Alternatively Spliced Focal Adhesion Kinase in Rat Brain with Increased Autophosphorylation Activity
1997; Elsevier BV; Volume: 272; Issue: 45 Linguagem: Inglês
10.1074/jbc.272.45.28720
ISSN1083-351X
AutoresFerran Burgaya, Madeleine Toutant, Jeanne‐Marie Studler, Alícia Costa, Marc Le Bert, Michèle Gelman, Jean‐Antoine Girault,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
Resumopp125 focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase transducing signals initiated by integrin engagement and G protein-coupled receptors, is highly expressed in brain. FAK from brain had a higher molecular weight and an increased autophosphorylation activity, than from other tissues. In addition to a 9-base insertion in the 3′-coding region, which defines FAK+, rat striatal FAK mRNAs contained several additional short exons, coding for peptides of 28, 6, and 7 residues, respectively (termed boxes 28, 6, and 7), surrounding the autophosphorylated Tyr-397. In transfected COS 7 cells, the presence of boxes 6 and 7 conferred an increased overall tyrosine phosphorylation, a higher phosphorylation of Tyr-397 assessed with a phosphorylation state-specific antibody, and a more active autophosphorylation in immune precipitates. The presence of box 28 did not alter further these parameters. Two-dimensional phosphopeptide maps of hippocampal FAK were identical to those of FAK+6,7. The presence of the various exons did not alter the interaction of FAK with c-Src, n-Src, or Fyn. Thus, several splice isoforms of FAK are preferentially expressed in rat brain, some of which have an increased autophosphorylation activity, suggesting that FAK may have specific properties in neurons. pp125 focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase transducing signals initiated by integrin engagement and G protein-coupled receptors, is highly expressed in brain. FAK from brain had a higher molecular weight and an increased autophosphorylation activity, than from other tissues. In addition to a 9-base insertion in the 3′-coding region, which defines FAK+, rat striatal FAK mRNAs contained several additional short exons, coding for peptides of 28, 6, and 7 residues, respectively (termed boxes 28, 6, and 7), surrounding the autophosphorylated Tyr-397. In transfected COS 7 cells, the presence of boxes 6 and 7 conferred an increased overall tyrosine phosphorylation, a higher phosphorylation of Tyr-397 assessed with a phosphorylation state-specific antibody, and a more active autophosphorylation in immune precipitates. The presence of box 28 did not alter further these parameters. Two-dimensional phosphopeptide maps of hippocampal FAK were identical to those of FAK+6,7. The presence of the various exons did not alter the interaction of FAK with c-Src, n-Src, or Fyn. Thus, several splice isoforms of FAK are preferentially expressed in rat brain, some of which have an increased autophosphorylation activity, suggesting that FAK may have specific properties in neurons. Focal adhesion kinase (FAK) 1The abbreviations used are: FAK, pp125 focal adhesion kinase; DOTAP,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium me- thylsulfate; PWR, Pro-Trp-Arg; SH2, Src-homology domain 2; SH3, Src-homology domain 3. 1The abbreviations used are: FAK, pp125 focal adhesion kinase; DOTAP,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium me- thylsulfate; PWR, Pro-Trp-Arg; SH2, Src-homology domain 2; SH3, Src-homology domain 3. is a 125-kDa cytoplasmic tyrosine kinase associated with focal adhesions in non-neuronal cells (see Refs. 1Schaller M.D. Parsons J.T. Curr. Opin. Cell Biol. 1994; 6: 705-710Crossref PubMed Scopus (493) Google Scholar, 2Schaller M.D. J. Endocrinol. 1996; 150: 1-7Crossref PubMed Scopus (74) Google Scholar, 3Guan J.L. Chen H.C. Int. Rev. Cytol. 1996; 168: 81-121Crossref PubMed Google Scholar, 4Hanks S.K. Polte T.R. BioEssays. 1997; 19: 137-145Crossref PubMed Scopus (440) Google Scholar, 5Ilic D. Damsky C.H. Yamamoto T. J. Cell Sci. 1997; 110: 401-407Crossref PubMed Google Scholar, for reviews). Although FAK does not have SH2 or SH3 domains, it is associated to many proteins, some of which bind to FAK phosphorylated on tyrosine via their own SH2 domains (1Schaller M.D. Parsons J.T. Curr. Opin. Cell Biol. 1994; 6: 705-710Crossref PubMed Scopus (493) Google Scholar, 2Schaller M.D. J. Endocrinol. 1996; 150: 1-7Crossref PubMed Scopus (74) Google Scholar, 3Guan J.L. Chen H.C. Int. Rev. Cytol. 1996; 168: 81-121Crossref PubMed Google Scholar, 4Hanks S.K. Polte T.R. BioEssays. 1997; 19: 137-145Crossref PubMed Scopus (440) Google Scholar, 5Ilic D. Damsky C.H. Yamamoto T. J. Cell Sci. 1997; 110: 401-407Crossref PubMed Google Scholar). FAK is phosphorylated on tyrosine in response to integrin engagement and to stimulation of various G protein-coupled receptors (1Schaller M.D. Parsons J.T. Curr. Opin. Cell Biol. 1994; 6: 705-710Crossref PubMed Scopus (493) Google Scholar, 2Schaller M.D. J. Endocrinol. 1996; 150: 1-7Crossref PubMed Scopus (74) Google Scholar, 3Guan J.L. Chen H.C. Int. Rev. Cytol. 1996; 168: 81-121Crossref PubMed Google Scholar, 4Hanks S.K. Polte T.R. BioEssays. 1997; 19: 137-145Crossref PubMed Scopus (440) Google Scholar, 5Ilic D. Damsky C.H. Yamamoto T. J. Cell Sci. 1997; 110: 401-407Crossref PubMed Google Scholar). The first step appears to be the autophosphorylation of Tyr-397 which allows the binding of the SH2 domains of Src (6Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1113) Google Scholar), Fyn (7Cobb B.S. Schaller M.D. Leu T.-H. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (483) Google Scholar), or phosphatidylinositol 3-kinase (8Chen H.-C. Appeddu P.A. Isoda H. Guan J.-L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). The recruitment of Src-family kinases results in the phosphorylation of neighboring proteins and at least 5 residues in FAK itself (9Calalb M.B. Polte T.R. Hanks S.K. Mol. Cell. Biol. 1995; 15: 954-963Crossref PubMed Google Scholar). Phosphorylation of some of these residues leads to the recruitment of Grb2 and the activation of the mitogen-activated protein kinase cascade (10Schlaepfer D.D. Hanks S.K. Hunter T. Van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1437) Google Scholar). The importance of FAK is attested by the embryonic lethality of the knock out of its gene (11Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Aizawa S. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1580) Google Scholar). FAK is thought to have various functions ranging from the regulation of focal adhesions turnover to the prevention of anoikis, a form of apoptosis induced by the detachment of cells from the extracellular matrix (12Hungerford J.E. Compton M.T. Matter M.L. Hoffstrom B.G. Otey C.A. J. Cell Biol. 1996; 135: 1383-1390Crossref PubMed Scopus (333) Google Scholar).FAK is highly expressed in the nervous tissue during development, a period at which it is enriched in neuronal growth cones (13Burgaya F. Menegon A. Menegoz M. Valtorta F. Girault J.-A. Eur. J. Neurosci. 1995; 7: 1810-1821Crossref PubMed Scopus (76) Google Scholar, 14Stevens G.R. Zhang C. Berg M.M. Lambert M.P. Barber K. Cantallops I. Routtenberg A. Klein W.L. J. Neurosci. Res. 1996; 46: 445-455Crossref PubMed Scopus (29) Google Scholar, 15Worley T.L. Holt C.E. Neuroreport. 1996; 7: 1133-1137Crossref PubMed Scopus (8) Google Scholar). In the brain of adult rats, FAK is expressed at higher levels than in most other tissues, especially in neurons of the hippocampus and the cerebral cortex (13Burgaya F. Menegon A. Menegoz M. Valtorta F. Girault J.-A. Eur. J. Neurosci. 1995; 7: 1810-1821Crossref PubMed Scopus (76) Google Scholar, 16Grant S.G.N. Karl K.A. Kiebler M.A. Kandel E.R. Genes Dev. 1995; 9: 1909-1921Crossref PubMed Scopus (130) Google Scholar). In brain, the major form of FAK is a splice variant termed FAK+, which contains a 3-amino acid insertion (Pro-Trp-Arg) at position 904 (17Burgaya F. Girault J.-A. Mol. Brain Res. 1996; 37: 63-73Crossref PubMed Scopus (35) Google Scholar, 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). In rat hippocampal slices, FAK+ phosphorylation is strongly stimulated by several neurotransmitters, including glutamate (19Siciliano J.C. Toutant M. Derkinderen P. Sasaki T. Girault J.-A. J. Biol. Chem. 1996; 271: 28942-28946Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) and acetylcholine, 2P. Derkinderen, J. C. Sicilians, M. Toutant, and J.-A. Girault, submitted for publication. 2P. Derkinderen, J. C. Sicilians, M. Toutant, and J.-A. Girault, submitted for publication. as well as lipid messengers anandamide, arachidonic acid (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), and lysophosphatidic acid.2 The sensitivity of FAK+ phosphorylation to neurotransmitters suggests that FAK+ transduces information generated by neuronal activity into phosphorylation of proteins, possibly resulting in changes in ion channel properties, cytoskeletal organization, or gene expression. Thus, FAK may be critical for the suggested role of protein tyrosine phosphorylation in synaptic plasticity (20O'Dell T.J. Kandel E.R. Grant S.G.N. Nature. 1991; 353: 558-560Crossref PubMed Scopus (469) Google Scholar, 21Boxall A.R. Lancaster B. Garthwaite J. Neuron. 1996; 16: 805-813Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). This hypothesis is supported by the observation that FAK phosphorylation is decreased in Fyn knock out mice (16Grant S.G.N. Karl K.A. Kiebler M.A. Kandel E.R. Genes Dev. 1995; 9: 1909-1921Crossref PubMed Scopus (130) Google Scholar) which display anomalous hippocampal development and long term potentiation (20O'Dell T.J. Kandel E.R. Grant S.G.N. Nature. 1991; 353: 558-560Crossref PubMed Scopus (469) Google Scholar, 22Kojima N. Wang J. Mansuy I.M. Grant S.G.N. Mayford M. Kandel E.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4761-4765Crossref PubMed Scopus (166) Google Scholar).As an approach to better understand the function of FAK in neurons, we have examined the biochemical properties of this kinase in rat brain. We show that the slightly larger size of FAK in brain as compared with other tissues, can be accounted for by the presence of additional exons surrounding the autophosphorylated tyrosine. The presence of two of these exons resulted in an increased autophosphorylation activity.DISCUSSIONThis study underlines the complexity of FAK structure and processing. Although it appears to be coded by a single gene, a number of transcriptional and post-transcriptional mechanisms generate multiple isoforms of FAK mRNA and protein. Two different polyadenylation sites and two different 5′ ends were found in rat, corresponding to those described in chick and mouse, respectively (17Burgaya F. Girault J.-A. Mol. Brain Res. 1996; 37: 63-73Crossref PubMed Scopus (35) Google Scholar). A very short alternative exon, coding for 3 amino acids defining FAK+, is found in rat, mouse (17Burgaya F. Girault J.-A. Mol. Brain Res. 1996; 37: 63-73Crossref PubMed Scopus (35) Google Scholar, 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), human (28Whitney G.S. Chan P.-Y. Blake J. Cosand W.L. Neubauer M.G. Aruffo A. Kanner S.B. DNA Cell Biol. 1993; 12: 823-830Crossref PubMed Scopus (90) Google Scholar), and Xenopus(26Hens M.D. DeSimone D.W. Dev. Biol. 1995; 170: 274-288Crossref PubMed Scopus (67) Google Scholar). A short form of FAK, called FAK-related non-kinase has been described in chick (29Schaller M.D. Borgman C.A. Parsons J.T. Mol. Cell. Biol. 1993; 13: 785-791Crossref PubMed Scopus (279) Google Scholar) and may also exist in human (30André E. Becker-André M. Biochem. Biophys. Res. Commun. 1993; 190: 140-147Crossref PubMed Scopus (85) Google Scholar). In the present study, we show that 3 additional exons can be found in rat and mouse FAK, corresponding to the region of the protein which surrounds the autophosphorylation site, at the junction between the amino-terminal and the catalytic domains of FAK. At least two of these exons (boxes 6 and 7) have been highly conserved during evolution, since they exist also in Xenopus (26Hens M.D. DeSimone D.W. Dev. Biol. 1995; 170: 274-288Crossref PubMed Scopus (67) Google Scholar). Assuming that all these variations of FAK mRNAs occur independently from the others, at least 512 different mRNAs coding for 64 different forms of full-length FAK protein could exist. This calculation does not take into account the short gene product FAK-related non-kinase and the possible additional exons which have been reported in human cDNAs (30André E. Becker-André M. Biochem. Biophys. Res. Commun. 1993; 190: 140-147Crossref PubMed Scopus (85) Google Scholar). Although it is likely that not all combinations are expressed at significant levels, the wealth of potential isoforms, many of which, and perhaps all, have been highly conserved during evolution, provide a mechanism by which FAK expression and properties could be finely tuned.It is noteworthy that exons which give rise to changes in the coding sequence (FAK+, boxes 28, 6, and 7) are all highly expressed in the nervous tissue. This may be linked to the preferential inclusion of small exons which has been suggested to occur during mRNA splicing in brain (31Black D.L. Cell. 1992; 69: 795-807Abstract Full Text PDF PubMed Scopus (147) Google Scholar). In fact, among the other tissues examined only testis contained high amounts of FAK+ (data not shown) and box 7 (this study). An important consequence of the existence of isoforms with differences in the coding region, is that they may provide neuronal FAK with specific properties. We found that FAK and FAK+ were indistinguishable concerning their amount of tyrosine phosphorylation, their autophosphorylation, and their interactions with Src and Fyn. This is perhaps not surprising since the Pro-Trp-Arg insertion of FAK+ is located in the carboxyl-terminal region of the protein, at a distance in the primary sequence from the kinase domain and the autophosphorylation site. In contrast, we found that the presence of boxes 6 and 7 which are located on either side of the autophosphorylated tyrosine, affected dramatically the tyrosine phosphorylation in cells and in immune precipitates. Using a site- and phosphorylation-specific antibody, we could show that the increased phosphorylation of FAK+6,7 could be accounted for, at least in part, by an increased phosphorylation of Tyr-397, the major autophosphorylation site. Boxes 6 and 7 were always found associated in striatal cDNAs and we do not know whether one, or the other, or the combination of the two is responsible for the observed effect. On the other hand, the presence of box 28, located amino-terminal of box 6, did not alter the autophosphorylation rate.Work from several laboratories has shown that autophosphorylation of FAK on Tyr-397 is a critical aspect of its regulation and function, since it is responsible for the binding of Src family kinases (6Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1113) Google Scholar, 7Cobb B.S. Schaller M.D. Leu T.-H. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (483) Google Scholar) and phosphatidylinositol 3-kinase (8Chen H.-C. Appeddu P.A. Isoda H. Guan J.-L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). Moreover, although in vitro FAK is able to phosphorylate several proteins, including paxillin and p130 cas, these are also substrates for Src which may be responsible for their phosphorylation in cells (see Refs. 4Hanks S.K. Polte T.R. BioEssays. 1997; 19: 137-145Crossref PubMed Scopus (440) Google Scholar and5Ilic D. Damsky C.H. Yamamoto T. J. Cell Sci. 1997; 110: 401-407Crossref PubMed Google Scholar). The available evidence suggests that FAK may function as an adaptor regulated by autophosphorylation, rather than as a protein kinase active on different substrates. Thus, an increased ability to autophosphorylate, provided by alternative splicing, could have important functional consequences. For instance, it could increase the phosphorylation of FAK in cells which express FAK6,7 in response to integrin engagement or stimulation of G protein-coupled receptors. It may thus play a role in the high responsiveness of FAK in hippocampus to neurotransmitters or lipid messengers (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, 19Siciliano J.C. Toutant M. Derkinderen P. Sasaki T. Girault J.-A. J. Biol. Chem. 1996; 271: 28942-28946Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Our studies of FAK isoforms gave important negative results concerning their interactions with Src family kinases. The presence of insertions did not alter the interaction of FAK with these kinases, at least in transfected COS 7 cells. Moreover we did not observe a preferential association of any of the isoforms of FAK with c-Src, n-Src, or Fyn.One possible functional consequence of increased tyrosine phosphorylation of FAK is suggested by knock out experiments which have revealed that FAK is more important for the turnover of focal adhesions than for their formation (11Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Aizawa S. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1580) Google Scholar). Interestingly, growth cones of neurons in culture do not have real focal adhesions, but display smaller structures, termed point contacts, which may be related to the high mobility of neuritic extensions (32Arregui C.O. Carbonetto S. McKerracher L. J. Neurosci. 1994; 14: 6967-6977Crossref PubMed Google Scholar). It will be important to determine whether the isoforms of FAK with a higher capacity to autophosphorylate are associated with these adhesion structures, which may have a high reversibility. It is striking that point contacts are also observed in transformed cells in which they may be related to an increased mobility or invasiveness (32Arregui C.O. Carbonetto S. McKerracher L. J. Neurosci. 1994; 14: 6967-6977Crossref PubMed Google Scholar). Since a high degree of FAK expression in malignant cells may be related to their invasiveness or their ability to create metastasis (33Malik R.K. Parsons J.T. Biochim. Biophys. Acta Rev. Cancer. 1996; 1287: 73-76Crossref PubMed Scopus (28) Google Scholar), our findings suggest that the expression of splice variants of FAK in such cells could have important consequences on these characteristics. Focal adhesion kinase (FAK) 1The abbreviations used are: FAK, pp125 focal adhesion kinase; DOTAP,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium me- thylsulfate; PWR, Pro-Trp-Arg; SH2, Src-homology domain 2; SH3, Src-homology domain 3. 1The abbreviations used are: FAK, pp125 focal adhesion kinase; DOTAP,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium me- thylsulfate; PWR, Pro-Trp-Arg; SH2, Src-homology domain 2; SH3, Src-homology domain 3. is a 125-kDa cytoplasmic tyrosine kinase associated with focal adhesions in non-neuronal cells (see Refs. 1Schaller M.D. Parsons J.T. Curr. Opin. Cell Biol. 1994; 6: 705-710Crossref PubMed Scopus (493) Google Scholar, 2Schaller M.D. J. Endocrinol. 1996; 150: 1-7Crossref PubMed Scopus (74) Google Scholar, 3Guan J.L. Chen H.C. Int. Rev. Cytol. 1996; 168: 81-121Crossref PubMed Google Scholar, 4Hanks S.K. Polte T.R. BioEssays. 1997; 19: 137-145Crossref PubMed Scopus (440) Google Scholar, 5Ilic D. Damsky C.H. Yamamoto T. J. Cell Sci. 1997; 110: 401-407Crossref PubMed Google Scholar, for reviews). Although FAK does not have SH2 or SH3 domains, it is associated to many proteins, some of which bind to FAK phosphorylated on tyrosine via their own SH2 domains (1Schaller M.D. Parsons J.T. Curr. Opin. Cell Biol. 1994; 6: 705-710Crossref PubMed Scopus (493) Google Scholar, 2Schaller M.D. J. Endocrinol. 1996; 150: 1-7Crossref PubMed Scopus (74) Google Scholar, 3Guan J.L. Chen H.C. Int. Rev. Cytol. 1996; 168: 81-121Crossref PubMed Google Scholar, 4Hanks S.K. Polte T.R. BioEssays. 1997; 19: 137-145Crossref PubMed Scopus (440) Google Scholar, 5Ilic D. Damsky C.H. Yamamoto T. J. Cell Sci. 1997; 110: 401-407Crossref PubMed Google Scholar). FAK is phosphorylated on tyrosine in response to integrin engagement and to stimulation of various G protein-coupled receptors (1Schaller M.D. Parsons J.T. Curr. Opin. Cell Biol. 1994; 6: 705-710Crossref PubMed Scopus (493) Google Scholar, 2Schaller M.D. J. Endocrinol. 1996; 150: 1-7Crossref PubMed Scopus (74) Google Scholar, 3Guan J.L. Chen H.C. Int. Rev. Cytol. 1996; 168: 81-121Crossref PubMed Google Scholar, 4Hanks S.K. Polte T.R. BioEssays. 1997; 19: 137-145Crossref PubMed Scopus (440) Google Scholar, 5Ilic D. Damsky C.H. Yamamoto T. J. Cell Sci. 1997; 110: 401-407Crossref PubMed Google Scholar). The first step appears to be the autophosphorylation of Tyr-397 which allows the binding of the SH2 domains of Src (6Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1113) Google Scholar), Fyn (7Cobb B.S. Schaller M.D. Leu T.-H. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (483) Google Scholar), or phosphatidylinositol 3-kinase (8Chen H.-C. Appeddu P.A. Isoda H. Guan J.-L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). The recruitment of Src-family kinases results in the phosphorylation of neighboring proteins and at least 5 residues in FAK itself (9Calalb M.B. Polte T.R. Hanks S.K. Mol. Cell. Biol. 1995; 15: 954-963Crossref PubMed Google Scholar). Phosphorylation of some of these residues leads to the recruitment of Grb2 and the activation of the mitogen-activated protein kinase cascade (10Schlaepfer D.D. Hanks S.K. Hunter T. Van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1437) Google Scholar). The importance of FAK is attested by the embryonic lethality of the knock out of its gene (11Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Aizawa S. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1580) Google Scholar). FAK is thought to have various functions ranging from the regulation of focal adhesions turnover to the prevention of anoikis, a form of apoptosis induced by the detachment of cells from the extracellular matrix (12Hungerford J.E. Compton M.T. Matter M.L. Hoffstrom B.G. Otey C.A. J. Cell Biol. 1996; 135: 1383-1390Crossref PubMed Scopus (333) Google Scholar). FAK is highly expressed in the nervous tissue during development, a period at which it is enriched in neuronal growth cones (13Burgaya F. Menegon A. Menegoz M. Valtorta F. Girault J.-A. Eur. J. Neurosci. 1995; 7: 1810-1821Crossref PubMed Scopus (76) Google Scholar, 14Stevens G.R. Zhang C. Berg M.M. Lambert M.P. Barber K. Cantallops I. Routtenberg A. Klein W.L. J. Neurosci. Res. 1996; 46: 445-455Crossref PubMed Scopus (29) Google Scholar, 15Worley T.L. Holt C.E. Neuroreport. 1996; 7: 1133-1137Crossref PubMed Scopus (8) Google Scholar). In the brain of adult rats, FAK is expressed at higher levels than in most other tissues, especially in neurons of the hippocampus and the cerebral cortex (13Burgaya F. Menegon A. Menegoz M. Valtorta F. Girault J.-A. Eur. J. Neurosci. 1995; 7: 1810-1821Crossref PubMed Scopus (76) Google Scholar, 16Grant S.G.N. Karl K.A. Kiebler M.A. Kandel E.R. Genes Dev. 1995; 9: 1909-1921Crossref PubMed Scopus (130) Google Scholar). In brain, the major form of FAK is a splice variant termed FAK+, which contains a 3-amino acid insertion (Pro-Trp-Arg) at position 904 (17Burgaya F. Girault J.-A. Mol. Brain Res. 1996; 37: 63-73Crossref PubMed Scopus (35) Google Scholar, 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). In rat hippocampal slices, FAK+ phosphorylation is strongly stimulated by several neurotransmitters, including glutamate (19Siciliano J.C. Toutant M. Derkinderen P. Sasaki T. Girault J.-A. J. Biol. Chem. 1996; 271: 28942-28946Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) and acetylcholine, 2P. Derkinderen, J. C. Sicilians, M. Toutant, and J.-A. Girault, submitted for publication. 2P. Derkinderen, J. C. Sicilians, M. Toutant, and J.-A. Girault, submitted for publication. as well as lipid messengers anandamide, arachidonic acid (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), and lysophosphatidic acid.2 The sensitivity of FAK+ phosphorylation to neurotransmitters suggests that FAK+ transduces information generated by neuronal activity into phosphorylation of proteins, possibly resulting in changes in ion channel properties, cytoskeletal organization, or gene expression. Thus, FAK may be critical for the suggested role of protein tyrosine phosphorylation in synaptic plasticity (20O'Dell T.J. Kandel E.R. Grant S.G.N. Nature. 1991; 353: 558-560Crossref PubMed Scopus (469) Google Scholar, 21Boxall A.R. Lancaster B. Garthwaite J. Neuron. 1996; 16: 805-813Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). This hypothesis is supported by the observation that FAK phosphorylation is decreased in Fyn knock out mice (16Grant S.G.N. Karl K.A. Kiebler M.A. Kandel E.R. Genes Dev. 1995; 9: 1909-1921Crossref PubMed Scopus (130) Google Scholar) which display anomalous hippocampal development and long term potentiation (20O'Dell T.J. Kandel E.R. Grant S.G.N. Nature. 1991; 353: 558-560Crossref PubMed Scopus (469) Google Scholar, 22Kojima N. Wang J. Mansuy I.M. Grant S.G.N. Mayford M. Kandel E.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4761-4765Crossref PubMed Scopus (166) Google Scholar). As an approach to better understand the function of FAK in neurons, we have examined the biochemical properties of this kinase in rat brain. We show that the slightly larger size of FAK in brain as compared with other tissues, can be accounted for by the presence of additional exons surrounding the autophosphorylated tyrosine. The presence of two of these exons resulted in an increased autophosphorylation activity. DISCUSSIONThis study underlines the complexity of FAK structure and processing. Although it appears to be coded by a single gene, a number of transcriptional and post-transcriptional mechanisms generate multiple isoforms of FAK mRNA and protein. Two different polyadenylation sites and two different 5′ ends were found in rat, corresponding to those described in chick and mouse, respectively (17Burgaya F. Girault J.-A. Mol. Brain Res. 1996; 37: 63-73Crossref PubMed Scopus (35) Google Scholar). A very short alternative exon, coding for 3 amino acids defining FAK+, is found in rat, mouse (17Burgaya F. Girault J.-A. Mol. Brain Res. 1996; 37: 63-73Crossref PubMed Scopus (35) Google Scholar, 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), human (28Whitney G.S. Chan P.-Y. Blake J. Cosand W.L. Neubauer M.G. Aruffo A. Kanner S.B. DNA Cell Biol. 1993; 12: 823-830Crossref PubMed Scopus (90) Google Scholar), and Xenopus(26Hens M.D. DeSimone D.W. Dev. Biol. 1995; 170: 274-288Crossref PubMed Scopus (67) Google Scholar). A short form of FAK, called FAK-related non-kinase has been described in chick (29Schaller M.D. Borgman C.A. Parsons J.T. Mol. Cell. Biol. 1993; 13: 785-791Crossref PubMed Scopus (279) Google Scholar) and may also exist in human (30André E. Becker-André M. Biochem. Biophys. Res. Commun. 1993; 190: 140-147Crossref PubMed Scopus (85) Google Scholar). In the present study, we show that 3 additional exons can be found in rat and mouse FAK, corresponding to the region of the protein which surrounds the autophosphorylation site, at the junction between the amino-terminal and the catalytic domains of FAK. At least two of these exons (boxes 6 and 7) have been highly conserved during evolution, since they exist also in Xenopus (26Hens M.D. DeSimone D.W. Dev. Biol. 1995; 170: 274-288Crossref PubMed Scopus (67) Google Scholar). Assuming that all these variations of FAK mRNAs occur independently from the others, at least 512 different mRNAs coding for 64 different forms of full-length FAK protein could exist. This calculation does not take into account the short gene product FAK-related non-kinase and the possible additional exons which have been reported in human cDNAs (30André E. Becker-André M. Biochem. Biophys. Res. Commun. 1993; 190: 140-147Crossref PubMed Scopus (85) Google Scholar). Although it is likely that not all combinations are expressed at significant levels, the wealth of potential isoforms, many of which, and perhaps all, have been highly conserved during evolution, provide a mechanism by which FAK expression and properties could be finely tuned.It is noteworthy that exons which give rise to changes in the coding sequence (FAK+, boxes 28, 6, and 7) are all highly expressed in the nervous tissue. This may be linked to the preferential inclusion of small exons which has been suggested to occur during mRNA splicing in brain (31Black D.L. Cell. 1992; 69: 795-807Abstract Full Text PDF PubMed Scopus (147) Google Scholar). In fact, among the other tissues examined only testis contained high amounts of FAK+ (data not shown) and box 7 (this study). An important consequence of the existence of isoforms with differences in the coding region, is that they may provide neuronal FAK with specific properties. We found that FAK and FAK+ were indistinguishable concerning their amount of tyrosine phosphorylation, their autophosphorylation, and their interactions with Src and Fyn. This is perhaps not surprising since the Pro-Trp-Arg insertion of FAK+ is located in the carboxyl-terminal region of the protein, at a distance in the primary sequence from the kinase domain and the autophosphorylation site. In contrast, we found that the presence of boxes 6 and 7 which are located on either side of the autophosphorylated tyrosine, affected dramatically the tyrosine phosphorylation in cells and in immune precipitates. Using a site- and phosphorylation-specific antibody, we could show that the increased phosphorylation of FAK+6,7 could be accounted for, at least in part, by an increased phosphorylation of Tyr-397, the major autophosphorylation site. Boxes 6 and 7 were always found associated in striatal cDNAs and we do not know whether one, or the other, or the combination of the two is responsible for the observed effect. On the other hand, the presence of box 28, located amino-terminal of box 6, did not alter the autophosphorylation rate.Work from several laboratories has shown that autophosphorylation of FAK on Tyr-397 is a critical aspect of its regulation and function, since it is responsible for the binding of Src family kinases (6Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1113) Google Scholar, 7Cobb B.S. Schaller M.D. Leu T.-H. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (483) Google Scholar) and phosphatidylinositol 3-kinase (8Chen H.-C. Appeddu P.A. Isoda H. Guan J.-L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). Moreover, although in vitro FAK is able to phosphorylate several proteins, including paxillin and p130 cas, these are also substrates for Src which may be responsible for their phosphorylation in cells (see Refs. 4Hanks S.K. Polte T.R. BioEssays. 1997; 19: 137-145Crossref PubMed Scopus (440) Google Scholar and5Ilic D. Damsky C.H. Yamamoto T. J. Cell Sci. 1997; 110: 401-407Crossref PubMed Google Scholar). The available evidence suggests that FAK may function as an adaptor regulated by autophosphorylation, rather than as a protein kinase active on different substrates. Thus, an increased ability to autophosphorylate, provided by alternative splicing, could have important functional consequences. For instance, it could increase the phosphorylation of FAK in cells which express FAK6,7 in response to integrin engagement or stimulation of G protein-coupled receptors. It may thus play a role in the high responsiveness of FAK in hippocampus to neurotransmitters or lipid messengers (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, 19Siciliano J.C. Toutant M. Derkinderen P. Sasaki T. Girault J.-A. J. Biol. Chem. 1996; 271: 28942-28946Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Our studies of FAK isoforms gave important negative results concerning their interactions with Src family kinases. The presence of insertions did not alter the interaction of FAK with these kinases, at least in transfected COS 7 cells. Moreover we did not observe a preferential association of any of the isoforms of FAK with c-Src, n-Src, or Fyn.One possible functional consequence of increased tyrosine phosphorylation of FAK is suggested by knock out experiments which have revealed that FAK is more important for the turnover of focal adhesions than for their formation (11Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Aizawa S. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1580) Google Scholar). Interestingly, growth cones of neurons in culture do not have real focal adhesions, but display smaller structures, termed point contacts, which may be related to the high mobility of neuritic extensions (32Arregui C.O. Carbonetto S. McKerracher L. J. Neurosci. 1994; 14: 6967-6977Crossref PubMed Google Scholar). It will be important to determine whether the isoforms of FAK with a higher capacity to autophosphorylate are associated with these adhesion structures, which may have a high reversibility. It is striking that point contacts are also observed in transformed cells in which they may be related to an increased mobility or invasiveness (32Arregui C.O. Carbonetto S. McKerracher L. J. Neurosci. 1994; 14: 6967-6977Crossref PubMed Google Scholar). Since a high degree of FAK expression in malignant cells may be related to their invasiveness or their ability to create metastasis (33Malik R.K. Parsons J.T. Biochim. Biophys. Acta Rev. Cancer. 1996; 1287: 73-76Crossref PubMed Scopus (28) Google Scholar), our findings suggest that the expression of splice variants of FAK in such cells could have important consequences on these characteristics. This study underlines the complexity of FAK structure and processing. Although it appears to be coded by a single gene, a number of transcriptional and post-transcriptional mechanisms generate multiple isoforms of FAK mRNA and protein. Two different polyadenylation sites and two different 5′ ends were found in rat, corresponding to those described in chick and mouse, respectively (17Burgaya F. Girault J.-A. Mol. Brain Res. 1996; 37: 63-73Crossref PubMed Scopus (35) Google Scholar). A very short alternative exon, coding for 3 amino acids defining FAK+, is found in rat, mouse (17Burgaya F. Girault J.-A. Mol. Brain Res. 1996; 37: 63-73Crossref PubMed Scopus (35) Google Scholar, 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), human (28Whitney G.S. Chan P.-Y. Blake J. Cosand W.L. Neubauer M.G. Aruffo A. Kanner S.B. DNA Cell Biol. 1993; 12: 823-830Crossref PubMed Scopus (90) Google Scholar), and Xenopus(26Hens M.D. DeSimone D.W. Dev. Biol. 1995; 170: 274-288Crossref PubMed Scopus (67) Google Scholar). A short form of FAK, called FAK-related non-kinase has been described in chick (29Schaller M.D. Borgman C.A. Parsons J.T. Mol. Cell. Biol. 1993; 13: 785-791Crossref PubMed Scopus (279) Google Scholar) and may also exist in human (30André E. Becker-André M. Biochem. Biophys. Res. Commun. 1993; 190: 140-147Crossref PubMed Scopus (85) Google Scholar). In the present study, we show that 3 additional exons can be found in rat and mouse FAK, corresponding to the region of the protein which surrounds the autophosphorylation site, at the junction between the amino-terminal and the catalytic domains of FAK. At least two of these exons (boxes 6 and 7) have been highly conserved during evolution, since they exist also in Xenopus (26Hens M.D. DeSimone D.W. Dev. Biol. 1995; 170: 274-288Crossref PubMed Scopus (67) Google Scholar). Assuming that all these variations of FAK mRNAs occur independently from the others, at least 512 different mRNAs coding for 64 different forms of full-length FAK protein could exist. This calculation does not take into account the short gene product FAK-related non-kinase and the possible additional exons which have been reported in human cDNAs (30André E. Becker-André M. Biochem. Biophys. Res. Commun. 1993; 190: 140-147Crossref PubMed Scopus (85) Google Scholar). Although it is likely that not all combinations are expressed at significant levels, the wealth of potential isoforms, many of which, and perhaps all, have been highly conserved during evolution, provide a mechanism by which FAK expression and properties could be finely tuned. It is noteworthy that exons which give rise to changes in the coding sequence (FAK+, boxes 28, 6, and 7) are all highly expressed in the nervous tissue. This may be linked to the preferential inclusion of small exons which has been suggested to occur during mRNA splicing in brain (31Black D.L. Cell. 1992; 69: 795-807Abstract Full Text PDF PubMed Scopus (147) Google Scholar). In fact, among the other tissues examined only testis contained high amounts of FAK+ (data not shown) and box 7 (this study). An important consequence of the existence of isoforms with differences in the coding region, is that they may provide neuronal FAK with specific properties. We found that FAK and FAK+ were indistinguishable concerning their amount of tyrosine phosphorylation, their autophosphorylation, and their interactions with Src and Fyn. This is perhaps not surprising since the Pro-Trp-Arg insertion of FAK+ is located in the carboxyl-terminal region of the protein, at a distance in the primary sequence from the kinase domain and the autophosphorylation site. In contrast, we found that the presence of boxes 6 and 7 which are located on either side of the autophosphorylated tyrosine, affected dramatically the tyrosine phosphorylation in cells and in immune precipitates. Using a site- and phosphorylation-specific antibody, we could show that the increased phosphorylation of FAK+6,7 could be accounted for, at least in part, by an increased phosphorylation of Tyr-397, the major autophosphorylation site. Boxes 6 and 7 were always found associated in striatal cDNAs and we do not know whether one, or the other, or the combination of the two is responsible for the observed effect. On the other hand, the presence of box 28, located amino-terminal of box 6, did not alter the autophosphorylation rate. Work from several laboratories has shown that autophosphorylation of FAK on Tyr-397 is a critical aspect of its regulation and function, since it is responsible for the binding of Src family kinases (6Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1113) Google Scholar, 7Cobb B.S. Schaller M.D. Leu T.-H. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (483) Google Scholar) and phosphatidylinositol 3-kinase (8Chen H.-C. Appeddu P.A. Isoda H. Guan J.-L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). Moreover, although in vitro FAK is able to phosphorylate several proteins, including paxillin and p130 cas, these are also substrates for Src which may be responsible for their phosphorylation in cells (see Refs. 4Hanks S.K. Polte T.R. BioEssays. 1997; 19: 137-145Crossref PubMed Scopus (440) Google Scholar and5Ilic D. Damsky C.H. Yamamoto T. J. Cell Sci. 1997; 110: 401-407Crossref PubMed Google Scholar). The available evidence suggests that FAK may function as an adaptor regulated by autophosphorylation, rather than as a protein kinase active on different substrates. Thus, an increased ability to autophosphorylate, provided by alternative splicing, could have important functional consequences. For instance, it could increase the phosphorylation of FAK in cells which express FAK6,7 in response to integrin engagement or stimulation of G protein-coupled receptors. It may thus play a role in the high responsiveness of FAK in hippocampus to neurotransmitters or lipid messengers (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, 19Siciliano J.C. Toutant M. Derkinderen P. Sasaki T. Girault J.-A. J. Biol. Chem. 1996; 271: 28942-28946Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Our studies of FAK isoforms gave important negative results concerning their interactions with Src family kinases. The presence of insertions did not alter the interaction of FAK with these kinases, at least in transfected COS 7 cells. Moreover we did not observe a preferential association of any of the isoforms of FAK with c-Src, n-Src, or Fyn. One possible functional consequence of increased tyrosine phosphorylation of FAK is suggested by knock out experiments which have revealed that FAK is more important for the turnover of focal adhesions than for their formation (11Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Aizawa S. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1580) Google Scholar). Interestingly, growth cones of neurons in culture do not have real focal adhesions, but display smaller structures, termed point contacts, which may be related to the high mobility of neuritic extensions (32Arregui C.O. Carbonetto S. McKerracher L. J. Neurosci. 1994; 14: 6967-6977Crossref PubMed Google Scholar). It will be important to determine whether the isoforms of FAK with a higher capacity to autophosphorylate are associated with these adhesion structures, which may have a high reversibility. It is striking that point contacts are also observed in transformed cells in which they may be related to an increased mobility or invasiveness (32Arregui C.O. Carbonetto S. McKerracher L. J. Neurosci. 1994; 14: 6967-6977Crossref PubMed Google Scholar). Since a high degree of FAK expression in malignant cells may be related to their invasiveness or their ability to create metastasis (33Malik R.K. Parsons J.T. Biochim. Biophys. Acta Rev. Cancer. 1996; 1287: 73-76Crossref PubMed Scopus (28) Google Scholar), our findings suggest that the expression of splice variants of FAK in such cells could have important consequences on these characteristics. Prof. Jacques Glowinski is gratefully acknowledged for encouragement and support throughout this study.
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