura K., Abe K., Shimada K. and Morohashi K. (2005) A novel isoform of Vinexin, Vinexin gamma, regulates Sox9 gene expression through activation of MAPK cascade in mouse fetal gonad. Genes Cells 10, 421–434. Mitsushima M., Sezaki T., Akahane R., Ueda K., Suetsugu S., Takenawa T. and Kioka N. (2006) Protein kinase A-dependent increase in WAVE2 expression induced by the focal adhesion protein vinexin. Genes Cells 11, 281–292. Mizutani K., Ito H., Iwamoto I., Morishita R., Deguchi T., Nozawa Y., Asano T. and Nagata K. I. (2007) Essential roles of ERK-mediated phosphorylation of vinexin in cell spreading, migration and anchorage-independent growth. Oncogene 26, 7122–7131. Morishita R., Shinohara H., Ueda H., Kato K. and Asano T. (1999) High expression of the gamma5 isoform of G protein in neuroepithelial cells and its replacement of the gamma2 isoform during neuronal differentiation in the rat brain. J. Neurochem. 73, 2369–2374. Nagata K. and Inagaki M. (2005) Cytoskeletal modification of Rho guanine nucleotide exchange factor activity: identification of a Rho guanine nucleotide exchange factor as a binding partner for Sept9b, a mammalian septin. Oncogene 24, 65–76. Nagata K., Puls A., Futter C., Aspenstrom P., Schaefer E., Nakata T., Hirokawa N. and Hall A. (1998) The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3. EMBO J. 17, 149–158.
Nagata K., Kawajiri A., Matsui S. et al. (2003) Filament formation of MSF-A, a mammalian septin, in human mammary epithelial cells depends on interactions with microtubules. J. Biol. Chem. 278, 18538–18543. Nagata K., Asano T., Nozawa Y. and Inagaki M. (2004) Biochemical and cell biological analyses of a mammalian septin complex, Sept7/9b/ 11. J. Biol. Chem. 279, 55895–55904. Pennuto M., Bonanomi D., Benfenati F. and Valtorta F. (2003) Synaptophysin I controls the targeting of VAMP2/synaptobrevin II to synaptic vesicles. Mol. Biol. Cell 14, 4909–4919. Shinohara H., Asano T., Kato K., Kameshima T. and Semba R. (1998) Localization of a G protein Gi2 in the cilia of rat ependyma, oviduct and trachea. Eur. J. Neurosci. 10, 699–707. Sudo K., Ito H., Iwamoto I., Morishita R., Asano T. and Nagata K. (2006) Identification of a cell polarity-related protein, Lin-7B, as a binding partner for a Rho effector, Rhotekin, and their possible interaction in neurons. Neurosci. Res. 56, 347–355. Sudo K., Ito H., Iwamoto I., Morishita R., Asano T. and Nagata K. (2007) SEPT9 sequence alterations causing hereditary neuralgic amyotrophy are associated with altered interactions with SEPT4/11 and resistance to Rho/Rhotekin-signaling. Hum. Mut. 28, 1005– 1013. Wakabayashi M., Ito T., Mitsushima M., Aizawa S., Ueda K., Amachi T. and Kioka N. (2003) Interaction of lp-dlg/KIAA0583, a membrane-associated guanylate kinase family protein, with vinexin and beta-catenin at sites of cell–cell contact. J. Biol. Chem. 278, 21709–21714.
2008 The Authors Journal Compilation 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 61–72
