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Identification of novel F-box proteins in Xenopus laevis

1999; Elsevier BV; Volume: 9; Issue: 20 Linguagem: Inglês

10.1016/s0960-9822(00)80006-8

1879-0445

Julie D Regan-Reimann, Quoc Vong Duong, Peter K. Jackson,

Cancer-related Molecular Pathways

The SCF family of ubiquitin ligases [1Feldman RM Correll CC Kaplan KB Deshaies RJ A complex of Cdc4p, Skp1p, and Cdc53p/cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1p.Cell. 1997; 91: 221-230Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar, 2Skowyra D Craig KL Tyers M Elledge SJ Harper JW F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex.Cell. 1997; 91: 209-219Abstract Full Text Full Text PDF PubMed Scopus (1001) Google Scholar] control many physiological processes including DNA replication [3Schwob E Bohm T Mendenhall MD Nasmyth K The B-type cyclin kinase inhibitor p40SIC1 controls the G1 to S transition in S. cerevisiae.Cell. 1994; 79: 233-244Abstract Full Text PDF PubMed Scopus (764) Google Scholar, 4Yew PR Kirschner MW Proteolysis and DNA replication: the CDC34 requirement in the Xenopus egg cell cycle.Science. 1997; 277: 1672-1676Crossref PubMed Scopus (59) Google Scholar], centrosome duplication [[5]Freed E Lacey KR Huie P Lyapina SA Deshaies RJ Stearns T Jackson PK Components of an SCF ubiquitin ligase localize to the centrosome and regulate the centrosome duplication cycle.Genes Dev. 1999; 13: 2242-2257Crossref PubMed Scopus (173) Google Scholar], kinetochore assembly [[6]Kaplan KB Hyman AA Sorger PK Regulating the yeast kinetochore by ubiquitin-dependent degradation and Skp1p-mediated phosphorylation.Cell. 1997; 91: 491-500Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar] and transcription [[7]Marti A Wirbelauer C Scheffner M Krek W Interaction between ubiquitin-protein ligase SCFSkp2 and E2F-1 underlies the regulation of E2F-1 degradation.Nat Cell Biol. 1999; 1: 14-19Crossref PubMed Scopus (279) Google Scholar]. SCFs are composed of four subunits: a protein subunit called a cullin binds two other subunits, one called Skp1 and the other, a ring-finger protein called Rbx1 [[8]Skowyra D Koepp DM Kamura T Conrad RC Conaway RC Conaway JW Elledge SJ Harper JW Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1.Science. 1999; 284: 662-665Crossref PubMed Scopus (350) Google Scholar]; Skp1 binds to a protein containing an F box, a 44–50 amino-acid motif found in a variety of proteins [9Winston JT Koepp DM Zhu C Elledge SJ Harper JW A family of mammalian F-box proteins.Curr Biol. 1999; 9: 1180-1182Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 10Carrano AC Eytan E Hershko A Pagano M SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27.Nat Cell Biol. 1999; 1: 193-199Crossref PubMed Scopus (1293) Google Scholar, 11Sutterluty H Chatelain E Marti A Wirbelauer C Senften M Muller U Krek W p45SKP2 promotes p27KIP1 degradation and induces S phase in quiescent cells.Nat Cell Biol. 1999; 1: 207-214Crossref PubMed Scopus (620) Google Scholar, 12Tsvetkov LM Yeh K-H Lee S-J Sun H Zhang H p27Kip1 ubiquitination and degradation is regulated by the SCFSkp2 complex through phosphorylated Thr187 in p27.Curr Biol. 1999; 9: 661-664Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar]. To understand the role of SCF complexes in the early embryonic cell cycle, we screened a Xenopus oocyte library (Clontech) for F-box proteins using human Skp1 as bait in a yeast two-hybrid screen (the human and Xenopus Skp1 proteins are identical). In two independent screens (5.3 × 106 clones), 449 positive clones were isolated, from which five novel F-box proteins were identified: Fbx26, Fbx27, Fbx28, Fbl5 and Fbl13. Each had an identifiable F box. A number of other proteins were also identified that interacted with Skp1 but did not fit the current consensus F-box sequence; it is possible that these may turn out to be a distinct class of F-box proteins. The sequences of the five Xenopus F-box proteins are aligned and their domain organizations are shown in Figure 1. Three of the proteins, Fbx26, Fbx27 and Fbx28, did not have recognizable domains outside the F-box motif. Fbx26, which did not bear significant homology to any protein currently in the database, was especially abundant in the library (88% of clones screened). BLAST searches indicated that there was some homology between Fbx27 and the kinetochore protein CENP-F and the retinoblastoma-p105Rb-interacting protein mitosin [[13]Goodwin RL Pabon-Pena LM Foster GC Bader D The cloning and analysis of LEK1 identifies variations in the LEK/centromere protein F/mitosin gene family.J Biol Chem. 1999; 274: 18597-18604Crossref PubMed Scopus (29) Google Scholar]. Fbx28 showed significant homology to a human protein, KIAA0483, whose function is unknown. Two of the proteins, Fbl5 and Fbl13, contained leucine-rich repeats [[14]Kobe B Deisenhofer J Proteins with leucine-rich repeats.Curr Opin Struct Biol. 1995; 5: 409-416Crossref PubMed Scopus (313) Google Scholar]. When the sequence of Xenopus Fbl5 was compared with those of the human F-box proteins [9Winston JT Koepp DM Zhu C Elledge SJ Harper JW A family of mammalian F-box proteins.Curr Biol. 1999; 9: 1180-1182Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 15Cenciarelli C Chiaur DS Guardavaccaro D Parks W Vidal M Pagano M Identification of a family of human F-box proteins.Curr Biol. 1999; 9: 1177-1179Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar], we found that the Xenopus protein was most similar to human Fbl5 (61% identity). A phylogenetic comparison (Figure 2) suggested that Fbl5 was related to, but distinct from, Skp2, the other known human Fbl. Skp2 was recently shown to be critical for destroying the cyclin-dependent kinase (Cdk) inhibitor p27KIP1 and the transcription factor E2F-1 at the G1–S transition of the cell cycle [7Marti A Wirbelauer C Scheffner M Krek W Interaction between ubiquitin-protein ligase SCFSkp2 and E2F-1 underlies the regulation of E2F-1 degradation.Nat Cell Biol. 1999; 1: 14-19Crossref PubMed Scopus (279) Google Scholar, 10Carrano AC Eytan E Hershko A Pagano M SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27.Nat Cell Biol. 1999; 1: 193-199Crossref PubMed Scopus (1293) Google Scholar, 11Sutterluty H Chatelain E Marti A Wirbelauer C Senften M Muller U Krek W p45SKP2 promotes p27KIP1 degradation and induces S phase in quiescent cells.Nat Cell Biol. 1999; 1: 207-214Crossref PubMed Scopus (620) Google Scholar, 12Tsvetkov LM Yeh K-H Lee S-J Sun H Zhang H p27Kip1 ubiquitination and degradation is regulated by the SCFSkp2 complex through phosphorylated Thr187 in p27.Curr Biol. 1999; 9: 661-664Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar]. Fbl13 is a novel protein related to, but distinct from, other known F-box proteins in the database that contain leucine-rich repeats (Figure 2). We have identified the first examples of F-box proteins in X. laevis. The ability to interfere with the function of proteins in the Xenopus embryo by expression of wild-type or dominant-negative versions of the proteins may allow us to identify new roles for SCF ubiquitin ligases in cell-cycle progression and early development. We thank Wade Harper and Michelle Pagano for sharing unpublished results. This work was supported by funding from the NIGMS (GM54811), a junior investigator award from the Howard Hughes Medical Institute (P.K.J.), a grant from the Stanford Cancer Council, and the MSTP training grant (GM07365) from NIGMS (J.D.R-R.). JD Regan-Reimann, QV Duong and PK Jackson, Departments of Pathology, Microbiology and Immunology, Stanford University School of Medicine, Palo Alto, California 94305, USA. E-mail address for PK Jackson: [email protected]