Abstract Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules orchestrates a vast number of processes in the cell. Consequently, post-translational modification pathways are considered major targets for therapeutic intervention of many diseases, including cancers. Neddylation is a post-translational mechanism like ubiquitination, but instead of ubiquitin, NEDD8 is conjugated to protein targets via an E1-E2-E3 multi-enzymatic cascade. Neddylation is upregulated in multiple cancer types and the development of therapeutics targeting neddylation pathways are gaining increasing popularity. UBE2M, one of the two E2 neddylation conjugation enzymes, takes part in two types of reactions: transthiolation-transfer of NEDD8 from a thioester to a thiol group and aminolysis-transfer of NEDD8 from a thioester to an amino group. These activities require both a 26-residue N-terminal docking peptide and a conserved E2 catalytic core domain, which is the basis for the transfer of NEDD8. Here, we describe findings in the quest to understand the UBE2M interaction with NEDD8 and effects on cancer cells by modifying UBE2M at the N-terminal docking peptide or at the catalytic site. Cancer cells were transfected with ΔN or catalytic site UBE2M mutant constructs and analyzed by immunoprecipitation for charging of NEDD8 onto UBE2M. The catalytic site mutants are deficient in charging (C111A) or discharging (C111S) of UBE2M by NEDD8, which leads to a decrease in neddylation of downstream substrates, such as CULLIN family members. Elimination of the N-terminal docking peptide by introduction of catalytically competent ΔN UBE2M mutant constructs resulted in reduced reaction kinetics. Structural modeling suggests that the loss of the N-terminal docking region likely weakens protein-protein interactions between UBE2M and the UBA3-APPBP1 E1 complex. Interestingly, ΔN UBE2M mutants were observed to be slightly charged with NEDD8, but appeared to have a defect in NEDD8 discharge, resulting in reduced neddylation of substrates. Moreover, further phenotypic evaluation of UBE2M mutant transfected cancer cells revealed a divergence in phenotypic effect: while both mutant types led to a deficiency in NEDD8 charge or discharge from UBE2M, only the manipulation of the active site of UBE2M led to a DNA re-replication phenotype with subsequent cell death. These observations reveal the complexity of the neddylation cascade and raise the question if slowing down discharge of NEDD8 from UBE2M is a more viable therapeutic option than slowing down the charging of UBE2M. Citation Format: Alex G. Batrouni, Jacob P. Matson, Andressa L. Mota, Archna Ravi, Marcus O'Hara, Micholas D. Smith, Jeremy C. Smith, Vivek K. Vishnudas, Anjan Thakurta, Stephane Gesta, Maria D. Nastke. In charge: Targeting neddylation of UBE2M for anticancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4694.

Microtubule-targeting agents (MTAs) are among the most effective chemotherapeutics used in the treatment of cancer. However, the clinical utility of current MTAs, such as paclitaxel and vinblastine, are often limited due to adverse side effects or multidrug resistance (MDR) driving the continuous pursuit for the development of novel microtubule interactors. Here, we report the development of a novel non P-gp substrate MTA that is metabolically stable, and that displays broad-spectrum anti-cancer activity in vitro and ex vivo. Anti-cancer activity was assessed by screening a panel of 102 cancer cell lines and in a multi-tumor type panel of patient-derived organoids (PDOs). In vitro, double to triple digit nanomolar (ranging from 17nM to 318 nM) potency was observed in 101 cell lines, while viability was reduced by more than 70% in 76 cell lines out of 102. Similarly, 23 out of 34 (68%) of ex vivo treated PDOs were highly or partially responsive as indicated by a 37-73% decrease in viability. Studies using spindle assembly checkpoint (SAC) inhibitors demonstrated a mechanism at or before microtubule-kinetochore attachment in M phase of the cell cycle. Subsequent microscopic evaluation of kinetochore assembly revealed a lack of proper mitotic spindle formation highlighted with an increase in Mad1 signal intensity emphasizing metaphase arrest. Furthermore, in vitro tubulin polymerization assays demonstrated that the anti-cancer effect is a result of cytoskeleton targeting to inhibit microtubule assembly. Additionally, the antimicrotubule agent inhibited the formation of EBI (N,N’-ethylene-bis(iodoacetamide)):β-tubulin adducts indicating an occupied colchicine-binding site of tubulin. Six multi-tumor preclinical oncology models were evaluated for in vivo anti-tumor efficacy by oral administration of the compound. Further, tumor growth inhibition (%) was demonstrated in preclinical murine models of Acute Myeloid Leukemia (AML) (MV-4-11, 51%), colorectal (COLO205, 35%), prostate (DU 145, 38%), and gastric cancer (HS746T, 74%; SNU-5, 48%). Moreover, tumor tissue analysis for prominent mitotic cell cycle markers, Cyclin B1 and pHH3, revealed a dose-dependent elevation of both markers revealing mitotic arrest phenotype. Together, our data supports further preclinical and clinical development of a novel oral MTA notably in Taxane resistant and/or Taxane sensitive tumors. Citation Format: Maria-Dorothea Nastke, Jacob Matson, Andressa Mota, Archna Ravi, Alex Batrouni, Marcus O'Hara, Shiva Kazerounian, Arcan Guven, Nicole Pellegrino, Kashni Grover, Kayleigh Gray, Anne Diers, Michael Kiebish, Vivek Vishnudas, Stephane Gesta. Development of a novel oral microtubule targeting agent with pan-cancer efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1663.

The ubiquitin-proteasome system (UPS) plays an important role in cellular processes for protein quality control and homeostasis and indeed, dysregulation within the pathway has implications in numerous diseases, especially cancer. Here, we introduce BRG399, a novel modulator of UBE2K - a unique E2 conjugating enzyme of the UPS, that we previously identified as a potential therapeutic target using BERG’s Interrogative Biology® platform. The anticancer potential of UBE2K modulation was validated by gene manipulation using siRNA mediated knockdown of UBE2K in several cancer cell models. Knockdown of UBE2K resulted in a 50% decrease in cell number in MIA PaCa2 cells and a 30% decrease in cell number in SKHEP1 and HepG2 cells at 96h post transfection. This effect was the result of a robust G2/M cell cycle arrest associated with increased CyclinB1 expression. In addition, a modest increase in apoptosis/necrosis (6-8%) was observed in cells with UBE2K knockdown. Using Fragment-Based Ligand Discovery and structure-based drug design, BRG399 was developed as a new chemical entity (NCE) which binds to UBE2K, resulting in modulation of several established canonical functions and an anti-cancer phenotype. In a panel of 102 cancer cell lines, BRG399 exhibited an anti-cancer activity in all cell lines tested as evident by extrapolation of IC50 and %Max Effect values from the dose response curves. In all but one cell line, double to triple digit nanomolar potency was observed ranging from 17nM to 318 nM and viability of 76 cell lines was reduced by more than 70%. The anti-cancer potency of BRG399 was also established in a multi-tumor type panel of patient-derived organoids (PDO) of which 68% were highly or partially responsive to BRG399 as indicated by 37-73% decrease in PDO viability. Mechanistic studies demonstrated that BRG399’s anti-cancer effect was a result of a robust G2/M cell cycle arrest and apoptosis mediated cell death. Molecular events associated with G2/M arrest included stabilization of CyclinB1 and sustained activation of cyclin-dependent kinase CDK1, while those associated with apoptosis involved decreased expression of anti-apoptotic proteins Bcl-XL and Mcl-1 and increased expression of cleaved PARP (cPARP) and Capase 3. Together, these results support a novel role for UBE2K in the regulation of the cell cycle in cancer cells and demonstrate that its modulation by BRG399 influences phenotypic end-points consistent with an anti-cancer effect. Citation Format: Maria-Dorothea Nastke, Marisa Permatteo, Pragalath Sundararajan, Shefali Sharma, Jacob Matson, Kaleigh Gray, Arcan Guven, Shiva Kazerounian, Anne Diers, Rangaprasad Sarangarajan, Niven R. Narain, Vivek K. Vishnudas, Stephane Gesta. BRG399: A novel potent small molecule modulator of UBE2K for the treatment of various cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5321.

The ubiquitin-proteasome system is essential for cell cycle progression. Cyclin F is a cell cycle-regulated substrate adapter F-box protein for the Skp1, CUL1, and F-box protein (SCF) family of E3 ubiquitin ligases. Despite its importance in cell cycle progression, identifying cyclin F-bound SCF complex (SCFCyclin F) substrates has remained challenging. Since cyclin F overexpression rescues a yeast mutant in the cdc4 gene, we considered the possibility that other genes that genetically modify cdc4 mutant lethality could also encode cyclin F substrates. We identified the mitochondrial and cytosolic deacylating enzyme sirtuin 5 (SIRT5) as a novel cyclin F substrate. SIRT5 has been implicated in metabolic processes, but its connection to the cell cycle is not known. We show that cyclin F interacts with and controls the ubiquitination, abundance, and stability of SIRT5. We show SIRT5 knockout results in a diminished G1 population and a subsequent increase in both S and G2/M. Global proteomic analyses reveal cyclin-dependent kinase (CDK) signaling changes congruent with the cell cycle changes in SIRT5 knockout cells. Together, these data demonstrate that SIRT5 is regulated by cyclin F and suggest a connection between SIRT5, cell cycle regulation, and metabolism.

Minichromosome maintenance protein 10 (MCM10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 variants in patients with distinctive, but overlapping, clinical phenotypes: natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of MCM10-associated disease, we modeled these variants in human cell lines. MCM10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of MCM10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in MCM10-deficient cells require endonucleolytic processing by MUS81, as MCM10 : MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic variants in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes observed in both NKD and RCM patients.

Chromatin flow cytometry for MCM loading cell cycle analysis, adaptable to any antibody measurements by chromatin flow cytometry.

R loops arise from hybridization of RNA transcripts with template DNA during transcription. Unrepaired R loops lead to transcription-replication collisions, causing DNA damage and genomic instability. In this issue of Genes & Development, Pérez-Calero and colleagues (pp. 898-912) identify UAP56 as a cotranscriptional RNA-DNA helicase that unwinds R loops. They found that UAP56 helicase activity is required to remove R loops formed from different sources and prevent R-loop accumulation genome-wide at actively transcribed genes.

To maintain tissue homeostasis, cells transition between cell cycle quiescence and proliferation. An essential G1 process is minichromosome maintenance complex (MCM) loading at DNA replication origins to prepare for S phase, known as origin licensing. A p53-dependent origin licensing checkpoint normally ensures sufficient MCM loading before S phase entry. We used quantitative flow cytometry and live cell imaging to compare MCM loading during the long first G1 upon cell cycle entry and the shorter G1 phases in the second and subsequent cycles. We discovered that despite the longer G1 phase, the first G1 after cell cycle re-entry is significantly underlicensed. Consequently, the first S phase cells are hypersensitive to replication stress. This underlicensing results from a combination of slow MCM loading with a severely compromised origin licensing checkpoint. The hypersensitivity to replication stress increases over repeated rounds of quiescence. Thus, underlicensing after cell cycle re-entry from quiescence distinguishes a higher-risk first cell cycle that likely promotes genome instability.

The earliest step in DNA replication is origin licensing, which is the DNA loading of minichromosome maintenance (MCM) helicase complexes. The Cdc10-dependent transcript 1 (Cdt1) protein is essential for MCM loading during the G1 phase of the cell cycle, but the mechanism of Cdt1 function is still incompletely understood. We examined a collection of rare Cdt1 variants that cause a form of primordial dwarfism (the Meier-Gorlin syndrome) plus one hypomorphic Drosophila allele to shed light on Cdt1 function. Three hypomorphic variants load MCM less efficiently than wild-type (WT) Cdt1, and their lower activity correlates with impaired MCM binding. A structural homology model of the human Cdt1-MCM complex positions the altered Cdt1 residues at two distinct interfaces rather than the previously described single MCM interaction domain. Surprisingly, one dwarfism allele (Cdt1-A66T) is more active than WT Cdt1. This hypermorphic variant binds both cyclin A and SCFSkp2 poorly relative to WT Cdt1. Detailed quantitative live-cell imaging analysis demonstrated no change in the stability of this variant, however. Instead, we propose that cyclin A/CDK inhibits the Cdt1 licensing function independent of the creation of the SCFSkp2 phosphodegron. Together, these findings identify key Cdt1 interactions required for both efficient origin licensing and tight Cdt1 regulation to ensure normal cell proliferation and genome stability.

The correct execution of DNA replication is critical for normal cell proliferation. The earliest step in DNA replication is known as origin licensing. Origins are licensed by the loading of MCM complexes, an event that is restricted to G1 phase. The Cdt1 protein is essential for MCM loading, yet the molecular mechanisms of its activity are still remarkably mysterious. Here, we examined a collection of rare Cdt1 variants associated with a specific form of human primordial dwarfism (Meier‐Gorlin syndrome), plus one hypomorphic conserved Drosophila allele, to shed light on Cdt1 function. Our results indicate that two dwarfism variants (Cdt1‐R462Q, Cdt1‐E468K) are expressed normally, but load less MCM than WT Cdt1. We found the lower origin licensing activity of these variants correlates with impaired MCM binding. Likewise, the hypomorphic Drosophila allele (Cdt1‐R210C) consistently induces less MCM loading, and this deficiency also correlates with impaired MCM binding. Interestingly, this variant impacts a previously uncharacterized MCM binding domain in Cdt1. To visualize these molecular interactions, we generated human Cdt1‐MCM structural homology models based on recent yeast cryo‐EM structures. Our models position the hypomorphic mutations at direct contact points in the interface between human Cdt1 and human MCM complex. These results suggest human Cdt1 uses two domains to make multiple contacts with the MCM hexamer rather than the previously‐described single Cdt1‐MCM interaction site. Furthermore, this newly‐discovered MCM binding domain can explain how the Cdt1 inhibitor protein, geminin, blocks Cdt1‐MCM interaction.Surprisingly, one dwarfism allele (Cdt1‐A66T) is more rather than less active than WT Cdt1, reproducibly inducing four‐fold more DNA re‐replication when overexpressed to the same degree. A66 is located next to the S phase CDK binding motif, and its mutational alteration inhibits Cyclin A binding and subsequent recognition by the E3 ubiquitin ligase, SCFSkp2. This variant is not more stable however and has identical degradation and accumulation kinetics to WT Cdt1, presumably because the CRL4Cdt2 E3 ubiquitin ligase interaction is intact. We postulate that Cyclin A acts as a direct inhibitor of Cdt1‐MCM binding, suggesting a novel Cyclin‐dependent mechanism of Cdt1 inhibition at the end of S phase and prior to cell division. Together, these findings reveal additional roles of Cyclin/Cdk regulation of Cdt1, as well as newly described Cdt1‐MCM molecular interactions. Overall, our study identifies two new aspects of Cdt1 interactions that ensure efficient but once‐and‐only once genome duplication and normal cell proliferation.Support or Funding InformationThis work was supported by the National Institutes of Health award to J.G.C. R01GM102413 and R01GM102413‐S1 and to the University of North Carolina Flow Cytometry Core Facility award P30CA016086. P.N.P. is supported by the National Institutes of Health F31GM121073.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Animal cells transition between periods of active cell division cycles and periods of quiescence without cell division. Prior to cell division, DNA replication initiates at thousands of origins across the genome during S phase of every cell cycle. Each origin that initiates must have been licensed for replication in the preceding G1 phase by the regulated loading of Minichromosome maintenance (MCM) complexes onto DNA. Cells with insufficient MCM loading are underlicensed, risk incomplete replication, and are hypersensitive to replication stress and DNA damage. Licensing is tightly restricted to G1 phase, but the length of G1 varies. How mammalian cells ensure sufficient licensing in cells with either long or short G1 phases is still poorly understood. We recently reported that cycling cells with naturally short G1 phases license origins faster than cells with long G1s and that the fast loading is important for a short G1. The first G1 phase of cells re‐entering the cell cycle from quiescence is longer than G1 phase in actively cycling cells. We hypothesized that the differences in G1 length during cell cycle re‐entry and G1 length in dividing cells will result in differences in the amount or rate of origin licensing in the different G1 phases. We used single cell flow cytometry and live cell imaging to explore the relationships among cell cycle re‐entry, G1 length, origin licensing, and the activity of the family of cyclin‐dependent kinases that drive the G1‐to‐S phase transition in untransformed human cells. Despite the extra time in G1 phase, cells re‐entering the cell cycle are significantly underlicensed in the first cell cycle but not the second or subsequent cell cycles. Moreover, cells in the first S phase are hypersensitive to replication stress. Thus, G1 phase upon cell cycle re‐entry is characterized by slow (or delayed) origin licensing combined with S phase entry before all cells are fully licensed. Overproducing cyclin E or cyclin A can uncouple G1 length from origin licensing by driving premature S phase entry. We predict that the normal coupling of origin licensing and Cdk2 activation is weaker at the first G1/S transition. Cells re‐entering the cell cycle from quiescence have increased levels of Cdk inhibitor proteins which may contribute to the uncoupling of licensing timing or speed and S phase entry. The transition from quiescence to active division is a particularly sensitive time for DNA damage and the number of transitions into and out of quiescence may play a role in long term cell viability and genome stability.Support or Funding InformationThis work was supported by a fellowship from the NSF (DGE‐1144081) to J.P.M and by the NIH to J.G.C. (GM083024 and GM102413). Additional funding was provided by the W.M. Keck Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

All cells respond to osmotic stress by implementing molecular signaling events to protect the organism. Failure to properly adapt can lead to pathologies such as hypertension and ischemia-reperfusion injury. Mitogen-activated protein kinases (MAPKs) are activated in response to osmotic stress, as well as by signals acting through G protein-coupled receptors (GPCRs). For proper adaptation, the action of these kinases must be coordinated. To identify second messengers of stress adaptation, we conducted a mass spectrometry-based global metabolomics profiling analysis, quantifying nearly 300 metabolites in the yeast S. cerevisiae. We show that three branched-chain amino acid (BCAA) metabolites increase in response to osmotic stress and require the MAPK Hog1. Ectopic addition of these BCAA derivatives promotes phosphorylation of the G protein α subunit and dampens G protein-dependent transcription, similar to that seen in response to osmotic stress. Conversely, genetic ablation of Hog1 activity or the BCAA-regulatory enzymes leads to diminished phosphorylation of Gα and increased transcription. Taken together, our results define a new class of candidate second messengers that mediate cross talk between osmotic stress and GPCR signaling pathways.

Cell proliferation is a fundamental requirement for organismal development and homeostasis. The mammalian cell division cycle is tightly controlled to ensure complete and precise genome duplication and segregation of replicated chromosomes to daughter cells. The onset of DNA replication marks an irreversible commitment to cell division, and the accumulated efforts of many decades of molecular and cellular studies have probed this cellular decision, commonly called the restriction point. Despite a long-standing conceptual framework of the restriction point for progression through G1 phase into S phase or exit from G1 phase to quiescence (G0), recent technical advances in quantitative single cell analysis of mammalian cells have provided new insights. Significant intercellular heterogeneity revealed by single cell studies and the discovery of discrete subpopulations in proliferating cultures suggests the need for an even more nuanced understanding of cell proliferation decisions. In this review, we describe some of the recent developments in the cell cycle field made possible by quantitative single cell experimental approaches.

KEAP1 is a substrate adaptor protein for a CUL3-based E3 ubiquitin ligase. Ubiquitylation and degradation of the antioxidant transcription factor NRF2 is considered the primary function of KEAP1; however, few other KEAP1 substrates have been identified. Because KEAP1 is altered in a number of human pathologies and has been proposed as a potential therapeutic target therein, we sought to better understand KEAP1 through systematic identification of its substrates. Toward this goal, we combined parallel affinity capture proteomics and candidate-based approaches. Substrate-trapping proteomics yielded NRF2 and the related transcription factor NRF1 as KEAP1 substrates. Our targeted investigation of KEAP1-interacting proteins revealed MCM3, an essential subunit of the replicative DNA helicase, as a new substrate. We show that MCM3 is ubiquitylated by the KEAP1-CUL3-RBX1 complex in cells and in vitro. Using ubiquitin remnant profiling, we identify the sites of KEAP1-dependent ubiquitylation in MCM3, and these sites are on predicted exposed surfaces of the MCM2-7 complex. Unexpectedly, we determined that KEAP1 does not regulate total MCM3 protein stability or subcellular localization. Our analysis of a KEAP1 targeting motif in MCM3 suggests that MCM3 is a point of direct contact between KEAP1 and the MCM hexamer. Moreover, KEAP1 associates with chromatin in a cell cycle-dependent fashion with kinetics similar to the MCM2-7 complex. KEAP1 is thus poised to affect MCM2-7 dynamics or function rather than MCM3 abundance. Together, these data establish new functions for KEAP1 within the nucleus and identify MCM3 as a novel substrate of the KEAP1-CUL3-RBX1 E3 ligase.

Abstract While the KEAP1-NRF2 axis is essential for maintaining redox homeostasis, whether KEAP1 has alternative functions and how this pathway crosstalks with other important cellular processes remains unknown. KEAP1 targets the NRF2 transcription factor for proteasomal degradation in a redox-sensitive manner. Thus, this pathway serves as the cell's primary response to elevated reactive oxygen species. Importantly, KEAP1-NRF2 are frequently mutated in cancer, most strikingly in non-small cell lung cancer, where KEAP1 or NRF2 are mutated in 20-30% of patient tumors. Though regulation of NRF2 has long been considered the only physiologically important role for the E3 ligase KEAP1, we have determined that KEAP1 binds the master cell cycle regulator, MCM3, a subunit of the hexameric DNA replication licensing complex, MCM2-7. Excitingly, our ubiquitination assay data establish MCM3 as a new substrate for KEAP1; however, KEAP1 intriguingly does not regulate total cellular levels of MCM3. Consistent with this, we determined that only a small pool of cellular MCM3 is bound to KEAP1, suggesting that KEAP1 may bind and ubiquitinate a highly specified pool of MCM3. To determine the function of KEAP1-dependent ubiquitination of MCM3, we recently applied a new proteomics technique to map the ubiquitinated residues within MCM3 and identify these lysines within the larger MCM2-7 helicase. This mapping and protein modeling has provided new insight into the structure-function relationship of ubiquitinated MCM3. As MCM2-7 chromatin loading is a highly coordinated, cell cycle-dependent process, we tested whether KEAP1 loaded concurrently onto chromatin. Strikingly, we found that KEAP1 indeed loads onto chromatin during G1 and unloads in late S phase in a similar fashion as the MCM complex, further suggesting KEAP1 regulates the function of this essential cell cycle regulator on chromatin. Given the role of MCM3 in cell cycle progression, we tested whether KEAP1 was required for normal G1 to S phase progression and saw that loss of KEAP1 retards S phase DNA synthesis, which is an MCM-dependent process. Intriguingly, primary, untransformed KEAP1 knockout fibroblasts show decreased growth and aberrant cell cycle patterns consistent with a defect in the G1 to S transition. Overall, these data suggest a novel function for KEAP1 in regulating the MCM complex and cell cycle progression. We postulate that KEAP1 promotes cell cycle progression in a redox-sensitive manner through its association with MCM3 and that this presents a novel mechanism by which cells may halt cell cycle to protect DNA from damage by reactive oxygen species. Citation Format: Kathleen M. Mulvaney, Jacob Matson, Feng Yan, Dennis Goldfarb, Jeannette Cook, Michael Benjamin Major. Elucidating the function of MCM3 ubiquitination by KEAP1: crosstalk between redox-sensing and cell cycle progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-081. doi:10.1158/1538-7445.AM2015-LB-081

Background: CRL4CDT2 mediates replication-coupled destruction during S phase. CRL4CDT2 substrates reaccumulate by an unexplored mechanism. Results: CDK1 activity blocks CRL4CDT2 by preventing chromatin recruitment of the substrate receptor, CDT2. Conclusion: CDK1 activity facilitates CRL4CDT2 substrate reaccumulation upon S phase exit; several of these substrates are then required for normal mitotic progression. Significance: We provide the first evidence that CDK1 regulates the activity of CRL4CDT2. Replication-coupled destruction of a cohort of cell cycle proteins ensures efficient and precise genome duplication. Three proteins destroyed during replication via the CRL4CDT2 ubiquitin E3 ligase, CDT1, p21, and SET8 (PR-SET7), are also essential or important during mitosis, making their reaccumulation after S phase a critical cell cycle event. During early and mid-S phase and during DNA repair, proliferating cell nuclear antigen (PCNA) loading onto DNA (PCNADNA) triggers the interaction between CRL4CDT2 and its substrates, resulting in their degradation. We have discovered that, beginning in late S phase, PCNADNA is no longer sufficient to trigger CRL4CDT2-mediated degradation. A CDK1-dependent mechanism that blocks CRL4CDT2 activity by interfering with CDT2 recruitment to chromatin actively protects CRL4CDT2 substrates. We postulate that deliberate override of replication-coupled destruction allows anticipatory accumulation in late S phase. We further show that (as for CDT1) de novo SET8 reaccumulation is important for normal mitotic progression. In this manner, CDK1-dependent CRL4CDT2 inactivation contributes to efficient transition from S phase to mitosis.

To the best of our knowledge, two phosphorylation sites have been reported previously, among 11 known Vaccinia virus phosphoproteins. Here, via phosphopeptide mass spectrometry, up to 189 phosphorylation sites were identified among 48 proteins in preparations of purified Vaccinia mature virus (MV). 8.5% of phospho-residues were pTyr. Viral phosphoproteins were found in diverse functional classes, including structural proteins, membrane proteins and RNA polymerase subunits. Among the nine identified membrane phosphoproteins, the sites in just one, namely A14L, were deduced to be internal with respect to the accompanying membrane. Examination of sites in known substrates of the Vaccinia-encoded protein kinase VPK2, indicated VPK2 to be a proline-dependent kinase. The MV phosphoproteome was enriched in potential substrates of cellular kinases belonging to the CDK2/CDK3, CK2, and p38 groups. Quantitative mass spectrometry identified several sites that became phosphorylated during intravirion kinase activation in vitro, each showing one of two distinct pH-dependency profiles.