Yeast growth selection system for detecting activity and inhibition of dimerization-dependent receptor tyrosine kinase

Abstract

Receptor tyrosine kinases (RTKs) play an important role in the control of fundamental cellular processes, including cell proliferation, migration, differentiation, and survival. Deregulated RTK signaling is critically involved in the development and progression of human cancer. Here, we present an assay for monitoring RTK activities in yeast, which provides an ideal heterologous cellular system to study these mammalian proteins in a null background environment. With our system, we have reconstituted aspects of the epidermal growth factor receptor (EGFR) signaling pathway as a model. Our approach is based on the Ras-recruitment system, in which membrane localization of a constitutively active human Ras achieved through protein-protein interactions can rescue growth of a temperature-sensitive yeast strain (cdc25-2). We show that co-expression of a dimerizing membrane-bound EGFR variant with specific adaptor proteins fused to the active Ras rescues growth of the cdc25-2 mutant yeast strain at the nonpermissive temperature. Using kinase-defective RTK mutants and selective EGFR kinase inhibitors, we demonstrate that growth rate of this yeast strain correlates with kinase activity of the EGFR derivatives. The RTK cellular assay presented here can be applied in high-throughput screens for selecting RTK-specific inhibitors that must be able to permeate the membrane and to function in an eukaryotic intrecellular environment.