Abstract
Non-receptor protein-tyrosine kinases regulate cellular responses to many external signals and are important drug discovery targets for cancer and infectious diseases. While many assays exist for the assessment of kinase activity in vitro, methods that report changes in tyrosine kinase activity in single cells have the potential to provide information about kinase responses at the cell population level. In this protocol, we combined bimolecular fluorescence complementation (BiFC), an established method for the assessment of protein-protein interactions, and immunofluorescence staining with phosphospecific antibodies to characterize changes in host cell tyrosine kinase activity in the presence of an HIV-1 virulence factor, Nef. Specifically, two Tec family kinases (Itk and Btk) as well as Nef were fused to complementary, non-fluorescent fragments of the Venus variant of YFP. Each kinase was expressed in 293T cells in the presence or absence of Nef and immunostained for protein expression and activity with anti-phosphotyrosine (pTyr) antibodies. Multi-color confocal microscopy revealed the interaction of Nef with each kinase (BiFC), kinase activity, and kinase protein expression. Strong BiFC signals were observed when Nef was co-expressed with both Itk and Btk, indicative of interaction, and a strong anti-pTyr immunoreactivity was also seen. The BiFC, pTyr, and kinase expression signals co-localized to the plasma membrane, consistent with Nef-mediated kinase activation in this subcellular compartment. Image analysis allowed calculation of pTyr-to-kinase protein ratios, which showed a range of responses in individual cells across the population that shifted upward in the presence of Nef and back down in the presence of a kinase inhibitor. This method has the potential to reveal changes in steady-state non-receptor tyrosine kinase activity and subcellular localization in a cell population in response to other protein-kinase interactions, information that is not attainable from immunoblotting or other in vitro methods.