Identification of potential protein interactors of Lrrk2
Introduction
Parkinson's disease (PD) characterized by resting tremor, rigidity and bradykinesia is a common neurodegenerative disorder affecting approximately 1–2% of the population over 60 years of age. Although the vast majority of PD cases are thought to be sporadic, the identification of mutations in several genes causative for rare familial cases has helped to elucidate the mechanisms involved in the development of disease [1].
Pathogenic mutations in the recently discovered gene LRRK2 have been shown to be one of the most important genetic causes of both familial and sporadic late-onset parkinsonism [1]. LRRK2 encodes a protein of approximately 286 kDa encompassing numerous functional domains [2], [3]. Separate studies suggest that enhanced Lrrk2 kinase activity due to mutations in the kinase domain might be involved in the mechanism leading to the disease [4], [5], [6]. However, little is known about the physiological function of the other Lrrk2 domains, their pathways and how they are involved in the development of parkinsonism [7], [8].
In silico models of the Lrrk2 protein suggest the presence of several functional domains associated with protein–protein interactions. These include an armadillo, ankyrin and leucine-rich-repeat domain at the N-terminal end and the WD40 domain following the Roc-COR-kinase domains at the C-terminal [3]. The identification of direct-interacting partners of Lrrk2 represents a key step in elucidating the normal function of Lrrk2, and the pathomechanism by which mutant Lrrk2 causes parkinsonism. We have coupled immunoprecipitation with tandem mass spectrometry to identify 14 potential Lrrk2 interactors that may help to reveal further insights into Lrrk2 biology.
Section snippets
Immunoprecipitation studies
HEK293T cells, maintained in Opti-mem 1 supplemented with fetal calf serum and penicillin/streptomycin (Gibco), were transfected with MOCK or a pcDNA3 vector encoding full length LRRK2-V5 using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Cells were harvested and lysed in 50 mM Tris/HCl, 150 mM NaCl and 0.1% Triton-X-100. For immunoprecipitation, processed lysates with equal protein concentrations were incubated with the respective antibody (anti-V5/Invitrogen,
Results
To identify potential protein interactors of Lrrk2, we adopted an immunoprecipitation approach followed by in-gel digestion and tandem mass spectrometry (MS/MS). False positive results are a common caveat of single-step immunoprecipitation procedures, due to the co-isolation of non-specific proteins. Thus, gels were screened for silver-stained protein bands that were only present when overexpressed Lrrk2-V5 was immunoprecipitated with V5- antibody. Negative controls included a “pull down” of
Discussion
Lrrk2 pathogenic substitutions have been demonstrated in Roc (R1441C/G/H), COR (Y1699C) and kinase (G2019S, I2020 T) domains, and a common risk factor (G2385R) has been identified in the WD40 domain [9], [10], [11]. Additional coding variants have been found throughout the protein and may also be associated with disease [12]. Protein interactions are likely to have a central role in the normal function of Lrrk2 and may play a role in the development of disease. A combination of
Acknowledgments
Mayo Clinic Jacksonville is a Morris K. Udall Parkinson's Disease Research Center of Excellence (NINDS P50 #NS40256). We thank Minnie Schreiber for technical assistance. We would like to thank all those who have contributed to our research.
References (15)
- et al.
LRRK2 in Parkinson's disease: protein domains and functional insights
Trends Neurosci
(2006) - et al.
Kinase activity is required for the toxic effects of mutant LRRK2/dardarin
Neurobiol Dis
(2006) - et al.
The familial Parkinsonism gene LRRK2 regulates neurite process morphology
Neuron
(2006) Genetics of Parkinson disease: paradigm shifts and future prospects
Nat Rev Genet
(2006)The Parkinson disease gene LRRK2: evolutionary and structural insights
Mol Biol Evol
(2006)- et al.
The Parkinson disease causing LRRK2 mutation I2020T is associated with increased kinase activity
Hum Mol Genet
(2006) - et al.
Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity
Proc Natl Acad Sci USA
(2005)
Cited by (67)
Heat Shock Protein 90 in Parkinson's Disease: Profile of a Serial Killer
2024, NeuroscienceFbxl18 targets LRRK2 for proteasomal degradation and attenuates cell toxicity
2017, Neurobiology of DiseaseNovel insights into the neurobiology underlying LRRK2-linked Parkinson's disease
2014, NeuropharmacologyCitation Excerpt :In either case, an increase in surface membrane localization of the dopamine D1 receptor upon overexpression of pathogenic LRRK2 in neuronal cells, or in the striatum of G2019S transgenic mice as compared to control, has recently been reported as well (Migheli et al., 2013), and it will be interesting to determine whether this is related to abnormal PKA signalling. Early studies identified AKAP8 as a LRRK2 interactor (Daechsel et al., 2007), further indicating the possibility that LRRK2, either directly or through interactions with additional AKAPs, may regulate the spatial control of PKA signalling. Taken together with the reported phosphorylation of LRRK2 by PKA (see above), LRRK2 seems to regulate, and be regulated by, PKA-mediated events, highlighting a complex signalling network which requires further investigations.
Parkinson Disease: Translating Insights from Molecular Mechanisms to Neuroprotection
2021, Pharmacological Reviews