The N-terminal regulatory domain of cyclin A contains redundant ubiquitination targeting sequences and acceptor sites.
Cyclin A is destroyed during mitosis by the ubiquitin-proteasome system. Like cyclin B, a destruction box (D-box) motif is required for the destruction of cyclin A. However, cyclin A degradation is more complicated than cyclin B because cyclin A's D-box motif is more extensive and proteolysis involves complex signaling in some organisms. In this study, we found that in addition to the D-box, the region between residues 123-157 also contributed to the ubiquitination and degradation of human cyclin A. Indeed, removal of the bulk of the N-terminal regulatory domain was needed to completely stabilize cyclin A and eliminate ubiquitination. A putative second RxxL motif around residue 138 played only a minor role in cyclin A degradation. To distinguish between sequences recognized by the ubiquitination machinery and the ubiquitin acceptor sites per se, we utilized a novel approach involving in vitro cleavage of cyclin A after ubiquitination. We found that several lysine residues proximal to the D-box (Lys37, Lys54, and Lys68) were ubiquitin acceptor sites. Cyclin A lacking the three lysine residues was degraded slower than the wild-type protein. Although these lysines were normally used, ubiquitination could shift to other cryptic sites when the preferred sites were unavailable, suggesting the exact positions of the ubiquitin chains also contributed to degradation. Together, these data revealed that ubiquitination does not occur randomly on cyclin A and open up questions on the precise function of the D-box.
Fung
Tsz Kan
TK
Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.
Yam
Cain H
CH
Poon
Randy Y C
RY
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D019925:Cyclin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008938:Mitosis; D008969:Molecular Sequence Data; D011485:Protein Binding; D016415:Sequence Alignment; D025801:Ubiquitin / Q000378:metabolism*
D019925:Cyclin A; D025801:Ubiquitin; D008239:Lysine