Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities.
The spindle checkpoint prevents chromosome mis-segregation by delaying sister chromatid separation until all chromosomes have achieved bipolar attachment to the mitotic spindle. Its operation is essential for accurate chromosome segregation, whereas its dysregulation can contribute to birth defects and tumorigenesis. The target of the spindle checkpoint is the anaphase-promoting complex (APC), a ubiquitin ligase that promotes sister chromatid separation and progression to anaphase. Using a short hairpin RNA screen targeting components of the ubiquitin-proteasome pathway in human cells, we identified the deubiquitinating enzyme USP44 (ubiquitin-specific protease 44) as a critical regulator of the spindle checkpoint. USP44 is not required for the initial recognition of unattached kinetochores and the subsequent recruitment of checkpoint components. Instead, it prevents the premature activation of the APC by stabilizing the APC-inhibitory Mad2-Cdc20 complex. USP44 deubiquitinates the APC coactivator Cdc20 both in vitro and in vivo, and thereby directly counteracts the APC-driven disassembly of Mad2-Cdc20 complexes (discussed in an accompanying paper). Our findings suggest that a dynamic balance of ubiquitination by the APC and deubiquitination by USP44 contributes to the generation of the switch-like transition controlling anaphase entry, analogous to the way that phosphorylation and dephosphorylation of Cdk1 by Wee1 and Cdc25 controls entry into mitosis.
Stegmeier
Frank
F
Howard Hughes Medical Institute, Department of Genetics, Harvard Partners Center for Genetics and Genomics,Boston, Massachusetts 02115, USA.
Rape
Michael
M
Draviam
Viji M
VM
Nalepa
Grzegorz
G
Sowa
Mathew E
ME
Ang
Xiaolu L
XL
McDonald
E Robert
ER
Li
Mamie Z
MZ
Hannon
Gregory J
GJ
Sorger
Peter K
PK
Kirschner
Marc W
MW
Harper
J Wade
JW
Elledge
Stephen J
SJ
D000705:Anaphase / Q000187:drug effects / Q000502:physiology*; D064173:Anaphase-Promoting Complex-Cyclosome; D002135:Calcium-Binding Proteins / Q000378:metabolism; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D010450:Endopeptidases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000187:drug effects / Q000378:metabolism; D064246:Mad2 Proteins; D017239:Paclitaxel / Q000494:pharmacology; D012097:Repressor Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism; D064570:Ubiquitin-Specific Proteases
D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D012097:Repressor Proteins; D025801:Ubiquitin; C577488:CDC20 protein, human; C105156:UBE2C protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D010450:Endopeptidases; D064570:Ubiquitin-Specific Proteases; D017239:Paclitaxel