14-3-3 phosphorylation inhibits 14-3-3θ's ability to regulate LRRK2 kinase activity and toxicity

被引:2
作者
Pattanayak, Rudradip [1 ]
Ekkatine, Roschongporn [1 ]
Petit, Chad M. [2 ]
Yacoubian, Talene A. [1 ]
机构
[1] Univ Alabama Birmingham, Ctr Neurodegenerat & Expt Therapeut, Heersink Sch Med, Dept Neurol, 1719 Sixth Ave South,Civitan Int Res Bldg 510, Birmingham, AL 35294 USA
[2] Univ Alabama Birmingham, Heersink Sch Med, Dept Biochem & Mol Genet, 720 20th St South,Kaul 452, Birmingham, AL 35294 USA
基金
美国国家卫生研究院;
关键词
Parkinson's disease; 14-3-3; theta; G2019S LRRK2; kinase activity; neurite length; DISEASE-ASSOCIATED MUTATIONS; PARKINSONS-DISEASE; LEWY BODY; PROTEINS; BINDING; DISRUPTS; GENE;
D O I
10.1093/hmg/ddae142
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
LRRK2 mutations are among the most common genetic causes for Parkinson's 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. Finally, we found that the S232D mutation failed to protect against G2019S LRRK2-induced neurite shortening in primary cultures, while the S232A mutation was protective. 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 and toxicity.
引用
收藏
页码:2071 / 2083
页数:13
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