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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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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 disrupts 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) Quantitative liquid YTH assays using CPRG as substrate for β-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.
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DDP337F6: 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 disrupts 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) Quantitative liquid YTH assays using CPRG as substrate for β-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.

Mentions: Numerous PARK8 mutations affect residues in the Roc-COR tandem domain (Fig. 6), but the pathogenic mechanism underlying 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 mutation Y1699C weakened the interaction of LRRK2 with DVL1, 2 and 3 as well as DVL1ΔDIX (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 DVL1ΔDIX, 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 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 interaction with DVL2.


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)

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 disrupts 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) Quantitative liquid YTH assays using CPRG as substrate for β-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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DDP337F6: 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 disrupts 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) Quantitative liquid YTH assays using CPRG as substrate for β-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.
Mentions: Numerous PARK8 mutations affect residues in the Roc-COR tandem domain (Fig. 6), but the pathogenic mechanism underlying 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 mutation Y1699C weakened the interaction of LRRK2 with DVL1, 2 and 3 as well as DVL1ΔDIX (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 DVL1ΔDIX, 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 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 interaction with DVL2.

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