Functional analysis of plant Snf1-related protein kinase signaling complexes using proteomics and reverse genetics in Arabidopsis

Snf1/AMPK蛋白激酶家族的保守成员在真核生物中作为细胞糖/能量传感器控制关键的代谢和信号通路。在这些激酶的三聚体核心中，催化Snf1/a和激活Snf4/g亚基由一种靶向Gal83/Sip/ß亚基的不同底物锚定。拟南芥编码三个a，两个ß和两个同构的g亚基，AtSNF4和AKINßg。通过通常只存在于ß亚基中的kiss结构域，AKINßg可以单独招募a亚基来执行植物特异性信号功能。我们发现，不同的拟南芥snf1相关激酶（SnRKs）是由亚基在体内组合组装形成的，它们出现在由激酶抑制剂PRL1蛋白、PAM1蛋白甲基化酶和剪接体组装因子RSP33组成的剪接体复合体中；蛋白酶体- scf泛素连接酶复合物；结合SnRK-和prl1相互作用激酶和No Apical分生系统类信号因子。本项目旨在通过监测相应基因和蛋白的时空调控，系统表征SnRK信号激酶组分；蛋白复合物的免疫亲和纯化及质谱分析以及通过确定敲除突变对snrk控制的信号通路的影响来分析其调控亚基的功能。

Conserved members of Snf1/AMPK protein kinase family function as cellular sugar/energy sensors controlling key metabolic and signaling pathways in eukaryotes. In the trimeric core of these kinases the catalytic Snf1/a and activating Snf4/g subunits are anchored by one of the different substrate targeting Gal83/Sip/ß subunits. Arabidopsis encodes three a, two ß and two isomorphic g subunits, AtSNF4 and AKINßg. Through a KIS-domain present normally only in ß subunits, AKINßg may recruit alone the a subunits to perform plant specific signaling functions. We found that different Arabidopsis Snf1-related kinases (SnRKs), formed by combinatorial assembly of the subunits in vivo, occur in a spliceosomal complex of kinase inhibitor PRL1 protein, PAM1 protein methylase and spliceosome assembly factor RSP33; in proteasome-SCF ubiquitin ligase complexes; and bound to SnRK- and PRL1-interacting kinases and No Apical Meristem class of signaling factors. The proposed project aims to systematically characterize the components of SnRK signaling kinases by monitoring the spatial and temporal regulation of corresponding genes and proteins; immunoaffinity purification and mass spectrometry analysis of protein complexes; and functional analysis of their regulatory subunits by determining the effects of knockout mutations on the SnRK-controlled signaling pathways.