Isolation of novel substrates using a tyrosine kinase overlay/in situ assay.
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.
Gelman
Irwin H
IH
Department of Medicine, Division of Infectious Diseases and Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY, USA.
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D005822:Genetic Vectors; D015151:Immunoblotting / Q000379:methods; D007313:Insecta; D051379:Mice; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013379:Substrate Specificity
D003598:Cytoskeletal Proteins; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse