Development of a microscopy-based assay for protein kinase Cζ activation in human breast cancer cells

Abstract Protein kinase Cζ (PKCζ) plays a critical role in cancer cell chemotaxis. Upon activation induced by epidermal growth factor (EGF) or chemoattractant SDF-1α, PKCζ redistributes from cytosol to plasma membrane. Based on this property, we developed a rapid cell-based assay for inhibitors of ligand-induced PKCζ activation. PKCζ green fluorescent protein (GFP) was transfected into human breast cancer cells, MDA-MB-231, to establish a stable cell line, PKCζ-GFP/MDA-MB-231. PKCζ-GFP/MDA-MB-231 maintained phenotypes, such as chemotaxis, adhesion, and cell migration, similar to those of its parental cell line. Therefore it could be used as a representative cancer cell line. EGF induced translocation of PKCζ-GFP to plasma membrane in a pattern similar to that of endogenous PKCζ, indicative of activation of PKCζ Translocation of PKCζ-GFP could be easily and directly recorded by an inverted fluorescence microscope. Inhibitors of chemotaxis also impaired the translocation of PKCζ-GFP, which further validated the biological relevance of our assay. Taken together, we have developed a simple, rapid, and reliable assay to detect the ligand-induced activation of PKCζ in human cancer cells. This assay can be used in screening for inhibitors of PKCζ activation, which is critically required for cancer cell chemotaxis.