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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. 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 a similar "dot" structure. Immunoprecipitation assays confirmed that these accumulated proteins are polyubiquitinated. Our results demonstrate that this large-scale application of cell-based arrays represents a novel global approach in identifying candidates of the polyubiquitinated proteins. Therefore, the technique utilized here will facilitate future research on ubiquitination-regulated cell signaling.

We implemented a proteomics approach to the systematical analysis of the alterations in the proteome of NIH3T3 cells transformed by oncogenic H-RasV12. Forty-four proteins associated with Ras-mediated transformation have been identified, and 28 proteins were not previously reported. RT-PCR analysis showed that approximately 44% of target proteins identified showed concomitant changes in mRNA abundance. A principal finding was the up-regulation of gankyrin, which was the first evidence to show that gankyrin pathway was implicated in Ras-activated transformation.