Lek Associates with and Is Activated by Kit in a Small Cell Lung Cancer Cell Line: Inhibition of SCF-mediated Growth by the Src Family Kinase Inhibitor PPI1

At least 70% of small cell lung cancers (SCLCs) express the Kit receptor tyrosine kinase and its ligand, stem cell factor (SCF). In an effort to define the signal transduction pathways activated In Kit in SCLC, we focused on Src family kinases and, in particular. Lek, a Src-related tyrosine kinase that is expressed in hemopoietic cells and certain tumors, including SCLC. SCF treatment of the H526 cell line induced a physical association between Kit and Lek that, in vitro, was dependent on phosphorylation of the juxtamemhrane domain of Kit. Stimulation of Kit with recombinant SCF resulted in a rapid 3-6-fold increase in the specific activity of Lek. which was similar in magnitude to the activation of Lek resulting from the cross-linking of the T-cell receptor complex of Jurkat cells. Lek activity peaked by 5 min after SCF addition, and the elevated activity persisted for at least 30 min in the presence of SCF, with kinetics similar to the activation of mitogen-activated protein kinase. PP1, an inhibitor of Src family kinases with selectivity for Lek, completely inhib ited SCF-mediated growth but had little effect on insulin-like growth factor-I-mediated growth. PPI antagonized both SCF-mediated prolifer ation and inhibition of apoptosis. PPI had no effect on Kit kinase activity but was shown to block total Lek activity by at least 90% by immune complex kinase assay. Low levels of Src, Hck, and Yes were also expressed in the H526 cell line; only Yes showed a consistent increase in specific activity, which was also inhibited by PPI following SCF treatment. These data demonstrate that, in the H526 SCLC cell line. Lek and, possibly. Yes are downstream of Kit in a signal transduction pathway; the inhibition by PPI of SCF-mediated proliferation and inhibition of apoptosis suggests that Src family kinases are intermediates in the signaling pathways that regulate these processes.