SU6656, a selective Src family kinase inhibitor, used to probe growth factor signaling

The use of small-molecule inhibitors to study molecular components of cellu lar signal transduction pathways provides a means of analysis complementary to currently used techniques, such as antisense, dominant-negative (interf ering) mutants and constitutively activated mutants. We have identified and characterized a small-molecule inhibitor, SU6656, which exhibits selective for Src and other members of the Src family. A related inhibitor, SU6657, inhibits many kinases, including Src and the platelet-derived growth factor (PDGF) receptor. The nse of SU6656 confirmed our previous findings that Sr c family kinases are required for both Myc induction and DNA synthesis in r esponse to PDGF stimulation of MDI 3T3 fibroblasts. By comparing PDGF stimu lated tyrosine phosphorylation events in untreated and SU6656-treated cells , we found that some substrates (for example, c-Cbl, and protein kinase C d elta) were Src family substrates whereas others (for example, phospholipase C-gamma) were not. One protein, the adaptor Shc, was a substrate for both Src family kinases ton tyrosines 239 and 240) and a distinct tyrosine kinas e ton tyrosine 317, which is perhaps phosphorylated by the PDGF receptor it self). Microinjection experiments demonstrated that a Shc molecule carrying mutations of tyrosines 239 and 240, in conjunction with an SH2 domain muta tion, interfered with PDGF-stimulated DNA synthesis. Deletion of the phosph otyrosine-binding domain also inhibited synthesis. These inhibitions were o vercome by heterologous expression of Myc, supporting the hypothesis that S hc functions in the Src pathway. SU6656 should prove a useful additional to ol for further dissecting the role of Src kinases in this and other signal transduction pathways.