Cdc48/p97–Ufd1–Npl4 antagonizes Aurora B during chromosome segregation in HeLa cells

Abstract

SummaryDuring exit from mitosis in Xenopus laevis egg extracts, the AAA+ ATPase Cdc48/p97 (also known as VCP in vertebrates) and its adapter Ufd1-Npl4 remove the kinase Aurora B from chromatin to allow nucleus formation. Here, we show that in HeLa cells Ufd1-Npl4 already antagonizes Aurora B on chromosomes during earlier mitotic stages and that this is crucial for proper chromosome segregation. Depletion of Ufd1-Npl4 by small interfering RNA (siRNA) caused chromosome alignment and anaphase defects resulting in missegregated chromosomes and multi-lobed nuclei. Ufd1-Npl4 depletion also led to increased levels of Aurora B on prometaphase and metaphase chromosomes. This increase was associated with higher Aurora B activity, as evidenced by the partial resistance of CENP-A phosphorylation to the Aurora B inhibitor hesperadin. Furthermore, low concentrations of hesperadin partially rescued chromosome alignment in Ufd1-depleted cells, whereas, conversely, Ufd1-depletion partially restored congression in the presence of hesperadin. These data establish Cdc48/p97-Ufd1-Npl4 as a crucial negative regulator of Aurora B early in mitosis of human somatic cells and suggest that the activity of Aurora B on chromosomes needs to be restrained to ensure faithful chromosome segregation.

Proliferating Cell Nuclear Antigen (PCNA)-binding Protein C1orf124 Is a Regulator of Translesion Synthesis

“…5, A and B). Studies have shown that VCP/p97 functions in regulating DNA metabolic processes by mediating proteasomal degradation or catalyzing the extraction of proteins or protein complexes from the chromatin (35,38). On the basis of this information and the data presented in our study, we propose that C1orf124 may function to stabilize Ub-PCNA and RAD18 on DNA damage sites by preventing their removal or extraction from the chromatin by VCP/p97 during TLS (Fig.…”

“…Recent studies imply that VCP acts by extracting protein complexes bound to chromatin rather than promoting protein degradation. For example, Aurora B has been shown to be extracted from mitotic chromosomes by VCP/p97 and its cofactors (35). Furthermore, VCP/p97 regulates DNA replication by mediating the removal of replication licensing factor Cdt1 that is bound to PCNA (36).…”

Distinct conformations of the protein complex p97-Ufd1-Npl4 revealed by electron cryomicroscopy

“…The emerging consensus is that p97 binds a small number of cofactors forming "core" complexes, one of which is Ufd1-Npl4. Under certain cellular conditions additional adaptors either recruit or bind to p97 core complexes (17). The p97-Ufd1-Npl4 structures presented here begin to explain how additional p97 adaptors could bind to a p97-Ufd1-Npl4 core complex.…”

“…There has been much speculation as to how p97 can interact with so many different adaptor proteins that contain common p97 interaction domains (17,38,44). The emerging consensus is that p97 binds a small number of cofactors forming "core" complexes, one of which is Ufd1-Npl4.…”

“…One of the best-studied adaptors is the heterodimer Ufd1-Npl4, the only essential cofactor in yeast (3)(4)(5)(6), which directs p97 into cellular processes regulated by ubiquitin-proteasome degradation such as endoplasmic reticulum-associated degradation (ERAD) (7)(8)(9)(10), mitotic progression (11,12), replication (13), and transcription factor activation (14,15). In ERAD (16), mitosis (17), and nucleus reformation (12), the p97-Ufd1-Npl4 complex has been shown to recognize ubiquitylated substrates associated with different cellular structures and, with the energy obtained from the binding and/or hydrolysis of ATP, to extract them into the cytosol to be recycled after deubiquitylation or degraded by the proteasome (10,16,18,19). However, it remains unclear how the ubiquitin-binding domains in p97-Ufd1-Npl4 are positioned to recognize the substrate and how the conformational changes in p97 are translated into mechanic force acting on substrate molecules.…”