ABSTRACT
The anaphase-promoting complex/cyclosome (APC/C) and the spindle assembly checkpoint (SAC), which inhibits the APC/C, are essential determinants of mitotic timing and faithful division of genetic material. Activation of the APC/C is known to depend on two APC/C-interacting E2 ubiquitin-conjugating enzymes-UBE2C and UBE2S. We show that APC/C activity in human cells is tuned by the combinatorial use of three E2s, namely UBE2C, UBE2S, and UBE2D. Genetic deletion of UBE2C and UBE2S, individually or in combination, leads to discriminative reduction in APC/C function and sensitizes cells to UBE2D depletion. Reduction of APC/C activity results in loss of switch-like metaphase-to-anaphase transition and, strikingly, renders cells insensitive to chemical inhibition of MPS1 and genetic ablation of MAD2, both of which are essential for the SAC. These results provide insights into the regulation of APC/C activity and demonstrate that the essentiality of the SAC is imposed by the strength of the APC/C.
Subject(s)
Anaphase-Promoting Complex-Cyclosome/genetics , Mad2 Proteins/genetics , Spindle Apparatus/metabolism , Ubiquitin-Conjugating Enzymes/genetics , Anaphase/drug effects , Anaphase-Promoting Complex-Cyclosome/antagonists & inhibitors , Anaphase-Promoting Complex-Cyclosome/metabolism , CRISPR-Cas Systems , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Survival/drug effects , Gene Deletion , Gene Expression , HCT116 Cells , Humans , M Phase Cell Cycle Checkpoints , Mad2 Proteins/deficiency , Metaphase/drug effects , Morpholines/pharmacology , Nocodazole/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Purines/pharmacology , Signal Transduction , Spindle Apparatus/drug effects , Spindle Apparatus/ultrastructure , Ubiquitin-Conjugating Enzymes/deficiency , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
During mitosis large alterations in cellular structures occur rapidly, which to a large extent is regulated by post-translational modification of proteins. Modification of proteins with the small ubiquitin-related protein SUMO2/3 regulates mitotic progression, but few mitotic targets have been identified so far. To deepen our understanding of SUMO2/3 during this window of the cell cycle, we undertook a comprehensive proteomic characterization of SUMO2/3 modified proteins in mitosis and upon mitotic exit. We developed an efficient tandem affinity purification strategy of SUMO2/3 modified proteins from mitotic cells. Combining this purification strategy with cell synchronization procedures and quantitative mass spectrometry allowed for the mapping of numerous novel targets and their dynamics as cells progressed out of mitosis. This identified RhoGDIα as a major SUMO2/3 modified protein, specifically during mitosis, mediated by the SUMO ligases PIAS2 and PIAS3. Our data provide a rich resource for further exploring the role of SUMO2/3 modifications in mitosis and cell cycle regulation.
