ULK3

ULK3 is a serine/threonine kinase that functions as a multi-pathway signaling node, coupling its catalytic activity to cytokinetic abscission control, Hedgehog-pathway transcription, epigenetic regulation, and autophagy (PMID:26011858, PMID:19878745, PMID:36797248, PMID:27717182). At the midbody, ULK3 phosphorylates ESCRT-III subunits IST1 and CHMP4C through its tandem MIT domains, an essential signal that sustains the abscission checkpoint and delays cell division in response to lagging chromosomes and midbody tension (PMID:26011858). In Sonic Hedgehog signaling, ULK3 directly phosphorylates and activates the transcription factors GLI1 and GLI2 and alters GLI1 localization, with its catalytic activity required for this positive regulatory role (PMID:19878745); this activity is gated by Suppressor of Fused (Sufu), which binds the ULK3 kinase domain, blocks autophosphorylation and GLI phosphorylation, and—when complexed with ULK3—favors the GLI2 repressor form, giving ULK3 both kinase-dependent and kinase-independent functions that are relieved upon SHH-induced complex destabilization (PMID:20643644). ULK3-driven GLI activation feeds into oncogenic and epigenetic programs, contributing to cancer-associated fibroblast conversion and to transcriptional upregulation of DNMT3A during autophagy (PMID:28877478, PMID:35226587). In keratinocytes and squamous cell carcinoma, nuclear ULK3 directly binds and regulates the histone arginine methyltransferases PRMT1 and PRMT5, controlling their chromatin association and histone H4 methylation to support proliferation and tumorigenicity (PMID:36797248). ULK3 also promotes autophagosome formation through an MTOR-insensitive mechanism, acting as part of the ULK–ATG13–FIP200 complex—a role conserved in the Drosophila ortholog ADUK, which requires Atg13 but not Atg1 (PMID:27717182, PMID:39171951, PMID:40831505). Crystallographic and biochemical analyses define a kinase domain with an active/inactive conformational switch, show that autophosphorylation does not control activity whereas phosphorylation of two kinase-domain residues by an unidentified kinase abolishes it, and establish SU6668 and related compounds as ATP-pocket-binding inhibitors with therapeutic relevance in neuronal autophagy and multiple myeloma models (PMID:30096229, PMID:34190988, PMID:39171951, PMID:40831505).

1. 2009 Medium

   Established ULK3 as a catalytically active kinase with a defined downstream substrate, placing it as a positive regulator within the SHH/GLI transcriptional pathway.

   Evidence In vitro kinase assays, GLI-luciferase reporters, immunofluorescence, and kinase-dead mutant analysis

   PMID:19878745

   Open questions at the time

   • GLI phosphorylation sites not mapped
   • in vivo SHH-pathway relevance not established
   • endogenous regulation of ULK3 activity unknown
2. 2010 Medium

   Identified Sufu as a direct upstream regulator that gates ULK3 activity, revealing a dual kinase-dependent and kinase-independent role for ULK3 in modulating GLI activator versus repressor balance.

   Evidence Reciprocal Co-IP, in vitro kinase assays, GLI-luciferase reporters, and siRNA knockdown

   PMID:20643644

   Open questions at the time

   • structural basis of Sufu-ULK3 interaction not defined
   • mechanism of SHH-induced complex destabilization unresolved
3. 2015 High

   Defined a distinct, non-Hedgehog role for ULK3 as the abscission-checkpoint kinase that phosphorylates ESCRT-III subunits via its MIT domains to time cytokinesis.

   Evidence Crystal structures, in vitro phosphorylation assays, Co-IP/binding, mutagenesis, and lagging-chromosome cell biology

   PMID:26011858

   Open questions at the time

   • how diverse stress signals (nuclear pore defects, tension) feed into ULK3 not detailed
   • regulation of ULK3 at the midbody unknown
4. 2016 Medium

   Showed the autophagy-inducing function of ULK3 is evolutionarily conserved and operates via an Atg13/FIP200-dependent but Atg1-independent route, distinguishing it from the canonical ULK1/2 autophagy machinery.

   Evidence Co-IP of the Drosophila ortholog ADUK with Atg13/FIP200, genetic loss-of-function, autophagy flux, and lifespan analysis

   PMID:27717182

   Open questions at the time

   • direct human ULK3 substrates in autophagy not identified
   • molecular role within the ATG13-FIP200 complex unclear
5. 2017 Medium

   Connected ULK3 to tumor stroma biology, showing CSL-controlled ULK3 drives GLI2-dependent CAF conversion alongside a separable GLI-independent autophagy function.

   Evidence Co-IP, siRNA knockdown, autophagy assays, CAF functional assays, and tumor co-injection models

   PMID:28877478

   Open questions at the time

   • mechanism separating GLI versus autophagy outputs not resolved
   • in vivo specificity of ULK3 silencing not established
6. 2018 Medium

   Revealed that ULK3 catalytic activity is controlled not by autophosphorylation but by inhibitory phosphorylation of two kinase-domain residues by an unidentified kinase, and that the annotated kinase domain alone is insufficient for activity.

   Evidence In vitro kinase assays, MS phosphosite mapping, site-directed mutagenesis, and inhibitor binding kinetics with SU6668

   PMID:30096229

   Open questions at the time

   • the upstream kinase phosphorylating ULK3 is unidentified
   • physiological trigger for inhibitory phosphorylation unknown
   • role of the 271-300 region structurally undefined
7. 2021 High

   Provided high-resolution structures of the ULK3 kinase domain defining an active/inactive conformational switch and a candidate substrate-recognition mechanism, and linked ULK3-GLI1 activity to epigenetic upregulation of DNMT3A during autophagy.

   Evidence X-ray crystallography with inhibitor co-complexes and fingerprinting; proximity ligation assay, ChIP, RT-qPCR, and siRNA knockdown

   PMID:34190988 PMID:35226587

   Open questions at the time

   • full-length ULK3 structure not determined
   • how the conformational switch is set in cells unknown
   • direct demonstration that ULK3 phosphorylates GLI1 in the DNMT3A context not shown
8. 2023 High

   Established a nuclear, chromatin-associated function for ULK3 as a direct binding partner and regulator of PRMT1/5, linking it to histone H4 arginine methylation and SCC tumorigenicity.

   Evidence Reciprocal Co-IP, chromatin fractionation, histone methylation assays, gene silencing, nuclear imaging, and orthotopic cancer models

   PMID:36797248

   Open questions at the time

   • whether ULK3 kinase activity is required for PRMT regulation not resolved
   • PRMT substrate specificity changes induced by ULK3 not detailed
9. 2024 Medium

   Demonstrated ULK3 as a functionally relevant, MTOR-insensitive autophagy-inducing kinase whose inhibition rescues neuronal and behavioral deficits caused by BIN1 deficiency.

   Evidence AAV-mediated RNAi, SU6668 pharmacological inhibition, autophagy flux, MRI volumetry, Barnes maze, and primary neuron imaging

   PMID:39171951

   Open questions at the time

   • molecular link between BIN1 loss and ULK3 activation unknown
   • ULK3 autophagy substrates in neurons not identified
10. 2025 Medium

    Positioned ULK3 within the ULK-ATG13-FIP200 complex as a survival dependency in multiple myeloma and validated ATP-pocket inhibitors as therapeutic tools.

    Evidence Co-crystallization with inhibitors, genetic knockdown/knockout, in vivo MM tumor models, ex vivo patient samples, and Co-IP of the complex (preprint)

    PMID:40831505

    Open questions at the time

    • preprint not yet peer-reviewed
    • mechanism by which ULK3 restores proteasome-inhibitor sensitivity unresolved
    • direct catalytic targets in MM not defined

• The integration of ULK3's multiple roles remains unresolved: how a single kinase is partitioned between midbody ESCRT-III phosphorylation, nuclear GLI/PRMT regulation, and autophagy initiation, and what upstream kinase sets its activity state, is unknown.
• identity of the inhibitory upstream kinase unknown
• no unified model of subcellular partitioning across functions
• substrate repertoire in autophagy initiation undefined