Protein Kinase A Contributes to the Negative Control of Snf1 Protein Kinase in Saccharomyces cerevisiae
Protein Kinase A Contributes to the Negative Control of Snf1 Protein Kinase in Saccharomyces cerevisiae
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DOI：
10.1128/ec.05061-11
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发表时间：
2012-02-01
期刊：
影响因子：
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通讯作者：
Kuchin, Sergei
中科院分区：
文献类型：
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作者：
Barrett, LaKisha;Orlova, Marianna;Kuchin, Sergei
Snf1 蛋白激酶调节对葡萄糖限制和其他压力的反应。 Snf1 激活需要部分冗余的上游激酶（Sak1、Tos3 和 Elm1）对其 T 环苏氨酸进行磷酸化。在有利的条件下，Snf1 会被 Reg1-Glc7 蛋白磷酸酶关闭。 reg1 突变导致 Snf1 激活增加和生长缓慢。为了识别 Snf1 通路的新成分，我们寻找了像 snf1 一样抑制 reg1 以获得缓慢生长表型的突变。除了编码已知通路成分（SNF1、SNF4 和 SAK1）的基因突变外，我们还发现了“快速”突变，称为 fst1 和 fst2。这里使用的 Sigma 1278b 菌株中突变体的异常形态帮助我们将 fst1 和 fst2 识别为 RasGAP 基因 IRA1 和 IRA2 的突变。缺乏 Ira1、Ira2 或 Bcy1（环 AMP (cAMP) 依赖性蛋白激酶 A (PKA) 的负调节亚基）的细胞表现出 Snf1 通路激活减少。相反，在缺乏 Gpr1 糖受体的细胞中，Snf1 激活升高，而 Gpr1 糖受体有助于 PKA 信号传导。我们发现 Snf1 激活激酶 Sak1 在体内在理想的 PKA 基序内的保守丝氨酸 (Ser1074) 上被磷酸化。然而，这种磷酸化本身似乎在调节中仅发挥有限的作用，并且 Sak1 并不是 PKA 途径的唯一相关靶标。总的来说，我们的结果表明，整合多种调控输入的 PKA 可以通过复杂的机制在各种条件下促进 Snf1 调控。我们的结果还支持这样的观点，即与哺乳动物的对应物 AMP 激活蛋白激酶 (AMPK) 一样，酵母 Snf1 参与协调生长与营养可用性的代谢检查点控制。
Snf1 protein kinase regulates responses to glucose limitation and other stresses. Snf1 activation requires phosphorylation of its T-loop threonine by partially redundant upstream kinases (Sak1, Tos3, and Elm1). Under favorable conditions, Snf1 is turned off by Reg1-Glc7 protein phosphatase. The reg1 mutation causes increased Snf1 activation and slow growth. To identify new components of the Snf1 pathway, we searched for mutations that, like snf1, suppress reg1 for the slow-growth phenotype. In addition to mutations in genes encoding known pathway components (SNF1, SNF4, and SAK1), we recovered "fast" mutations, designated fst1 and fst2. Unusual morphology of the mutants in the Sigma 1278b strains employed here helped us identify fst1 and fst2 as mutations in the RasGAP genes IRA1 and IRA2. Cells lacking Ira1, Ira2, or Bcy1, the negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA), exhibited reduced Snf1 pathway activation. Conversely, Snf1 activation was elevated in cells lacking the Gpr1 sugar receptor, which contributes to PKA signaling. We show that the Snf1-activating kinase Sak1 is phosphorylated in vivo on a conserved serine (Ser1074) within an ideal PKA motif. However, this phosphorylation alone appears to play only a modest role in regulation, and Sak1 is not the only relevant target of the PKA pathway. Collectively, our results suggest that PKA, which integrates multiple regulatory inputs, could contribute to Snf1 regulation under various conditions via a complex mechanism. Our results also support the view that, like its mammalian counterpart, AMP-activated protein kinase (AMPK), yeast Snf1 participates in metabolic checkpoint control that coordinates growth with nutrient availability.