Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation.
ABSTRACT Eukaryotic cells rely on a surveillance mechanism known as the spindle checkpoint to ensure accurate chromosome segregation. The spindle checkpoint prevents sister chromatids from separating until all kinetochores achieve bipolar attachments to the mitotic spindle. Checkpoint proteins tightly inhibit the anaphase-promoting complex (APC), a ubiquitin ligase required for chromosome segregation and progression to anaphase. Unattached kinetochores promote the binding of checkpoint proteins Mad2 and BubR1 to the APC-activator Cdc20, rendering it unable to activate APC. Once all kinetochores are properly attached, however, cells inactivate the checkpoint within minutes, allowing for the rapid and synchronous segregation of chromosomes. How cells switch from strong APC inhibition before kinetochore attachment to rapid APC activation once attachment is complete remains a mystery. Here we show that checkpoint inactivation is an energy-consuming process involving APC-dependent multi-ubiquitination. Multi-ubiquitination by APC leads to the dissociation of Mad2 and BubR1 from Cdc20, a process that is reversed by a Cdc20-directed de-ubiquitinating enzyme. The mutual regulation between checkpoint proteins and APC leaves the cell poised for rapid checkpoint inactivation and ensures that chromosome segregation promptly follows the completion of kinetochore attachment. In addition, our results suggest a mechanistic basis for how cancer cells can have a compromised spindle checkpoint without corresponding mutations in checkpoint genes.
- SourceAvailable from: Laura A Díaz-Martínez[Show abstract] [Hide abstract]
ABSTRACT: The antimitotic anti-cancer drugs, including taxol, perturb spindle dynamics, and induce prolonged, spindle checkpoint-dependent mitotic arrest in cancer cells. These cells then either undergo apoptosis triggered by the intrinsic mitochondrial pathway or exit mitosis without proper cell division in an adaptation pathway. Using a genome-wide small interfering RNA (siRNA) screen in taxol-treated HeLa cells, we systematically identify components of the mitotic apoptosis and adaptation pathways. We show that the Mad2 inhibitor p31comet actively promotes mitotic adaptation through cyclin B1 degradation and has a minor separate function in suppressing apoptosis. Conversely, the pro-apoptotic Bcl2 family member, Noxa, is a critical initiator of mitotic cell death. Unexpectedly, the upstream components of the mitochondrial apoptosis pathway and the mitochondrial fission protein Drp1 contribute to mitotic adaption. Our results reveal crosstalk between the apoptosis and adaptation pathways during mitotic arrest.The EMBO Journal 07/2014; DOI:10.15252/embj.201487826 · 10.75 Impact Factor
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ABSTRACT: The ubiquitin-conjugating enzymes 2C (UBE2C) is an integral component of the ubiquitin proteasome system. UBE2C consists of a conserved core domain containing the catalytic Cys residue and an N-terminal extension. The core domain is required for ubiquitin adduct formation by interacting with the ubiquitin-fold domain in the E1 enzyme, and contributes to the E3 enzyme binding. UBE2C N-terminal extension regulates E3 enzyme activity as a part of an intrinsic inhibitory mechanism. UBE2C is required for the destruction of mitotic cyclins and securin, which are essential for spindle assembly checkpoint and mitotic exit. The UBE2C mRNA and/or protein levels are aberrantly increased in many cancer types with poor clinical outcomes. Accumulation of UBE2C stimulates cell proliferation and anchorage-independent growth. UBE2C transgenic mice are prone to develop spontaneous tumors and carcinogen-induced tumor with evidence of chromosome aneuploidy.The international journal of biochemistry & cell biology 12/2013; DOI:10.1016/j.biocel.2013.11.023 · 4.24 Impact Factor
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ABSTRACT: The anaphase-promoting complex or cyclosome (APC/C) is a conserved, multisubunit E3 ubiquitin (Ub) ligase that is active both in dividing and in postmitotic cells. Its contributions to life are especially well studied in the domain of cell division, in which the APC/C lies at the epicenter of a regulatory network that controls the directionality and timing of cell cycle events. Biochemical and structural work is shedding light on the overall organization of APC/C subunits and on the mechanism of substrate recognition and Ub chain initiation and extension as well as on the molecular mechanisms of a checkpoint that seizes control of APC/C activity during mitosis. Here, we review how these recent advancements are modifying our understanding of the APC/C.The Journal of Cell Biology 04/2013; 201(2):177-89. DOI:10.1083/jcb.201301130 · 9.69 Impact Factor