Phosphorylation-induced SUMOylation promotes Ulk4 condensation at ciliary tip to transduce Hedgehog signal

Hedgehog (Hh) signaling controls embryonic development and adult tissue homeostasis through the Gli family of transcription factors. In vertebrates, Hh signal transduction depends on the primary cilium where Gli is thought to be activated at the ciliary tip, but the underlying mechanism has remained poorly understood. Our previous study showed that two U nc-51-like k inase (Ulk) family members Ulk4 and Stk36 colocalize with Gli2 at ciliary tip to promote Gli2 phosphorylation upon Shh stimulation, and that ciliary tip localization of Ulk4/Stk36 depends on phosphorylation of Ulk4 by Stk36 (Zhou et al. 2023). Here we show that phosphorylation of Ulk4 drives its phase separation to form biomolecular condensates that recruit Stk36 and Gli2. Mechanistically, Stk36-mediated phosphorylation of Ulk4 promotes its SUMOylation in response to Shh, and subsequent interaction between SUMO and a S UMO-Interacting- M otif (SIM) in the C-terminal region of Ulk4 drives Ulk4 self-assembly to form biomolecular condensates. Phospho-mimetic mutation of Ulk4 sufficed to drive Ulk4/Stk36/Gli2 condensation at ciliary tip, resulting in constitutive Shh pathway activation. Taken together, our results suggest that phosphorylation-dependent SUMOylation of Ulk4 promotes kinase-substrate condensation at ciliary tip to transduce the Hh signal. We speculate that phase separation driven by phosphorylation-dependent SUMOylation may represent a general mechanism for kinase-mediated signaling.