Identification of the autophosphorylation sites of LRRK2.
Parkinson's disease (PD) is a major adult-onset neurodegenerative disorder affecting the extrapyramidal motor system. A subset of patients develop PD as an autosomal dominant trait, of which PARK8 caused by mutations in the leucine-rich repeat kinase 2 (LRRK2) gene is highlighted because of its high frequency and clinicopathological similarity to sporadic PD. Previous studies have suggested that overactivation of LRRK2 caused by missense mutations leads to neuronal toxicity in PARK8, although the regulatory mechanism that governs the kinase activity of LRRK2 remains unknown. In this study, we expressed the carboxyl-half fragments of LRRK2 (DeltaN-LRRK2) that harbors the kinase as well as the ras-like (ROC) domains in Sf9 cells, subjected them to in vitro phosphorylation reaction, and analyzed the autophosphorylation by matrix assisted laser desorption/ionization- time of flight (MALDI-TOF) mass spectrometer. We identified Ser1403, Thr1404, Thr1410, Thr1491 located within the ROC domain, as well as Thr1967 and Thr1969 in the kinase domain, as the autophosphorylation sites. Substitution of Thr1967, an autophosphorylation site located within the kinase domain, to Ala caused a significant decrease in the kinase activity, implicating Thr1967 in the kinase activity of LRRK2. Phosphospecific antibodies to the autophosphorylation sites specifically recognized full-length LRRK2 subjected to in vitro phosphorylation reaction, indicating that the autophosphorylation takes place in holoproteins. Further analysis of autophosphorylation will clarify the mechanism of activation of LRRK2, as well as the pathomechanism of PD in relation to overactivation of LRRK2.
Kamikawaji
Shogo
S
Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo, 113-0033 Japan.
Ito
Genta
G
Iwatsubo
Takeshi
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000906:Antibodies / Q000276:immunology; D055162:Biocatalysis; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism; D010300:Parkinson Disease / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010769:Phosphothreonine / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera; D013912:Threonine / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D000906:Antibodies; D020035:Holoenzymes; D008840:Microfilament Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010769:Phosphothreonine; C071058:moesin; D013912:Threonine; D012694:Serine; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases