Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 May 15;348(Pt 1):119–128.

Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms.

M Toutant 1, J M Studler 1, F Burgaya 1, A Costa 1, P Ezan 1, M Gelman 1, J A Girault 1
PMCID: PMC1221044  PMID: 10794722

Abstract

In brain, focal adhesion kinase (FAK) is regulated by neurotransmitters and has a higher molecular mass than in other tissues, due to alternative splicing. Two exons code for additional peptides of six and seven residues ('boxes' 6 and 7), located on either side of Tyr(397), which increase its autophosphorylation. Using in situ hybridization and a monoclonal antibody (Mab77) which does not recognize FAK containing box 7, we show that, although mRNAs coding for boxes 6 and 7 have different patterns of expression in brain, FAK+6,7 is the main isoform in forebrain neurons. The various FAK isoforms fused to green fluorescent protein were all targeted to focal adhesions in non-neuronal cells. Phosphorylation-state-specific antibodies were used to study in detail the phosphorylation of Tyr(397), a critical residue for the activation and function of FAK. The presence of boxes 6 and 7 increased autophosphorylation of Tyr(397) independently and additively, whereas they had a weak effect on FAK kinase activity towards poly(Glu,Tyr). Src-family kinases were also able to phosphorylate Tyr(397) in cells, but this phosphorylation was decreased in the presence of box 6 or 7, and abolished in the presence of both. Thus the additional exons characteristic of neuronal isoforms of FAK do not alter its targeting, but change dramatically the phosphorylation of Tyr(397). They increase its autophosphorylation in vitro and in transfected COS-7 cells, whereas they prevent its phosphorylation when co-transfected with Src-family kinases.

Full Text

The Full Text of this article is available as a PDF (362.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. André E., Becker-André M. Expression of an N-terminally truncated form of human focal adhesion kinase in brain. Biochem Biophys Res Commun. 1993 Jan 15;190(1):140–147. doi: 10.1006/bbrc.1993.1022. [DOI] [PubMed] [Google Scholar]
  2. Bellis S. L., Miller J. T., Turner C. E. Characterization of tyrosine phosphorylation of paxillin in vitro by focal adhesion kinase. J Biol Chem. 1995 Jul 21;270(29):17437–17441. doi: 10.1074/jbc.270.29.17437. [DOI] [PubMed] [Google Scholar]
  3. Black D. L. Activation of c-src neuron-specific splicing by an unusual RNA element in vivo and in vitro. Cell. 1992 May 29;69(5):795–807. doi: 10.1016/0092-8674(92)90291-j. [DOI] [PubMed] [Google Scholar]
  4. Boussif O., Lezoualc'h F., Zanta M. A., Mergny M. D., Scherman D., Demeneix B., Behr J. P. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7297–7301. doi: 10.1073/pnas.92.16.7297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burgaya F., Girault J. A. Cloning of focal adhesion kinase, pp125FAK, from rat brain reveals multiple transcripts with different patterns of expression. Brain Res Mol Brain Res. 1996 Apr;37(1-2):63–73. doi: 10.1016/0169-328x(95)00273-u. [DOI] [PubMed] [Google Scholar]
  6. Burgaya F., Menegon A., Menegoz M., Valtorta F., Girault J. A. Focal adhesion kinase in rat central nervous system. Eur J Neurosci. 1995 Aug 1;7(8):1810–1821. doi: 10.1111/j.1460-9568.1995.tb00700.x. [DOI] [PubMed] [Google Scholar]
  7. Burgaya F., Toutant M., Studler J. M., Costa A., Le Bert M., Gelman M., Girault J. A. Alternatively spliced focal adhesion kinase in rat brain with increased autophosphorylation activity. J Biol Chem. 1997 Nov 7;272(45):28720–28725. doi: 10.1074/jbc.272.45.28720. [DOI] [PubMed] [Google Scholar]
  8. Calalb M. B., Polte T. R., Hanks S. K. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol Cell Biol. 1995 Feb;15(2):954–963. doi: 10.1128/mcb.15.2.954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Calalb M. B., Zhang X., Polte T. R., Hanks S. K. Focal adhesion kinase tyrosine-861 is a major site of phosphorylation by Src. Biochem Biophys Res Commun. 1996 Nov 21;228(3):662–668. doi: 10.1006/bbrc.1996.1714. [DOI] [PubMed] [Google Scholar]
  10. Chen H. C., Appeddu P. A., Isoda H., Guan J. L. Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3-kinase. J Biol Chem. 1996 Oct 18;271(42):26329–26334. doi: 10.1074/jbc.271.42.26329. [DOI] [PubMed] [Google Scholar]
  11. Chen H. C., Appeddu P. A., Parsons J. T., Hildebrand J. D., Schaller M. D., Guan J. L. Interaction of focal adhesion kinase with cytoskeletal protein talin. J Biol Chem. 1995 Jul 14;270(28):16995–16999. doi: 10.1074/jbc.270.28.16995. [DOI] [PubMed] [Google Scholar]
  12. Cobb B. S., Schaller M. D., Leu T. H., Parsons J. T. Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK. Mol Cell Biol. 1994 Jan;14(1):147–155. doi: 10.1128/mcb.14.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Czernik A. J., Girault J. A., Nairn A. C., Chen J., Snyder G., Kebabian J., Greengard P. Production of phosphorylation state-specific antibodies. Methods Enzymol. 1991;201:264–283. doi: 10.1016/0076-6879(91)01025-w. [DOI] [PubMed] [Google Scholar]
  14. Derkinderen P., Siciliano J., Toutant M., Girault J. A. Differential regulation of FAK+ and PYK2/Cakbeta, two related tyrosine kinases, in rat hippocampal slices: effects of LPA, carbachol, depolarization and hyperosmolarity. Eur J Neurosci. 1998 May;10(5):1667–1675. doi: 10.1046/j.1460-9568.1998.00174.x. [DOI] [PubMed] [Google Scholar]
  15. Derkinderen P., Toutant M., Burgaya F., Le Bert M., Siciliano J. C., de Franciscis V., Gelman M., Girault J. A. Regulation of a neuronal form of focal adhesion kinase by anandamide. Science. 1996 Sep 20;273(5282):1719–1722. doi: 10.1126/science.273.5282.1719. [DOI] [PubMed] [Google Scholar]
  16. Eide B. L., Turck C. W., Escobedo J. A. Identification of Tyr-397 as the primary site of tyrosine phosphorylation and pp60src association in the focal adhesion kinase, pp125FAK. Mol Cell Biol. 1995 May;15(5):2819–2827. doi: 10.1128/mcb.15.5.2819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Evans G. J., Pocock J. M. Modulation of neurotransmitter release by dihydropyridine-sensitive calcium channels involves tyrosine phosphorylation. Eur J Neurosci. 1999 Jan;11(1):279–292. doi: 10.1046/j.1460-9568.1999.00427.x. [DOI] [PubMed] [Google Scholar]
  18. Girault J. A., Costa A., Derkinderen P., Studler J. M., Toutant M. FAK and PYK2/CAKbeta in the nervous system: a link between neuronal activity, plasticity and survival? Trends Neurosci. 1999 Jun;22(6):257–263. doi: 10.1016/s0166-2236(98)01358-7. [DOI] [PubMed] [Google Scholar]
  19. Girault J. A., Labesse G., Mornon J. P., Callebaut I. Janus kinases and focal adhesion kinases play in the 4.1 band: a superfamily of band 4.1 domains important for cell structure and signal transduction. Mol Med. 1998 Dec;4(12):751–769. [PMC free article] [PubMed] [Google Scholar]
  20. Girault J. A., Labesse G., Mornon J. P., Callebaut I. The N-termini of FAK and JAKs contain divergent band 4.1 domains. Trends Biochem Sci. 1999 Feb;24(2):54–57. doi: 10.1016/s0968-0004(98)01331-0. [DOI] [PubMed] [Google Scholar]
  21. Grant S. G., Karl K. A., Kiebler M. A., Kandel E. R. Focal adhesion kinase in the brain: novel subcellular localization and specific regulation by Fyn tyrosine kinase in mutant mice. Genes Dev. 1995 Aug 1;9(15):1909–1921. doi: 10.1101/gad.9.15.1909. [DOI] [PubMed] [Google Scholar]
  22. Hanks S. K., Calalb M. B., Harper M. C., Patel S. K. Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8487–8491. doi: 10.1073/pnas.89.18.8487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Harte M. T., Hildebrand J. D., Burnham M. R., Bouton A. H., Parsons J. T. p130Cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase. J Biol Chem. 1996 Jun 7;271(23):13649–13655. doi: 10.1074/jbc.271.23.13649. [DOI] [PubMed] [Google Scholar]
  24. Hens M. D., DeSimone D. W. Molecular analysis and developmental expression of the focal adhesion kinase pp125FAK in Xenopus laevis. Dev Biol. 1995 Aug;170(2):274–288. doi: 10.1006/dbio.1995.1214. [DOI] [PubMed] [Google Scholar]
  25. Hildebrand J. D., Schaller M. D., Parsons J. T. Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125FAK, to cellular focal adhesions. J Cell Biol. 1993 Nov;123(4):993–1005. doi: 10.1083/jcb.123.4.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hildebrand J. D., Schaller M. D., Parsons J. T. Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase. Mol Biol Cell. 1995 Jun;6(6):637–647. doi: 10.1091/mbc.6.6.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ilić D., Almeida E. A., Schlaepfer D. D., Dazin P., Aizawa S., Damsky C. H. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. J Cell Biol. 1998 Oct 19;143(2):547–560. doi: 10.1083/jcb.143.2.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ilić D., Furuta Y., Kanazawa S., Takeda N., Sobue K., Nakatsuji N., Nomura S., Fujimoto J., Okada M., Yamamoto T. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature. 1995 Oct 12;377(6549):539–544. doi: 10.1038/377539a0. [DOI] [PubMed] [Google Scholar]
  29. Law S. F., Estojak J., Wang B., Mysliwiec T., Kruh G., Golemis E. A. Human enhancer of filamentation 1, a novel p130cas-like docking protein, associates with focal adhesion kinase and induces pseudohyphal growth in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Jul;16(7):3327–3337. doi: 10.1128/mcb.16.7.3327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lev S., Moreno H., Martinez R., Canoll P., Peles E., Musacchio J. M., Plowman G. D., Rudy B., Schlessinger J. Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions. Nature. 1995 Aug 31;376(6543):737–745. doi: 10.1038/376737a0. [DOI] [PubMed] [Google Scholar]
  31. Menegon A., Burgaya F., Baudot P., Dunlap D. D., Girault J. A., Valtorta F. FAK+ and PYK2/CAKbeta, two related tyrosine kinases highly expressed in the central nervous system: similarities and differences in the expression pattern. Eur J Neurosci. 1999 Nov;11(11):3777–3788. doi: 10.1046/j.1460-9568.1999.00798.x. [DOI] [PubMed] [Google Scholar]
  32. Polte T. R., Hanks S. K. Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas. Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10678–10682. doi: 10.1073/pnas.92.23.10678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Richardson A., Parsons T. A mechanism for regulation of the adhesion-associated proteintyrosine kinase pp125FAK. Nature. 1996 Apr 11;380(6574):538–540. doi: 10.1038/380538a0. [DOI] [PubMed] [Google Scholar]
  34. Sasaki H., Nagura K., Ishino M., Tobioka H., Kotani K., Sasaki T. Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily. J Biol Chem. 1995 Sep 8;270(36):21206–21219. doi: 10.1074/jbc.270.36.21206. [DOI] [PubMed] [Google Scholar]
  35. Schaller M. D., Borgman C. A., Cobb B. S., Vines R. R., Reynolds A. B., Parsons J. T. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5192–5196. doi: 10.1073/pnas.89.11.5192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schaller M. D., Borgman C. A., Parsons J. T. Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK. Mol Cell Biol. 1993 Feb;13(2):785–791. doi: 10.1128/mcb.13.2.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schaller M. D., Hildebrand J. D., Shannon J. D., Fox J. W., Vines R. R., Parsons J. T. Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol Cell Biol. 1994 Mar;14(3):1680–1688. doi: 10.1128/mcb.14.3.1680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schaller M. D., Otey C. A., Hildebrand J. D., Parsons J. T. Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains. J Cell Biol. 1995 Sep;130(5):1181–1187. doi: 10.1083/jcb.130.5.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schlaepfer D. D., Hanks S. K., Hunter T., van der Geer P. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature. 1994 Dec 22;372(6508):786–791. doi: 10.1038/372786a0. [DOI] [PubMed] [Google Scholar]
  40. Schlaepfer D. D., Hunter T. 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. 1996 Oct;16(10):5623–5633. doi: 10.1128/mcb.16.10.5623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schlaepfer D. D., Hunter T. Integrin signalling and tyrosine phosphorylation: just the FAKs? Trends Cell Biol. 1998 Apr;8(4):151–157. doi: 10.1016/s0962-8924(97)01172-0. [DOI] [PubMed] [Google Scholar]
  42. Serpente N., Birling M. C., Price J. The regulation of the expression, phosphorylation, and protein associations of pp125FAK during rat brain development. Mol Cell Neurosci. 1996 May;7(5):391–403. doi: 10.1006/mcne.1996.0028. [DOI] [PubMed] [Google Scholar]
  43. Siciliano J. C., Gelman M., Girault J. A. Depolarization and neurotransmitters increase neuronal protein tyrosine phosphorylation. J Neurochem. 1994 Mar;62(3):950–959. doi: 10.1046/j.1471-4159.1994.62030950.x. [DOI] [PubMed] [Google Scholar]
  44. Siciliano J. C., Toutant M., Derkinderen P., Sasaki T., Girault J. A. Differential regulation of proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) and pp125(FAK) by glutamate and depolarization in rat hippocampus. J Biol Chem. 1996 Nov 15;271(46):28942–28946. doi: 10.1074/jbc.271.46.28942. [DOI] [PubMed] [Google Scholar]
  45. Songyang Z., Carraway K. L., 3rd, Eck M. J., Harrison S. C., Feldman R. A., Mohammadi M., Schlessinger J., Hubbard S. R., Smith D. P., Eng C. Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. Nature. 1995 Feb 9;373(6514):536–539. doi: 10.1038/373536a0. [DOI] [PubMed] [Google Scholar]
  46. Taylor J. M., Hildebrand J. D., Mack C. P., Cox M. E., Parsons J. T. 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. 1998 Apr 3;273(14):8063–8070. doi: 10.1074/jbc.273.14.8063. [DOI] [PubMed] [Google Scholar]
  47. Thomas J. W., Ellis B., Boerner R. J., Knight W. B., White G. C., 2nd, Schaller M. D. SH2- and SH3-mediated interactions between focal adhesion kinase and Src. J Biol Chem. 1998 Jan 2;273(1):577–583. doi: 10.1074/jbc.273.1.577. [DOI] [PubMed] [Google Scholar]
  48. Whitney G. S., Chan P. Y., Blake J., Cosand W. L., Neubauer M. G., Aruffo A., Kanner S. B. Human T and B lymphocytes express a structurally conserved focal adhesion kinase, pp125FAK. DNA Cell Biol. 1993 Nov;12(9):823–830. doi: 10.1089/dna.1993.12.823. [DOI] [PubMed] [Google Scholar]
  49. Xing Z., Chen H. C., Nowlen J. K., Taylor S. J., Shalloway D., Guan J. L. Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain. Mol Biol Cell. 1994 Apr;5(4):413–421. doi: 10.1091/mbc.5.4.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Xiong W. C., Macklem M., Parsons J. T. Expression and characterization of splice variants of PYK2, a focal adhesion kinase-related protein. J Cell Sci. 1998 Jul 30;111(Pt 14):1981–1991. doi: 10.1242/jcs.111.14.1981. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES