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Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways.

Sancho RM, Law BM, Harvey K - Hum. Mol. Genet. (2009)

Bottom Line: Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, The School of Pharmacy, Brunswick Square, London, UK.

ABSTRACT
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 intramolecular 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.

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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 (DVL1ΔDIX) 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 FLAG vector.
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DDP337F1: 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 (DVL1ΔDIX) 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 FLAG vector.

Mentions: In order to identify LRRK2 accessory proteins potentially regulating kinase activity, we screened an embryonic human brain cDNA library (Clontech) using the LexA yeast two-hybrid (YTH) system and the LRRK2 Roc-COR 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 consistently weaker compared with DVL2 or DVL3 (Fig. 1A). However, deletion constructs lacking the N-terminal DVL1 DIX domain (DVL1ΔDIX) 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) demonstrated 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).


Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways.

Sancho RM, Law BM, Harvey K - Hum. Mol. Genet. (2009)

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 (DVL1ΔDIX) 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 FLAG vector.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2748899&req=5

DDP337F1: 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 (DVL1ΔDIX) 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 FLAG vector.
Mentions: In order to identify LRRK2 accessory proteins potentially regulating kinase activity, we screened an embryonic human brain cDNA library (Clontech) using the LexA yeast two-hybrid (YTH) system and the LRRK2 Roc-COR 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 consistently weaker compared with DVL2 or DVL3 (Fig. 1A). However, deletion constructs lacking the N-terminal DVL1 DIX domain (DVL1ΔDIX) 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) demonstrated 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).

Bottom Line: Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, The School of Pharmacy, Brunswick Square, London, UK.

ABSTRACT
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 intramolecular 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.

Show MeSH
Related in: MedlinePlus