Mutations in the LRRK2 Roc-COR tandem
domain link Parkinson’s disease to Wnt
Rosa M. Sancho, Bernard M.H. Law and Kirsten Harvey?
Department of Pharmacology, The School of Pharmacy, 29-39 Brunswick Square, London, UK
Received June 9, 2009; Revised and Accepted July 20, 2009
Mutations in PARK8, encoding LRRK2, are the most common known cause of Parkinson’s disease. The
LRRK2 Roc-COR tandem domain exhibits GTPase activity controlling LRRK2 kinase activity via an intramo-
lecular process. We report the interaction of LRRK2 with the dishevelled family of phosphoproteins (DVL1-3),
key regulators of Wnt (Wingless/Int) signalling pathways important for axon guidance, synapse formation
and neuronal maintenance. Interestingly, DVLs can interact with and mediate the activation of small
GTPases with structural similarity to the LRRK2 Roc domain. The LRRK2 Roc-COR domain and the DVL1
DEP domain were necessary and sufficient for LRRK2–DVL1 interaction. Co-expression of DVL1 increased
LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain. Strikingly, LRRK2–
DVL1-3 interactions were disrupted by the familial PARK8 mutation Y1699C, whereas pathogenic mutations
at residues R1441 and R1728 strengthened LRRK2–DVL1 interactions. Co-expression of DVL1 with LRRK2 in
mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293
and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y
cells. This is the first report of the modulation of a key LRRK2-accessory protein interaction by PARK8 Roc-
COR domain mutations segregating with Parkinson’s disease. Since the DVL1 DEP domain is known to be
involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity,
and (ii) Roc-COR domain mutations modulating LRRK2–DVL interactions indirectly influence kinase activity.
Our findings also link LRRK2 to Wnt signalling pathways, suggesting novel pathogenic mechanisms and new
targets for genetic analysis in Parkinson’s disease.
The PARK8 locus encodes LRRK2, a 2527 amino acid cytoso-
lic protein kinase. Mutations in PARK8 are the most common
known cause of Parkinson’s disease, with missense mutations
found in patients with familial as well as apparently idiopathic
Parkinson’s disease (1–5). LRRK2 belongs to the ROCO
family of proteins which are characterized by the unique com-
bination of a Roc (Ras of complex proteins) domain with
intrinsic GTPase activity and a COR (C-terminal of Roc)
domain. The Roc-COR tandem domain controls LRRK2
kinase activity via an intramolecular process (6–12). The
modification of LRRK2 GTPase and kinase activity by
PARK8 mutations affecting residues in the Roc, COR and
kinase domains is believed to lead to neuronal cell death,
but the pathways involved remain elusive (1,2,7,8,11–16).
The combination of GTPase activity mediated via the
Roc-COR tandem domain and kinase activity of the mitogen-
activated protein kinase kinase kinase domain suggests a
complex role for LRRK2 in cell signalling. Additional
protein–protein interaction domains, such as LRR (Leucine
rich repeat) and WD40 propeller motifs provide additional
complexity and could potentially localize LRRK2 to different
subcellular compartments. The Roc domain shares sequence
similarity with all five subfamilies of the Ras-related super-
family of small GTPases (Ras, Rho, Rab, Sar/Arf and Ran),
?To whom correspondence should be addressed at: Department of Pharmacology, The School of Pharmacy, London WC1N 1AX, UK. Tel: þ44
2077535888; Fax: þ44 2077535902; Email: email@example.com
# 2009 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is
Human Molecular Genetics, 2009, Vol. 18, No. 20
Advance Access published on July 22, 2009
has conserved amino acids involved in GTP binding/hydroly-
sis and exhibits intrinsic GTPase activity. Evidence suggests
that the COR domain forms dimers, resulting in juxtaposition
of the associated Roc domains (10). Since the isolated LRRK2
kinase domain was shown to be catalytically inactive (16,17),
it is clear that the Roc and COR domains are vital for kinase
activity and/or protein stabilization. One current theory
suggests that mutations in the Roc and COR domains
reduce GTPase activity, leading to higher kinase activity.
This suggestion is based on reports that the R1441C/
R1441G substitutions in the Roc domain reduce GTPase
activity (9,10,14,15), whereas these mutations as well as
the Y1699C mutation in the COR domain increase kinase
Thus, the LRRK2 Roc domain is likely to serve as a mol-
ecular switch, regulating kinase activity by cycling between
GDP-bound and GTP-bound states (6–17). However, it is cur-
rently unclear how this activity is controlled in vivo. Normally,
guanine nucleotide exchange factors (GEFs) facilitate GTP
binding and effector activation, whereas GTPase-activating
proteins (GAPs) increase the intrinsic rate of GTP hydrolysis
to terminate signalling (18). Interestingly, the GTPase activity
of a Roc-COR tandem domain from Chlorobium tepidum, a
prokaryotic homologue of LRRK2, shows a low affinity for
nucleotide and fast GDP dissociation, suggesting that
LRRK2 may not require a classical RhoGEF for GTPase
activity (10). Rather, Roc GTPase activity was proposed to
be stimulated solely by COR dimerization. However, this
model seems simplistic, as it would not allow for up- or down-
regulation of GTPase and kinase activity. Thus, the identifi-
cation of proteins involved in regulating LRRK2 GTPase
activity and mediating downstream signalling is of fundamen-
tal importancein understanding
Parkinson’s disease. Since the Roc-COR tandem domain con-
trols LRRK2 kinase activity, we searched for interactors of the
Roc-COR domain with likely relevance for GTPase and kinase
activity. Here, we describe the discovery and characterization
of the interaction of LRRK2 with all members of the dishev-
elled (DVL) family of phosphoproteins in yeast and mamma-
lian cell systems. DVLs have a modular architecture
consisting of DIX (Dishevelled/Axin), PDZ (PSD-95, DLG,
ZO1) and DEP (Dishevelled, EGL-10, Pleckstrin) domains.
Importantly, DVL proteins are key regulators of Wnt signal-
ling pathways leading to multiple downstream effects (19),
including the activation of small GTPases such as Rac1 that
are structurally similar to the LRRK2 Roc domain (20).
Recent studies have underlined the importance of DVLs in
key processes in neuronal development, such as axon gui-
dance, synapse formation and neuronal maintenance (19,21–
27). The identification of DVL proteins as interactors of
LRRK2 suggests a plausible physiological role for LRRK2
in these processes. DVL proteins altered the subcellular distri-
bution of LRRK2 and co-localized in neurites and growth
cones of differentiated dopaminergic SH-SY5Y cells. The
LRRK2-DVL1 interaction stabilizes steady-state levels of
LRRK2, but importantly LRRK2-DVL interactions are
decreased or increased by selected PARK8 Roc-COR domain
mutations segregating with Parkinson’s disease.
Further functional characterization of LRRK2-DVL inter-
encountered in purifying intact DVL proteins, reproducibility
and sensitivity of LRRK2 GTPase and kinase assays, and
the lack of commercially available antibodies against
LRRK2 and DVL1-3 proteins that function in immunoprecipi-
tation experiments. Nonetheless, the dissemination of our
results to a wider audience at this stage will highlight valuable
leads for further research into the endogenous control of
LRRK2 GTPase and kinase activity. We also suggest the rel-
evance of Wnt signalling pathways to Parkinson’s disease,
underpin the importance of microtubule dynamics in neurode-
generation (28–34) and identify DVL proteins as therapeutic
and genetic targets for future therapeutic and genetic research.
LRRK2 associates with DVL family proteins
via a Roc-COR–DEP domain interaction
In order to identify LRRK2 accessory proteins potentially
regulating kinase activity, we screened an embryonic
human brain cDNA library (Clontech) using the LexA yeast
tandem domain (Roc-COR) as ‘bait’. This resulted in the
identification of several overlapping partial cDNAs encoding
DVL2 and DVL3, members of the dishevelled family of
phosphoproteins (Fig. 1A), which contain single DIX, PDZ
and DEP domains. None of the encoded proteins harboured
an intact DIX domain, suggesting that this motif was not
necessary for LRRK2 binding. Q-PCRs confirmed that
DVL1–DVL3 transcripts are detectable in the adult human
brain, including the substantia nigra (Supplementary Material,
Fig. S1). DVLs were considered promising candidates for
further analysis since they are known to interact with and
mediate the activation of small GTPases, such as Rac1 and
RhoA. Interestingly, the DVL1/DVL2 DEP domain alone is
sufficient for Rac1 activation, whereas both PDZ and DEP
domains are required for RhoA activation (20,35). Further
analysis demonstrated that LRRK2 interacts with full-length
DVL1-3 proteins in yeast (Fig. 1A) and HEK293 cells, as
demonstrated by co-immunoprecipitation of myc-tagged
LRRK2 and FLAG-tagged DVL constructs (Fig. 1B). It is
also noteworthy that the DVLs appear to differ in the
nature of their interaction with LRRK2. Although the
Roc-COR bait demonstrated an interaction with all three full-
length DVL proteins (DVL1-3; Fig. 1A), the interaction
between the LRRK2 Roc-COR domain and DVL1 was con-
sistently weaker compared with DVL2 or DVL3 (Fig. 1A).
However, deletion constructs lacking the N-terminal DVL1
DIX domain (DVL1DDIX) showed a robust interaction
with the Roc-COR bait, equivalent to DVL2 or DVL3
(Fig. 1A). Expressing selected subdomains or deletions of
DVL1 in yeast (Fig. 2A) or HEK293 cells (Fig. 2B) demon-
strated that removal of the DIX and/or PDZ domains did not
abolish DVL interactions with the LRRK2 Roc-COR tandem
domain, whereas constructs lacking the DEP domain were no
longer associated with LRRK2 (Fig. 2A and B). Hence,
DVL1 is capable of interacting with the LRRK2 Roc-COR
region in yeast and mammalian cells via the DEP domain,
which can interact with and mediate the activation of small
GTPases such as Rac1 (20).
3956Human Molecular Genetics, 2009, Vol. 18, No. 20
DVL co-expression alters the subcellular distribution
Transfection of C-terminally TAP-tagged LRRK2 in HEK293
cells results in cytoplasmic distribution of the protein (Fig. 3B)
and perinuclear aggregates in 5–10% of the transfected cells
similar to that previously reported for myc- or EGFP-tagged
LRRK2 (13). In contrast, expression of FLAG-tagged DVL1
results in cytoplasmic round or doughnut-shaped protein
aggregates (Fig. 3C). Interestingly, the co-expression of
TAP-LRRK2 and FLAG-DVL1 or FLAG-DVL3 results in a
redistribution of LRRK2 to DVL aggregates and changes in
cell morphology (Fig. 3D–I), a result that was not observed
using the TAP epitope tag alone (Fig. 3J–L). A similar redis-
tribution of myc-LRRK2 to FLAG-DVL1 aggregates was also
observed in non-differentiated dopaminergic SH-SY5Y cells
(Fig. 3O–Q). As expected, this redistribution relies on
LRRK2 Roc-COR-dishevelled interactions, since the full-
length myc-tagged LRRK2 (Fig. 4A–C) and the myc-tagged
Roc-COR tandem domain (Fig. 4D–F) both redistributed to
FLAG-DVL1 aggregates. The unusual distribution of DVL
Figure 1. The LRRK2 Roc-COR tandem domain interacts with dishevelled proteins DVL1, DVL2 and DVL3. (A) Full-length or partial DVL1, DVL2 and DVL3
preys were tested for interactions with the LRRK2 Roc-COR tandem domain bait in the YTH system using LacZ freeze-fracture assays. All negative controls
show yeast growth but no blue colouration in the LacZ assay, demonstrating that the co-expression of bait and prey plasmids with empty prey or bait vectors does
not result in transcription of reporter genes, i.e. no autoactivation was observed. Note that deletion of the DVL1 DIX domain (DVL1DDIX) increases the strength
of the Roc-COR-DVL1 interaction. (B) Co-immunoprecipitation of LRRK2 and DVL1, DVL2 and DVL3 in HEK293 cells co-transfected with full-length myc-
tagged LRRK2 and full-length FLAG-tagged DVL1, DVL2 or DVL3 constructs. Note that myc-LRRK2 is present in the cell lysates (CL) and FLAG-DVL1,
FLAG-DVL2 and FLAG-DVL3 immunoprecipitation (IP) samples purified using FLAG beads, but not in IP samples from cells co-transfected with the empty
Human Molecular Genetics, 2009, Vol. 18, No. 203957
proteins relies on the N-terminal DIX domain, since deletion
of this region resulted in the loss of cytoplasmic DVL1 aggre-
gates and the redistribution of FLAG-DVL1DDIX and
myc-LRRK2 throughout the cytoplasm with some enrichment
at the cell membrane (Fig. 4G–I). However, experiments in
yeast suggested that neither the Roc nor COR domains alone
interacted with DVL1 and DVL2 (Fig. 4J), suggesting that
an intact tandem domain is required for LRRK2-DVL inter-
actions. We confirmed this by co-immunoprecipitation exper-
iments showing that FLAG-tagged DVL1-3 were able to
co-precipitate the myc-tagged LRRK2 Roc-COR tandem
domain from co-transfected HEK293 cells (Fig. 4K).
Figure 2. The LRRK2 Roc-COR tandem domain interacts with the DVL1 DEP domain. (A) DVL1 deletion constructs lacking key domains (DDIX, DPDZ or
DDEP), or encoding individual DIX, PDZ or DEP domains were tested for interactions with the LRRK2 Roc-COR tandem domain bait using the YTH system.
LacZ freeze-fracture assays demonstrate that only constructs expressing the DVL1 DEP domain are able to interact with the Roc-COR bait, whereas the DIX and
PDZ domains were dispensable. (B) Confirmation of the LRRK2-binding site on DVL1 by co-immunoprecipitation in HEK293 cells co-transfected with con-
structs encoding the myc-tagged LRRK2 Roc-COR tandem domain and FLAG-tagged DVL1 variants. Note that the myc-tagged LRRK2 Roc-COR tandem
domain is present in the cell lysates (CL) and FLAG-DVL1, FLAG-DVL1DDIX, FLAG-DVL1DPDZ immunoprecipitation (IP) samples purified using
FLAG beads, but not in IP samples from cells co-transfected with FLAG-DVL1DDEP.
3958 Human Molecular Genetics, 2009, Vol. 18, No. 20
DVL1 stabilizes LRRK2 protein expression
In order to assess the functional effects of DVL1 on LRRK2
activity, we attempted both LRRK2 kinase assays (13) and
GTPase (14) assays, although these have been unsuccessful
to date. As might have been predicted, given the involvement
of DVLs in diverse Wnt signalling pathways (19–27), immu-
noprecipitation of FLAG-DVLs alone from transfected
HEK293 cells resulted in GTPase and kinase activity that is
likely to be unrelated to LRRK2 (data not shown). Exper-
iments with recombinant LRRK2 and DVL proteins will be
required to resolve this issue. However, we were able to
show clear effects of FLAG-DVL1 on the stability of the myc-
tagged Roc-COR domain in cell lysates from co-transfected
cells (Fig. 5A), with quantification showing that co-expression
of FLAG-DVL1 increased levels of myc-Roc-COR by
approximately 4-fold (Fig. 5C). This effect was lost upon
co-expression ofthe myc-Roc-CORprotein with
FLAG-DVL1DDEP, implying that the DEP domain is
crucial for mediating this effect (Fig. 5B and D).
PARK8 mutations segregating with Parkinson’s disease
alter LRRK2–DVL interactions
Numerous PARK8 mutations affect residues in the Roc-COR
tandem domain (Fig. 6), but the pathogenic mechanism under-
lying these changes is as yet unclear. Most mutations are
assumed to alter the folding or intrinsic GTPase activity of
the Roc-COR tandem domain, so in turn influencing LRRK2
kinase activity (9–16). For this reason, we decided to assess
whether selected familial Parkinson’s disease mutations
(I1371V, R1441C, R1441G, R1441H, R1514Q, Y1699C,
R1728H, R1728L or M1869T) in the Roc-COR domain
(Fig. 6A) have any influence on LRRK2 interactions with
DVL1, DVL2 or DVL3. Surprisingly, the COR domain
Figure 3. Redistribution of LRRK2 to cytoplasmic aggregates containing DVL proteins and changes in cell morphology. Confocal microscopy showing the
distributions of (A) TAP-epitope, (B) TAP-tagged full-length LRRK2 and (C) FLAG-tagged full-length DVL1. Note that the TAP epitope has a predominantly
nuclear and cytoplasmic distribution, whereas TAP-LRRK2 is distributed evenly in the cytoplasm including cell processes. In contrast, FLAG-DVL1 is
expressed in the cytoplasm forming round structures that represent DVL protein polymers (36). TAP-LRRK2 re-distributes to FLAG-DVL1 (D–F) and
FLAG-DVL3 (G–I) aggregates upon co-transfection, and cells show unique changes in morphology, spreading and flattening out to cover a larger surface
area. In contrast, the TAP epitope tag does not co-distribute with FLAG-DVL1 aggregates (J–L), and changes in cell morphology are not observed. Similar
results were obtained with myc-LRRK2, LRRK2-EGFP and LRRK2-V5-His (data not shown), suggesting that the redistribution of LRRK2 to DVL aggregates
is independent on the nature of the epitope tag. A similar distribution of myc-LRRK2 (M) and FLAG-DVL1 (N) and the redistribution of myc-LRRK2 to
FLAG-DVL1 aggregates (O–Q) are seen in non-differentiated dopaminergic SH-SY5Y cells, demonstrating that this effect also occurs in different cell
lines. Scale bars: 10 mm.
Human Molecular Genetics, 2009, Vol. 18, No. 20 3959
mutation Y1699C weakened the interaction of LRRK2 with
DVL1, 2 and 3 as well as DVL1DDIX (Fig. 6B–F). From
freeze-fracture filters (Fig. 6B), this effect is most evident
for DVL1, although quantitative YTH assays revealed that
Y1699C also influences DVL2 and DVL3 interactions
(Fig. 6E and F). In contrast, all known substitutions at residues
R1441 and R1728 appear to strengthen the LRRK2–DVL1
interaction (Fig. 6B and C). Curiously, these mutations do
not influence Roc-COR bait interactions with DVL1DDIX,
DVL2 or DVL3 preys (Fig. 6B and D–F), i.e. the observed
effect is specific for full-length DVL1. Other substitutions
including I1371V, R1514Q and M1869Q did not alter the
Figure 4. The cytoplasmic distribution of DVL–LRRK2 complexes is dependent on the DVL1 DIX domain and the intact LRRK2 Roc-COR tandem domain.
Confocal microscopy showing the co-distribution of FLAG-tagged full-length DVL1 with myc-tagged full-length LRRK2 in cytoplasmic DVL protein polymers
(A–C). Since a myc-tagged Roc-COR construct shows similar targeting to cytoplasmic FLAG-DVL1 aggregates, the Roc-COR tandem domain is sufficient for
this interaction (D–F). Deleting the DIX domain by site-directed mutagenesis (DVL1DDIX) resulted in the loss of cytoplasmic DVL1 aggregates and the redis-
tribution of FLAG-DVL1DDIX and myc-LRRK2 throughout the cytoplasm, but with apparent enrichment at the cell membrane (G–I). Scale bars: 10 mm. (J)
LacZ freeze-fracture assays demonstrate that the intact Roc-COR tandem domain, but not individual Roc or COR domains, binds DVL1 and DVL2 effectively.
(K) Co-immunoprecipitation of the myc-tagged LRRK2 Roc-COR tandem domain by FLAG-tagged DVLs confirms that the LRRK2 Roc-COR tandem domain
is sufficient for DVL binding. Note that the myc-tagged LRRK2 Roc-COR tandem domain is present in the cell lysates (CL) and enriched in FLAG-bead purified
immunoprecipitation (IP) samples from co-transfections with FLAG-DVL1, FLAG-DVL2 and FLAG-DVL3, but not in control experiments using empty FLAG
3960 Human Molecular Genetics, 2009, Vol. 18, No. 20
interaction between the LRRK2 Roc-COR domain and any of
the DVL1 or DVL3 baits. However, the R1514Q substitution
caused a small but significant reduction of the Roc-COR inter-
action with DVL2.
DVL1 and LRRK2 co-localize in neurites and growth
cones of differentiated SH-SY5Y cells
Since DVLs were previously shown to stabilize microtubules
in nocodazole-treated differentiated neurones (22), we exam-
ined whether (i) a similar effect could be observed upon trans-
fection of DVL1 into differentiated SH-SY5Y cells and (ii)
whether co-expression of LRRK2 enhances or opposes
DVL-induced microtubule stabilization. The formation of a
stable microtubule network was assessed by staining for
acetylatedtubulin(22).As predicted, differentiating
abundance of stable microtubules and neurite projections
(Fig. 7A–C) that were resistant to nocodazole treatment
(Fig. 7D–F). In contrast, SH-SY5Y cells expressing myc-
tagged full-length LRRK2 contained acetylated tubulin
(Fig. 7G–I), but this was not resistant to nocodazole treatment
(Fig. 7J–L). Nocodazole-resistant acetylated tubulin was also
detected in cells co-expressing FLAG-DVL1 and myc-LRRK2
(Fig. 7M–T). Because of the low co-transfection efficiency in
these experiments, we could not assess whether LRRK2-
DVL1 co-expression affected the length or number of neur-
onal processes. However, DVL1 still appeared to be able to
stabilize microtubules against nocodazole treatment in the
presence of LRRK2. After differentiation with retinoic acid
treatment, myc-LRRK2 is observed in the cytoplasm and neur-
onal processes (visualized by labelling for tubulin) and appears
Figure 5. Co-expression of DVL1 stabilizes the expression of the LRRK2 Roc-COR tandem domain. (A) Co-expression of the myc-tagged Roc-COR tandem
domain with FLAG-DVL1 results in a stabilization of the Roc-COR domain in cell lysates. (B) The stabilization of LRRK2 is dependent on the DVL1 DEP
domain interaction with the Roc-COR tandem domain, since DVL1 lacking the interacting DEP domain (FLAG-DVL1DDEP) was not capable of stabilizing
myc-Roc-COR. (C and D) Quantification of these effects after normalization to levels of cellular actin shows that FLAG-DVL1 increases myc-Roc-COR
?4-fold compared with controls with an empty FLAG vector or FLAG-DVL1DDEP. Statistical significance was determined using a Student’s t-test (two-tailed).
Error bars represent the standard deviation of the mean.???P , 0.001.
Human Molecular Genetics, 2009, Vol. 18, No. 203961
Figure 6. Modulation of the interaction between the LRRK2 Roc-COR tandem domain and dishevelled proteins (DVL1-3) by familial Parkinson’s disease
mutations. (A) Locations of amino acids affected by familial Parkinson’s disease mutations in the Roc-COR tandem domain. (B) LacZ freeze-fracture assays
of LRRK2-DVL interactions. Using this semi-quantitative assay, the most striking effect observed is that the Y1699C mutation in the COR domain clearly dis-
rupts the LRRK2–DVL1 interaction. All negative controls show yeast growth but no blue colour in the LacZ assay, demonstrating that the co-expression of bait
and prey plasmids with empty prey or empty bait vectors does not result in transcription of reporter genes, i.e. no autoactivation was observed. (C–F) Quan-
titative liquid YTH assays using CPRG as substrate for b-galactosidase expression reveal that substitutions at R1441 and R1728 show a strengthened interaction
between the Roc-COR domain bait and DVL1 prey, whereas Y1699C disrupted interactions between the Roc-COR domain bait and DVL1, DVL2 and DVL3
preys. Statistical significance was determined using a Student’s t-test (two-tailed). Error bars represent the standard deviation of the mean.???P , 0.001,??P ,
0.01,?P , 0.05.
3962 Human Molecular Genetics, 2009, Vol. 18, No. 20
antibodies directed against GAP43 (Fig. 8A–F), a neuronal
growth cone protein thought to be involved in pathfinding.
Co-transfection of myc-LRRK2 and FLAG-DVL1 resulted in
co-localization of both proteins in puncta within neurites
(Fig. 8G–J) and growth cones (Fig. 8K–R).
The aim of our study was to characterize potential modulators
of LRRK2 GTPase and kinase activity by identifying interac-
tors of the LRRK2 Roc-COR tandem domain. Using the YTH
system, we found that the LRRK2 Roc-COR tandem domain
interacts with dishevelled family proteins (DVL1, DVL2,
DVL3). We found that DVL1 interacts with LRRK2 via the
DEP domain, which is found in various signalling proteins
such as GEFs, GAPs and Roco family proteins (6). DEP
domains have also been shown to interact with and mediate
the activation of small GTPases such as Rac1 and RhoA,
which share structural similarity with the LRRK2 Roc
domain (20,35). Quantitative PCRs confirmed that transcripts
for DVL1-3 are expressed in brain and detectable in substantia
nigra. Co-immunoprecipitation of epitope-tagged constructs
confirmed that LRRK2 associates with DVL1, DVL2 and
DVL3 in mammalian cells and that DVL proteins increase
the steady-state level of the LRRK2 Roc-COR domain.
Although the exact mechanism responsible for this obser-
vation remains to be determined, it is tempting to speculate
that DVLs stabilize the Roc-COR dimer, which according to
current models (10) would enhance LRRK2 GTPase activity.
DVLs may also hinder the binding of the E3 ubiquitin ligase
CHIP (36,37) which interacts with the Roc domain and
decreases LRRK2 levels via ubiquitination and proteasome-
Interestingly, DVL proteins show a distinct subcellular
localization when expressed in mammalian cells, forming
doughnut-shaped cytoplasmic aggregates that are thought to
represent DVL polymers that are able to mediate Wnt signal-
ling (38). This unique expression pattern appears to be
mediated by the DVL DIX domain, which is necessary for
DVL polymerization. Co-expression of LRRK2 and DVLs
resulted in a re-distribution of LRRK2 to DVL aggregates
and changes in cell morphology, supporting a direct inter-
action of LRRK2 with DVL polymers. Co-localization of
LRRK2 and DVL1 aggregates was also observed in neurites
and growth cones of differentiated dopaminergic SH-SY5Y
cells. These findings support the idea of an important inter-
play between LRRK2 and DVLs in the development and
maintenance of neurites via Wnt signalling pathways. In
humans, 19 spatially and temporally expressed Wnt ligands
signal through their Frizzled (FZ) receptors and their
co-receptors in the cell membrane, leading to hyperphosphor-
ylation of DVLs and the subsequent downstream activation of
one of three Wnt signalling pathways (19). These include the
canonical Wnt pathway, the non-canonical or planar cell
polarity cascade and the Wnt/Ca2þpathway. These different
pathways control cell fate and tissue polarity and affect
dance, neurite outgrowth/branching and synapse formation
(19–27). DVL proteins are also involved in the differentiation
of dopaminergic cells in the ventral midbrain, and in the
release and recycling of dopaminergic vesicles at presynaptic
sites (24–27) and have been linked to neurodegeneration
Figure 7. DVL1, but not LRRK2, stabilizes microtubules treated with nocodazole. (A–L) Confocal microscopy showing stable microtubules stained for acetyl-
ated tubulin in SH-SY5Y cells 5 days after treatment with retinoic acid transfected with FLAG-DVL1 or myc-LRRK2. Note that in cells transfected with
FLAG-DVL1, stable microtubules were observed both before (A–C) and after (D–F) nocodazole treatment. However, while acetylated tubulin was observed
in cells expressing myc-LRRK2 before nocodazole treatment (G–I), LRRK2 alone was unable to protect microtubules from nocodazole treatment (J–L). Inter-
estingly, cells co-transfected with FLAG-DVL1 and myc-LRRK2 showed stable microtubules both before (M–P) and after (Q–T) nocodazole treatment. Scale
bars: 10 mm.
Human Molecular Genetics, 2009, Vol. 18, No. 203963
(39–41). A potential link between LRRK2 and Wnt signalling
was also suggested by a recent LRRK2 RNAi study (42),
which revealed that LRRK2 knockdown resulted in the
up-regulation of several genes involved in the canonical Wnt/
b-catenin signalling pathway, including DVL2.
A key finding of this study is the modulation of a LRRK2–
accessory protein interaction of significance for LRRK2
GTPase and kinase activity by PARK8 Roc-COR domain
mutations segregating with Parkinson’s disease. For example,
the Y1699C substitution in the COR domain, resulting from
Figure 8. Co-localization of LRRK2 and DVL1 in the cytoplasm, neurites and growth cones in differentiated dopaminergic cells. (A–F) Confocal microscopy
showing expression of myc-tagged LRRK2 in SH-SY5Y cells 5 days after treatment with retinoic acid. Note that LRRK2 co-localizes with tubulin staining in
neurites (A–C) and GAP43 immunostaining in growth cones (D–F). Co-expression of FLAG-tagged DVL1 with myc-tagged LRRK2 in differentiated SH-SY5Y
cells shows co-localization of both proteins in multiple cytoplasmic aggregates (G–J). (J) Magnification of the process shown in (G–I). Co-localization of
LRRK2 and DVL1 in an extended neuronal terminal, suggestive of a growth cone (K–N) and in growth cones co-stained for GAP43 (O–R). Scale bars: 10 mm.
3964 Human Molecular Genetics, 2009, Vol. 18, No. 20
a key familial PARK8 mutation (5), disrupts LRRK2 inter-
actions with DVL1, DVL2 and DVL3. This could result
from direct disruption of the DVL1-binding site, or alterna-
tively Y1699C might alter the conformation of the Roc-COR
dimer, making the structure less accessible to DVL1-3.
Little is known about the influence of the Y1699C mutation
on LRRK2 GTPase or kinase activity (7,10,12,13,16), but
individuals with the Y1699C mutation show unique clinical
aspects including a ‘prominent behavioural disorder’ with
unexpectedly high prevalence of anxiety and depression that
can pre-date the onset of Parkinson’s disease symptoms (5).
The Y1699C mutation also seems fully penetrant, which is
not the case for other pathogenic mutations in LRRK2 such
as G2019S, suggesting a more severe genetic defect for
Y1699C (1–5). In contrast, the LRRK2 Roc-COR interaction
with DVL1 is strengthened by pathogenic mutations causing
R1441C/G/H changes and the R1728H/L substitutions.
Although the R1728H/L substitutions have not been proved
to be pathogenic, the likelihood that two different substitutions
occur at exactly the same amino acid position is low, unless
these are disease-causing. Our study supports a potential
pathogenic mechanism for R1728H/L (4). Interestingly, the
effects of the R1441C/G/H and R1728H/L substitutions were
not observed for DVL1DDIX, DVL2 and DVL3. These
results imply that DVL1 multimerization has a subtle effect
on DVL1–LRRK2 interactions, and that DVL1 appears to
interact differently with LRRK2 compared with DVL2 and
DVL3. Intriguingly, substitutions that do not show clear segre-
gation with Parkinson’s disease (e.g. R1514Q and M1869T
(3,4)) did not show clear-cut effects on interactions with
DVL1 or DVL3, although a slight reduction in DVL2–
LRRK2 interactions was observed for the R1514Q mutant.
In summary, all investigated pathogenic mutations (Y1699C,
R1441C/G/H and R1728H/L) in the Roc-COR tandem
domain have a specific influence on the interaction of
LRRK2 with DVL1, but only the Y1699C substitution alters
interactions with DVL1, DVL2 and DVL3. If DVL proteins
enhance LRRK2 GTPase activity, these results imply that
Y1699C would reduce intrinsic GTPase activity mediated by
DVL1–DVL3, whereas R1441C/G/H and R1728H/L would
stabilize LRRK2–DVL1 interactions. The effect of such a
stable complex on GTPase and kinase activity remains uncer-
tain, as this could either reduce or enhance LRRK2 activity.
Since dishevelled proteins are known to co-precipitate with
small GTPases such as Rac1 and RhoA (20,35), studies with
recombinant LRRK2 and DVL proteins will be required to
provide clear evidence of an influence of DVLs on LRRK2
GTPase and kinase activity.
Lastly, this study also suggests that the dishevelled genes
(DVL1, DVL2 and DVL3) represent excellent candidates for
genetic screening in individuals with familial and idiopathic
Parkinson’s disease. Interestingly, DVL1 knockout mice are
viable and fertile and demonstrate no gross physical abnorm-
alities. However, they show an impaired social behaviour phe-
notype (43), with lower huddling behaviour, nest building,
social dominance and whisker trimming. These behaviours
could be interpreted as reflecting increased anxiety in social
contexts, and whether this phenotype mirrors the ‘prominent
behavioural disorder’ seen in patients with the LRRK2
Y1699C mutation remains to be seen. In contrast, both
DVL2 and DVL3 knockout mice exhibit cardiovascular
outflow tract defects, although DVL2 knockouts also exhibit
vertebral and rib malformations and neural tube closure
defects (44,45). Although DVL1 has been assessed as a candi-
date gene in Alzheimer’s disease (40), as yet, no human
disease has been associated with human DVL1, DVL2 or
DVL3. Given our results, we suggest that it would be timely
to re-examine this gene family in unresolved cases of familial
or idiopathic Parkinson’s disease. In particular, we note that
the DVL1 gene maps to chromosome 1p36 (http://genome.
ucsc.edu/), a region of the human genome enriched in Parkin-
son’s disease genes, including PARK6 (PINK1), PARK7
(DJ-1) and PARK9 (ATP13A2). In addition, several studies
have implicated DVL proteins in dopaminergic synapse for-
mation and transmitter release (21,24–27). Over-expression
of Parkinson’s disease-associated LRRK2 mutations in neur-
onal cultures induces a progressive ‘reduction’ in neurite
length and branching, especially for axons, the longest neur-
onal processes (28,29). Similar effects are observed when
DVL1 is down-regulated via RNAi, which results in abroga-
tion of axon differentiation (23). Lastly, DVL1 is one of a
total of 24 genes that have been found to display a signifi-
cantly higher rate of protein evolution in primates than in
rodents, implying a specialized function in the human
In summary, we propose that dishevelled proteins are key
LRRK2 interactors, since they have the potential to influence
LRRK2 GTPase and kinase activity. Since mutations in the
Roc-COR tandem domain segregating with Parkinson’s
disease either weaken or strengthen LRRK2-DVL1 inter-
actions, this implies that the correct level of LRRK2 activity
is key to the health of dopaminergic neurones. We also
suggest that DVL proteins and other components of Wnt sig-
nalling pathways may represent novel therapeutic targets in
the treatment of Parkinson’s disease, whereas the DVL1
gene on chromosome 1p36 should be considered a high-
priority candidate for genetic screening. Our data also link
LRRK2 to Wnt signalling pathways that control the develop-
ment and maintenance of neuronal processes, suggesting
novel potential mechanisms in the pathogenesis of Parkinson’s
MATERIALS AND METHODS
Cloning of LRRK2/DVL expression constructs
and site-directed mutagenesis
LRRK2 and DVL cDNAs were amplified from human whole-
brain first-strand cDNA (Clontech) and cloned into the YTH
bait vector pDS-BAIT (pDS; Dualsystems Biotech), the prey
vector pACT2 (Clontech) or the mammalian expression
vectors pRK5myc or pRK5FLAG. This results in an
in-frame fusion of the LexA-BD, GAL4-AD, myc or FLAG
epitope tags to the N-termini of LRRK2 or DVL proteins,
respectively. Mutations were made in different constructs
using the QuikChange site-directed mutagenesis kit (Strata-
gene). Amplifications were performed using Pfx DNA poly-
merase, and all plasmids were sequenced to confirm the
introduction of the desired mutations.
Human Molecular Genetics, 2009, Vol. 18, No. 203965
The yeast strain L40 (Invitrogen) was co-transformed with
various pDS-LRRK2 and pACT2-DVL constructs. Transform-
ations were plated on selective dropout media lacking either
leucine, tryptophan and histidine supplemented with 0.5 mM
3-AT (for suppression of ‘leaky’ histidine expression) for
nutritional selection, or leucine and tryptophan for transform-
ation controls. After incubation at 308C for 3–6 days to allow
prototropic colonies to emerge, LacZ reporter gene assays
were performed as described previously (47). Quantitative
YTH assays were performed by resuspending cell pellets in
Z-buffer containing 40 mM b-mercaptoethanol, followed by
lysis in 0.1% (w/v) SDS and 0.1% (v/v) chloroform (48).
All protein interactions were assayed in three to four indepen-
dent experiments in triplicate. After the addition of chloro-
pheno-red-b-D-galactopyranoside (CPRG), the colour change
was recorded at 540 nm and readings adjusted for turbidity
of the yeast suspension at 620 nm. The background signal
(bait plus empty pACT2 vector) was subtracted from each
reading and values were normalized to the wild-type
Roc-COR response, which was set at 100%. Statistical signifi-
cance was determined using a Student’s t-test (two-tailed).
Error bars represent the standard deviation of the mean.
Cell culture and immunocytochemistry
HEK293 cells (ATCC CRL1573) were grown in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10%
(v/v) fetal bovine serum, 2 mM glutamine, 100 U/ml penicil-
lin G and 100 mg/ml streptomycin at 378C in 95% air25%
CO2 (49). Exponentially growing cells were transfected
with constructs encoding epitope-tagged DVL and LRRK2
using Lipofectamine LTX reagent (Invitrogen). After 24 h,
cells were fixed in 4% (w/v) PFA and stained with anti-
bodies recognizing myc (Sigma) and FLAG (Sigma) and
secondary anti-mouse and anti-rabbit antibodies conjugated
to Alexa 488, Alexa 546 or Cy5 fluorochromes (Molecular
Co-immunoprecipitation and Western blotting
HEK293 cells were transfected as described earlier and har-
vested 48 h post-transfection, lysed in a solution containing
50 mM NaCl/complete protease inhibitor cocktail (Roche)
and homogenized using a tissue grinder. Following centrifu-
gation (48C, 40 min, 100 000g), cell lysates were recovered
and the concentration of NaCl was increased to 150 mM.
One millilitre of cell lysate containing ?700 mg of protein
was added to 40 ml of anti-FLAG M2 affinity gel (Sigma)
and incubated overnight at 48C on a turning disk in order to
purify the FLAG-tagged proteins. The affinity gel was sub-
jected to centrifugation (48C, 100g, 3 min), followed by two
washes in 50 mM NaCl, 50 mM Tris, 0.1% Triton X-100,
two washes in 150 mM NaCl, PBS, 0.1% Triton X-100 and
two washes in PBS, 0.1% Triton X-100. The FLAG fusion
proteins were eluted with 150 ng of 3? FLAG peptide
(Sigma) for 30 min at RT. The eluates were analysed by
SDS–PAGE and immunoblotting. Approximately 10 mg of
protein was loaded into 4–12% (w/v) BisTris pre-cast gels
(Invitrogen). Proteins were transferred to polyvinylidine fluor-
ide membranes (Millipore) and non-specific bands blocked
with 5% (w/v) skimmed milk in PBS plus 0.1% (v/v) Tween
20 or with 20% (v/v) horse serum in PBS. Anti-myc antibody
(Sigma) was used at 1:2000, and anti-FLAG antibody
(Sigma) was used at 1:3000 at 48C overnight. For detection,
an HRP-conjugated anti-rabbit secondary antibody (Santa
Cruz) was used at a final dilution of 1:2000, together with
the SuperSignal West Pico Chemiluminescent Substrate
Culture and immunocytochemistry of SH-SY5Y cells
SH-SY5Y cells were cultured in DMEM:F12 (1:1 Gibco)
supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/
streptomycin. SH-SY5Y cells were differentiated by treatment
with 10 mM retinoic acid and subsequent incubation for 4–7
days in neurobasal medium supplemented with 2 mM of
L-glutamine, 1% (v/v) penicillin/streptomycin and B27 sup-
plement (Gibco). SH-SY5Y cells were transiently transfected
using Neuromag (Oz Biosciences) or FuGene 6 (Roche).
After 48 h, cells were fixed in methanol and stained with anti-
bodies recognizing GAP-43 (mAb347; Chemicon), NeuN
(mAb377; Chemicon), beta-tubulin (NB600-1470; Novus Bio-
logicals) or acetylated tubulin (6-11B-1; Sigma). In order to
destabilize microtubules, cells were treated with 5 mM
nocodazole (Sigma) for 30 min prior to fixation and
Confocal microscopy and image analysis
Confocal microscopy was performed using a Zeiss LSM 510
META. All images were taken with a ?63 objective. Fluor-
escence excited by the 488, 543 and 633 nm laser lines of
argon and helium/neon lasers was detected separately using
only one laser at the time (multitrack function) and a combi-
nation of band pass filters (BP 505-530, BP 560-615), long
pass (LP 560) filters and meta function (649–798) dependent
on the combination of fluorochromes used.
expression in substantia nigra was performed using the com-
parative method (DDCT), commercially available TaqMan
primer sets and an ABI 7500 real-time PCR system (Applied
Biosystems, Warrington, UK). Total RNA was converted
into first-strand cDNA using a high-capacity cDNA reverse
transcription kit (Applied Biosystems). Cycling conditions
were 508C for 2 min, 958C for 10 min and 40 cycles of
958C for 15 s, followed by 608C for 1 min. Data were analysed
using the SDS 7500 system software (v1.3.1, Applied Biosys-
tems) before being exported into Microsoft Excel. Exper-
iments were repeated four times in triplicate using GAPDH
and PPIA as endogenous controls for normalizing gene
3966Human Molecular Genetics, 2009, Vol. 18, No. 20
Supplementary Material is available at HMG online.
K.H. thanks Dr Mark R. Cookson and Professor Robert
J. Harvey for constructive comments and suggestions on the
Conflict of Interest statement. None declared.
This work was supported by grants from the Royal Society, the
British Medical Association (Dawkins and Lawson award) and
the Wellcome Trust (WT088145) to K.H. and two School of
Pharmacy PhD studentships. The funders had no role in
study design, data collection and analysis, decision to
publish or preparation of the manuscript. Funding to pay the
Open Access publication charges for this article was provided
by The School of Pharmacy.
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