Regulation of a cyclin-CDK-CDK inhibitor complex by inositol pyrophosphates

Abstract

In budding yeast, phosphate starvation triggers inhibition of the Pho80-Pho85 cyclin-cyclin-dependent kinase (CDK) complex by the CDK inhibitor Pho81, leading to expression of genes involved in nutrient homeostasis. We isolated myo-d-inositol heptakisphosphate (IP7) as a cellular component that stimulates Pho81-dependent inhibition of Pho80-Pho85. IP7 is necessary for Pho81-dependent inhibition of Pho80-Pho85 in vitro. Moreover, intracellular concentrations of IP7 increased upon phosphate starvation, and yeast mutants defective in IP7 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.

Fig. 1

Purification of a cellular component that inactivates Pho80-Pho85 in a Pho81-dependent manner. (A) Phosphorylation of recombinant Pho4 by Pho80-Pho85-Pho81 immunopurified from P_i-rich (High) and P_i-starved (Low) cells expressing Pho80–hemagglutinin tag (3HA). (B) Effects of cell fractions on Pho80-Pho85 activity. Extracts from cells (3 × 10⁹ cells per ml extract) grown in high-P_i medium (High P_i extract) or starved for phosphate (Low P_i extract) were added either directly or after filtration (molecular weight cut-off 3 kD; Low and High indicate low– and high–molecular weight fractions, respectively) to Pho80-Pho85-Pho81 immunopurified from P_i-rich cells, and kinase assays were done as in (A). U, untreated Pho80-Pho85-Pho81 from the same gel. (C) The low-P_i low–molecular weight fraction in (B) was serially diluted and added to Pho80-Pho85 immunopurified from wild-type (WT) and pho81Δ cells that had been grown in P_i-rich medium. Kinase assays were then performed using recombinant Pho4 as a substrate. (D) Structures of relevant IP₇ isomers. 4-PP-IP₅ and 6-PP-IP₅ are mirror images of each other. P, phosphate; PP, pyrophosphate.

Fig. 2

Inhibition of Pho80-Pho85-Pho81 by synthetic IP₇. (A) Synthetic IP₇ was serially diluted and added to immunopurified Pho80-Pho85 isolated from wild-type (WT) or pho81Δ cells that had been grown in P_i-rich medium (Fig. 1C). Kinase activity was then measured. (B) Serial dilutions of inositol, myo-d-inositol 1,4,5-triphosphate (IP₃), IP₆, or IP₇ were added to Pho80-Pho85-Pho81 immunopurified from wild-type cells grown in P_i-rich medium, and kinase activity was measured. (C) Synthetic IP₇ was added to recombinant Pho80-Pho85 or Pho80-Pho85–Pho81-MD complex (50 nM Pho80-Pho85 and 250 nM Pho81-MD) and incubated 20 min at room temperature. Kinase activity was then measured.

Fig. 3

Increase in IP₇ upon P_i starvation. Strong anion exchange chromatograms of extracts derived from wild-type (WT) cells labeled with [³H]-inositol grown in medium containing high P_i (10 mM) or low P_i (5 μM). Cells were starved for 2 hours at 30°C before lysis and analysis. CPM, counts per minute.

Fig. 4

Effect of disrupted inositol polyphosphate metabolism on Pho80-Pho85 activity, monitored by Pho4 localization. (A) Schematic diagram showing inositol polyphosphate metabolism in S. cerevisiae. (B) Wild-type (WT), kcs1Δ, or vip1Δ cell extracts from P_i-starved cells were added to immunopurified Pho80-Pho85 from P_i-rich wild-type (top) or pho81Δ (bottom) cells, and kinase activity was measured. U, untreated. (C) Fluorescence microscopic (100× magnification) analysis of wild-type, ipk1Δ, kcs1Δ, vip1Δ, or ddp1Δ cells expressing Pho4–green fluorescent protein, grown in medium containing high (top) or low (bottom) concentrations of P_i. (D) Isomer specificity of the IP₇-mediated Pho80-Pho85 inactivation. Pho80-Pho85 (8 nM) in the presence (top) and the absence (bottom) of His₆–Pho81-MD (250 nM) was incubated with IP₇ isomers synthesized with recombinant Vip1 (top) and human IP6 kinase 1 (bottom), and kinase activity was measured.