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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

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LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation

April Reynolds1, Elizabeth A Doggett1, Steve M Riddle2

  • 1Parkinson's Institute Sunnyvale, CA, USA.

Frontiers in Molecular Neuroscience
|July 11, 2014
PubMed

Insights

Parkinson's disease (PD) genetics reveal that Leucine-Rich Repeat protein Kinase 2 (LRRK2) mutations enhance kinase activity and alter cellular phosphorylation. Analyzing LRRK2 phosphosites offers distinct measures of its biological activity.

Area of Science:

  • Neuroscience
  • Genetics
  • Biochemistry

Background:

  • Missense mutations in the Leucine-Rich Repeat protein Kinase 2 (LRRK2) gene are a primary genetic risk factor for Parkinson's disease (PD).
  • LRRK2, a serine/threonine kinase, is implicated in neuronal toxicity, but its precise pathogenic mechanisms remain unclear.
  • LRRK2 exhibits autophosphorylation and is constitutively phosphorylated at specific cellular sites (Ser910, Ser935, Ser955, Ser973), potentially regulated by upstream kinases.

Purpose of the Study:

  • To investigate the phosphoregulation of LRRK2 at key sites in response to disease-associated mutations and specific inhibitors.
  • To analyze the impact of mutations in the Roc-COR domains and alanine substitutions of phosphosites on LRRK2 kinase activity.
  • To elucidate the distinct roles of Ser1292 and cellular site phosphorylation in measuring LRRK2 activity.

Main Methods:

  • Analysis of disease-associated LRRK2 mutations (e.g., Arg1441Cys, Gly2019Ser) and alanine substitutions at phosphosites (Ser910, 935, 955, 973).
  • Assessment of LRRK2 autophosphorylation at Ser1292 and cellular site phosphorylation under various conditions, including LRRK2 inhibition.
  • Investigation of the effects of increased cAMP levels and specific phosphatases (Calyculin A, Okadaic acid) on LRRK2 phosphorylation.

Main Results:

  • Roc-COR domain mutants (e.g., Arg1441Cys, Tyr1699Cys) enhance LRRK2 kinase activity and induce dephosphorylation of cellular sites.
  • While individual cellular site mutations had no effect, Ser910/935/955/973Ala mutations showed a trend toward increased LRRK2 kinase activity.
  • LRRK2 kinase inhibition dephosphorylated Ser1292, while cellular sites were dephosphorylated by Calyculin A-sensitive phosphatases.

Conclusions:

  • Specific LRRK2 mutations can modulate its kinase activity and affect cellular phosphorylation patterns.
  • Phosphorylation at Ser1292 and the cellular sites (Ser910/935/955/973) represent distinct and valuable indicators of LRRK2 activity.
  • Comparative analysis of these phosphorylation sites provides a comprehensive understanding of LRRK2's biological function in vitro and in vivo.
Keywords:
GTPaseLRRK2Parkinson’s diseasekinasekinase inhibitorphosphorylation

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