Abstract
Identifying substrates of receptor and non-receptor protein tyrosine kinases (PTK), and how phosphorylation of these substrates affects signaling and cytoskeletal pathways, has been a key step in understanding the role of PTK in differentiation, mitogenesis and oncogenesis. However, it has been difficult to distinguish substrates phosphorylated directly by PTK vs those phosphorylated by PTK-activated kinases. The following describes an in situ/overlay technique in which purified PTK (in our case, FAK) can be used to identify potential substrates from filter lifts of protein produced from a cDNA expression library.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hunter, T. and Sefton, B. M. (1980) Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc. Natl. Acad. Sci. USA 77, 1311–1315.
Robinson, D. R., Wu, Y. M., and Lin, S. F. (2000) The protein tyrosine kinase family of the human genome. Oncogene 19, 5548–5557.
Thomas, S. M. and Brugge, J. S. (1997) Cellular functions regulated by Src family kinases. Ann. Rev. Cell Dev. Biol. 13, 513–609.
Guan, J. L., Trevithick, J. E., and Hynes, R. O. (1991) Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein. Cell. Regul. 2, 951–964.
Chan, P. Y., Kanner, S. B., Whitney, G., and Aruffo, A. (1994) A transmembrane-anchored chimeric focal adhesion kinase is constitutively activated and phosphorylated at tyrosine residues identical to pp125FAK. J. Biol. Chem. 269, 20,567–20,574.
Lipfert, L., Haimovich, B., Schaller, M. D., Cobb, B. S., Parsons, J. T., and Brugge, J. S. (1992) Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets. J. Cell. Biol. 119, 905–912.
Burridge, K. and Chrzanowska-Wodnicka, M. (1996) Focal adhesions, contractility, and signaling. Ann. Rev. Cell Dev. Biol. 12, 463–519.
Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., et al. (1995) Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 377, 539–544.
Schaller, M. D., Hildebrand, J. D., and Parsons, J. T. (1999) Complex formation with focal adhesion kinase: A mechanism to regulate activity and subcellular localization of Src kinases. Molev. Biol. Cell 10, 3489–3505.
Fincham, V. J. and Frame, M. C. (1998) The catalytic activity of Src is dispensable for translocation to focal adhesions but controls the turnover of these structures during cell motility. EMBO J. 17, 81–92.
Schlaepfer, D. D., Hauck, C. R., and Sieg, D. J. (1999) Signaling through focal adhesion kinase. Prog. Biophys. Mol. Biol. 71, 435–478.
Kumar, C. C. (1998) Signaling by integrin receptors. Oncogene 17, 1365–1373.
Cary, L. A. and Guan, J. L. (1999) Focal adhesion kinase in integrin-mediated signaling. Front. Biosci. 4, D102–D113.
Chen, H. C. and Guan, J. L. (1994) Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase. Proc. Natl. Acad. Sci. USA 91, 10,148–10,152.
Ruest, P. J., Shin, N. Y., Polte, T. R., Zhang, X., and Hanks, S. K. (2001) Mechanisms of CAS substrate domain tyrosine phosphorylation by FAK and Src. Mol. Cell. Biol. 21, 7641–7652.
Izaguirre, G., Aguirre, L., Hu, Y. P., Lee, H. Y., Schlaepfer, D. D., Aneskievich, B. J., and Haimovich, B. (2001) The cytoskeletal/non-muscle isoform of α-actinin is phosphorylated on its actin-binding domain by the focal adhesion kinase. J. Biol. Chem. 276, 28,676–28,685.
Withers, B. E., Keller, P. R., and Fry, D. W. (1996) Expression, purification and characterization of focal adhesion kinase using a baculovirus system. Prot. Expr. Purif. 7, 12–18.
Brown, M. T. and Cooper, J. A. (1996) Regulation, substrates and functions of src. Biochim. Biophys. Acta Rev. Cancer 1287, 121–149.
Lock, P., Abram, C. L., Gibson, T., and Courtneidge, S. A. (1998) A new method for isolating tyrosine kinase substrates used to identify Fish, an SH3 and PX domain-containing protein, and Src substrate. EMBO J. 17, 4346–4357.
Fukunaga, R. and Hunter, T. (1997) MNK1, a new MAP kinase-activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates. EMBO J. 16, 1921–1933.
Lin, X., Tombler, E., Nelson, P. J., Ross, M., and Gelman, I. H. (1996) A novel src-and ras-suppressed protein kinase C substrate associated with cytoskeletal architecture. J. Biol. Chem. 271, 28,430–28,438.
Guan, J. L. (1997) Role of focal adhesion kinase in integrin signaling. Int. J. Biochem. Cell Biol. 29, 1085–1096.
Lacoste, J., Ma, A., and Parsons, J. T. (1998) Assay and purification of focal adhesion kinase. Methods Enzymol. 298, 89–102.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Gelman, I.H. (2003). Isolation of Novel Substrates Using a Tyrosine Kinase Overlay/In Situ Assay. In: Terrian, D.M. (eds) Cancer Cell Signaling. Methods in Molecular Biology™, vol 218. Humana Press. https://doi.org/10.1385/1-59259-356-9:133
Download citation
DOI: https://doi.org/10.1385/1-59259-356-9:133
Publisher Name: Humana Press
Print ISBN: 978-1-58829-075-5
Online ISBN: 978-1-59259-356-9
eBook Packages: Springer Protocols