title: Differential protein-protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways.
abstract: Genetic studies show that LRRK2, and not its closest paralogue LRRK1, is linked to Parkinson's disease. To gain insight into the molecular and cellular basis of this discrepancy, we searched for LRRK1- and LRRK2-specific cellular processes by identifying their distinct interacting proteins. A protein microarray-based interaction screen was performed with recombinant 3xFlag-LRRK1 and 3xFlag-LRRK2 and, in parallel, co-immunoprecipitation followed by mass spectrometry was performed from SH-SY5Y neuroblastoma cell lines stably expressing 3xFlag-LRRK1 or 3xFlag-LRRK2. We identified a set of LRRK1- and LRRK2-specific as well as common interactors. One of our most prominent findings was that both screens pointed to epidermal growth factor receptor (EGF-R) as a LRRK1-specific interactor, while 14-3-3 proteins were LRRK2-specific. This is consistent with phosphosite mapping of LRRK1, revealing phosphosites outside of 14-3-3 consensus binding motifs. To assess the functional relevance of these interactions, SH-SY5Y-LRRK1 and -LRRK2 cell lines were treated with LRRK2 kinase inhibitors that disrupt 14-3-3 binding, or with EGF, an EGF-R agonist. Redistribution of LRRK2, not LRRK1, from diffuse cytoplasmic to filamentous aggregates was observed after inhibitor treatment. Similarly, EGF induced translocation of LRRK1, but not of LRRK2, to endosomes. Our study confirms that LRRK1 and LRRK2 can carry out distinct functions by interacting with different cellular proteins. LRRK1 and LRRK2 (leucine-rich repeat kinase) interaction partners were identified by two different protein-protein interaction screens. These confirmed epidermal growth factor receptor (EGR-R) as a LRRK1-specific interactor, while 14-3-3 proteins were LRRK2-specific. Functional analysis of these interactions and the pathways they mediate shows that LRRK1 and LRRK2 signaling do not intersect, reflective of the differential role of both LRRKs in Parkinson's disease.
authors.affiliation: Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven, Leuven, Belgium.
authors.forename: Lauran
authors.initials: L
authors.lastname: Reyniers
authors.affiliation: Department of Biomedical Sciences, University of Sassari, Sassari, Italy.
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Del Giudice
authors.affiliation: Department of Biology, University of Padova, Padova, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Civiero
authors.affiliation: Department of Biology, University of Padova, Padova, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Belluzzi
authors.affiliation: Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven, Leuven, Belgium.
authors.forename: Evy
authors.initials: E
authors.lastname: Lobbestael
authors.affiliation: Laboratory of Neurogenetics, National Institute on Aging, NIH, Bethesda, Maryland, USA.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Beilina
authors.affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy.|Proteomics Center of Padova University, Padova, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Arrigoni
authors.affiliation: Department of Cellular and Molecular Medicine, KU Leuven, Laboratory of protein phosphorylation and proteomics, Leuven, Belgium.
authors.forename: Rita
authors.initials: R
authors.lastname: Derua
authors.affiliation: Department of Cellular and Molecular Medicine, KU Leuven, Laboratory of protein phosphorylation and proteomics, Leuven, Belgium.
authors.forename: Etienne
authors.initials: E
authors.lastname: Waelkens
authors.affiliation: Protein/Peptide Sequencing Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Biomedical Sciences, University of Sassari, Sassari, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Crosio
authors.affiliation: Department of Biomedical Sciences, University of Sassari, Sassari, Italy.
authors.forename: Ciro
authors.initials: C
authors.lastname: Iaccarino
authors.affiliation: Laboratory of Neurogenetics, National Institute on Aging, NIH, Bethesda, Maryland, USA.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Cookson
authors.affiliation: Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven, Leuven, Belgium.
authors.forename: Veerle
authors.initials: V
authors.lastname: Baekelandt
authors.affiliation: Department of Biology, University of Padova, Padova, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Greggio
authors.affiliation: Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven, Leuven, Belgium.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Taymans
mesh_terms: D045744:Cell Line, Tumor; D057809:HEK293 Cells; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: C510751:LRRK1 protein, human; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases
keywords: LRRK1; LRRK2; Parkinson's disease; protein-protein interactions; signaling networks