Regulation of Interleukin-10 Receptor Ubiquitination and Stability by Beta-TrCP-Containing Ubiquitin E3 Ligase

Interleukin-10 (IL-10) initiates potent anti-inflammatory effects via activating its cell surface receptor, composed of IL-10R1 and IL-10R2 subunits. The level of IL-10R1 is a major determinant of the cells' responsiveness to IL-10. Here, via a series of biochemical analyses using 293T cells reconstituted with IL-10R1, we identify the latter as a novel substrate of βTrCP-containing ubiquitin E3 ligase. Within the intracellular tail of IL-10R1, a canonical (318DpSGFGpS) and a slightly deviated (369DpSGICLQEP) βTrCP recognition motif can additively recruit βTrCP in a phosphorylation-dependent manner. βTrCP recruitment leads to ubiquitination, endocytosis and degradation of IL-10R1, subsequently reducing the cellular responsiveness to IL-10. Our study uncovers a novel negative regulatory mechanism that may potentially affect IL-10 function in target cells under physiological or pathological conditions

βTrCP controls the lysosome-mediated degradation of CDK1, whose accumulation correlates with tumor malignancy

n mammals, cell cycle progression is controlled by cyclin-dependent kinases, among which CDK1 plays important roles in the regulation of the G2/M transition, G1 progression and G1/S transition. CDK1 is highly regulated by its association to cyclins, phosphorylation and dephosphorylation, changes in subcellular localization, and by direct binding of CDK inhibitor proteins. CDK1 steady-state protein levels are held constant throughout the cell cycle by a coordinated regulation of protein synthesis and degradation. We show that CDK1 is ubiquitinated by the E3 ubiquitin ligase SCFβTrCP and degraded by the lysosome. Furthermore, we found that DNA damage not only triggers the stabilization of inhibitory phosphorylation sites on CDK1 and repression of CDK1 gene expression, but also regulates βTrCP-induced CDK1 degradation in a cell type-dependent manner. Specifically, treatment with the chemotherapeutic agent doxorubicin in certain cell lines provokes CDK1 degradation and induces apoptosis, whereas in others it inhibits destruction of the protein. These observations raise the possibility that different tumor types, depending on their pathogenic spectrum mutations, may display different sensitivity to βTrCP-induced CDK1 degradation after DNA damage. Finally, we found that CDK1 accumulation in patients’ tumors shows a negative correlation with βTrCP and a positive correlation with the degree of tumor malignancy.España, Ministerio de Economía y Competitividad SAF2011-30003Junta de Andalucía, Dirección General de Investigación, Tecnología y Empresa P08- CVI-03603 and P10-CTS-6243

Small molecule and peptide inhibitors of βTrCP and the βTrCP–NRF2 protein–protein interaction

The E3 ligase beta-transducin repeat-containing protein (βTrCP) is an essential component of the ubiquitin–proteasome system that is responsible for the maintenance of cellular protein levels in human cells. Key target substrates for degradation include inhibitor of nuclear factor kappa B, programmed cell death protein 4 and forkhead box protein O3, alongside the transcription factor nuclear factor erythroid-2-related factor 2 (NRF2) that is responsible for cellular protection against oxidative damage. The tumour suppressive nature of many of its substrates and the overexpression of βTrCP observed in various cancers support a potential therapeutic role for inhibitors in the treatment of cancer. A small molecule substituted pyrazolone, GS143, and the natural product erioflorin have been identified as inhibitors of βTrCP and protect its targets from proteasomal degradation. Modified peptides based on the sequences of native substrates have also been reported with KD values in the nanomolar range. This review describes the current status of inhibitors of this E3 ligase. The scope for further inhibitor design and the development of PROTAC and molecular glue-type structures is explored in the context of βTrCP as an example of WD40 domain-containing proteins that are gaining attention as drug targets

The role of BST2/tetherin in feline retrovirus infection

Pathogenic retroviral infections of mammals have induced the evolution of cellular anti-viral restriction factors and have shaped their biological activities. This intrinsic immunity plays an important role in controlling viral replication and imposes a barrier to viral cross-species transmission. Well-studied examples of such host restriction factors are TRIM5α, an E3 ubiquitin ligase that binds incoming retroviral capsids in the cytoplasm via its C-terminal PRY/SPRY (B30.2) domain and targets them for proteasomal degradation, and APOBEC3 proteins, cytidine deaminases that induce hypermutation and impair viral reverse transcription. Tetherin (BST-2, CD317) is an interferon-inducible transmembrane protein that potently inhibits the release of nascent retrovirus particles in single-cycle replication assays. However, whether the primary biological activity of tetherin in vivo is that of a restriction factor remains uncertain as recent studies on human tetherin suggest that it is unable to prevent spreading infection of human immunodeficiency virus type 1 (HIV-1). The feline tetherin homologue resembles human tetherin in amino acid sequence, protein topology and anti-viral activity. Transiently expressed feline tetherin displays potent inhibition of feline immunodeficiency virus (FIV) and HIV-1 particle release. However, stable ectopic expression of feline tetherin in a range of feline cell lines has no inhibitory effect on the growth of either primary or cell culture-adapted strains of FIV. By comparing and contrasting the activities of the felid and primate tetherins against their respective immunodeficiency-causing lentiviruses we may gain insight into the contribution of tetherins to the control of lentiviral replication and the evolution of lentiviral virulence

Regulation of Autophosphorylation Controls PLK4 Self-Destruction and Centriole Number

The deposited article is a post-print version and has been submitted to peer review.This publication hasn't any creative commons license associated.This deposit is composed by the main article plus the supplementary materials of the publication.Polo-like kinase 4 (PLK4) is a major player in centriole biogenesis: in its absence centrioles fail to form, while in excess leads to centriole amplification. The SCF-Slimb/βTrCP-E3 ubiquitin ligase controls PLK4 levels through recognition of a conserved phosphodegron. SCF-Slimb/βTrCP substrate binding and targeting for degradation is normally regulated by phosphorylation cascades, controlling complex processes, such as circadian clocks and morphogenesis. Here, we show that PLK4 is a suicide kinase, autophosphorylating in residues that are critical for SCF-Slimb/βTrCP binding. We demonstrate a multisite trans-autophosphorylation mechanism, likely to ensure that both a threshold of PLK4 concentration is attained and a sequence of events is observed before PLK4 can autodestruct. First, we show that PLK4 trans-autophosphorylates other PLK4 molecules on both Ser293 and Thr297 within the degron and that these residues contribute differently for PLK4 degradation, the first being critical and the second maximizing auto-destruction. Second, PLK4 trans-autophosphorylates a phospho-cluster outside the degron, which regulates Thr297 phosphorylation, PLK4 degradation, and centriole number. Finally, we show the importance of PLK4-Slimb/βTrCP regulation as it operates in both soma and germline. As βTrCP, PLK4, and centriole number are deregulated in several cancers, our work provides novel links between centriole number control and tumorigenesis.Fundação para a Ciência e Tecnologia grants: (SFRH/BD/33213/2007, PTDC/SAU-OBD/73194/2006, PTDC/SAU-OBD/105616/2008); ERC grant:(ERC-2010-StG grant number 261344); EMBO installation grant.info:eu-repo/semantics/publishedVersio

Cigarette-Specific Carcinogen Induces Stabilization of DNA methyltransferases through AKT/GSK3β/βTrCP Pathway in Lung Cancer

[[abstract]]Background: Most lung cancer cases are caused by cigarette-specific carcinogens, such as nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Previous studies have shown that NNK induces promoter hypermethylation of several tumor suppressor genes (TSGs) in mouse models. However, the mechanism involved in the promoter hypermethylation induced by NNK remains unclear. DNA methylation is carried out by DNA methyltransferases (DNMTs), which have been shown to be overexpressed in human cancers including lung cancer. Purpose: Our previous study showed that smoking lung cancer patients have a significant high level of DNMTs expression. Therefore, the present study aims to investigate what mechanisms involved in DNMTs overexpression induced by a cigarette carcinogen, NNK, in lung cancer by cell, animal, and clinical models. Results: Western blot assays indicated that DNMT1 and DNMT3b increased after treated with pro-carcinogen, nicotine for 6 h in IMR90, A549, and H1299 lung cells. In addition, nicotine-derived carcinogen, NNK treatment for 2 h increased endogenous and exogenous DNMT1 protein levels but not mRNA expression level in A549 and H1299 lung cells, suggesting that NNK-induced DNMT expression occurs at the post-translational level. Treatment with translation inhibitor cycloheximide with or without combining treatment of NNK confirmed that NNK indeed prolonged DNMT1 protein half-life. In addition, NNKactivated phosphorylation of AKT, NFκB, ERK1/2, and p38 pathways in IMR90 and A549 cells. Note that AKT pathway was significantly stimulated by NNK between 15 and 120 min in these cells. To evaluate the involvement of AKT signaling pathway in DNMT1 protein accumulation after NNK treatment, cells were treated with AKT inhibitor, LY294002, or AKT siRNA knock down oligos. The results indicated that NNK increased DNMT1 protein level can be abolished by both AKT inhibition treatments. In addition, immunoprecipitation assay suggested that NNK increased DNMT1 protein stability through AKT signaling pathway and was associated with ubiquitination protein degradation system. This conclusion was validated by combining treatment of AKT inhibitor and proteasome inhibitor MG132 in A549 and IMR90, which abolished the DNMT1 degradation induced by AKT inhibitor. Since AKT downstream GSK3β/βTrCP ubiqutin-proteasome pathway has been implicated in degradation of many proteins. We investigated whether GSK3β/βTrCP ubiqutin-proteasome pathway was involved in NNK-induced DNMT1 protein stability. We treated the cells with GSK3β inhibitor, SB415286, in the presence of NNK. In addition, cells overexpressing different levels of GSK3β or βTrCP construct were analyzed for DNMT1 protein level. The data indicated that NNK induced DNMT1 protein stability resulted from attenuation of GSK3β/βTrCP-mediated DNMT1 protein degradation system. In addition, we found that DNMT1 protein interacted with GSK3β and βTrCP by immunoprecipitation assay. Furthermore, chromatin-immunoprecipitationPCR and methylation-specific PCR assays showed that increased DNMT1 indeed bound to methylated TSG promoters after treated with NNK. In animal model data, immunohistochemical staining assay showed that NNK increased protein expression level of DNMT1, DNMT3B, p-AKT, and inactive form of p-GSK3β (ser9) in mice adenoma lung tissue treated with NNK. However, protein expression level of βTrCP was reduced in NNK-treated mice adenoma lung tissue. In clinical data, immunohistochemical staining for the DNMT1 protein expression was performed on 109 NSCLC tumor samples with smoking status data available. The results indicated that DNMT1 protein expression level was significantly higher in smoking patients compared to non-smoking patients including ex-smokers and never smokers (P<0.001). Interestingly, the DNMT1 protein of ex-smoke patients were expressed at a significantly lower level in tumor nuclear compared to that found in still-smoking patients (P=0.001). Conclusion: These data suggest that deregulation of DNMTs is associated with the NNK-induced DNMT stability by AKT/GSK3β/βTrCP pathway and results in epigenetic alteration of target TSGs and ultimately leads to lung cancer.

Die Rolle der AAA+ -ATPase p97 in der Regulation der Aktivierung des Zellzykluskontrollpunkt nach DNA Schaden

Der Zellzyklus ist ein streng regulierter Prozess, welcher die exakte Replikation und Segregation des genetisches Materials in zwei Tochterzellen reguliert. Um einen geordneten Verlauf durch den Zellzyklus und damit der Bewahrung der genomischen Integrität zu gewährleisten, enthält der Zellzyklus in Säugetieren sogenannte Kontrollpunkte. Die Aktivierung der Zellzyklus Kontrollpunkte hängt dabei nicht nur von zahlreichen Phosphorylierungsereignissen ab, sondern auch vom koordinierten Abbau der Zellzyklus regulierenden Proteine durch das Ubiquitin-Proteasom-System (UPS). Ein zentraler Regulator im Ubiquitin-Proteasome-System ist die AAA+-ATPase p97, welche ubiquitinierte Substrate erkennt und sie für den proteasomalen Abbau überführt. Zusammen mit dem Ko-Faktor Ufd1-Npl4, reguliert p97 zahlreiche Ereignisse innerhalb des Zellzyklus und beeinflusst dadurch den Fortschritt des Zellzyklus unter physiologischen Bedingungen als auch in Reaktion auf DNA Schaden. Die Depletierung von p97Ufd1-Npl4 führt zu schwerwiegenden Segregationsdefekten in der Mitose, welche nicht vollständig durch die Funktion von p97 in der Mitose erklärt werden können. Vielmehr kann eine Anzahl dieser Segregationsdefekte aus der Funktion von p97 in verschiedenen Interphase Prozessen resultieren. Daher versuchten wir im Rahmen dieser Arbeit die Relevanz der Interphaseprozesse, welche durch p97 reguliert werden, auf die Ausprägung von Segregationsdefekten in der Mitose zu untersuchen. Darüber hinaus untersuchten wir den molekularen Mechanismus des p97Ufd1-Npl4 Komplexes, welcher die Aktivität des G2/M Kontrollpunktes nach DNA Schaden in humanen, somatischen Zellen reguliert. In dieser Arbeit zeigen wir, dass p97 notwendig ist für die vollständige Aktivierung des G2/M Kontrollpunktes nach ionisierender Bestrahlung und den Eintritt in die Mitose in Anwesenheit von DNA Schaden und die daraus resultierende Ausprägung chromosomaler Instabilität verhindert. Die Depletion von p97Ufd1-Npl4 in bestrahlten Zellen führt zu einer Zunahme an Segregationsdefekten, die durch den Transfer von DNA Schäden in die Mitose entstanden sind. Durch die Analyse der Hauptregulatoren der DNA Schadensantwort, konnten wir einen verzögerten Abbau von CDC25A in p97Ufd1-Npl4 depletierten Zellen nachweisen. Darüber hinaus konnten wir mit quantitativen FACS Experimenten zeigen, dass p97Ufd1-Npl4 depletierte Zellen eine verminderte Aktivierung des DNA Schadenkontrollpunktes aufweisen und ein deutlicher Teil der Zellen sich trotz Bestrahlung in Mitose befindet. Ausschlaggebend für den Defekt in der Aktivierung des Kontrollpunktes ist dabei die Stabilisierung von CDC25A, da eine zusätzliche Inhibierung der CDC25 Phosphatasen den Kontrollpunkt vollständig wiederherstellt. Des Weiteren konnten wir zeigen, dass p97Ufd1-Npl4 mit dem F-box Protein βTrCP der SCFβTrCP E3 Ligase interagiert. Zusammenfassend etablieren unsere Ergebnisse eine neue Funktion des p97Ufd1-Npl4 Komplexes innerhalb der DNA Schadensantwort durch die Interaktion mit der SCFβTrCP E3 Ligase und der Vermittlung des Abbaus von ubiquitinierten CDC25A. Die vorliegende Arbeit belegt dadurch eine weitere wichtige Rolle von p97 in der Erhaltung der genomischen Stabilität und Integrität.The cell cycle is a tightly regulated process that governs faithful replication and segregation of the DNA material into two daughter cells during proliferation. In order to ensure ordered cell cycle progression, and thus maintain genomic integrity, the mammalian cell cycle harbours a number of checkpoints. The activation of cell cycle checkpoints as well as the regulation of the DNA damage response signalling pathway strongly depends not only on multiple phosphorylation events, but also on the timely degradation of cell cycle regulators by the ubiquitin-proteasome system (UPS). A central component of the UPS is the AAA+-type ATPase p97, which recognizes ubiquitylated substrates and targets them for proteasomal degradation. Together with one of its co-factors, Ufd1-Npl4, p97 acts at multiple stages during cell cycle progression, including mitosis and S phase, thus ensuring ordered progression under physiological conditions and, importantly, in response to DNA damage. Depletion of p97Ufd1-Npl4 leads to severe defects in chromosome segregation during mitosis, which might not be fully explained by its mitotic function but may as well be caused by its interphase functions. Therefore, this work aimed at elucidating the relevance of p97Ufd1-Npl4 interphase functions on the manifestation of chromosome segregation defects in mitosis. Furthermore, we investigated the molecular basis of the role of p97Ufd1-Npl4 complex in modulating the G2/M checkpoint after DNA damage induction in human somatic cells. Here, we show that p97 is required to ensure robust activation of the G2/M checkpoint after ionizing irradiation (IR), protecting cells from entering mitosis despite DNA damage and thus suppressing the manifestation of chromosomal instability. Depletion of p97Ufd1-Npl4 in cells exposed to IR, led to an increase in segregation defects, which are caused by pre-mitotic errors not being repaired before entering mitosis, indicating a defective DNA damage response. Indeed, analysing the key regulators and effectors of the DNA damage response pathway, we found a delayed CDC25A degradation in Ufd1-Npl4 depleted cells. Moreover, using a quantitative FACS approach, we showed that depletion of the p97Ufd1-Npl4 complex led to an impaired G2/M checkpoint after irradiation with a considerable fraction of mitotic cells. Importantly, we showed that p97Ufd1-Npl4 ensures proper degradation of CDC25A and that persistent activity of the stabilized CDC25A causes a negligent G2/M checkpoint activation, as this phenotype was rescued by additional inhibition of CDC25 phosphatases. Moreover, we showed that p97Ufd1-Npl4 physically interacts with the SCF E3 ligase F-box protein βTrCP, confirming the emerging evidence for the connection of p97 to multiple E3 ligase substrates. These results establish a novel function of the p97Ufd1-Npl4 complex in facilitating CDC25A degradation downstream of ubiquitination by βTrCP and highlight a crucial aspect of p97 function for maintaining genome stability and integrity

Interplay between Polo kinase, LKB1-activated NUAK1 kinase, PP1β^MYPT1phosphatase complex and the SCF^βTrCP E3 ubiquitin ligase

NUAK1 (NUAK family SnF1-like kinase-1) and NUAK2 protein kinases are activated by the LKB1 tumour suppressor and have been implicated in regulating multiple processes such as cell survival, senescence, adhesion and polarity. In the present paper we present evidence that expression of NUAK1 is controlled by CDK (cyclin-dependent kinase), PLK (Polo kinase) and the SCF(βTrCP) (Skp, Cullin and F-box(βTrCP)) E3 ubiquitin ligase complex. Our data indicate that CDK phosphorylates NUAK1 at Ser(445), triggering binding to PLK, which subsequently phosphorylates NUAK1 at two conserved non-catalytic serine residues (Ser(476) and Ser(480)). This induces binding of NUAK1 to βTrCP, the substrate-recognition subunit of the SCF(βTrCP) E3 ligase, resulting in NUAK1 becoming ubiquitylated and degraded. We also show that NUAK1 and PLK1 are reciprocally controlled in the cell cycle. In G(2)–M-phase, when PLK1 is most active, NUAK1 levels are low and vice versa in S-phase, when PLK1 expression is low, NUAK1 is more highly expressed. Moreover, NUAK1 inhibitors (WZ4003 or HTH-01-015) suppress proliferation by reducing the population of cells in S-phase and mitosis, an effect that can be rescued by overexpression of a NUAK1 mutant in which Ser(476) and Ser(480) are mutated to alanine. Finally, previous work has suggested that NUAK1 phosphorylates and inhibits PP1β(MYPT1) (where PP1 is protein phosphatase 1) and that a major role for the PP1β(MYPT1) complex is to inhibit PLK1 by dephosphorylating its T-loop (Thr(210)). We demonstrate that activation of NUAK1 leads to a striking increase in phosphorylation of PLK1 at Thr(210), an effect that is suppressed by NUAK1 inhibitors. Our data link NUAK1 to important cell-cycle signalling components (CDK, PLK and SCF(βTrCP)) and suggest that NUAK1 plays a role in stimulating S-phase, as well as PLK1 activity via its ability to regulate the PP1β(MYPT1) phosphatase