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.