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bioRxiv · 10.1101/2023.05.27.542591

14-3-3 phosphorylation inhibits 14-3-3theta's ability to regulate LRRK2 kinase activity

Pattanayak, R.·Petit, C. M.·Yacoubian, T. A.
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Abstract

LRRK2 mutations are among the most common genetic causes for Parkinsons disease (PD), and toxicity is associated with increased kinase activity. 14-3-3 proteins are key interactors that regulate LRRK2 kinase activity. Phosphorylation of the 14-3-3{theta} isoform at S232 is dramatically increased in human PD brains. Here we investigate the impact of 14-3-3{theta} phosphorylation on its ability to regulate LRRK2 kinase activity. Both wildtype and the non-phosphorylatable S232A 14-3-3{theta} mutant reduced the kinase activity of wildtype and G2019S LRRK2, whereas the phosphomimetic S232D 14-3-3{theta} mutant had minimal effects on LRRK2 kinase activity, as determined by measuring autophosphorylation at S1292 and T1503 and Rab10 phosphorylation. However, wildtype and both 14-3-3{theta} mutants similarly reduced the kinase activity of the R1441G LRRK2 mutant. 14-3-3{theta} phosphorylation did not promote global dissociation with LRRK2, as determined by co-immunoprecipitation and proximal ligation assays. 14-3-3s interact with LRRK2 at several phosphorylated serine/threonine sites, including T2524 in the C-terminal helix, which can fold back to regulate the kinase domain. Interaction between 14-3-3{theta} and phosphorylated T2524 LRRK2 was important for 14-3-3{theta}s ability to regulate kinase activity, as wildtype and S232A 14-3-3{theta} failed to reduce the kinase activity of G2019S/T2524A LRRK2. Molecular modeling showed that 14-3-3{theta} phosphorylation causes a partial rearrangement of its canonical binding pocket, thus affecting the interaction between 14-3-3{theta} and the C-terminus of LRRK2. We conclude that 14-3-3{theta} phosphorylation destabilizes the interaction of 14-3-3{theta} with LRRK2 at T2524, which consequently promotes LRRK2 kinase activity.

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Pattanayak, R., Petit, C. M., Yacoubian, T. A.. 2023-05-30. 14-3-3 phosphorylation inhibits 14-3-3theta's ability to regulate LRRK2 kinase activity. https://doi.org/10.1101/2023.05.27.542591

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