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Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
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Endogenous Rab38 regulates LRRK2's membrane recruitment and substrate Rab phosphorylation in melanocytes

Alexandra Unapanta1, Farbod Shavarebi1, Jacob Porath1

  • 1Department of Pathology, University of California San Diego, San Diego, California, USA.

The Journal of Biological Chemistry
|August 25, 2023
PubMed

Insights

Rab38 is identified as a key regulator of leucine-rich repeat kinase 2 (LRRK2) in melanocytes, impacting Parkinson's disease pathways. This finding reveals a novel physiologic mechanism controlling LRRK2 activity and substrate phosphorylation.

Area of Science:

  • Cell Biology
  • Neuroscience
  • Genetics

Background:

  • Point mutations in leucine-rich repeat kinase 2 (LRRK2) are linked to Parkinson's disease, increasing its kinase activity.
  • Endogenous cellular pathways that physiologically enhance LRRK2 kinase function remain largely unidentified.
  • While Rab29 can recruit LRRK2 to Golgi membranes when overexpressed, its role in endogenous LRRK2 regulation is unclear.

Purpose of the Study:

  • To identify novel physiological regulators of LRRK2 kinase activity in melanocytes.
  • To investigate the role of Rab GTPases, specifically Rab38, Rab32, and Rab29, in modulating LRRK2 function.
  • To elucidate the mechanism by which Rab38 influences LRRK2 localization and activity.

Main Methods:

  • Knockdown and CRISPR knockout of Rab38, Rab32, and Rab29 in mouse melanocytes.
  • Assessment of LRRK2 substrate phosphorylation (e.g., Rab10, Rab12) using Western blotting.
  • Analysis of LRRK2 membrane association and pericentriolar recruitment in B16-F10 melanoma cells.
  • Investigation of the role of BLOC-3 (guanine nucleotide exchange factor) and LRRK2's Rab38-binding site.

Main Results:

  • Knockdown of Rab38, but not Rab32 or Rab29, significantly decreased LRRK2 substrate phosphorylation in mouse melanocytes.
  • Rab38 was found to drive LRRK2 membrane association and pericentriolar recruitment in melanoma cells.
  • This LRRK2 recruitment by Rab38 was dependent on the presence of endogenous Rab38 and BLOC-3.
  • Disruption of the LRRK2 Rab38-binding site impaired LRRK2 membrane association and phosphorylation activity.

Conclusions:

  • Rab38 is identified as a novel physiological regulator of LRRK2 kinase activity in melanocytes.
  • LRRK2 plays a significant role in coordinating Rab GTPase function in vesicular trafficking.
  • These findings provide insights into the molecular mechanisms underlying LRRK2 regulation in health and Parkinson's disease.
Keywords:
BLOC-3LROLRRK2Parkinson's diseaseRab10Rab32Rab38melanocytesmelanosomes

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