Affinage

FBXL3

F-box/LRR-repeat protein 3 · UniProt Q9UKT7

Length
428 aa
Mass
48.7 kDa
Annotated
2026-04-28
27 papers in source corpus 11 papers cited in narrative 11 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

FBXL3 is the substrate-recognition subunit of the SCF(FBXL3) E3 ubiquitin ligase complex and a central regulator of mammalian circadian period, acting primarily through ubiquitin-dependent degradation of CRY1 and CRY2 transcriptional repressors. Structural studies show that FBXL3 captures CRY2 by inserting its C-terminal tail into the FAD-binding pocket while occluding the PER-binding interface, with FAD and PER proteins competing for CRY binding to gate ubiquitination; SCF(FBXL3) complex assembly itself is substrate-dependent, requiring CRY interaction for efficient SKP1–CUL1 recruitment (PMID:23503662, PMID:24085301). Beyond the clock, CRY proteins serve as substrate-recruiting cofactors that direct FBXL3-mediated ubiquitination toward non-clock targets: CRY2 specifically recruits phospho-T58 c-MYC for degradation, and both CRYs recruit the kinase TLK2 (PMID:27840026, PMID:30655559). FBXL3 also ubiquitinates CRY-independent substrates including TCF12 in muscle satellite cells to restrain myogenic differentiation, and GLDC in tumor cells—following SRC-mediated Y306 phosphorylation of FBXL3—to promote K63-linked ubiquitination that suppresses MHC-I transcription and facilitates immune evasion (PMID:40755783, PMID:41728086).

Mechanistic history

Synthesis pass · year-by-year structured walk · 9 steps
  1. 2007 High

    The identification of FBXL3 as the F-box protein responsible for CRY degradation established the first molecular link between targeted proteolysis and circadian period determination in mammals.

    Evidence ENU mutagenesis screen yielding Afh (C358S) mice with ~27-h periods, combined with CRY degradation assays in vivo and in vitro

    PMID:17463252

    Open questions at the time
    • Structural basis for FBXL3–CRY recognition unknown
    • Whether FBXL3 directly binds CRY or acts through intermediaries not resolved
    • Role of individual CRY paralogs in period determination unclear
  2. 2013 High

    Crystal structures of the FBXL3–SKP1–CRY2 complex resolved how FBXL3 recognizes CRY via FAD-pocket insertion and PER-interface burial, revealing a competitive regulatory mechanism where FAD and PER antagonize CRY ubiquitination.

    Evidence X-ray crystallography of apo CRY2, FAD-bound CRY2, and FBXL3–SKP1–CRY2 ternary complex with functional mutagenesis

    PMID:23503662

    Open questions at the time
    • Whether FAD/PER competition operates quantitatively in vivo not shown
    • Structural basis for CRY1 versus CRY2 specificity differences unknown
  3. 2013 High

    Genetic epistasis studies demonstrated that FBXL3 controls circadian period exclusively through CRY proteins and also regulates a second feedback loop involving Rev-Erbα:HDAC3, while revealing CRY-dosage-dependent and paralog-specific contributions to period setting.

    Evidence Combinatorial Fbxl3(Afh) × Cry1-KO, Cry2-KO, Cry1/2-DKO, and Rev-erbα-KO mice with behavioral and SCN bioluminescence assays

    PMID:23471982 PMID:23616524

    Open questions at the time
    • Whether FBXL3 directly targets Rev-Erbα or acts indirectly via CRY not distinguished
    • Mechanism of CRY1 versus CRY2 differential repressor potency unclear
  4. 2013 High

    The discovery that SCF(FBXL3) complex assembly requires CRY substrate binding revealed an unusual substrate-dependent activation mechanism for this E3 ligase.

    Evidence Co-immunoprecipitation and in vitro reconstitution showing FBXL3 fails to associate with SKP1/CUL1 without CRY1; CRY-binding-deficient FBXL3 mutant also fails to form SCF complex

    PMID:24085301

    Open questions at the time
    • Whether substrate-dependent assembly is unique to FBXL3 or shared among FBXL family members unknown
    • Structural mechanism of how CRY binding promotes SKP1/CUL1 recruitment not resolved
  5. 2016 High

    Showing that CRY2 (but not CRY1) recruits phospho-T58 c-MYC to SCF(FBXL3) for ubiquitination expanded the function of CRY proteins from clock substrates to substrate-recruiting cofactors with paralog-specific target selectivity.

    Evidence Co-immunoprecipitation, ubiquitination assays, siRNA knockdown, and phosphomimetic mutant analysis in mammalian cells

    PMID:27840026

    Open questions at the time
    • Structural basis for CRY2 specificity in c-MYC recruitment unknown
    • Full scope of CRY-dependent non-clock substrates not mapped
  6. 2019 Medium

    Identification of TLK2 as a second CRY-dependent substrate of SCF(FBXL3) demonstrated that the cofactor-mediated targeting mechanism generalizes beyond c-MYC.

    Evidence Affinity purification mass spectrometry, co-IP, and genetic overexpression/deletion of CRY1/CRY2

    PMID:30655559

    Open questions at the time
    • Ubiquitin chain linkage type on TLK2 not characterized
    • Physiological consequences of TLK2 regulation by FBXL3 not tested in vivo
    • Single-lab finding awaiting independent replication
  7. 2025 Medium

    Discovery that YAP-TEAD signaling transcriptionally upregulates FBXL3 to accelerate CRY degradation provided the first upstream regulatory axis linking mechanotransduction to circadian clock attenuation.

    Evidence RNA-seq, ATAC-seq, ChIP-PCR, and YAP-TEAD inhibitor treatment in iPSC embryoid bodies

    PMID:40413171

    Open questions at the time
    • Whether YAP-TEAD regulation of FBXL3 operates in adult SCN or peripheral tissues not tested
    • Direct versus indirect TEAD binding to FBXL3 promoter not fully resolved
  8. 2025 Medium

    Identification of TCF12 as a CRY-independent FBXL3 substrate in muscle satellite cells established FBXL3 as a negative regulator of myogenic differentiation, with in vivo loss-of-function enhancing muscle regeneration in dystrophic mice.

    Evidence Satellite cell-specific Fbxl3 knockout (Pax7-CreER), ubiquitination assays, ChIP-PCR, and AAV-mediated silencing in mdx mice

    PMID:40554051 PMID:40755783

    Open questions at the time
    • Whether TCF12 ubiquitination is K48-linked and proteasomally degraded not explicitly shown
    • Contribution of circadian CRY regulation versus TCF12 regulation to muscle phenotype not separated
    • Single-lab finding in one disease model
  9. 2026 Medium

    Demonstration that EGFR/SRC-mediated Y306 phosphorylation of FBXL3 enables K63-linked ubiquitination of nuclear GLDC to suppress MHC-I transcription revealed a non-degradative ubiquitin signaling function for FBXL3 in tumor immune evasion.

    Evidence Co-IP, K63-linkage-specific ubiquitination assays, site-directed mutagenesis, ChIP assays, and in vivo tumor models with anti-PD-1

    PMID:41728086

    Open questions at the time
    • Whether CRY proteins are involved in GLDC recruitment not addressed
    • K63-linked activity represents a new chain-type specificity for SCF(FBXL3) not reconciled with canonical K48-linked activity
    • Single-lab finding awaiting independent validation

Open questions

Synthesis pass · forward-looking unresolved questions
  • The full repertoire of FBXL3 substrates, the structural basis for CRY paralog-specific cofactor activity, and how FBXL3 switches between K48- and K63-linked ubiquitination remain unresolved.
  • Comprehensive substrate profiling (e.g., ubiquitin-remnant proteomics in FBXL3-depleted cells) not performed
  • No structural comparison of CRY1 vs CRY2 cofactor complexes with non-clock substrates
  • Mechanism governing ubiquitin chain-type specificity (K48 vs K63) unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140096 catalytic activity, acting on a protein 6
Localization
GO:0005634 nucleus 1
Pathway
R-HSA-392499 Metabolism of proteins 7 R-HSA-9909396 Circadian clock 4 R-HSA-168256 Immune System 1
Partners
CRY1CRY2CUL1GLDCMYCSKP1TCF12TLK2
Complex memberships
SCF(FBXL3)

Evidence

Reading pass · 11 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2007 FBXL3 (F-box protein with leucine-rich repeats) is required for CRY protein degradation in the mammalian circadian clock; the Afh mutation (Cys358Ser substitution) delays CRY protein degradation and results in ~27-hour free-running circadian rhythms in homozygous mice. ENU mutagenesis screen, in vivo circadian behavioral assays, Per2::Luciferase tissue slices, in vitro degradation assays Science High 17463252
2013 Crystal structures of CRY2 in apo, FAD-bound, and FBXL3-SKP1-complexed forms reveal that FBXL3 captures CRY2 by inserting its conserved C-terminal tail into the FAD-binding pocket and simultaneously burying the PER-binding interface; FAD and PER proteins can compete with FBXL3 for CRY2 binding, providing a regulatory mechanism for CRY ubiquitination. X-ray crystallography, in vitro binding assays, structural mutagenesis Nature High 23503662
2013 FBXL3 regulates two circadian feedback loops: it promotes CRY ubiquitination/degradation to control E-box-driven gene expression amplitude, and it regulates Rev-Erbα:HDAC3 corepressor complex activity to control RRE-mediated transcription; deletion of Rev-erbα in Fbxl3-deficient mice rescues the long-period phenotype. Genetic interaction screen with single and double mutant mice, circadian behavioral assays, transcriptional reporter assays Proceedings of the National Academy of Sciences of the United States of America High 23471982
2013 SCF(FBXL3) complex formation is substrate-dependent in vivo: Fbxl3 does not associate with Skp1 and Cul1 efficiently in cells unless its substrate CRY1 is co-expressed; a mutant Fbxl3 unable to bind CRY1 also fails to form an SCF complex, indicating CRY interaction is required for SCF(FBXL3) assembly. Co-immunoprecipitation in transfected mammalian cells, in vitro reconstitution with recombinant proteins, domain-swap analysis The Journal of biological chemistry High 24085301
2013 Stabilization of endogenous CRY proteins by the Fbxl3(Afh) mutation lengthens circadian period in a CRY-dosage-dependent manner in the SCN; CRY1 is more potent than CRY2 as a transcriptional repressor, and the Afh mutation acts exclusively through CRYs (has no effect in CRY1/CRY2 double-knockout SCN). Genetic epistasis using Fbxl3(Afh) combined with Cry1 and Cry2 knockout mice, SCN bioluminescence recording, wheel-running behavioral assays The Journal of neuroscience High 23616524
2016 CRY2 functions as an essential cofactor in the SCF(FBXL3) E3 ligase complex to recruit T58-phosphorylated c-MYC for ubiquitylation and degradation; CRY1 cannot substitute for CRY2 in this function, revealing substrate-specific cofactor roles for the two CRY paralogs. Co-immunoprecipitation, ubiquitination assays, protein degradation assays, siRNA knockdown, phosphomimetic mutants Molecular cell High 27840026
2019 Both CRY1 and CRY2 recruit the cell cycle kinase TLK2 (in a TLK2 kinase-activity-dependent manner) to SCF(FBXL3) for ubiquitination; CRY overexpression or deletion correspondingly decreases or increases TLK2 protein abundance. Affinity purification mass spectrometry, co-immunoprecipitation, genetic deletion and overexpression of CRY1/CRY2 Scientific reports Medium 30655559
2025 YAP-TEAD mechanosensing cascade directly transcriptionally upregulates FBXL3, which then promotes CRY protein degradation, attenuating circadian clock gene oscillations in iPSC embryoid bodies under shaking culture conditions. RNA-seq, ATAC-seq, ChIP-PCR, verteporfin (YAP-TEAD inhibitor) treatment, circadian reporter assays Cell death discovery Medium 40413171
2025 FBXL3 in muscle satellite cells promotes ubiquitination and degradation of TCF12, a transcription factor that activates MEF2C expression; loss of FBXL3 activates TCF12→MEF2C→myogenin axis, augmenting myogenic differentiation and muscle regeneration. Satellite cell-specific knockout (Pax7-CreER), RNA-seq, GSEA, ChIP-PCR, dual-luciferase reporter assay, co-immunoprecipitation/ubiquitination assays Frontiers in immunology Medium 40755783
2025 Satellite cell-specific deletion of FBXL3 in mdx mice increases myogenin expression and enhances muscle regeneration, identifying FBXL3 as a negative regulator of muscle repair; AAV-mediated FBXL3 silencing in gastrocnemius muscle also increases myogenin and muscle mass. Satellite cell-specific conditional knockout in mdx mice, AAV-mediated gene silencing, histological analysis, grip strength/endurance functional tests Biochemical and biophysical research communications Medium 40554051
2026 EGFR activation triggers SRC-mediated phosphorylation of FBXL3 at Y306, enabling nuclear FBXL3 to interact with GLDC and catalyze K63-linked polyubiquitination at GLDC K636; ubiquitinated GLDC recruits SMARCE1/DMAP1 to inhibit STAT1-driven MHC-I gene transcription, promoting tumor immune evasion. Co-immunoprecipitation, ubiquitination assays with K63-linkage-specific analysis, site-directed mutagenesis (Y306, K636), ChIP assays, in vivo tumor models with anti-PD-1 Cell insight Medium 41728086

Source papers

Stage 0 corpus · 27 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2007 The after-hours mutant reveals a role for Fbxl3 in determining mammalian circadian period. Science (New York, N.Y.) 387 17463252
2013 SCF(FBXL3) ubiquitin ligase targets cryptochromes at their cofactor pocket. Nature 192 23503662
2016 CRY2 and FBXL3 Cooperatively Degrade c-MYC. Molecular cell 168 27840026
1996 Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors. Journal of virology 112 8892898
2017 miR-181d and c-myc-mediated inhibition of CRY2 and FBXL3 reprograms metabolism in colorectal cancer. Cell death & disease 63 28749470
1993 Multiplicity of virus-encoded helper T-cell epitopes expressed on FBL-3 tumor cells. Journal of virology 56 7687300
2013 Distinct and separable roles for endogenous CRY1 and CRY2 within the circadian molecular clockwork of the suprachiasmatic nucleus, as revealed by the Fbxl3(Afh) mutation. The Journal of neuroscience : the official journal of the Society for Neuroscience 51 23616524
2013 Dual roles of FBXL3 in the mammalian circadian feedback loops are important for period determination and robustness of the clock. Proceedings of the National Academy of Sciences of the United States of America 41 23471982
2013 Substrate binding promotes formation of the Skp1-Cul1-Fbxl3 (SCF(Fbxl3)) protein complex. The Journal of biological chemistry 27 24085301
1993 Rejection of an IA+ variant line of FBL-3 leukemia by cytotoxic T lymphocytes with CD4+ and CD4-CD8- T cell receptor-alpha beta phenotypes generated in CD8-depleted C57BL/6 mice. Journal of immunology (Baltimore, Md. : 1950) 27 8496592
1994 Fine structure of a virus-encoded helper T-cell epitope expressed on FBL-3 tumor cells. Journal of virology 26 7525983
2019 The circadian E3 ligase complex SCFFBXL3+CRY targets TLK2. Scientific reports 25 30655559
2019 Biallelic variants in FBXL3 cause intellectual disability, delayed motor development and short stature. Human molecular genetics 18 30481285
2018 FBXL3 is regulated by miRNA-4735-3p and suppresses cell proliferation and migration in non-small cell lung cancer. Pathology, research and practice 15 30594330
1995 A single retroviral gag precursor signal peptide recognized by FBL-3 tumor-specific cytotoxic T lymphocytes. Journal of virology 13 7474084
1989 Characterization of a CD4(L3T4)-positive cytotoxic T cell clone that is restricted by class I major histocompatibility complex antigen on FBL-3 tumor cell. Immunobiology 13 2483152
2022 A Zebrafish Model for a Rare Genetic Disease Reveals a Conserved Role for FBXL3 in the Circadian Clock System. International journal of molecular sciences 8 35216494
2020 lncRNA CASC2 suppresses the growth of hemangioma cells by regulating miR-18a-5p/FBXL3 axis. Journal of biological regulators and homeostatic agents 7 32138500
1998 Induction of cross-reactivity in an endogenous viral peptide non-reactive to FBL-3 tumor-specific helper T-cell clones. Microbiology and immunology 4 9719100
1994 Effects of non-MHC background genes on the induction of CD4+ T cells that prevent rejection of a highly immunogenic tumor, FBL-3. International immunology 3 7916205
2025 Shaking culture attenuates circadian rhythms in induced pluripotent stem cells during osteogenic differentiation through the TEAD-Fbxl3-CRY axis. Cell death discovery 1 40413171
2024 [Tumor-associated fibroblasts promotes proliferation and migration of prostate cancer cells by suppressing FBXL3 via upregulating hsa-miR-18b-5p]. Nan fang yi ke da xue xue bao = Journal of Southern Medical University 1 39051074
2026 Phosphorylation of FBXL3 mediates GLDC polyubiquitination to suppress MHC-I expression and promote cancer immune evasion. Cell insight 0 41728086
2025 Fbxl3 deletion mitigates myopathy in mdx mice through upregulation of myogenin. Biochemical and biophysical research communications 0 40554051
2025 FBXL3 serves as a suppressor of regenerative myogenesis. Frontiers in immunology 0 40755783
2010 [Establishment of an erythroleukemia model in CB6F1 mice by transplant with haploidentical mouse leukemic cell line FBL-3]. Zhongguo shi yan xue ye xue za zhi 0 21129245
1980 Regulation of the in vitro secondary cell-mediated cytotoxic response against syngeneic FBL-3 leukemia by macrophages. Microbiology and immunology 0 6968022