A QUICK screen for Lrrk2 interaction partners--leucine-rich repeat kinase 2 is involved in actin cytoskeleton dynamics.
Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinson's disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement, and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function.
Meixner
Andrea
A
Department of Protein Science, Helmholtz Zentrum München-German Research Center for Environmental Health (GmbH), 85764 Neuherberg, Germany.
Boldt
Karsten
K
Van Troys
Marleen
M
Askenazi
Manor
M
Gloeckner
Christian J
CJ
Bauer
Matthias
M
Marto
Jarrod A
JA
Ampe
Christophe
C
Kinkl
Norbert
N
Ueffing
Marius
M
D000199:Actins / Q000378:metabolism*; D000818:Animals; D003599:Cytoskeleton / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D016086:Lentivirus / Q000235:genetics; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D008636:Mesencephalon / Q000201:enzymology; D051379:Mice; D041681:NIH 3T3 Cells; D016501:Neurites / Q000378:metabolism; D058105:Polymerization; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D015203:Reproducibility of Results; D015398:Signal Transduction
D000199:Actins; D012333:RNA, Messenger; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; C495279:Lrrk2 protein, mouse; D017346:Protein Serine-Threonine Kinases; D004298:Dopamine