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• W2030544662 abstract "The Aurora B kinase is the enzymatic core of the chromosomal passenger complex, which is a critical regulator of mitosis. To identify novel regulators of Aurora B, we performed a genome-wide screen for suppressors of a temperature-sensitive lethal allele of the C. elegans Aurora B kinase AIR-2. This screen uncovered a member of the Afg2/Spaf subfamily of Cdc48-like AAA ATPases as an essential inhibitor of AIR-2 stability and activity. Depletion of CDC-48.3 restores viability to air-2 mutant embryos and leads to abnormally high AIR-2 levels at the late telophase/G1 transition. Furthermore, CDC-48.3 binds directly to AIR-2 and inhibits its kinase activity from metaphase through telophase. While canonical p97/Cdc48 proteins have been assigned contradictory roles in the regulation of Aurora B, our results identify a member of the Afg2/Spaf AAA ATPases as a critical in vivo inhibitor of this kinase during embryonic development. The Aurora B kinase is the enzymatic core of the chromosomal passenger complex, which is a critical regulator of mitosis. To identify novel regulators of Aurora B, we performed a genome-wide screen for suppressors of a temperature-sensitive lethal allele of the C. elegans Aurora B kinase AIR-2. This screen uncovered a member of the Afg2/Spaf subfamily of Cdc48-like AAA ATPases as an essential inhibitor of AIR-2 stability and activity. Depletion of CDC-48.3 restores viability to air-2 mutant embryos and leads to abnormally high AIR-2 levels at the late telophase/G1 transition. Furthermore, CDC-48.3 binds directly to AIR-2 and inhibits its kinase activity from metaphase through telophase. While canonical p97/Cdc48 proteins have been assigned contradictory roles in the regulation of Aurora B, our results identify a member of the Afg2/Spaf AAA ATPases as a critical in vivo inhibitor of this kinase during embryonic development. Eukaryotes have evolved complex regulatory mechanisms to ensure that the cell cycle progresses in a timely and accurate manner. Key components of these pathways are protein kinases that are critical for the proper timing of each cell cycle phase. Preeminent among these proteins are the cyclin-dependent kinases (Cdks), which upon binding to cyclins, phosphorylate numerous targets to trigger cell cycle progression (Norbury and Nurse, 1991Norbury C. Nurse P. Cyclins and cell cycle control.Curr. Biol. 1991; 1: 23-24Abstract Full Text PDF PubMed Scopus (21) Google Scholar). In addition to Cdk1/cyclin B, members of the Aurora/Ipl1 kinase family are also key regulators of mitosis (Ducat and Zheng, 2004Ducat D. Zheng Y. Aurora kinases in spindle assembly and chromosome segregation.Exp. Cell Res. 2004; 301: 60-67Crossref PubMed Scopus (154) Google Scholar, Vagnarelli and Earnshaw, 2004Vagnarelli P. Earnshaw W.C. Chromosomal passengers: the four-dimensional regulation of mitotic events.Chromosoma. 2004; 113: 211-222Crossref PubMed Scopus (277) Google Scholar). These proteins, which include Aurora A and B, are serine/threonine kinases that are essential for cell division events such as spindle assembly, chromosome segregation, and cytokinesis (Andrews et al., 2003Andrews P.D. Knatko E. Moore W.J. Swedlow J.R. Mitotic mechanics: the auroras come into view.Curr. Opin. Cell Biol. 2003; 15: 672-683Crossref PubMed Scopus (251) Google Scholar). While Aurora A localizes to mitotic centrosomes and is required for centrosome maturation and the formation of a functional bipolar mitotic spindle (Ducat and Zheng, 2004Ducat D. Zheng Y. Aurora kinases in spindle assembly and chromosome segregation.Exp. Cell Res. 2004; 301: 60-67Crossref PubMed Scopus (154) Google Scholar, Hannak et al., 2001Hannak E. Kirkham M. Hyman A.A. Oegema K. Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans.J. Cell Biol. 2001; 155: 1109-1116Crossref PubMed Scopus (349) Google Scholar, Vagnarelli and Earnshaw, 2004Vagnarelli P. Earnshaw W.C. Chromosomal passengers: the four-dimensional regulation of mitotic events.Chromosoma. 2004; 113: 211-222Crossref PubMed Scopus (277) Google Scholar), Aurora B is the catalytic core of the highly conserved chromosomal passenger complex (CPC) (Ruchaud et al., 2007Ruchaud S. Carmena M. Earnshaw W.C. The chromosomal passenger complex: one for all and all for one.Cell. 2007; 131: 230-231Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The CPC includes, in addition to Aurora B, three regulatory subunits: the inner centromeric protein (INCENP), Survivin, and Borealin/Dasra-B (Ruchaud et al., 2007Ruchaud S. Carmena M. Earnshaw W.C. The chromosomal passenger complex: one for all and all for one.Cell. 2007; 131: 230-231Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Beginning in prophase, the CPC localizes to condensing chromosomes and progressively concentrates at the inner centromere where one function is to correct improper spindle-kinetochore attachments (Vagnarelli and Earnshaw, 2004Vagnarelli P. Earnshaw W.C. Chromosomal passengers: the four-dimensional regulation of mitotic events.Chromosoma. 2004; 113: 211-222Crossref PubMed Scopus (277) Google Scholar). At the onset of anaphase, the CPC redistributes to the central spindle and cleavage furrow to regulate the completion of cytokinesis (Vagnarelli and Earnshaw, 2004Vagnarelli P. Earnshaw W.C. Chromosomal passengers: the four-dimensional regulation of mitotic events.Chromosoma. 2004; 113: 211-222Crossref PubMed Scopus (277) Google Scholar). Importantly, the other passenger proteins directly influence Aurora B localization (Ruchaud et al., 2007Ruchaud S. Carmena M. Earnshaw W.C. The chromosomal passenger complex: one for all and all for one.Cell. 2007; 131: 230-231Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, Jeyaprakash et al., 2007Jeyaprakash A.A. Klein U.R. Lindner D. Ebert J. Nigg E.A. Conti E. Structure of a Survivin-Borealin-INCENP core complex reveals how chromosomal passengers travel together.Cell. 2007; 131: 271-285Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), and phosphorylation of conserved residues in the C terminus of INCENP substantially increases Aurora B kinase activity (Bishop and Schumacher, 2002Bishop J.D. Schumacher J.M. Phosphorylation of the carboxyl terminus of inner centromere protein (INCENP) by the Aurora B Kinase stimulates Aurora B kinase activity.J. Biol. Chem. 2002; 277: 27577-27580Crossref PubMed Scopus (206) Google Scholar, Honda et al., 2003Honda R. Korner R. Nigg E.A. Exploring the functional interactions between Aurora B, INCENP, and survivin in mitosis.Mol. Biol. Cell. 2003; 14: 3325-3341Crossref PubMed Scopus (418) Google Scholar). Aurora B levels peak in early mitosis and then dramatically decline at mitotic exit. In vertebrates, this drop is mediated in part by Aurora B ubiquitination via the anaphase-promoting complex (APC/C-Cdh1), and subsequent degradation by the proteasome (Nguyen et al., 2005Nguyen H.G. Chinnappan D. Urano T. Ravid K. Mechanism of Aurora-B degradation and its dependency on intact KEN and A-boxes: identification of an aneuploidy-promoting property.Mol. Cell. Biol. 2005; 25: 4977-4992Crossref PubMed Scopus (125) Google Scholar, Stewart and Fang, 2005Stewart S. Fang G. Destruction box-dependent degradation of aurora B is mediated by the anaphase-promoting complex/cyclosome and Cdh1.Cancer Res. 2005; 65: 8730-8735Crossref PubMed Scopus (122) Google Scholar). Recent reports have linked the Cdc48/p97 AAA ATPase with the regulation of Aurora B and the chromosomal passenger complex. In one study, p97 and its cofactors Npl4 and Ufd1 copurified with Survivin isolated from Xenopus egg extracts (Vong et al., 2005Vong Q.P. Cao K. Li H.Y. Iglesias P.A. Zheng Y. Chromosome alignment and segregation regulated by ubiquitination of survivin.Science. 2005; 310: 1499-1504Crossref PubMed Scopus (192) Google Scholar). Ufd1 was shown to be required for Survivin ubiquitination, and for the localization of Survivin and Aurora B to centromeres. Conversely, the deubiquitinating enzyme hFAM was required for the disassociation of Survivin and Aurora B from anaphase chromosomes (Vong et al., 2005Vong Q.P. Cao K. Li H.Y. Iglesias P.A. Zheng Y. Chromosome alignment and segregation regulated by ubiquitination of survivin.Science. 2005; 310: 1499-1504Crossref PubMed Scopus (192) Google Scholar). Hence, this study concluded that p97/Ufd1/Npl4 is a positive regulator of the CPC, as it is required for the localization of Survivin and Aurora B to metaphase centromeres (Vong et al., 2005Vong Q.P. Cao K. Li H.Y. Iglesias P.A. Zheng Y. Chromosome alignment and segregation regulated by ubiquitination of survivin.Science. 2005; 310: 1499-1504Crossref PubMed Scopus (192) Google Scholar). Surprisingly, a recent study contradicts these findings, suggesting that p97 is required for the disassociation of Aurora B from chromosomes, which is in turn a prerequisite for nuclear envelope reformation at the end of mitosis (Ramadan et al., 2007Ramadan K. Bruderer R. Spiga F.M. Popp O. Baur T. Gotta M. Meyer H.H. Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.Nature. 2007; 450: 1258-1262Crossref PubMed Scopus (200) Google Scholar). p97 is required for mitotic spindle disassembly and nuclear envelope reformation in Xenopus egg extracts (Cao et al., 2003Cao K. Nakajima R. Meyer H.H. Zheng Y. The AAA-ATPase Cdc48/p97 regulates spindle disassembly at the end of mitosis.Cell. 2003; 115: 355-367Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, Hetzer et al., 2001Hetzer M. Meyer H.H. Walther T.C. Bilbao-Cortes D. Warren G. Mattaj I.W. Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly.Nat. Cell Biol. 2001; 3: 1086-1091Crossref PubMed Scopus (275) Google Scholar). However, inhibition or depletion of Aurora B relieved this requirement, suggesting that Aurora B is a key target of p97 in this pathway (Kelly et al., 2007Kelly A.E. Sampath S.C. Maniar T.A. Woo E.M. Chait B.T. Funabiki H. Chromosomal enrichment and activation of the aurora B pathway are coupled to spatially regulate spindle assembly.Dev. Cell. 2007; 12: 31-43Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, Ramadan et al., 2007Ramadan K. Bruderer R. Spiga F.M. Popp O. Baur T. Gotta M. Meyer H.H. Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.Nature. 2007; 450: 1258-1262Crossref PubMed Scopus (200) Google Scholar). Indeed, p97 physically interacted with ubiquitinated Aurora B and was required to extract the kinase from chromatin (Ramadan et al., 2007Ramadan K. Bruderer R. Spiga F.M. Popp O. Baur T. Gotta M. Meyer H.H. Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.Nature. 2007; 450: 1258-1262Crossref PubMed Scopus (200) Google Scholar). Chromosome release resulted in a corresponding drop in kinase activity, arguably due to dissemination of the kinase from activating clusters (Kelly et al., 2007Kelly A.E. Sampath S.C. Maniar T.A. Woo E.M. Chait B.T. Funabiki H. Chromosomal enrichment and activation of the aurora B pathway are coupled to spatially regulate spindle assembly.Dev. Cell. 2007; 12: 31-43Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Consistent findings were found upon depletion of the two Cdc48/p97 orthologs in C. elegans (Ramadan et al., 2007Ramadan K. Bruderer R. Spiga F.M. Popp O. Baur T. Gotta M. Meyer H.H. Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.Nature. 2007; 450: 1258-1262Crossref PubMed Scopus (200) Google Scholar). cdc-48.1 and cdc-48.2(RNAi) resulted in defects in chromosome decondensation and nuclear envelope reassembly, as well as the retention of the Aurora B kinase AIR-2 on anaphase chromosomes (Ramadan et al., 2007Ramadan K. Bruderer R. Spiga F.M. Popp O. Baur T. Gotta M. Meyer H.H. Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.Nature. 2007; 450: 1258-1262Crossref PubMed Scopus (200) Google Scholar). In addition, RNAi of either cdc-48.1 or cdc-48.2 partially rescued a hypomorphic temperature-sensitive (ts) allele of air-2, and resulted in an increase in the phosphorylation of histone H3, a conserved target of the Aurora B kinases (Ramadan et al., 2007Ramadan K. Bruderer R. Spiga F.M. Popp O. Baur T. Gotta M. Meyer H.H. Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.Nature. 2007; 450: 1258-1262Crossref PubMed Scopus (200) Google Scholar). The disparate conclusions reached by these studies raise a number of questions regarding the cellular pathways that control Aurora B kinase activity and functions. To elucidate the regulation of the Aurora B kinase in an unbiased fashion, we undertook a C. elegans genome-wide screen for loss-of-function suppressors of the same air-2 allele used in the study described above, air-2(or207ts) (Severson et al., 2000Severson A.F. Hamill D.R. Carter J.C. Schumacher J. Bowerman B. The aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis.Curr. Biol. 2000; 10: 1162-1171Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Although we did not recover either of the canonical CDC-48 family members in our screen, we did find, among a handful of reproducible suppressors, a member of the Afg2/Spaf subfamily of Cdc48/p97 AAA+ ATPases. K04G2.3/CDC-48.3 is closely related to yeast Afg2 and mammalian Spaf, which form a distinct subgroup of AAA+ ATPases that also includes an uncharacterized Drosophila protein (Frohlich, 2001Frohlich K.U. An AAA family tree.J. Cell Sci. 2001; 114: 1601-1602PubMed Google Scholar). In contrast to canonical Cdc48 and p97, little is known regarding the specific functions of the Afg2/Spaf proteins. The only reported function of S. cerevisiae Afg2 (ATPase family gene) is the release and recycling of nucleolar shuttling factors from pre-60S ribosomal particles (Pertschy et al., 2007Pertschy B. Saveanu C. Zisser G. Lebreton A. Tengg M. Jacquier A. Liebminger E. Nobis B. Kappel L. van der Klei I. et al.Cytoplasmic recycling of 60S preribosomal factors depends on the AAA protein Drg1.Mol. Cell. Biol. 2007; 27: 6581-6592Crossref PubMed Scopus (73) Google Scholar). Murine Spaf was first identified due to increased expression in an epidermal chemical carcinogenesis model (Liu et al., 2000Liu Y. Black J. Kisiel N. Kulesz-Martin M.F. SPAF, a new AAA-protein specific to early spermatogenesis and malignant conversion.Oncogene. 2000; 19: 1579-1588Crossref PubMed Scopus (24) Google Scholar). Spaf (spermatogenesis-associated factor) is highly expressed in testis, and is enriched in the cytoplasm of spermatagonia and early spermatocytes (Liu et al., 2000Liu Y. Black J. Kisiel N. Kulesz-Martin M.F. SPAF, a new AAA-protein specific to early spermatogenesis and malignant conversion.Oncogene. 2000; 19: 1579-1588Crossref PubMed Scopus (24) Google Scholar); however, the functional role of Spaf in the epidermis or sperm development is not known. We here report that C. elegans CDC-48.3 is an essential inhibitor of the Aurora B kinase AIR-2. In vitro, CDC-48.3 binds directly to and inhibits AIR-2 kinase activity in an ATPase-dependent manner. In vivo, CDC-48.3 inhibits AIR-2 activity from metaphase through telophase, and is required for the characteristic drop in AIR-2 expression at mitotic exit. Importantly, loss of CDC-48.3 in wild-type embryos results in mitotic spindle and chromosome segregation defects as well as significant delays in mitotic progression. In sum, these results reveal that a member of the highly conserved Afg2/SPAF subfamily of AAA ATPases is essential for timely and accurate cell division and is a critical regulator of the AIR-2 Aurora B kinase. To isolate inhibitors of the C. elegans Aurora B kinase AIR-2, a genome-wide RNAi screen for suppressors of a ts air-2 allele, air-2(or207ts)(Severson et al., 2000Severson A.F. Hamill D.R. Carter J.C. Schumacher J. Bowerman B. The aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis.Curr. Biol. 2000; 10: 1162-1171Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), was performed. The or207 mutation replaces a conserved proline (P265) within the predicted kinase domain with lysine, resulting in undetectable kinase activity in vitro (Bishop and Schumacher, 2002Bishop J.D. Schumacher J.M. Phosphorylation of the carboxyl terminus of inner centromere protein (INCENP) by the Aurora B Kinase stimulates Aurora B kinase activity.J. Biol. Chem. 2002; 277: 27577-27580Crossref PubMed Scopus (206) Google Scholar). At the permissive temperature, 15°C, air-2(or207ts) embryos are nearly 100% viable and are phenotypically indistinguishable from wild-type (wt). When shifted to restrictive temperatures, air-2(or207ts) hermaphrodites produce dead polyploid one-cell embryos with gross defects in chromosome segregation and cytokinesis, a phenotype highly reminiscent of air-2(RNAi) embryos (Schumacher et al., 1998bSchumacher J.M. Golden A. Donovan P.J. AIR-2: An Aurora/Ipl1-related protein kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caenorhabditis elegans embryos.J. Cell Biol. 1998; 143: 1635-1646Crossref PubMed Scopus (261) Google Scholar, Severson et al., 2000Severson A.F. Hamill D.R. Carter J.C. Schumacher J. Bowerman B. The aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis.Curr. Biol. 2000; 10: 1162-1171Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). To identify suppressors of air-2(or207ts) lethality, air-2(or207ts) larvae were fed E. coli transformed with an RNAi feeding library representing 86.9% of all C. elegans open reading frames (Kamath et al., 2003Kamath R.S. Fraser A.G. Dong Y. Poulin G. Durbin R. Gotta M. Kanapin A. Le Bot N. Moreno S. Sohrmann M. et al.Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.Nature. 2003; 421: 231-237Crossref PubMed Scopus (2578) Google Scholar). To optimize the number of suppressors uncovered, the screen was performed at a semipermissive temperature, 22°C, which is the lowest temperature that yields ∼100% air-2(or207ts) lethality (Figure 1A). Suppressors were identified by the presence of any surviving larvae. Fifty-seven candidate suppressors were recovered after screening the entire RNAi library, and retesting confirmed four independent and reproducible suppressors. The characterization of the strongest of these suppressors, K04G2.3, is presented here; analysis of the other three suppressors will be presented elsewhere. K04G2.3(RNAi) restored air-2(or207ts) embryonic viability to 72.3% versus 1% for controls at 20°C, and 21.3% versus 0% at 22°C. K04G2.3 encodes a homolog of the Afg2/Spaf subfamily of Cdc48-like AAA+ ATPases (see Figure S1 available online). The closest C. elegans relatives of K04G2.3 encode redundant canonical Cdc48 ATPases, CDC-48.1 and CDC-48.2 (Poteryaev et al., 2005Poteryaev D. Squirrell J.M. Campbell J.M. White J.G. Spang A. Involvement of the actin cytoskeleton and homotypic membrane fusion in ER dynamics in Caenorhabditis elegans.Mol. Biol. Cell. 2005; 16: 2139-2153Crossref PubMed Scopus (97) Google Scholar, Sasagawa et al., 2007Sasagawa Y. Yamanaka K. Nishikori S. Ogura T. Caenorhabditis elegans p97/CDC-48 is crucial for progression of meiosis I.Biochem. Biophys. Res. Commun. 2007; 358: 920-924Crossref PubMed Scopus (23) Google Scholar, Yamauchi et al., 2006Yamauchi S. Yamanaka K. Ogura T. Comparative analysis of expression of two p97 homologues in Caenorhabditis elegans.Biochem. Biophys. Res. Commun. 2006; 345: 746-753Crossref PubMed Scopus (17) Google Scholar). Since the K04G2.3 gene product is closely related to these proteins, we named this gene cdc-48.3 (accession number: NP_492211). To confirm that cdc-48.3(RNAi) suppression of air-2(or207ts) lethality was specific, we assayed whether cdc-48.3(RNAi) could suppress additional embryonic lethal ts mutants. Indeed, of four mutants examined (Gomes et al., 2001Gomes J.E. Encalada S.E. Swan K.A. Shelton C.A. Carter J.C. Bowerman B. The maternal gene spn-4 encodes a predicted RRM protein required for mitotic spindle orientation and cell fate patterning in early C. elegans embryos.Development. 2001; 128: 4301-4314PubMed Google Scholar, Meneghini et al., 1999Meneghini M.D. Ishitani T. Carter J.C. Hisamoto N. Ninomiya-Tsuji J. Thorpe C.J. Hamill D.R. Matsumoto K. Bowerman B. MAP kinase and Wnt pathways converge to downregulate an HMG-domain repressor in Caenorhabditis elegans.Nature. 1999; 399: 793-797Crossref PubMed Scopus (231) Google Scholar, O'Rourke et al., 2007O'Rourke S.M. Dorfman M.D. Carter J.C. Bowerman B. Dynein modifiers in C. elegans: light chains suppress conditional heavy chain mutants.PLoS Genet. 2007; 3: e128Crossref PubMed Scopus (63) Google Scholar, Severson et al., 2000Severson A.F. Hamill D.R. Carter J.C. Schumacher J. Bowerman B. The aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis.Curr. Biol. 2000; 10: 1162-1171Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), cdc-48.3(RNAi) only restored significant viability to air-2(or207ts) embryos (Figure S2A). To test whether loss of the other Cdc48 homologs could also suppress air-2(or207ts) lethality, RNAi of cdc-48.1 and cdc-48.2 alone or simultaneously (via coinjection of cdc-48.1 and cdc-48.2 dsRNAs) was performed. Neither cdc-48.1(RNAi) nor cdc-48.2(RNAi) alone or in combination could suppress air-2(or207ts) lethality (Table S1). Cdc48 regulates various cellular processes via association with a number of conserved cofactors (Cao et al., 2003Cao K. Nakajima R. Meyer H.H. Zheng Y. The AAA-ATPase Cdc48/p97 regulates spindle disassembly at the end of mitosis.Cell. 2003; 115: 355-367Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, Isaacson et al., 2007Isaacson R.L. Pye V.E. Simpson P. Meyer H.H. Zhang X. Freemont P.S. Matthews S. Detailed structural insights into the p97-Npl4-Ufd1 interface.J. Biol. Chem. 2007; 282: 21361-21369Crossref PubMed Scopus (50) Google Scholar, Vong et al., 2005Vong Q.P. Cao K. Li H.Y. Iglesias P.A. Zheng Y. Chromosome alignment and segregation regulated by ubiquitination of survivin.Science. 2005; 310: 1499-1504Crossref PubMed Scopus (192) Google Scholar). However, RNAi of the C. elegans homologs of the Cdc48 cofactors Ufd1, Npl4, and Ubx did not suppress air-2(or207ts) lethality (Table S1). Altogether, these data suggest that cdc-48.3 is a specific negative regulator of the air-2 kinase pathway during C. elegans embryogenesis, and may act independently of known Cdc48 cofactors. air-2(or207ts) embryos display defects in chromosome segregation and cytokinesis at restrictive temperatures (Severson et al., 2000Severson A.F. Hamill D.R. Carter J.C. Schumacher J. Bowerman B. The aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis.Curr. Biol. 2000; 10: 1162-1171Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). The mutant AIR-2 protein (AIR-2ts) is still expressed at these temperatures but fails to dissociate from anaphase chromosomes and localize to the spindle midzone and midbody. The mutant protein has no detectable kinase activity in vitro (Bishop and Schumacher, 2002Bishop J.D. Schumacher J.M. Phosphorylation of the carboxyl terminus of inner centromere protein (INCENP) by the Aurora B Kinase stimulates Aurora B kinase activity.J. Biol. Chem. 2002; 277: 27577-27580Crossref PubMed Scopus (206) Google Scholar); hence, kinase activity may potentiate AIR-2 localization dynamics (Kamath et al., 2003Kamath R.S. Fraser A.G. Dong Y. Poulin G. Durbin R. Gotta M. Kanapin A. Le Bot N. Moreno S. Sohrmann M. et al.Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.Nature. 2003; 421: 231-237Crossref PubMed Scopus (2578) Google Scholar). Given that cdc-48.3(RNAi) suppressed air-2(or207ts) lethality, we examined the extent to which cdc-48.3(RNAi) could rescue the localization of the AIR-2ts protein and air-2(or207ts) mitotic defects. At 22°C, AIR-2ts localizes to chromosomes from early prophase through metaphase in both control and cdc-48.3(RNAi) treated air-2(or207ts) embryos (Figure 1B). At anaphase, AIR-2ts remained at least partially localized to chromosomes in the majority of control treated embryos (Figure 1B, Table 1), but was no longer associated with anaphase chromosomes in most cdc-48.3(RNAi) treated embryos. At telophase, AIR-2ts localized around chromosomes in a nuclear envelope-like pattern in control treated embryos, whereas it was associated with the midbody in the majority of cdc-48.3(RNAi) treated embryos. Hence, upon depletion of CDC-48.3, appropriate AIR-2 localization is restored in air-2(or207ts) embryos reared at restrictive temperatures. Furthermore, DAPI staining revealed that while chromosomes segregated properly in approximately 22% of control treated air-2(or207ts) embryos, successful chromosome segregation occurred in approximately 87% of cdc-48.3(RNAi) embryos (Figure 1B, Table 1). Altogether, these findings suggest that suppression of air-2(or207ts) lethality by cdc-48.3(RNAi) is due in part to the restoration of AIR-2 localization, which contributes to increased mitotic fidelity.Table 1Rescue of air-2(or207ts) Mitotic Defects by CDC-48.3 DepletionStageMidzone/Midbody AIR-2Chromosomal/NE AIR-2AIR-2 at BothaBoth, chromosomes and midzone/midbody.Chromosome SegregationAnaphasecontrol(RNAi)(n = 23)8.7%39.1%52.2%21.7%cdc-48.3(RNAi)(n = 21)47.7%19.0%33.6%85.7%Telophasecontrol(RNAi)(n = 25)12.0%68.0%20.0%28.0%cdc-48.3(RNAi)(n = 22)59.1%13.6%27.3%86.3%Late telophase/G1control(RNAi)(n = 23)17.4%82.6%N/A17.4%cdc-48.3(RNAi)(n = 26)88.5%11.5%N/A88.5%Control(RNAi): T7(RNAi);air-2(or207ts) (22°C); cdc-48.3(RNAi): cdc-48.3(RNAi);air-2(or207ts) (22°C). NE, nuclear envelope.a Both, chromosomes and midzone/midbody. Open table in a new tab Control(RNAi): T7(RNAi);air-2(or207ts) (22°C); cdc-48.3(RNAi): cdc-48.3(RNAi);air-2(or207ts) (22°C). NE, nuclear envelope. One conserved Cdc48 function is to target ubiquitinated proteins to the 26S proteasome for degradation (Cao et al., 2003Cao K. Nakajima R. Meyer H.H. Zheng Y. The AAA-ATPase Cdc48/p97 regulates spindle disassembly at the end of mitosis.Cell. 2003; 115: 355-367Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Given this and the genetic interaction between cdc-48.3 and air-2, we assayed whether CDC-48.3 regulates AIR-2 stability. Western analysis revealed that AIR-2 levels are significantly upregulated (∼3–4-fold) in extracts from cdc-48.3(RNAi) treated embryos as compared to wt and air-2(or207ts) embryos treated with control RNAi (Figure 2A). To assess the affect of CDC-48.3 depletion on the temporal and spatial localization of AIR-2 during the cell cycle, early embryos from control and cdc-48.3(RNAi) treated wt hermaphrodites were immunostained with tubulin and AIR-2-specific antibodies. There were no detectable differences in AIR-2 intensity or localization in cdc-48.3(RNAi) versus control embryos from early prophase through telophase (Figures 2B and 2C). However, at late-telophase/G1, marked accumulation of AIR-2 immunostaining was present at the spindle midbody of cdc-48.3(RNAi) embryos as compared to controls. Note that there is no discernible difference in the length of the mitotic spindle in control versus cdc-48.3(RNAi) embryos (Figure S2B). A similar pattern was found in subsequent cell cycles and in air-2(or207ts); cdc-48.3(RNAi) versus control treated air-2(or207ts) embryos (Figures S2C and S2D). To visualize the effects of cdc-48.3(RNAi) on AIR-2 dynamics in real-time, live imaging of GFP-tagged AIR-2 in early embryos was performed. GFP-AIR-2 intensity and localization were similar in control and cdc-48.3(RNAi) embryos from pronuclear meeting through early telophase of the first mitotic division (Movies S1 and S2). In control embryos, the GFP-AIR-2 signal dissipated after cleavage furrow ingression at ∼12.5 min post pronuclear meeting. However, in all cdc-48.3(RNAi) embryos examined, a robust GFP-AIR-2 signal was present at the spindle midbody following cleavage furrow ingression and persisted into the next mitotic cycle. Cdc48 directly interacts with target proteins to extricate them from protein complexes and cellular structures, as well as for delivery of targets to the 26S proteasome (Ye, 2006Ye Y. Diverse functions with a common regulator: ubiquitin takes command of an AAA ATPase.J. Struct. Biol. 2006; 156: 29-40Crossref PubMed Scopus (170) Google Scholar). To determine whether AIR-2 and CDC-48.3 physically associate, AIR-2 was immunoprecipitated from extracts made from transgenic animals expressing a GFP-CDC-48.3 fusion protein. This tagged line was used since attempts at creating CDC-48.3 antibodies have failed (T.R.H., unpublished data). GFP-CDC-48.3 is present throughout the cytoplasm in small puncta and is greatly reduced upon treatment with cdc-48.3(RNAi) (Figure S2D). GFP-CDC-48.3 is present in AIR-2 immunocomplexes isolated from control RNAi treated animals, but not from air-2(RNAi) or cdc-48.3(RNAi) treated animals (Figure 3A). To determine whether AIR-2 and CDC-48.3 directly interact, in vitro binding assays were conducted (Figure 3B). This analysis revealed that AIR-2 readily interacts with full-length CDC-48.3 but not with CDC-48.1 or glutathione beads. Structural studies have determined that Cdc48 forms a barrel-like hexamer with a substrate/cofactor-binding N-domain “lid” followed" @default.
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• W2030544662 date "2008-10-01" @default.
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• W2030544662 title "An Afg2/Spaf-Related Cdc48-like AAA ATPase Regulates the Stability and Activity of the C. elegans Aurora B Kinase AIR-2" @default.
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• W2030544662 doi "https://doi.org/10.1016/j.devcel.2008.08.005" @default.
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