RAG2 Is Down-regulated by Cytoplasmic Sequestration and Ubiquitin-dependent Degradation

Abstract

Periodic accumulation and degradation of RAG2 (recombination-activating gene 2) protein controls the cellcycle-dependent V(D)J recombination of lymphocyte antigen receptor genes. Here we show the molecular mechanism of RAG2 degradation. The RAG2 protein is translocated from the nucleus to the cytoplasm and degraded through the ubiquitin/proteasome system. RAG2 translocation is mediated by the Thr-490 phosphorylation of RAG2. Inhibition of this phosphorylation by p27Kip1 stabilizes the RAG2 protein in the nucleus. These results suggest that RAG2 sequestration in the cytoplasm and its subsequent degradation by the ubiquitin/proteasome system upon entering the S phase is an integral part of G0/G1-specific V(D)J recombination.During B and T cell development, the genes encoding the variable region of immunoglobulin (Ig) and the T cell receptor (TCR) are assembled from germ line-variable (V), diversity (D), and joining (J) gene segments by V(D)J recombination (1-3). V(D)J recombination is initiated by the introduction of sitespecific double-strand breaks (DSBs) between two recombining gene segments and their flanking recombination signal sequences (RSSs). The essential components of this reaction are RAG1 and RAG2 (recombination-activating genes 1 and 2) proteins, which are expressed specifically in the developing B and T lineage cells under V(D)J recombination (4, 5). Subsequent reaction steps employ more generally expressed proteins involved in non-homologous end joining (6, 7): the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the Ku70/80 dimer, XRCC4, and DNA Ligase IV. More proteins may be involved in V(D)J recombination to ensure the strict regulation of this system. Several lines of evidence suggest that V(D)J recombination is restricted to the G0/G1 stage of the cell cycle and that periodic accumulation and destruction of the RAG2 protein regulate cell-cycle-dependent V(D)J recombination (8). According to Lee and Desiderio, Thr-490 of RAG2 is phosphorylated by cyclin A/CDK2 when the cells are at the G1-S transition of the cell cycle, thereby triggering the rapid degradation of RAG2 (9). However, the precise molecular mechanism of the degradation process remains elusive.The ubiquitin/proteasome system plays a major role in target-specific protein degradation and in the regulation of protein expression levels (10, 11). The formation of ubiquitin-protein conjugates proceeds via a three-step cascade. First, a ubiquitinactivating enzyme (E1) activates ubiquitin, which is then transferred by a ubiquitin-conjugating enzyme (E2) to a ubiquitin ligase (E3) with which the substrate protein is associated. Finally, E3 catalyzes the conjugation of ubiquitin to the substrate protein. Proteins polyubiquitinated by these enzymes are subjected to degradation by the 26S proteasome. Recent reports have suggested that many proteins, such as p53, IB, ␤-catenin, and p27Kip1, are degraded by the ubiquitin/proteasome pathway (10 -13).p27Kip1, a cyclin-dependent kinase inhibitor, increases RAG2 stability by inhibiting ...

Alternative Pathways for the Repair of RAG-Induced DNA Breaks

“…Substitution of a phosphorylation-defective RAG2 (RAG2-T490A) did not, however, affect the level of HDR (data not shown). Phosphorylation has been shown to enhance ubiquitin-dependent degradation of RAG2 by promoting the transit of the protein from the nucleus to the cytoplasm (30). The requirement for T490 phosphorylation in ubiquitination is not absolute, however, implying that it is not the sole arbiter of RAG2 stability.…”

B7-CD28 Interaction Promotes Proliferation and Survival but Suppresses Differentiation of CD4−CD8− T Cells in the Thymus

“…This is due to the fact that this transition requires RAG activity restricted to the G 0 /G 1 phase of the cell cycle (50,51). Indeed, RAG-2 protein is accumulated at G 0 /G 1 , and its expression level decreases rapidly at the G 1 -S transition of the cell cycle by cytoplasmic sequestration and ubiquitin-dependent degradation (52). Our analysis of RAG-2 protein and TCR gene expression clearly demonstrated that DN from B7-deficient mice have increased accumulation of RAG-2 protein and increased expression of rearranged TCR genes.…”

MMP13 mutation causes spondyloepimetaphyseal dysplasia, Missouri type (SEMDMO)

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