Cdc4 Phosphodegron Research Articles

Abstract B007: Investigation of the mechanism of PADI2 regulation by the FBXW7 tumor suppressor

Abstract Serous endometrial cancer (SEC) is an aggressive form of cancer that frequently exhibits recurrent somatic mutations in FBXW7 at residues 465, 479, and 505. FBXW7 is a tumor suppressor and is part of a Skp1-Cullin-F-box containing (SCF) complex that targets various proteins, including certain oncoproteins, for ubiquitin-mediated proteasomal degradation. FBXW7 interacts with Cdc4 phosphodegron (CPD) motif(s) within substrate proteins. We previously reported upregulation of peptidyl arginine deiminase 2 (PADI2), a potentially druggable protein, in FBXW7-mutant SEC cell lines compared to the isogenic FBXW7-wildtype cells. The purpose of this study is to investigate the mechanism by which FBXW7 regulates PADI2 levels. We searched for the CPD motif in the PADI2 protein sequence. We used co-immunoprecipitation and immunoblotting to study FBXW7-PADI2 interactions after transiently transfecting HEK293 cells with constructs expressing MYC-tagged FBXW7 and/or HA-tagged PADI2, in the presence or absence of bortezomib. We identified three putative CPD motifs in PADI2, leading us to hypothesize that wildtype FBXW7 binds PADI2 and thus targets PADI2 for degradation via a proteasome-dependent mechanism. Our experimental data show that FBXW7 interacts with PADI2, in the presence of bortezomib, a proteasome inhibitor, in transiently transfected HEK293 cells. Additionally, we have shown that mutation of the putative CPD motifs of PADI2 abrogates the PADI2-FBXW7 interaction. Our findings implicate PADI2 as a novel substrate of the FBXW7 tumor suppressor. Citation Format: Chandra Mani Kafle, Daphne W. Bell. Investigation of the mechanism of PADI2 regulation by the FBXW7 tumor suppressor [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B007.

Negative feedback by conserved kinases patterns the degradation of Caenorhabditiselegans Raf in vulval fate patterning.

Activation of a canonical EGFR-Ras-Raf-ERK cascade initiates patterning of multipotent vulval precursor cells (VPCs) of Caenorhabditis elegans We have previously shown that this pathway includes a negative-feedback component in which MPK-1/ERK activity targets the upstream kinase LIN-45/Raf for degradation by the SEL-10/FBXW7 E3 ubiquitin ligase. This regulation requires a Cdc4 phosphodegron (CPD) in LIN-45 that is conserved in BRAF. Here, we identify and characterize the minimal degron that encompasses the CPD and is sufficient for SEL-10-mediated, MPK-1-dependent protein degradation. A targeted screen of conserved protein kinase-encoding genes yielded gsk-3 (an ortholog of human GSK3B) and cdk-2 (a CDK2-related kinase) as required for LIN-45 degron-mediated turnover. Genetic analysis revealed that LIN-45 degradation is blocked at the second larval stage due to cell cycle quiescence, and that relief of this block during the third larval stage relies on activation of CDKs. Additionally, activation of MPK-1 provides spatial pattern to LIN-45 degradation but does not bypass the requirement for gsk-3 and cdk-2 This analysis supports a model whereby MPK-1/ERK, GSK-3/GSK3 and CDK-2/CDK2, along with SEL-10/FBXW7, constitute a regulatory network that exerts spatial and temporal control of LIN-45/Raf degradation during VPC patterning.

SCFFbxw7 ubiquitylates KLF7 for degradation in a manner dependent on GSK-3-mediated phosphorylation.

The biological relation between ubiquitin ligases and their substrates has been largely unclear. We previously developed a method-differential proteomics-based identification of ubiquitylation substrates (DiPIUS)-for the comprehensive identification of substrates for a given ubiquitin ligase. We have now applied DiPIUS to the F-box protein Fbxw7 in three cell lines (mHepa, Neuro2A and C2C12) and thereby identified Krüppel-like factor 7 (KLF7) as a candidate substrate of the SCFFbxw7 ubiquitin ligase complex. KLF7 was shown to interact with Fbxw7 and to undergo Fbxw7-mediated polyubiquitylation. The stability of KLF7 was increased by depletion of Fbxw7, mutation of a putative Cdc4 phosphodegron (CPD) of KLF7 or exposure to inhibitors of glycogen synthase kinase-3 (GSK-3). Over-expression of Fbxw7 in Neuro2A cells down-regulated expression of the p21Cip1 gene, which is a transcriptional target of KLF7 in neuronal differentiation and maintenance. Despite the presence of an almost identical CPD sequence in KLF6, the closest paralog of KLF7, mutation of this sequence affected neither the interaction of KLF6 with Fbxw7 nor its half-life. Our results suggest that KLF7, but not KLF6, is a bona fide substrate of SCFFbxw7 , and that control of KLF7 abundance by SCFFbxw7 might contribute to the regulation of neuronal differentiation and maintenance.

Abstract 2825: PIN1-SCFFBW7 proteolytic signaling regulates the Fanconi anemia pathway

Abstract Genome instability caused by defective DNA repair mechanisms is a hallmark of cancer and drives tumorigenesis. The Fanconi anemia (FA) DNA repair pathway contributes to the integrity of the genome by resolving DNA interstrand cross-links (ICLs) encountered during DNA replication. Deregulation of the FA pathway is associated with cancer predisposition and affects therapeutic outcomes against DNA-damaging cytotoxic chemotherapy. FANCD2 monoubiquitination by a multi-subunit ubiquitin E3 ligase, the FA core complex, is an essential gateway that connects the DNA damage response to enzymatic steps of DNA ICL repair. We have previously identified FAAP20 as a critical component of the FA pathway; and demonstrated that the phosphorylation of the Cdc4 phospho-degron (CPD) motif in FAAP20 by GSK3β is required for its proteasomal degradation mediated by the E3 ligase, SCFFBW7 complex. However, the upstream signaling that governs the FAAP20 phosphorylation status and its detailed mode of action for FAAP20 degradation remain elusive. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, is a key regulator of the integrity of the FA core complex and, therefore, FA pathway activity as a whole. By catalyzing a proline cis-trans isomerization of the phosphorylated backbone and thereby converting the substrate into a conformation that is favorable or refractory to downstream proteolytic signaling, PIN1 modulates the rate of protein turnover. We demonstrate that PIN1 interacts and catalyzes a phosphorylated Ser48-Pro49 motif of FAAP20 and by inducing a conformational change of FAAP20, PIN1 enhances the affinity of FAAP20 with PP2A (Protein phosphatase 2), the phosphatase of the CPD motif in FAAP20 and thereby prevents its degradation from the GSK3β-FBW7 mediated proteolytic signaling. Collectively, our studies uncover PIN1-dependent isomerization as a new regulatory mechanism for DNA ICL repair. Given that PIN1 overexpression is prevalent in diverse human cancers, identifying a way to regulate PIN1 activity in the FA pathway may help develop a PIN1 inhibitor as a chemosensitizer for cytotoxic chemotherapy to increase its therapeutic efficacy. Citation Format: Jingming Wang, Bryan Chan, Michael Tong, Markus Seeliger, Hyungjin Kim. PIN1-SCFFBW7 proteolytic signaling regulates the Fanconi anemia pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2825.

FBW7 targets KLF10 for ubiquitin-dependent degradation

FBW7, a key component of SCFFBW7 E3 ubiquitin ligase, targets various proteins for degradation via the conserved Cdc4 phosphodegron (CPD) in substrates. In this study, we report that KLF10 is degraded by FBW7 via a conserved CPD. Through systematic analysis of the degradation of KLF transcription factors by FBW7, we identified KLF10 as a novel degradation target of FBW7. Ectopic expression of FBW7 markedly promoted the degradation of KLF10 while knockdown of endogenous FBW7 increased the protein levels of KLF10. In addition, simultaneous mutations of both threonine 82 (T82) and serine 86 (S86) significantly reduced the FBW7-mediated KLF10 degradation. Moreover, KLF10 containing a conserved putative CPD (TPPXSP) from amino acids 82 to 87, directly interacted with WD40 domain of FBW7 in a phosphorylation-dependent manner. Importantly, FBW7 could reverse the KLF10-mediated inhibition of Smad7 activity. Thus, our study uncovers a novel regulatory mechanism underlying which KLF10 stability and its biological function are mediated by FBW7.

Highlights of This Issue

Using fluorescence microscopy coupled with inhibitors/siRNA knockdowns, this study demonstrates that extracellular ATP is actively internalized by cancer cells through micropinocytosis and other endocytic processes in vitro and in vivo. This ATP internalization elevates the intracellular ATP levels by more than 50% within 24 hours. These new findings provide insight into key questions of the Warburg effect: why cancer cells upregulate glycolysis and how cancer cells manage to meet their energy (ATP) requirements even under hypoxic conditions. This ATP sharing among cancer cells performs multiple and previously unrecognized important functions and offers a novel anticancer target.Development of new androgen receptor (AR) inhibitors and recognition that AR is expressed in approximately 80% of all breast cancers (BCa) has led to revived interest in AR as a therapeutic target. However, the role of AR in estrogen receptor-positive (ER+) BCa is controversial. In multiple ER+/AR+ cell lines and patient-derived xenograft (PDX) models, the AR antagonist enzalutamide decreased estrogen-induced tumor growth, synergized with anti-estrogens tamoxifen and fulvestrant, and inhibited viability of tamoxifen-resistant tumors. Mechanistically, anti-androgens that inhibit AR nuclear localization diminished ER chromatin binding following estradiol treatment. The preclinical data support further testing of new generation AR antagonists in ER+ BCa, alone or in combination with ER-targeting agents, particularly in tumors with high AR or those resistant to anti-estrogens.Deregulated expression of CDX2 has differential effects ranging from tumor suppressive to oncogenic in colorectal cancers; however, the underlying mechanism has remained unexplored. Kumar and colleagues demonstrate that the E3 ubiquitin ligase, Fbw7, regulates CDX2 protein stability and functions in a GSK3β-dependent manner where phosphorylation of CDX2 at two potential Cdc4 Phosphodegron (CPD) motifs primes it to be recognized and ubiquitinated by Fbw7. Very importantly, the data also show that Fbw7-mediated ubiquitination of CDX2 can be antagonized by USP28. Collectively, these data provide a detailed molecular mechanism of CDX2 regulation by the Fbw7-GSK3β-USP28 axis which can be targeted in colorectal cancer for better therapeutics.The advent of new selective MMP9-inhibtory agents preluded a renaissance of MMP9 as a therapeutic target for aggressive gastrointestinal tumors. Here, a clinically relevant pancreatic cancer mouse model was used to determine the consequences of genetic ablation of MMP9. Selective long-term loss of MMP9 functions, dramatically promoted invasive growth of pancreatic cancer triggered by elevated systemic levels of IL-6. Abrogation of MMP9-dependent SCF-signaling in the bone marrow led it to secrete IL-6, which activated the pro-invasive IL-6/IL6R/STAT3 axis in pancreatic tumor cells. This fatal inter-organ communication represents an important caveat for the use of systemically administered selective MMP9 inhibitors in cancer.

Tumor Suppressor HIPK2 Regulates Malignant Growth via Phosphorylation of Notch1.

The receptor Notch1 plays an important role in malignant progression of many cancers, but its regulation is not fully understood. In this study, we report that the kinase HIPK2 is responsible for facilitating the Fbw7-dependent proteasomal degradation of Notch1 by phosphorylating its intracellular domain (Notch1-IC) within the Cdc4 phosphodegron motif. Notch1-IC expression was higher in cancer cells than normal cells. Under genotoxic stress, Notch1-IC was phosphorylated constitutively by HIPK2 and was maintained at a low level through proteasomal degradation. HIPK2 phosphorylated the residue T2512 in Notch1-IC. Somatic mutations near this residue rendered Notch1-IC resistant to degradation, as induced either by HIPK2 overexpression or adriamycin treatment. In revealing an important mechanism of Notch1 stability, the results of this study could offer a therapeutic strategy to block Notch1-dependent progression in many types of cancer. Cancer Res; 76(16); 4728-40. ©2016 AACR.

Regulation of BRAF protein stability by a negative feedback loop involving the MEK–ERK pathway but not the FBXW7 tumour suppressor

The V600EBRAF oncogenic mutation is detected in a wide range of human cancers and induces hyperactivation of the downstream MEK–ERK signalling cascade. Although output of the BRAF–MEK–ERK pathway is regulated by feed-forward RAF activity, feedback control also plays an important role. One such feedback pathway has been identified in Caenorhabditis elegans and involves ERK-mediated phosphorylation of BRAF within a CDC4 phosphodegron (CPD), targeting BRAF for degradation via CDC4 (also known as FBXW7), a component of the SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase complex. Here we investigate this pathway in mammalian cells. Short-term expression of autochthonous V600EBRAF in mouse embryonic fibroblasts (MEFs) leads to down-regulation of BRAF protein levels in a proteasome-dependent manner and V600EBRAF has a reduced half-life compared to WTBRAF in HEK293T cells. These effects were reversed by treatment with the MEK inhibitor PD184352. We have identified the equivalent CPD at residues 400–405 in human BRAF and have found that mutation of ERK phosphorylation sites at residues T401 and S405 in V600EBRAF increases the half-life of the protein. While BRAF and FBXW7 co-immunoprecipitated, the overexpression of FBXW7 did not influence the half-life of either WTBRAF or V600EBRAF. Furthermore, disruption of the substrate-binding site of mouse FBXW7 using the R482Q mutation did not affect the interaction with BRAF and the expression levels of WTBRAF and V600EBRAF were not altered in MEFs derived from mice with the homozygous knockin R482QFBXW7 mutation. Overall these data confirm the existence of a negative feedback pathway by which BRAF protein stability is regulated by ERK. However, unlike the situation in C. elegans, FBXW7 does not play a unique role in mediating subsequent BRAF degradation.

Ubiquitin ligase Fbw7 restricts the replication of hepatitis C virus by targeting NS5B for ubiquitination and degradation

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) and responsible for replicating the whole HCV genome with help of viral and cellular proteins. However, how cellular factors influence NS5B and, in turn, regulating HCV replication are still poorly defined. The well known tumor suppressor Fbw7, a component of E3 ubiquitin ligase SCFFbw7, targets oncoproteins or cellular regulatory proteins for ubiquitin-mediated degradation through a highly conserved binding site called a Cdc4 phosphodegron (CPD). But little is known about whether Fbw7 plays a role in regulation of viral proteins. In this study, we revealed that the conserved CPD is shared by NS5B of almost all genotype of HCV and our data demonstrated that NS5B is a bona fide substrate of Fbw7. Forced expression of Fbw7 promoted the ubiquination of NS5B and negatively regulated its turnover in the proteasome–dependent manner. We further revealed the interaction between NS5B and Fbw7, which resulted in the relocation of Fbw7 from nucleus to cytoplasm. During HCV replication, ectopic expression of Fbw7 could strongly down-regulate NS5B level and consequently inhibited the virus replication. When endogenous Fbw7 was knocked down, both NS5B protein abundance and HCV replication were remarkably up-regulated. The results provide more insights into the interplay of HCV and cellular factors and shed light on molecular mechanisms of HCV replication and pathogenesis.

Rictor Undergoes Glycogen Synthase Kinase 3 (GSK3)-dependent, FBXW7-mediated Ubiquitination and Proteasomal Degradation

Rictor, an essential component of mTOR complex 2 (mTORC2), plays a pivotal role in regulating mTOR signaling and other biological functions. Posttranslational regulation of rictor (e.g. via degradation) and its underlying mechanism are largely undefined and thus are the focus of this study. Chemical inhibition of the proteasome increased rictor ubiquitination and levels. Consistently, inhibition of FBXW7 with various genetic means including knockdown, knock-out, and enforced expression of a dominant-negative mutant inhibited rictor ubiquitination and increased rictor levels, whereas enforced expression of FBXW7 decreased rictor stability and levels. Moreover, we detected an interaction between FBXW7 and rictor. Hence, rictor is degraded through an FBXW7-mediated ubiquitination/proteasome mechanism. We show that this process is dependent on glycogen synthase kinase 3 (GSK3): GSK3 was associated with rictor and directly phosphorylated the Thr-1695 site in a putative CDC4 phospho-degron motif of rictor; mutation of this site impaired the interaction between rictor and FBXW7, decreased rictor ubiquitination, and increased rictor stability. Finally, enforced activation of Akt enhanced rictor levels and increased mTORC2 activity as evidenced by increased formation of mTORC2 and elevated phosphorylation of Akt, SGK1, and PKCα. Hence we suggest that PI3K/Akt signaling may positively regulate mTORC2 signaling, likely through suppressing GSK3-dependent rictor degradation.

Regulation of GATA-binding Protein 2 Levels via Ubiquitin-dependent Degradation by Fbw7: INVOLVEMENT OF CYCLIN B-CYCLIN-DEPENDENT KINASE 1-MEDIATED PHOSPHORYLATION OF THR176 IN GATA-BINDING PROTEIN 2

A GATA family transcription factor, GATA-binding protein 2 (GATA2), participates in cell growth and differentiation of various cells, such as hematopoietic stem cells. Although its expression level is controlled by transcriptional induction and proteolytic degradation, the responsible E3 ligase has not been identified. Here, we demonstrate that F-box/WD repeat-containing protein 7 (Fbw7/Fbxw7), a component of Skp1, Cullin 1, F-box-containing complex (SCF)-type E3 ligase, is an E3 ligase for GATA2. GATA2 contains a cell division control protein 4 (Cdc4) phosphodegron (CPD), a consensus motif for ubiquitylation by Fbw7, which includes Thr(176). Ectopic expression of Fbw7 destabilized GATA2 and promoted its proteasomal degradation. Substitution of threonine 176 to alanine in GATA2 inhibited binding with Fbw7, and the ubiquitylation and degradation of GATA2 by Fbw7 was suppressed. The CPD kinase, which mediates the phosphorylation of Thr(176), was cyclin B-cyclin-dependent kinase 1 (CDK1). Moreover, depletion of endogenous Fbw7 stabilized endogenous GATA2 in K562 cells. Conditional Fbw7 depletion in mice increased GATA2 levels in hematopoietic stem cells and myeloid progenitors at the early stage. Increased GATA2 levels in Fbw7-conditional knock-out mice were correlated with a decrease in a c-Kit high expressing population of myeloid progenitor cells. Our results suggest that Fbw7 is a bona fide E3 ubiquitin ligase for GATA2 in vivo.

Fbw7 targets GATA3 through cyclin-dependent kinase 2-dependent proteolysis and contributes to regulation of T-cell development.

Proper development of T cells depends on lineage-specific regulators controlled transcriptionally and posttranslationally to ensure precise levels at appropriate times. Conditional inactivation of F-box protein Fbw7 in mouse T-cell development resulted in reduced thymic CD4 single-positive (SP) and splenic CD4(+) and CD8(+) cell proportions. Fbw7 deficiency skewed CD8 SP lineage differentiation, which exhibited a higher incidence of apoptosis. Similar perturbations during development of CD8-positive cells were reported with transgenic mice, which enforced GATA3 (T-cell differentiation regulator) expression throughout T-cell development. We observed augmented GATA3 in CD4/CD8 double negative (DN) stage 4, CD4 SP, and CD8 SP lineages in Fbw7-deficient thymocytes. Using overexpressed proteins in cultured cells, we demonstrated that Fbw7 bound to, ubiquitylated, and destabilized GATA3. Two Cdc4 phosphodegron (CPD) candidate sequences, consensus Fbw7 recognition domains, were identified in GATA3, and phosphorylation of Thr-156 in CPD was required for Fbw7-mediated ubiquitylation and degradation. Phosphorylation of GATA3 Thr-156 was detected in mouse thymocytes, and cyclin-dependent kinase 2 (CDK2) was identified as a respondent for phosphorylation at Thr-156. These observations suggest that Fbw7-mediated GATA3 regulation with CDK2-mediated phosphorylation of CPD contributes to the precise differentiation of T-cell lineages.

The canonical wnt signal restricts the glycogen synthase kinase 3/fbw7-dependent ubiquitination and degradation of eya1 phosphatase.

Haploinsufficiency of Eya1 causes the branchio-oto-renal (BOR) syndrome, and abnormally high levels of Eya1 are linked to breast cancer progression and poor prognosis. Therefore, regulation of Eya1 activity is key to its tissue-specific functions and oncogenic activities. Here, we show that Eya1 is posttranslationally modified by ubiquitin and that its ubiquitination level is self-limited to prevent premature degradation. Eya1 has an evolutionarily conserved CDC4 phosphodegron (CPD) signal, a target site of glycogen synthase kinase 3 (GSK3) kinase and Fbw7 ubiquitin ligase, which is required for Eya1 ubiquitination. Genetic deletion of Fbw7 and pharmacological inhibition of GSK3 significantly decrease Eya1 ubiquitination. Conversely, activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the canonical Wnt signal suppresses Eya1 ubiquitination. Compound Eya1(+/-); Wnt9b(+/-) mutants exhibit an increased penetrance of renal defect, indicating that they function in the same genetic pathway in vivo. Together, these findings reveal that the canonical Wnt and PI3K/Akt signal pathways restrain the GSK3/Fbw7-dependent Eya1 ubiquitination, and they further suggest that dysregulation of this novel axis contributes to tumorigenesis.

SCF(FBXW7α) modulates the intra-S-phase DNA-damage checkpoint by regulating Polo like kinase-1 stability.

The intra-S-checkpoint is essential to control cell progression through S phase under normal conditions and in response to replication stress. When DNA lesions are detected, replication fork progression is blocked allowing time for repair to avoid genomic instability and the risk of cancer. DNA replication initiates at many origins of replication in eukaryotic cells, where a series of proteins form pre-replicative complexes (pre-RCs) that are activated to become pre-initiation complexes and ensure a single round of replication in each cell cycle. PLK1 plays an important role in the regulation of DNA replication, contributing to the regulation of pre-RCs formation by phosphorylating several proteins, under both normal and stress conditions. Here we report that PLK1 is ubiquitinated and degraded by SCFFBXW7α/proteasome. Moreover, we identified a new Cdc4 phosphodegron in PLK1, conserved from yeast to humans, whose mutation prevents PLK1 destruction. We established that endogenous SCFFBXW7α degrades PLK1 in the G1 and S phases of an unperturbed cell cycle and in S phase following UV irradiation. Furthermore, we showed that FBXW7α overexpression or UV irradiation prevented the loading of proteins onto chromatin to form pre-RCs and, accordingly, reduced cell proliferation. We conclude that PLK1 degradation mediated by SCFFBXW7α modulates the intra-S-phase checkpoint.

F-box and WD Repeat Domain-containing-7 (Fbxw7) Protein Targets Endoplasmic Reticulum-anchored Osteogenic and Chondrogenic Transcriptional Factors for Degradation

Although identification of substrates for an enzyme is a key step in elucidation of its biological functions, detection of the interaction between enzymes and substrates remains challenging. We recently developed a new approach, termed differential proteomics-based identification of ubiquitylation substrates (DiPIUS), for the discovery of substrates of ubiquitin ligases. We have now applied this approach to Fbxw7, the F-box protein component of an Skp1-Cul1-F-box protein-type ubiquitin ligase and, thereby, identified two similar transcription factors, old astrocyte specifically induced substance (OASIS) and BBF2 human homolog on chromosome 7 (BBF2H7), as candidate substrates. Coimmunoprecipitation analysis confirmed that the α and γ isoforms of Fbxw7 interact with OASIS and BBF2H7 in vivo. Sustained overexpression of Fbxw7 resulted in marked down-regulation of OASIS and BBF2H7, whereas RNAi-mediated Fbxw7 depletion stabilized both proteins. Mutation of a putative Cdc4 phosphodegron in OASIS and BBF2H7 attenuated their association with Fbxw7 and resulted in their stabilization. Depletion of Fbxw7 promoted the differentiation of mouse C2C12 mesenchymal cells into osteoblasts in association with the accumulation of OASIS. Conversely, overexpression of Fbxw7 in C2C12 cells resulted in down-regulation of Col1A1 mRNA, a target of OASIS. Conditional ablation of Fbxw7 in primary mouse mesenchymal cells promoted chondrogenesis in association with up-regulation of BBF2H7, whereas overexpression of Fbxw7 inhibited chondrogenesis in ATDC5 cells. Collectively, our results suggest that OASIS and BBF2H7 are bona fide substrates of Fbxw7 and that Fbxw7 controls osteogenesis and chondrogenesis by targeting OASIS and BBF2H7, respectively, for degradation.

Glycogen synthase kinase 3 regulates expression of nuclear factor-erythroid-2 related transcription factor-1 (Nrf1) and inhibits pro-survival function of Nrf1

Nuclear factor E2-related factor-1 (Nrf1) is a basic leucine zipper transcription factor that is known to regulate antioxidant and cytoprotective gene expression. It was recently shown that Nrf1 is regulated by SCF–Fbw7 ubiquitin ligase. However our knowledge of upstream signals that targets Nrf1 for degradation by the UPS is not known. We report here that Nrf1 expression is negatively regulated by glycogen synthase kinase 3 (GSK3) in Fbw7-dependent manner. We show that GSK3 interacts with Nrf1 and phosphorylates the Cdc4 phosphodegron domain (CPD) in Nrf1. Mutation of serine residue in the CPD of Nrf1 to alanine (S350A), blocks Nrf1 from phosphorylation by GSK3, and stabilizes Nrf1. Knockdown of Nrf1 and expression of a constitutively active form of GSK3 results in increased apoptosis in neuronal cells in response to ER stress, while expression of the GSK3 phosphorylation resistant S350A–Nrf1 attenuates apoptotic cell death. Together these data suggest that GSK3 regulates Nrf1 expression and cell survival function in response to stress activation.

SEL-10/Fbw7-dependent negative feedback regulation of LIN-45/Braf signaling in C. elegans via a conserved phosphodegron

The conserved E3 ubiquitin ligase component named SEL-10 in Caenorhabditis elegans and Fbw7 in mammals targets substrates for ubiquitin-mediated degradation through a high-affinity binding site called a Cdc4 phosphodegron (CPD). As many known substrates of Fbw7 are oncoproteins, the identification of new substrates may offer insight into cancer biology as well as aspects of proteome regulation. Here, we evaluated whether the presence of an evolutionarily conserved CPD would be a feasible complement to proteomics-based approaches for identifying new potential substrates. For functional assessments, we focused on LIN-45, a component of the signal transduction pathway underlying vulval induction and the ortholog of human Braf, an effector of Ras in numerous cancers. Our analysis demonstrates that LIN-45 behaves as a bona fide substrate of SEL-10, with mutation of the CPD or loss of sel-10 resulting in increased activity and protein stability in vivo. Furthermore, during vulval induction, the downstream kinase MPK-1/ERK is also required for LIN-45 protein degradation in a negative feedback loop, resulting in degradation of LIN-45 where ERK is highly active. As the CPD consensus sequence is conserved in human Braf, we propose that Fbw7 may also regulate Braf stability in some cell contexts. We discuss the implications of our findings for vulval development in C. elegans, the potential applicability to human Braf, and the value of a CPD-based predictive approach for human Fbw7 substrates.

Abstract 2034: The Fbw7 tumor suppressor: Genetic and biochemical approaches to study a dimeric ubiquitin ligase

Abstract Fbw7, the human ortholog of yeast Cdc4, is the substrate-recognition component of a cullin-RING ubiquitin ligase and regulates a number of oncoprotein substrates including cyclin E, c-Myc, Notch, and c-Jun. A growing number of additional pro-growth and pro-survival factors have been more recently described as Fbw7 targets. Accordingly, Fbw7 is a tumor suppressor and is mutated in approximately ten percent of human malignancies. Its loss leads to chromosomal instability and tumorigenesis. Fbw7 binds its substrates after they become phosphorylated within a consensus sequence called a Cdc4 phospho-degron (CPD). CPD phosphorylation creates a high-affinity binding site for Fbw7, which brings the ligase machinery into proximity with the substrate, leading to ubiquitylation and proteasomal degradation of the substrate. Interestingly, Fbw7 contains a conserved dimerization domain and we can show the protein primarily forms dimers in cells. One Fbw7 dimer contains two substrate-binding interfaces and may make two independent CPD contacts, but the physiologic function of Fbw7 dimerization is still elusive. Using gene-targeting, we studied the effects of ablating Fbw7 dimerization in vivo and found endogenous cyclin E and c-Myc regulation are moderately affected. Using purified Fbw7 monomers or dimers in in vitro ubiquitylation assays, we observed that a dimeric ligase is able to conjugate ubiquitin moieties onto a wider variety of substrate lysine residues than can the monomeric ligase. This is likely due to the greater spatial reach of the dimer. Additionally, preliminary data shows that the dimer may be capable of catalyzing the formation of non-K48-linked polyubiquitin chains, whereas the monomer is not. Cancer-associated mutations in Fbw7 most often target three arginine residues that form a pocket for binding phosphorylated substrate CPDs. Mutation of any of these arginines render Fbw7 unable to bind substrates and properly regulate their abundance. However, these mutations occur typically in only one allele. Dimerization of Fbw7 may explain why such heterozygous mutations are sufficient for tumorigenesis: the mutant form may dominantly inhibit enzyme function of the wild-type allele through dimerization. We have generated hetero-typic Fbw7 dimers containing one wild-type and one mutant protein in each dimer and are currently studying its binding and ubiquitylation capabilities. Given the importance of Fbw7 and its substrates in tumor-progression pathways, the results of this study will further our understanding of human tumor suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2034. doi:1538-7445.AM2012-2034

The Fbw7 Tumor Suppressor Regulates Nuclear Factor E2-related Factor 1 Transcription Factor Turnover through Proteasome-mediated Proteolysis

Nuclear factor E2-related factor 1 (Nrf1) is a basic leucine zipper transcription factor that plays important roles in cellular stress response and development. Currently, the mechanism regulating Nrf1 expression is poorly understood. We report here that Nrf1 is a short-lived protein that is targeted by F-box protein Fbw7, which is the substrate-specifying component of SCF (Skp1-Cul1-Fbox protein-Rbx1)-type ubiquitin ligase for degradation via the ubiquitin-proteasome pathway. We show that Fbw7 directly binds Nrf1 through a Cdc4 phosphodegron and that enforced expression of Fbw7 promotes the ubiquitination and degradation of Nrf1. Conversely, depletion of endogenous Fbw7 leads to decreased Nrf1 ubiquitination and accumulation of Nrf1 protein. Accordingly, expression of Fbw7 leads to down-regulation of antioxidant response element-driven gene activation, whereas disruption of Fbw7-mediated destabilization of Nrf1 leads to increased antioxidant response element-driven gene expression. Together, these data identify Fbw7 as a regulator of Nrf1 expression and reveal a novel function of Fbw7 in cellular stress response.

BCL-3 Degradation Involves Its Polyubiquitination through a FBW7-independent Pathway and Its Binding to the Proteasome Subunit PSMB1

The oncogenic protein BCL-3 activates or represses gene transcription through binding with the NF-kappaB proteins p50 and p52 and is degraded through a phospho- and GSK3-dependent pathway. However, the mechanisms underlying its degradation remain poorly understood. Yeast two-hybrid analysis led to the identification of the proteasome subunit PSMB1 as a BCL-3-associated protein. The binding of BCL-3 to PSMB1 is required for its degradation through the proteasome. Indeed, PSMB1-depleted cells are defective in degrading polyubiquitinated BCL-3. The N-terminal part of BCL-3 includes lysines 13 and 26 required for the Lys(48)-linked polyubiquitination of BCL-3. Moreover, the E3 ligase FBW7, known to polyubiquitinate a variety of substrates phosphorylated by GSK3, is dispensable for BCL-3 degradation. Thus, our data defined a unique motif of BCL-3 that is needed for its recruitment to the proteasome and identified PSMB1 as a key protein required for the proteasome-mediated degradation of a nuclear and oncogenic IkappaB protein.