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Hedgehog (Hh) signaling culminates in the conversion of the latent transcription factor Cubitus interruptus (Ci)/Gli from a repressor form (Ci(R)/Gli(R)) into an activator form (Ci(A)/Gli(A)). While sequential phosphorylation of Ci/Gli by protein kinase A(PKA), glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1) is essential for its proteolytic processing that generates Ci(R)/Gli(R), sequential phosphorylation of Ci/Gli by the Fused (Fu)/Unc-51 like kinase (Ulk) family kinases Fu/Ulk3/Stk36 and CK1 contributes to the formation of Ci(A)/Gli(A). Fu/Ulk3/Stk36-mediated phosphorylation of Ci/Gli is stimulated by Hh, leading to altered interaction between Ci/Gli and the Hh pathway repressor Sufu. Here we describe both in vitro and in vivo assays that determine Ci/Gli phosphorylation by the Fu/Ulk family kinases and its regulation by Hh.

Hedgehog (Hh) is a morphogen that binds to its receptor Patched 1 and activates Smoothened (SMO), thereby governing embryonic development and postnatal tissue homeostasis. Cholesterol can bind and covalently conjugate to the luminal cysteine-rich domain (CRD) of human SMO at the D95 residue (D99 in mouse). The reaction mechanism and biological function of SMO cholesterylation have not been elucidated. Here, we show that the SMO-CRD undergoes auto-cholesterylation which is boosted by calcium and involves an intramolecular ester intermediate. In cells, Hh stimulation elevates local calcium concentration in the SMO-localized endosomes through store-operated calcium entry. In addition, we identify the signaling-incompetent SMO D95E mutation, and the D95E mutant SMO can bind cholesterol but cannot be modified or activated by cholesterol. The homozygous Smo

The GPCR-family protein Smoothened (Smo) is an obligatory signal transducer of the Hedgehog (Hh) signaling pathway. Binding of Hh to its receptor Patched (Ptc) alleviates Ptc-mediated inhibition of Smo, allowing Smo to activate the Cubitus interruptus (Ci)/Gli family of zinc finger transcription factors. The activation of Smo is an early and crucial event in Hh signal transduction. Studies have shown that Hh induces cell surface/ciliary accumulation and phosphorylation of Smo by multiple kinases, including protein kinase A (PKA), casein kinase 1 (CK1), casein kinase 2 (CK2), G protein-coupled receptor kinase 2 (Gprk2/GRK2), and atypical PKC (aPKC). Here, we describe the assays used to examine the phosphorylation and activity of Smo, including in vitro kinase assay, phospho-specific antibodies, luciferase reporter assay, cell surface accumulation, and ciliary localization assays. These assays provide powerful tools to study Smo phosphorylation and activation, leading to mechanistic insight into Smo regulation.

The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis by regulating the abundance, localization, and activity of the GPCR family protein Smoothened (Smo). Smo trafficking and subcellular accumulation are controlled by multiple posttranslational modifications (PTMs) including phosphorylation, ubiquitination, and sumoylation, which appears to be conserved from Drosophila to mammals. Smo ubiquitination is dynamically regulated by E3 ubiquitin ligases and deubiquitinases (dubs) and is opposed by Hh signaling. By contrast, Smo sumoylation is stimulated by Hh, which counteracts Smo ubiquitination by recruiting the dub USP8. We describe cell-base assays for Smo ubiquitination and its regulation by Hh and the E3 ligases in Drosophila. We also describe assays for Smo sumoylation in both Drosophila and mammalian cultured cells.

Graded Hedgehog (Hh) signaling is mediated by graded Cubitus interruptus (Ci)/Gli transcriptional activity, but how the Hh gradient is converted into the Ci/Gli activity gradient remains poorly understood. Here, we show that graded Hh induces a progressive increase in Ci phosphorylation at multiple Fused (Fu)/CK1 sites including a cluster located in the C-terminal Sufu-binding domain. We demonstrated that Fu directly phosphorylated Ci on S1382, priming CK1 phosphorylation on adjacent sites, and that Fu/CK1-mediated phosphorylation of the C-terminal sites interfered with Sufu binding and facilitated Ci activation. Phosphorylation at the N-terminal, middle, and C-terminal Fu/CK1 sites occurred independently of one another and each increased progressively in response to increasing levels of Hh or increasing amounts of Hh exposure time. Increasing the number of phospho-mimetic mutations of Fu/CK1 sites resulted in progressively increased Ci activation by alleviating Sufu-mediated inhibition. We found that the C-terminal Fu/CK1 phosphorylation cluster is conserved in Gli2 and contributes to its dose-dependent activation. Our study suggests that the Hh signaling gradient is translated into a Ci/Gli phosphorylation gradient that activates Ci/Gli by gradually releasing Sufu-mediated inhibition.

Summary The Fused (Fu) kinase is a key transducer of Hedgehog signaling, but its relevant substrates have remained obscured due to the difficulty of obtaining active Fu for in vitro kinase assay. Based on the mechanism of Fu activation in vivo, we engineered a constitutively active Fu and expressed it in Sf9 cells using the baculovirus system. The kinase was affinity purified and applied for in vitro kinase assay using recombinant GST-fusion proteins as substrates to identify Fu-specific phosphorylation sites. For complete details on the use and execution of this protocol, please refer to Han et al. (2019)., Graphical Abstract, Highlights • Purification of constitutively active Fu from insect cells for in vitro kinase assay • Priming phosphorylation by Fu can allow secondary in vitro kinase assay • High-purity protein elution with Flag M2 affinity agarose and 3X Flag peptide • Sensitive phospho-protein detection via pIMAGO-biotin kit or specific antibodies, The Fused (Fu) kinase is a key transducer of Hedgehog signaling but its relevant substrates have remained obscured due to the difficulty of obtaining active Fu for in vitro kinase assay. Based on the mechanism of Fu activation in vivo, we engineered a constitutively active Fu and expressed it in Sf9 cells using the baculovirus system. The kinase was affinity purified and applied for in vitro kinase assay using recombinant GST-fusion proteins as substrates to identify Fu-specific phosphorylation sites.

Hedgehog (Hh) signaling culminates in the conversion of the latent transcription factor Cubitus interruptus (Ci)/Gli into its activator form (Ci(A)/Gli(A)), but the underlying mechanism remains poorly understood. Here we demonstrate that Hh stimulates the phosphorylation of Ci by the Ser/Thr kinase Fused (Fu) and that Fu-mediated phosphorylation of Ci promotes its activation. We find that Fu directly phosphorylates Ci on Ser218 and Ser1230, which primes its further phosphorylation by CK1 on adjacent sties. These phosphorylation events alter Ci binding to the pathway inhibitor Suppressor of fused (Sufu) and facilitate the recruitment of Transportin and the transcriptional coactivator CBP. Furthermore, we provide evidence that Sonic hedgehog (Shh) activates Gli2 by stimulating its phosphorylation on conserved sites through the Fu family kinases ULK3 and mFu/STK36 in a manner depending on Gli2 ciliary localization. Hence, Fu family kinase-mediated phosphorylation of Ci/Gli serves as a conserved mechanism that activates the Hh pathway transcription factor.

The tumor suppressor protein Suppressor of fused (Sufu) plays a conserved role in the Hedgehog (Hh) signaling pathway by inhibiting Cubitus interruptus (Ci)/Glioma-associated oncogene homolog (Gli) transcription factors, but the molecular mechanism by which Sufu inhibits Ci/Gli activity remains poorly understood. Here we show that Sufu can bind Ci/Gli through a C-terminal Sufu-interacting site (SIC) in addition to a previously identified N-terminal site (SIN), and that both SIC and SIN are required for optimal inhibition of Ci/Gli by Sufu. We show that Sufu can sequester Ci/Gli in the cytoplasm through binding to SIN while inhibiting Ci/Gli activity in the nucleus depending on SIC. We also find that binding of Sufu to SIC and the middle region of Ci can impede recruitment of the transcriptional coactivator CBP by masking its binding site in the C-terminal region of Ci. Indeed, moving the CBP-binding site to an "exposed" location can render Ci resistant to Sufu-mediated inhibition in the nucleus. Hence, our study identifies a previously unidentified and conserved Sufu-binding motif in the C-terminal region of Ci/Gli and provides mechanistic insight into how Sufu inhibits Ci/Gli activity in the nucleus.

Hedgehog (Hh) signaling in vertebrates depends on primary cilia. Upon stimulation, Hh pathway components, including Gli transcription factors, accumulate at primary cilia to transduce the Hh signal, but the mechanisms underlying their ciliary targeting remains largely unknown. Here, we show that the PY-type nuclear localization signal (PY-NLS)/karyopherinβ2 (Kapβ2) nuclear import system regulates Gli ciliary localization and Hh pathway activation. Mutating the PY-NLS in Gli or knockdown of Kapβ2 diminished Gli ciliary localization. Kapβ2 is required for the formation of Gli activator (GliA) in wild-type but not in Sufu mutant cells. Knockdown of Kapβ2 affected Hh signaling in zebrafish embryos, as well as in vitro cultured cerebellum granule neuron progenitors (CGNPs) and SmoM2-driven medulloblastoma cells. Furthermore, Kapβ2 depletion impaired the growth of cultured medulloblastoma cells, which was rescued by Gli overexpression. Interestingly, Kapβ2 is a transcriptional target of the Hh pathway, thus forming a positive feedback loop for Gli activation. Our study unravels the molecular mechanism and cellular machinery regulating Gli ciliary localization and identifies Kapβ2 as a critical regulator of the Hh pathway and a potential drug target for Hh-driven cancers., Author summary The secreted Hedgehog (Hh) protein plays an evolutionarily conserved role in both embryonic development and adult tissue homeostasis. Malfunction of Hh signaling activity contributes to a wide range of human diseases, including birth defects and cancer. Hh signaling in vertebrates critically depends on the primary cilium, a microtubule-based plasma membrane protrusion present on the surface of most mammalian cells. Upon ligand stimulation, Hh pathway components, including the seven-transmembrane protein Smoothened (Smo) and Gli transcription factors, accumulate at primary cilia to transduce the Hh signal, but the mechanisms underlying their ciliary targeting are still poorly understood. Here, we discover that the PY-type nuclear localization signal (PY-NLS) and the nuclear import factor karyopherinβ2 (Kapβ2) regulate Gli ciliary localization and Hh pathway activity. Mutating the PY-NLS in Gli or knockdown of Kapβ2 diminished Gli ciliary localization without affecting Smo ciliary accumulation in response to Hh. Kapβ2 regulates the formation of the active form of Gli, which is required for proper Hh signaling in zebrafish embryos and cultured cerebellum granule neuron progenitors (CGNPs). Kapβ2 depletion impaired the growth of medulloblastoma cells driven by an oncogenic form of Smo. Finally, Kapβ2 is a transcriptional target of the Hh pathway, forming a positive feedback loop to promote Gli activation. Our study reveals the molecular mechanism underlying the regulation of Gli ciliary targeting and identifies Kapβ2 as a potential cancer drug target.

Hedgehog (Hh) signaling controls embryonic development and adult tissue homeostasis through the G protein coupled receptor (GPCR)-family protein Smoothened (Smo). Upon stimulation, Smo accumulates on the cell surface in Drosophila or primary cilia in vertebrates, which is thought to be essential for its activation and function, but the underlying mechanisms remain poorly understood. Here we show that Hh stimulates the binding of Smo to a plasma membrane-associated kinase Gilgamesh (Gish)/CK1γ and that Gish fine-tunes Hh pathway activity by phosphorylating a Ser/Thr cluster (CL-II) in the juxtamembrane region of Smo carboxyl-terminal intracellular tail (C-tail). We find that CL-II phosphorylation is promoted by protein kinase A (PKA)-mediated phosphorylation of Smo C-tail and depends on cell surface localization of both Gish and Smo. Consistent with CL-II being critical for high-threshold Hh target gene expression, its phosphorylation appears to require higher levels of Hh or longer exposure to the same level of Hh than PKA-site phosphorylation on Smo. Furthermore, we find that vertebrate CK1γ is localized at the primary cilium to promote Smo phosphorylation and Sonic hedgehog (Shh) pathway activation. Our study reveals a conserved mechanism whereby Hh induces a change in Smo subcellular localization to promote its association with and activation by a plasma membrane localized kinase, and provides new insight into how Hh morphogen progressively activates Smo., A study of both fruit flies and vertebrate cells shows that the plasma membrane-associated kinase Gish/CK1γ promotes transduction of the Hedgehog signal by phosphorylating Smoothened., Author Summary The secreted glycoprotein Hedgehog (Hh) plays a conserved role in embryonic development and adult tissue homeostasis in species ranging from Drosophila to humans. Deregulation of Hh signal transduction contributes to a wide range of human disorders, including birth defects and cancer. The seven-transmembrane protein Smoothened (Smo) is an obligatory and conserved Hh signal transducer, but how Hh stimulates its activity remains unclear. Here we identify a plasma membrane associated kinase, Gilgamesh (Gish)/CK1γ, as a positive regulator of the Hh signaling activity. We find that Gish activates Hh signaling by phosphorylating a specific site in the Smo C-terminal intracellular tail. Phosphorylation of Smo by Gish is required for maximal activation of Smo and depends on membrane association of Gish and prior phosphorylation of Smo by protein kinase A (PKA). We also find that Hh stimulates the association of Smo with Gish after it travels to the plasma membrane, thus facilitating its phosphorylation by Gish. Finally, we provide evidence that CK1γ is found at the primary cilium in mammals and phosphorylates Smo to activate the Hh pathway. Our results uncover a conserved role of Gish/CK1γ in the regulation of Smo phosphorylation and provide new insight into the molecular underpinning of how Hh signal is transduced across the plasma membrane.