14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization.
LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients, but still little is understood about how it is regulated or functions. In the present study we have demonstrated that 14-3-3 protein isoforms interact with LRRK2. Consistent with this, endogenous LRRK2 isolated from Swiss 3T3 cells or various mouse tissues is associated with endogenous 14-3-3 isoforms. We have established that 14-3-3 binding is mediated by phosphorylation of LRRK2 at two conserved residues (Ser910 and Ser935) located before the leucine-rich repeat domain. Our results suggests that mutation of Ser910 and/or Ser935 to disrupt 14-3-3 binding does not affect intrinsic protein kinase activity, but induces LRRK2 to accumulate within discrete cytoplasmic pools, perhaps resembling inclusion bodies. To investigate links between 14-3-3 binding and Parkinson's disease, we studied how 41 reported mutations of LRRK2 affected 14-3-3 binding and cellular localization. Strikingly, we found that five of the six most common pathogenic mutations (R1441C, R1441G, R1441H, Y1699C and I2020T) display markedly reduced phosphorylation of Ser910/Ser935 thereby disrupting interaction with 14-3-3. We have also demonstrated that Ser910/Ser935 phosphorylation and 14-3-3 binding to endogenous LRRK2 is significantly reduced in tissues of homozygous LRRK2(R1441C) knock-in mice. Consistent with 14-3-3 regulating localization, all of the common pathogenic mutations displaying reduced 14-3-3-binding accumulated within inclusion bodies. We also found that three of the 41 LRRK2 mutations analysed displayed elevated protein kinase activity (R1728H, ~2-fold; G2019S, ~3-fold; and T2031S, ~4-fold). These results provide the first evidence suggesting that 14-3-3 regulates LRRK2 and that disruption of the interaction of LRRK2 with 14-3-3 may be linked to Parkinson's disease.
Nichols
R Jeremy
RJ
University of Dundee, Scotland, UK. jnichols@parkinsonsinstitute.org
Dzamko
Nicolas
N
Morrice
Nicholas A
NA
Campbell
David G
DG
Deak
Maria
M
Ordureau
Alban
A
Macartney
Thomas
T
Tong
Youren
Y
Shen
Jie
J
Prescott
Alan R
AR
Alessi
Dario R
DR
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000378:metabolism; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010300:Parkinson Disease / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013154:Spleen / Q000378:metabolism; D041701:Swiss 3T3 Cells
D048948:14-3-3 Proteins; D049452:Green Fluorescent Proteins; D012694:Serine; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; C495279:Lrrk2 protein, mouse; D017346:Protein Serine-Threonine Kinases