| Protein tyrosine kinases, which include at least 50 peptide growth factor receptors, play a central role in the regulation of cell proliferation and differentiation. While considerable effort has been directed to the study of intracellular signals mediated by some of these receptor tyrosine kinases, relatively little information concerning the protein substrates of fibroblast growth factor (FGF) receptors has been reported. Among the FGFs, keratinocyte growth factor (KGF/FGF-7) is unique because of its target-specificity for epithelial cells, the cell type in which most human cancers originate.; In order to identify tyrosine-phosphorylated proteins that participate in the cellular response to KGF, phosphotyrosine levels in known tyrosine kinase substrates have been assessed using an anti-phosphotyrosine antibody. The results show that a number of cellular proteins become phosphorylated on tyrosine residues in response to KGF treatment. Included among these are phospholipase C {dollar}sb{lcub}gamma{rcub}{dollar}, components of the Ras signaling pathway, and several substrates of the Src tyrosine kinase. Ras guanosine triphosphatase activating protein (RasGAP) is not phosphorylated in a KGF-dependent manner, which distinguishes the response to this growth factor from those to epidermal growth factor (EGF) and to platelet-derived growth factor {dollar}beta{dollar}-subunit-homodimers (PDGF BB).; In addition to this approach, an expression cloning strategy has been implemented for the purpose of isolating novel tyrosine kinase substrates. Polyclonal antisera raised against anti-phosphotyrosine-immunoaffinity-purified cellular proteins, which have been used as probes for the screening of mouse skin and NIH 3T3 cDNA libraries, detect a number of clones encoding {dollar}alpha{dollar}-actinin and nucleolin. Western blots of anti-phosphotyrosine-immunoprecipitated proteins from KGF receptor-expressing cell lines indicate that both {dollar}alpha{dollar}-actinin and nucleolin become phosphorylated on tyrosine residues when cells are exposed to KGF. Five different DNAs that encode hitherto unknown proteins were also isolated by this cloning method, all of which are possibly tyrosine-phosphorylated signaling-intermediates involved in the cellular response to KGF. |
