MASARYKOVA UNIVERZITA PŘÍRODOVĚDECKÁ FAKULTA · MASARYKOVA UNIVERZITA PŘÍRODOVĚDECKÁ...

MASARYKOVA UNIVERZITA

PŘÍRODOVĚDECKÁ FAKULTA

Ústav experimentální biologie

Oddělení fyziologie a imunologie živočichů

Dishevelled – functional and molecular analysis

HABILITAČNÍ PRÁCE Obor: Fyziologie živočichů

BRNO 2014 VÍTĚZSLAV BRYJA

ToLenkaandMax5.

AtthisplaceIwouldliketothanktoallpeoplewhomadethishabilitationthesispossible.TheseincludeErnestArenas,mypostdocsupervisorwhointroducedmetothefieldofWntsignaling,mycolleaguesandfellowsbothinSwedenandinBrno,mycollaboratorsfromallaroundtheworld,MasarykUniversity,whichprovidedmewiththeenvironmentrequiredformyprofessionalgrowthandmyformerandcurrentstudentsandpostdocs,whoformedan efficient bryjalab team. Further Iwant to thank Bára Valnohová for helpwith theformalpartsof the thesisandOndraBernatíkand IgorGreif forassistancewith figures.ThisworkwouldnotbepossiblewithoutacontinuoussupportfrommywifeLenkaandthewholefamily.

TABLE OF CONTENTS

Abstract .............................................................................................................................. 2

Abstrakt .............................................................................................................................. 2

1. Background of the thesis – Wnt signaling pathway ......................................................... 3

2. Dishevelled ..................................................................................................................... 4

2.1. STRUCTURE OF DVL……………………………………………………………………………………………………...........5

2.2. DVL‐ASSOCIATED KINASES AND CONSEQUENCES OF DVL PHOSPHORYLATIONS…………………………………….7

2.3. REGULATION OF DVL BY UBIQUITINATION………………………………………………………………………………..10

2.4. DVL IN THE WNT/Β‐CATENIN AND WNT/PCP PATHWAY – KEY POINTS…………………………………………..12

3. Future directions ........................................................................................................... 14

4. References .................................................................................................................... 16

5. List of primary research articles representing author´s contribution to the understanding

of Dvl function in the Wnt pathways (attachments) .......................................................... 23

Vítězslav Bryja, 2014

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Abstract

Wnt signaling pathway is a crucial language for intercellular communication between cells in

the multicellular organisms. Wnt signaling mediates a relay of information between closely

adjacent cells or conversely, Wnt ligands can form morphogenic gradients and as such act

over longer distances. Dysfunction or deregulation of Wnt signaling accounts for a number

of developmental defects, inherited diseases and many types of cancer. Dishevelled (Dvl) is a

key cytoplasmic protein required for signal transduction in two main branches of the Wnt

signaling ‐ Wnt/β‐catenin pathway and non‐canonical Wnt pathway. Dvl mediates contact

between Wnt receptors and cytoplasmic effectors. As such Dvl was shown to interact with

many other proteins including Wnt pathway receptors, cytoplasmic components and

proteins connected with other signaling pathways. Following ligand stimulation Dvl gets

phosphorylated and contributes to the full activation of receptor complexes. Dvl is probably

also the last common point between Wnt/β‐catenin and non‐canonical Wnt pathways and

Dvl proteins have the capacity to act as a switch between these Wnt pathways. This thesis

summarizes briefly our current understanding of Dvl biochemistry and biology and specifies

the contribution of the author to this topic.

Abstrakt

Signální dráha Wnt je základní jazyk sloužící ke komunikaci mezi buňkami v kontextu

mnohobuněčného organismu. Wnt signalizace zprostředkovává přenos informací mezi těsně

sousedícími buňkami buněk nebo může naopak vytvářet morfogenní gradienty a působit na

delší vzdálenosti. Dysfunkce nebo deregulace Wnt signalizace vede k řadě vývojových vad,

dědičných chorob a mnoha typům nádorů. Protein Dishevelled (Dvl), je klíčový

cytoplazmatický protein potřebný pro přenos signálu ve dvou hlavních větvích signalizace

Wnt – ve Wnt/β‐kateninové dráze a v nekanonické Wnt dráze. Dvl zprostředkovává kontakt

mezi Wnt receptory a cytoplazmatickými efektory. Jako takový Dvl interaguje s mnoha

dalšími proteiny jako jsou Wnt receptory, cytoplazmatické komponenty Wnt dráhy proteiny

spojenými s jinými signálními drahami. Po stimulaci ligandem je Dvl fosforylován a přispívá k

úplné aktivaci receptorových komplexů. Dvl je pravděpodobně také poslední společný bod

mezi Wnt/β‐kateninovou a nekanonickou Wnt drahou a Dvl proteiny mají schopnost

fungovat jako přepínač mezi jednotlivými větvemi Wnt dráhy. Tato práce stručně shrnuje

naše současné poznání biochemie a biologie Dvl a definuje přínos autora k tomuto tématu.


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1. Background of the thesis – Wnt signaling pathway

The behavior of each cell in the complex environment of the human body is

controlled by the extracellular factors, which are secreted in a time‐ and space‐restricted

manner by other cells in the organism. These extracellular factors drive cell fate decisions,

differentiation and migration, and ultimately shape the whole embryo during development

and control homeostasis in the adult. The major such pathway is the Wnt signaling pathway.

Wnt signaling mediates a relay of information between closely adjacent cells or conversely,

Wnt ligands can form morphogenic gradients and as such act over longer distances.

Dysfunction or deregulation of Wnt signaling accounts for a number of developmental

defects, inherited diseases and many types of cancer (Clevers, 2006).

Several “branches” of the Wnt pathway (Fig. 1) are crucial for cell‐to‐cell

communication, differentiation and morphogenesis in embryonic development. The best

studied Wnt/β‐catenin pathway, also known as “canonical” pathway, depends on β‐catenin

and members of the LEF/TCF (lymphoid enhancer‐binding factor/T‐cell factor) family of

transcriptional factors (further referred to as TCFs). In the absence of Wnt ligand, the

intracellular level of free β‐catenin is constantly low due to the activity of a degradation

complex composed of adenomatous polypolis coli (Apc), Axin and two serine/threonine

kinases ‐ glycogen synthase kinase (GSK) 3β and casein kinase (CK) 1α. β‐Catenin recruited to

the destruction complex gets phosphorylated and subsequently degraded by the ubiquitin‐

proteasome mechanism. The signaling is initiated by interaction of Wnt proteins with the

seven‐span transmembrane receptor of the Frizzled (Fz) family and with the co‐receptors

LRP5 and LRP6. Wnt binding to the receptor complex leads to the membrane sequestration

of Axin followed by disruption of the β‐catenin degradation complex. Subsequently, β‐

catenin accumulates in the cytoplasm and in the nucleus, where it associates with TCFs and

drives expression of Wnt‐responsive genes such as c‐myc or Cyclin D1. Excessive Wnt

signaling can cause the onset of diseases, particularly cancer. Conversely, low levels of the

Wnt/β‐catenin signaling activity underlie tissue degenerative conditions (Angers and Moon,

2009).

However, Wnts can activate other, so called non‐canonical Wnt pathways, which

are ‐catenin‐independent and biochemically distinct from canonical Wnt signaling. Recent

evidence suggests that several such pathways exist (for complete overview see (Semenov, et

al, 2007)). Among those, the best known appears to be the pathway that acts via small

GTPases Rac1 and RhoA and regulates planar cell polarity (PCP). Wnt/PCP pathway was

originally described in Drosophila (Seifert and Mlodzik, 2007). Vertebrate homologs of

Drosophila PCP proteins are well conserved and have been implicated in several vertebrate

processes such as convergent extension movements during gastrulation, in neurulation and

in the regulation of the polarity of hairy cells in the inner ear (Seifert and Mlodzik, 2007;

Torban, et al, 2004). Apart from the Wnt/PCP pathway several other Wnt‐related signaling

mechanisms have been defined. These include the Wnt/Ca2+ pathway that is mediated by

trimeric G‐proteins (Kuhl, 2004) or the Wnt/Ryk pathway involved in the axon guidance.


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Finally, Wnts can signal via the atypical receptor tyrosine kinases Ror1/2 (Semenov, et al,

2007; Schambony and Wedlich, 2007).

All known Wnt‐induced pathways are transduced via seven transmembrane

receptors Frizzleds (Fzd) and via phosphoprotein dishevelled (Dvl). There is a general

agreement based on genetic experiments that Dvl plays a crucial role as a signaling hub both

in Wnt/‐catenin and Wnt/PCP pathways (Wallingford and Habas, 2005). It is intriguing that

despite well documented importance of Dvl in the Wnt signaling, the molecular mechanisms

activating Dvl in response to Wnt and molecular mechanisms used by Dvl to activate

signaling cascades downstream are almost unknown.

2. Dishevelled

Dishevelled (Dvl) is a cytoplasmic phosphoprotein required for signal transduction in

the Wnt /β‐catenin pathway and in the non‐canonical Wnt pathways. Dvl was named based

on the phenotype of the Drosophila mutant with the defects in planar cell polarity (PCP)

manifested by disoriented body and wing hairs (Fahmy and Fahmy, 1959). Later on Dvl was

identified as a component of Wnt/β‐catenin pathway influencing segment polarity of

Drosophila larvae (Klingensmith, et al, 1994; Krasnow, et al, 1995; Noordermeer, et al, 1994;

Theisen, et al, 1994). Already these early genetic experiments in flies demonstrated the dual

role of Dvl.

Figure 1: The Wnt signaling pathways


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There is only one Dvl gene in Drosophila, in contrast to three genes (Dvl1, Dvl2 and

Dvl3) in vertebrate genomes (Klingensmith, et al, 1996; Sussman, et al, 1994; Tsang, et al,

1996). Dvl protein sequence similarity is around 50% between Drosophila and mouse Dvls,

consistent with the conserved function between species. The identity of the three mouse Dvl

proteins is around 60% and Dvl transgene rescue experiments in knock‐out mice suggest

that individual Dvls are largely redundant in vivo (Etheridge, et al, 2008) although the

phenotypes of individual Dvl knockouts differ. Dvl1‐/‐ mice were viable with no obvious

developmental malformations but showed social deficits due to impaired synaptic assembly,

neurotransmiter release and dendritic branching (Ahmad‐Annuar, et al, 2006; Lijam, et al,

1997; Rosso, et al, 2005). Phenotypes of Dvl2‐/‐ and Dvl3‐/‐ mouse were much more severe

and included perinatal lethality, abnormal cardiac morphogenesis due to neural crest defects

and skeletal malformations as a result of improper somite formation (Etheridge, et al, 2008;

Hamblet, et al, 2002; Kioussi, et al, 2002). The phenotypes typically associated with the PCP

defects such as craniorachischisis and defects in the organ of Corti were observed frequently

in compound mutants of two Dvl isoforms (the most severe in Dvl2‐/‐;Dvl3‐/‐ mice) (Etheridge,

et al, 2008). Interestingly, double Dvl mutants lacked phenotypes typically associated with

the Wnt/β‐catenin pathway defects and did not show global changes in the TCF/LEF‐driven

transcription (Etheridge, et al, 2008), which suggests that even low levels of the remaining

single Dvl isoform are capable to mediate physiological Wnt/β‐catenin signaling during

development.

2.1. Structure of Dvl

Dvl is a modular protein that contains three well defined structural domains linked

by the intrinsically disordered regions, which are predicted to have no secondary structure.

N terminal DIX (Dishevelled, Axin; aminoacids) domain is connected with the central PDZ

(Postsynaptic density, Discs large, Zonula occludens) domain by the serine (Ser)/threonine

(Thr)‐rich unstructured region with the conserved stretch of basic aminoacids (S/T region).

PDZ domain and the C‐terminal DEP domain (Dvl, Egl‐10, Pleckstrin) are linked by the region

enriched in proline (Pro‐rich region). C‐terminus is formed by relatively large but poorly

conserved disordered region (Fig. 2). Rescue analysis using Dvl deletion mutants suggested

that DIX and PDZ domains are necessary for Wnt/β‐catenin pathway, whereas PDZ and DEP

domains are required for Wnt/PCP pathway (Axelrod, et al, 1998; Boutros, et al, 1998).

Individual domains have been crystalized and share interesting functional and

structural features, which are summarized below:

a) DIX domain (Fig. 2B): Mutational studies of Dvl2 DIX domain and modeling based on the

crystal structure of the Axin DIX domain (Schwarz‐Romond, et al, 2007) have convincingly

demonstrated that the key feature of DIX domain is its ability to form head‐to‐tail polymers.

DIX domain mutants that lack the ability to polymerize were unable to form Dvl polymers

visible as cytoplasmatic Dvl punctae (when overexpressed) and were inactive in the Wnt/β‐

catenin pathway (Schwarz‐Romond, et al, 2007). It has been proposed that DIX‐domain


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mediated Dvl polymers are a physical platform for the complexes of Dvl and other signaling

components, called signalosomes, required for Lrp6 phosphorylation (Bilic, et al, 2007). The

requirement of DIX domain for the phosphorylation of Lrp6 and its capacity to dimerize

seems to be the molecular reason for the absolute requirement of DIX domain in the Wnt/β‐

catenin signaling.

b) PDZ domain (Fig. 2C): PDZ domain is commonly found in various proteins, where it serves

as a universal interaction and docking platform. Dvl PDZ domain seems to be inherently

more flexible than PDZ domains found in other proteins (Zhang, et al, 2009). PDZ domain

functions as an important scaffolding region of Dvl protein and it was shown to be required

for the interaction with many Dvl binding partners (for review see Gao and Chen, 2010).

Figure 2: Schematic representation of Dvl structure.

A. Aminoacid (aa) sequence identity (%) based on alignment of Drosophila Dsh, and mouse (m) and human (h)Dvl isoforms 1,2 and 3). Positions of aa bordering individual regions is based on hDvl3. 3D models of individual domains (hDvl3) are provided either as ribbon models (i) or surface models (ii). Electrostatic surface charge is depicted as negative in red and positive in blue.

B. DIX domain: (i) Residues mutated in Dvl polymerization mutants corresponding to F43S, V67A/K68A, and Y27D in mDvl2 are depicted in orange (Schwarz‐Romond, et al, 2007). (ii). Regions of DIX domain required for head to tail polymerization are marked by arrows.

C. PDZ domain: (i) α‐helix and β‐sheet forming the Fzd binding groove are indicated in green (Wong, et al, 2003). (ii) Fzd binding groove is indicated by a green line.

D. DEP domain: (i, ii) Residues and surface required for membrane binding are indicated in yellow (Simons, et al, 2009) and residues required for Fzd binding are shown in blue (Tauriello, et al, 2012). Position of K435 corresponding to K417 mutated in Drosophila Dsh1 mutation (Axelrod, et al, 1998) is indicated. (i‘,ii’): Residues (S464, T480, T485, S487, Y491, Y492) phosphorylated following Fzd overexpression (Yanfeng, et al, 2011) are indicated in red (i’). (ii’) Phosphomimicking mutations of these residues change electrostatic surface potential and disrupt the positive charge of the membrane binding region. Please note also the change caused by the mutation K435M (Dsh1, arrow). (from Bryja and Bernatik, 2014)


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Despite PDZ domain of Dvl has been implicated in many protein‐protein interactions, not

much is known about the molecular details and the significance of the individual

interactions. Notable exception is the NMR characterization of the interaction between PDZ

domain (mDvl1) and internal PDZ‐binding motif of FZD7 (Wong, et al, 2003). The same

binding cleft is bound by natural Wnt pathway inhibitors such as proteins from Dact family

(homologues of fly Dapper/Frodo) (Wong, et al, 2003) and serves also as a target for

development of novel drugs targeting FZD‐Dvl interaction (Shan, et al, 2012).

c) DEP domain (Fig. 2D): DEP domain of Dvl is responsible for the recruitment of Dvl to the

membrane and for the signal transduction in the non‐canonical Wnt/PCP pathway (Axelrod,

et al, 1998; Boutros, et al, 1998). The crystal structure of the DEP domain of Dvl is known

(Wong, et al, 2000). Positively charged region of Dvl DEP domain is essential for the binding

to the negatively charged lipids in the plasma membrane and mutation of the positively

charged residues abolished recruitment of Dvl to plasma membrane and caused PCP

phenotypes in Drosophila (Simons, et al, 2009). It was proposed that ion pumps (such as

Nhe2, Na+/H+ exchanger) in the plasma membrane control Dvl membrane recruitment by

regulating local pH (Simons, et al, 2009). It should be however noted that Fzd‐induced

phosphorylation within the DEP domain (Yanfeng, et al, 2011), which also changes the

charge of the surface‐interacting region from positive to negative, may have similar

consequences (see model in Fig. 2Di´). Furthermore, it was recently proposed that DEP

domain of Dvl is the second interaction interface (in addition to the PDZ domain) mediating

interaction with FZD (Tauriello, et al, 2012). Last but not least, currently there are only two

known point mutations which completely disrupt PCP signaling. Both mutations in the fully

conserved Lys (Dsh1 mutant K417M Drosophila Dvl) and Tyr (Y473A) are in the DEP domain.

2.2. Dvl‐associated kinases and consequences of Dvl phosphorylations

Dvl is a dynamically phosphorylated protein. Precise mapping and functional

analysis of Dvl phosphorylation sites is complicated by the fact, that approximately 15‐20%

(depends on the isoform) of Dvl is composed by Ser, Thr or tyrosine (Tyr). Treatment with

both classes of Wnt ligands triggers phosphorylation‐dependent mobility shift of Dvl on SDS‐

PAGE (phosphorylated and shifted Dvl; PS‐Dvl) (Bryja, et al, 2007; Gonzalez‐Sancho, et al,

2004). Numerous studies have analyzed phosphorylation of Dvl by using various approaches.

Comprehensive summary of identified Dvl phosphorylations and their known function is

provided in Fig. 3 and in the text below.

Among several Dvl kinases the best described and the first one to mention is casein

kinase (CK) 1 δ/ε. CK1δ and CK1ε are redundant in most of their functions in Dvl biology, and

in further text we will thus refer only to CK1ε. CK1ε is both required and sufficient for PS‐Dvl

formation. Following Wnt stimulation CK1ε gets activated by a poorly known mechanism,

which requires dephosphorylation of the inhibitory residues in its C‐terminus (Swiatek, et al,

2004) and cooperation with the DDX3 protein (Cruciat, et al, 2013). CK1ε‐mediated

phosphorylation of Dvl subsequently triggers interaction of Dvl with endogenous Axin1 and


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activates downstream Wnt/β‐catenin signaling (Bernatik, et al, 2011; Kishida, et al, 2001;

Peters, et al, 1999).

Apart from these clearly positive effects, CK1ε dissolves (and CK1 inhibition

promotes formation of) DIX‐domain dependent Dvl polymers, which are required for the

phosphorylation of Lrp6 and downstream signaling (Bilic, et al, 2007; Bryja, et al, 2007; Cong,

et al, 2004). We have shown that these opposing functions of CK1ε require distinct domains

of Dvl – activation requires phosphorylation in the PDZ domain (and likely also elsewhere),

whereas inhibitory activity requires Dvl C‐terminus (Bernatik, et al, 2011). Further detailed

molecular analysis supports the view that CK1ε acts as the component of the intrinsic

negative feedback loop, which by numerous sequential phosphorylations activates and

subsequently inactivates Dvl (Bernatik, et al, 2014). It seems that CK1ε acts as (i) the

activator of downstream Wnt/‐catenin signaling via phosphorylation of distinct S/T residues (phosphorylation of S280/S311 in the PDZ domain) as well as (ii) the inactivating

kinase affecting Dvl polymerization via phosphorylation of the residues in the C‐terminus of

Dvl3 (Bernatik, et al, 2014).

The negative role of Dvl C‐terminus in the Wnt/β‐catenin signaling was established

by our earlier work. We have proposed that Dvl C‐terminus acts as the CK1‐controlled negative regulator (Bernatik, et al, 2011; Witte, et al, 2010). In the most recent study

(Bernatik, et al, 2014) we identified specific residues phosphorylated by CK1ε in the C‐

Figure 3: Phosphorylation of Dvl. Summary of Dvl posttranslational modifications mapped onto hDvl3.

Position of detected phosphorylations () are shown. The bottom part of the scheme shows phosphorylation known from the literature, the upper part shows CK1ε‐mediated phosphorylation events discovered by our group. Color coding indicates the phosphorylation dynamics. Functionally relevant modifications are specified in detail. The figure is based on the following studies: 1 Klimowski, et al (2006), 2 Kikuchi, et al (2010), 3 Cong, et al (2004), 4 Yokoyama, et al (2012), 5 Singh, et al (2010), 6 Bernatik, et al (2014)


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terminus of Dvl3. We demonstrate that these residues control PS‐Dvl formation and are

critical for CK1ε‐induced changes in Dvl3 subcellular localization. Although the C‐terminal

residues are conserved only in Dvl3, multiple S/T residues are present also in the C‐terminal

region of Dvl1 and Dvl2. This opens the possibility that function of phosphorylated C‐

terminus is conserved despite the differences in the primary sequence. This possibility is

supported by recent findings by Gonzalez‐Sancho and colleagues (Gonzalez‐Sancho, et al,

2013), which demonstrated that formation of PS‐Dvl2 and its even localization depends on

other residues of hDvl2 C‐terminus. The residues are conserved in Dvl3 (S578 and S581 in

hDvl3) but were found constitutively phosphorylated in our study (Bernatik, et al, 2014).

The second important kinase is casein kinase 2 (CK2), which was identifed as the

first Dvl‐associated kinase during the search for the kinase activity co‐immunoprecipitating

with Dvl (Lee, et al, 1999; Willert, et al, 1997). CK2 is not structurally related to CK1 and

functions as a tetramer with 2 catalytic α (or α’) and 2 regulatory β subunits. When

compared to dynamic activation of CK1ε, CK2 behaves with respect to Dvl as a rather

constitutive kinase, which is not regulated by Wnt ligands although CK2 activity is required

for the PS‐Dvl activation and downstream signaling in both Wnt/β‐catenin and Wnt/PCP

pathway (Bernatik, et al, 2011; Bryja, et al, 2008).

Several other kinases were shown to bind and phosphorylate Dvl. However, their

role is according to the current knowledge restricted to specific model systems or limited to

one report only. These kinases include (i) PAR‐1 (in Xenopus, homologous to

MAP/microtubule affinity‐regulating kinase (MARK) family of kinases in mammals), which

were shown to activate canonical Wnt signaling as well as gastrulation movements, a

process controlled by non‐canonical Wnt signaling (Kusakabe and Nishida, 2004; Ossipova, et

al, 2005; Sun, et al, 2001), (ii) metastasis associated kinase 1 (MAK1), homologous to the

mammalian HUNK, a member of sucrose non fermenting related kinase 1, SNF1, family)

(Kibardin, et al, 2006), (iii) Polo‐like kinase (Plk1) a mitosis associated kinase, which via Dvl

phosphorylation at T206 affects division plane during mitosis (Kikuchi, et al, 2010) and (iv)

PKCδ regulating non‐canonical Wnt signaling (Chen, et al, 2003; Kinoshita, et al, 2003). The

last important kinase is tyrosine kinase Abl, identified in the screen for molecules required

for membrane localization of Dvl. Abl phosphorylated Dvl on several Tyr residues in the DEP

domain, and it was shown that both mutation of a critical Tyr (Y473F) or Abl depletion

caused PCP phenotypes but not Wnt/β‐catenin phenotypes in Drosophila (Singh, et al, 2010).

The information about the function of Abl‐driven phosphorylation of Dvl in vertebrates is

missing.

Recent progress in proteomics allowed to perform comprehensive studies

attempting to describe complete Dvl phosphorylation pattern. So far, two such studies are

available ‐ first focused on the identification of Dvl phosphorylation following Frizzled

overexpression in Drosophila model (Yanfeng, et al, 2011), the second (from our group)

analyzed phosphorylation of Dvl3 induced by overexpression of the most relevant Dvl kinase

– CK1ε (Bernatik, et al, 2014). Availability of these data allowed comparison of Fzd and CK1‐


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induced phosphorylation patterns. When we mutated CK1ε‐phosphorylated

serines/threonines to alanines in the Dvl3 C‐terminus, we observed deficiency in CK1ε‐

induced mobility shift or subcellular localization change. However, Fzd5‐induced mobility

shift or Fzd5‐induced subcellular localization in these Dvl3 mutants were indistinguishable

from wild type Dvl3. Moreover, when we generated and implemented anti‐pS643‐Dvl3

phospho‐specific antibody (recognizing residues phosphorylated by CK1ε), it recognized only

Dvl3 modified by CK1ε but not by Fzd5 overexpression. This data suggest to our surprise that

CK1ε is not directly downstream of Fzd receptor but rather represents independent, possibly

parallel, molecular mechanism of activation/de‐activation of Dvl (Bernatik, et al, 2014).

2.3. Regulation of Dvl by ubiquitination

Dvl is a key protein, which controls direction and amplitude of downstream Wnt

signaling. As such Dvl levels are tightly regulated. Several proteins, which differ both in the

route used for Dvl degradation and in the ability to target specific subcellular pool of Dvl,

were shown to trigger degradation of Dvl. Dvl is degraded both via proteasome and

lysosome/autophagy pathway. Lysosomal pathway, which degrades Dvl mainly in the

starved cells, is dependent on Von Hippel‐Lindau protein (Gao, et al, 2010) and was shown

to be promoted by the Wnt inhibitor Dapper (Zhang, et al, 2006).

Proteosomal degradation of Dvl is either unspecific ‐ triggered by an E3 ubiquitin

ligase NEDL1, associated with the amyotrophic lateral sclerosis (Miyazaki, et al, 2004) or by

KLHL12, an adaptor protein linking Dvl to Cullin3 E3 ubiquitin ligase (Angers, et al, 2006).

Complete degradation of Dvl then leads both to inhibition of Wnt/β‐catenin pathway, and to

disruption of convergent extension controlled by non‐canonical Wnt pathway. Alternatively,

in order to more precisely control Dvl function there are mechanisms that control

degradation of specific pools of Dvl. The examples include inversin (Invs), a protein mutated

in the cystic renal disease nephronopthisis, which enhances degradation of cytoplasmic but

not membrane‐bound Dvl (Simons, et al, 2005), myristoylated Naked2, which on the

contrary promotes degradation of Dvl bound to the cytoplasmatic membrane (Hu, et al,

2010), Rpgrip1l, which is essential for stabilization of Dvl at the base of cilium (Mahuzier, et

al, 2012) and Itch, HECT‐domain containing ubiquitin ligase that specifically promotes

degradation of phosphorylated and activated PS‐Dvl (Wei, et al, 2012). Unique function was

proposed for nucleoredoxin (NXN), which prevents KLHL12‐mediated degradation of the

inactive Dvl pool (Funato, et al, 2010).

Ubiquitination does not only target proteins for degradation but it also has a

potential to regulate the signaling abilities of modified protein by differential linkages (eg.

via ubiquitin K63) of ubiquitin side chains (Chen and Sun, 2009). We have recently found that

HECT domain E3 ligase Huwe1 promoted the formation of K63‐linked poly‐ubiquitin chains

on lysine residues within the DIX domain of Dvl (De Groot, et al, 2014). Huwe1 binds and

ubiquitinates Dvl in a Wnt3a‐ and CK1‐dependent manner. Importantly, K63‐linked poly‐

ubiquitination of Dvl does not target Dvl for degradation. Instead, we found that Huwe1


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inhibits Dvl multimerization via ubiquitination of the DIX domain and acts as a negative

regulator of Wnt/β‐catenin signaling (De Groot, et al, 2014).

Our data suggest that Huwe1‐mediated ubiquitination is dynamic and an enzymatic

system removing K63‐linked ubiquitin chains from Dvl has to exist. In a recent report, the de‐

ubiquitinating (DUB) enzyme USP14 was identified as a positive regulator of Wnt signaling.

This fact makes USP14 a likely counterpart to the activity of Huwe1 on Dvl. USP14 interacts

with a motif in the carboxy‐terminus of Dvl that is conserved among vertebrates.

Interestingly, USP14 has been shown to remove K63‐linked poly‐ubiquitin chains from Dvl

(Jung, et al, 2013) and to activate Wnt/β‐catenin signaling. The link to the Huwe1 mediated

K63‐poly‐ubiquitin chains from the DIX domain however remains to be established. Apart

from USP14 another DUB ‐ the tumor suppresor CYLD ‐ was found to be the deubiquitination

enzyme capable of removal of K63‐linked polyubiquitin chains. However, CYLD

downregulation and increased level of K63‐Ubi‐modified Dvl leads to the activation of

Wnt/β‐catenin pathway (Tauriello, et al, 2010) and it is thus likely that CYLD is not the

functional counterpart of Huwe1. It rather seems that CYLD acts together with Dvl in the

processes not directly linked to the regulation of Wnt signaling pathway such as regulation

of mitosis and cell cycle (Yang, et al, 2014).

Our study and work of others (summarized in Fig. 4) illustrated the importance of

ubiquitin mediated regulation of Dvl signaling in the Wnt pathway. However, so far only

Huwe1 described by us, represents Dvl ubiquitin ligase or de‐ubiquitinating enzyme with a

role in Wnt signaling that is evolutionary conserved in both vertebrates (human) and

invertebrates (Caenorhabditis elegans). This indicates that Huwe1 may represent an

ancestral mechanism of Dvl regulation.

Figure 4: Ubiquitination of Dvl. Summary of Dvl ubiquitinations mapped onto hDvl3. Position of detected

ubiquitinations () are shown. The bottom part of the scheme shows ubiquitinated sites known from the literature, the upper part shows Huwe1‐mediated phosphorylation events discovered by our group. Functionally relevant modifications are specified in detail. The figure is based on the following studies: 1Tauriello, et al (2010), 2 Kim, et al (2011) 3 De Groot, et al (2014)


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2.4. Dvl in the Wnt/β‐catenin and Wnt/PCP pathway – how the decision is made?

As mentioned several times earlier Dvl serves as a branching point between Wnt/β‐

catenin and Wnt/PCP signaling. It is natural that more than 15 years from this discovery a lot

of attention has been paid to uncover the mechanisms, which control Dvl function and

define the direction of downstream signaling. The answer is not completely solved yet but it

is obvious that the decision is made based on the combination of several mutually inter‐

connected factors, which involve (i) Dvl subcellular localization, (ii) Dvl polymerization, (iii)

Dvl phosphorylation, and (iv) Dvl binding partners/receptors.

In the Wnt/β‐catenin pathway (Fig. 5A) membrane localization of Dvl is not required

(Park, et al, 2005) in contrast to the intact DIX domain. DIX domains mediate Dvl

polymerization, which is crucial for formation large protein complexes, called signalosomes,

composed of polymerized Dvl, Axin and other Wnt pathway components (Bilic, et al, 2007;

Schwarz‐Romond, et al, 2007). The formation of signalosome complex is required for the

activating phosphorylation of Lrp6, subsequent recruitment of Axin and inhibition of the

destruction complex (Bilic, et al, 2007). Interaction interface between two DIX domains,

which mediates Dvl polymerization, is subject to post‐translational modifications – namely

K63‐linked polyubiquitination, which disrupt Dvl multimer formation and efficiently inhibits

function of Dvl in Wnt/β‐catenin signaling (De Groot, et al, 2014). This mechanism is part of

the negative feedback loop induced by ubiquitination of activated Dvl by an E3 ligase Huwe1

(De Groot, et al, 2014).

Precise sequence of individual events following addition of a ligand, typically Wnt3a,

is not precisely known. Current view is that following ligand binding Dvl gets activated by a

poorly known mechanism involving Fzd and CK1ε‐controlled phosphorylation (Bernatik, et al,

2011), which subsequently triggers production of phosphatidylinositol 4,5‐bisphosphates

(PtdIns(4,5)P2) (Pan, et al, 2008). The locally produced of PtdIns(4,5)P2 is recognized by the

scaffolding protein Amer1 (Tanneberger, et al, 2011) in complex with the Dvl‐binding protein

β‐arrestin (Kriz, et al, 2014). Our data suggest that following activation β‐arrestin acts as a

switch, which translocates PtdIns(4,5)P2‐producing kinases from Dvl towards Lrp6

phosphorylating complex. This allows efficient phosphorylation of Lrp6 at the signalosome

platform fed by the local production of PtdIns(4,5)P2.

In the Wnt/PCP pathway, DEP domain and membrane‐localization of Dvl is the

absolute prerequisite for the signaling activity (Fig. 5B). So far, all the Wnt/PCP‐deficient

mutants of Dvl also failed to be recruited to the membrane by Fzds (Axelrod, et al, 1998;

Simons, et al, 2009; Singh, et al, 2010) and even wild type Dvl constitutively trafficked to

other compartment such as outer mitochondrial membrane (Park, et al, 2005) was inactive

in the Wnt/PCP pathway. Various mechanisms, which control membrane localization of Dvl,

and subsequently favor PCP pathway over Wnt/β‐catenin, were described. They include

regulation of local pH (Simons, et al, 2009), PCP‐specific Dvl phosphorylation by Abl (Singh,

et al, 2010) or the targeted degradation of cytoplasmic Dvl by Inversin (Simons, et al, 2005).


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Further specificity to the pathway is provided by the dedicated membrane co‐

receptors. Several proteins mainly from the family of atypical receptor tyrosine kinases,

namely Ror1, Ror2, Ryk and PTK, dictate beta‐catenin‐independent direction of signaling and

cooperate with Dvl in non‐canonical pathway (Lu, et al, 2004; Shnitsar and Borchers, 2008;

Witte, et al, 2010). Lrp6, a coreceptor dedicated to the Wnt/ β‐catenin is not positively

involved in the PCP pathway (albeit is involved as a negative regulator (Bryja, et al, 2009)).

Following, non‐canonical Wnt pathway‐specific, activation Dvl directs signaling

further downstream mainly towards the molecular machineries controlling cytoskeleton, cell

shape and cell polarity. Key players in this process are mainly small GTPases from Rho family,

Rac1 and RhoA. These small monomeric G‐proteins are activated by the strictly regulated

exchange of GDP for GTP. This process is actively controlled and mediated by dedicated

proteins named guanine exchange factors (GEFs). Evidence for the employment of specific

GEF(s) in Wnt/β‐catenin‐independent signaling has only recently begun to emerge. WGEF,

p114‐RhoGEF, and GEF‐H1, have been proposed as GEFs for RhoA (Tanegashima, et al, 2008;

Tsuji, et al, 2010). However, GEF(s) involved in the Wnt5a/Dvl‐mediated activation of Rac1

have not been described until recently. Only in 2013 we were able to identify a long

searched protein responsible for Wnt5a/Dvl‐mediated activation of Rac1 in the non‐

canonical Wnt pathway as a specific Rac1 GEF, Tiam1 (Cajanek, et al, 2013). Our work

Figure 5: Key structural elements required for the function of Dvl in the main Wnt pathways (from Bryja and Bernatik, 2014).

A: Scheme of Dvl function in Wnt/β‐catenin pathway. Dvl is required for Lrp6 phosphorylation and subsequent inhibition of the destruction complex function. Polymerization via DIX domains but not membrane localization is required for this function.

B: Scheme of Dvl function in Wnt/PCP pathway. Dvl is recruited to the membrane (DEP‐domain dependently), where it interacts with Fzd and other co‐receptors (Ror1/2, Ryk, PTK7) and activates small GTPases Rho and Rac, which subsequently control re‐organization of cytoskeleton. C‐terminus of Dvl is required for binding to Ror2


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demonstrated that Tiam1 can interact with Dvl and enhances the Dvl‐Rac1 interaction. Most

importantly, Tiam1 is functionally required for the activation of Rac1 by Wnt5a or Dvl and for

Rac1‐dependent downstream cellular processes.

Interestingly, we showed that the Dvl‐Tiam1 interaction is strictly negatively

regulated by CK1 (Cajanek, et al, 2013). These findings are in full agreement with earlier

reports, which showed that phosphorylation of Dvl by CK1 is inhibitory for the non‐canonical Wnt/Dvl/Rac1/JNK pathway (Bryja, et al, 2008; Cong, et al, 2004). Thus, CK1 can act as a switch that directs signaling away from the Wnt/Rac1/JNK branch of non‐canonical

signaling towards the Wnt/‐catenin and PS‐Dvl‐dependent non‐canonical pathways. This fact might have clinically important consequences – for example we were able to show that

breast cancer‐specific mutations in CK1 produce a kinase inactive and dominant negative

variant of the kinase (Foldynova‐Trantirkova, et al, 2010). Such dominant negative CK1ε

variants, naturally occurring in breast carcinoma (Fuja, et al, 2004) efficiently bind, but fail to

phosphorylate Dvl, and act as loss‐of‐function in the Wnt/‐catenin pathway. At the same

time these mutants increase in a Dvl‐dependent manner the level of Rac1‐GTP and promote

cell migration and invasion (Foldynova‐Trantirkova, et al, 2010). Combined, these results

suggest that CK1, in addition to the functions discussed above, controls changes in protein‐protein interactions between Dvl and its binding partners, which contribute to the activation

of Wnt/β‐catenin or on the other side inactivation of the non‐canonical Wnt/Rac1 pathway.

Such “switch” function of CK1, which was first identified in Xenopus and cell culture experiments, is physiologically relevant and may contribute to cancer progression in

pathological conditions where CK1 is mutated.

3. Future directions

Dvls are intensively studied proteins, which despite the efforts of numerous labs still resist

our understanding and hide most of their secrets. The following section proposes a

subjective view of the aspects of Dvl biology, which require most attention in near future,

and which will be crucial for our full understanding of Dvl function.

Dvl in the receptor complex: The biggest challenge for the near future will be to describe the

sequence and relevance of events following interaction of the Wnt ligands with their

receptors. The lines of research, which will at the endogenous level define the dynamics of

Dvl post‐translational modifications, Dvl interaction partners and Dvl subcellular localization

following activation of discrete Wnt pathways will answer these questions. It is probable that

detailed mechanistic understanding of Dvl biology will shed light not only on the mechanism

discriminating between β‐catenin and PCP downstream signaling but also onto details of

various non‐canonical Wnt pathways, which diversified during the course of evolution and

differ in humans substantially from the core PCP pathway postulated in fly.


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Paradox of Dvl: Dvl has been studied extensively and nowadays more than 70 binding

partners and 40+ phosphorylation sites have been reported (Bernatik, et al, 2014; Gao and

Chen, 2010; Yanfeng, et al, 2011). The list of Dvl interaction partners and post‐translational

modifications is constantly growing but our understanding is not ‐ this phenomenon we call

“Dishevelled paradox”. It is very likely that cells regulate both post‐translational modification

as well as the composition of Dvl‐based complexes in a delicate manner, and that

endogenous Dvl exists in several well separated pools. The earlier efforts to sort the large

amount of available Dvl‐related data and to propose a robust model of biochemical details

of Dvl action have always failed. The main reason is that (to our surprise) the field is missing

the basic information about endogenous Dvl. Specifically, we lack the basic endogenous Dvl

coordinates such as information about the subcellular localization, protein amount (mainly

in comparison with other major Dvl partners), basic structural information and definition of

direct binding surfaces with other proteins.

Dvl out of the Wnt pathways: It is becoming obvious that Dvl controls processes, which are

not directly linked with Wnt/β‐catenin and Wnt/PCP pathway. Accumulating body of

evidence pointed to the role of Dvl in the biology of cilia. Dvl was shown in numerous

contexts to interact with centriolar proteins and to be required for the positioning of the

basal body and the proper function of cilia (Hashimoto, et al, 2010; Chaki, et al, 2012; Park,

et al, 2008). This function of Dvl is evolutionary conserved from planarians (Almuedo‐

Castillo, et al, 2011). Recently, Dvl was shown to be involved not only in the biogenesis of

cilia but also in the process of cilia disassembly (Lee, et al, 2012). The last exciting avenue is

the unexpected role of Dvl in the regulation of cell cycle. Very recent evidence suggests that

Dvl2 can regulate spindle orientation in mitosis via its interaction with Polo‐like kinase 1

(Kikuchi, et al, 2010) and cytokinesis by the regulation of midbody dynamics (Fumoto, et al,

2012). In order to fully understand the role of Dvl in these, often cell type‐specific processes,

the field will need to generate cell lines fully deficient in all three mammalian Dvls. This

possibility has become recently possible by development of novel gene editing technologies

– such as or the type II bacterial Clustered Regularly Interspaced Short Palindromic Repeats

(CRISPR)/CRISPR‐associated (Cas) enzymatic systems (Mali, et al, 2013).

All the diverse aspects of Dvl biology described above are tightly controlled and provide

unique outcome in each cell and each developmental process. The mechanism of such error‐

resistant integration of all Dvl functions remains the biggest mystery.


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5. List of primary research articles representing author´s contribution to the understanding of Dvl function in the Wnt pathways (attachments)

The list of attachments summarized below represents a selection of 20 primary research

articles (out of 67 author´s records available on Pubmed on Feb 21, 2014), which are directly

related to the topic of the habilitation. These attachments provide comprehensive overview

of V. Bryja´s contribution to the biology of Dishevelled. Individual articles are presented

without supplementary information in the printed version of the thesis. Full version is

available on the attached CD.

1. Schulte G, Bryja V, Rawal N, Castelo‐Branco G, Sousa KM, Arenas E (2005): Purified HA‐Wnt5a

increases differentiation of dopaminergic precursor cells. J. Neurochem 92:1550‐3.

2. Bryja V, Schulte G, Arenas E (2007): Wnt‐3a utilizes a novel low dose and rapid pathway that

does not require casein kinase 1‐mediated phosphorylation of Dvl to activate ‐catenin. Cell. Signal. 19: 610‐616.

3. Bryja V, Schulte G, Rawal N, Grahn A, Arenas E (2007): Wnt‐5a induces Dishevelled

phosphorylation and dopaminergic differentiation via a CK1‐dependent mechanism. J. Cell

Sci. 120: 586‐595.

4. Bryja V, Čajánek L, Grahn A, Schulte G (2007): Inhibition of endocytosis blocks Wnt signalling to

‐catenin by promoting dishevelled degradation. Acta Physiol.(Oxford) 190 (1): 53‐59

5. Bryja V, Gradl D, Schambony A, Arenas E, Schulte G (2007): ‐arrestin is a necessary component

of Wnt/‐catenin signaling in vitro and in vivo. Proc. Natl. Acad. Sci. USA 104: 6690‐6695.

6. Bryja V, Schambony A, Čajánek L, Dominguez I, Arenas E, Schulte G (2008): beta‐Arrestin and

casein kinase 1/2 define distinct branches of non‐canonical WNT signalling pathways.

EMBO Rep. 9(12): 1244‐50. Featured article.

7. Bryja V, Andersson ER, Schambony A, Esner M, Bryjová L, Biris KK, Hall AC, Kraft B, Cajanek L,

Yamaguchi TP, Buckingham M, Arenas E. (2009): The Extracellular Domain of Lrp5/6

Inhibits Non‐Canonical Wnt Signaling in vivo. Mol Biol Cell. 20: 924‐936. Cover article.

8. Andersson T, Södersten E, Duckworth JK, Cascante A, Fritz N, Sacchetti P, Cervenka I, Bryja V,

Hermanson O (2009): CXXC5 is a novel BMP4‐regulated modulator of Wnt‐signaling in

neural stem cells. J Biol Chem. 284(6):3672‐81.

9. Witte F, Bernatik O, Kirchner K, Masek J, Mahl A, Krejci P, Mundlos S, Schambony A, Bryja V,

Stricker S. (2010): Negative regulation of Wnt signaling mediated by CK1‐phosphorylated

Dishevelled via Ror2. FASEB J. 24(7):2417‐26.

10. Foldynova‐Trantirkova S, Sekyrova P, Tmejova K, Brumovska E, Bernatik O, Blankenfeldt W, Krejci

P, Kozubik A, Dolezal T, Trantirek L, Bryja V. (2010): Breast cancer‐specific mutations in

CK1epsilon inhibit Wnt/beta‐catenin and activate the Wnt/Rac1/JNK and NFAT pathways

to decrease cell adhesion and promote cell migration. Breast Cancer Res. 2010 May

27;12(3):R30.


24

11. K. Tanneberger, A.S. Pfister, K. Brauburger, J. Schneikert, M.V. Hadjihannas, V. Kriz, G. Schulte, V.

Bryja and J. Behrens (2011): Amer1/WTX couples Wnt‐induced formation of PtdIns(4,5)P2

to LRP6 phosphorylation. EMBO J. 30: 1433‐1443.

12. Bernatik O, Sri Ganji R, Cervenka I, Polonio T, Schulte G, Bryja V (2011): Sequential activation and

inactivation of Dishevelled in the Wnt/‐catenin pathway by casein kinases. J. Biol. Chemistry 286: 10396‐10410.

13. Chaki M, Airik R, Ghosh AK, Giles RH, Chen R, Slaats GG, Wang H, Hurd TW, Zhou W, Cluckey A,

Gee HY, Ramaswami G, Hong CJ, Hamilton BA, Cervenka I, Ganji RS, Bryja V, Arts HH, van

Reeuwijk J, Oud MM, Letteboer SJ, Roepman R, Husson H, Ibraghimov‐Beskrovnaya O,

Yasunaga T, Walz G, Eley L, Sayer JA, Schermer B, Liebau MC, Benzing T, Le Corre S,

Drummond I, Janssen S, Allen SJ, Natarajan S, O'Toole JF, Attanasio M, Saunier S, Antignac

C, Koenekoop RK, Ren H, Lopez I, Nayir A, Stoetzel C, Dollfus H, Massoudi R, Gleeson JG,

Andreoli SP, Doherty DG, Lindstrad A, Golzio C, Katsanis N, Pape L, Abboud EB, Al‐Rajhi

AA, Lewis RA, Omran H, Lee EY, Wang S, Sekiguchi JM, Saunders R, Johnson CA, Garner E,

Vanselow K, Andersen JS, Shlomai J, Nurnberg G, Nurnberg P, Levy S, Smogorzewska A,

Otto EA, Hildebrandt F. (2012): Exome Capture Reveals ZNF423 and CEP164 Mutations,

Linking Renal Ciliopathies to DNA Damage Response Signaling. Cell. 150(3): 533‐48.

14. L. Čajánek, R. Sri Ganji, C. Henriques‐Oliveira, P. Koník, S. Theofilopoulos, V. Bryja, E. Arenas

(2013): Tiam1 regulates the Wnt/Dvl/Rac1 signaling pathway and the differentiation of

midbrain dopaminergic neurons. Mol. Cell. Biol. 33(1):59‐70.

15. A. Soldano, Z. Okray, P. Janovská, K. Tmejová, E. Reynaud, A. Claeys, J. Yan, B. De Strooper, J.‐M.

Dura, V. Bryja, B. A. Hassan (2013): The Amyloid Precursor Proteins are conserved

modulators of the Wnt/PCP pathway required for robustness of axonal outgrowth. PLoS

Biol. 11(5):e1001562.

16. Kriz V, Pospichalova V, Masek J, Kilander MB, Slavik J, Tanneberger K, Schulte G, Machala M,

Kozubik A, Behrens J, Bryja V. (2014): β‐arrestin promotes Wnt‐induced Lrp6

phosphorylation via increased membrane recruitment of Amer1. J Biol Chem. 289 (2):

1128 –1141.

17. K. Seitz, V. Dürsch, J. Harnoš, V. Bryja, M. Gentzel, A. Schambony (2014): β‐Arrestin interacts with

the beta/gamma subunits of trimeric G proteins and Dishevelled in the Wnt‐/Ca2+

pathway in Xenopus gastrulation. Plos ONE 9(1):e87132.

18. R. de Groot; R. Sri Ganji; O. Bernatik; B. Lloyd‐Lewis; K. Seipel; K. Šedová; Z. Zdráhal; V.M. Dhople;

T. Dale; H. Korswagen*, V. Bryja*. Huwe1‐mediated ubiquitylation of Dvl defines a novel

negative feedback loop in the Wnt signaling pathway. Sci. Signal. (in press).

19. M.B.C Kilander, J. Petersen, J. Dahlström, R. Sri Ganji, J. Schuster, N. Dahl, V. Bryja, G. Schulte.

Preassembly of human Frizzled 6 with heterotrimeric Gαi2 is selectively impaired by the

pathogenic Frizzled 6 Arg511Cys mutation and is regulated by Disheveled. FASEB J. (fj.13‐

246363. Published online February 7, 2014).

20. O. Bernatík, K. Šedová, R. Sri Ganji, I. Červenka, L. Trantírek, Z. Zdráhal, V. Bryja. Functional

analysis of Dishevelled‐3 phosphorylation identifies distinct mechanisms driven by Casein

Kinase 1e and Fzd5 (J. Biol. Chem., in revision).

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