SPRED3

SPRED3 is a SPRED-family negative regulator of receptor tyrosine kinase-driven Ras/RAF/MAPK (ERK) signaling whose suppressive activity is encoded by its C-terminal cysteine-rich Sprouty-related (SPR) domain rather than the kinase-binding domain, and which lacks a functional c-kit binding domain (PMID:12646235). SPRED3 dampens ERK activation downstream of growth factor receptors in several contexts: it suppresses FGF-induced ERK phosphorylation and lens fiber differentiation (PMID:29501879), and its loss activates the Ras/RAF/MAPK pathway to confer EGFR-TKI (erlotinib) resistance and increased migration in NSCLC cells, a phenotype reversed by ERK1/2 inhibition (PMID:35117614). The same SPR domain directs membrane localization (PMID:12646235) and mediates S-acylation by the palmitoyl acyltransferase zDHHC17 (HIP14) through a zDABM-independent interaction that does not require the enzyme's ankyrin-repeat domain (PMID:24705354, PMID:36442513). Beyond canonical MAPK suppression, SPRED3 has context-specific roles in the thyroid, where it acts as a positive regulator of NF-κB to promote thyroid cancer cell growth (PMID:39227612), and where its knockout in mice produces primary hypothyroidism accompanied by altered thyroidal ERK signaling and dysregulated autophagy markers (PMID:40806788).

1. 2003 Medium

   Established that SPRED3 is a brain-expressed MAPK suppressor and localized the suppressive and membrane-targeting activity to the SPR domain rather than the kinase-binding domain, distinguishing it functionally within the SPRED family.

   Evidence Chimeric Spred-3/Spred-1 protein analysis with overexpression and ERK activation assays; RT-PCR expression profiling

   PMID:12646235

   Open questions at the time

   • Membrane localization inferred from domain function, not from direct imaging or fractionation
   • No identification of the direct molecular target within the Ras/RAF/MAPK cascade
   • Mechanism of ERK suppression by the SPR domain not resolved
2. 2006 Low

   Showed SPRED3 is post-translationally ubiquitinated upon RTK stimulation, hinting at regulated turnover, but the modification was not characterized.

   Evidence Ubiquitination assay in HEK293T cells with EGF/pervanadate stimulation and Western blotting

   PMID:17094949

   Open questions at the time

   • E3 ligase not identified
   • Functional consequence of ubiquitination unknown
   • Observed incidentally in a SPRED2-focused study
3. 2014 Medium

   Identified the first enzyme modifying SPRED3, establishing that it is a palmitoylation substrate of zDHHC17 (HIP14).

   Evidence Yeast two-hybrid interactome screen plus palmitoylation assay

   PMID:24705354

   Open questions at the time

   • Palmitoylation sites on SPRED3 not mapped
   • Functional impact on localization or MAPK suppression not tested
4. 2015 Low

   Extended SPRED3 negative regulation beyond ERK to TGFβ-induced EMT in lens epithelial cells.

   Evidence Plasmid overexpression in rat lens epithelial explants with TGFβ treatment, morphological scoring, and α-SMA immunolabeling

   PMID:25576668

   Open questions at the time

   • Overexpression-only without loss-of-function
   • Direct molecular link between SPRED3 and the TGFβ pathway not defined
   • Single explant system
5. 2018 Low

   Confirmed SPRED3 suppresses FGF/RTK-driven ERK signaling and the downstream differentiation program in lens cells.

   Evidence Transfection of lens epithelial explants, FGF stimulation, ERK1/2 phosphorylation assay, morphological elongation scoring

   PMID:29501879

   Open questions at the time

   • Overexpression-based; endogenous role not tested
   • Limited mechanistic depth specific to SPRED3
6. 2020 Medium

   Provided reciprocal genetic evidence that endogenous SPRED3 restrains the Ras/RAF/MAPK pathway, with loss driving EGFR-TKI resistance and migration in lung cancer.

   Evidence Whole-exome sequencing, CRISPR/Cas9 knockout and overexpression in HCC827 cells, p-ERK Western blotting, IC50/migration assays, and ERK-inhibitor epistasis

   PMID:35117614

   Open questions at the time

   • Direct biochemical target in the cascade not identified
   • Single cell-line context
   • Whether the c.120delG variant occurs in patient tumors not addressed
7. 2021 Medium

   Placed SPRED3 in a miRNA-regulated Raf1/MAPK axis in lung disease, with miR-342-5p directly repressing Spred3 and recombinant Spred3 worsening hyperoxia-induced pathology in vivo.

   Evidence 3'UTR luciferase reporter assay, transgenic mouse models, and recombinant protein treatment in murine hyperoxia models

   PMID:33434946

   Open questions at the time

   • Mechanism by which extracellular recombinant Spred3 acts not defined
   • Direction of Spred3's effect on Raf1 in this model not fully reconciled with its suppressor role
8. 2022 High

   Defined the molecular basis of SPRED3 S-acylation, showing the SPR domain mediates a zDABM-independent interaction with zDHHC17 that bypasses the enzyme's ankyrin-repeat domain.

   Evidence Mutational dissection of the SPR domain, co-immunoprecipitation with zDHHC17 domain mutants, and S-acylation assays

   PMID:36442513

   Open questions at the time

   • Functional consequence of S-acylation for MAPK suppression not established
   • In vivo relevance of the modification untested
9. 2024 Medium

   Revealed a context-dependent, non-canonical role in which SPRED3 positively regulates NF-κB to promote thyroid cancer growth, contrasting its MAPK-suppressing role elsewhere.

   Evidence Reciprocal Flag-SPRED3 overexpression and knockout, CCK8/colony formation assays, in vivo tumor model, and NF-κB luciferase reporter

   PMID:39227612

   Open questions at the time

   • Mechanism linking SPRED3 to NF-κB activation unknown
   • How a MAPK suppressor activates NF-κB not reconciled
10. 2025 Medium

    Demonstrated a physiological requirement for SPRED3 in thyroid homeostasis, with knockout mice developing primary hypothyroidism alongside altered thyroidal ERK and autophagy markers.

    Evidence SPRED3 knockout mouse hormonal profiling (TSH, T4), X-Gal promoter staining, and immunoblotting for ERK and autophagy regulators

    PMID:40806788

    Open questions at the time

    • Causal link between altered ERK/autophagy and hypothyroidism not established
    • Cell-type responsible for the phenotype within the thyroid not resolved

• How SPRED3 mechanistically engages the Ras/RAF/MAPK cascade, and how it switches between MAPK suppression and NF-κB activation in a tissue-specific manner, remains unresolved.
• No direct MAPK-pathway binding partner or substrate identified
• Functional role of S-acylation and palmitoylation in SPRED3 activity unknown
• Molecular basis for the thyroid-specific NF-κB-activating role undefined