14-3-3 phosphorylation inhibits 14-3-3θ's ability to regulate LRRK2 kinase activity.
14-3-3 phosphorylation inhibits 14-3-3θ's ability to regulate LRRK2 kinase activity.
复制标题
14-3-3 磷酸化抑制 14-3-3α 调节 LRRK2 激酶活性的能力。
DOI：
10.1101/2023.05.27.542591
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Yacoubian,TaleneA
中科院分区：
文献类型：
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作者：
Pattanayak,Rudradip;Petit,ChadM;Yacoubian,TaleneA
LRRK 2突变是帕金森病（PD）最常见的遗传原因之一，毒性与激酶活性增加有关。14-3-3蛋白是调节LRRK 2激酶活性的关键相互作用物。14-3-3θ亚型在S232处的磷酸化在人类PD脑中显著增加。在这里，我们研究了14-3-3θ磷酸化对其调节LRRK 2激酶活性的能力的影响。野生型和不可磷酸化的S232 A 14-3-3θ突变体都降低了野生型和G2019 S LRRK 2的激酶活性，而磷酸化模拟物S232 D 14-3-3θ突变体对LRRK 2激酶活性的影响最小，如通过测量S1292和T1503处的自磷酸化和Rab 10磷酸化所确定的。然而，野生型和两种14-3-3θ突变体类似地降低了R1441 G LRRK 2突变体的激酶活性。14-3-3θ磷酸化不促进与LRRK 2的整体解离，如通过免疫共沉淀和近端连接测定所确定的。14-3-3s在几个磷酸化丝氨酸/苏氨酸位点与LRRK 2相互作用，包括C-末端螺旋中的T2524，其可以折叠以调节激酶结构域。14-3-3θ和磷酸化T2524 LRRK 2之间的相互作用对于14-3-3θ调节激酶活性的能力是重要的，因为野生型和S232 A 14-3-3θ不能降低G2019 S/T2524 A LRRK 2的激酶活性。分子模拟表明，14-3-3θ磷酸化导致其典型结合口袋的部分重排，从而影响14-3-3θ与LRRK 2的C末端之间的相互作用。我们的结论是，14-3-3θ磷酸化使14-3-3θ与LRRK 2在T2524的相互作用不稳定，从而促进LRRK 2激酶活性。
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 14-3-3θ isoform at S232 is dramatically increased in human PD brains. Here we investigate the impact of 14-3-3θ phosphorylation on its ability to regulate LRRK2 kinase activity. Both wildtype and the non-phosphorylatable S232A 14-3-3θ mutant reduced the kinase activity of wildtype and G2019S LRRK2, whereas the phosphomimetic S232D 14-3-3θ mutant had minimal effects on LRRK2 kinase activity, as determined by measuring autophosphorylation at S1292 and T1503 and Rab10 phosphorylation. However, wildtype and both 14-3-3θ mutants similarly reduced the kinase activity of the R1441G LRRK2 mutant. 14-3-3θ 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 14-3-3θ and phosphorylated T2524 LRRK2 was important for 14-3-3θ’s ability to regulate kinase activity, as wildtype and S232A 14-3-3θ failed to reduce the kinase activity of G2019S/T2524A LRRK2. Molecular modeling showed that 14-3-3θ phosphorylation causes a partial rearrangement of its canonical binding pocket, thus affecting the interaction between 14-3-3θ and the C-terminus of LRRK2. We conclude that 14-3-3θ phosphorylation destabilizes the interaction of 14-3-3θ with LRRK2 at T2524, which consequently promotes LRRK2 kinase activity.