ABSTRACT
Lipid-derived inositol phosphates (IPs) are a complex group of second messengers generated by the sequential phosphorylation of inositol 1,4,5-trisphosphate (IP(3)). Synthetic pathways leading from IP(3) to the formation of inositol tetrakisphosphate IP(4), inositol pentakisphosphate IP(5), inositol hexakisphosphate IP(6), and inositol pyrophosphates PP-IPs have been elucidated in eukaryotes from yeast to human. Studies have attributed a variety of cellular functions to IPs, highlighting the importance of understanding how the pathways for their synthesis are regulated. This chapter summarizes experimental techniques for the biochemical characterization of the key inositol phosphate kinases IPKs necessary for producing the diverse array of IP species.
Subject(s)
Inositol 1,4,5-Trisphosphate/metabolism , Inositol Phosphates/metabolism , Phosphotransferases/metabolism , Arabidopsis/enzymology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Chromatography, High Pressure Liquid/methods , Chromatography, Thin Layer/methods , Inositol 1,4,5-Trisphosphate/chemistry , Inositol 1,4,5-Trisphosphate/genetics , Magnetic Resonance Spectroscopy/methods , Phosphorylation , Phosphotransferases/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
Inositol pyrophosphates are a diverse group of high-energy signaling molecules whose cellular roles remain an active area of study. We report a previously uncharacterized class of inositol pyrophosphate synthase and find it is identical to yeast Vip1 and Asp1 proteins, regulators of actin-related protein-2/3 (ARP 2/3) complexes. Vip1 and Asp1 acted as enzymes that encode inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase activities. Alterations in kinase activity led to defects in cell growth, morphology, and interactions with ARP complex members. The functionality of Asp1 and Vip1 may provide cells with increased signaling capacity through metabolism of IP6.
Subject(s)
Cytoskeletal Proteins/metabolism , Phosphotransferases (Phosphate Group Acceptor)/metabolism , Phytic Acid/metabolism , Saccharomyces cerevisiae/enzymology , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/enzymology , Sequence Alignment , Actin-Related Protein 3/metabolism , Amino Acid Sequence , Animals , Catalytic Domain , Chromatography, High Pressure Liquid , Conserved Sequence , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/isolation & purification , Humans , Inositol Phosphates/metabolism , Molecular Sequence Data , Multifunctional Enzymes , Phosphorylation , Phosphotransferases (Phosphate Group Acceptor)/chemistry , Phosphotransferases (Phosphate Group Acceptor)/genetics , Phosphotransferases (Phosphate Group Acceptor)/isolation & purification , Protein Structure, Tertiary , Pyrophosphatases , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification , Saccharomyces cerevisiae Proteins/metabolism , Schizosaccharomyces/cytology , Schizosaccharomyces/genetics , Schizosaccharomyces/growth & development , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/isolation & purification , Substrate Specificity , TemperatureABSTRACT
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.
