Identification of novel F-box proteins in Xenopus laevis
نویسندگان
چکیده
The SCF family of ubiquitin ligases [1,2] control many physiological processes including DNA replication [3,4], centrosome duplication [5], kinetochore assembly [6] and transcription [7]. 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]; Skp1 binds to a protein containing an F box, a 44–50 aminoacid motif found in a variety of proteins [9–12]. 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-p105Rbinteracting protein mitosin [13]. 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]. When the sequence of Xenopus Fbl5 was compared with those of the human F-box proteins [9,15], 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 [7,10–12]. 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.
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ورودعنوان ژورنال:
- Current Biology
دوره 9 شماره
صفحات -
تاریخ انتشار 1999