Regulation of a Cyclin-CDK-CDK Inhibitor Complex by Inositol Pyrophosphates

Abstract

In budding yeast, phosphate starvation triggers inhibition of the Pho80-Pho85 cyclin-cyclindependent kinase (CDK) complex by the CDK inhibitor Pho81, leading to expression of genes involved in nutrient homeostasis. We isolated myo-D-inositol heptakisphosphate (IP 7 ) as a cellular component that stimulates Pho81-dependent inhibition of Pho80-Pho85. IP 7 is necessary for Pho81-dependent inhibition of Pho80-Pho85 in vitro. Moreover, intracellular concentrations of IP 7 increased upon phosphate starvation, and yeast mutants defective in IP 7 production failed to inhibit Pho80-Pho85 in response to phosphate starvation. These observations reveal regulation of a cyclin-CDK complex by a metabolite and suggest that a complex metabolic network mediates signaling of phosphate availability.In response to nutrient limitation, cells increase transcription of starvation response genes. Many proteins that function in nutrient response systems have been identified, but the molecular details of their regulation are not well understood (1). The Saccharomyces cerevisiae Pho80-Pho85 cyclin-CDK complex is a key regulator in the phosphate (P i )-responsive (PHO) signaling pathway (2,3). When cells are grown in medium that is rich in P i , Pho80-Pho85 is active and phosphorylates the transcription factor Pho4 (4), resulting in its export from the nucleus (5). Upon P i starvation, Pho80-Pho85 kinase activity is decreased, leading to nuclear accumulation of Pho4 and transcription of PHO genes. In vivo, the Pho81 CDK inhibitor (CKI) is bound to Pho80-Pho85 regardless of P i conditions and is required for the inhibition of Pho80-Pho85 activity in response to P i -limitation (6).We used a biochemical strategy to identify cellular components that influence Pho80-Pho85-Pho81 activity. The Pho80-Pho85-Pho81 complex immunopurified from cells grown in P i -rich conditions actively phosphorylated Pho4 in vitro, whereas the complex isolated from P i -starved cells was less active (6) (Fig. 1A). To identify regulators of Pho80-Pho85-Pho81, we added extracts from cells grown in P i -rich and P i -starved conditions to active (from P i -rich) and inactive (from P i -starved) immunopurified Pho80-Pho85-Pho81 complexes and performed kinase assays with recombinant Pho4 serving as the substrate. Extracts from P i -starved cells efficiently inactivated Pho80-Pho85-Pho81, whereas the P i -rich cell extract had little effect (Fig. 1B). When the extract of P i -starved cells was fractionated into fractions of large (>3 kD) and small (<3 kD) molecular size, most inhibitory activity was found in the fractions of small molecular size. This activity had little effect on Pho80-Pho85 purified from pho81Δ cells (Fig. 1C), indicating that, as is true in vivo (6), the inhibitory activity requires Pho81 for its function.

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

“…Recent evidence shows that inositol polyphosphate species play a particular role in this regulation. More specifically, isoform synthesized by Vip1 was found to bind non-covalently with Pho80-Pho85-Pho81, thereby inducing additional interactions between Pho81 and Pho80-Pho85 that prevent substrates from accessing the kinase active site (Lee et al 2007;Lee et al 2008b). Intriguingly, under phosphate starvation conditions, Pho4-dependent antisense and intragenic RNA transcription in the KCS1 locus induces downregulation of the Kcs1 activity, leading to a positive feedback loop for activation of the PHO genes (Nishizawa et al 2008a).…”

Inositol pyrophosphates modulate cell cycle independently of alteration in telomere length

“…There are numerous ways how inositol pyrophosphates may regulate cell cycle progression, this may include events on vacuole biogenesis and cell wall integrity (Dubois et al, 2002), proper endocytic trafficking (Saiardi et al, 2002), cyclin-CDK-CDK inhibitor complex (Lee et al, 2007), chromatin remodelling (Steger et al, 2003) or RNA polymerase Imediated rRNA transcription (Thota et al, 2015) and for all of them understanding of structural analysis of diphosphoinositol polyphosphate kinase is crucial (Shears et al, 2013). Furthermore, above all of them, two might be of particular significance.…”

“…(PP) 2 -IP 4 (IP 8 ) is generated by either Kcs1-mediated phosphorylation of 1-PP-IP 5 or Vip1-mediated phosphorylation of 5-PP-IP 5 (Tsui and York, 2010). Kcs1 has been reported to modulate telomere length by generation of 5-PP-IP 4 (Saiardi et al, 2005;York et al, 2005), and Vip1-generated IP 7 binds to the cyclin-CDK-CDK inhibitor complex to regulate transcription of the yeast phosphate (Pi)-responsive (PHO) genes (Lee et al, 2007).…”

Inositol pyrophosphates as multifaceted metabolites in the regulation of mammalian signaling networks

“…Mechanistically, the dynamic, high-affinity binding of 5-IP7 to the polybasic region of Syt1 appears to hold Syt1 in a nonfunctional configuration . In budding yeast, 1-IP7 but not 5-IP7 interacts selectively with Pho80-Pho85-Pho81 in the regulation of phosphate homeostasis (Lee et al 2007). No 1-IP7-specific protein targets in mammals have been discovered yet.…”