A flow cytometric assay for the study of E3 ubiquitin ligase activity

Abstract

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. ' 2009 International Society for Advancement of Cytometry Key termsGFP; herpes simplex virus type 1; infected cell protein 0; E3 ubiquitin ligase; promyelocytic leukemia; FACS EXAMINING the E3 ubiquitin (Ub) ligase activity in live mammalian cells has proven challenging, as most methods to examine this activity result in the loss of cell viability. Thus, there is a need to develop a simple and sensitive assay to monitor and quantify this activity in real time. E3 ubiquitin ligases are cellular and pathogenic proteins that play pivotal roles in diverse cellular processes [Reviewed in (1)]. Typically, E3 Ub ligases are components of distinct biochemical pathways that attach and polymerize Ub (a 76 amino acid post-translational modification) to target proteins, marking them for degradation by the proteasome. Genetic, cell biology, and biochemical studies have demonstrated that E3 Ub ligases play key roles in controlling cellular events such as proliferation, apoptosis, and viral replication. The process of ubiquitination requires several proteins that must sequentially coordinate their activities in a specific pathway. Ubiquitination begins when an E1 Ub activating enzyme (Uba) forms a bond with Ub and then transfers Ub to an E2 Ub conjugating enzyme (Ubc). In most instances, interaction of the E2 Ubc with an E3 Ub ligase is required to target specific proteins for degradation. E3 Ub ligase proteins or complexes recognize target proteins and catalyze the transfer of Ub from the E2 Ubcs to form polymers on these targets. These polyubiquitinated proteins are recognized by the proteasome and consequently degraded.One E3 Ub ligase that has been recently characterized is herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0). ICP0 is expressed directly after viral infection (2,...

Two Overlapping Regions within the N-Terminal Half of the Herpes Simplex Virus 1 E3 Ubiquitin Ligase ICP0 Facilitate the Degradation and Dissociation of PML and Dissociation of Sp100 from ND10

“…examined exogenous PML-GFP fluorescence in a human embryonic cell line by flow cytometry, a method successfully used by our laboratory to monitor ICP0's E3 ubiquitin ligase activity (40). To examine and quantify the loss of PML-GFP fluorescence induced by WT HSV-1 (strain KOS) and each ICP0 mutant virus, cells were mock infected or infected with KOS or each ICP0 mutant, and the fluorescence signal of each sample was examined at 6 and 12 hpi ( Fig.…”

“…HEL-299 (human embryonic lung) cells and Vero (African green monkey kidney) cells were maintained at 37°C in 5% CO 2 and cultured in either alpha minimum essential medium (␣MEM) for HEL-299 cells or Dulbecco's modified Eagle medium (DMEM) for Vero cells, supplemented with penicillin (100 U/ml), streptomycin (100 g/ ml), and 2 mM L-glutamine and 10% or 5% fetal bovine serum (FBS). A human embryonic lung-derived cell line, WI-38ϩPML-green fluorescent protein (GFP) cells, and L7 cells (Vero cells stably transformed with the ICP0 gene) were grown and maintained as previously described (40,41). Wild-type (WT) HSV-1 (strain KOS), strain 7134 (an ICP0 null mutant), and ICP0 truncation mutants (n212, n428, n525, n680, and n720) were propagated as previously described (1,3).…”