From tumorigenesis to the establishment of local or metastatic high-grade tumours, an integral part of the cellular lifespan relies on various signalling pathways. Particular pathways that allow cells to proliferate by creating a network of new blood vessels have been documented, whereas other pathways are primarily involved with a migration to distant body parts, partially through the process of epithelial-mesenchymal transition (EMT). This review will discuss the different signalling pathways, such as TGF-β, Cripto-1, Wnt pathways, Hedgehog, Notch and NF-κB pathways, and how they promote tumour initiation and progression by influencing diverse cellular processes and EMT in general and in benign and malignant prostate tumours. This review will discuss only the critical pathways. Therefore, many other types of signalling pathways which are related to prostate cancer will not be discussed. Possibilities for further investigation will be mentioned, as many underlying mechanisms involved in these pathways have potential as targets in future tumour therapy. This review will also introduce some novel clinical trials relating to the inhibition of signalling pathways and their clinical outcomes.

• Klíčová slova
• EMT, Hedgehog, NF-κB, Notch, Prostate cancer, TGF, WNT/β-catenin, castration resistance, signalling pathways, therapeutic target, β,
• MeSH
• epitelo-mezenchymální tranzice fyziologie   MeSH
• lidé MeSH
• nádory prostaty * patologie   metabolismus   terapie   farmakoterapie   MeSH
• NF-kappa B metabolismus   MeSH
• proteiny hedgehog metabolismus   MeSH
• signální transdukce * fyziologie   MeSH
• transformující růstový faktor beta metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• NF-kappa B MeSH
• proteiny hedgehog MeSH
• transformující růstový faktor beta MeSH

Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.

• Klíčová slova
• DVL2, Wnt signaling, biochemistry, biomolecular condensates, cell biology, chemical biology, dishevelled, human, paralogs,
• MeSH
• beta-katenin metabolismus   genetika   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• multimerizace proteinu MeSH
• protein dishevelled * metabolismus   genetika   MeSH
• signální dráha Wnt * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-katenin MeSH
• DVL2 protein, human MeSH Prohlížeč
• protein dishevelled * MeSH

Polyglutamylation is a reversible posttranslational modification that is catalyzed by enzymes of the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation that is initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Dishevelled 3 (DVL3), thereby changing the interactome of DVL3. Polyglutamylation increases the capacity of DVL3 to get phosphorylated, to undergo phase separation, and to act in the noncanonical Wnt pathway. Both carboxy-terminal polyglutamylation and the resulting reduction in phase separation capacity of DVL3 can be reverted by the deglutamylating enzyme CCP6, demonstrating a causal relationship between TTLL11-mediated polyglutamylation and phase separation. Thus, C-terminal polyglutamylation represents a new type of posttranslational modification, broadening the range of proteins that can be modified by polyglutamylation and providing the first evidence that polyglutamylation can modulate protein phase separation.

• Klíčová slova
• Dishevelled 3, Noncanonical Wnt Signaling, Polyglutamylation, Protein Condensates, TTLL11,
• MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• kyselina polyglutamová metabolismus   analogy a deriváty   MeSH
• lidé MeSH
• peptidsynthasy * metabolismus   genetika   MeSH
• posttranslační úpravy proteinů * MeSH
• protein dishevelled * metabolismus   genetika   MeSH
• separace fází MeSH
• signální dráha Wnt MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DVL3 protein, human MeSH Prohlížeč
• kyselina polyglutamová MeSH
• peptidsynthasy * MeSH
• protein dishevelled * MeSH
• tubulin polyglutamylase MeSH Prohlížeč

Dishevelled (DVL) is the central signal transducer in both Wnt/β-catenin-dependent and independent signalling pathways. DVL is required to connect receptor complexes and downstream effectors. Since proximal Wnt pathway components and DVL itself are upregulated in many types of cancer, DVL represents an attractive therapeutic target in the Wnt-addicted cancers and other disorders caused by aberrant Wnt signalling. Here, we discuss progress in several approaches for the modulation of DVL function and hence inhibition of the Wnt signalling. Namely, we sum up the potential of modulation of enzymes that control post-translational modification of DVL - such as inhibition of DVL kinases or promotion of DVL ubiquitination and degradation. In addition, we discuss research directions that can take advantage of direct interaction with the protein domains essential for DVL function: the inhibition of DIX- and DEP-domain mediated polymerization and interaction of DVL PDZ domain with its ligands.

• Klíčová slova
• Casein kinase 1, DIX oligomerization, Dishevelled, PDZ inhibitors, Wnt signalling-related diseases,
• MeSH
• adaptorové proteiny signální transdukční MeSH
• fosfoproteiny * metabolismus   MeSH
• lidé MeSH
• protein dishevelled * metabolismus   MeSH
• signální dráha Wnt * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• fosfoproteiny * MeSH
• protein dishevelled * MeSH

Wnt and BMP signaling pathways are two key molecular machineries regulating development and homeostasis. The efficient coordination of Wnt and BMP is essential in many developmental processes such as establishment of antero-posterior and dorso-ventral body axis, regulation of convergent extension, or development of various organ systems. SMAD ubiquitination regulatory factor (Smurf) family of E3 ubiquitin ligases are important and evolutionary conserved regulators of TGF-β/BMP signaling pathways. Smurf2 has been previously shown to regulate Wnt/planar cell polarity (PCP) signaling pathway by ubiquitinating Prickle1, one of the key components of PCP. We explored the role of Smurf2 in Wnt pathways in further detail and identified that Smurf2 is also a ubiquitin ligase of Dishevelled (DVL), the key cytoplasmic signal transducer in the Wnt pathway. Interestingly, the Smurf2 and DVL relationship expands beyond substrate-E3 ligase. We can show that DVL activates Smurf2, which allows Smurf2 to ubiquitinate its substrates from Wnt/PCP (Prickle1) as well as TGF-β/BMP (Smad2) pathways more efficiently. Using SMAD7 as an example of Smurf2 activator we show that DVL and SMAD7 both activates Smurf2 activity. In HEK293 cells the deficiency of DVL phenocopies absence of Smurf2 and leads to the increased phosphorylation of R-Smads. Smurf2-DVL connection provides a novel and intriguing point of crosstalk for Wnt and BMP pathways.

• Klíčová slova
• Smurf2, TGF-β/BMP signaling, Wnt signaling, dishevelled, prickle, ubiquitination,
• MeSH
• biologické modely MeSH
• HEK293 buňky MeSH
• kostní morfogenetické proteiny metabolismus   MeSH
• lidé MeSH
• nádorové supresorové proteiny metabolismus   MeSH
• protein dishevelled metabolismus   MeSH
• proteiny s doménou LIM metabolismus   MeSH
• proteolýza MeSH
• signální dráha Wnt * MeSH
• signální transdukce MeSH
• transformující růstový faktor beta metabolismus   MeSH
• ubikvitinace MeSH
• ubikvitinligasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kostní morfogenetické proteiny MeSH
• nádorové supresorové proteiny MeSH
• PRICKLE1 protein, human MeSH Prohlížeč
• protein dishevelled MeSH
• proteiny s doménou LIM MeSH
• SMURF1 protein, human MeSH Prohlížeč
• SMURF2 protein, human MeSH Prohlížeč
• transformující růstový faktor beta MeSH
• ubikvitinligasy MeSH

Primary cilia are organelles necessary for proper implementation of developmental and homeostasis processes. To initiate their assembly, coordinated actions of multiple proteins are needed. Tau tubulin kinase 2 (TTBK2) is a key player in the cilium assembly pathway, controlling the final step of cilia initiation. The function of TTBK2 in ciliogenesis is critically dependent on its kinase activity; however, the precise mechanism of TTBK2 action has so far not been fully understood due to the very limited information about its relevant substrates. In this study, we demonstrate that CEP83, CEP89, CCDC92, Rabin8, and DVL3 are substrates of TTBK2 kinase activity. Further, we characterize a set of phosphosites of those substrates and CEP164 induced by TTBK2 in vitro and in vivo. Intriguingly, we further show that identified TTBK2 phosphosites and consensus sequence delineated from those are distinct from motifs previously assigned to TTBK2. Finally, we show that TTBK2 is also required for efficient phosphorylation of many S/T sites in CEP164 and provide evidence that TTBK2-induced phosphorylations of CEP164 modulate its function, which in turn seems relevant for the process of cilia formation. In summary, our work provides important insight into the substrates-TTBK2 kinase relationship and suggests that phosphorylation of substrates on multiple sites by TTBK2 is probably involved in the control of ciliogenesis in human cells.

• MeSH
• aminokyselinové motivy MeSH
• cilie metabolismus   MeSH
• fosforylace MeSH
• fosfoserin metabolismus   MeSH
• fosfothreonin metabolismus   MeSH
• HEK293 buňky MeSH
• kaseinkinasa I metabolismus   MeSH
• lidé MeSH
• multiproteinové komplexy metabolismus   MeSH
• organogeneze * MeSH
• protein-serin-threoninkinasy chemie   metabolismus   MeSH
• substrátová specifita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoserin MeSH
• fosfothreonin MeSH
• kaseinkinasa I MeSH
• multiproteinové komplexy MeSH
• protein-serin-threoninkinasy MeSH
• tau-tubulin kinase MeSH Prohlížeč

BACKGROUND: Dishevelled (DVL) is an essential component of the Wnt signaling cascades. Function of DVL is controlled by phosphorylation but the molecular details are missing. DVL3 contains 131 serines and threonines whose phosphorylation generates complex barcodes underlying diverse DVL3 functions. In order to dissect the role of DVL phosphorylation we analyzed the phosphorylation of human DVL3 induced by previously reported (CK1ε, NEK2, PLK1, CK2α, RIPK4, PKCδ) and newly identified (TTBK2, Aurora A) DVL kinases. METHODS: Shotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on immunoprecipitates from HEK293T cells was used to identify and quantify phosphorylation of DVL3 protein induced by 8 kinases. Functional characterization was performed by in-cell analysis of phospho-mimicking/non-phosphorylatable DVL3 mutants and supported by FRET assays and NMR spectroscopy. RESULTS: We used quantitative mass spectrometry and calculated site occupancies and quantified phosphorylation of > 80 residues. Functional validation demonstrated the importance of CK1ε-induced phosphorylation of S268 and S311 for Wnt-3a-induced β-catenin activation. S630-643 cluster phosphorylation by CK1, NEK2 or TTBK2 is essential for even subcellular distribution of DVL3 when induced by CK1 and TTBK2 but not by NEK2. Further investigation showed that NEK2 utilizes a different mechanism to promote even localization of DVL3. NEK2 triggered phosphorylation of PDZ domain at S263 and S280 prevents binding of DVL C-terminus to PDZ and promotes an open conformation of DVL3 that is more prone to even subcellular localization. CONCLUSIONS: We identify unique phosphorylation barcodes associated with DVL function. Our data provide an example of functional synergy between phosphorylation in structured domains and unstructured IDRs that together dictate the biological outcome. Video Abtract.

• Klíčová slova
• CK1, DVL3, Dishevelled, Kinase, Mass spectrometry, NEK2, Phosphorylation, TTBK2, Wnt,
• MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• hmotnostní spektrometrie MeSH
• kinasy NEK metabolismus   MeSH
• konformace proteinů MeSH
• kultivované buňky MeSH
• lidé MeSH
• protein dishevelled chemie   metabolismus   MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• DVL3 protein, human MeSH Prohlížeč
• kinasy NEK MeSH
• NEK2 protein, human MeSH Prohlížeč
• protein dishevelled MeSH

Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.

• MeSH
• analýza jednotlivých buněk metody   MeSH
• biosenzitivní techniky MeSH
• enzymatické testy metody   MeSH
• fluorescenční mikroskopie metody   MeSH
• fosforylace fyziologie   MeSH
• frizzled receptory metabolismus   MeSH
• genový knockout MeSH
• HEK293 buňky MeSH
• kaseinkinasa Iepsilon genetika   metabolismus   MeSH
• lidé MeSH
• mutageneze cílená MeSH
• oocyty MeSH
• PDZ domény fyziologie   MeSH
• protein dishevelled genetika   metabolismus   MeSH
• rezonanční přenos fluorescenční energie MeSH
• signální dráha Wnt fyziologie   MeSH
• simulace molekulární dynamiky MeSH
• Xenopus laevis MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DVL3 protein, human MeSH Prohlížeč
• frizzled receptory MeSH
• FZD6 protein, human MeSH Prohlížeč
• kaseinkinasa Iepsilon MeSH
• protein dishevelled MeSH

Frizzleds (FZDs) are receptors for secreted lipoglycoproteins of the Wingless/Int-1 (WNT) family, initiating an important signal transduction network in multicellular organisms. FZDs are G protein-coupled receptors (GPCRs), which are well known to be regulated by phosphorylation, leading to specific downstream signaling or receptor desensitization. The role and underlying mechanisms of FZD phosphorylation remain largely unexplored. Here, we investigated the phosphorylation of human FZD6 Using MS analysis and a phospho-state- and -site-specific antibody, we found that Ser-648, located in the FZD6 C terminus, is efficiently phosphorylated by casein kinase 1 ϵ (CK1ϵ) and that this phosphorylation requires the scaffolding protein Dishevelled (DVL). In an overexpression system, DVL1, -2, and -3 promoted CK1ϵ-mediated FZD6 phosphorylation on Ser-648. This DVL activity required an intact DEP domain and FZD-mediated recruitment of this domain to the cell membrane. Substitution of the CK1ϵ-targeted phosphomotif reduced FZD6 surface expression, suggesting that Ser-648 phosphorylation controls membrane trafficking of FZD6 Phospho-Ser-648 FZD6 immunoreactivity in human fallopian tube epithelium was predominantly apical, associated with cilia in a subset of epithelial cells, compared with the total FZD6 protein expression, suggesting that FZD6 phosphorylation contributes to asymmetric localization of receptor function within the cell and to epithelial polarity. Given the key role of FZD6 in planar cell polarity, our results raise the possibility that asymmetric phosphorylation of FZD6 rather than asymmetric protein distribution accounts for polarized receptor signaling.

• Klíčová slova
• Dishevelled, Frizzled, G protein-coupled receptor (GPCR), GRK, WNT, casein kinase 1, cell polarity, cell signaling, phosphorylation, receptor regulation, scaffold protein, serine/threonine protein kinase,
• MeSH
• buněčná membrána metabolismus   MeSH
• epitel metabolismus   MeSH
• fosfoproteiny imunologie   MeSH
• fosforylace MeSH
• frizzled receptory chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• hmotnostní spektrometrie MeSH
• kaseinkinasa I metabolismus   MeSH
• lidé MeSH
• protein dishevelled chemie   fyziologie   MeSH
• protilátky imunologie   MeSH
• sekvence aminokyselin MeSH
• serin metabolismus   MeSH
• signální transdukce MeSH
• vejcovody metabolismus   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoproteiny MeSH
• frizzled receptory MeSH
• FZD6 protein, human MeSH Prohlížeč
• kaseinkinasa I MeSH
• protein dishevelled MeSH
• protilátky MeSH
• serin 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