Functional analysis of MLPK, a membrane-anchored cytosolic protein kinase, in Brassica self-incompatibility signaling

MLPK（一种膜锚定胞质蛋白激酶）在芸苔属自交不亲和性信号传导中的功能分析

基本信息

批准号：
18380069
负责人：
TAKAYAMA Seiji
金额：
$ 10.79万
依托单位：
Nara Institute of Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

The aim of the present study was to evaluate the functional role of MLPK, a membrane-anchored cytosolic protein kinase found as a causal factor of a self-compatible Brassica mutant. The major findings are summarized as follows.1. Molecular function of MLPK.We identified two different MLPK transcripts, MLPKf1 and MLPKf2, which are produced by using alternative transcriptional initiation sites and encode two isoforms that differ only at the N-termini. Both MLPKf1 and MLPKf2 are expressed in papilla cells and localize to the plasma membrane by different molecular mechanisms. Although both MLPKPf1 and MLPKf2 could independently complement the mlpk/mlpk mutation, their mutant forms that lack the plasma membrane localization motifs failed to complement the mutation. Furthermore, a bimolecular fluorescence complementation (BiFC) assay revealed direct interactions between SRK and the MLPK isoforms in planta. These results suggest that MLPK isoforms localize to the papilla cell membrane and interact directly with SRK to transduce SI signaling.2.Target molecule of MLPKWe searched for MLPK-interacting stigmatic proteins by yeast two-hybrid screening and found several candidates. Some candidates were found to selectively interact with active form of MLPK, and were shown to be specifically phosphorylated by the recombinant MLPK in vitro. We are now trying to elucidate the physiological function of these candidate molecules.Arabidopsis thaliana is a self-compatible Brassica plant having an MLPK ortholog, APK1b. To test for biological functions of APK1b aside from SI signal transduction, we analyzed a T-DNA knockout line for APK1b: we failed, however, to detect any phenotypic alterations including fertility. The sole function of APK1b in SI signal transduction or the redundancy with regard to homologous kinases might explain the lack of aberrant phenotype in knockout mutant of APK1b.

本研究的目的是评估MLPK的功能作用，MLPK是一种膜锚定的胞质蛋白激酶，被发现是自兼容的胸前突变体的因子。主要发现总结如下1。 MLPK的分子功能。我们鉴定了两个不同的MLPK转录本MLPKF1和MLPKF2，它们是通过使用替代转录起始位点产生的，并编码了仅在N-Termini上不同的两个同工型。 MLPKF1和MLPKF2均在乳头细胞中表达，并通过不同的分子机制定位于质膜。尽管MLPKPF1和MLPKF2均可独立地补充MLPK/MLPK突变，但缺乏质膜定位基序的突变形式未能补充突变。此外，双分子荧光互补（BIFC）测定显示SRK与Planta中MLPK同工型之间的直接相互作用。这些结果表明，MLPK同工型定位于乳头细胞膜并与SRK直接相互作用以传递SI信号。2。MLPKWE的核心分子搜索了通过酵母两种杂交筛查的MLPK-Interactracting污名蛋白，并发现了几位候选者。发现一些候选者可以选择性地与MLPK的活性形式相互作用，并被证明在体外被重组MLPK特别磷酸化。现在，我们正在尝试阐明这些候选分子的生理功能。阿拉比迪托米斯（Arabidopsis thaliana）是一种具有MLPK直系古学APK1B的自兼容的胸前植物。为了测试APK1B的生物学功能，除了SI信号转导外，我们分析了APK1B的T-DNA敲除线：但是，我们未能检测到包括生育能力在内的任何表型改变。 APK1b在SI信号转导或同源激酶的冗余中的唯一功能可能解释了APK1B的基因敲除突变体中缺乏异常表型。