Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1 Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers.

Cell cycle gene expression programs fuel proliferation and are universally dysregulated in cancer. The retinoblastoma (RB)-family of proteins, RB1, RBL1/p107, and RBL2/p130, coordinately represses cell cycle gene expression, inhibiting proliferation, and suppressing tumorigenesis. Phosphorylation of RB-family proteins by cyclin-dependent kinases is firmly established. Like phosphorylation, ubiquitination is essential to cell cycle control, and numerous proliferative regulators, tumor suppressors, and oncoproteins are ubiquitinated. However, little is known about the role of ubiquitin signaling in controlling RB-family proteins. A systems genetics analysis of CRISPR/Cas9 screens suggested the potential regulation of the RB-network by cyclin F, a substrate recognition receptor for the SCF family of E3 ligases. We demonstrate that RBL2/p130 is a direct substrate of SCFcyclin F. We map a cyclin F regulatory site to a flexible linker in the p130 pocket domain, and show that this site mediates binding, stability, and ubiquitination. Expression of a mutant version of p130, which cannot be ubiquitinated, severely impaired proliferative capacity and cell cycle progression. Consistently, we observed reduced expression of cell cycle gene transcripts, as well a reduced abundance of cell cycle proteins, analyzed by quantitative, iterative immunofluorescent imaging. These data suggest a key role for SCFcyclin F in the CDK-RB network and raise the possibility that aberrant p130 degradation could dysregulate the cell cycle in human cancers.

Cullin-RING ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes, and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. We demonstrate that one substrate, NUSAP1, is an SCF(Cyclin F) substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology and provides a set of proteins likely to be key indicators of cellular physiology.

Methylation of histone H3 lysine 36 (H3K36me) by yeast Set2 is critical for the maintenance of chromatin structure and transcriptional fidelity. However, we do not know the full range of Set2/H3K36me functions or the scope of mechanisms that regulate Set2-dependent H3K36 methylation. Here, we show that the APC/CCDC20 complex regulates Set2 protein abundance during the cell cycle. Significantly, absence of Set2-mediated H3K36me causes a loss of cell cycle control and pronounced defects in the transcriptional fidelity of cell cycle regulatory genes, a class of genes that are generally long, hence highly dependent on Set2/H3K36me for their transcriptional fidelity. Because APC/C also controls human SETD2, and SETD2 likewise regulates cell cycle progression, our data imply an evolutionarily conserved cell cycle function for Set2/SETD2 that may explain why recurrent mutations of SETD2 contribute to human disease.

For chromosomes to congress and segregate during cell division, kinetochores must form stable attachments with spindle microtubules. We find that the centrosome protein, xCep57, localizes to kinetochores and interacts with the kinetochore proteins Zwint, Mis12, and CLIP-170. Immunodepletion of xCep57 from egg extracts yields weakened and elongated bipolar spindles which fail to align chromosomes. In the absence of xCep57, tension is lost between sister kinetochores, and spindle microtubules are no longer resistant to low doses of nocodazole. xCep57 inhibition on isolated mitotic chromosomes inhibits kinetochore-microtubule binding in vitro. xCep57 also interacts with gamma-tubulin. In xCep57 immunodepleted extracts, sperm centrosomes nucleate with normal kinetics, but are unable maintain microtubule anchorage. This characterization places xCep57 in a novel class of proteins required for stable microtubule attachments at the kinetochore and at the centrosome and suggests that the mechanism of microtubule binding at these two places is mechanistically similar.

The interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains. However, the potential coordination between these steps in ubiquitin chain formation remains undefined. While numerous studies have unveiled how RING E3s stimulate individual E2s for Ub transfer, here we change perspective to describe a case where the chain-elongating E2 UBE2S feeds back and directly stimulates the E3 APC/C to promote substrate priming and subsequent multiubiquitination by UBE2C. Our work reveals an unexpected model for the mechanisms of RING E3-dependent ubiquitination and for the diverse and complex interrelationship between components of the ubiquitination cascade.

Targeted protein degradation by the ubiquitin-proteasome system is an essential mechanism regulating cellular division. The kinase PLK1 coordinates protein degradation at the G2/M phase of the cell cycle by promoting the binding of substrates to the E3 ubiquitin ligase SCFβTrCP. However, the magnitude to which PLK1 shapes the mitotic proteome has not been characterized. Combining deep, quantitative proteomics with pharmacologic PLK1 inhibition (PLK1i), we identified more than 200 proteins whose abundances were increased by PLK1i at G2/M. We validate many new PLK1-regulated proteins, including several substrates of the cell cycle E3 SCFCyclin F, demonstrating that PLK1 promotes proteolysis through at least two distinct SCF-family E3 ligases. Further, we found that the protein kinase A anchoring protein AKAP2 is cell cycle regulated and that its mitotic degradation is dependent on the PLK1/βTrCP-signaling axis. Interactome analysis revealed that the strongest interactors of AKAP2 function in signaling networks regulating proliferation, including MAPK, AKT, and Hippo. Altogether, our data demonstrate that PLK1 coordinates a widespread program of protein breakdown at G2/M. We propose that dynamic proteolytic changes mediated by PLK1 integrate proliferative signals with the core cell cycle machinery during cell division. This has potential implications in malignancies where PLK1 is aberrantly regulated.

The outer kinetochore binds microtubules to control chromosome movement. Outer kinetochore assembly is restricted to mitosis, whereas the inner kinetochore remains tethered to centromeres throughout the cell cycle. The cues that regulate this transient assembly are unknown. We find that inhibition of Aurora B kinase significantly reduces outer kinetochore assembly in Xenopus laevis and human tissue culture cells, frog egg extracts, and budding yeast. In X. leavis M phase extracts, preassembled kinetochores disassemble after inhibiting Aurora B activity with either drugs or antibodies. Kinetochore disassembly, induced by Aurora B inhibition, is rescued by restraining protein phosphatase 1 (PP1) activity. PP1 is necessary for kinetochores to disassemble at the exit from M phase, and purified enzyme is sufficient to cause disassembly on isolated mitotic nuclei. These data demonstrate that Aurora B activity is required for kinetochore maintenance and that PP1 is necessary and sufficient to disassemble kinetochores. We suggest that Aurora B and PP1 coordinate cell cycle-dependent changes in kinetochore assembly though phosphorylation of kinetochore substrates.

E2F8 is a transcriptional repressor that antagonizes E2F1 at the crossroads of the cell cycle, apoptosis, and cancer. Previously, we discovered that E2F8 is a direct target of the APC/C ubiquitin ligase. Nevertheless, it remains unknown how E2F8 is dynamically controlled throughout the entirety of the cell cycle. Here, using newly developed human cell-free systems that recapitulate distinct inter-mitotic and G1 phases and a continuous transition from prometaphase to G1, we reveal an interlocking dephosphorylation switch coordinating E2F8 degradation with mitotic exit and the activation of APC/CCdh1. Further, we uncover differential proteolysis rates for E2F8 at different points within G1 phase, accounting for its accumulation in late G1 while APC/CCdh1 is still active. Finally, we demonstrate that the F-box protein Cyclin F regulates E2F8 in G2-phase. Altogether, our data define E2F8 regulation throughout the cell cycle, illuminating an extensive coordination between phosphorylation, ubiquitination and transcription in mammalian cell cycle.

The transcription factor FOXM1 contributes to cell cycle progression and is significantly upregulated in basal-like breast cancer (BLBC). Despite its importance in normal and cancer cell cycles, we lack a complete understanding of mechanisms that regulate FOXM1. We identified USP21 in an RNAi-based screen for deubiquitinases that control FOXM1 abundance. USP21 increases the stability of FOXM1, and USP21 binds and deubiquitinates FOXM1 in vivo and in vitro, indicating a direct enzyme-substrate relationship. Depleting USP21 downregulates the FOXM1 transcriptional network and causes a significant delay in cell cycle progression. Significantly, USP21 depletion sensitized BLBC cell lines and mouse xenograft tumors to paclitaxel, an anti-mitotic, frontline therapy in BLBC treatment. USP21 is the most frequently amplified deubiquitinase in BLBC patient tumors, and its amplification co-occurs with the upregulation of FOXM1 protein. Altogether, these data suggest a role for USP21 in the proliferation and potentially treatment of FOXM1-high, USP21-high BLBC.

Substrate polyubiquitination drives a myriad of cellular processes, including the cell cycle, apoptosis and immune responses. Polyubiquitination is highly dynamic, and obtaining mechanistic insight has thus far required artificially trapped structures to stabilize specific steps along the enzymatic process. So far, how any ubiquitin ligase builds a proteasomal degradation signal, which is canonically regarded as four or more ubiquitins, remains unclear. Here we present time-resolved cryogenic electron microscopy studies of the 1.2 MDa E3 ubiquitin ligase, known as the anaphase-promoting complex/cyclosome (APC/C), and its E2 co-enzymes (UBE2C/UBCH10 and UBE2S) during substrate polyubiquitination. Using cryoDRGN (Deep Reconstructing Generative Networks), a neural network-based approach, we reconstruct the conformational changes undergone by the human APC/C during polyubiquitination, directly visualize an active E3-E2 pair modifying its substrate, and identify unexpected interactions between multiple ubiquitins with parts of the APC/C machinery, including its coactivator CDH1. Together, we demonstrate how modification of substrates with nascent ubiquitin chains helps to potentiate processive substrate polyubiquitination, allowing us to model how a ubiquitin ligase builds a proteasomal degradation signal.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.

Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)-conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the N-terminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix-turn-helix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase-Promoting Complex/Cyclosome binding to the UBE2SHTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3-dependent activation mechanism. Altogether, we postulate the E2HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2-E3-mediated polyubiquitination.

Protein deubiquitinases play critical pathophysiological roles in cancer. Among all deubiquitinases, an oncogenic function for OTUD7B has been established in genetic NSCLC murine models. However, few deubiquitinase inhibitors have been developed due to technical challenges. Here, we report a putative small molecule OTUD7B inhibitor obtained from an AI-aided screen of a 4 million compound library. We validated the effects of the OTUD7B inhibitor (7Bi) in reducing Akt-pS473 signals in multiple NSCLC and HEK293 cells by blocking OTUD7B-governed GβL deubiquitination in cells, as well as inhibiting OTUD7B-mediated cleavage of K11-linked di-ub in an in vitro enzyme assay. Furthermore, we report in leukemia cells, either genetic depletion or 7Bi-mediated pharmacological inhibition of OTUD7B reduces Akt-pS473 via inhibiting the OTUD7B/GβL signaling axis. Together, our study identifies the first putative OTUD7B inhibitor showing activities both in cells and in vitro, with promising applications as a therapeutic agent in treating cancer with OTUD7B overexpression.

The oncogenic AKT kinase is a key regulator of apoptosis, cell growth, and cell-cycle progression. Despite its important role in proliferation, it remains largely unknown how AKT is mechanistically linked to the cell cycle. We show here that cyclin F, a substrate receptor F-box protein for the SCF (Skp1/Cul1/F-box) family of E3 ubiquitin ligases, is a bona fide AKT substrate. Cyclin F expression oscillates throughout the cell cycle, a rare feature among the 69 human F-box proteins, and all of its known substrates are involved in proliferation. AKT phosphorylation of cyclin F enhances its stability and promotes assembly into productive E3 ligase complexes. Importantly, expression of mutant versions of cyclin F that cannot be phosphorylated by AKT impair cell-cycle entry. Our data suggest that cyclin F transmits mitogen signaling through AKT to the core cell-cycle machinery. This discovery has potential implications for proliferative control in malignancies where AKT is activated.

Technological advances have enabled the analysis of cellular protein and RNA levels with unprecedented depth and sensitivity, allowing for an unbiased re-evaluation of gene regulation during fundamental biological processes. Here, we have chronicled the dynamics of protein and mRNA expression levels across a minimally perturbed cell cycle in human myeloid leukemia cells using centrifugal elutriation combined with mass spectrometry-based proteomics and RNA-Seq, avoiding artificial synchronization procedures. We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ∼6000 genes individually across the cell cycle, revealing complex, gene-specific patterns. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/). DOI: http://dx.doi.org/10.7554/eLife.01630.001.