ZNRF2

ZNRF2 is an N-myristoylated RING-type E3 ubiquitin ligase that operates at intracellular membranes to control substrate ubiquitination, lysosomal function, and mTORC1 signaling (PMID:22797923, PMID:27244671). Membrane targeting via N-myristoylation enhances its ligase activity, and it engages substrates through its UBZ domain while recruiting E2 enzymes—predominantly Ubc13/Uev1a—through its RING domain to catalyze Lys63-linked ubiquitination of the Na+/K+ATPase α1 subunit (PMID:22797923). ZNRF2 is governed by a reverse myristoyl switch: growth factor/insulin signaling drives its phosphorylation, 14-3-3 binding, and release from membranes into the cytosol (PMID:22797923). It interacts with mTOR and the V-ATPase on lysosomal membranes, acting upstream of the Rag GTPases to promote amino acid-stimulated mTORC1 lysosomal translocation and activation while preserving lysosomal acidity; in turn, active mTORC1 phosphorylates ZNRF2 on Ser145 (reversed by protein phosphatase 6) to trigger its cytosolic release, forming a negative feedback loop, with ZNRF2 loss elevating mTOR activity (PMID:27244671, PMID:40402410). Beyond Na+/K+ATPase, ZNRF2 ubiquitinates SLC3A2 at K147 to drive its plasma membrane localization and suppress ferroptosis, and mediates proteasome-dependent degradation of CD-M6PR to impair lysosomal enzyme trafficking in senescent cells (PMID:40999004, PMID:42065825). ZNRF2 transcription is activated by CREB1, and ZNRF2 mediates CREB1's pro-proliferative effect in breast cancer (PMID:37165255).

1. 2012 High

   Established ZNRF2 as a membrane-targeted E3 ligase whose activity and localization are switch-controlled, answering how a RING ligase couples growth-factor signaling to substrate ubiquitination.

   Evidence N-myristoylation analysis, Co-IP, domain mutagenesis, in vitro Lys63 ubiquitination assay, fractionation, and knockdown of Na+/K+ATPase α1 in cells

   PMID:22797923

   Open questions at the time

   • The kinase phosphorylating ZNRF2 to trigger the reverse myristoyl switch is not identified here
   • Whether Na+/K+ATPase α1 is the principal physiological substrate is not resolved
2. 2016 High

   Placed ZNRF2 in the amino acid sensing machinery upstream of Rag GTPases and the V-ATPase, defining a phosphorylation-dependent negative feedback loop with mTORC1.

   Evidence Co-IP (mTOR, V-ATPase), Ser145 phosphosite mapping, PP6 identification, fractionation, and knockdown with cell size/proliferation/mTORC1 readouts

   PMID:27244671

   Open questions at the time

   • The ubiquitination substrate by which ZNRF2 promotes mTORC1 translocation is not defined
   • Mechanism linking ZNRF2 to V-ATPase regulation of acidity is unresolved
3. 2023 Medium

   Identified an upstream transcriptional input, showing CREB1 directly activates ZNRF2 to drive proliferation, connecting ZNRF2 to oncogenic signaling.

   Evidence ChIP/promoter binding assay and ZNRF2 knockdown rescue of CREB1-driven proliferation in breast cancer cells

   PMID:37165255

   Open questions at the time

   • Single lab, single cancer context
   • Which ZNRF2 catalytic activity mediates the proliferative effect is not shown
4. 2025 Medium

   Extended ZNRF2 substrate repertoire to SLC3A2, linking site-specific ubiquitination to membrane transport, ferroptosis suppression, and tumor growth.

   Evidence K147 ubiquitination site mapping, Co-IP, membrane fractionation, ferroptosis and peptide-blocking assays, in vitro and in vivo tumor growth

   PMID:40999004

   Open questions at the time

   • Ubiquitin chain linkage type on SLC3A2 not specified
   • Single lab finding
5. 2025 Medium

   Demonstrated an in vivo physiological role for ZNRF2 as a negative regulator of mTOR-mediated neuroinflammation, consistent with the mTORC1 feedback model.

   Evidence ZNRF2 knockdown in MPTP Parkinson's mouse model with behavioral, cytokine, and mTOR-inhibitor rescue readouts

   PMID:40402410

   Open questions at the time

   • Direct molecular target of ZNRF2 in neurons not identified
   • Single lab, model-specific
6. 2026 Medium

   Defined an mTORC1–ZNRF2–CD-M6PR axis in senescence, showing stress-elevated ZNRF2 degrades CD-M6PR to impair lysosomal function.

   Evidence Structural prediction plus experimental validation of ZNRF2–CD-M6PR interaction, proteasome-inhibitor ubiquitination experiments, siRNA, senescence and lysosomal assays, aged mouse/human tissue analysis

   PMID:42065825

   Open questions at the time

   • Ubiquitin chain architecture directing proteasomal versus lysosomal fate not detailed
   • Single lab

• How ZNRF2 substrate selection and chain-type output are governed across its membrane substrates, and whether its dual role (mTORC1 promotion versus mTOR feedback inhibition) is context-dependent, remain unresolved.
• No unifying structural model of substrate recognition
• Reconciliation of pro- and anti-mTOR roles across tissues is unestablished