The N-terminal Regulatory Domain of Cyclin A Contains Redundant Ubiquitination Targeting Sequences and Acceptor Sites

Abstract

ACKNOWLEDGEMENTSWe are grateful for Jane Endicott, Tim Hunt and Michele Pagano for generous gifts of reagents. We thank members of the Poon Lab for constructive criticism on the manuscript. This work was supported in part by the Research Grants Council grants HKUST6135/03M and the HKUST grant HIA03/ 04.SC01 to R.Y.C.P.. ReportThe N-Terminal Regulatory Domain of Cyclin A Contains Redundant Ubiquitination Targeting Sequences and Acceptor Sites ABSTRACT 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.

Cyclin A Degradation Employs Preferentially Used Lysines and a Cyclin Box Function other than Cdk1 Binding

“…6D and E). Although similar results have been obtained recently with huCycA2, 43 we now could show that the C-terminal half, most likely the cyclin box, confers instability onto a heterologous protein when fused with it. This indicates that the all-important contribution for bypassing the checkpoint control is provided by the cyclin box, although the N-terminal signals also participate in checkpoint turnover and make additive contributions to it (Fig.…”

“…HuCycA2 also does not require its second D-box like sequence for turnover. 43 The elusive signal did not seem to be at the N-terminus, since the whole N-terminal half (CycA 1-170) underwent impaired proteolysis (Fig. 6A, B and I).…”

Generation of an indestructible cyclin B1 by caspase-6-dependent cleavage during mitotic catastrophe

“…Lysates were prepared and 200 mg was subjected to immunoprecipitation with an antiserum against FLAG. The immunoprecipitates were treated with 3C protease as described earlier (Fung et al, 2005) to remove the FLAG tag. The immunoprecipitates and the original lysates were subjected to immunoblotting for cyclin B1 and the FLAG epitope.…”

“…Plasmids expressing FLAG-cyclin B1 (Fung et al, 2005), ND88 (Chan et al, 2008a) and histone H2B-GFP (Chow et al, 2003b) were constructed or obtained from sources as described earlier. Cyclin B1 cDNA was amplified by PCR with the oligonucleotides 5 0 -TCCATGGATACTGCCTCTCCAAG CC-3 0 (for FLAG-cyclin B1(ND122)) or 5 0 -AGCCATGG GAGCTGATCCAAACC-3 0 (for FLAG-cyclin B1(ND160)) and a cyclin B1 reverse primer (5 0 -CGGATCCTTA CACCTTTGCCAC-3 0 ); the PCR products were cut with NcoI Figure 8 Mitotic catastrophe does not require the cleavage of cyclin B1, but cyclin B1D may enhance cell death during mitotic catastrophe.…”

“…and EcoRI, and ligated into pUHD-P1 (Yam et al, 1999a) or pUHD-P1/3C (Fung et al, 2005). FLAG-cyclin B1 in pUHD-P1 was amplified by PCR with vector forward primer and 5 0 -GATCAGCTCCATTTTCTGCATC-3 0 ; the PCR product was cut with NcoI, and ligated into pUHD-P1 cut with NcoI and BamHI (blunt with Klenow fragment) to produce FLAGcyclin B1(CD160) in pUHD-P1.…”