ABSTRACT
Mutations in leucine-rich repeat kinase 2 (LRRK2) are prevalent causes of late-onset Parkinson's disease. Here, we show that LRRK2 binds to MAPK kinases (MKK) 3, 6, and 7, and that LRRK2 is able to phosphorylate MKK3, 6 and 7. Over-expression of LRRK2 and MKK6 increased the steady state levels of each protein beyond that observed with over-expression of either protein alone. Co-expression increased levels of MKK6 in the membrane more than in the cytoplasm. The increased expression of LRRK2 and MKK6 requires MKK6 activity. The disease-linked LRRK2 mutations, G2019S, R1441C and I2020T, enhance binding of LRRK2 to MKK6. This interaction was further supported by in vivo studies in C. elegans. RNAi knockdown in C. elegans of the endogenous orthologs for MKK6 or p38, sek-1 and pmk-1, abolishes LRRK2-mediated protection against mitochondrial stress. These results were confirmed by deletion of sek-1 in C. elegans. These data demonstrate that MKKs and LRRK2 function in similar biological pathways, and support a role for LRRK2 in modulating the cellular stress response.
Subject(s)
Gene Expression Regulation/physiology , MAP Kinase Kinase 6/metabolism , Protein Serine-Threonine Kinases/metabolism , Amyloid beta-Protein Precursor/metabolism , Animals , Animals, Genetically Modified , Behavior, Animal , Caenorhabditis elegans , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line, Transformed , Gene Expression Regulation/genetics , Humans , Immunoprecipitation/methods , Insecticides/toxicity , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , MAP Kinase Kinase 6/genetics , Mortality , Mutation/genetics , Phosphorylation/drug effects , Protein Binding/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/pharmacology , RNA Interference/physiology , Rotenone/toxicity , Subcellular Fractions/metabolism , Transfection/methodsABSTRACT
Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant familial Parkinson's disease. We generated lines of Caenorhabditis elegans expressing neuronally directed human LRRK2. Expressing human LRRK2 increased nematode survival in response to rotenone or paraquat, which are agents that cause mitochondrial dysfunction. Protection by G2019S, R1441C, or kinase-dead LRRK2 was less than protection by wild-type LRRK2. Knockdown of lrk-1, the endogenous ortholog of LRRK2 in C. elegans, reduced survival associated with mitochondrial dysfunction. C. elegans expressing LRRK2 showed rapid loss of dopaminergic markers (DAT::GFP fluorescence and dopamine levels) beginning in early adulthood. Loss of dopaminergic markers was greater for the G2019S LRRK2 line than for the wild-type line. Rotenone treatment induced a larger loss of dopamine markers in C. elegans expressing G2019S LRRK2 than in C. elegans expressing wild-type LRRK2; however, loss of dopaminergic markers in the G2019S LRRK2 nematode lines was not statistically different from that in the control line. These data suggest that LRRK2 plays an important role in modulating the response to mitochondrial inhibition and raises the possibility that mutations in LRRK2 selectively enhance the vulnerability of dopaminergic neurons to a stressor associated with Parkinson's disease.
