The yeast AMP-activated protein kinase Snf1p phosphorylates the inositol polyphosphate kinase Kcs1p. The yeast AMP-activated protein kinase Snf1p phosphorylates the inositol polyphosphate kinase Kcs1p

The yeast Snf1p/AMP-activated kinase (AMPK) maintains energy homeostasis, controlling metabolic processes and glucose derepression in response to nutrient levels and environmental cues. Under conditions of nitrogen or glucose limitation, Snf1p regulates pseudohyphal growth, a morphological transition characterized by the formation of extended multicellular filaments. During pseudohyphal growth, Snf1p is required for wild-type levels of inositol polyphosphate (InsP), soluble phosphorylated species of the six-carbon cyclitol inositol that function as conserved metabolic second messengers. InsP levels are established through the activity of a family of inositol kinases, including the inositol polyphosphate kinase Kcs1p, which principally generates pyrophosphorylated forms of InsP7 and InsP8. Here, we report that Snf1p regulates Kcs1p, affecting Kcs1p phosphorylation and inositol kinase activity. A snf1 kinase-defective mutant exhibits decreased Kcs1p phosphorylation, and Kcs1p is phosphorylated in vivo at Ser residues 537 and 646 during pseudohyphal growth. By in vitro analysis, Snf1p directly phosphorylates Kcs1p, predominantly at amino acids 537 and 646. A yeast strain carrying kcs1 encoding Ser-to-Ala point mutations at these residues (kcs1-S537A,S646A) shows elevated levels of pyrophosphorylated InsP7, comparable to InsP7 levels observed upon deletion of SNF1. The kcs1-S537A,S646A mutant exhibits decreased pseudohyphal growth, invasive growth, and cell elongation. Transcriptional profiling indicates extensive perturbation of metabolic pathways in kcs1-S537A,S646A. Growth of kcs1-S537A,S646A is affected on medium containing glycerol and antimycin A, consistent with decreased Snf1p signaling. This work identifies Snf1p phosphorylation of Kcs1p, collectively highlighting the interconnectedness of AMPK activity and InsP signaling in coordinating nutrient availability, energy homoeostasis, and cell growth. Overall design: We inoculated three single colonies each of a control strain (SSY128: kcs1 delta/delta with pRS416-KCS1) and kcs1 phosphodefective strain (SSY146: kcs1delta/delta with pRS416-kcs1-S537A,S646A) in 5 ml of SC -Ura media at 30 degrees C with shaking (250 rpm). Cultures were grown to saturation and were subsequently diluted in 50 ml of low-nitrogen media to an absorbance (optical density at 600 nm) of 0.05. Cultures were grown at 30 degrees C with shaking for roughly 18-20 hours to an optical density of approximately 0.5 at 600 nm. We subsequently harvested cells from each culture and stored the cell pellets at -80 degrees C. We isolated RNA from the frozen pellets using the RiboPure Yeast Kit (Thermo Fisher Scientific). RNA samples were treated with DNase, and RNA concentrations were determined using the NanoDrop microvolume spectrophotometer.

酵母Snf1p/AMP激活激酶（AMPK）可维持能量稳态，响应营养水平与环境信号调控代谢过程及葡萄糖去阻遏作用。在氮源或葡萄糖限制条件下，Snf1p可调控假菌丝生长——这是一种以延伸多细胞丝状体形成为特征的形态转变过程。假菌丝生长过程中，Snf1p是维持野生型肌醇多磷酸（inositol polyphosphate, InsP）水平所必需的；InsP是六碳环醇肌醇的可溶性磷酸化衍生物，作为保守的代谢第二信使发挥功能。InsP水平由一类肌醇激酶家族的活性调控，其中包括肌醇多磷酸激酶Kcs1p，其主要催化生成InsP7与InsP8的焦磷酸化形式。本研究发现Snf1p可调控Kcs1p，影响Kcs1p的磷酸化修饰与肌醇激酶活性。snf1激酶缺陷型突变体的Kcs1p磷酸化水平降低；假菌丝生长过程中，Kcs1p在体内的丝氨酸残基537与646位点发生磷酸化。体外实验分析显示，Snf1p可直接磷酸化Kcs1p，主要作用位点为氨基酸537与646。携带上述位点丝氨酸突变为丙氨酸点突变的kcs1基因（即kcs1-S537A,S646A）的酵母菌株，其焦磷酸化InsP7水平升高，该表型与SNF1基因缺失菌株中观察到的InsP7水平相当。kcs1-S537A,S646A突变体的假菌丝生长、侵入生长与细胞伸长能力均出现下降。转录组分析显示，kcs1-S537A,S646A突变体的代谢通路存在广泛失调。kcs1-S537A,S646A突变体在含甘油与抗霉素A的培养基上的生长受到影响，这与Snf1p信号通路活性降低的表型一致。本研究证实了Snf1p对Kcs1p的磷酸化修饰，整体揭示了AMPK活性与InsP信号通路之间的关联性，二者共同参与协调营养供给、能量稳态与细胞生长过程。
整体实验设计：我们分别挑取对照菌株（SSY128：kcs1Δ/Δ搭配pRS416-KCS1质粒）与kcs1磷酸化缺陷型菌株（SSY146：kcs1Δ/Δ搭配pRS416-kcs1-S537A,S646A质粒）的三个单菌落，接种至5 mL SC-Ura培养基中，于30℃、250 rpm摇床培养。待培养液生长至饱和后，将其转接至50 mL低氮培养基中，调整菌液浓度至OD600为0.05。将菌液置于30℃、摇床培养约18-20小时，至OD600约为0.5。随后收集各培养物的细胞，将细胞沉淀保存于-80℃冰箱。我们使用RiboPure酵母RNA提取试剂盒（RiboPure Yeast Kit，赛默飞世尔科技，Thermo Fisher Scientific）从冷冻细胞沉淀中提取总RNA。RNA样品经DNase处理后，使用NanoDrop微量分光光度计测定RNA浓度。