Small ubiquitin-related modifiers (SUMO) are evolutionarily conserved ubiquitin-like proteins that regulate several cellular processes including cell cycle progression, intracellular trafficking, protein degradation and apoptosis. Despite the importance of protein SUMOylation in different biological pathways, the global identification of acceptor sites in complex cell extracts remains a challenge. Here we generate a monoclonal antibody that enriches for peptides containing SUMO remnant chains following tryptic digestion. We identify 954 SUMO3-modified lysine residues on 538 proteins and profile by quantitative proteomics the dynamic changes of protein SUMOylation following proteasome inhibition. More than 86% of these SUMOylation sites have not been reported previously, including 5 sites on the tumour suppressor parafibromin (CDC73). The modification of CDC73 at K136 affects its nuclear retention within PML nuclear bodies on proteasome inhibition. In contrast, a CDC73 K136R mutant translocates to the cytoplasm under the same conditions, further demonstrating the effectiveness of our method to characterize the dynamics of lysine SUMOylation.
Tyrosine phosphorylation is closely associated with cell proliferation. During the cell cycle, serine and threonine phosphorylation plays the leading role, and such phosphorylation events are most dynamic during the mitotic phase of the cell cycle. However, mitotic phosphotyrosine is not well characterized. Although a few functionally-relevant mitotic phosphotyrosine sites have been characterized, evidence suggests that this modification may be more prevalent than previously appreciated. Here, we examined tyrosine phosphorylation in mitotic human cells including those on spindle-associated proteins.? Database mining confirmed ~2000 mitotic phosphotyrosine sites, and network analysis revealed a number of subnetworks that were enriched in tyrosine-phosphorylated proteins, including components of the kinetochore or spindle and SRC family kinases. We identified Polo-like kinase 1 (PLK1), a major signaling hub in the spindle subnetwork, as phosphorylated at the conserved Tyr in the kinase domain. Substitution of Tyr with a phosphomimetic residue eliminated PLK1 activity in vitro and in cells. Further analysis showed that Tyr phosphorylation reduced the phosphorylation of Thr in the activation loop, a phosphorylation event necessary for PLK1 activity. Our data indicate that mitotic tyrosine phosphorylation regulated a key serine/threonine kinase hub in mitotic cells and suggested that spatially separating tyrosine phosphorylation events can reveal previously unrecognized regulatory events and complexes associated with specific structures of the cell cycle.
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