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Assaying the Kinase Activity of LRRK2 in vitro
06:09

Assaying the Kinase Activity of LRRK2 in vitro

Published on: January 18, 2012

The Parkinson's disease associated LRRK2 exhibits weaker in vitro phosphorylation of 4E-BP compared to

Azad Kumar1, Elisa Greggio, Alexandra Beilina

  • 1Cell Biology and Gene Expression Unit, Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, Maryland, United States of America.

Plos One
|January 22, 2010
PubMed

Insights

Leucine-rich repeat kinase 2 (LRRK2) mutations cause inherited Parkinson's disease. This study found that while LRRK2 can phosphorylate 4E-BP in vitro, it is a poor substrate, suggesting other kinases mediate this effect in cells.

Area of Science:

  • Neuroscience
  • Molecular Biology
  • Biochemistry

Background:

  • Mutations in Leucine-rich repeat kinase 2 (LRRK2) are a leading genetic cause of Parkinson's disease (PD).
  • Identifying LRRK2's physiological substrates is crucial for understanding its role in PD pathogenesis.
  • 4E-binding protein (4E-BP) has been proposed as a potential LRRK2 substrate.

Purpose of the Study:

  • To investigate whether 4E-binding protein (4E-BP) is a direct substrate of Leucine-rich repeat kinase 2 (LRRK2).
  • To compare the phosphorylation activity of wild-type LRRK2 and a Parkinson's disease-associated mutant (G2019S) on 4E-BP.
  • To identify other kinases that may phosphorylate 4E-BP.

Main Methods:

  • In vitro kinase assays using purified LRRK2 (wild-type and G2019S mutant) and 4E-BP.
  • Testing phosphorylation of 4E-BP by other kinases, including MAPK14/p38alpha.
  • Cell-based assays using inducible LRRK2 expression in HEK cell lines to assess 4E-BP phosphorylation in a cellular context.

Main Results:

  • LRRK2 demonstrated in vitro kinase activity towards 4E-BP, with the G2019S mutant showing approximately 2-fold higher activity than wild-type.
  • LRRK2 autophosphorylation was more robust than 4E-BP phosphorylation, even with an excess of 4E-BP.
  • MAPK14/p38alpha efficiently phosphorylated 4E-BP at the same site as LRRK2 in vitro.
  • No changes in 4E-BP phosphorylation were observed upon inducible LRRK2 expression in HEK cells.
  • MAPK14/p38alpha, but not LRRK2, phosphorylated 4E-BP in transient overexpression experiments.

Conclusions:

  • 4E-BP is a relatively poor direct substrate for LRRK2.
  • MAPK14/p38alpha is a more efficient kinase for 4E-BP phosphorylation in vitro and in cellular models.
  • Observed increases in 4E-BP phosphorylation in some disease models may be attributed to p38-mediated cellular stress rather than direct LRRK2 activity.

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