DNA Damage Modulates Nucleolar Interaction of the Werner Protein with the AAA ATPase p97/VCP

Abstract

We report a novel nucleolar interaction between the AAA ATPase p97/VCP and the Werner protein (WRNp), a member of the RecQ helicase family. p97/VCP mediates several important cellular functions in eucaryotic cells, including membrane fusion of the endoplasmic reticulum and Golgi and ubiquitin-dependent protein degradation. Mutations in the WRN gene cause Werner syndrome, a genetic disorder characterized by premature onset of aging symptoms, a higher incidence of cancer, and a high susceptibility to DNA damage caused by topoisomerase inhibitors. We observed that both WRNp and valosin-containing protein (VCP) were present in the nucleoplasm and in nucleolar foci in mammalian cells and that WRNp and p97/VCP physically interacted in the nucleoli. Importantly, the nucleolar WRNp/VCP complex was dissociated by treatment with camptothecin, an inhibitor of topoisomerase I, whereas other WRNp-associated protein complexes, such as WRNp/Ku 80, were not dissociated by this drug. Because WRN syndrome cells are sensitive to topoisomerase inhibitors, these observations suggest that the VCP/WRNp interaction plays an important role in WRN biology. We propose a novel role for VCP in the DNA damage response pathway through modulation of WRNp availability. INTRODUCTIONThe ATPases associated with diverse cellular activities (AAA) proteins are a common family of Mg 2ϩ -dependent ATPases that contain one or two conserved ATP-binding domains (Ogura and Wilkinson, 2001). These domains, or AAA cassettes , consist of a conserved sequence of 230-amino acid residues that include the Walker A and B motifs. Another sequence-conserved domain, the second region of homology, is found in part of the AAA cassette (Confalonieri and Duguet, 1995) but is not present in the closely related AAAϩ family (Neuwald et al., 1999) of proteins. Cdc48p, p97, and valosin-containing protein (VCP) are 92-to 97-kDa orthologous members of a subfamily of AAA ATPases originally defined in yeast, Xenopus, and mammals, respectively (Moir et al., 1982;Peters et al., 1990;Frohlich et al., 1991). This subfamily has a known propensity to oligomerize and form homohexamers (Peters et al., 1992) that are a part of multiprotein complexes (Ogura and Wilkinson, 2001).Cdc48p/p97/VCP is involved in two major and distinct cellular pathways: homotypic membrane fusion of endoplasmic reticulum (ER) and Golgi fragments and ubiquitindependent protein degradation. This versatility is achieved through different sets of adaptor proteins . In ER membrane fusion, Cdc48p/p97/VCP is complexed with Ufe1p and Shp1p (Latterich et al., 1995;Lin et al., 2001) or with their vertebrate orthologs p47 and syntaxin 5 in Golgi membrane fusion (Acharya et al., 1995;Rabouille et al., 1995). It is likely that the role of Cdc48p in ER membrane fusion is to remove a fusion inhibitor, thus permitting membrane fusion to occur (Lin et al., 2001). Cdc48p/p97/VCP also participates in the degradation of ubiquitinated proteins in yeast via Ufd1p and Npl4p and in mammals (Ghislain et al., 1996;Dai et al., 1998;Meyer ...

Akt-mediated Valosin-containing Protein 97 Phosphorylation Regulates Its Association with Ubiquitinated Proteins

“…Several lines of evidence suggest that binding of alternative cofactors determines the specific role of VCP. Thus far, a number of VCP cofactors have been identified and regulate various VCP-mediated functions (35)(36)(37)(38)(39). Here we identify VCP as an Akt-binding protein and as an Akt substrate, suggesting a role for Akt in the regulation of VCP function.…”

Cell cycle progression requires the CDC-48 UFD−1/NPL−4 complex for efficient DNA replication

“…At present it is unclear how CDC-48 affects replication but recent studies reported that p97 binds to DNA replication factors (31,32). Interestingly, p97 associates with the human Werner RecQ helicase (33) and C. elegans CDC-48.1 with HIM-6, a Bloom Syndrome helicase homolog (34). As both RecQ helicases are involved in DNA replication to prevent accelerated aging, it is conceivable to speculate about an evolutionarily conserved requirement of CDC-48/p97 in DNA synthesis linked to cancer and longevity.…”

Age‐specific hormonal decline is accompanied by transcriptional changes in human sebocytes in vitro

“…VCP has been implicated in a number of cellular events that are regulated during mitosis, including homotypic membrane fusion, spindle polar body function, and ubiquitindependent protein degradation. A proposed novel role for VCP is in the DNA damage response pathway through modulation of Werner protein (WRNp) availability in the nucleolus (Partridge et al, 2003). TREX1 is also involved in DNA repair mechanisms.…”