title: Expression of constitutively active CDK1 stabilizes APC-Cdh1 substrates and potentiates premature spindle assembly and checkpoint function in G1 cells.
abstract: Mitotic progression in eukaryotic cells depends upon the activation of cyclin-dependent kinase 1 (CDK1), followed by its inactivation through the anaphase-promoting complex (APC)/cyclosome-mediated degradation of M-phase cyclins. Previous work revealed that expression of a constitutively active CDK1 (CDK1AF) in HeLa cells permitted their division, but yielded G1 daughter cells that underwent premature S-phase and early mitotic events. While CDK1AF was found to impede the sustained activity of APC-Cdh1, it was unknown if this defect improperly stabilized mitotic substrates and contributed to the occurrence of these premature M phases. Here, we show that CDK1AF expression in HeLa cells improperly stabilized APC-Cdh1 substrates in G1-phase daughter cells, including mitotic kinases and the APC adaptor, Cdc20. Division of CDK1AF-expressing cells produced G1 daughters with an accelerated S-phase onset, interrupted by the formation of premature bipolar spindles capable of spindle assembly checkpoint function. Further characterization of these phenotypes induced by CDK1AF expression revealed that this early spindle formation depended upon premature CDK1 and Aurora B activities, and their inhibition induced rapid spindle disassembly. Following its normal M-phase degradation, we found that the absence of Wee1 in these prematurely cycling daughter cells permitted the endogenous CDK1 to contribute to these premature mitotic events, since expression of a non-degradable Wee1 reduced the number of cells that exhibited premature cyclin B1oscillations. Lastly, we discovered that Cdh1-ablated cells could not be forced into a premature M phase, despite cyclin B1 overexpression and proteasome inhibition. Together, these results demonstrate that expression of constitutively active CDK1AF hampers the destruction of critical APC-Cdh1 targets, and that this type of condition could prevent newly divided cells from properly maintaining a prolonged interphase state. We propose that this more subtle type of defect in activity of the APC-driven negative-feedback loop may have implications for triggering genome instability and tumorigenesis.
authors.affiliation: Department of Biology, Indiana University, Bloomington, Indiana, United States of America.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ma
authors.forename: Xi
authors.initials: X
authors.lastname: Yuan
authors.forename: William R
authors.initials: WR
authors.lastname: Wyatt
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Pomerening
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome; D064107:Aurora Kinase B; D064127:Aurora Kinases; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly; D056744:Cyclin B1 / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004795:Enzyme Stability; D059585:G1 Phase Cell Cycle Checkpoints / Q000502:physiology*; D015870:Gene Expression; D020868:Gene Silencing; D042822:Genomic Instability; D006367:HeLa Cells; D006801:Humans; D059566:M Phase Cell Cycle Checkpoints; D064246:Mad2 Proteins; D008938:Mitosis; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D061988:Proteasome Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*
chemical_list: D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D056744:Cyclin B1; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D061988:Proteasome Inhibitors; D012097:Repressor Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; C576921:AURKB protein, human; D064107:Aurora Kinase B; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase