Non-canonical MAPK signaling in yeast

项目摘要/摘要：丝裂原活化蛋白激酶(MAPK)是关键 在其激活过程中通过TXY基序的磷酸化而激活的信号酶 循环。虽然双特异性MAPK激酶激活了许多MAPK(规范信号)， 非典型的激酶和自动磷酸化也可以激活MAPK(非典型的 信令)。关于典型信号通路在健康中的作用，人们知道很多。 和疾病。人们对非规范信号的了解要少得多。Smk1是减数分裂- 酵母中控制配子形成(产孢子)的特异性MAPK 通过非规范的信号通路产生不同形式的MAPK 在减数分裂的不同阶段。首先，CDK激活的激酶，Cak1，磷酸化 随着减数分裂的进行，SMK1‘S激活了T-残基。这 单磷酸化形式的Smk1分布在整个细胞内。晚些时候 程序(在后期II)，一个减数分裂特异蛋白Ssp2与Smk1结合并激活 其Y-残基的顺式自动磷酸化。该反应发生在特定的 使特化细胞中产生的单倍体细胞化的膜状结构 组织。因此，Ssp2将双磷酸化的MAPK的脉冲传递到 在这一发展计划的“正确时间”放置“。这种时空特异性是 通过Ssp2蛋白的模块化实现；它由靶向结构域(TD)定位和 它通过一个蛋白激活域(KAD)激活MAPK的顺式自动磷酸化。 Ssp2KAD本身由结合Smk1和调控RNA的片段组成 识别基序(RRM)。本提案是以Ssp2KAD/Smk1反应为模型 在发育系统的背景下研究MAPKs的自动激活。具体的 本建议的目的是：1-阐明非规范MAPK的分子机制 使用细菌重组系统和纯化蛋白的信号传递，2.测试a SMK1激活的基于竞争的开关位点模型，以及3-识别RNA(S) 被Ssp2中的RNA识别基序识别。

Project Summary/Abstract: Mitogen activated protein kinases (MAPKs) are key signaling enzymes that are activated by phosphorylation of a TXY motif in their activation loops. While dual-specificity MAPK kinases activate many MAPKs (canonical signaling), atypical kinases and autophosphorylation can also activate MAPKs (non-canonical signaling). A great deal is known about the role of canonical signaling pathways in health and disease. Much less is known about non-canonical signaling. Smk1 is a meiosis- specific MAPK in the yeast S. cerevisiae that controls gamete formation (sporulation) through a non-canonical signaling pathway that generates different forms of the MAPK at different stages of meiosis. First, the CDK activating kinase, Cak1, phosphorylates Smk1's activating T-residue as the meiotic divisions are taking place. This monophosphorylated form of Smk1 is distributed throughout the cell. Later in the program (at anaphase II), a meiosis-specific protein, Ssp2, binds to Smk1 and activates the cis-autophosphorylation of its Y-residue. This reaction occurs at specific membraneous structures that cellularize the haploids generated in this specialized cell division. Thus, Ssp2 delivers a pulse of the doubly phosphorylated MAPK to the “right place” at the “right time” in this developmental program. This spatiotemporal specificity is achieved by modularity of the Ssp2 protein; it is localized by a targeting domain (TD) and it activates cis-autophosphorylation of the MAPK via a kinase activating domain (KAD). The Ssp2KAD is itself composed of segments that bind Smk1 and a regulatory RNA recognition motif (RRM). This proposal is to use the Ssp2KAD/Smk1 reaction as a model to study autoactivation of MAPKs in the context of a developmental system. The specific aims of this proposal are: 1- Elucidate molecular mechanisms of non-canonical MAPK signaling using a bacterial reconstitution system and purified proteins, 2-Test a competition-based switch-site model for Smk1 activation, and 3- Identify the RNA(s) recognized by the RNA recognition motif in Ssp2.