Laboratory of Molecular Genetics, Frontier Research Unit, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.
Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
J Biol Chem. 2023 Apr;299(4):104569. doi: 10.1016/j.jbc.2023.104569. Epub 2023 Mar 2.
To cope with an increased external osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK) through the high-osmolarity glycerol (HOG) pathway, which governs adaptive responses to osmostress. In the HOG pathway, two apparently redundant upstream branches, termed SLN1 and SHO1, activate cognate MAP3Ks (MAPKK kinase) Ssk2/22 and Ste11, respectively. These MAP3Ks, when activated, phosphorylate and thus activate the Pbs2 MAP2K (MAPK kinase), which in turn phosphorylates and activates Hog1. Previous studies have shown that protein tyrosine phosphatases and the serine/threonine protein phosphatases type 2C negatively regulate the HOG pathway to prevent its excessive and inappropriate activation, which is detrimental to cell growth. The tyrosine phosphatases Ptp2 and Ptp3 dephosphorylate Hog1 at Tyr-176, whereas the protein phosphatase type 2Cs Ptc1 and Ptc2 dephosphorylate Hog1 at Thr-174. In contrast, the identities of phosphatases that dephosphorylate Pbs2 remained less clear. Here, we examined the phosphorylation status of Pbs2 at the activating phosphorylation sites Ser-514 and Thr-518 (S514 and T518) in various mutants, both in the unstimulated and osmostressed conditions. Thus, we found that Ptc1-Ptc4 collectively regulate Pbs2 negatively, but each Ptc acts differently to the two phosphorylation sites in Pbs2. T518 is predominantly dephosphorylated by Ptc1, while S514 can be dephosphorylated by any of Ptc1-4 to an appreciable extent. We also show that Pbs2 dephosphorylation by Ptc1 requires the adaptor protein Nbp2 that recruits Ptc1 to Pbs2, thus highlighting the complex processes involved in regulating adaptive responses to osmostress.
为了应对外部渗透压的增加，芽殖酵母酿酒酵母通过高渗透压甘油（HOG）途径激活 Hog1 丝裂原激活蛋白激酶（MAPK），该途径控制着对渗透压应激的适应性反应。在 HOG 途径中，两个明显冗余的上游分支，分别称为 SLN1 和 SHO1，分别激活同源的 MAP3Ks（MAPKK 激酶）Ssk2/22 和 Ste11。这些 MAP3Ks 被激活后，磷酸化并激活 Pbs2 MAP2K（MAPK 激酶），后者反过来磷酸化并激活 Hog1。先前的研究表明，蛋白酪氨酸磷酸酶和丝氨酸/苏氨酸蛋白磷酸酶 2C 型负调控 HOG 途径，以防止其过度和不适当的激活，这对细胞生长是有害的。酪氨酸磷酸酶 Ptp2 和 Ptp3 在 Tyr-176 处去磷酸化 Hog1，而蛋白磷酸酶 2C 型 Ptc1 和 Ptc2 在 Thr-174 处去磷酸化 Hog1。相比之下，去磷酸化 Pbs2 的磷酸酶的身份仍然不太清楚。在这里，我们检查了各种突变体中 Pbs2 在激活磷酸化位点 Ser-514 和 Thr-518（S514 和 T518）的磷酸化状态，无论是在未刺激还是渗透压胁迫条件下。因此，我们发现 Ptc1-Ptc4 共同负调控 Pbs2，但每个 Ptc 对 Pbs2 的两个磷酸化位点的作用不同。T518 主要由 Ptc1 去磷酸化，而 S514 可以由 Ptc1-4 中的任何一个去磷酸化到相当大的程度。我们还表明，Ptc1 对 Pbs2 的去磷酸化需要衔接蛋白 Nbp2，该蛋白将 Ptc1 招募到 Pbs2，从而突出了调节渗透压应激适应性反应的复杂过程。
