Department of Molecular and Cell Biology, Division of Biochemistry, Biophysics and Structural Biology, University of California, Berkeley, CA 94720, USA.
Yaffe Laboratory, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Biomolecules. 2021 Oct 17;11(10):1530. doi: 10.3390/biom11101530.
Eukaryotes utilize distinct mitogen/messenger-activated protein kinase (MAPK) pathways to evoke appropriate responses when confronted with different stimuli. In yeast, hyperosmotic stress activates MAPK Hog1, whereas mating pheromones activate MAPK Fus3 (and MAPK Kss1). Because these pathways share several upstream components, including the small guanosine-5'-triphosphate phosphohydrolase (GTPase) cell-division-cycle-42 (Cdc42), mechanisms must exist to prevent inadvertent cross-pathway activation. Hog1 activity is required to prevent crosstalk to Fus3 and Kss1. To identify other factors required to maintain signaling fidelity during hypertonic stress, we devised an unbiased genetic selection for mutants unable to prevent such crosstalk even when active Hog1 is present. We repeatedly isolated truncated alleles of , a Cdc42-specific GTPase-activating protein (GAP), each lacking its C-terminal catalytic domain, that permit activation of the mating MAPKs under hyperosmotic conditions despite Hog1 being present. We show that Rga1 down-regulates Cdc42 within the high-osmolarity glycerol (HOG) pathway, but not the mating pathway. Because induction of mating pathway output via crosstalk from the HOG pathway takes significantly longer than induction of HOG pathway output, our findings suggest that, under normal conditions, Rga1 contributes to signal insulation by limiting availability of the GTP-bound Cdc42 pool generated by hypertonic stress. Thus, Rga1 action contributes to squelching crosstalk by imposing a type of "kinetic proofreading". Although Rga1 is a Hog1 substrate in vitro, we eliminated the possibility that its direct Hog1-mediated phosphorylation is necessary for its function in vivo. Instead, we found first that, like its paralog Rga2, Rga1 is subject to inhibitory phosphorylation by the cyclin-dependent protein kinase 1 (Cdk1) ortholog Cdc28 and that hyperosmotic shock stimulates its dephosphorylation and thus Rga1 activation. Second, we found that Hog1 promotes Rga1 activation by blocking its Cdk1-mediated phosphorylation, thereby allowing its phosphoprotein phosphatase 2A (PP2A)-mediated dephosphorylation. These findings shed light on why Hog1 activity is required to prevent crosstalk from the HOG pathway to the mating pheromone response pathway.
真核生物利用不同的有丝分裂原/信使激活的蛋白激酶（MAPK）途径来对不同的刺激做出适当的反应。在酵母中，高渗胁迫激活 MAPK Hog1，而交配信息素则激活 MAPK Fus3（和 MAPK Kss1）。由于这些途径共享几个上游成分，包括小 G 蛋白-5'-三磷酸磷酸水解酶（GTPase）细胞分裂周期-42（Cdc42），因此必须存在机制来防止交叉途径的意外激活。Hog1 的活性对于防止与 Fus3 和 Kss1 的交叉对话是必需的。为了确定在高渗胁迫期间维持信号保真度所需的其他因素，我们设计了一种无偏遗传选择，用于鉴定即使在有活性的 Hog1 存在的情况下也无法防止这种串扰的突变体。我们反复分离出一种 Cdc42 特异性 GTP 酶激活蛋白（GAP）的截断等位基因，该基因缺失其 C 端催化结构域，尽管 Hog1 存在，但允许在高渗条件下激活交配 MAPK。我们表明，Rga1 在高渗透压甘油（HOG）途径中下调 Cdc42，但不在交配途径中。由于通过 HOG 途径的串扰诱导交配途径输出所需的时间明显长于诱导 HOG 途径输出所需的时间，因此我们的发现表明，在正常条件下，Rga1 通过限制由高渗应激产生的结合 GTP 的 Cdc42 池的可用性，有助于信号隔离。因此，Rga1 通过施加一种“动力学校验”来抑制串扰。尽管 Rga1 是体外 Hog1 的底物，但我们排除了其在体内功能中直接由 Hog1 介导的磷酸化的可能性。相反，我们首先发现，像它的同源物 Rga2 一样，Rga1 受到细胞周期蛋白依赖性蛋白激酶 1（Cdk1）同源物 Cdc28 的抑制性磷酸化，并且高渗休克刺激其去磷酸化，从而激活 Rga1。其次，我们发现 Hog1 通过阻断其 Cdk1 介导的磷酸化来促进 Rga1 的激活，从而允许其磷酸蛋白磷酸酶 2A（PP2A）介导的去磷酸化。这些发现揭示了为什么 Hog1 活性对于防止 HOG 途径与交配信息素反应途径的串扰是必需的。
