Absence Of Hh Research Articles

Phosphatidic acid binding to Patched contributes to the inhibition of Smoothened and Hedgehog signaling in Drosophila wing development.

Hedgehog (Hh) signaling controls growth and patterning during embryonic development and homeostasis in adult tissues. Hh binding to the receptor Patched (Ptc) elicits intracellular signaling by relieving Ptc-mediated inhibition of the transmembrane protein Smoothened (Smo). We uncovered a role for the lipid phosphatidic acid (PA) in the regulation of the Hh pathway in Drosophila melanogaster. Deleting the Ptc C-terminal tail or mutating the predicted PA-binding sites within it prevented Ptc from inhibiting Smo in wing discs and in cultured cells. The C-terminal tail of Ptc directly interacted with PA in vitro, an association that was reduced by Hh, and increased the amount of PA at the plasma membrane in cultured cells. Smo also interacted with PA in vitro through a binding pocket located in the transmembrane region, and mutating residues in this pocket reduced Smo activity in vivo and in cells. By genetically manipulating PA amounts in vivo or treating cultured cells with PA, we demonstrated that PA promoted Smo activation. Our findings suggest that Ptc may sequester PA in the absence of Hh and release it in the presence of Hh, thereby increasing the amount of PA that is locally available to promote Smo activation.

Gli Phosphorylation Code in Hedgehog Signal Transduction.

Hedgehog (Hh) family of secreted proteins governs many key processes in embryonic development and adult tissue homeostasis in species ranging from insects to human. Deregulation of Hh signaling has been implicated in a wide range of human diseases including birth defect and cancer. Hh signaling pathway culminates in the conversion of the latent transcription factor Cubitus interruptus (Ci)/Gli from a repressor form (CiR/GliR) into an activator form (CiA/GliA). Both the production of CiR/GliR in the absence of Hh and the formation of CiA/GliA in response to Hh are regulated by phosphorylation. Whereas previous studies demonstrated that sequential phosphorylation by protein kinase A (PKA), glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1) at multiple Ser/Thr clusters in the C-terminal region of Ci/Gli targets it for proteolytic processing to generate CiR/GliR, recent studies revealed that phosphorylation of Ci/Gli by the Fused (Fu)/Unc-51 like kinase (Ulk) family kinases Fu/Ulk3/Stk36 and other kinases contributes to Ci/Gli activation. 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 review our current understanding of how various Ci/Gli phosphorylation events are regulated and how they influence Hh signal transduction.

Drosophila hedgehog can act as a morphogen in the absence of regulated Ci processing.

Extracellular Hedgehog (Hh) proteins induce transcriptional changes in target cells by inhibiting the proteolytic processing of full-length Drosophila Ci or mammalian Gli proteins to nuclear transcriptional repressors and by activating the full-length Ci or Gli proteins. We used Ci variants expressed at physiological levels to investigate the contributions of these mechanisms to dose-dependent Hh signaling in Drosophila wing imaginal discs. Ci variants that cannot be processed supported a normal pattern of graded target gene activation and the development of adults with normal wing morphology, when supplemented by constitutive Ci repressor, showing that Hh can signal normally in the absence of regulated processing. The processing-resistant Ci variants were also significantly activated in the absence of Hh by elimination of Cos2, likely acting through binding the CORD domain of Ci, or PKA, revealing separate inhibitory roles of these two components in addition to their well-established roles in promoting Ci processing.

Glycolysis regulates Hedgehog signalling via the plasma membrane potential

Changes in cell metabolism and plasma membrane potential have been linked to shifts between tissue growth and differentiation, and to developmental patterning. How such changes mediate these effects is poorly understood. Here, we use the developing wing of Drosophila to investigate the interplay between cell metabolism and a key developmental regulator—the Hedgehog (Hh) signalling pathway. We show that reducing glycolysis both lowers steady‐state levels of ATP and stabilizes Smoothened (Smo), the 7‐pass transmembrane protein that transduces the Hh signal. As a result, the transcription factor Cubitus interruptus accumulates in its full‐length, transcription activating form. We show that glycolysis is required to maintain the plasma membrane potential and that plasma membrane depolarization blocks cellular uptake of N‐acylethanolamides—lipoprotein‐borne Hh pathway inhibitors required for Smo destabilization. Similarly, pharmacological inhibition of glycolysis in mammalian cells induces ciliary translocation of Smo—a key step in pathway activation—in the absence of Hh. Thus, changes in cell metabolism alter Hh signalling through their effects on plasma membrane potential.

Dual functions of Rack1 in regulating Hedgehog pathway

Hedgehog (Hh) pathway plays multiple roles in many physiological processes and its dysregulation leads to congenital disorders and cancers. Hh regulates the cellular localization of Smoothened (Smo) and the stability of Cubitus interruptus (Ci) to fine-tune the signal outputs. However, the underlying mechanisms are still unclear. Here, we show that the scaffold protein Rack1 plays dual roles in Hh signaling. In the absence of Hh, Rack1 promotes Ci and Cos2 to form a Ci–Rack1–Cos2 complex, culminating in Slimb-mediated Ci proteolysis. In the presence of Hh, Rack1 dissociates from Ci–Rack1–Cos2 complex and forms a trimeric complex with Smo and Usp8, leading to Smo deubiquitination and cell surface accumulation. Furthermore, we find the regulation of Rack1 on Hh pathway is conserved from Drosophila to mammalian cells. Our findings demonstrate that Rack1 plays dual roles during Hh signal transduction and provide Rack1 as a potential drug target for Hh-related diseases.

PKAc-directed interaction and phosphorylation of Ptc is required for Hh signaling inhibition in Drosophila

Ptc is a gatekeeper to avoid abnormal Hh signaling activation, but the key regulators involved in Ptc-mediated inhibition remain largely unknown. Here, we identify PKAc as a key regulator required for Ptc inhibitory function. In the absence of Hh, PKAc physically interacts with Ptc and phosphorylates Ptc at Ser-1150 and -1183 residues. The presence of Hh unleashes PKAc from Ptc and activates Hh signaling. By combining both in vitro and in vivo functional assays, we demonstrate that such Ptc–PKAc interaction and Ptc phosphorylation are both important for Ptc inhibitory function. Interestingly, we further demonstrate that PKAc is subjected to palmitoylation, contributing to its kinase activity on plasma membrane. Based on those novel findings, we establish a working model on Ptc inhibitory function: In the absence of Hh, PKAc interacts with and phosphorylates Ptc to ensure its inhibitory function; and Hh presence releases PKAc from Ptc, resulting in Hh signaling activation.

Synaptojanin regulates Hedgehog signalling by modulating phosphatidylinositol 4-phosphate levels

In Hedgehog (Hh) signalling, Hh ligand concentration gradient is effectively translated into a spatially distinct transcriptional program to give precisely controlled context dependent developmental outcomes. In the absence of Hh, the receptor Patched (Ptc) inhibits the signal transducer Smoothened (Smo) by maintaining low phosphatidylinositol 4-phosphate (PI(4)P) levels. Binding of Hh to its receptor Ptc promotes PI(4)P production, which in turn activates Smo. Using wingdiscs of Drosophila melanogaster, this study shows that Synaptojanin (Synj), a dual phosphatase, modulates PI(4)P levels and affects Smo activation, and thereby functions as an additional regulatory step in the Hh pathway. Reducing the levels of Synj in the wing-discs caused enhancement of a Hh dominant gain-of-function Moonrat phenotype in the adult wings. Synj downregulation augmented Hh signalling, which was associated with elevated PI(4)P levels and Smo activation. Synj did not control the absolute pathway activity but rather fine-tuned the response since its downregulation increased expression of decapentaplegic (dpp), a low-threshold target of the pathway while the high-threshold targets remained unaffected. This is the first report that identifies Synj as a negative regulator of Hh signalling, implying its importance and an additional regulatory step in Hh signal transduction.

The deubiquitinase UCHL5/UCH37 positively regulates Hedgehog signaling by deubiquitinating Smoothened.

The Hedgehog (Hh) signaling pathway plays important roles in developmental processes including pattern formation and tissue homeostasis. The seven-pass transmembrane receptor Smoothened (Smo) is the pivotal transducer in the pathway; it, and thus the pathway overall, is regulated by ubiquitin-mediated degradation, which occurs in the absence of Hh. In the presence of Hh, the ubiquitination levels of Smo are decreased, but the molecular basis for this outcome is not well understood. Here, we identify the deubiquitinase UCHL5 as a positive regulator of the Hh pathway. We provide both genetic and biochemical evidence that UCHL5 interacts with and deubiquitinates Smo, increasing stability and promoting accumulation at the cell membrane. Strikingly, we find that Hh enhances the interaction between UCHL5 and Smo, thereby stabilizing Smo. We also find that proteasome subunit RPN13, an activator of UCHL5, could enhance the effect of UCHL5 on Smo protein level. More importantly, we find that the mammalian counterpart of UCHL5, UCH37, plays the same role in the regulation of Hh signaling by modulating hSmo ubiquitination and stability. Our findings thus identify UCHL5/UCH37 as a critical regulator of Hh signaling and potential therapeutic target for cancers.

Glypican-6 promotes the growth of developing long bones by stimulating Hedgehog signaling.

Autosomal-recessive omodysplasia (OMOD1) is a genetic condition characterized by short stature, shortened limbs, and facial dysmorphism. OMOD1 is caused by loss-of-function mutations of glypican 6 (GPC6). In this study, we show that GPC6-null embryos display most of the abnormalities found in OMOD1 patients and that Hedgehog (Hh) signaling is significantly reduced in the long bones of these embryos. The Hh-stimulatory activity of GPC6 was also observed in cultured cells, where this GPC increased the binding of Hh to Patched 1 (Ptc1). Consistent with this, GPC6 interacts with Hh through its core protein and with Ptc1 through its glycosaminoglycan chains. Hh signaling is triggered at the primary cilium. In the absence of Hh, we observed that GPC6 is localized outside of the cilium but moves into the cilium upon the addition of Hh. We conclude that GPC6 stimulates Hh signaling by binding to Hh and Ptc1 at the cilium and increasing the interaction of the receptor and ligand.

Smoothened regulation in response to Hedgehog stimulation.

The Hedgehog (Hh) signaling pathway play critical roles in embryonic development and adult tissue homeostasis. A critical step in Hh signal transduction is how Hh receptor Patched (Ptc) inhibits the atypical G protein-coupled receptor Smoothened (Smo) in the absence of Hh and how this inhibition is release by Hh stimulation. It is unlikely that Ptc inhibits Smo by direct interaction. Here we discuss how Hh regulates the phosphorylation and ubiquitination of Smo, leading to cell surface and ciliary accumulation of Smo in Drosophila and vertebrate cells, respectively. In addition, we discuss how PI(4)P phospholipid acts in between Ptc and Smo to regulate Smo phosphorylation and activation in response to Hh stimulation.

Contributions of Costal 2-Fused interactions to Hedgehog signaling in Drosophila.

The Drosophila kinesin-family protein Costal 2 (Cos2) and its mammalian ortholog Kif7 play dual roles in Hedgehog (Hh) signaling. In the absence of Hh, Cos2 and Kif7 contribute to proteolytic processing and silencing of the Hh-regulated transcription factors, Drosophila Cubitus interruptus (Ci) and mammalian Gli proteins. Cos2 and Kif7 are also necessary for full activation of full-length Ci-155 and Gli transcription factors in response to Hh proteins. Here, we use classical fused alleles and transgenic Cos2 products deficient for Fused (Fu) association to show that Cos2 must bind to Fu to support efficient Ci-155 processing. Residual Ci-155 processing in the absence of Cos2-Fu interaction did not require Suppressor of Fused, which has been implicated in processing mammalian Gli proteins. We also provide evidence that Cos2 binding to the CORD domain of Ci-155 contributes to both Ci-155 processing and Ci-155 silencing in the absence of Hh. In the presence of Hh, Ci-155 processing is blocked and Cos2 now promotes activation of Ci-155, which requires Fu kinase activity. Here, we show that normal Ci-155 activation by Hh requires Cos2 binding to Fu, supporting the hypothesis that Cos2 mediates the apposition of Fu molecules suitable for cross-phosphorylation and consequent full activation of Fu kinase. We also find that phosphorylation of Cos2 by Fu at two previously mapped sites, S572 and S931, which is thought to mediate Ci-155 activation, is not required for normal activation of Ci-155 by Hh or by activated Fu.

Drosophila Vps36 regulates Smo trafficking in Hedgehog signaling

The hedgehog (Hh) signaling pathway plays a very important role in metazoan development by controlling pattern formation. Malfunction of the Hh signaling pathway leads to numerous serious human diseases, including congenital disorders and cancers. The seven-transmembrane domain protein Smoothened (Smo) is a key transducer of the Hh signaling pathway, and mediates the graded Hh signal across the cell plasma membrane, thereby inducing the proper expression of downstream genes. Smo accumulation on the cell plasma membrane is regulated by its C-tail phosphorylation and the graded Hh signal. The inhibitory mechanism for Smo membrane accumulation in the absence of Hh, however, is still largely unknown. Here, we report that Vps36 of the ESCRT-II complex regulates Smo trafficking between the cytosol and plasma membrane by specifically recognizing the ubiquitin signal on Smo in the absence of Hh. Furthermore, in the absence of Hh, Smo is ubiquitylated on its cytoplasmic part, including its internal loops and C-tail. Taken together, our data suggest that the ESCRT-II complex, especially Vps36, has a special role in controlling Hh signaling by targeting the membrane protein Smo for its trafficking in the absence of Hh, thereby regulating Hh signaling activity.

Drosophila Vps36 is involved in Hh signaling by regulating Smo trafficking

The hedgehog (Hh) signaling pathway plays a very important role in metazoan development by controlling pattern formation. Malfunction of the Hh signaling pathway leads to numerous serious human diseases, including congenital disorders and cancers. The seven-transmembrane domain protein Smoothened (Smo) is a key transducer of the Hh signaling pathway, and mediates the graded Hh signal across the cell plasma membrane, thereby inducing the proper expression of downstream genes. Smo accumulation on the cell plasma membrane is regulated by its C-tail phosphorylation and the graded Hh signal. The inhibitory mechanism for Smo membrane accumulation in the absence of Hh, however, is still largely unknown. Here, we report that Vps36 of the ESCRT-II complex regulates Smo trafficking between the cytosol and plasma membrane by specifically recognizing the ubiquitin signal on Smo in the absence of Hh. Furthermore, in the absence of Hh, Smo is ubiquitylated on its cytoplasmic part, including its internal loops and C-tail. Taken together, our data suggest that the ESCRT-II complex, especially Vps36, has a special role in controlling Hh signaling by targeting the membrane protein Smo for its trafficking in the absence of Hh, thereby regulating Hh signaling activity.

The extracellular loops of Smoothened play a regulatory role in control of Hedgehog pathway activation

The Hedgehog (Hh) signaling pathway plays an instructional role during development, and is frequently activated in cancer. Ligand-induced pathway activation requires signaling by the transmembrane protein Smoothened (Smo), a member of the G-protein-coupled receptor (GPCR) superfamily. The extracellular (EC) loops of canonical GPCRs harbor cysteine residues that engage in disulfide bonds, affecting active and inactive signaling states through regulating receptor conformation, dimerization and/or ligand binding. Although a functional importance for cysteines localized to the N-terminal extracellular cysteine-rich domain has been described, a functional role for a set of conserved cysteines in the EC loops of Smo has not yet been established. In this study, we mutated each of the conserved EC cysteines, and tested for effects on Hh signal transduction. Cysteine mutagenesis reveals that previously uncharacterized functional roles exist for Smo EC1 and EC2. We provide in vitro and in vivo evidence that EC1 cysteine mutation induces significant Hh-independent Smo signaling, triggering a level of pathway activation similar to that of a maximal Hh response in Drosophila and mammalian systems. Furthermore, we show that a single amino acid change in EC2 attenuates Hh-induced Smo signaling, whereas deletion of the central region of EC2 renders Smo fully active, suggesting that the conformation of EC2 is crucial for regulated Smo activity. Taken together, these findings are consistent with loop cysteines engaging in disulfide bonds that facilitate a Smo conformation that is silent in the absence of Hh, but can transition to a fully active state in response to ligand.

The extracellular loops of Smoothened play a regulatory role in control of Hedgehog pathway activation

The Hedgehog (Hh) signaling pathway plays an instructional role during development, and is frequently activated in cancer. Ligand-induced pathway activation requires signaling by the transmembrane protein Smoothened (Smo), a member of the G-protein-coupled receptor (GPCR) superfamily. The extracellular (EC) loops of canonical GPCRs harbor cysteine residues that engage in disulfide bonds, affecting active and inactive signaling states through regulating receptor conformation, dimerization and/or ligand binding. Although a functional importance for cysteines localized to the N-terminal extracellular cysteine-rich domain has been described, a functional role for a set of conserved cysteines in the EC loops of Smo has not yet been established. In this study, we mutated each of the conserved EC cysteines, and tested for effects on Hh signal transduction. Cysteine mutagenesis reveals that previously uncharacterized functional roles exist for Smo EC1 and EC2. We provide in vitro and in vivo evidence that EC1 cysteine mutation induces significant Hh-independent Smo signaling, triggering a level of pathway activation similar to that of a maximal Hh response in Drosophila and mammalian systems. Furthermore, we show that a single amino acid change in EC2 attenuates Hh-induced Smo signaling, whereas deletion of the central region of EC2 renders Smo fully active, suggesting that the conformation of EC2 is crucial for regulated Smo activity. Taken together, these findings are consistent with loop cysteines engaging in disulfide bonds that facilitate a Smo conformation that is silent in the absence of Hh, but can transition to a fully active state in response to ligand.

Protein kinase A acts at the basal body of the primary cilium to prevent Gli2 activation and ventralization of the mouse neural tube

Protein kinase A (PKA) is an evolutionarily conserved negative regulator of the hedgehog (Hh) signal transduction pathway. PKA is known to be required for the proteolytic processing event that generates the repressor forms of the Ci and Gli transcription factors that keep target genes off in the absence of Hh. Here, we show that complete loss of PKA activity in the mouse leads to midgestation lethality and a completely ventralized neural tube, demonstrating that PKA is as strong a negative regulator of the sonic hedgehog (Shh) pathway as patched 1 (Ptch1) or suppressor of fused (Sufu). Genetic analysis shows that although PKA is important for production of the repressor form of Gli3, the principal function of PKA in the Shh pathway in neural development is to restrain activation of Gli2. Activation of the Hh pathway in PKA mutants depends on cilia, and the catalytic and regulatory subunits of PKA are localized to a compartment at the base of the primary cilia, just proximal to the basal body. The data show that PKA does not affect cilia length or trafficking of smoothened (Smo) in the cilium. Instead, we find that there is a significant increase in the level of Gli2 at the tips of cilia of PKA-null cells. The data suggest a model in which PKA acts at the base of the cilium after Gli proteins have transited the primary cilium; in this model the sequential movement of Gli proteins between compartments in the cilium and at its base controls accessibility of Gli proteins to PKA, which determines the fates of Gli proteins and the activity of the Shh pathway.

Regulation of mammalian Gli proteins by Costal 2 and PKA in Drosophila reveals Hedgehog pathway conservation

Hedgehog (Hh) signaling activates full-length Ci/Gli family transcription factors and prevents Ci/Gli proteolytic processing to repressor forms. In the absence of Hh, Ci/Gli processing is initiated by direct Pka phosphorylation. Despite those fundamental similarities between Drosophila and mammalian Hh pathways, the differential reliance on cilia and some key signal transduction components had suggested a major divergence in the mechanisms that regulate Ci/Gli protein activities, including the role of the kinesin-family protein Costal 2 (Cos2), which directs Ci processing in Drosophila. Here, we show that Cos2 binds to three regions of Gli1, just as for Ci, and that Cos2 functions to silence mammalian Gli1 in Drosophila in a Hh-regulated manner. Cos2 and the mammalian kinesin Kif7 can also direct Gli3 and Ci processing in fly, underscoring a fundamental conserved role for Cos2 family proteins in Hh signaling. We also show that direct PKA phosphorylation regulates the activity, rather than the proteolysis of Gli in Drosophilia, and we provide evidence for an analogous action of PKA on Ci.

Osteopontin: A missing link between hedgehog signaling and fibrosis in nonalcoholic steatohepatitis

Osteopontin: A missing link between hedgehog signaling and fibrosis in nonalcoholic steatohepatitis

Casein Kinase 2 Promotes Hedgehog Signaling by Regulating both Smoothened and Cubitus Interruptus

Casein kinase 2 (CK2) is a typical serine/threonine kinase consisting of α and β subunits and has been implicated in many cellular and developmental processes. In this study, we demonstrate that CK2 is a positive regulator of the Hedgehog (Hh) signal transduction pathway. We found that inactivation of CK2 by CK2β RNAi enhances the loss-of-Hh wing phenotype induced by a dominant negative form of Smoothened (Smo). CK2β RNAi attenuates Hh-induced Smo accumulation and down-regulates Hh target gene expression, whereas increasing CK2 activity by coexpressing CK2α and CK2β increases Smo accumulation and induces ectopic Hh target gene expression. We identified the serine residues in Smo that can be phosphorylated by CK2 in vitro. Mutating these serine residues attenuates the ability of Smo to transduce high level Hh signaling activity in vivo. Furthermore, we found that CK2 plays an additional positive role downstream of Smo by regulating the stability of full-length Cubitus interruptus (Ci). CK2β RNAi promotes Ci degradation whereas coexpressing CK2α and CK2β increases the half-life of Ci. We showed that CK2 prevents Ci ubiquitination and degradation by the proteasome. Thus, CK2 promotes Hh signaling activity by regulating multiple pathway components.

Kinetics of Hedgehog-Dependent Full-Length Gli3 Accumulation in Primary Cilia and Subsequent Degradation

Hedgehog (Hh) signaling in vertebrates depends on intraflagellar transport (IFT) within primary cilia. The Hh receptor Patched is found in cilia in the absence of Hh and is replaced by the signal transducer Smoothened within an hour of Hh stimulation. By generating antibodies capable of detecting endogenous pathway transcription factors Gli2 and Gli3, we monitored their kinetics of accumulation in cilia upon Hh stimulation. Localization occurs within minutes of Hh addition, making it the fastest reported readout of pathway activity, which permits more precise temporal and spatial localization of Hh signaling events. We show that the species of Gli3 that accumulates at cilium tips is full-length and likely not protein kinase A phosphorylated. We also confirmed that phosphorylation and betaTrCP/Cul1 are required for endogenous Gli3 processing and that this is inhibited by Hh. Surprisingly, however, Hh-dependent inhibition of processing does not lead to accumulation of full-length Gli3, but instead renders it labile, leading to its proteasomal degradation via the SPOP/Cul3 complex. In fact, full-length Gli3 disappears with faster kinetics than the Gli3 repressor, the latter not requiring SPOP/Cul3 or betaTrCP/Cul1. This may contribute to the increased Gli3 activator/repressor ratios found in IFT mutants.