Identification Of Ubiquitin Target Proteins Using Cell-based Arrays

Ubiquitylation emerges as a signal involved in the regulation of a variety of biological processes. A global understanding of ubiquitinated proteins in vivo is key to unraveling the biological significance of ubiquitination. There are, however, a few effective screening methods for rapid analysis of ubiquitinated proteins. In the current study, we designed a cell-based cDNA expression array combined with cell imaging for the rapid identification of polyubiquitinated proteins, which normally accumulate to form the unique "dot" structure following inhibition of ubiquitin proteasomes. The array consisted of 112 cDNAs encoding key components of major cellular pathways and potential targets of polyubiquitination. Among them, 40 proteins formed accumulation dots in response to Proteasome inhibitor, MG-132, treatment. It suggested that they were routinely regulated through ubiquitin-proteasomes. More importantly, 24 of those 40 proteins, such as MAPKAPK3, NLK and RhoGDI2, are previously not known as the targets of ubiquitin. We further validated our findings by examining the endogenous counterparts of some of these proteins and found that those endogenous proteins form the similar "dot" structure. Immunoprecipitation assays confirmed that these accumulated proteins are polyubiquitinated. Some proteins requiring additional stimuli for their polyubiquitylation, such as signal of phosphorylation stimulated by cytokines, were negatively screened on this array.Our results demonstrate that this large-scale application of cell-based arrays represents a novel global approach in identifying candidates of the polyubiquitinated proteins. This live-imaging technique as well as other proteomics approaches to visualize ubiquitin target networks in vivo would empower a more comprehensive analysis of cellular signaling events governed by ubiquitylation. Therefore, the technique utilized here will facilitate future research on ubiquitination-regulated cell signaling.