A flow cytometric assay for the study of E3 ubiquitin ligase activity.
Current methods for monitoring E3 ubiquitin ligase activity in cell culture or in vivo are limited. As a result, the degradation of cellular targets by many E3 ubiquitin ligases in live cells has not yet been examined. For this study, a target of an E3 ubiquitin ligase was expressed as a fluorescently labeled protein in cell culture. If the E3 ubiquitin ligase mediates the degradation of a target protein in cell culture, it is expected that the target will show a reduced fluorescence signal by FCM analysis. We initially used the E3 ubiquitin ligase, herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) and one of its targets, promyelocytic leukemia (PML) protein, to determine the feasibility of our approach. Cells expressing a PML-GFP fusion protein were selected by cell sorting and infected with an adenoviral vector expressing ICP0. In contrast to mock-infected cells, only PML-GFP-expressing cells infected with the ICP0 adenoviral vector led to a significant decrease in the fluorescence signal of PML-GFP when examined by fluorescence microscopy and FCM analysis. Our results suggest that it is possible to examine the live activity of an E3 ubiquitin ligase (via one of its targets) in cell culture by FCM analysis.
Hilliard
Joshua G
JG
Department of Molecular Biosciences, University of Kansas, Lawrence, USA.
Cooper
Anne L
AL
Slusser
Joyce G
JG
Davido
David J
DJ
D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D000071156:Promyelocytic Leukemia Protein; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human; D049452:Green Fluorescent Proteins; D044767:Ubiquitin-Protein Ligases; C051804:Vmw110 protein, Human herpesvirus 1