SU6656, a Selective Src Family Kinase Inhibitor, Used To Probe Growth Factor Signaling

Abstract

The use of small-molecule inhibitors to study molecular components of cellular signal transduction pathways provides a means of analysis complementary to currently used techniques, such as antisense, dominant-negative (interfering) mutants and constitutively activated mutants. We have identified and characterized a small-molecule inhibitor, SU6656, which exhibits selectivity 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 use of SU6656 confirmed our previous findings that Src family kinases are required for both Myc induction and DNA synthesis in response to PDGF stimulation of NIH 3T3 fibroblasts. By comparing PDGF-stimulated tyrosine phosphorylation events in untreated and SU6656-treated cells, we found that some substrates (for example, c-Cbl, and protein kinase C δ) were Src family substrates whereas others (for example, phospholipase C-γ) were not. One protein, the adaptor Shc, was a substrate for both Src family kinases (on tyrosines 239 and 240) and a distinct tyrosine kinase (on tyrosine 317, which is perhaps phosphorylated by the PDGF receptor itself). Microinjection experiments demonstrated that a Shc molecule carrying mutations of tyrosines 239 and 240, in conjunction with an SH2 domain mutation, interfered with PDGF-stimulated DNA synthesis. Deletion of the phosphotyrosine-binding domain also inhibited synthesis. These inhibitions were overcome by heterologous expression of Myc, supporting the hypothesis that Shc functions in the Src pathway. SU6656 should prove a useful additional tool for further dissecting the role of Src kinases in this and other signal transduction pathways.

ACKNOWLEDGMENTS

We thank Tom Yu for peptide synthesis, James Rodda for assistance with PDGFR kinetic studies, all members of Screening Operations at Sugen for biochemical IC₅₀ measurements, Yossi Schlessinger for the human Shc cDNA, David Markby for Myc RPA oligos, Valerie Abbott for technical help, Chris Liang for comments on SU6656 binding, and members of Sugen's Drug Discovery Biochemistry Group (Terrence Hui, Rachael Hawtin, Deb Moshinsky, and Audie Rice) for the panel of purified GST-kinase fusion proteins.

Robert A. Blake and Martin A. Broome contributed equally to this work.