Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein's C-Terminal Helix.

The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms

Hst3 is turned over by a replication stress-responsive SCFCdc4 phospho-degron

Hst3 is the histone deacetylase that removes histone H3K56 acetylation. H3K56 acetylation is a cell-cycle- and damage-regulated chromatin marker, and proper regulation of H3K56 acetylation is important for replication, genomic stability, chromatin assembly, and the response to and recovery from DNA damage. Understanding the regulation of enzymes that regulate H3K56 acetylation is of great interest, because the loss of H3K56 acetylation leads to genomic instability. HST3 is controlled at both the transcriptional and posttranscriptional level. Here, we show that Hst3 is targeted for turnover by the ubiquitin ligase SCF(Cdc4) after phosphorylation of a multisite degron. In addition, we find that Hst3 turnover increases in response to replication stress in a Rad53-dependent way. Turnover of Hst3 is promoted by Mck1 activity in both conditions. The Hst3 degron contains two canonical Cdc4 phospho-degrons, and the phosphorylation of each of these is required for efficient turnover both in an unperturbed cell cycle and in response to replication stress

Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein's C-Terminal Helix.

The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms

DNA Damage Regulates Translation through β-TRCP Targeting of CReP

<div><p>The Skp1-Cul1-F box complex (SCF) associates with any one of a number of F box proteins, which serve as substrate binding adaptors. The human F box protein βTRCP directs the conjugation of ubiquitin to a variety of substrate proteins, leading to the destruction of the substrate by the proteasome. To identify βTRCP substrates, we employed a recently-developed technique, called Ligase Trapping, wherein a ubiquitin ligase is fused to a ubiquitin-binding domain to “trap” ubiquitinated substrates. 88% of the candidate substrates that we examined were bona fide substrates, comprising twelve previously validated substrates, eleven new substrates and three false positives. One βTRCP substrate, CReP, is a Protein Phosphatase 1 (PP1) specificity subunit that targets the translation initiation factor eIF2α to promote the removal of a stress-induced inhibitory phosphorylation and increase cap-dependent translation. We found that CReP is targeted by βTRCP for degradation upon DNA damage. Using a stable CReP allele, we show that depletion of CReP is required for the full induction of eIF2α phosphorylation upon DNA damage, and contributes to keeping the levels of translation low as cells recover from DNA damage.</p></div

CReP ubiquitination is dependent on CRLs and turnover is regulated by a βTRCP consensus degron.

<p>(A) Ubiquitinated CReP precipitated by the βTRCP ligase trap depends on cullin activity. Tagged CReP was transiently expressed in the βTRCP or negative control ligase trap cell lines, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005292#pgen.1005292.g002" target="_blank">Fig 2B</a>. Where indicated, 1 μM MLN4924 was added 4 hours before cell collection to inhibit cullin activity. (B) A near-consensus βTRCP degron in CReP, compared to well-validated degrons. (C) CReP turnover depends on βTRCP consensus sites. Two consensus sites in CReP were mutated to generate the 11A mutant. Wildtype or mutant CReP was expressed transiently in 293 cells, which were then treated with 100 μg/mL cycloheximide for the time indicated to monitor degradation in the absence of new protein synthesis. Where indicated, cells were treated with 1 μM MLN4924 coincident with cycloheximide addition. (D) Quantitation of the average of two independent replicates of (B).</p

Establishing Ligase Trapping in human cells.

<p>(A) The SCF includes the scaffolds Skp1 (unlabeled, in red) and Cul1, which connect the E2-binding protein Roc1 to an F box protein such as βTRCP, which recruits substrates. Ligase Trapping is a two-step process in which ubiquitinated substrates are first precipitated under native conditions by a ubiquitin ligase fused to a UBA domain and then purified further under denaturing conditions via a 6xHis tag on ubiquitin. (B) βTRCP Ligase Trap purifies ubiquitinated species of the known substrate ATF4. Stable cell lines expressing the βTRCP Ligase Trap or a negative control (FBXO24 or Fbw7) were induced to express 6xHisUb for 3 days, transfected with 5xHA-tagged ATF4 for 24 hours, treated with 5 μM MG132 for 4 hours, lysed and subjected to a two-step precipitation. First, the Ligase Traps were purified under native conditions with anti-Flag antibody and eluted with Flag peptide. Then, the eluate was denatured in 6M urea and ubiquitinated proteins purified with NiNTA beads and eluted with imidazole. Loading was 1X input (In), 250X 1st step (1), and 5,000X 2nd step (2). (C) The interaction between the βTRCP Ligase Trap and the known substrate β-catenin depends on conserved substrate-binding regions in βTRCP. The pulldown in B was repeated, but without MG132 and with the substrate β-catenin as prey and both wt and mutant βTRCP as bait. (D) Fbw7 Ligase Trap specifically purifies ubiquitinated species of the known Fbw7 substrate MED13. Performed as in Fig 1B.</p

CReP ubiquitination is dependent on CRLs and turnover is regulated by a βTRCP consensus degron.

<p>(A) Ubiquitinated CReP precipitated by the βTRCP ligase trap depends on cullin activity. Tagged CReP was transiently expressed in the βTRCP or negative control ligase trap cell lines, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005292#pgen.1005292.g002" target="_blank">Fig 2B</a>. Where indicated, 1 μM MLN4924 was added 4 hours before cell collection to inhibit cullin activity. (B) A near-consensus βTRCP degron in CReP, compared to well-validated degrons. (C) CReP turnover depends on βTRCP consensus sites. Two consensus sites in CReP were mutated to generate the 11A mutant. Wildtype or mutant CReP was expressed transiently in 293 cells, which were then treated with 100 μg/mL cycloheximide for the time indicated to monitor degradation in the absence of new protein synthesis. Where indicated, cells were treated with 1 μM MLN4924 coincident with cycloheximide addition. (D) Quantitation of the average of two independent replicates of (B).</p

Ubiquitin ligase binding and turnover of a subset of novel βTRCP substrates.

<p>(A) βTRCP binds to its candidate substrates <i>in vivo</i>. HEK293 cells were transfected with 3xFlag-tagged F box proteins and 5xHA-tagged substrates for 1 day, lysed and subjected to a one-step precipitation. The F box proteins were purified under native conditions with anti-Flag antibody and eluted with Flag peptide. Loading was 1X input (In) and 75.3X IP for CReP, and 1X input (In) and 83.7X IP for other substrates. (B) Effect of βTRCP knockdown on candidate substrate half-life. HEK293 cells were co-transfected with a negative control plasmid, or a plasmid encoding an shRNA targeting βTRCP1 and 2, and a plasmid encoding a tagged βTRCP candidate substrate. Cells were treated with 100 μg/mL cycloheximide for the indicated time before collection.</p

Regulation of CReP turnover and impact on eIF2α phosphorylation.

<p>(A) CReP is depleted upon DNA damage but not proteostatic stress. Cells were treated with 1 μM thapsigargin, 3 μg/mL camptothecin, or 300 J/m² UV for the indicated time; all samples not treated with UV were mock-treated and all samples were given the same total volume of the solvent DMSO. (B) CReP turnover upon DNA damage depends at least in part on βTRCP. Cells were transfected for 48 hours with an empty vector or shRNA targeting βTRCP1 and 2, then irradiated with 300 J/m² UV-C. CReP levels are quantitated below, and the half-life calculated from the linear (0–2 hr.) part of the timecourse. (C) CReP depletion and full eIF2α phosphorylation in UV depends on CRLs. Cells were treated with UV with or without MLN4924 for the times indicated. (D) CReP depletion in primary human fibroblasts depends on CRLs. Primary human fibroblasts were treated with 1 μg/mL camptothecin for 6 hours, with 1 μM MLN4924 where indicated. (E) The 31A allele of CReP is stable even upon treatment with DNA damage and cycloheximide. Cells were transfected with wildtype or mutant CReP, then pre-treated for 2 hours with 3 μg/mL camptothecin before addition of cycloheximide. (F) Expression of a stable allele of CReP prevents phosphorylation of eIF2α in response to UV treatment. Cells were transfected with tagged wild type or mutant CReP, then treated with UV for the indicated times.</p