Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK's kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.

• Klíčová slova
• FGFR, ICK, cilia length, fibroblast growth factor, intestinal cell kinase,
• MeSH
• buňky NIH 3T3 MeSH
• cilie metabolismus   MeSH
• CRISPR-Cas systémy MeSH
• fibroblastové růstové faktory metabolismus   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• modely u zvířat MeSH
• myši knockoutované MeSH
• myši MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• proteiny hedgehog metabolismus   MeSH
• proteomika MeSH
• receptor fibroblastových růstových faktorů, typ 1 metabolismus   MeSH
• receptor fibroblastových růstových faktorů, typ 3 genetika   metabolismus   MeSH
• receptor fibroblastových růstových faktorů, typ 4 metabolismus   MeSH
• receptory fibroblastových růstových faktorů genetika   metabolismus   MeSH
• signální transdukce MeSH
• simulace molekulového dockingu MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• CILK1 protein, human MeSH Prohlížeč
• Cilk1 protein, mouse MeSH Prohlížeč
• FGFR1 protein, human MeSH Prohlížeč
• FGFR3 protein, human MeSH Prohlížeč
• FGFR4 protein, human MeSH Prohlížeč
• fibroblastové růstové faktory MeSH
• protein-serin-threoninkinasy MeSH
• proteiny hedgehog MeSH
• receptor fibroblastových růstových faktorů, typ 1 MeSH
• receptor fibroblastových růstových faktorů, typ 3 MeSH
• receptor fibroblastových růstových faktorů, typ 4 MeSH
• receptory fibroblastových růstových faktorů MeSH

Cilia project from almost every cell integrating extracellular cues with signaling pathways. Constitutive activation of FGFR3 signaling produces the skeletal disorders achondroplasia (ACH) and thanatophoric dysplasia (TD), but many of the molecular mechanisms underlying these phenotypes remain unresolved. Here, we report in vivo evidence for significantly shortened primary cilia in ACH and TD cartilage growth plates. Using in vivo and in vitro methodologies, our data demonstrate that transient versus sustained activation of FGF signaling correlated with different cilia consequences. Transient FGF pathway activation elongated cilia, while sustained activity shortened cilia. FGF signaling extended primary cilia via ERK MAP kinase and mTORC2 signaling, but not through mTORC1. Employing a GFP-tagged IFT20 construct to measure intraflagellar (IFT) speed in cilia, we showed that FGF signaling affected IFT velocities, as well as modulating cilia-based Hedgehog signaling. Our data integrate primary cilia into canonical FGF signal transduction and uncover a FGF-cilia pathway that needs consideration when elucidating the mechanisms of physiological and pathological FGFR function, or in the development of FGFR therapeutics.

• MeSH
• achondroplazie genetika   patofyziologie   MeSH
• buňky NIH 3T3 MeSH
• chondrocyty metabolismus   MeSH
• chrupavka metabolismus   MeSH
• cilie patologie   fyziologie   MeSH
• ciliopatie genetika   patofyziologie   MeSH
• fenotyp MeSH
• fibroblastové růstové faktory metabolismus   MeSH
• lidé MeSH
• myši MeSH
• primární buněčná kultura MeSH
• receptor fibroblastových růstových faktorů, typ 3 genetika   metabolismus   MeSH
• růstová ploténka metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• thanatoforní dysplazie genetika   patofyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• FGFR3 protein, human MeSH Prohlížeč
• fibroblastové růstové faktory MeSH
• receptor fibroblastových růstových faktorů, typ 3 MeSH

Wnt signaling cascade has developed together with multicellularity to orchestrate the development and homeostasis of complex structures. Wnt pathway components - such as β-catenin, Dishevelled (DVL), Lrp6, and Axin-- are often dedicated proteins that emerged in evolution together with the Wnt signaling cascade and are believed to function primarily in the Wnt cascade. It is interesting to see that in recent literature many of these proteins are connected with cellular functions that are more ancient and not limited to multicellular organisms - such as cell cycle regulation, centrosome biology, or cell division. In this review, we summarize the recent literature describing this crosstalk. Specifically, we attempt to find the answers to the following questions: Is the response to Wnt ligands regulated by the cell cycle? Is the centrosome and/or cilium required to activate the Wnt pathway? How do Wnt pathway components regulate the centrosomal cycle and cilia formation and function? We critically review the evidence that describes how these connections are regulated and how they help to integrate cell-to-cell communication with the cell and the centrosomal cycle in order to achieve a fine-tuned, physiological response.

• Klíčová slova
• Wnt, cell cycle, centrosome, cilium, crosstalk, planar cell polarity,
• MeSH
• buněčný cyklus * MeSH
• centrozom metabolismus   MeSH
• lidé MeSH
• mezibuněčná komunikace MeSH
• polarita buněk MeSH
• signální dráha Wnt * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Mammalian limb development is driven by the integrative input from several signaling pathways; a failure to receive or a misinterpretation of these signals results in skeletal defects. The brachydactylies, a group of overlapping inherited human hand malformation syndromes, are mainly caused by mutations in BMP signaling pathway components. Two closely related forms, Brachydactyly type B2 (BDB2) and BDB1 are caused by mutations in the BMP antagonist Noggin (NOG) and the atypical receptor tyrosine kinase ROR2 that acts as a receptor in the non-canonical Wnt pathway. Genetic analysis of Nog and Ror2 functional interaction via crossing Noggin and Ror2 mutant mice revealed a widening of skeletal elements in compound but not in any of the single mutants, thus indicating genetic interaction. Since ROR2 is a non-canonical Wnt co-receptor specific for Wnt-5a we speculated that this phenotype might be a result of deregulated Wnt-5a signaling activation, which is known to be essential for limb skeletal elements growth and patterning. We show that Noggin potentiates activation of the Wnt-5a-Ror2-Disheveled (Dvl) pathway in mouse embryonic fibroblast (MEF) cells in a Ror2-dependent fashion. Rat chondrosarcoma chondrocytes (RCS), however, are not able to respond to Noggin in this fashion unless growth arrest is induced by FGF2. In summary, our data demonstrate genetic interaction between Noggin and Ror2 and show that Noggin can sensitize cells to Wnt-5a/Ror2-mediated non-canonical Wnt signaling, a feature that in cartilage may depend on the presence of active FGF signaling. These findings indicate an unappreciated function of Noggin that will help to understand BMP and Wnt/PCP signaling pathway interactions.

• Klíčová slova
• BMP signaling, Ror2, Wnt5a, brachydactyly, noggin, non-canonical Wnt pathways,
• Publikační typ
• časopisecké články MeSH

Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways.

• MeSH
• acetofenony farmakologie   MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• fosforylace účinky léků   MeSH
• myší embryonální kmenové buňky účinky léků   metabolismus   MeSH
• myši MeSH
• NADPH-oxidasy genetika   metabolismus   MeSH
• oniové sloučeniny farmakologie   MeSH
• oxidační stres účinky léků   MeSH
• proliferace buněk účinky léků   MeSH
• proteiny Wnt metabolismus   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• synergismus léků MeSH
• transkripční faktor STAT3 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetofenony MeSH
• acetovanillone MeSH Prohlížeč
• diphenyleneiodonium MeSH Prohlížeč
• extracelulárním signálem regulované MAP kinasy MeSH
• fosfatidylinositol-3-kinasy MeSH
• NADPH-oxidasy MeSH
• oniové sloučeniny MeSH
• proteiny Wnt MeSH
• protoonkogenní proteiny c-akt MeSH
• reaktivní formy kyslíku MeSH
• transkripční faktor STAT3 MeSH

β-Arrestin is a scaffold protein that regulates signal transduction by seven transmembrane-spanning receptors. Among other functions it is also critically required for Wnt/β-catenin signal transduction. In the present study we provide for the first time a mechanistic basis for the β-arrestin function in Wnt/β-catenin signaling. We demonstrate that β-arrestin is required for efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling. β-Arrestin regulates Lrp6 phosphorylation via a novel interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled-associated PtdIns(4,5)P2 production to the phosphorylation of Lrp6. Using fluorescence recovery after photobleaching we show here that β-arrestin is required for the Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally, we show that β-arrestin interacts with PtdIns kinases PI4KIIα and PIP5KIβ. Importantly, cells lacking β-arrestin showed higher steady-state levels of the relevant PtdInsP and were unable to increase levels of these PtdInsP in response to Wnt3a. In summary, our data show that β-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of the membrane dynamics of Amer1. We propose that β-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5)P2, which is produced locally upon Wnt3a stimulation by β-arrestin- and Dishevelled-associated kinases.

• Klíčová slova
• Amer1/WTX/FAM123B, Dvl, Lrp6 Phosphorylation, Membrane Lipids, Phosphatidylinositol Kinase, Phosphatidylinositol Phosphate Kinase, Phosphatidylinositol Signaling, Wnt Signaling, β-Arrestin, β-Catenin,
• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• arrestiny genetika   metabolismus   MeSH
• beta arrestiny MeSH
• buněčná membrána metabolismus   MeSH
• embryo savčí cytologie   MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• fosfoproteiny genetika   metabolismus   MeSH
• fosforylace MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• konfokální mikroskopie MeSH
• kultivované buňky MeSH
• LDL receptor related protein 6 genetika   metabolismus   MeSH
• lidé MeSH
• myši knockoutované MeSH
• myši MeSH
• nádorové supresorové proteiny genetika   metabolismus   MeSH
• protein dishevelled MeSH
• protein Wnt3A genetika   metabolismus   MeSH
• RNA interference MeSH
• vazba proteinů MeSH
• vedlejší histokompatibilní antigeny MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1-phosphatidylinositol-4-phosphate 5-kinase MeSH Prohlížeč
• adaptorové proteiny signální transdukční MeSH
• AMER1 protein, human MeSH Prohlížeč
• arrestiny MeSH
• beta arrestiny MeSH
• fosfatidylinositol-4,5-difosfát MeSH
• fosfoproteiny MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• LDL receptor related protein 6 MeSH
• LRP6 protein, human MeSH Prohlížeč
• nádorové supresorové proteiny MeSH
• phosphatidylinositol phosphate 4-kinase MeSH Prohlížeč
• protein dishevelled MeSH
• protein Wnt3A MeSH
• vedlejší histokompatibilní antigeny MeSH