pyrophosphate and 1-diphosphoinositol-pentakisphosphate

Diphosphoinositol polyphosphates, also known as inositol pyrophosphates, are a family of water soluble inositol phosphates that possess diphosphate or pyrophosphate moieties. In the presence of divalent cations such as Mg(2+), the "high energy" beta phosphate can be transferred from the inositol pyrophosphates, InsP(7) and InsP(8), to prephosphorylated serine residues on proteins, to form pyrophosphoserine. This chapter provides detailed methods to identify proteins that are substrates for pyrophosphorylation by InsP(7), conduct phosphorylation assays on purified protein, and detect protein pyrophosphorylation.

Topics: Amino Acid Sequence; Autoradiography; Biochemistry; Blotting, Western; Diphosphates; Escherichia coli; Gene Expression; Inositol Phosphates; Molecular Sequence Data; Phosphorylation; Proteins; Recombinant Fusion Proteins; Saccharomyces cerevisiae

High-energy inositol pyrophosphates, such as IP(7) (diphosphoinositol pentakisphosphate), can directly donate a beta-phosphate to a prephosphorylated serine residue generating pyrophosphorylated proteins. Here, we show that the beta subunit of AP-3, a clathrin-associated protein complex required for HIV-1 release, is a target of IP(7)-mediated pyrophosphorylation. We have identified Kif3A, a motor protein of the kinesin superfamily, as an AP3B1-binding partner and demonstrate that Kif3A, like the AP-3 complex, is involved in an intracellular process required for HIV-1 Gag release. Importantly, IP(7)-mediated pyrophosphorylation of AP3B1 modulates the interaction with Kif3A and, as a consequence, affects the release of HIV-1 virus-like particles. This study identifies a cellular process that is regulated by IP(7)-mediated pyrophosphorylation.

Topics: Adaptor Protein Complex 3; Adaptor Protein Complex beta Subunits; Adaptor Proteins, Vesicular Transport; Diphosphates; Gene Products, gag; HIV-1; Humans; Inositol Phosphates; Kinesins; Phosphorylation

Eukaryotic cells produce a family of diverse inositol polyphosphates (IPs) containing pyrophosphate bonds. Inositol pyrophosphates have been linked to a wide range of cellular functions, and there is growing evidence that they act as second messengers. Inositol hexakisphosphate kinase (IP6K) is able to convert the natural substrates inositol pentakisphosphate (IP 5) and inositol hexakisphosphate (IP 6) to several products with an increasing number of phospho-anhydride bonds. In this study, we structurally analyzed IPs synthesized by three mammalian isoforms of IP6K from IP 5 and IP 6. The NMR and mass analyses showed a number of products with diverse, yet specific, stereochemistry, defined by the architecture of IP6K's active site. We now report that IP6K synthesizes both pyrophosphate (diphospho) as well as triphospho groups on the inositol ring. All three IP6K isoforms share the same activities both in vitro and in vivo.

Topics: Cacodylic Acid; Chromatography, High Pressure Liquid; Diphosphates; Inositol Phosphates; Isoenzymes; Mass Spectrometry; Phosphotransferases (Phosphate Group Acceptor); Phytic Acid; Polyphosphates; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism; Substrate Specificity