Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, Buffalo, NY, USA.
Department of Biological Sciences, State University of New York at Buffalo, 532 Cooke Hall, Buffalo, NY, 14260-1300, USA.
Sci Rep. 2020 Dec 17;10(1):22184. doi: 10.1038/s41598-020-78470-4.
MAPK pathways regulate different responses yet can share common components. Although core regulators of MAPK pathways are well known, new pathway regulators continue to be identified. Overexpression screens can uncover new roles for genes in biological processes and are well suited to identify essential genes that cannot be evaluated by gene deletion analysis. In this study, a genome-wide screen was performed to identify genes that, when overexpressed, induce a reporter (FUS1-HIS3) that responds to ERK-type pathways (Mating and filamentous growth or fMAPK) but not p38-type pathways (HOG) in yeast. Approximately 4500 plasmids overexpressing individual yeast genes were introduced into strains containing the reporter by high-throughput transformation. Candidate genes were identified by measuring growth as a readout of reporter activity. Fourteen genes were identified and validated by re-testing: two were metabolic controls (HIS3, ATR1), five had established roles in regulating ERK-type pathways (STE4, STE7, BMH1, BMH2, MIG2) and seven represent potentially new regulators of MAPK signaling (RRN6, CIN5, MRS6, KAR2, TFA1, RSC3, RGT2). MRS6 encodes a Rab escort protein and effector of the TOR pathway that plays a role in nutrient signaling. MRS6 overexpression stimulated invasive growth and phosphorylation of the ERK-type fMAPK, Kss1. Overexpression of MRS6 reduced the osmotolerance of cells and phosphorylation of the p38/HOG MAPK, Hog1. Mrs6 interacted with the PAK kinase Ste20 and MAPKK Ste7 by two-hybrid analysis. Based on these results, Mrs6 may selectively propagate an ERK-dependent signal. Identifying new regulators of MAPK pathways may provide new insights into signal integration among core cellular processes and the execution of pathway-specific responses.
MAPK 途径调节不同的反应，但可以共享共同的成分。尽管 MAPK 途径的核心调节剂是众所周知的，但新的途径调节剂仍在不断被发现。过表达筛选可以揭示基因在生物过程中的新作用，非常适合识别不能通过基因缺失分析评估的必需基因。在这项研究中，进行了全基因组筛选，以鉴定在酵母中过表达时会诱导报告基因（FUS1-HIS3）的基因，该报告基因响应 ERK 型途径（交配和丝状生长或 fMAPK），但不响应 p38 型途径（HOG）。通过高通量转化将大约 4500 个过表达单个酵母基因的质粒引入含有报告基因的菌株中。通过测量作为报告基因活性的读出值的生长来鉴定候选基因。通过重新测试鉴定并验证了 14 个基因：两个是代谢控制基因（HIS3、ATR1），五个在调节 ERK 型途径中具有既定作用（STE4、STE7、BMH1、BMH2、MIG2），七个代表 MAPK 信号转导的潜在新调节剂（RRN6、CIN5、MRS6、KAR2、TFA1、RSC3、RGT2）。MRS6 编码一种 Rab 伴侣蛋白和 TOR 途径的效应物，在营养信号中起作用。MRS6 的过表达刺激了侵袭性生长和 ERK 型 fMAPK、Kss1 的磷酸化。MRS6 的过表达降低了细胞的耐渗性和 p38/HOG MAPK、Hog1 的磷酸化。Mrs6 通过双杂交分析与 PAK 激酶 Ste20 和 MAPKK Ste7 相互作用。基于这些结果，Mrs6 可能选择性地传播依赖 ERK 的信号。鉴定 MAPK 途径的新调节剂可能为核心细胞过程之间的信号整合以及途径特异性反应的执行提供新的见解。
