Cullin-RING Ubiquitin Ligase Regulatory Circuits: A Quarter Century Beyond the F-Box Hypothesis.

Cullin-RING ubiquitin ligases (CRLs) are dynamic modular platforms that regulate myriad biological processes through target-specific ubiquitylation. Our knowledge of this system emerged from the F-box hypothesis, posited a quarter century ago: Numerous interchangeable F-box proteins confer specific substrate recognition for a core CUL1-based RING E3 ubiquitin ligase. This paradigm has been expanded through the evolution of a superfamily of analogous modular CRLs, with five major families and over 200 different substrate-binding receptors in humans. Regulation is achieved by numerous factors organized in circuits that dynamically control CRL activation and substrate ubiquitylation. CRLs also serve as a vast landscape for developing small molecules that reshape interactions and promote targeted ubiquitylation-dependent turnover of proteins of interest. Here, we review molecular principles underlying CRL function, the role of allosteric and conformational mechanisms in controlling substrate timing and ubiquitylation, and how the dynamics of substrate receptor interchange drives the turnover of selected target proteins to promote cellular decision-making.

Germline variants in tumor suppressor FBXW7 lead to impaired ubiquitination and a neurodevelopmental syndrome.

Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.

The F-box Protein KIB1 Mediates Brassinosteroid-Induced Inactivation and Degradation of GSK3-like Kinases in Arabidopsis.

The glycogen synthase kinase-3 (GSK3) family kinases are central cellular regulators highly conserved in all eukaryotes. In Arabidopsis, the GSK3-like kinase BIN2 phosphorylates a range of proteins to control broad developmental processes, and BIN2 is degraded through unknown mechanism upon receptor kinase-mediated brassinosteroid (BR) signaling. Here we identify KIB1 as an F-box E3 ubiquitin ligase that promotes the degradation of BIN2 while blocking its substrate access. Loss-of-function mutations of KIB1 and its homologs abolished BR-induced BIN2 degradation and caused severe BR-insensitive phenotypes. KIB1 directly interacted with BIN2 in a BR-dependent manner and promoted BIN2 ubiquitination in vitro. Expression of an F-box-truncated KIB1 caused BIN2 accumulation but dephosphorylation of its substrate BZR1 and activation of BR responses because KIB1 blocked BIN2 binding to BZR1. Our study demonstrates that KIB1 plays an essential role in BR signaling by inhibiting BIN2 through dual mechanisms of blocking substrate access and promoting degradation.

Potential role of Fbxo22 in resistance to endocrine therapy in breast cancer with invasive lobular carcinoma.

BACKGROUND: Invasive lobular carcinoma (ILC) is distinct from invasive ductal carcinoma (IDC) in terms of their hormonal microenvironments that may require different therapeutic strategies. We previously reported that selective estrogen receptor modulator (SERM) function requires F-box protein 22 (Fbxo22). Here, we investigated the role of Fbxo22 as a potential biomarker contributing to the resistance to endocrine therapy in ILC. METHODS: A total of 302 breast cancer (BC) patients including 150 ILC were recruited in the study. Fbxo22 expression and clinical information were analyzed to elucidate whether Fbxo22 negativity could be a prognostic factor or there were any correlations among clinical variables and SERM efficacy. RESULTS: Fbxo22 negativity was significantly higher in ILC compared with IDC (58.0% vs. 27.0%, P < 0.001) and higher in postmenopausal patients than premenopausal patients (64.1% vs. 48.2%, P = 0.041). In the ILC cohort, Fbxo22-negative patients had poorer overall survival (OS) than Fbxo22-positive patients, with 10-year OS rates of 77.4% vs. 93.6% (P = 0.055). All patients treated with SERMs, Fbxo22 negativity resulted in a poorer outcome, with 10-year OS rates of 81.3% vs. 92.3% (P = 0.032). In multivariate analysis regarding recurrence-free survival (RFS) in ILC patients, Fbxo22 status was independently predictive of survival as well as lymph node metastasis. CONCLUSION: Fbxo22 negativity significantly impacts on survival in BC patients with IDC and ILC, and the disadvantage was enhanced among ILC postmenopausal women or patients treated with SERMs. The findings suggest that different therapeutic strategies might be needed according to the different histopathological types when considering adjuvant endocrine therapy.

Regulatory networks of the F-box protein FBX206 and OVATE family proteins modulate brassinosteroid biosynthesis to regulate grain size and yield in rice.

F-box proteins participate in the regulation of many processes, including cell division, development, and plant hormone responses. Brassinosteroids (BRs) regulate plant growth and development by activating core transcriptional and other multiple factors. In rice, OVATE family proteins (OFPs) participate in BR signalling and regulate grain size. Here we identified an F-box E3 ubiquitin ligase, FBX206, that acts as a negative factor in BR signalling and regulates grain size and yield in rice. Suppressed expression of FBX206 by RNAi leads to promoted plant growth and increased grain yield. Molecular analyses showed that the expression levels of BR biosynthetic genes were up-regulated, whereas those of BR catabolic genes were down-regulated in FBX206-RNAi plants, resulting in the accumulation of 28-homoBL, one of the bioactive BRs. FBX206 interacted with OsOFP8, a positive regulator in BR signalling, and OsOFP19, a negative regulator in BR signalling. SCFFBX206 mediated the degradation of OsOFP8 but suppressed OsOFP19 degradation. OsOFP8 interacted with OsOFP19, and the reciprocal regulation between OsOFP8 and OsOFP19 required the presence of FBX206. FBX206 itself was ubiquitinated and degraded, but interactions of OsOFP8 and OsOFP19 synergistically suppressed the degradation of FBX206. Genetic interactions indicated an additive effect between FBX206 and OsOFP8 and epistatic effects of OsOFP19 on FBX206 and OsOFP8. Our study reveals the regulatory networks of FBX206, OsOFP8, and OsOFP19 in BR signalling that regulate grain size and yield in rice.

The F-box protein ZmFBL41 negatively regulates disease resistance to Rhizoctonia solani by degrading the abscisic acid synthase ZmNCED6 in maize.

KEY MESSAGE: The maize F-box protein ZmFBL41 targets abscisic acid synthase 9-cis-epoxycarotenoid dioxygenase 6 for degradation, and this regulatory module is exploited by Rhizoctonia solani to promote infection. F-box proteins are crucial regulators of plant growth, development, and responses to abiotic and biotic stresses. Previous research identified the F-box gene ZmFBL41 as a negative regulator of maize (Zea mays) defenses against Rhizoctonia solani. However, the precise mechanisms by which F-box proteins mediate resistance to R. solani remain poorly understood. In this study, we show that ZmFBL41 interacts with an abscisic acid (ABA) synthase, 9-cis-epoxycarotenoid dioxygenase 6 (ZmNCED6), promoting its degradation via the ubiquitination pathway. We discovered that the ectopic overexpression of ZmNCED6 in rice (Oryza sativa) inhibited R. solani infection by activating stomatal closure, callose deposition, and jasmonic acid (JA) biosynthesis, indicating that ZmNCED6 enhances plant immunity against R. solani. Natural variation at ZmFBL41 across different maize haplotypes did not affect the ZmFBL41-ZmNCED6 interaction. These findings suggest that ZmFBL41 targets ZmNCED6 for degradation, leading to a decrease in ABA levels in maize, in turn, inhibiting ABA-mediated disease resistance pathways, such as stomatal closure, callose deposition, and JA biosynthesis, ultimately facilitating R. solani infection.

The E3 ligase subunit FBXO45 binds the interferon-λ receptor and promotes its degradation during influenza virus infection.

Influenza remains a major public health challenge, as the viral infection activates multiple biological networks linked to altered host innate immunity. Following infection, IFN-λ, a ligand crucial for the resolution of viral infections, is known to bind to its cognate receptor, IFNLR1, in lung epithelia. However, little is known regarding the molecular expression and regulation of IFNLR1. Here, we show that IFNLR1 is a labile protein in human airway epithelia that is rapidly degraded after influenza infection. Using an unbiased proximal ligation biotin screen, we first identified that the Skp-Cullin-F box E3 ligase subunit, FBXO45, binds to IFNLR1. We demonstrate that FBXO45, induced in response to influenza infection, mediates IFNLR1 protein polyubiquitination and degradation through the ubiquitin-proteasome system by docking with its intracellular receptor domain. Furthermore, we found ectopically expressed FBXO45 and its silencing in cells differentially regulated both IFNLR1 protein stability and interferon-stimulated gene expression. Mutagenesis studies also indicated that expression of a K319R/K320R IFNLR1 variant in cells exhibited reduced polyubiquitination, yet greater stability and proteolytic resistance to FBXO45 and influenza-mediated receptor degradation. These results indicate that the IFN-λ-IFNLR1 receptor axis is tightly regulated by the Skp-Cullin-F box ubiquitin machinery, a pathway that may be exploited by influenza infection as a means to limit antiviral responses.

Oxygen-dependent regulation of E3(SCF)ubiquitin ligases and a Skp1-associated JmjD6 homolog in development of the social amoeba Dictyostelium.

E3-SCF (Skp1/cullin-1/F-box protein) polyubiquitin ligases activate the proteasomal degradation of over a thousand proteins, but the evolutionary diversification of the F-box protein (FBP) family of substrate receptor subunits has challenged their elucidation in protists. Here, we expand the FBP candidate list in the social amoeba Dictyostelium and show that the Skp1 interactome is highly remodeled as cells transition from growth to multicellular development. Importantly, a subset of candidate FBPs was less represented when the posttranslational hydroxylation and glycosylation of Skp1 was abrogated by deletion of the O2-sensing Skp1 prolyl hydroxylase PhyA. A role for this Skp1 modification for SCF activity was indicated by partial rescue of development, which normally depends on high O2 and PhyA, of phyA-KO cells by proteasomal inhibitors. Further examination of two FBPs, FbxwD and the Jumonji C protein JcdI, suggested that Skp1 was substituted by other factors in phyA-KO cells. Although a double-KO of jcdI and its paralog jcdH did not affect development, overexpression of JcdI increased its sensitivity to O2. JcdI, a nonheme dioxygenase shown to have physiological O2 dependence, is conserved across protists with its F-box and other domains, and is related to the human oncogene JmjD6. Sensitization of JcdI-overexpression cells to O2 depended on its dioxygenase activity and other domains, but not its F-box, which may however be the mediator of its reduced levels in WT relative to Skp1 modification mutant cells. The findings suggest that activation of JcdI by O2 is tempered by homeostatic downregulation via PhyA and association with Skp1.

Fbxo22 inhibits metastasis in triple-negative breast cancer through ubiquitin modification of KDM5A and regulation of H3K4me3 demethylation.

The importance of Fbxo22 in carcinogenesis has been highly documented. Here, we discussed downstream regulatory factors of Fbxo22 in TNBC. RNA-sequencing was conducted for identifying differentially expressed genes, followed by construction of a regulatory network. Expression patterns of Fbxo22/KDM5A in TNBC were determined by their correlation with the prognosis analyzed. Then, regulation mechanisms between Fbxo22 and KDM5A as well as between KDM5A and H3K4me3 were assayed. After silencing and overexpression experiments, the significance of Fbxo22 in repressing tumorigenesis in vitro and in vivo was explored. Fbxo22 was poorly expressed, while KDM5A was highly expressed in TNBC. Patients with elevated Fbxo22, decreased KDM5A, or higher p16 had long overall survival. Fbxo22 reduced the levels of KDM5A by ubiquitination. KDM5A promoted histone H3K4me3 demethylation to downregulate p16 expression. Fbxo22 reduced KDM5A expression to enhance p16, thus inducing DNA damage as well as reducing tumorigenesis and metastasis in TNBC. Our study validated FBXO22 as a tumor suppressor in TNBC through ubiquitination of KDM5A and regulation of p16.

Genome-Wide Analysis of the FBA Subfamily of the Poplar F-Box Gene Family and Its Role under Drought Stress.

F-box proteins are important components of eukaryotic SCF E3 ubiquitin ligase complexes, which specifically determine protein substrate proteasomal degradation during plant growth and development, as well as biotic and abiotic stress. It has been found that the FBA (F-box associated) protein family is one of the largest subgroups of the widely prevalent F-box family and plays significant roles in plant development and stress response. However, the FBA gene family in poplar has not been systematically studied to date. In this study, a total of 337 F-box candidate genes were discovered based on the fourth-generation genome resequencing of P. trichocarpa. The domain analysis and classification of candidate genes revealed that 74 of these candidate genes belong to the FBA protein family. The poplar F-box genes have undergone multiple gene replication events, particularly in the FBA subfamily, and their evolution can be attributed to genome-wide duplication (WGD) and tandem duplication (TD). In addition, we investigated the P. trichocarpa FBA subfamily using the PlantGenIE database and quantitative real-time PCR (qRT-PCR); the results showed that they are expressed in the cambium, phloem and mature tissues, but rarely expressed in young leaves and flowers. Moreover, they are also widely involved in the drought stress response. At last, we selected and cloned PtrFBA60 for physiological function analysis and found that it played an important role in coping with drought stress. Taken together, the family analysis of FBA genes in P. trichocarpa provides a new opportunity for the identification of P. trichocarpa candidate FBA genes and elucidation of their functions in growth, development and stress response, thus demonstrating their utility in the improvement of P. trichocarpa.

The F-box protein SHORT PRIMARY ROOT modulates primary root meristem activity by targeting SEUSS-LIKE protein for degradation in rice.

Root meristem activity is essential for root morphogenesis and adaptation, but the molecular mechanism regulating root meristem activity is not fully understood. Here, we identify an F-box family E3 ubiquitin ligase named SHORT PRIMARY ROOT (SHPR) that regulates primary root (PR) meristem activity and cell proliferation in rice. SHPR loss-of-function mutations impair PR elongation in rice. SHPR is involved in the formation of an SCF complex with the Oryza sativa SKP1-like protein OSK1/20. We show that SHPR interacts with Oryza sativa SEUSS-LIKE (OsSLK) in the nucleus and is required for OsSLK polyubiquitination and degradation by the ubiquitin 26S-proteasome system (UPS). Transgenic plants overexpressing OsSLK display a shorter PR phenotype, which is similar to the SHPR loss-of-function mutants. Genetic analysis suggests that SHPR promotes PR elongation in an OsSLK-dependent manner. Collectively, our study establishes SHPR as an E3 ubiquitin ligase that targets OsSLK for degradation, and uncovers a protein ubiquitination pathway as a mechanism for modulating root meristem activity in rice.

Host F-Box Protein 22 Enhances the Uptake of Brucella by Macrophages and Drives a Sustained Release of Proinflammatory Cytokines through Degradation of the Anti-Inflammatory Effector Proteins of Brucella.

Brucella species are intracellular bacterial pathogens, causing the worldwide zoonotic disease brucellosis. Brucella invades professional and nonprofessional phagocytic cells, followed by resisting intracellular killing and establishing a replication permissive niche. Brucella also modulates the innate and adaptive immune responses of the host for its chronic persistence. The complex intracellular cycle of Brucella depends in a major way on multiple host factors, but limited information is available on host and bacterial proteins that play an essential role in the invasion, intracellular replication, and modulation of host immune responses. By employing a small interfering RNA (siRNA) screening, we identified a role for the host protein FBXO22 in the Brucella-macrophage interaction. FBXO22 is the key element in the SCF E3 ubiquitination complex, where it determines the substrate specificity for ubiquitination and degradation of various host proteins. Downregulation of FBXO22 by siRNA or the CRISPR-Cas9 system resulted in diminished uptake of Brucella into macrophages, which was dependent on NF-κB-mediated regulation of phagocytic receptors. FBXO22 expression was upregulated in Brucella-infected macrophages, which resulted in induction of phagocytic receptors and enhanced production of proinflammatory cytokines through NF-κB. Furthermore, we found that FBXO22 recruits the effector proteins of Brucella, including the anti-inflammatory proteins TcpB and OMP25, for degradation through the SCF complex. We did not observe any role for another F-box-containing protein of the SCF complex, ß-TrCP, in the Brucella-macrophage interaction. Our findings unravel novel functions of FBXO22 in host-pathogen interaction and its contribution to pathogenesis of infectious diseases.

Structural basis for assembly of the CBF3 kinetochore complex.

Eukaryotic chromosomes contain a specialised region known as the centromere, which forms the platform for kinetochore assembly and microtubule attachment. The centromere is distinguished by the presence of nucleosomes containing the histone H3 variant, CENP-A. In budding yeast, centromere establishment begins with the recognition of a specific DNA sequence by the CBF3 complex. This in turn facilitates CENP-ACse4 nucleosome deposition and kinetochore assembly. Here, we describe a 3.6 Å single-particle cryo-EM reconstruction of the core CBF3 complex, incorporating the sequence-specific DNA-binding protein Cep3 together with regulatory subunits Ctf13 and Skp1. This provides the first structural data on Ctf13, defining it as an F-box protein of the leucine-rich-repeat family, and demonstrates how a novel F-box-mediated interaction between Ctf13 and Skp1 is responsible for initial assembly of the CBF3 complex.

The Kelch-F-box protein SMALL AND GLOSSY LEAVES 1 (SAGL1) negatively influences salicylic acid biosynthesis in Arabidopsis thaliana by promoting the turn-over of transcription factor SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1).

Salicylic acid (SA) is a key phytohormone regulating plant immunity. Although the transcriptional regulation of SA biosynthesis has been well-studied, its post-translational regulation is largely unknown. We report that a Kelch repeats-containing F-box (KFB) protein, SMALL AND GLOSSY LEAVES 1 (SAGL1), negatively influences SA biosynthesis in Arabidopsis thaliana by mediating the proteolytic turnover of SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1), a master transcription factor that directly drives SA biosynthesis during immunity. Loss of SAGL1 resulted in characteristic growth inhibition. Combining metabolomic, transcriptional and phenotypic analyses, we found that SAGL1 represses SA biosynthesis and SA-mediated immune activation. Genetic crosses to mutants that are deficient in SA biosynthesis blocked the SA overaccumulation in sagl1 and rescued its growth. Biochemical and proteomic analysis identified that SAGL1 interacts with SARD1 and promotes the degradation of SARD1 in a proteasome-dependent manner. These results unravelled a critical role of KFB protein SAGL1 in maintaining SA homeostasis via controlling SARD1 stability.

Fbxw17 is dispensable for viability and fertility in mice.

BACKGROUND: Skp1-Cullin-F-box (SCF) E3 ligase complex plays an important role in regulating spermatogenesis and fertility in mice. As a member of F-box proteins, the function of F-box and WD-40 domain protein 17 (Fbxw17) during spermatogenesis and fertility is unclear. In this study, we illustrate its function for spermatogenesis and fertility. METHODS AND RESULTS: Here, we generated the Fbxw17 knockout (KO) mouse model by using the CRISPR/Cas9 system and analyzed the meiotic process and the fertility. Then, our results demonstrated that testis and sperm in the Fbxw17 KO mice had normal morphology. The testis weight, sperm count and fertility of Fbxw17 KO mice showed no significant difference compared with the wild-type mice. Subsequently, histological analysis of Fbxw17 KO mice revealed apparently normal germ cells of all stages and mature spermatozoa. Meanwhile, nuclear spread analysis showed that the synaptonemal complex formation and DSB repair proceeded normally in Fbxw17-deficient spermatocytes. Furthermore, we didn't find defects in the meiotic prophase I spermatocytes and germ cells showed no apparent apoptosis in Fbxw17 KO mice. CONCLUSIONS: Our results show that Fbxw17 is dispensable for fertility in mice.

F-box only protein 9 and its role in cancer.

The F-box proteins (FBP), substrate recognition subunit of the SCF (Skp1-Cullin1-F-box protein complex) E3 ligase, play important roles in the ubiquitylation and subsequent degradation of the target proteins from several cellular processes. Disorders of F-box protein-mediated proteolysis lead to human malignancies. FBP plays an important role in many cellular processes, including cell proliferation, cell cycle, apoptosis, migration, invasion, and metastasis, suggesting that it can be associated with tumorigenesis, cancer development and progression. However, the expression and function of FBXO9 (F-box only protein 9) differ in various types of human cancer. Due to the ability to regulate the stability and activity of oncogenes and tumor-suppressor genes, and the physiological functions of many of the F-box proteins remain subtle, further genetic and mechanistic studies will elaborate and help define FBXO9's role. Targeting F-box protein or F-box protein signaling pathways could be an effective strategy for preventing or treating human cancer. This review is presented to summarize the part of FBXO9 in different types of human cancer and its regulation mechanism, and to pave the way to design FBXO9-targeting anticancer therapies.

F-box protein 17 promotes glioma progression by regulating glycolysis pathway.

F-box protein 17 (FBXO17) is associated with high-grade glioma and acted as a promotor of glioma development. This study investigated the effect and underlying pathway of FBXO17 on glioma. The Cancer Genome Atlas database was applied to analyze FBXO17 expression information in glioma. First, high FBXO17 expressions are associated with glioma and poor prognosis. Then, FBXO17 was upregulated in glioma cells. Meanwhile, knock-down of FBXO17 inhibited cell proliferation, migration, and invasion, but increased the cell apoptosis. Besides, knock-down of FBXO17 inhibited mitochondrial membrane potential and increased reactive oxygen species. Furthermore, knock-down of FBXO17 decreased level of adenosine triphosphate, glucose, lactate, GLUT1, HK2, PFKP, PKM2, and LDHA. In conclusion, FBXO17 was high expression in glioma, and FBXO17 regulates glioma by regulating glycolysis pathway, providing novel theoretical for the treatment of glioma.

The proteasome regulator PI31 is required for protein homeostasis, synapse maintenance, and neuronal survival in mice.

Proteasome-mediated degradation of intracellular proteins is essential for cell function and survival. The proteasome-binding protein PI31 (Proteasomal Inhibitor of 31kD) promotes 26S assembly and functions as an adapter for proteasome transport in axons. As localized protein synthesis and degradation is especially critical in neurons, we generated a conditional loss of PI31 in spinal motor neurons (MNs) and cerebellar Purkinje cells (PCs). A cKO of PI31 in these neurons caused axon degeneration, neuronal loss, and progressive spinal and cerebellar neurological dysfunction. For both MNs and PCs, markers of proteotoxic stress preceded axonal degeneration and motor dysfunction, indicating a critical role for PI31 in neuronal homeostasis. The time course of the loss of MN and PC function in developing mouse central nervous system suggests a key role for PI31 in human neurodegenerative diseases.

MicroRNAs Are Involved in Regulating Plant Development and Stress Response through Fine-Tuning of TIR1/AFB-Dependent Auxin Signaling.

Auxin, primarily indole-3-acetic acid (IAA), is a versatile signal molecule that regulates many aspects of plant growth, development, and stress response. Recently, microRNAs (miRNAs), a type of short non-coding RNA, have emerged as master regulators of the auxin response pathways by affecting auxin homeostasis and perception in plants. The combination of these miRNAs and the autoregulation of the auxin signaling pathways, as well as the interaction with other hormones, creates a regulatory network that controls the level of auxin perception and signal transduction to maintain signaling homeostasis. In this review, we will detail the miRNAs involved in auxin signaling to illustrate its in planta complex regulation.

The F-box protein FBXL16 up-regulates the stability of C-MYC oncoprotein by antagonizing the activity of the F-box protein FBW7.

F-box proteins, such as F-box/WD repeat-containing protein 7 (FBW7), are essential components of the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. They bind to S-phase kinase-associated protein 1 (SKP1) through the F-box motif and deliver their protein substrate to the E3 ligase complex for ubiquitination and subsequent degradation. F-box and leucine-rich repeat protein 16 (FBXL16) is a poorly studied F-box protein. Because it does not interact with the scaffold protein cullin 1 (CUL1), we hypothesized that FBXL16 might not form a functional SCF-E3 ligase complex. In the present study, we found that FBXL16 up-regulates the levels of proteins targeted by SCF-E3 ligases, such as C-MYC, ß-catenin, and steroid receptor coactivator 3 (SRC-3). Focusing on C-MYC, a well-known oncoprotein overexpressed in most human cancers, we show that FBXL16 stabilizes C-MYC by antagonizing FBW7-mediated C-MYC ubiquitination and degradation. Further, we found that, although FBXL16 does not interact with CUL1, it interacts with SKP1 via its N-terminal F-box domain and with its substrate C-MYC via its C-terminal leucine-rich repeats (LRRs) domain. We found that both the F-box domain and the LRR domain are important for FBXL16-mediated C-MYC stabilization. In line with its role in up-regulating the levels of the C-MYC and SRC-3 oncoproteins, FBXL16 promoted cancer cell growth and migration and colony formation in soft agar. Our findings reveal that FBXL16 is an F-box protein that antagonizes the activity of another F-box protein, FBW7, and thereby increases C-MYC stability, resulting in increased cancer cell growth and invasiveness.