scholarly journals Time-resolved proteomics profiling of the ciliary Hedgehog response

2021 ◽  
Vol 220 (5) ◽  
Author(s):  
Elena A. May ◽  
Marian Kalocsay ◽  
Inès Galtier D’Auriac ◽  
Patrick S. Schuster ◽  
Steven P. Gygi ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Regulatory Subunit ◽  
Time Resolved ◽  
Pathway Activation ◽  
Dependent Protein ◽  
A Cell ◽  
Cell Type Specific ◽  
Rapid Characterization ◽  
G Protein Coupled

The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid, and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein–coupled receptor GPR161 in response to Hedgehog, and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the phosphatase Paladin as a cell type–specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.

scholarly journals Time-resolved proteomic profiling of the ciliary Hedgehog response reveals that GPR161 and PKA undergo regulated co-exit from cilia

2020 ◽  
Author(s):  
Elena A. May ◽  
Marian Kalocsay ◽  
Inès Galtier D’Auriac ◽  
Steven P. Gygi ◽  
Maxence V. Nachury ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Regulatory Subunit ◽  
Proteomic Profiling ◽  
Time Resolved ◽  
Pathway Activation ◽  
Dependent Protein ◽  
A Cell ◽  
Cell Type Specific ◽  
Rapid Characterization

ABSTRACTThe primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein-coupled receptor GPR161 in response to Hedgehog; and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the putative phosphatase Paladin as a cell type-specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.

scholarly journals Activation of Cilia-Independent Hedgehog/GLI1 Signaling as a Novel Concept for Neuroblastoma Therapy

Cancers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1908
Author(s):  
Anke Koeniger ◽  
Anna Brichkina ◽  
Iris Nee ◽  
Lukas Dempwolff ◽  
Anna Hupfer ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Survival Rates ◽  
Genetic Alterations ◽  
Novel Therapy ◽  
Therapeutic Concept ◽  
Pathway Activation ◽  
Starting Point ◽  
Novel Concept ◽  
Neural Crest Differentiation

Although being rare in absolute numbers, neuroblastoma (NB) represents the most frequent solid tumor in infants and young children. Therapy options and prognosis are comparably good for NB patients except for the high risk stage 4 class. Particularly in adolescent patients with certain genetic alterations, 5-year survival rates can drop below 30%, necessitating the development of novel therapy approaches. The developmentally important Hedgehog (Hh) pathway is involved in neural crest differentiation, the cell type being causal in the etiology of NB. However, and in contrast to its function in some other cancer types, Hedgehog signaling and its transcription factor GLI1 exert tumor-suppressive functions in NB, rendering GLI1 an interesting new candidate for anti-NB therapy. Unfortunately, the therapeutic concept of pharmacological Hh/GLI1 pathway activation is difficult to implement as NB cells have lost primary cilia, essential organelles for Hh perception and activation. In order to bypass this bottleneck, we have identified a GLI1-activating small molecule which stimulates endogenous GLI1 production without the need for upstream Hh pathway elements such as Smoothened or primary cilia. This isoxazole compound potently abrogates NB cell proliferation and might serve as a starting point for the development of a novel class of NB-suppressive molecules.

scholarly journals Hypoxia-Induced Gene Expression Occurs Solely through the Action of Hypoxia-Inducible Factor 1α (HIF-1α): Role of Cytoplasmic Trapping of HIF-2α

2003 ◽  
Vol 23 (14) ◽  
pp. 4959-4971 ◽  
Author(s):  
Sang-ki Park ◽  
Agnes M. Dadak ◽  
Volker H. Haase ◽  
Lucrezia Fontana ◽  
Amato J. Giaccia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activity ◽  
Hypoxia Inducible Factor ◽  
Low Oxygen ◽  
Hypoxic Conditions ◽  
Show Evidence ◽  
Dependent Protein ◽  
A Cell ◽  
Cell Type Specific ◽  
Inducible Genes

ABSTRACT The hypoxia-inducible factors 1α (HIF-1α) and 2α (HIF-2α) have extensive structural homology and have been identified as key transcription factors responsible for gene expression in response to hypoxia. They play critical roles not only in normal development, but also in tumor progression. Here we report on the differential regulation of protein expression and transcriptional activity of HIF-1α and -2α by hypoxia in immortalized mouse embryo fibroblasts (MEFs). We show that oxygen-dependent protein degradation is restricted to HIF-1α, as HIF-2α protein is detected in MEFs regardless of oxygenation and is localized primarily to the cytoplasm. Endogenous HIF-2α remained transcriptionally inactive under hypoxic conditions; however, ectopically overexpressed HIF-2α translocated into the nucleus and could stimulate expression of hypoxia-inducible genes. We show that the factor inhibiting HIF-1 can selectively inhibit the transcriptional activity of HIF-1α but has no effect on HIF-2α-mediated transcription in MEFs. We propose that HIF-2α is not a redundant transcription factor of HIF-1α for hypoxia-induced gene expression and show evidence that there is a cell type-specific modulator(s) that enables selective activation of HIF-1α but not HIF-2α in response to low-oxygen stress.

Abstract LB-028: Cell density sensing alters TGF-beta signaling in a cell type-specific manner, independent from Hippo pathway activation

2015 ◽  
Author(s):  
Flore Nallet-Staub ◽  
Xueqian Yin ◽  
Cristèle Gilbert ◽  
Véronique Marsaud ◽  
Saber Ben Mimoun ◽  
...  
Keyword(s):  
Cell Density ◽  
Hippo Pathway ◽  
Tgf Beta ◽  
Cell Type ◽  
Pathway Activation ◽  
Specific Manner ◽  
A Cell ◽  
Cell Type Specific

scholarly journals Ciliary and extraciliary Gpr161 pools repress hedgehog signaling in a tissue-specific manner

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Sun-Hee Hwang ◽  
Bandarigoda N Somatilaka ◽  
Kevin White ◽  
Saikat Mukhopadhyay
Keyword(s):  
G Protein ◽  
Neural Tube ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Camp Signaling ◽  
Tissue Specific ◽  
Specific Manner ◽  
G Protein Coupled ◽  
Ciliary Localization

The role of compartmentalized signaling in primary cilia during tissue morphogenesis is not well understood. The cilia-localized G-protein-coupled receptor—Gpr161 represses hedgehog pathway via cAMP signaling. We engineered a knock-in at Gpr161 locus in mice to generate a variant (Gpr161mut1), which was ciliary localization defective but cAMP signaling competent. Tissue phenotypes from hedgehog signaling depend on downstream bifunctional Gli transcriptional factors functioning as activators/repressors. Compared to knockout (ko), Gpr161mut1/ko had delayed embryonic lethality, moderately increased hedgehog targets and partially down-regulated Gli3-repressor. Unlike ko, the Gpr161mut1/ko neural tube did not show Gli2-activator-dependent expansion of ventral-most progenitors. Instead, the intermediate neural tube showed progenitor expansion that depends on loss of Gli3-repressor. Increased extraciliary receptor (Gpr161mut1/mut1) prevented ventralization. Morphogenesis in limb buds and midface requires Gli-repressor; these tissues in Gpr161mut1/mut1 manifested hedgehog hyperactivation phenotypes—polydactyly and midfacial widening. Thus, ciliary and extraciliary Gpr161 pools likely establish tissue-specific Gli-repressor thresholds in determining morpho-phenotypic outcomes.

scholarly journals KIF14 controls ciliogenesis via regulation of Aurora A and is important for Hedgehog signaling

2020 ◽  
Vol 219 (6) ◽  
Author(s):  
Petra Pejskova ◽  
Madeline Louise Reilly ◽  
Lucia Bino ◽  
Ondrej Bernatik ◽  
Linda Dolanska ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Primary Cilium ◽  
Hedgehog Signaling ◽  
Cell Cycle Progression ◽  
Primary Cilia ◽  
Aurora A ◽  
Pathway Activation ◽  
Cilia Formation ◽  
Tightly Coupled ◽  
Hh Signaling

Primary cilia play critical roles in development and disease. Their assembly and disassembly are tightly coupled to cell cycle progression. Here, we present data identifying KIF14 as a regulator of cilia formation and Hedgehog (HH) signaling. We show that RNAi depletion of KIF14 specifically leads to defects in ciliogenesis and basal body (BB) biogenesis, as its absence hampers the efficiency of primary cilium formation and the dynamics of primary cilium elongation, and disrupts the localization of the distal appendage proteins SCLT1 and FBF1 and components of the IFT-B complex. We identify deregulated Aurora A activity as a mechanism contributing to the primary cilium and BB formation defects seen after KIF14 depletion. In addition, we show that primary cilia in KIF14-depleted cells are defective in response to HH pathway activation, independently of the effects of Aurora A. In sum, our data point to KIF14 as a critical node connecting cell cycle machinery, effective ciliogenesis, and HH signaling.

Requirements for the adenylyl cyclases in the development of Dictyostelium

Development ◽  
2001 ◽  
Vol 128 (18) ◽  
pp. 3649-3654 ◽  
Author(s):  
Christophe Anjard ◽  
Fredrik Söderbom ◽  
William F. Loomis
Keyword(s):  
Adenylyl Cyclase ◽  
Intracellular Signaling ◽  
Adenylyl Cyclases ◽  
Fruiting Bodies ◽  
Dependent Protein ◽  
A Cell ◽  
Express Cell ◽  
Cell Type Specific ◽  
Cell Densities ◽  
Mutant Cells

It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA− mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA− PKA-Cover strain. When developed at high cell densities, acrA−acaA− PKA-Cover cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high.

scholarly journals Plexins Promote Hedgehog Signaling Through Their Cytoplasmic GAP Activity

2021 ◽  
Author(s):  
Justine M Pinskey ◽  
Tyler M Hoard ◽  
Xiao-Feng Zhao ◽  
Nicole E Franks ◽  
Zoe C Frank ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Hedgehog Pathway ◽  
Loss Of Function ◽  
3T3 Fibroblasts ◽  
Pathway Activation ◽  
Gli Transcription Factors ◽  
Full Complement ◽  
Positive Regulators ◽  
Nih 3T3

Hedgehog signaling controls tissue patterning during embryonic and postnatal development and continues to play important roles throughout life. Characterizing the full complement of Hedgehog pathway components is essential to understanding its wide-ranging functions. Previous work has identified Neuropilins, established Semaphorin receptors, as positive regulators of Hedgehog signaling. Neuropilins require Plexin co-receptors to mediate Semaphorin signaling, but a role for Plexins in Hedgehog signaling has not yet been explored. Here, we provide evidence that multiple Plexins promote Hedgehog signaling in NIH/3T3 fibroblasts and that Plexin loss-of-function in these cells results in significantly reduced Hedgehog pathway activity. Catalytic activity of the Plexin GTPase activating protein (GAP) domain is required for Hedgehog signal promotion, and constitutive activation of the GAP domain further amplifies Hedgehog signaling. Additionally, we demonstrate that Plexins promote Hedgehog signaling at the level of GLI transcription factors and that this promotion requires intact primary cilia. Finally, we find that Plexin loss-of-function significantly reduces the response to Hedgehoga pathway activation in the mouse dentate gyrus. Together, these data identify Plexins as novel components of the Hedgehog pathway and provide insight into their mechanism of action.

A cell-based time-resolved fluorescence assay for selection of antibody reagents for G protein-coupled receptor immunohistochemistry

2004 ◽  
Vol 291 (1-2) ◽  
pp. 123-135 ◽  
Author(s):  
Jui-Lan Su ◽  
Jim Fornwald ◽  
Philip Rivers ◽  
Susan Goldsworthy ◽  
Noeleen A. Looney ◽  
...  
Keyword(s):  
G Protein ◽  
Fluorescence Assay ◽  
Time Resolved Fluorescence ◽  
G Protein Coupled Receptor ◽  
Time Resolved ◽  
A Cell ◽  
G Protein Coupled ◽  
Selection Of

scholarly journals Cell Density Sensing Alters TGF-β Signaling in a Cell-Type-Specific Manner, Independent from Hippo Pathway Activation

2015 ◽  
Vol 32 (5) ◽  
pp. 640-651 ◽  
Author(s):  
Flore Nallet-Staub ◽  
Xueqian Yin ◽  
Cristèle Gilbert ◽  
Véronique Marsaud ◽  
Saber Ben Mimoun ◽  
...  
Keyword(s):  
Cell Density ◽  
Hippo Pathway ◽  
Cell Type ◽  
Pathway Activation ◽  
Specific Manner ◽  
A Cell ◽  
Cell Type Specific
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