Dual Role of Fyn in the Regulation of FAK+6,7 by Cannabinoids in Hippocampus
2001; Elsevier BV; Volume: 276; Issue: 41 Linguagem: Inglês
10.1074/jbc.m105630200
ISSN1083-351X
AutoresPascal Derkinderen, Madeleine Toutant, Gress Kadaré, Jean‐Antoine Girault, Catherine Ledent, Marc Parmentier,
Tópico(s)Sleep and Wakefulness Research
ResumoIn hippocampus endocannabinoids modulate synaptic function and plasticity and increase tyrosine phosphorylation of several proteins, including focal adhesion kinase (FAK). Autophosphorylation of FAK on Tyr-397 is generally a critical step for its activation, allowing the recruitment of Src family kinases, and phosphorylation of FAK and associated proteins. We have examined the mechanisms of the regulation of FAK by cannabinoids in rat and mouse hippocampal slices. Anandamide and 2-arachidonoylglycerol, two endocannabinoids, and Δ9-tetrahydrocannabinol, stimulated tyrosine phosphorylation of FAK+6,7, a neuronal splice isoform of FAK, on several residues including Tyr-397. Cannabinoids increased phosphorylation of p130-Cas, a protein associated with FAK, but had no effect on PYK2, a tyrosine kinase related to FAK and enriched in hippocampus. Pharmacological experiments and the use of knockout mice demonstrated that the effects of cannabinoids were mediated through CB1 receptors. These effects were sensitive to manipulation of cAMP-dependent protein kinase, suggesting that they were mediated by inhibition of a cAMP pathway. PP2, an Src family kinase inhibitor, prevented the effects of cannabinoids on p130-Cas and on FAK+6,7 tyrosines 577 and 925, but not 397, indicating that FAK autophosphorylation was upstream of Src family kinases in response to CB1-R stimulation. Endocannabinoids increased the association of Fyn, but not Src, with FAK+6,7. In hippocampal slices from Fyn −/− mice, the levels of p130-Cas were increased, and the effects of endocannabinoids on tyrosine phosphorylation, including of Tyr-397, were completely abolished. These results demonstrate the specific functional association of Fyn with FAK+6,7 in a pathway regulated by endocannabinoids, in which Fyn may play roles dependent and independent of its catalytic activity. In hippocampus endocannabinoids modulate synaptic function and plasticity and increase tyrosine phosphorylation of several proteins, including focal adhesion kinase (FAK). Autophosphorylation of FAK on Tyr-397 is generally a critical step for its activation, allowing the recruitment of Src family kinases, and phosphorylation of FAK and associated proteins. We have examined the mechanisms of the regulation of FAK by cannabinoids in rat and mouse hippocampal slices. Anandamide and 2-arachidonoylglycerol, two endocannabinoids, and Δ9-tetrahydrocannabinol, stimulated tyrosine phosphorylation of FAK+6,7, a neuronal splice isoform of FAK, on several residues including Tyr-397. Cannabinoids increased phosphorylation of p130-Cas, a protein associated with FAK, but had no effect on PYK2, a tyrosine kinase related to FAK and enriched in hippocampus. Pharmacological experiments and the use of knockout mice demonstrated that the effects of cannabinoids were mediated through CB1 receptors. These effects were sensitive to manipulation of cAMP-dependent protein kinase, suggesting that they were mediated by inhibition of a cAMP pathway. PP2, an Src family kinase inhibitor, prevented the effects of cannabinoids on p130-Cas and on FAK+6,7 tyrosines 577 and 925, but not 397, indicating that FAK autophosphorylation was upstream of Src family kinases in response to CB1-R stimulation. Endocannabinoids increased the association of Fyn, but not Src, with FAK+6,7. In hippocampal slices from Fyn −/− mice, the levels of p130-Cas were increased, and the effects of endocannabinoids on tyrosine phosphorylation, including of Tyr-397, were completely abolished. These results demonstrate the specific functional association of Fyn with FAK+6,7 in a pathway regulated by endocannabinoids, in which Fyn may play roles dependent and independent of its catalytic activity. Δ9-tetrahydrocannabinol artificial cerebrospinal fluid 2-arachidonoylglycerol cannabinoid type 1 receptor focal adhesion kinase isobutylmethylxanthine lysophosphatidic acid long term potentiation 130-kDa Crk-associated substrate cAMP-dependent protein kinase proline-rich tyrosine kinase 2 tetrodotoxin 8-bromo-cAMP monoclonal antibody Although cannabis has been used for several centuries as a therapeutic and recreational drug (1Dewey W.L. Pharmacol. Rev. 1986; 38: 151-178PubMed Google Scholar, 2Voth E.A. Schwartz R.H. Ann. Intern. Med. 1997; 126: 791-798Crossref PubMed Scopus (148) Google Scholar), the mechanism of action of Δ9-tetrahydrocannabinol (THC),1 its main active compound, is only beginning to be elucidated. THC acts through two G protein-coupled receptor subtypes, the CB1 and CB2 receptors (3Matsuda L.A. Lolait S.J. Brownstein M.J. Young A.C. Bonner T.I. Nature. 1990; 346: 561-564Crossref PubMed Scopus (4191) Google Scholar, 4Munro S. Thomas K.L. Abu-Shaar M. Nature. 1993; 365: 61-65Crossref PubMed Scopus (4121) Google Scholar). CB1 receptors (CB1-R) are mainly expressed in the central nervous system and are particularly abundant in basal ganglia, hippocampus, and cerebellum (5Herkenham M. Lynn A.B. Little M.D. Johnson M.R. Melvin L.S. De Costa B.R. Rice K.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1932-1936Crossref PubMed Scopus (1914) Google Scholar). In contrast, CB2 receptors are expressed outside of the nervous system, mostly in lymphoid organs (4Munro S. Thomas K.L. Abu-Shaar M. Nature. 1993; 365: 61-65Crossref PubMed Scopus (4121) Google Scholar). Endogenous ligands of CB1-R (endocannabinoids) include anandamide and 2-arachidonoylglycerol (2-AG) (6Devane W.A. Hanus L. Breuer A. Pertwee R.G. Stevenson L.A. Griffin G. Gibson D. Mandelbaum A. Etinger A. Mechoulam R. Science. 1992; 258: 1946-1949Crossref PubMed Scopus (4658) Google Scholar, 7Di Marzo V. Fontana A. Cadas H. Schinelli S. Cimino G. Schwartz J.-C. Piomelli D. Nature. 1994; 372: 686-691Crossref PubMed Scopus (1345) Google Scholar, 8Mechoulam R. Ben-Shabat S. Hanus L. Ligumsky M. Kaminski N.E. Schatz A.R. Gopher A. Almog S. Martin B.R. Compton D.R. Pertwee R.G. Griffin G. Bayewitch M. Barg J. Vogel Z. Biochem. Pharmacol. 1995; 50: 83-90Crossref PubMed Scopus (2339) Google Scholar, 9Stella N. Schweitzer P. Piomelli D. Nature. 1997; 388: 773-778Crossref PubMed Scopus (1240) Google Scholar). These arachidonic acid derivatives are not stored in vesicles, but their phospholipid precursors are synthesized and hydrolyzed following calcium influx into neurons or stimulation of neurotransmitter receptors (7Di Marzo V. Fontana A. Cadas H. Schinelli S. Cimino G. Schwartz J.-C. Piomelli D. Nature. 1994; 372: 686-691Crossref PubMed Scopus (1345) Google Scholar, 9Stella N. Schweitzer P. Piomelli D. Nature. 1997; 388: 773-778Crossref PubMed Scopus (1240) Google Scholar,10Giuffrida A. Parsona L.H. Kerr T.M. Rodriguez de Fonseca F. Navarro M. Piomelli D. Nat. Neurosci. 1999; 2: 358-363Crossref PubMed Scopus (673) Google Scholar). In hippocampus electrical stimulation of Schaeffer collaterals increases the production of 2-AG (9Stella N. Schweitzer P. Piomelli D. Nature. 1997; 388: 773-778Crossref PubMed Scopus (1240) Google Scholar). CB1-R modulates synaptic plasticity in the hippocampus by decreasing long term potentiation (LTP) and depression (11Misner D.L. Sullivan J.M. J. Neurosci. 1999; 19: 6795-6805Crossref PubMed Google Scholar). Recent work (12Wilson R.I. Nicoll R.A. Nature. 2001; 410: 588-592Crossref PubMed Scopus (1254) Google Scholar, 13Ohno-Shosaku T. Maejima T. Kano M. Neuron. 2001; 29: 729-738Abstract Full Text Full Text PDF PubMed Scopus (684) Google Scholar) has demonstrated that endocannabinoids are retrograde synaptic messengers mediating depolarization-induced suppression of inhibition. The electrophysiological effects of endocannabinoids appear to result from the ability of CB1-R to decrease neurotransmitter release (11Misner D.L. Sullivan J.M. J. Neurosci. 1999; 19: 6795-6805Crossref PubMed Google Scholar, 12Wilson R.I. Nicoll R.A. Nature. 2001; 410: 588-592Crossref PubMed Scopus (1254) Google Scholar, 13Ohno-Shosaku T. Maejima T. Kano M. Neuron. 2001; 29: 729-738Abstract Full Text Full Text PDF PubMed Scopus (684) Google Scholar, 14Katona I. Sperlágh B. Sı́k A. Käfalvi A. Vizi E.S. Mackie K. Freund T.F. J. Neurosci. 1999; 19: 4544-4558Crossref PubMed Google Scholar, 15Hajos N. Katona I. Naiem S.S. MacKie K. Ledent C. Mody I. Freund T.F. Eur. J. Neurosci. 2000; 12: 3239-3249Crossref PubMed Scopus (435) Google Scholar). In addition to the recent progress in understanding the action of endocannabinoids, the possible therapeutic effects of drugs affecting transmission by cannabinoids have received a great deal of interest in various conditions (see Ref. 16Piomelli D. Giuffrida A. Calignano A. Rodriguez de Fonseca F. Trends Pharmacol. Sci. 2000; 21: 218-224Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar for a recent review), underscoring the importance of identifying the actions of CB1-R. The signal transduction pathways regulated by the activation of CB1-R include inhibition of adenylyl cyclase, activation of mitogen-activated protein kinases and phosphoinositide 3-kinase, induction of immediate early genes, and stimulation of nitric-oxide synthase (see Ref. 17Howlett A.C. Mukhopadhyay S. Chem. Phys. Lipids. 2000; 1: 53-70Crossref Scopus (200) Google Scholar for a recent review). Many of these results were obtained in cells in culture, often non-neuronal, and the signaling pathways activated by CB1-R in the brain are still poorly characterized. We have shown previously that anandamide increases protein tyrosine phosphorylation in rat hippocampus (18Derkinderen P. Toutant M. Burgaya F. Le Bert M. Siciliano J.C. De Franciscis V. Gelman M. Girault J.A. Science. 1996; 273: 1719-1722Crossref PubMed Scopus (158) Google Scholar). One of the proteins phosphorylated in response to anandamide was a splice isoform of FAK characterized by the presence of a 3-amino acid insertion in the C-terminal region, termed FAK+ (18Derkinderen P. Toutant M. Burgaya F. Le Bert M. Siciliano J.C. De Franciscis V. Gelman M. Girault J.A. Science. 1996; 273: 1719-1722Crossref PubMed Scopus (158) Google Scholar). Further work has revealed that the major neuronal isoform of FAK, termed FAK+6,7, contains additional alternatively spliced exons, coding for two short peptides of 6 and 7 amino acids on either side of Tyr-397 (19Burgaya F. Toutant M. Studler J.M. Costa A. Le Bert M. Gelman M. Girault J.A. J. Biol. Chem. 1997; 272: 28720-28725Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The presence of these latter peptides results in a dramatic increase in FAK autophosphorylation on Tyr-397 (19Burgaya F. Toutant M. Studler J.M. Costa A. Le Bert M. Gelman M. Girault J.A. J. Biol. Chem. 1997; 272: 28720-28725Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 20Toutant M. Studler J.M. Burgaya F. Costa A. Ezan P. Gelman M. Girault J.A. Biochem. J. 2000; 348: 119-128Crossref PubMed Google Scholar), a residue that is essential for FAK function. 2FAK+ corresponds to the presence of three additional residues inserted at position 903; FAK6 corresponds to six additional residues at position 392; FAK7 corresponds to seven additional residues at position 411 and a frameshift mutation that causes a Thr to Ala residue substitution (19Burgaya F. Toutant M. Studler J.M. Costa A. Le Bert M. Gelman M. Girault J.A. J. Biol. Chem. 1997; 272: 28720-28725Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). To avoid confusion, in this report we use the numbering of FAK tyrosine residues corresponding to the "standard" FAK isoform, without additional exons, although tyrosines 397, 576, 577, and 925 correspond to positions 403, 589, 590, and 941, respectively, in FAK+6,7.2FAK+ corresponds to the presence of three additional residues inserted at position 903; FAK6 corresponds to six additional residues at position 392; FAK7 corresponds to seven additional residues at position 411 and a frameshift mutation that causes a Thr to Ala residue substitution (19Burgaya F. Toutant M. Studler J.M. Costa A. Le Bert M. Gelman M. Girault J.A. J. Biol. Chem. 1997; 272: 28720-28725Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). To avoid confusion, in this report we use the numbering of FAK tyrosine residues corresponding to the "standard" FAK isoform, without additional exons, although tyrosines 397, 576, 577, and 925 correspond to positions 403, 589, 590, and 941, respectively, in FAK+6,7. FAK transduces messages generated by integrin-mediated cell contacts and also by soluble factors acting on G protein-coupled receptors or on receptor tyrosine kinases (see Refs. 21Schlaepfer D.D. Hauck C.R. Sieg D.J. Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1031) Google Scholar and 22Schlaepfer D.D. Hunter T. Trends Cell Biol. 1998; 8: 151-157Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar for reviews). FAK is upstream of several signaling pathways, and its functional roles include control of focal adhesions turnover, cell motility, and adhesion-dependent survival. In the context of the adult nervous system, several lines of evidence suggest that FAK may be involved in neuronal plasticity and survival (23Girault J.A. Costa A. Derkinderen P. Studler J.M. Toutant M. Trends Neurosci. 1999; 22: 257-263Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Although there are exceptions to this rule, tyrosine phosphorylation of FAK occurs generally in two steps. First, Tyr-397 is autophosphorylated, allowing the binding of SH2 domain-containing proteins including Src family kinases (24Cobb B.S. Schaller M.D. Leu T.-H. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (483) Google Scholar), phosphatidylinositol 3-kinase (25Chen H.C. Appeddu P.A. Isoda H. Guan J.L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar), and phospholipase Cγ (26Zhang X. Chattopadhyay A. Ji Q.S. Owen J.D. Ruest P.J. Carpenter G. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9021-9026Crossref PubMed Scopus (158) Google Scholar). Src family kinases phosphorylate other residues in FAK and in neighboring proteins, including p130-Cas, a protein associated with FAK in many cell types (see Ref. 27O'Neill G.M. Fashena S.J. Golemis E.A. Trends Cell Biol. 2000; 10: 111-119Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar for a review). Thus, FAK behaves as an adapter protein regulated by autophosphorylation and structurally, as well as functionally, closely associated with Src family kinases. Hippocampus is an excellent model for studying the regulation of FAK by cannabinoids, since it is a brain region where CB1-R is abundantly expressed (14Katona I. Sperlágh B. Sı́k A. Käfalvi A. Vizi E.S. Mackie K. Freund T.F. J. Neurosci. 1999; 19: 4544-4558Crossref PubMed Google Scholar, 28Tsou K. Brown S. Sañudo-Peña M.C. Mackie K. Walker J.M. Neuroscience. 1998; 83: 393-411Crossref PubMed Scopus (1343) Google Scholar, 29Tsou K. Mackie K. Sanudo-Pena M.C. Walker J.M. Neuroscience. 1999; 93: 969-975Crossref PubMed Scopus (286) Google Scholar), and where their physiological effects are well characterized (11Misner D.L. Sullivan J.M. J. Neurosci. 1999; 19: 6795-6805Crossref PubMed Google Scholar, 14Katona I. Sperlágh B. Sı́k A. Käfalvi A. Vizi E.S. Mackie K. Freund T.F. J. Neurosci. 1999; 19: 4544-4558Crossref PubMed Google Scholar, 30Sullivan J.M. J. Neurophysiol. 1999; 82: 1286-1294Crossref PubMed Scopus (150) Google Scholar, 31Hoffman A.F. Lupica C.R. J. Neurosci. 2000; 20: 2470-2479Crossref PubMed Google Scholar). In addition, the use of hippocampal slices provides insights on the regulation and function of signaling pathways in the context of mature brain tissue. Therefore, the aim of the present study was to dissect the mechanism of FAK activation by cannabinoids in rat and mouse hippocampus. We studied the effects on protein tyrosine phosphorylation pathways of various cannabinoid agonists, including 2-AG, the major endocannabinoid in hippocampus (9Stella N. Schweitzer P. Piomelli D. Nature. 1997; 388: 773-778Crossref PubMed Scopus (1240) Google Scholar), and THC. We examined the role of CB1-R and of inhibition of adenylyl cyclase in the activation of FAK and p130-Cas. Finally, we investigated the mechanisms of activation of FAK by cannabinoids in hippocampal slices, by assessing the contribution of FAK autophosphorylation and the role of Src family kinases. Anandamide, 8 bromo-3′-5′-cyclic adenosine monophosphate (8-Br-cAMP), THC, 3-isobutyl-1-methylxanthine (IBMX), lysophosphatidic acid (LPA), protein A-Sepharose, tetrodotoxin (TTX), WIN 55212-2, and 2-arachidonoylglycerol were from Sigma. CP 55940 was from Pfizer. PP2 was from Calbiochem. SR 141716A was from Sanofi (Sanofi Research, Montpellier, France). Artificial cerebrospinal fluid (ACSF) contained (in mm) NaCl, 125; KCl, 2.4; MgCl2, 0.83; CaCl2, 1.1; KH2PO4, 0.5; Na2SO4, 0.5; NaHCO3, 27; glucose, 10; Hepes, pH 7.4, 110. Ca2+-free ACSF had the same composition except for MgCl2, 1.93 mm. The following commercially available phosphospecific antibodies were used for Western blotting: antiphospho-Tyr-577 FAK (BIOSOURCE, diluted 1:1,000) and antiphospho-Tyr-925 FAK (BIOSOURCE, diluted 1:1,000). Antiphospho-Tyr-397 FAK was produced and affinity-purified as described previously (20Toutant M. Studler J.M. Burgaya F. Costa A. Ezan P. Gelman M. Girault J.A. Biochem. J. 2000; 348: 119-128Crossref PubMed Google Scholar) or was from BIOSOURCE (diluted 1:1,000). Anti-Fyn antibodies were from Transduction Laboratories (mouse monoclonal for Western blot, 1:250) or from Santa Cruz Biotechnology (rabbit polyclonal for immunoprecipitation, 20 μl per sample). Anti-Src antibodies were from Calbiochem (rabbit polyclonal for Western blot, 1:500) or from Upstate Biotechnology, Inc. (mouse monoclonal for immunoprecipitation, 7 μl per sample). Anti-p130-Cas antibodies were from Transduction Laboratories (mouse monoclonal for Western blot, 1:500) or from Santa Cruz Biotechnology (rabbit polyclonal for immunoprecipitation, 20 μl per sample). For immunoprecipitation, polyclonal anti-FAK+and anti-PYK2 were produced as described previously (18Derkinderen P. Toutant M. Burgaya F. Le Bert M. Siciliano J.C. De Franciscis V. Gelman M. Girault J.A. Science. 1996; 273: 1719-1722Crossref PubMed Scopus (158) Google Scholar, 32Derkinderen P. Siciliano J. Toutant M. Girault J.A. Eur. J. Neurosci. 1998; 10: 1667-1675Crossref PubMed Scopus (59) Google Scholar). Rabbit polyclonal antibody against CB1-R was a gift of Dr. Mackie. Monoclonal antibodies to phosphotyrosine (4G10) were from Upstate Biotechnology, Inc. (diluted 1:4,000). FAK-transfected COS-7 cells were prepared as described (20Toutant M. Studler J.M. Burgaya F. Costa A. Ezan P. Gelman M. Girault J.A. Biochem. J. 2000; 348: 119-128Crossref PubMed Google Scholar). Rat hippocampal slices (300-μm thick) were prepared from young male Harlan Sprague-Dawley rats (150–200 g) with a McIlwain tissue chopper, as described previously (33Siciliano J.C. Gelman M. Girault J.A. J. Neurochem. 1994; 62: 950-959Crossref PubMed Scopus (48) Google Scholar). Briefly, slices were dissected in ice-cold, Ca2+-free ACSF and were placed for 10 min in polypropylene tubes (three slices per tube) containing 1 ml of Ca2+-free ACSF at 35 °C, equilibrated at pH 7.4 in O2/CO2 (95:5, v/v). They were then incubated at 35 °C in 900 μl of ACSF containing 1.1 mmCa2+ and 1 μm TTX for 50 min before pharmacological treatment. Treatment of control slices was carried out by the addition of vehicle. TTX was added to prevent indirect effects due to neuronal firing and had no effect on tyrosine phosphorylation by itself (data not shown). At the end of the experiment, ACSF was aspirated, and slices were immediately frozen on dry ice and kept at −80 °C. Mice hippocampal slices from wild type, CB1 knockout mice (34Ledent C. Valverde O. Cossu C. Petitet F. Aubert L.F. Beslot F. Böhme G.A. Imperato A. Pedrazzini T. Roques B.P. Vassart G. Fratta W. Parmentier M. Science. 1999; 283: 401-404Crossref PubMed Scopus (1096) Google Scholar), and Fyn knockout mice (35Grant S.G.N. O'Dell T.J. Karl K.A. Stein P.L. Soriano P. Kandel E.R. Science. 1992; 258: 1903-1910Crossref PubMed Scopus (965) Google Scholar) (The Jackson Laboratories) were prepared as rat slices except for the use of Vibratome instead of McIlwain chopper. Protein A-Sepharose with 10% v/v Sephacryl was washed in PB+ buffer (2.7 mm KCl, 1.2 mm KH2PO4, 137 mm NaCl, 8.3 mm NaH2PO4, 5 mmEDTA, 5 mm EGTA, 10 mm sodium pyrophosphate, 50 mm NaF) containing 10 mg/ml bovine serum albumin, equilibrated in PB+ buffer with 1% (w/v) Triton X-100 and used as a 50% (v/v) slurry. Homogenized samples (∼500 μg of proteins in 1% SDS in the presence of 1 mm sodium orthovanadate) were equilibrated in PB+ buffer with 5% (w/v) Triton X-100 and precleared with 100 μl of Sephacryl G-10. Samples were then incubated for 2 h at 4 °C with 80 μl of protein A slurry and 25 μl of FAK+, PYK2, or p130-Cas antisera. The protein A-Sepharose-bound immune complexes were washed three times in PB+ buffer as follows: once with Triton (1% w/v), once with Triton and 0.5m NaCl, and once without Triton. For coimmunoprecipitation, slices (500–600 μg of protein) were homogenized with a Heidolph tissue homogenizer in ice-cold modified CRIPA buffer (150 mm NaCl, 50 mm Tris, pH 7.4, 5 mmEDTA, 50 mm NaF) containing 1 mmNa+ orthovanadate, 1%(v/v) Triton X-100, 0.5% deoxycholate, 0.1% SDS, and protease inhibitors (complete, Roche Molecular Biochemicals). Samples were precleared with 100 μl of Sephacryl beads, and immunoprecipitations were carried out with polyclonal antibodies anti-Fyn or with monoclonal antibodies anti-Src preadsorbed, respectively, on protein A- or protein G-Sepharose beads. In all cases, immunoprecipitation pellets were heated at 100 °C for 5 min in 70 μl of sample buffer for SDS-polyacrylamide gel electrophoresis. Slices were lysed by sonication in 1% SDS (v/v) containing 1 mm Na+ orthovanadate at 100 °C. Equal amounts of slices lysates (60 μg) were separated by SDS-polyacrylamide gel electrophoresis (8% weight/vol) prior to electrophoretic transfer onto nitrocellulose membrane (Hybond Pure,Amersham Pharmacia Biotech). Membranes were blocked 1 h at room temperature in Tris-buffered saline (100 mm NaCl, 10 mm Tris, pH 7.5) with 0.1% Tween 20 or 5% nonfat dry milk for the detection of phosphorylated and non-phosphorylated proteins, respectively. The membranes were then incubated overnight at 4 °C with the primary antibodies. After three short washes, membranes were incubated for 2 h at room temperature with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies (Amersham Pharmacia Biotech, diluted 1:4,000). Bound antibodies were visualized by enhanced chemiluminescence detection (ECL, Amersham Pharmacia Biotech). When necessary, membranes were stripped in buffer (containing 100 mm glycine pH 2.5, 200 mm NaCl, 0.1% Tween 20 (v/v) and 0.1% (v/v) β-mercaptoethanol) for 45 min at room temperature, followed by extensive washing in TBS before reblocking and reprobing. Quantification was achieved by scanning the autoradiograms and measurement of relative optical density with NIH Image. We compared the effects on FAK of 2-AG, the endocannabinoid produced in hippocampal slices following electrical stimulation (9Stella N. Schweitzer P. Piomelli D. Nature. 1997; 388: 773-778Crossref PubMed Scopus (1240) Google Scholar), to those of anandamide previously shown to increase protein tyrosine phosphorylation in rat hippocampal slices (18Derkinderen P. Toutant M. Burgaya F. Le Bert M. Siciliano J.C. De Franciscis V. Gelman M. Girault J.A. Science. 1996; 273: 1719-1722Crossref PubMed Scopus (158) Google Scholar). We also examined the effects of THC, the widely abused cannabinoid agonist. Immunoprecipitation with antibodies specific for FAK+ revealed that 2-AG (1 μm) and THC (0.1 μm) increased its tyrosine phosphorylation in rat hippocampal slices, as anandamide (1 μm) (Fig.1A). Since autophosphorylation of FAK on Tyr-397 is the critical step in its activation (24Cobb B.S. Schaller M.D. Leu T.-H. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (483) Google Scholar, 36Xing Z. Chen H.-C. Nowlen J.K. Taylor S.J. Shalloway D. Guan J.-L. Mol. Biol. Cell. 1994; 5: 413-421Crossref PubMed Scopus (284) Google Scholar, 37Schlaepfer D.D. Broome M.A. Hunter T. Mol. Cell. Biol. 1997; 17: 1702-1713Crossref PubMed Scopus (399) Google Scholar), we measured the phosphorylation of this residue with a phosphorylation state-specific antibody (19Burgaya F. Toutant M. Studler J.M. Costa A. Le Bert M. Gelman M. Girault J.A. J. Biol. Chem. 1997; 272: 28720-28725Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 20Toutant M. Studler J.M. Burgaya F. Costa A. Ezan P. Gelman M. Girault J.A. Biochem. J. 2000; 348: 119-128Crossref PubMed Google Scholar). The increased phosphorylation of Tyr-397 in response to cannabinoid agonists matched that of FAK total tyrosine phosphorylation (Fig. 1A). The effects of endocannabinoids and THC on total tyrosine phosphorylation of FAK and on phosphorylation of Tyr-397 were completely prevented by a pretreatment of slices with a high concentration (100 μm) of SR 141716A, a specific inhibitor of CB1-R (38Rinaldi-Carmona M. Barth F. Héaulme M. Shir D. Calandra B. Congy C. Martinez S. Maruani J. Néliat G. Caput D. Ferrara P. Soubrié P. Brelière J.C. Le Fur G. FEBS Lett. 1994; 350: 240-244Crossref PubMed Scopus (1635) Google Scholar) (Fig. 1A). In contrast, the effects of lysophosphatidic acid (LPA), a lipidic messenger unrelated to cannabinoids and known to increase the tyrosine phosphorylation of FAK in hippocampus (32Derkinderen P. Siciliano J. Toutant M. Girault J.A. Eur. J. Neurosci. 1998; 10: 1667-1675Crossref PubMed Scopus (59) Google Scholar), persisted in the presence of SR 141716A (data not shown). In addition, synthetic CB1 agonists CP 55940 and WIN 55212-2 also increased tyrosine phosphorylation of FAK, and their effects were blocked by the specific CB1 antagonist SR 141716A (data not shown). However, endocannabinoids have a potential for acting on other targets beside CB1 and CB2 receptors (17Howlett A.C. Mukhopadhyay S. Chem. Phys. Lipids. 2000; 1: 53-70Crossref Scopus (200) Google Scholar), and the concentrations of WIN 55212-2 and SR 141716A required to achieve their effects were high (100 μm), raising the possibility that they could exert unspecific effects. To assess the exact contribution of CB1-R in the effects of anandamide and 2-AG, we used hippocampal slices prepared from genetically altered mice lacking CB1-R (34Ledent C. Valverde O. Cossu C. Petitet F. Aubert L.F. Beslot F. Böhme G.A. Imperato A. Pedrazzini T. Roques B.P. Vassart G. Fratta W. Parmentier M. Science. 1999; 283: 401-404Crossref PubMed Scopus (1096) Google Scholar). In wild type mice, as in rats, anandamide and 2-AG increased FAK total tyrosine phosphorylation and Tyr-397 phosphorylation (Fig. 1B). These effects were completely prevented in CB1-R −/− mice, although the expression levels of FAK were unchanged (Fig. 1B). However, FAK could still be activated by LPA in CB1-R −/− mice, demonstrating that the other components of the pathway were not altered in these mutant mice (Fig. 1B). Combined with the pharmacological experiments described above, the results in CB1-R knockout mice demonstrate that the effects of endocannabinoids on FAK in hippocampus are mediated through activation of CB1-R. Finally, we examined the concentration and time dependence of the effects of 2-AG on phosphorylation of FAK on Tyr-397. The effects of 2-AG were detected at very low concentration (half-maximal effect at ∼20 nm, Fig. 1C) and were rapid (half-maximal effect at ∼1 min, Fig. 1D). We have shown previously (18Derkinderen P. Toutant M. Burgaya F. Le Bert M. Siciliano J.C. De Franciscis V. Gelman M. Girault J.A. Science. 1996; 273: 1719-1722Crossref PubMed Scopus (158) Google Scholar, 19Burgaya F. Toutant M. Studler J.M. Costa A. Le Bert M. Gelman M. Girault J.A. J. Biol. Chem. 1997; 272: 28720-28725Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 20Toutant M. Studler J.M. Burgaya F. Costa A. Ezan P. Gelman M. Girault J.A. Biochem. J. 2000; 348: 119-128Crossref PubMed Google Scholar) that FAK+6,7 is the major isoform of FAK expressed in forebrain. Several lines of evidence indicate that the isoform of FAK in hippocampal slices was indeed FAK+6,7. It was recognized by anti-FAK+antibodies (Fig. 1A). In SDS-polyacrylamide gel electrophoresis it migrated with an apparent molecular weight slightly higher than FAK+, FAK+6, or FAK+7 expressed in COS-7 cells, and comigrated with FAK+6,7 (Fig.2A). It was not recognized by monoclonal antibody 77 (mAb 77, Fig. 2A) whose interaction with FAK is prevented by the presence of box 7 (20Toutant M. Studler J.M. Burgaya F. Costa A. Ezan P. Gelman M. Girault J.A. Biochem. J. 2000; 348: 119-128Crossref PubMed Google Scholar). On the basis of its apparent molecular weight and of the lack of detection by mAb77, this isoform of FAK was found in neurons but not in astrocytes or microglial cells in culture (Fig. 2B). The form of FAK phosphorylated on Tyr-397 in response to anandamide had the same characteristics (Fig. 2C). These results provide strong evidence that the isoform of FAK regulated by cannabinoids in hippocampal slices was FAK+6,7 and that the observed regulations were taking place in neurons. FAK belongs to a family of two highly related kinases, including PYK2/RAFTK/cell adhesion kinase β/CADTK (see Ref. 39Avraham H. Park S.Y. Schinkmann K. Avraham S. Cell. Signal. 2000; 12: 123-133Crossref PubMed Scopus (409) Google Scholar for a review), which is highly expressed in hippocampus (40Menegon A. Burgaya F. Baudot P. Dunlap D.D. Girault J.A. Valtorta F. Eur. J. Neurosci. 1999; 11: 3777-3788Crossref PubMed Scopus (72) Google Scholar) and is involved in LTP (41Huang Y. Lu
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