Development of a microscopy-based assay for protein kinase Czeta activation in human breast cancer cells.
Protein kinase Czeta (PKCzeta) plays a critical role in cancer cell chemotaxis. Upon activation induced by epidermal growth factor (EGF) or chemoattractant SDF-1alpha, PKCzeta redistributes from cytosol to plasma membrane. Based on this property, we developed a rapid cell-based assay for inhibitors of ligand-induced PKCzeta activation. PKCzeta green fluorescent protein (GFP) was transfected into human breast cancer cells, MDA-MB-231, to establish a stable cell line, PKCzeta-GFP/MDA-MB-231. PKCzeta-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 PKCzeta-GFP to plasma membrane in a pattern similar to that of endogenous PKCzeta, indicative of activation of PKCzeta Translocation of PKCzeta-GFP could be easily and directly recorded by an inverted fluorescence microscope. Inhibitors of chemotaxis also impaired the translocation of PKCzeta-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 PKCzeta in human cancer cells. This assay can be used in screening for inhibitors of PKCzeta activation, which is critically required for cancer cell chemotaxis.
Zhao
Caijie
C
Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology and State Key Laboratory of Molecular Dynamic and Stable Structures, College of Chemistry, Peking University, Beijing 100871, China.
Cai
Mi
M
Zhang
Yao
Y
Liu
Ying
Y
Sun
Ronghua
R
Zhang
Ning
N
D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002448:Cell Adhesion / Q000502:physiology; D045744:Cell Line, Tumor; D002633:Chemotaxis / Q000502:physiology; D004789:Enzyme Activation; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000099221:Protein Kinase C zeta
D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D011993:Recombinant Fusion Proteins; D000099221:Protein Kinase C zeta