鞭毛运输马达蛋白KIF3B的去磷酸化机制研究

Cilia/flagella are cellular organism with essential functions of providing motility, sensing and signaling transduction, and mutations of genes related with ciliary structure or function cause multiple human diseases termed ciliopathies. The ciliary length is precise regulated and determined by its assembly rate. Intraflagellar transport (IFT) is the main mechanism of protein transport required for ciliary assembly, and made of IFT particles and protein motors that driven IFT particles transport between ciliary base and tip. Based on our previous work, the anterograde motor subunit KIF3B is phosphorylated by CaMKII at Serine 663, and the phosphate state changes in response to ciliary length changes, that the phosphorylation is decreased induced by deflagellation and increased back as the flagellar assembly complete, which works as a ciliary length signal. The robust dephosphorylation depends on an (or several) unknown phosphatase(s). In this proposal, we propose to study the dephosphorylation of KIF3B S663 and the mechanism of ciliary length regulation, including the following aspects: 1) To indentify the phosphatase of KIF3B S663 by immnopecipited and mass spec analysis. 2) Verify the dephophorytion of by PP1A using inhibitor and phosphatase defect mutants. 3) To reveal the function of PP1A and its regulatory factors in ciliary assembly by interfacing gene expression. Based on these work, we will reveal the functions of phosphatase in ciliary assembly and the mechanisms of organelle size controls by signals like phosphorylation state and finally to understand how the cell maintains ciliary length and organelle size, providing guidance on possible diagnostics and treatment for ciliopathies diseases.

纤毛是细胞表面具有运动、感知和信号传递功能的细胞器，其结构或功能的变异可导致多种疾病。纤毛的长度具有细胞特异性，纤毛组装时细胞可感知纤毛长度信号、调节组装速率进而维持一定纤毛长度。纤毛的组装依赖于纤毛内蛋白运输（IFT），IFT由正反向马达蛋白和IFT颗粒构成。我们的研究发现正向马达蛋白KIF3B S663的磷酸化与纤毛长度密切相关，值得注意的是纤毛丢失后KIF3B立即去磷酸化，说明胞内存在迅速去磷酸化机制，即磷酸酶的作用，但是目前关于磷酸酶调控纤毛长度的研究完全空白，亟待展开。我们拟开展以下研究：(1)系统鉴定KIF3B S663的磷酸酶及其复合物。(2)验证磷酸酶1A（PP1A）对KIF3B S663的调控。(3)研究PP1A及其调控因子参与纤毛组装的功能。本项目的研究将揭示磷酸酶调控纤毛长度的机理，有利于加深人们对纤毛组装机制的认识，为纤毛疾病的预防与治疗提供理论基础。