Quantitative proteomics reveals a Gα/MAPK signaling hub that controls pheromone-induced cellular polarization in yeast
Quantitative proteomics reveals a Gα/MAPK signaling hub that controls pheromone-induced cellular polarization in yeast
复制标题
定量蛋白质组学揭示了控制酵母中信息素诱导的细胞极化的 Gα/MAPK 信号中枢
DOI：
10.1016/j.jprot.2019.103467
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发表时间：
2019
影响因子：
3.3
通讯作者：
Metodiev, Metodi V.
中科院分区：
文献类型：
--
作者：
Waszczak, Nicholaz;DeFlorio, Reagan;Ismael, Amber;Cheng, Naiyuan;Stone, David E.;Metodiev, Metodi V.
交配特异性酵母Gα通过隔离Gβγ和调节Fus 3 MAP激酶来控制信息素信号传导。破坏Gα-Fus 3相互作用导致向化性的严重缺陷。由于Gα集中在两个已知靶标磷酸化所需的Fus 3的趋化性生长位点，我们筛选了磷酸化依赖于信息素刺激和Gα-Fus 3相互作用的其他蛋白质。使用Fus 3结合严重缺陷的Gα突变形式、GαDSD和定量质谱，鉴定了14种蛋白质作为Gα募集的Fus 3的潜在靶点，其中10种先前与细胞极性和形态发生有关。为了探索这些发现的生物学相关性，我们将重点放在Spa 2极性体蛋白上，该蛋白在信息素处理的Gα DSD细胞中的多个丝氨酸残基上低磷酸化。对六个位点进行诱变以产生Spa 26 XSA突变蛋白。Spa 26 XSA对Fus 3的亲和力增加，这与激酶-底物相互作用一致，Spa 26 XSAc细胞在梯度感应和受精卵形成方面表现出明显的缺陷。这些结果表明，Gα促进了信息素刺激的细胞皮层的Fus 3对Spa 2的磷酸化，并且这种机制在趋化性中起作用。Gα-Fus 3信号枢纽如何影响其他推定的目标，在这里确定还有待确定。SignificancePreviously，之间的相互作用的G α蛋白，Gpa 1，和MAPK的信息素反应途径，Fus 3，被证明是重要的有效感应的信息素梯度和维持细胞极性在交配过程中。在这里，我们表明，潜在的分子机制涉及磷酸化的特定皮质目标的Gpa 1/Fus 3。这些已被确定的定量磷酸蛋白质组学使用的突变体Gpa 1，这是有缺陷的相互作用与Fus 3。这些目标之一是极性体蛋白Spa 2。Gpa 1/Fus 3靶向的Spa 2磷酸化位点的丙氨酸取代导致信息素梯度传感和合子形成的显著缺陷。这些结果揭示了G α蛋白和MAPK如何在原型模型系统中控制细胞极性。我们的研究结果具有更广泛的意义，因为类似的机制存在于高等真核生物中，并涉及重要的生物学，如神经元发育，免疫和癌细胞转移。
The mating-specific yeast Gα controls pheromone signaling by sequestering Gβγ and by regulating the Fus3 MAP kinase. Disrupting Gα-Fus3 interaction leads to severe defects in chemotropism. Because Gα concentrates at the chemotropic growth site where Fus3 is required for the phosphorylation of two known targets, we screened for additional proteins whose phosphorylation depends on pheromone stimulation and Gα-Fus3 interaction. Using a mutant form of Gα severely defective in Fus3-binding, GαDSD, and quantitative mass spectrometry, fourteen proteins were identified as potential targets of Gα-recruited Fus3, ten of which were previously implicated in cell polarity and morphogenesis. To explore the biological relevance of these findings, we focused on the Spa2 polarisome protein, which was hypophosphorylated on multiple serine residues in pheromone-treated GαDSDcells. Six sites were mutagenized to create the Spa26XSAmutant protein. Spa26XSAexhibited increased affinity for Fus3, consistent with a kinase-substrate interaction, and Spa26XSAcells exhibited dramatic defects in gradient sensing and zygote formation. These results suggest that Gα promotes the phosphorylation of Spa2 by Fus3 at the cortex of pheromone-stimulated cells, and that this mechanism plays a role in chemotropism. How the Gα-Fus3 signaling hub affects the other putative targets identified here has yet to be determined.SignificancePreviously, interaction between the G alpha protein, Gpa1, and the MAPK of the pheromone response pathway, Fus3, was shown to be important for efficient sensing of the pheromone gradient and for the maintenance of cell polarity during mating. Here we show that the underlying molecular mechanisms involve the phosphorylation of specific cortical targets of Gpa1/Fus3. These have been identified by quantitative phosphoproteomics using a mutant of Gpa1, which is defective in interacting with Fus3. One of these targets is the polarisome protein Spa2. Alanine substitution of the Spa2 phosphorylation sites targeted by Gpa1/Fus3 lead to a dramatic defect in pheromone gradient sensing and zygote formation. These results reveal how the G alpha protein and the MAPK control cell polarity in a prototypical model system. Our results have wider significance as similar mechanisms exist in higher eukaryotes and are involved in important biological such as neuron development, immunity, and cancer cell metastasis.