Genome-wide ubiquitin & Ubl modification profiling in mitosis

Mammalian posttranslational modification networks have been mainly inferred from large-scale mass spectrometry (MS) and yeast two-hybrid data as well as protein microarrays. However, most methods usually lack the ability to monitor multiple cellular perturbations or Ubl modifications. Thus, we sought a strategy which will allow us to systematically perturb cellular conditions and monitor cellular post-translational modification (PTM) responses. To that end, we developed an assay to profile PTMs on a genome-wide scale using mammalian cell extracts and protein microarrays. We used mitotic arrest and release as a perturbation and explored the changes in poly-ubiquitination upon mitotic release. In our initial study we looked for known and putative substrates of the anaphase-promoting complex (APC). Using our assay we were able to identify 11 out of 16 known substrates as well as additional novel substrates, some of which we have validated using traditional assay of protein degradation in mitotic extracts.;We then expanded our study to other Ubls (SUMO1, SUMO2/3, NEDD8, FAT10, UFM1 and ISG15) and mapped their reactivity profiles upon release of the mitotic checkpoint. The Ubl network architecture suggested that: 1) our in vitro system exhibited specificity in Ubl-substrate interactions and 2) non-random and highly independent target specificities have evolved for these parallel Ubl pathways. Lastly, we have tried to identify Ubl substrates that are differentially regulated upon release of the mitotic checkpoint. Among the seven Ubl tested, FAT10 and ubiquitin were the two Ubls that showed the most significant changes. The vast decrease in FAT10 substrate reactivities upon release of the mitotic checkpoint lead us to investigate the possible role of the pathway in mitotic regulation. We found that blocking FAT10ylation caused a prolonged arrest in mitosis followed by cell death. Additionally, we found that the FATlOylation E2-conjugating enzyme expression is regulated in the cell cycle and degrades at the end of mitosis in an APC-dependent degradation.;The work described herein can provide a direct and broadly-applicable systematic approach to study PTMs in mammalian cells under different cellular condition and should ultimately lead to better understanding of cellular PTM regulation.