14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization

Biochem J. 2010 Sep 15;430(3):393-404. doi: 10.1042/BJ20100483.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / genetics
  • 14-3-3 Proteins / metabolism*
  • Amino Acid Sequence
  • Animals
  • Binding Sites / genetics
  • Brain / metabolism
  • Cell Line
  • Cytoplasm / metabolism*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Immunoprecipitation
  • Kidney / metabolism
  • Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
  • Mice
  • Mice, Knockout
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Mutation*
  • Parkinson Disease / genetics
  • Phosphorylation
  • Protein Binding
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Sequence Homology, Amino Acid
  • Serine / genetics
  • Serine / metabolism
  • Spleen / metabolism
  • Swiss 3T3 Cells

Substances

  • 14-3-3 Proteins
  • Green Fluorescent Proteins
  • Serine
  • Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
  • Lrrk2 protein, mouse
  • Protein-Serine-Threonine Kinases