Specific interactions of neuronal focal adhesion ... - Wiley Online Library

Journal of Neurochemistry, 2003, 84, 253–265

Specific interactions of neuronal focal adhesion kinase isoforms with Src kinases and amphiphysin Samantha Messina,*,1,2 Franco Onofri,*,1 Lucilla Bongiorno-Borbone,*, Silvia Giovedı`,* Flavia Valtorta,à Jean-Antoine Girault and Fabio Benfenati* *Department of Experimental Medicine, Section of Human Physiology, University of Genova, Genova, Italy INSERM/UPMC U536, Institut du Fer a` Moulin, Paris, France àDepartment of Neuroscience, San Raffaele Scientific Institute, Milano, Italy

Abstract Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that activates Src family kinases via SH2- and SH3-mediated interactions. Specific FAK isoforms (FAK+), responsive to depolarization and neurotransmitters, are enriched in neurons. We analyzed the interactions of endogenous FAK+ and recombinant FAK+ isoforms containing amino acid insertions (boxes 6,7,28) with an array of SH3 domains and the c-Src SH2/SH3 domain tandem. Endogenous FAK+ bound specifically to the SH3 domains of c-Src (but not n-Src), Fyn, Yes, phosphtidylinositol-3 kinase, amphiphysin II, amphiphysin I, phospholipase Cc and NH2-terminal Grb2. The inclusion of boxes 6,7 was associated with a significant decrease in the binding of FAK+ to the c-Src and Fyn SH3 domains, and a significant increase in the binding to the Src SH2 domain, as a

consequence of the higher phosphorylation of Tyr-397. The novel interaction with the amphiphysin SH3 domain, involving the COOH-terminal proline-rich region of FAK, was confirmed by coimmunoprecipitation of the two proteins and a closely similar response to stimuli affecting the actin cytoskeleton. Moreover, an impairment of endocytosis was observed in synaptosomes after internalization of a proline-rich peptide corresponding to the site of interaction. The data account for the different subcellular distribution of FAK and Src kinases and the specific regulation of the transduction pathways linked to FAK activation in the brain and implicate FAK in the regulation of membrane trafficking in nerve terminals. Keywords: amphiphysin, nerve terminals, SH2 domains, SH3 domains, tyrosine phosphorylation. J. Neurochem. (2003) 84, 253–265.

Focal adhesion kinase (FAK) is a widely expressed, cytoplasmic tyrosine kinase that transduces signals generated by integrin engagement and G protein-coupled receptors. The FAK molecule comprises three domains: a regulatory NH2-terminal domain related to band 4.1 domains, a central catalytic domain and a COOH-terminal region that contains a sequence (FAT) necessary to target the kinase to focal adhesions in non-neuronal cells (Schaller 1996; Girault et al. 1999). Activation of FAK promotes autophosphorylation of Tyr397 that recruits Src family kinases and phosphatidylinositol-3 kinase (PI3K) through an SH2 domain-mediated interaction (Schaller et al. 1994; Cobb et al. 1994; Calalb et al. 1995). Engagement of the SH2 domain activates Src kinases that phosphorylate FAK on multiple tyrosine residues in the catalytic (Tyr407, Tyr576/577) and COOH-terminal (Tyr871, Tyr925) domains as well as multiple substrates including paxillin, p130cas and PI3K (for review, see Girault et al. 1999). While phosphorylation of the catalytic domain

of FAK increases its catalytic activity, phosphorylation of Tyr925 creates a high affinity binding site for the SH2

Received June 19, 2002; revised manuscript received September 27, 2002; accepted October 6, 2002. Address correspondence and reprint requests to Dr Fabio Benfenati, Department of Experimental Medicine, Section of Human Physiology, University of Genova, Viale Benedetto XV 3, 16132 Genova, Italy. E-mail: [email protected] 1 The first two authors contributed equally to the experimental work. 2 Present address: Department of Experimental Medicine and Pathology, INM Neuromed, University of Rome La Sapienza, Roma, Italy. Abbreviations used: COOH-Grb2 SH3 domain, COOH-terminal SH3 domain of Grb2; DMEM, Dulbecco’s modified Eagle’s medium; FAK, focal adhesion kinase; FAK+, neuronal FAK isoforms; GST, glutathioneS-transferase; NH2-Grb2 SH3 domain, NH2-terminal SH3 domain of Grb2; p47phox, NADPH oxidase factor p47phox; PI3K, p85 subunit of phosphatidylinositol-3 kinase, PLC-c, phospholipase Cc; RasGAP, Ras GTPase activating protein; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SH2 domains, Src homology-2 domains; SH3 domains, Src homology-3 domains.

2003 International Society for Neurochemistry, Journal of Neurochemistry, 84, 253–265

253

254 S. Messina et al.

domain of Grb2 and can activate the Ras-dependent MAP kinase pathway (Schlaepfer et al. 1994; Schlaepfer and Hunter 1996). Thus, the possibility exists that the main role of FAK is to act as a regulated adapter protein that recruits and activates Src kinases (Schaller 1996; Schlaepfer and Hunter 1998). The finding that FAK interacts with SH3 domain-containing proteins further emphasizes this putative role. FAK contains three major proline-rich regions, two in the COOHterminal domain (encompassing Pro712/715 and Pro878, respectively) and one in the NH2-terminal domain in the vicinity of the autophosphorylated tyrosine (encompassing Pro371/374). These regions represent binding sites for various SH3 domain-containing proteins such as c-Src (Thomas et al. 1998), PI3K (Guinebault et al. 1995), p130cas (Polte and Hanks 1995; Harte et al. 1996), GRAF (Taylor et al. 1998) and HEF (Law et al. 1996). FAK is highly expressed in brain, where it exists as various isoforms that are enriched or exclusively present in the nervous tissue (Burgaya et al. 1995; Burgaya et al. 1997). The brain FAK isoforms have a higher molecular mass than non-neuronal FAK, due to the insertion of short peptides coded by the use of alternative exons (Burgaya and Girault 1996; Burgaya et al. 1997). All the brain-specific isoforms share a three amino acid insertion (Pro-Trp-Arg) at position 904 in the COOH-terminal domain (FAK+, as compared with the non-neuronal FAK with no additional exons or FAK0). The alternative insertion of peptides of six, seven or 28 amino acids in length (termed boxes 6, 7 and 28) coded by additional exons on either side of Tyr397 (at position 393 for boxes 28 and 6 and at position 412 for box 7) generates a series of FAK+ isoforms that are differentially expressed in brain, with the isoform including both boxes 6 and 7 (FAK+6,7) being the major isoform in forebrain neurons (Toutant et al. 2000). The additional exons typical of neuronal FAK isoforms have been shown to affect the functional properties of FAK without altering its targeting (Menegon et al. 1999; Toutant et al. 2000; A. Contestabile and F. Valtorta, unpublished results). The presence of boxes 6 and 7 increased autophosphorylation of Tyr397 independently and additively, while it decreased phosphorylation of Tyr397 by Src-family kinases (Burgaya et al. 1997; Toutant et al. 2000). The increased autophosphorylation results from an alteration in the mechanism of the reaction (Toutant et al. 2002). In the brain, FAK phosphorylation and activation are regulated by depolarization, neurotransmitters such as glutamate and acetylcholine, as well as by lipid messengers (Siciliano et al. 1994; Derkinderen et al. 1996; Siciliano et al. 1996; Derkinderen et al. 1998) and the kinase has been suggested to be involved in neurite outgrowth and synaptic plasticity (Burgaya et al. 1995; Grant et al. 1995; Derkinderen et al. 1998; Lauri et al. 2000). In view of the essential role of multiple interactions with SH2 and SH3 domain-containing proteins for FAK-mediated signal trans-

duction, it seemed important to gain insight into the specificity of the interactions between neuronal FAK isoforms and SH3 domains and to identify new potential FAK partners in the nervous tissue. To this aim, we have analyzed the binding specificity of endogenous brain FAK+ and of recombinant FAK+ isoforms with an array of SH3 domains belonging to proteins implicated in signal transduction or cytoskeleton assembly and with the c-Src SH3/SH2 domain tandem. The results suggest the existence of novel interactions of FAK with the SH3 domains of amphiphysins and of specific changes in the SH2- and SH3-mediated interactions of FAK with Src kinases due to the presence of boxes 6 and 7.

Experimental procedures Materials Sprague–Dawley rats (body weight 150–250 g) were from Harlan Italy (S. Pietro al Natisone, Italy). Fetal calf serum (FCS), trypsin– EDTA, and penicillin/streptomycin solutions were purchased from HyClone Europe Ltd. (Cramlington, UK); Dulbecco’s modified Eagle’s medium (DMEM) from Life Technologies (Milan, Italy); sodium orthovanadate, protease inhibitors, Triton-X 100, dithiothreitol (DTT), cytochalasin B, phalloidin, NADP and glutamate dehydrogenase from Sigma (Milan, Italy); the Src kinase inhibitor PP2, the tyrosine phosphatase inhibitor phenylarsine oxide (PAO) and the actin depolymerizing agent mycalolide B from Calbiochem (La Jolla, CA, USA); the styryl dye N-(3-triethylammoniumpropyl)4-(4-(diethylamino)styryl) pyridinium dibromide (FM 2-10) (FM 2-10) from Molecular Probes (Eugene, OR, USA). The Renaissance enhanced chemiluminescence detection system was from New England Nuclear (Brussels, Belgium). Glutathione–sepharose, Percoll and pGEX-2T were from Amersham-Pharmacia (Milan, Italy). The SH3 domain pGEX-2T constructs used in this study were: chicken c-Src SH3 domain (amino acids 84–148), bovine PI3K SH3 domain (amino acids 1–86), human full-length Grb2 (amino acids 1–217), NH2-Grb2 and COOH-Grb2 SH3 domains (amino acids 1–58 and 159–217, respectively), human Ras GTPase activating protein (RasGAP) SH3 domain (amino acids 275–345), human phospholipase Cc (PLCc) SH3 domain (amino acids 792–851), chicken a-spectrin SH3 domain (amino acids 967–1025), Crk SH3 domain (amino acids 369–429), COOH-terminal SH3 domain of the p47phox subunit of NADPH oxidase (amino acids 226–286), generously provided by Dr I. Gout (Ludwig Institute for Cancer Research, University College, London, UK); amphiphysins I and II SH3 domains (amino acids 588–695 and 516–612, respectively), generously provided by Dr P. De Camilli (Yale University, New Haven, CT, USA); Fyn, Yes and n-Src SH3 domains, generously provided by Dr J. Brugge (Harvard Medical School, Boston, MA, USA). The pGEX-2T constructs of SH2 domains and SH2/SH3 domain tandems, generously provided by Dr G. Superti-Furga (European Molecular Biology Laboratory, Heidelberg, Germany), were: Abl SH2 domain, Src SH2 domain, wild-type c-Src SH2/SH3 domain tandem, Ser94 fi Pro/Arg95 fi Trp c-Src-SH2/SH3 tandem (mutation in SH3 domain, SH2 intact), Arg175 fi Lys c-Src SH2/SH3 tandem (mutation in SH2 domain, SH3 intact). The antiFAK polyclonal antibodies A-17 (directed against residues 2–18)


Binding of neuronal FAK to SH3/SH2 domains 255

and C-20 (directed against residues 1033–1052) and the anti-GFP monoclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-phosphotyrosine monoclonal antibody (clone 4G10) and the rabbit polyclonal anti-FAK [pY397] phosphospecific antibody were from Upstate Biotechnology (Lake Placid, NY, USA) and Biosource International (Camarillo, CA, USA), respectively. The L42 and L56 sera recognizing the nonneuronal and neuronal forms of FAK were raised in rabbits and used for immunoprecipitation. The antibodies against amphiphysin I (mouse mAbs #4) and against amphiphysins I/II (rabbit CD9 antibody) were kindly provided by Drs P. De Camilli and O. Cremona (Yale University, New Haven, MC, USA). The peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were from Bio-Rad (Milan, Italy). The pBK-CMV and pEGFP-C1 vectors were purchased from Stratagene (La Jolla, CA, USA) and Clontech (Palo Alto, CA, USA), respectively. The peptide DPSSPPKKPPRPG, corresponding to the second COOH-terminal proline-rich region (PR2, FAK868–880), and the respective control peptide in which G was substituted for P (DGAAGGKKGGRGG) were synthesized by Sigma-Genosys (Cambridge, UK) and purified by HPLC. Protein expression and purification Bacterial cells were transformed to ampicillin resistance by electroporation with constructs containing pGEX-2T in frame with sequences encoding for SH3 and SH2 domains. Large-scale cultures of Luria broth containing ampicillin (100 lg/mL) were inoculated with small overnight cultures grown at 37C to log phase and induced with isopropyl b-O-thiogalactopyranoside (IPTG, 100 lM) for 3–5 h. GST and GST-SH3/SH2 domain fusion proteins were extracted from bacterial lysates and purified to homogeneity by affinity chromatography on glutathione–sepharose and dialyzed against 25 mM Tris-Cl, 50 mM NaCl, pH 7.4 (Onofri et al. 2000). Preparation of brain tissue extracts Subcellular fractionation of rat cerebral cortex was performed as previously described (Huttner et al. 1983). Crude synaptosomal pellets (P2) were resuspended in 10 volumes of extraction buffer (50 mM Tris-Cl, pH 7.4, 2% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and 1 lg/mL pepstatin), extracted for 1 h on ice and centrifuged at 400 000 g for 1 h in a Beckman TL-100 ultracentrifuge. The soluble fraction was then immediately subjected to binding assays (see below). Highly purified synaptosomes were obtained by centrifugation of the resuspended P2 fraction at 32 500 g for 5 min on a discontinuous four-step 3–23% Percoll gradient and by collecting the third and fourth interfaces, as previously described (Dunkley et al. 1986). Cell culture and transfection COS-7 simian fibroblasts were grown in DMEM medium containing 10% FCS. Cells (8 · 104) were plated 16–20 h before transfection on uncoated 100 mm-diameter culture dishes and transfected 16–20 h after plating by calcium phosphate precipitation with 30 lg of DNA/ dish (Sambrook et al. 1989). Forty-eight hours after transfection, cells were lysed for 15 min in ice cold RIPA buffer (1% Triton X-100, 0.5% deoxycholete, 50 mM Tris pH 7.6, 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM EDTA, 1 mM phenyl-

methylsulfonyl fluoride, 1 lg/mL aprotinin, 1 lg/mL leupeptin, 1 lg/mL pepstatin). Cell lysates were clarified at 18 000 g for 10 min at + 4C and the supernatant fractions were immediately frozen in liquid N2 for further analysis. Aliquots of the cell extracts were subjected to quantitative FAK immunoblotting in order to evaluate the expression of the various FAK isoforms after transfection. SH3 and SH2 binding assays The binding of GST-SH3 domains, GST-SH2 domains or GST-SH2/ SH3 domain tandems to extracts of brain synaptosomes or of transfected COS-7 cells was assessed by affinity precipitation experiments as previously described (Onofri et al. 2000). GST or GST-fusion proteins were coupled to prewashed glutathione– sepharose (0.1 nmol fusion protein per lL settled sepharose beads) in binding buffer (10 mM Hepes, 150 mM NaCl, 1% v/v Triton X-100, pH 7.4). After extensive washing, 50 lL of fusion proteincoupled beads were incubated for 3–5 h at 4C in 350–500 lL (final volume) of binding buffer with either P2 extract (400 lg total protein) or RIPA lysates of transfected COS-7 cells (approximately 300–400 lg total protein, containing reproducible amounts of recombinant FAK). After the incubation, the beads were pelleted by centrifugation, washed three times in binding buffer, twice in detergent-free binding buffer, resuspended in SDS stop buffer (Laemmli 1970) and boiled for 5 min. Immunoprecipitation Nonidet P-40 (2% v/v) extracts of Percoll-purified synaptosomes or RIPA extracts of transfected COS-7 cells were incubated for 3 h at 4C with each of the following antibodies: anti-pan-FAK antibody directed against the NH2-terminal domain of FAK (L56 serum, 20 lL), anti-FAK+ antibody directed against the COOH-terminal [Pro-Trp-Arg] insert of FAK+ (L42 serum, 20 lL), control rabbit serum (20 lL), anti-amphiphysin I monoclonal antibody (mAb #4, 5 lg purified IgG) and control mouse IgG (5 lg). Immunoprecipitation was performed as previously described (Derkinderen et al. 1996) using protein G-sepharose (25 lL settled prewashed beads) pre-adsorbed with the respective antibodies to precipitate the protein complexes. The immune complex kinase assays on recombinant FAK isoforms from transfected COS-7 cells were performed as previously described (Toutant et al. 2000). Treatments and subfractionation of synaptosomes Percoll-purified synaptosomes (400 lg protein/200 lL sample) were resuspended in Hepes-buffered medium (HBM; 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1.3 mM MgSO4, 1 mM Na2HPO4, 20 mM Hepes, pH 7.4) containing glucose, pre-incubated at 37C for 10 min and incubated for additional 10 min with either cytochalasin B (20–200 lM), mycalolide B (20 lM), PP2 (20 lM) or PAO (1 mM) solubilized in dimethyl sulfoxide. Dimethyl sulfoxide was added as vehicle to control samples and its concentration in the synaptosomal samples never exceeded 0.5%. In the case of phalloidin, the membrane-impermeant compound was internalized into synaptosomes by homogenization in buffered sucrose containing 50–100 lM phalloidin (Bernstein and Bamburg 1989). After the treatments, synaptosomes were chilled at 4C, washed once in HBM buffer, osmotically lysed in 20 mM Hepes pH 7.4 and spun at 400 000 g for 20 min in a Beckman TL-100 ultracentrifuge (Bongiorno-Borbone et al. 2002).



Assays of exo-endocytosis in synaptosomes Percoll-purified synaptosomes were homogenized in the presence of either the proline-rich peptide corresponding to the FAK PR2 region (PRP) or a control glycine-rich peptide (GRP) 1 mM in the homogenization medium; Raiteri et al. 2000. The release of glutamate was assessed by a previously described fluorometric technique (Nicholls and Sihra 1986). Synaptosomes (1 mg protein) were incubated in 1 mL of HBM containing 50 U glutamate dehydrogenase, 1 mM NADP, 10 mM glucose and either 1.2 mM CaCl2 or 3 mM EGTA for 10 min at 37C before KCl (30 mM)induced depolarization. Ca2+-dependent release was calculated as the difference between the samples run in the presence of Ca2+ and those run in the presence of EGTA. Synaptic vesicle endocytosis was assayed as previously described (Marks and McMahon 1998; Di Paolo et al. 2002). Briefly, synaptosomes (1 mg protein) were incubated in 1 mL of HBM containing 10 mM glucose and 0.1 mM FM 2–10 in the presence of either 1.2 mM CaCl2 or 3 mM EGTA for 3 min at 37C before vesicle cycling was induced by the addition of KCl (30 mM) for 2 min. After depolarization, synaptosomes were washed twice in HBM containing 1 mg/mL bovine serum albumin to remove the non-internalized dye, osmotically lysed in 20 mM Hepes pH 7.4 for 10 min and centrifuged at 125 000 g for 5 min. After centrifugation, the supernatant (LS1 fraction) containing synaptic vesicles was removed and measured for FM 2–10 fluorescence. Immunoblotting assays Aliquots of glutathione–sepharose eluates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; Laemmli 1970) on 7% polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. For immunoblotting, membranes were blocked in Tris-buffered saline (50 mM TrisCl, 200 mM NaCl, pH 7.4)/5% (w/w) non-fat dry milk or bovine serum albumin (in the case of phospho-specific antibodies) and incubated overnight at 4C with 0.2 lg/mL of the affinity-purified primary antibodies, as previously described (Benfenati et al. 1992). After wash and incubation with horseradish peroxidase-conjugated secondary antibodies (1 : 3000 dilution), immunoreactivity was analyzed by using the chemiluminescence detection system. Miscellaneous procedures Protein concentrations were determined using the Bradford (BioRad, Milan, Italy) assay using bovine serum albumin as standard. Quantitation was carried out by laser scanning densitometry (Ultroscan XL, LKB, Bromma, Sweden) of the exposed films obtained in the linear range of the emulsion response by interpolation of the values into a suitable standard curve made with various amounts of brain homogenate. Statistical analysis was carried out by one-way ANOVA followed by the Student’s t-test or the Duncan’s multiple comparison test. The correlation between translocation of FAK and translocation of amphiphysins was assessed by the Pearson’s correlation coefficient.

Results

Binding of brain FAK isoforms to SH3 domains We analyzed the interactions of endogenous brain FAK as well as its brain-specific splice isoforms overexpressed in

COS-7 cells with an array of SH3 domains of signaling or adapter proteins such as NH2-terminal and COOH-terminal Grb2, c-Src, n-Src, Fyn, Yes, PI3K, Ras-GAP, PLCc, Crk, p47 and amphiphysins. All these domains were expressed in Escherichia coli as fusion proteins with GST and their interactions with the proline-rich regions of FAK were examined by affinity chromatography assays. The SH3 binding of endogenous brain FAK was highly specific. The binding was high with the SH3 domains of c-Src, Fyn, Yes, PI3K and amphiphysin II and moderate with the SH3 domains of NH2-terminal Grb2, PLCc and amphiphysin I. GST and the SH3 domains of COOH-terminal Grb2, Crk, p47, RasGAP and n-Src exhibited no or negligible binding (Fig. 1a). While the site of insertion of the COOH-terminal tripeptide that is shared by all the neuron-specific FAK isoforms (FAK+) does not overlap with proline-rich regions, the site of insertion of boxes 6, 7 and 28, generated by the alternative use of various neuronal exons, occurs in the vicinity of the sites involved in the interaction with the SH2 and SH3 domains of Src kinases within the NH2-terminal regulatory domain (Burgaya et al. 1997; Thomas et al. 1998). Thus, it was of interest to ascertain whether the presence of these inserts may either lead to quantitative variations in the known interactions with SH3 and/or SH2 domains or promote interactions with previously unrecognized partners. To this aim, we overexpressed neuronal FAK (FAK+) as well as its splicing isoforms FAK+6,7 and FAK+6,7,28 in COS-7 simian fibroblasts, which do not naturally express the neuronal isoforms of FAK. Lysates of the transfected cells were used as a source of FAK+ isoforms to analyze, by affinity chromatography, the specific properties of the various isoforms in the interactions with SH3 and SH2 domains. FAK+ isoforms were overexpressed either as single polypeptides or as fusion proteins with GFP (see Experimental procedures). Both FAK+ and FAK+GFP isoforms were used in the binding experiments with indistinguishable results. In fact, the presence of GFP did not significantly alter the FAK binding pattern to either SH3 or SH2 domains and GFP expressed alone did not exhibit any detectable interaction with SH3 and/or SH2 domains even when used in a 5–10-fold molar excess with respect to FAK+GFP (Fig. 1b). As the expression levels of FAK+GFP were generally much higher than those of FAK+, FAK isoforms fused with GFP were preferably used. Similarly to endogenous FAK, the recombinant FAK+ isoforms exhibited high binding to the SH3 domains of Src kinases, PI3K and amphiphysin II and very low or negligible binding to the SH3 domains of n-Src, COOH-terminal Grb2, Crk, RasGAP and p47 (Fig. 1a). However, the interactions with the amphiphysin I and PLCc SH3 domains were less pronounced for endogenous FAK, probably because of the presence of high affinity interactors in synaptosomal extracts. Interestingly, the splice isoform FAK+ displayed an overall



(a)

(b)

(c)

higher binding than FAK+6,7 with the SH3 domains of c-Src, Fyn and PI3K. The effect was particularly pronounced in the case of the c-Src SH3 domain with a dramatic and highly significant 60% decrease in binding due to the insertion of boxes 6 and 7 (Fig. 1c). The further inclusion of box 28 in the FAK+6,7 molecule (FAK+6,7,28) did not significantly affect the SH3 binding pattern with respect to FAK+6,7 (data not shown), suggesting that the effect observed with the NH2-terminal splice variants of FAK+, is relatively independent of the length of the peptide insert.

Fig. 1 Binding of endogenous brain FAK and of recombinant FAK isoforms to SH3 domains. (a) The binding of endogenous FAK from synaptosomal extracts as well as of recombinant FAK isoforms (FAK+ and FAK+6,7) overexpressed in COS-7 cells as fusion proteins with GFP to the indicated GST-SH3 domains or to GST alone was analyzed by affinity chromatography on glutathione–sepharose followed by immunoblotting with anti-FAK specific antibodies. Equal amounts of immunoreactive FAK and equimolar amounts of SH3 domains were added to the incubation mixture. The amounts of endogenous brain FAK (End. FAK), FAK+ or FAK+6,7 bound to the SH3 domains were analyzed by quantitative immunoblotting with anti-FAK antibodies (three top panels). The amounts of GST or SH3 domain-GST fusion proteins present in the assay are shown as Coomassie staining of the gels (bottom panel). The molecular mass standards shown on the left indicate the slower electrophoretic mobility of FAK+GFP with respect to native FAK+ and the slightly higher apparent molecular mass of FAK+6,7GFP with respect to FAK+GFP. (b) FAK+GFP (upper panel), equimolar amounts of GFP (middle panel) or a fivefold excess GFP (lower panel) overexpressed in COS-7 cells were analyzed for binding to GST or to the indicated SH2 and/or SH3 domains fused to GST by affinity chromatography on glutathione–sepharose. The coprecipitated proteins were detected using anti-GFP antibodies. Molecular mass standards are shown on the left. f.l. Grb2, full length Grb2; Total, total amounts loaded. (c) The binding of endogenous brain FAK (open bars), FAK+ (grey bars) and FAK+6,7 (black bars) to the SH3 domains was quantitatively analyzed by densitometric scanning of the immunoblots. Binding values (means ± SEM; n ¼ 5) are expressed in percentage of the binding to full length Grb2 (containing two SH3 and one SH2 domains) taken as internal standard. The statistical analysis was carried out by comparing the binding of FAK+6,7 to the various SH3 ligands with that of FAK+ (*p 0.05; **p < 0.01; Student’s t-test for paired samples).

Binding of brain FAK isoforms to SH2 domains and to the c-Src SH2/SH3 domain tandem The vicinity of the SH3 binding site for c-Src to the FAK autophosphorylation/SH2 binding site and the possibility that the recruitment and activation of Src-family kinases is mediated by a dual interaction with SH2 and SH3 domains (Mayer 1997; Thomas et al. 1998) prompted us to investigate the effect of the inclusion of boxes 6 and 7 on the binding to the isolated SH2 domain and SH2/SH3 domain tandem of c-Src. The SH2 domains of Abl and Src, the tandem of c-Src SH2/SH3 domains, as well as the tandem of c-Src SH2/SH3 domains bearing point mutations in either the SH2 (Arg175 fi Lys) or the SH3 domain (Ser94 fi Pro/Arg95 fi Trp) that abolish binding were expressed as fusion proteins with GST and their binding to endogenous and recombinant FAK was examined by affinity chromatography. While the interaction with the Abl SH2 domain was negligible, the binding of both endogenous brain FAK and recombinant FAK+ isoforms to the Src SH2 domain was approximately fivefold higher than the binding to the c-Src SH3 domain (Fig. 2a), consistent with previous observations (Thomas et al. 1998). Accordingly, the binding of



Fig. 2 Binding of endogenous brain FAK and of recombinant FAK isoforms to c-Src SH2/SH3 domains. (a) The binding of endogenous brain FAK as well as of recombinant FAK isoforms (FAK+ and FAK+6,7) overexpressed in COS-7 cells as fusion proteins with GFP to the indicated SH2 domains, SH3 domains and c-Src SH2/SH3 domain tandem expressed as GST fusion proteins was evaluated as described in the legend to Fig. 1. The binding of FAK to the c-Src SH2/ SH3 domain tandem was also evaluated in the presence of point mutations in either the SH2 (Arg175 fi Lys; c-Src mSH2/SH3) or the SH3 (Ser94 fi Pro/Arg95 fi Trp; c-Src SH2/mSH3) domain. Equal amounts of immunoreactive FAK and equimolar amounts of SH2/SH3 domains were added to the incubation mixture. The bound amounts of endogenous brain FAK (End. FAK), FAK+ or FAK+6,7 were analyzed by quantitative immunoblotting with anti-FAK antibodies (three top panels). The amounts of GST or GST fusion proteins present in the assay are shown as Coomassie staining of the gels (bottom panel). Molecular mass standards are shown on the left. (b) The immunoblots from five independent experiments were quantitatively analyzed by laser scanning densitometry. The binding of endogenous brain FAK (open bars), FAK+ (hatched bars) and FAK+6,7 (black bars) to the various SH2 and/or SH3 ligands is expressed in percentage of the binding to full length Grb2 taken as internal standard (means ± SEM). The statistical analysis was carried out by comparing the binding of FAK+6,7 with the various ligands with that of FAK+ (*p < 0.05; **p < 0.01; Student’s t-test for paired samples).

(a)

(b)

endogenous brain FAK and recombinant FAK+ isoforms to the wild-type c-Src SH2/SH3 domain tandem was only slightly higher than that of the Src SH2 domain alone and was not greatly affected by the point mutation in the SH3 domain, while it was dramatically reduced by the point mutation in the SH2 domain, that decreased the binding to the level of the isolated c-Src SH3 domain (Figs 2a and b). Interestingly, the binding to the Src SH2 domain was significantly higher for the FAK+6,7 isoform than for the FAK+ isoform; moreover, the point mutation in the SH2 domain practically abolished the binding of FAK+6,7 to the c-Src SH2/SH3 domain, consistent with the impaired ability of this FAK isoform to interact with the c-Src SH3 domain (Fig. 2b). As only the autophosphorylated form of FAK binds to the Src SH2 domain, the SH2 domain binding pattern suggests that both endogenous brain FAK and recombinant FAK isoforms are constitutively phosphorylated on tyrosine and that inclusion of the 6,7 boxes affects the levels of basal

phosphorylation. Indeed, immunoblotting with anti-phosphotyrosine antibodies of extracts of synaptosomes and COS-7 cells overexpressing either FAK+ or FAK+6,7 fused to GFP revealed that both endogenous FAK and recombinant FAK isoforms are phosphorylated on tyrosine residues including Tyr397 and that FAK+6,7 isoform is consistently more phosphorylated than FAK+ under basal conditions (Fig. 3a). The higher level of basal phosphorylation of the FAK+6,7 isoform with respect to the FAK+ isoform was associated, under our experimental conditions, with a higher autophosphorylation activity in immune complex kinase assays (Fig. 3b), in agreement with previous reports (Burgaya et al. 1997; Toutant et al. 2000). Role of PR2 in the association of FAK with amphiphysin The novel interaction of both endogenous brain FAK and recombinant neuronal FAK isoforms with the SH3 domains of amphiphysins may be functionally important in nerve terminals. In an attempt to identify the FAK proline-rich region involved in this interaction, we transfected COS-7 cells with either wild-type FAK+ or a truncated mutant of FAK (FAK D3) lacking a COOH-terminal fragment (residues 841–1054) including the COOH-terminal proline-rich region (PR2 region, encompassing Pro878) and analyzed the corresponding cell extracts for binding to the SH3 domains of PI3K, reported to bind, at least in part, to the more NH2terminal proline-rich region (PR1 region, encompassing Pro712/715; Schaller 1996) and amphiphysin II. As shown in Fig. 4a, the deletion of the COOH-terminal fragment



(a)

(b)

Fig. 3 FAK+6,7 is constitutively more phosphorylated and has a higher autophosphorylation activity than FAK+. (a) Extracts of brain synaptosomes (End. FAK; 100 lg total protein) and of COS-7 cells overexpressing either FAK+ or FAK+6,7 fused to GFP (12 lg total protein) were subjected to immunoblotting with anti-FAK (upper panel), anti-phosphotyrosine antibody (middle panel) and phosphospecific anti-FAK P-Tyr397 (lower panel) antibodies. (b) Extracts of COS-7 cells overexpressing either FAK+ or FAK+6,7 fused to GFP (200 lg total protein) were subjected to immunoprecipitation with antiFAK antibodies (L42 serum) followed by immune complex kinase assay in the presence of 10 lM ATP and immunoblotting with anti-FAK (upper panel), anti-phosphotyrosine (middle panel) and phosphospecific anti-FAK P-Tyr397 (lower panel) antibodies. Molecular mass standards are shown on the left.

decreased the binding to the PI3K SH3 domain and abolished the binding to the amphiphysin SH3 domain, suggesting an involvement of the PR2 region in the interaction. In addition, an excess of a synthetic peptide corresponding to the PR2 region (DPSSPPKKPPRPG, FAK868–880) significantly inhibited the binding of FAK+ to the SH3 domains of amphiphysins I and II, PI3K and PLCc when compared with a peptide in which G was substituted for P (DGAAGGKKGGRGG), while it did not affect the binding to the SH3 domains of Src kinases (Fig. 4b). These data indicate that not only amphiphysins bind to the PR2 region of FAK, but that PI3K and PLCc also have affinity for this site of interaction.

Association of amphiphysin with FAK in intact brain synaptosomes To determine whether an association between FAK and amphiphysins occurs in intact brain, we analyzed whether the two proteins co-distribute in brain tissue. In the rat forebrain, FAK and amphiphysin I exhibited a similar subcellular distribution, with the only exception of the cytosolic fraction S3 and of the S3-containing fraction S2 in which FAK was significantly more enriched than amphiphysin I, suggesting a more widespread role for FAK in neuronal cells (Figs 5a and b). Not considering the latter two fractions, a highly significant correlation was found between the subcellular distributions of FAK and amphiphysin immunoreactivities (Fig. 5c). As a closely similar distribution of FAK and amphiphysin was observed in synaptosomal subfractions, we performed coimmunoprecipitations from detergent extracts of purified synaptosomes using two distinct anti-FAK antibodies (the anti-FAK+ L42 serum and the anti-pan-FAK+ L56 serum) or a non-specific rabbit serum and analyzed the immunoprecipitates for the presence of amphiphysins I and II. As shown in Fig. 6, western blot analysis of the immunoprecipitates demonstrated that significant amounts of amphiphysins I and II are associated with FAK in the complexes irrespective of the anti-FAK antibody used for immunoprecipitation, whereas no detectable amounts of either FAK or amphiphysin were recovered when the non-specific serum was used. When both amphiphysins I and II were immunoprecipitated with a specific monoclonal antibody (mAb #4), small but significant amounts of FAK were coimmunoprecipitated (Fig. 6), consistent with the lower abundance of FAK in synaptosomes as compared with amphiphysins and other abundant amphiphysin partners such as dynamin and synaptojanin. The association of FAK with the plasma membrane in nerve terminals is regulated by its phosphorylation on tyrosine residues and by the state of assembly of the actin cytoskeleton (Bongiorno-Borbone et al. 2002). Thus, we investigated whether treatments of purified synaptosomes with agents affecting the actin cytoskeleton (the actin depolymerizing agents cytochalasin B and mycalolide B and the F-actin stabilizing agent phalloidin) or FAK tyrosine phosphorylation (the Src kinase inhibitor PP2 or the tyrosine phosphatase inhibitor PAO) were able to induce similar changes in the compartmentalization of FAK and amphiphysin. Interestingly, the response of amphiphysin I to the treatments was closely similar to that of FAK, with decreased association of amphiphysin I with the plasma membrane after disruption of the actin cytoskeleton or inhibition of FAK phosphorylation (Figs 7a and b). The translocations of the two proteins between the membrane and the synaptosol were strictly correlated (Fig. 7c), suggesting that FAK is indeed associated with amphiphysin and contributes to its compartmentalization in nerve terminals.



Fig. 4 The amphiphysin SH3 domains bind to the COOH-terminal proline-rich region (PR2) of FAK. (a) Binding of recombinant FAK+ and of its COOH-terminal truncation mutant FAK+ D3 to PI3K and amphiphysin SH3 domains. FAK+ and its truncation mutant FAK D3 lacking the COOH-terminal fragment (residues 841–1054) were overexpressed in COS-7 cells. RIPA extracts of mock transfected (pBK-CMV vector alone) and of FAK transfected cells were assayed for binding to equimolar amounts of GST or of the GST-SH3 domains of PI3K and amphiphysin II by affinity chromatography on glutathione–sepharose. The recombinant FAK+ and FAK D3 bound to the SH3 domains were analyzed by quantitative immunoblotting with NH2-terminal-specific anti-FAK antibodies (upper panel). The amounts of GST and GST fusion proteins present in the assay are shown as Coomassie staining of the gels (bottom panel). The molecular mass standards shown on the left indicate the higher electrophoretic mobility of FAK D3 with respect to wild-type FAK+. The COOH-terminal truncation of FAK+ virtually abolished the binding to the amphiphysin II SH3 domain, whereas it only decreased the binding to the PI3K SH3 domain. (b) Inhibitory effects of a peptide corresponding to the FAK PR2 region. The binding of recombinant FAK isoforms FAK+, overexpressed in COS-7 cells as fusion protein with GFP, to GST or to the indicated SH3 domain-GST fusion proteins was evaluated as described in the legend to Fig. 1. Five hundred micromoles of either the proline-rich peptide corresponding to the FAK PR2 region (PRP) or a control peptide in which glycine was substituted for proline (GRP) were present in the assay. Equal amounts of immunoreactive FAK and equimolar amounts of SH3 domains were added to the incubation mixture. The respective amounts of bound FAK+ were detected by immunoblotting with anti-FAK antibodies. In the bottom panel, the amounts of GST or GST fusion proteins present in the assay are shown as Coomassie staining of the gels. Molecular mass standards are shown on the left.

(a)

(b)

Amphiphysins are key actors of synaptic vesicle endocytosis (Cremona and De Camilli 1997; Wigge and McMahon 1998; Di Paolo et al. 2002) and their activity is mediated by interactions between their SH3 domain and proline-rich regions of other nerve terminal partners such as dynamin (Shupliakov et al. 1997; Zhang and Zelhof 2002). In order to ascertain whether the interaction with FAK plays a role in amphiphysin function, we introduced into nerve terminals a synthetic peptide (PRP) corresponding to the site of interaction in FAK and demonstrated to inhibit the FAK– amphiphysin interaction in vitro (see above) and assayed its effects on exocytosis and endocytosis. While PRP internalization did not affect Ca2+-dependent glutamate release with respect to a control peptide (Figs 8a and b), it significantly inhibited the Ca2+-dependent endocytotic trapping of the extracellular styryl dye FM 2–10 stimulated by depolarization (Fig. 8c). Discussion

Several FAK isoforms generated by the transcription of additional short exons are highly expressed in the central nervous system and this diversity may serve distinct functional properties (Burgaya et al. 1997). The major neuronal isoform is FAK+6,7 (Toutant et al. 2000; Derkinderen et al.

2001). Remarkably, the insertion of boxes 6 and 7 occurs in the proximity of the putative binding sequences for the Src SH2 and SH3 domains (Thomas et al. 1998), thus raising the possibility that the various brain isoforms display distinct patterns of interaction with Src kinases and/or are coupled to distinct signal transduction cascades. We have for the first time investigated the specific interactions of neuron-specific FAK with SH3 and SH2 domains and the presence of changes in the interaction pattern of the differentially spliced brain FAK isoforms using endogenous FAK from brain tissue as well as pure recombinant neuronal FAK+ isoforms overexpressed in COS-7 cells. The data presented here indicate that: (i) the SH3 domains of all Src-family kinases tested, with the notable exception of n-Src, are good ligands for both endogenous brain FAK and recombinant neuronal FAK isoforms; (ii) FAK+6,7, the most prominent brain splice isoform of FAK, exhibits a markedly decreased binding to the SH3 domain of c-Src (and, to a lesser degree, of Fyn and PI3K); (iii) the FAK+6,7 isoform exhibits an increased interaction with the Src SH2 domain associated with increased levels of basal tyrosine phosphorylation and autophosphorylation activity; (iv) neuronal FAK isoforms display previously unknown



(a)

(b)

(c)

Fig. 5 Subcellular distribution of FAK and amphiphysin in the rat forebrain. The distribution of FAK and amphiphysin I in subcellular fractions of rat forebrain (S1, postnuclear supernatant; S2, supernatant of P2; P2, crude synaptosomes; S3, cytosol; P3, microsomes; LS1, supernatant of LP1; LP1, crude synaptic membranes after osmotic lysis of P2; LS2, synaptosol; LP2, crude synaptic vesicles) was analyzed by quantitative immunoblotting (a). In (b), the enrichment factors of FAK (gray bars) and amphiphysin I (black bars) with respect to total forebrain homogenate are reported as means ± SEM of four independent experiments. In (c), the correlation between the enrichment factors of FAK and amphiphysin I was analyzed by linear regression and Pearson’s correlation coefficient (r). The regression function and the 95% confidence intervals excluding the S2 and S3 fractions are shown as solid and dotted lines, respectively. r ¼ 0.99; p < 0.001.

interactions with the SH3 domains of amphiphysins, a family of proteins involved in endocytosis and cytoskeletal assembly, PLCc and the NH2-terminal Grb2 SH3 domain; and (v)

Fig. 6 FAK and amphiphysin I associate in extracts of purified synaptosomal fractions. Nonidet P-40 (2% v/v) extracts of Percoll-purified synaptosomes prepared from rat forebrain were incubated for 3 h at 4C with the following antibodies: rabbit anti-pan-FAK antibody (L56 serum, 20 lL), rabbit anti-FAK+ antibody (L42 serum, 20 lL), control rabbit serum (20 lL), anti-amphiphysin I monoclonal antibody (mAb #4, 5 lg purified IgG) and control mouse IgG (5 lg). The protein complexes were sedimented with protein G–sepharose pre-adsorbed with the respective antibodies and the samples were resolved by SDS–PAGE and transferred to nitrocellulose membranes. Immunoblotting was performed with primary anti-FAK (C-20), anti-amphiphysin I (mAb #4) and anti-amphiphysins I/II (CD9) antibodies, followed by the appropriate peroxidase-conjugated secondary antibodies and the chemiluminescence detection system.

FAK and amphiphysins are associated in nerve terminals and this interaction may contribute to the subcellular distribution of amphiphysins and to their role in endocytosis. A direct involvement of SH3 and SH2 domains in presynaptic physiology was originally suggested by the demonstration that several proteins that play a pivotal role in the exo-endocytotic cycle of synaptic vesicles and in signal transduction at synaptic level such as amphiphysin, endophilin, dynamin, synaptojanin and synapsin contain SH3 domains or are excellent ligands for SH3 domains (McPherson et al. 1994; Cremona and De Camilli 1997; Onofri et al. 1997; Onofri et al. 2000). The novel SH3-mediated interaction between neuronal FAK isoforms and amphiphysins may be of physiological importance. Both proteins have been reported to be present in the presynaptic compartment in soluble and membrane-associated pools (Siciliano et al. 1996; Lichte et al. 1992). Here, we demonstrate that FAK and amphiphysin share a similar subcellular distribution, that amphiphysin can be efficiently coimmunoprecipitated with FAK+ from intact nerve terminals and that amphiphysin translocation parallels FAK translocation in response to stimuli affecting FAK tyrosine phosphorylation and the



(a)

(b)

(c)

Fig. 7 Effects of changes in actin assembly and tyrosine phosphorylation on the membrane association of FAK and amphiphysin in brain synaptosomes. Synaptosomes were treated with either vehicle (CONT), phalloidin (PHALL, internalized during homogenization), cytochalasin B (CYTO B, 20 lM), mycalolide B (MYC B, 20 lM), PP2 (20 lM) or PAO (1 mM). After osmotic lysis and high-speed centrifugation, corresponding aliquots of total synaptosomes (T) and of the respective membrane fractions (M) were subjected to SDS–PAGE followed by immunoblotting with anti-FAK and anti-amphiphysin I antibodies. (a) The results of a representative experiment are shown. (b) The effects of the various treatments on the membrane association of FAK and amphiphysin I were quantitatively analyzed and expressed as percentages of the corresponding values found in control, untreated synaptosomes (means ± SEM of 6–10 independent experiments). *p < 0.05; **p < 0.01 versus control group; Duncan’s multiple comparison test. (c) The correlation between the membrane association of FAK and that of amphiphysin I was analyzed by linear regression and Pearson’s correlation coefficient (r). The regression function and the 95% confidence intervals are shown as solid and dotted lines, respectively. r ¼ 0.99; p < 0.001.

Fig. 8 Effects of a proline-rich peptide corresponding to the FAK PR2 region on exo-endocytosis in synaptosomes. (a) Fluorometric tracings of KCl-induced glutamate release from Percoll-purified synaptosomes in which the proline-rich peptide corresponding to the FAK PR2 region (PRP) or a control glycine-rich peptide (GRP) was internalized during homogenization. (b) Ca2+-dependent glutamate release was measured 10 min after depolarization as the difference between the release observed in the presence of extracellular Ca2+ and that in the presence of EGTA (n ¼ 8). (c) Ca2+-dependent FM 2–10 loading in synaptosomes in which either PRP or GRP was internalized during homogenization. The difference between the amount of FM 2–10 trapped into synaptic vesicles after a first round of stimulation with 30 mM KCl in the presence of extracellular Ca2+ and that trapped in the presence of EGTA was analyzed in a low-speed supernatant after osmotic lysis of synaptosomes. **p < 0.01, Student’s t-test.



assembly of the actin cytoskeleton. The disruption of the FAK–amphiphysin interaction by internalization of a prolinerich peptide corresponding to the FAK site brought about a defect in endocytosis. Although it is possible that the FAK proline-rich peptide could interfere with other interactions of amphiphysin and/or with the functions of other SH3 domain protein implicated in endocytosis, the data suggests an involvement of FAK in the recruitment of amphiphysins to the plasma membrane and in the regulation of their interactions with the actin cytoskeleton and other proline-rich partners. Various studies suggested that amphiphysin family members are implicated in the regulation of the dynamics of actin cytoskeleton. Thus, amphiphysin is colocalized with actin patches in both axonal growth cones and transfected fibroblasts (Butler et al. 1997; Mundigl et al. 1998) and amphiphysin mutants in yeast or decreased amphiphysin expression in hippocampal neurons are associated with defects in actin cytoskeleton organization (Munn et al. 1995; Sivadon et al. 1995; Mundigl et al. 1998). In non-neuronal cells, FAK interacts with components of the actin cytoskeleton such as talin, a-actinin and paxillin and is part of the complex that anchors the F-actin cytoskeleton to the plasma membrane (Guinebault et al. 1995; Hynes 1999; Critchley 2000). Thus, the FAK/amphiphysin interaction may represent one of the potential mechanisms for the effects of amphiphysin on actin physiology and synaptic vesicle trafficking. FAK interacts with Src in most cells studied. However, it has been reported recently that in hippocampal slices induced to express long-term potentiation or stimulated by endocannabinoids FAK interacts preferentially with Fyn (Lauri et al. 2000; Derkinderen et al. 2001). FAK basal phosphorylation is impaired in Fyn knockout but not in Src or Yes knockout mice (Grant et al. 1995) and endocannabinoidinduced phosphorylation is also prevented in Fyn mutant mice (Derkinderen et al. 2001). The fact that FAK does not bind to the SH3 domain of n-Src, a Src isoform abundantly expressed in neurons, and that the major brain FAK isoform FAK+6,7 has a dramatically reduced binding to the SH3 domain of c-Src may explain the preferential association of FAK with Fyn in brain. Moreover, the decreased association of c-Src SH3 domain and, to a lesser extent of Fyn SH3 domain, with FAK+6,7 provides a possible explanation for the lack of direct phosphorylation of Tyr397 by Src family kinases in FAK+6,7/Src cotransfected cells (Toutant et al. 2000) and for the strikingly different subcellular distribution of FAK and Src kinases in brain tissue (Burgaya et al. 1995; Siciliano et al. 1996; Bongiorno-Borbone et al. 2002). The current hypothesis regarding the activation of nonneuronal FAK holds that FAK autophosphorylation is promoted in a Src-independent manner by extracellular signals; subsequently inactive Src becomes activated by a dual SH2/SH3 interaction with autophosphorylated FAK and mediates the downstream phosphorylation cascade (Schaller 1996; Girault et al. 1999). Our data showing that FAK+6,7

exhibits both a markedly decreased interaction with the c-Src SH3 domain and an increased interaction with the Src SH2 domain suggest that the predominant brain FAK isoform may not be permanently associated with Src (or Fyn) and that this association, triggered by FAK autophosphorylation, may promote a partial activation of Src (or Fyn). Thus, autophosphorylation of Tyr397 may represent a potent molecular switch by which Src family kinases recruit the major brain FAK isoform to the membrane in neurons. In this respect, we have recently reported that a significant correlation exists between autophosphorylation and translocation of FAK from the cytosol to the membrane in rat brain synaptosomes (Bongiorno-Borbone et al. 2002). Although FAK is highly enriched in neurons and activated by depolarization and neurotransmitters, its precise function in neuronal physiology remains elusive. It is known that tyrosine phosphorylation plays a critical role in synaptic function and that tyrosine kinases inhibitors block two widely studied forms of synaptic plasticity, long-term potentiation and long-term depression (Boxall and Lancaster 1998). Activation of Src has been found to be required for longterm potentiation induction (Lu et al. 1998). Hippocampal long-term potentiation is severely impaired in mice lacking the tyrosine kinase Fyn (Grant et al. 1992) and FAK phosphorylation is severely impaired in Fyn knock-out mice (Grant et al. 1995; Derkinderen et al. 2001). These observations make FAK an excellent candidate for coupling neuronal activity to changes in synaptic efficacy. Our findings showing that the multiple brain FAK isoforms display distinct patterns of autophosphorylation and interaction with neuronal proteins involved in signal transduction and membrane trafficking further underline the multifunctional role of FAK in synaptic transmission in the central nervous system. Acknowledgements We thank Drs I. Gout (Ludwig Institute for Cancer Research, University College, London, UK), P. De Camilli and O. Cremona (Yale University Medical School, New Haven, CT), G. Superti-Furga (EMBL, Heidelberg, Germany) and J. Brugge (Harvard University Medical School, Boston, CT, USA) for providing SH3 and SH2 domain constructs and antibodies. This work was mainly supported by the grant ÔNon-receptor tyrosine kinases in neuronal communication during development and synaptic plasticityÕ (PL 970526) from the European Communities (to FB, FV and J-AG) and by grants from MURST-Italy Cofin 2000, AIRC and Fisher Foundation for Alzheimer Disease (to FB). The support of Telethon Italy (grant 1131 to FB and 1000 to FV) is also gratefully acknowledged.

References Benfenati F., Valtorta F., Rubenstein J. L., Gorelick F., Greengard P. and Czernik A. J. (1992) Synaptic vesicle-associated Ca2+/calmodulindependent protein kinase II is a binding protein for synapsin I. Nature 359, 417–420.



Bernstein B. W. and Bamburg J. R. (1989) Cycling of actin assembly in synaptosomes and neurotransmitter release. Neuron 3, 257–265. Bongiorno-Borbone L., Onofri F., Giovedı` S., Ferrari R., Girault J.-A. and Benfenati F. (2002) The translocation of focal adhesion kinase in isolated nerve terminals is regulated by phosphorylation and actin assembly. J. Neurochem. 81, 1212–1222. Boxall A. R. and Lancaster B. (1998) Tyrosine kinases and synaptic transmission. Eur. J. Neurosci. 10, 2–7. Burgaya F. and Girault J.-A. (1996) Cloning of focal adhesion kinase from rat striatum reveals multiple transcripts. Mol. Brain. Res. 37, 63–73. Burgaya F., Menegon A., Menegoz M., Valtorta F. and Girault J.-A. (1995) Focal adhesion kinase in rat central nervous system. Eur. J. Neurosci. 7, 1810–1821. Burgaya F., Toutant M., Studler J. M., Costa A., Le Bert M., Gelman M. and Girault J.-A. (1997) Alternatively spliced focal adhesion kinase in rat brain with increased autophosphorylation activity. J. Biol. Chem. 272, 28720–28725. Butler M. H., David C., Ochoa G. C., Freyberg Z., Daniell L., Grabs D., Cremona O. and De Camilli P. (1997) Amphiphysin II (SH3P9; BIN1), a member of the amphiphysin/RVS family, is localized in the cortical cytomatrix of axon initial segments and nodes of Ranvier in brain and around T-tubules in skeletal muscle. J. Cell Biol. 137, 1355–1367. Calalb M. B., Polte T. R. and Hanks S. K. (1995) Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol. Cell. Biol. 15, 954–963. Cobb B. S., Schaller M. D., Leu T.-H. and Parsons J. T. (1994) Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK. Mol. Cell. Biol. 14, 147–155. Cremona O. and De Camilli P. (1997) Synaptic vesicle endocytosis. Curr. Op. Neurobiol. 7, 323–330. Critchley D. R. (2000) Focal adhesions – the cytoskeletal connection. Curr. Op. Cell Biol. 12, 133–139. Derkinderen P., Toutant M., Burgaya F., Le Bert M., Siciliano J. C., De Franciscis V., Gelman M. and Girault J.-A. (1996) Regulation of a neuronal form of focal adhesion kinase by anandamide. Science 273, 1719–1722. Derkinderen P., Siciliano J., Toutant M. and Girault J.-A. (1998) Differential regulation of FAK+ and PYK2/Cakb, two related tyrosine kinases, in rat hippocampal slices: effects of LPA, carbachol, depolarization and hyperosmolarity. Eur. J. Neurosci. 10, 1667– 1675. Derkinderen P., Toutant M., Kadare´ G., Ledent C., Parmentier M. and Girault J.-A. (2001) Dual role of Fyn in the regulation of FAK+6,7 by cannabinoids in hippocampus. J. Biol. Chem. 276, 38289– 38296. Di Paolo G., Sankaranarayanan S., Wenk M. R., Daniell L., Perucco E., Caldarone B. J., Flavell R., Picciotto M. R., Ryan T. A., Cremona O. and De Camilli P. (2002) Decreased synaptic vesicle recycling efficiency and cognitive deficits in amphiphysin 1 knockout mice. Neuron 33, 789–804. Dunkley P. R., Jarvie P. E., Heath J. W., Kidd G. J. and Rostas J. A. P. (1986) A rapid method for isolation of synaptosomes on Percoll gradients. Brain Res. 372, 115–129. Girault J.-A., Costa A., Derkinderen P., Studler J. M. and Toutant M. (1999) FAK and PYK2/CAKb in the nervous system: a link between neuronal activity, plasticity and survival? Trends Neurosci. 22, 257–263. Grant S. G. N., O’Dell T. J., Karl K. A., Stein P. L., Soriano P. and Kandel E. R. (1992) Impaired long-term potentiation, spatial learning, and hippocampal development in fyn mutant mice. Science 258, 1903–1910.

Grant S. G. N., Karl K. A., Kiebler M. A. and Kandel E. R. (1995) Focal adhesion kinase in the brain: novel subcellular localization and specific regulation by Fyn tyrosine kinase in mutant mice. Genes Dev. 9, 1909–1921. Guinebault C., Payrastre B., Racaud-Sultan C., Mazarguil H., Breton M., Mauco G., Plantavid M. and Chap H. (1995) Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase. J. Cell Biol. 129, 831–842. Harte M. T., Hildebrand J. D., Burnham M. R., Bouton A. H. and Parsons J. T. (1996) p130Cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase. J. Biol. Chem. 271, 13649–13655. Huttner W. B., Schiebler W., Greengard P. and De Camilli P. (1983) Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation. J. Cell Biol. 96, 1374–1388. Hynes R. O. (1999) Cell adhesion: old and new questions. Trends Cell Biol. 9, M33–M37. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lauri S. E., Taira T. and Rauvala H. (2000) High-frequency synaptic stimulation induces association of fyn and c-src to distinct phosphorylated components. Neuroreport 11, 997–1000. Law S. F., Estojak J., Wang B. L., Mysliwiec T., Kruh G. and Golemis E. A. (1996) Human enhancer of filamentation 1, a novel p 130caslike docking protein, associates with focal adhesion kinase and induces pseudohyphal growth in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 3327–3337. Lichte B., Veh R. W., Meyer H. E. and Kilimann M. W. (1992) Amphiphysin, a novel protein associated with synaptic vesicles. EMBO J. 11, 2521–2530. Lu Y. M., Roder J. C., Davidow J. and Salter M. W. (1998) Src activation in the induction of long-term potentiation in CA1 hippocampal neurons. Science 279, 1363–1367. Marks B. and McMahon H. T. (1998) Calcium triggers calcineurindependent synaptic vesicle recycling in mammalian nerve terminals. Curr. Biol. 8, 740–749. Mayer B. J. (1997) Signal transduction: clamping down on Src activity. Curr. Biol. 7, R295–R298. McPherson P. S., Czernik A. J., Chilcote T. J., Onofri F., Benfenati F., Greengard P., Schlessinger J. and De Camilli P. (1994) Interaction of Grb2 via its Src homology 3 domains with synaptic proteins including synapsin I. Proc. Natl Acad. Sci. USA 91, 6486–6490. Menegon A., Burgaya F., Baudot P., Dunlap D. D., Girault J. A. and Valtorta F. (1999) FAK+ and PYK2/Cak, two related tyrosine kinases highly expressed in the central nervous system: Similarities and differences in the expression pattern. Eur. J. Neurosci. 11, 3777–3788. Mundigl O., Ochoa G. C., David C., Slepnev V. I., Kabanov A. and De Camilli P. (1998) Amphiphysin I antisense oligonucleotides inhibit neurite outgrowth in cultured hippocampal neurons. J. Neurosci. 18, 93–103. Munn A. L., Stevenson B. J., Geli M. I. and Riezman H. (1995) end5, end6, and end7: mutations that cause actin delocalization and block the internalization step of endocytosis in Saccharomyces cerevisiae. Mol. Biol. Cell 6, 1721–1742. Nicholls D. G. and Sihra T. S. (1986) Synaptosomes possess an exocytotic pool of glutamate. Nature 321, 772–775. Onofri F., Giovedı` S., Vaccaro P., Czernik A. J., Valtorta F., De Camilli P. and Greengard P. and. Benfenati F. (1997) Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity. Proc. Natl Acad. Sci. USA 94, 12168–12173.



Onofri F., Giovedı` S., Kao H.-T., Valtorta F., Bongiorno Borbone L., De Camilli P., Greengard P. and Benfenati F. (2000) Specificity of the binding of synapsin I to Src homology-3 domains. J. Biol. Chem. 275, 29857–29867. Polte T. R. and Hanks S. K. (1995) Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas. Proc. Natl Acad. Sci. USA 92, 10678–10682. Raiteri M., Sala R., Fassio A., Rossetto O. and Bonanno G. (2000) Entrapping of impermeant probes of different size into nonpermeabilized synaptosomes as a method to study presynaptic mechanisms. J. Neurochem. 74, 423–431. Sambrook J., Fritsch E. F. and Maniatis T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schaller M. D. (1996) The focal adhesion kinase. J. Endocrinol. 150, 1–7. Schaller M. D., Hildebrand J. D., Shannon J. D., Fox J. W., Vines R. R. and Parsons J. T. (1994) Autophosphorylation of the focal adhesion kinase pp125FAK directs SH2-dependent binding of pp60src. Mol. Cell Biol. 14, 1680–1688. Schlaepfer D. D. and Hunter T. (1996) Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases. Mol. Cell Biol. 16, 5623–5633. Schlaepfer D. D. and Hunter T. (1998) Integrin signalling and tyrosine phosphorylation: just the FAKs? Trends Cell Biol. 8, 151–157. Schlaepfer D. D., Hanks S. K., Hunter T. and van der Geer P. (1994) Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature 372, 786–791. Shupliakov O., Low P., Grabs D., Gad H., Chen H., David C., Takai K. and De Camilli P. (1997) Synaptic vesicle endocytosis impaired by disruption of dynamin–SH3 domain interactions. Science 276, 259–263.

Siciliano J. C., Gelman M. and Girault J.-A. (1994) Depolarization and neurotransmitters increase neuronal protein tyrosine phosphorylation. J. Neurochem. 62, 950–959. Siciliano J. C., Toutant M., Derkinderen P., Sasaki T. and Girault J.-A. (1996) Differential regulation of proline-rich tyrosine kinase 2 cell adhesion kinase (PYK2/CAKb) and pp125FAK by glutamate and depolarization in rat hippocampus. J. Biol. Chem. 271, 28942– 28946. Sivadon P., Bauer F., Aigle M. and Crouzet M. (1995) Actin cytoskeleton and budding pattern are altered in the yeast rvs161 mutant: the Rvs161 protein shares common domains with the brain protein amphiphysin. Mol. Gen. Genet. 246, 485–495. Taylor J. M., Hildebrand J. D., Mack C. P., Cox M. E. and Parsons J. T. (1998) Characterization of Graf, the GTPase-activating protein for Rho associated with focal adhesion kinase-phosphorylation and possible regulation by mitogen-activated protein kinase. J. Biol. Chem. 273, 8063–8070. Thomas J. W., Ellis B., Boerner R. J., Knight W. B., White G. C. 2nd and Schaller M. D. (1998) SH2- and SH3-mediated interactions between focal adhesion kinase and Src. J. Biol. Chem. 273, 577– 583. Toutant M., Studler J. M., Burgaya F., Costa A., Ezan P., Gelman M. and Girault J.-A. (2000) Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms. Biochem. J. 348, 119–128. Toutant M., Costa A., Studler J. M., Kadare´ G., Carnaud M. and Girault J. A. (2002) Alternative splicing controls the mechanisms of FAK autophosphorylation. Mol. Cell Biol. 22, 7731–7743. Wigge P. and McMahon H. T. (1998) The amphiphysin family of proteins and their role in endocytosis at the synapse. Trends Neurosci. 21, 339–344. Zhang B. and Zelhof A. C. (2002) Amphiphysins: raising the BAR for synaptic vesicle recycling and membrane dynamics. Traffic 3, 452– 460.
