Mouse ribonucleotide reductase R2 protein: a new target for anaphase-promoting complex-Cdh1-mediated proteolysis - PubMed (original) (raw)

Mouse ribonucleotide reductase R2 protein: a new target for anaphase-promoting complex-Cdh1-mediated proteolysis

Anna Lena Chabes et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Ribonucleotide reductase consists of two nonidentical proteins, R1 and R2, and catalyzes the rate-limiting step in DNA precursor synthesis: the reduction of ribonucleotides to deoxyribonucleotides. A strictly balanced supply of deoxyribonucleotides is essential for both accurate DNA replication and repair. Therefore, ribonucleotide reductase activity is under exquisite control both transcriptionally and posttranscriptionally. In proliferating mammalian cells, enzyme activity is regulated by control of R2 protein stability. This control, which responds to DNA damage, is effective until cells pass into mitosis. We demonstrate that the mitotic degradation and hence the overall periodicity of R2 protein levels depends on a KEN box sequence, recognized by the Cdh1-anaphase-promoting complex. The mouse R2 protein specifically binds Cdh1 and is polyubiquitinated in an in vitro ubiquitin assay system. Mutating the KEN signal stabilizes the R2 protein during mitosisG(1) in R2 protein-overexpressing cells. The degradation process, which blocks deoxyribonucleotide production during G(1), may be an important mechanism protecting the cell against unscheduled DNA synthesis. The newly discovered p53-induced p53R2 protein that lacks a KEN box may supply deoxyribonucleotides for DNA repair during G(0)G(1).

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Figures

Figure 1

Figure 1

The KEN sequence is conserved in human, mouse, hamster, and guinea pig (GenBank accession no. AY209181) R2 proteins. A comparison of the N-terminal amino acid sequence in the human, mouse, hamster, and guinea pig R2 proteins is shown. The conserved KEN sequence and the uniquely phosphorylated Ser-20 are indicated in bold.

Figure 2

Figure 2

Wild-type mouse R2 protein but not the AAN mutant R2 protein specifically binds to Cdh1. _In vitro_-translated 35S-labeled wild-type R2 protein (R2 WT), the AAN mutant R2 protein (R2 AAN), the S20A mutant R2 protein (R2 S20A), or the S20D mutant R2 protein (R2 S20D) was incubated in the presence of cold _in vitro_-translated myc-tagged Cdh1 (MT-Cdh1) or Cdc20 (MT-Cdc20) bound to α-myc beads or in the presence of myc tag (MT) alone bound to α-myc beads. The left-most lane in each image indicates 10% of the in vitro translation material supplied in the binding assay.

Figure 3

Figure 3

Wild-type mouse R2 protein but not the AAN mutant R2 protein is recognized as a substrate by Cdh1–APC. Incubation of 35S-labeled _in vitro_-translated R2 protein in an in vitro ubiquitination assay containing immunopurified Xenopus APC (E3); and baculovirus-expressed and purified Cdh1, E1, UBC4/UBCx (E2), ubiquitin, and an energy-regenerating system. Lane 1, wild-type R2 protein input; lane 2, wild-type R2 protein after a 1-h incubation; lane 3, AAN mutant R2 protein input; lane 4, AAN mutant R2 protein after a 1-h incubation. Lower shows a lighter exposure where the quantitative loss of the unmodified protein can be seen in lane 2 but not in lane 4. The arrow indicates the position of monoubiquitinated R2 protein.

Figure 4

Figure 4

Mutating the KEN signal stabilizes the R2 protein during mitosis and G1. (A) Stably transformed mouse Balb/3T3 fibroblasts overexpressing wild-type R2 protein (clone D2) or the AAN mutant R2 protein (clone MK6) were explanted on 5-cm dishes (2 × 105 cells per dish) and allowed to grow for 24 h in DMEM containing 10% horse serum. Then the cells were synchronized in DMEM containing 1% horse serum for 45 h and finally released by the addition of DMEM containing 20% horse serum. At different time points after serum readdition (19–27 h), cells overexpressing wild-type R2 protein (lanes 1–5) or the AAN mutant R2 protein (lanes 6–10) were harvested for flow cytometry and immunoblotting. (B) Stably transformed cells overexpressing native or AAN-mutated R2 protein (clones D4 and MK 2, respectively) were explanted on 5-cm dishes and allowed to grow for 24 h in DMEM containing 10% horse serum. After harvesting four dishes for flow cytometry and immunoblotting, the medium was changed to DMEM containing 1% serum and cells were harvested as before after 24 and 36 h. Finally, the cells were released from starvation with DMEM containing 20% serum and harvested 4 h after serum readdition. To make sure that the same amount of protein (0.3 μg) was loaded in each lane, the protein concentration of each cell extract was determined by the Bio-Rad protein assay before loading. Lane 1, logarithmically growing, wild-type R2 protein-overexpressing cells; lane 2, the same after 24 h of serum starvation; lane 3, the same after 36 h of serum starvation; lane 4, the same 4 h after serum readdition; lane 5, logarithmically growing, AAN mutant R2 protein-overexpressing cells; lane 6, the same after 24 h of serum starvation; lane 7, the same after 36 h of serum starvation; lane 8, the same 4 h after serum readdition. In both A and B, Lower shows the flow cytometry profile corresponding to each time point.

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