The PP2A-Integrator-CDK9 axis fine-tunes transcription and can be targeted therapeutically in cancer.

Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.

Selective PP2A Enhancement through Biased Heterotrimer Stabilization.

Impairment of protein phosphatases, including the family of serine/threonine phosphatases designated PP2A, is essential for the pathogenesis of many diseases, including cancer. The ability of PP2A to dephosphorylate hundreds of proteins is regulated by over 40 specificity-determining regulatory "B" subunits that compete for assembly and activation of heterogeneous PP2A heterotrimers. Here, we reveal how a small molecule, DT-061, specifically stabilizes the B56α-PP2A holoenzyme in a fully assembled, active state to dephosphorylate selective substrates, such as its well-known oncogenic target, c-Myc. Our 3.6 Å structure identifies molecular interactions between DT-061 and all three PP2A subunits that prevent dissociation of the active enzyme and highlight inherent mechanisms of PP2A complex assembly. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for therapeutic targeting, and aid in the development of phosphatase-based therapeutics tailored against disease specific phospho-protein targets.

Allosteric Activators of Protein Phosphatase 2A Display Broad Antitumor Activity Mediated by Dephosphorylation of MYBL2.

Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates.

B-Cell-Specific Diversion of Glucose Carbon Utilization Reveals a Unique Vulnerability in B Cell Malignancies.

B cell activation during normal immune responses and oncogenic transformation impose increased metabolic demands on B cells and their ability to retain redox homeostasis. While the serine/threonine-protein phosphatase 2A (PP2A) was identified as a tumor suppressor in multiple types of cancer, our genetic studies revealed an essential role of PP2A in B cell tumors. Thereby, PP2A redirects glucose carbon utilization from glycolysis to the pentose phosphate pathway (PPP) to salvage oxidative stress. This unique vulnerability reflects constitutively low PPP activity in B cells and transcriptional repression of G6PD and other key PPP enzymes by the B cell transcription factors PAX5 and IKZF1. Reflecting B-cell-specific transcriptional PPP-repression, glucose carbon utilization in B cells is heavily skewed in favor of glycolysis resulting in lack of PPP-dependent antioxidant protection. These findings reveal a gatekeeper function of the PPP in a broad range of B cell malignancies that can be efficiently targeted by small molecule inhibition of PP2A and G6PD.

IntS6 and the Integrator phosphatase module tune the efficiency of select premature transcription termination events.

The metazoan-specific Integrator complex catalyzes 3' end processing of small nuclear RNAs (snRNAs) and premature termination that attenuates the transcription of many protein-coding genes. Integrator has RNA endonuclease and protein phosphatase activities, but it remains unclear if both are required for complex function. Here, we show IntS6 (Integrator subunit 6) over-expression blocks Integrator function at a subset of Drosophila protein-coding genes, although having no effect on snRNAs or attenuation of other loci. Over-expressed IntS6 titrates protein phosphatase 2A (PP2A) subunits, thereby only affecting gene loci where phosphatase activity is necessary for Integrator function. IntS6 functions analogous to a PP2A regulatory B subunit as over-expression of canonical B subunits, which do not bind Integrator, is also sufficient to inhibit Integrator activity. These results show that the phosphatase module is critical at only a subset of Integrator-regulated genes and point to PP2A recruitment as a tunable step that modulates transcription termination efficiency.

SPT5 stabilizes RNA polymerase II, orchestrates transcription cycles, and maintains the enhancer landscape.

Transcription progression is governed by multitasking regulators including SPT5, an evolutionarily conserved factor implicated in virtually all transcriptional steps from enhancer activation to termination. Here we utilize a rapid degradation system and reveal crucial functions of SPT5 in maintaining cellular and chromatin RNA polymerase II (Pol II) levels. Rapid SPT5 depletion causes a pronounced reduction of paused Pol II at promoters and enhancers, distinct from negative elongation factor (NELF) degradation resulting in short-distance paused Pol II redistribution. Most genes exhibit downregulation, but not upregulation, accompanied by greatly impaired transcription activation, altered chromatin landscape at enhancers, and severe Pol II processivity defects at gene bodies. Phosphorylation of an SPT5 linker at serine 666 potentiates pause release and is antagonized by Integrator-PP2A (INTAC) targeting SPT5 and Pol II, while phosphorylation of the SPT5 C-terminal region links to 3' end termination. Our findings position SPT5 as an essential positive regulator of global transcription.

Protein phosphatases in the RNAPII transcription cycle: erasers, sculptors, gatekeepers, and potential drug targets.

The transcription cycle of RNA polymerase II (RNAPII) is governed at multiple points by opposing actions of cyclin-dependent kinases (CDKs) and protein phosphatases, in a process with similarities to the cell division cycle. While important roles of the kinases have been established, phosphatases have emerged more slowly as key players in transcription, and large gaps remain in understanding of their precise functions and targets. Much of the earlier work focused on the roles and regulation of sui generis and often atypical phosphatases-FCP1, Rtr1/RPAP2, and SSU72-with seemingly dedicated functions in RNAPII transcription. Decisive roles in the transcription cycle have now been uncovered for members of the major phosphoprotein phosphatase (PPP) family, including PP1, PP2A, and PP4-abundant enzymes with pleiotropic roles in cellular signaling pathways. These phosphatases appear to act principally at the transitions between transcription cycle phases, ensuring fine control of elongation and termination. Much is still unknown, however, about the division of labor among the PPP family members, and their possible regulation by or of the transcriptional kinases. CDKs active in transcription have recently drawn attention as potential therapeutic targets in cancer and other diseases, raising the prospect that the phosphatases might also present opportunities for new drug development. Here we review the current knowledge and outstanding questions about phosphatases in the context of the RNAPII transcription cycle.

Increases in cyclin A/Cdk activity and in PP2A-B55 inhibition by FAM122A are key mitosis-inducing events.

Entry into mitosis has been classically attributed to the activation of a cyclin B/Cdk1 amplification loop via a partial pool of this kinase becoming active at the end of G2 phase. However, how this initial pool is activated is still unknown. Here we discovered a new role of the recently identified PP2A-B55 inhibitor FAM122A in triggering mitotic entry. Accordingly, depletion of the orthologue of FAM122A in C. elegans prevents entry into mitosis in germline stem cells. Moreover, data from Xenopus egg extracts strongly suggest that FAM122A-dependent inhibition of PP2A-B55 could be the initial event promoting mitotic entry. Inhibition of this phosphatase allows subsequent phosphorylation of early mitotic substrates by cyclin A/Cdk, resulting in full cyclin B/Cdk1 and Greatwall (Gwl) kinase activation. Subsequent to Greatwall activation, Arpp19/ENSA become phosphorylated and now compete with FAM122A, promoting its dissociation from PP2A-B55 and taking over its phosphatase inhibition role until the end of mitosis.

CHK1 Inhibitor Blocks Phosphorylation of FAM122A and Promotes Replication Stress.

While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A/PABIR1 confers cellular resistance to CHK1 inhibitors (CHK1is) and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase that dephosphorylates the WEE1 protein and rescues WEE1 from ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1is. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1i plus a WEE1 inhibitor can overcome CHK1i resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1i sensitivity or resistance.

The diverging role of CDC14B: from mitotic exit in yeast to cell fate control in humans.

CDC14, originally identified as crucial mediator of mitotic exit in budding yeast, belongs to the family of dual-specificity phosphatases (DUSPs) that are present in most eukaryotes. Contradicting data have sparked a contentious discussion whether a cell cycle role is conserved in the human paralogs CDC14A and CDC14B but possibly masked due to redundancy. Subsequent studies on CDC14A and CDC14B double knockouts in human and mouse demonstrated that CDC14 activity is dispensable for mitotic progression in higher eukaryotes and instead suggested functional specialization. In this review, we provide a comprehensive overview of our current understanding of how faithful cell division is linked to phosphorylation and dephosphorylation and compare functional similarities and divergences between the mitotic phosphatases CDC14, PP2A, and PP1 from yeast and higher eukaryotes. Furthermore, we review the latest discoveries on CDC14B, which identify this nuclear phosphatase as a key regulator of gene expression and reveal its role in neuronal development. Finally, we discuss CDC14B functions in meiosis and possible implications in other developmental processes.

A bifunctional kinase-phosphatase module balances mitotic checkpoint strength and kinetochore-microtubule attachment stability.

Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the kinetochore-microtubule error-correction pathway keeps this attachment process free from errors. We demonstrate here that the optimal strength and dynamics of these processes are set by a kinase-phosphatase pair (PLK1-PP2A) that engage in negative feedback from adjacent phospho-binding motifs on the BUB complex. Uncoupling this feedback to skew the balance towards PLK1 produces a strong checkpoint, hypostable microtubule attachments and mitotic delays. Conversely, skewing the balance towards PP2A causes a weak checkpoint, hyperstable microtubule attachments and chromosome segregation errors. These phenotypes are associated with altered BUB complex recruitment to KNL1-MELT motifs, implicating PLK1-PP2A in controlling auto-amplification of MELT phosphorylation. In support, KNL1-BUB disassembly becomes contingent on PLK1 inhibition when KNL1 is engineered to contain excess MELT motifs. This elevates BUB-PLK1/PP2A complex levels on metaphase kinetochores, stabilises kinetochore-microtubule attachments, induces chromosome segregation defects and prevents KNL1-BUB disassembly at anaphase. Together, these data demonstrate how a bifunctional PLK1/PP2A module has evolved together with the MELT motifs to optimise BUB complex dynamics and ensure accurate chromosome segregation.

PHOSPHATASE 2A dephosphorylates PHYTOCHROME INTERACTING FACTOR3 to modulate photomorphogenesis in Arabidopsis.

The phytochrome (phy) family of sensory photoreceptors modulates developmental programs in response to ambient light. Phys also control gene expression in part by directly interacting with the bHLH class of transcription factors, PHYTOCHROME-INTERACTING FACTORS (PIFs), and inducing their rapid phosphorylation and degradation. Several kinases have been shown to phosphorylate PIFs and promote their degradation. However, the phosphatases that dephosphorylate PIFs are less understood. Here, we describe four regulatory subunits of the Arabidopsis (Arabidopsis thaliana) protein PHOSPHATASE 2A (PP2A) family (B'α, B'ß, B''α and B''ß) that interact with PIF3 in yeast two-hybrid, in vitro and in vivo assays. The pp2ab''αß and b''αß/b'αß mutants displayed short hypocotyls, while the overexpression of the B subunits induced longer hypocotyls compared to the wild type under red light. The light-induced degradation of PIF3 was faster in the b''αß/b'αß quadruple mutant compared to in the wild type. Consistently, immunoprecipitated PP2A A and B subunits directly dephosphorylated PIF3-MYC in vitro. RNA-seq analyses showed that B''α and B''ß alter global gene expression in response to red light. PIFs (PIF1, PIF3, PIF4 and PIF5) are epistatic to these B subunits in regulating hypocotyl elongation under red light. Collectively, these data show an essential function of PP2A in dephosphorylating PIF3 to modulate photomorphogenesis in Arabidopsis.

Phosphorylated transcription factor PuHB40 mediates ROS-dependent anthocyanin biosynthesis in pear exposed to high light.

Plants are increasingly vulnerable to environmental stresses because of global warming and climate change. Stress-induced reactive oxygen species (ROS) accumulation results in plant cell damage and even cell death. Anthocyanins are important antioxidants that scavenge ROS to maintain ROS homeostasis. However, the mechanism underlying ROS-induced anthocyanin accumulation is unclear. In this study, we determined that the HD-Zip I family member transcription factor PuHB40 mediates ROS-dependent anthocyanin biosynthesis under high-light stress in pear (Pyrus ussuriensis). Specifically, PuHB40 induces the PuMYB123-like-PubHLH3 transcription factor complex for anthocyanin biosynthesis. PuHB40-mediated transcriptional activation depends on its phosphorylation level, which is regulated by protein phosphatase PP2A. Elevated ROS content maintains high PuHB40 phosphorylation levels, while also enhancing PuHB40-induced PuMYB123-like transcription by decreasing PuPP2AA2 expression, ultimately leading to increased anthocyanin biosynthesis. Our study reveals a pathway regulating ROS-induced anthocyanin biosynthesis in pear, further clarifying the mechanism underlying abiotic stress-induced anthocyanin biosynthesis, which may have implications for improving plant stress tolerance.

Chemogenetic profiling reveals PP2A-independent cytotoxicity of proposed PP2A activators iHAP1 and DT-061.

Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT-061 have recently been reported to selectively stabilize specific PP2A-B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT-061 on PP2A-B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome-wide CRISPR-Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT-061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT-061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A-B56 biology.

The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30.

Transcription of the Ebola virus genome depends on the viral transcription factor VP30 in its unphosphorylated form, but the underlying molecular mechanism of VP30 dephosphorylation is unknown. Here we show that the Ebola virus nucleoprotein (NP) recruits the host PP2A-B56 protein phosphatase through a B56-binding LxxIxE motif and that this motif is essential for VP30 dephosphorylation and viral transcription. The LxxIxE motif and the binding site of VP30 in NP are in close proximity, and both binding sites are required for the dephosphorylation of VP30. We generate a specific inhibitor of PP2A-B56 and show that it suppresses Ebola virus transcription and infection. This work dissects the molecular mechanism of VP30 dephosphorylation by PP2A-B56, and it pinpoints this phosphatase as a potential target for therapeutic intervention.

Phosphorylation Dynamics in a flg22-Induced, G Protein-Dependent Network Reveals the AtRGS1 Phosphatase.

The microbe-associated molecular pattern flg22 is recognized in a flagellin-sensitive 2-dependent manner in root tip cells. Here, we show a rapid and massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome in WT and a mutant deficient in heterotrimeric G-protein-coupled signaling. flg22-induced changes fall on proteins comprising a subset of this proteome, the heterotrimeric G protein interactome, and on highly-populated hubs of the immunity network. Approximately 95% of the phosphorylation changes in the heterotrimeric G-protein interactome depend, at least partially, on a functional G protein complex. One member of this interactome is ATBα, a substrate-recognition subunit of a protein phosphatase 2A complex and an interactor to Arabidopsis thaliana Regulator of G Signaling 1 protein (AtRGS1), a flg22-phosphorylated, 7-transmembrane spanning modulator of the nucleotide-binding state of the core G-protein complex. A null mutation of ATBα strongly increases basal endocytosis of AtRGS1. AtRGS1 steady-state protein level is lower in the atbα mutant in a proteasome-dependent manner. We propose that phosphorylation-dependent endocytosis of AtRGS1 is part of the mechanism to degrade AtRGS1, thus sustaining activation of the heterotrimeric G protein complex required for the regulation of system dynamics in innate immunity. The PP2A(ATBα) complex is a critical regulator of this signaling pathway.

The luciferase-based in vivo protein-protein interaction assay revealed that CHK1 promotes PP2A and PME-1 interaction.

Protein phosphatase 2A (PP2A) is an essential serine/threonine protein phosphatase, and its dysfunction is involved in the onset of cancer and neurodegenerative disorders. PP2A functions as a trimeric holoenzyme whose composition is regulated by the methyl-esterification (methylation) of the PP2A catalytic subunit (PP2Ac). Protein phosphatase methylesterase-1 (PME-1) is the sole PP2Ac methylesterase, and the higher PME-1 expression is observed in various cancer and neurodegenerative diseases. Apart from serving as a methylesterase, PME-1 acts as a PP2A inhibitory protein, binding directly to PP2Ac and suppressing its activity. The intricate function of PME-1 hinders drug development by targeting the PME-1/PP2Ac axis. This study applied the NanoBiT system, a bioluminescence-based protein interaction assay, to elucidate the molecular mechanism that modulates unknown PME-1/PP2Ac protein-protein interaction (PPI). Compound screening identified that the CHK1 inhibitors inhibited PME-1/PP2Ac association without affecting PP2Ac methylation levels. CHK1 directly phosphorylates PP2Ac to promote PME-1 association. Phospho-mass spectrometry identified multiple phospho-sites on PP2Ac, including the Thr219, that affect PME-1 interaction. An anti-phospho-Thr219 PP2Ac antibody was generated and showed that CHK1 regulates the phosphorylation levels of this site in cells. On the contrary, in vitro phosphatase assay showed that CHK1 is the substrate of PP2A, and PME-1 hindered PP2A-mediated dephosphorylation of CHK1. Our data provides novel insights into the molecular mechanisms governing the PME-1/PP2Ac PPI and the triad relationship between PP2A, PME-1, and CHK1.

Biochemical characterization of the Eya and PP2A-B55α interaction.

The eyes absent (Eya) proteins were first identified as co-activators of the six homeobox family of transcription factors and are critical in embryonic development. These proteins are also re-expressed in cancers after development is complete, where they drive tumor progression. We have previously shown that the Eya3 N-terminal domain (NTD) contains Ser/Thr phosphatase activity through an interaction with the protein phosphatase 2A (PP2A)-B55α holoenzyme and that this interaction increases the half-life of Myc through pT58 dephosphorylation. Here, we showed that Eya3 directly interacted with the NTD of Myc, recruiting PP2A-B55α to Myc. We also showed that Eya3 increased the Ser/Thr phosphatase activity of PP2A-B55α but not PP2A-B56α. Furthermore, we demonstrated that the NTD (∼250 amino acids) of Eya3 was completely disordered, and it used a 38-residue segment to interact with B55α. In addition, knockdown and phosphoproteomic analyses demonstrated that Eya3 and B55α affected highly similar phosphosite motifs with a preference for Ser/Thr followed by Pro, consistent with Eya3's apparent Ser/Thr phosphatase activity being mediated through its interaction with PP2A-B55α. Intriguingly, mutating this Pro to other amino acids in a Myc peptide dramatically increased dephosphorylation by PP2A. Not surprisingly, MycP59A, a naturally occurring mutation hotspot in several cancers, enhanced Eya3-PP2A-B55α-mediated dephosphorylation of pT58 on Myc, leading to increased Myc stability and cell proliferation, underscoring the critical role of this phosphosite in regulating Myc stability.

Structural characterization of methylation-independent PP2A assembly guides alphafold2Multimer prediction of family-wide PP2A complexes.

Dysregulation of phosphorylation-dependent signaling is a hallmark of tumorigenesis. Protein phosphatase 2 (PP2A) is an essential regulator of cell growth. One scaffold subunit (A) binds to a catalytic subunit (C) to form a core AC heterodimer, which together with one of many regulatory (B) subunits forms the active trimeric enzyme. The combinatorial number of distinct PP2A complexes is large, which results in diverse substrate specificity and subcellular localization. The detailed mechanism of PP2A assembly and regulation remains elusive and reports about an important role of methylation of the carboxy terminus of PP2A C are conflicting. A better understanding of the molecular underpinnings of PP2A assembly and regulation is critical to dissecting PP2A function in physiology and disease. Here, we combined biochemical reconstitution, mass spectrometry, X-ray crystallography, and functional assays to characterize the assembly of trimeric PP2A. In vitro studies demonstrated that methylation of the carboxy-terminus of PP2A C was dispensable for PP2A assembly in vitro. To corroborate these findings, we determined the X-ray crystal structure of the unmethylated PP2A Aα-B56ε-Cα trimer complex to 3.1 Å resolution. The experimental structure superimposed well with an Alphafold2Multimer prediction of the PP2A trimer. We then predicted models of all canonical PP2A complexes providing a framework for structural analysis of PP2A. In conclusion, methylation was dispensable for trimeric PP2A assembly and integrative structural biology studies of PP2A offered predictive models for all canonical PP2A complexes.

Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop.

The anaphase-promoting complex/cyclosome (APC/C), a multi-subunit ubiquitin ligase essential for cell cycle control, is regulated by reversible phosphorylation. APC/C phosphorylation by cyclin-dependent kinase 1 (Cdk1) promotes Cdc20 co-activator loading in mitosis to form active APC/C-Cdc20. However, detailed phospho-regulation of APC/C dynamics through other kinases and phosphatases is still poorly understood. Here, we show that an interplay between polo-like kinase (Plx1) and PP2A-B56 phosphatase on a flexible loop domain of the subunit Apc1 (Apc1-loop500 ) controls APC/C activity and mitotic progression. Plx1 directly binds to the Apc1-loop500 in a phosphorylation-dependent manner and promotes the formation of APC/C-Cdc20 via Apc3 phosphorylation. Upon phosphorylation of loop residue T532, PP2A-B56 is recruited to the Apc1-loop500 and differentially promotes dissociation of Plx1 and PP2A-B56 through dephosphorylation of Plx1-binding sites. Stable Plx1 binding, which prevents PP2A-B56 recruitment, prematurely activates the APC/C and delays APC/C dephosphorylation during mitotic exit. Furthermore, the phosphorylation status of the Apc1-loop500 is controlled by distant Apc3-loop phosphorylation. Our study suggests that phosphorylation-dependent feedback regulation through flexible loop domains within a macromolecular complex coordinates the activity and dynamics of the APC/C during the cell cycle.