Cellular delivery and photochemical release of a caged inositol-pyrophosphate induces PH-domain translocation in cellulo

Pavlovic, Igor; Thakor, Divyeshsinh T.; Vargas, Jessica R.; McKinlay, Colin J.; Hauke, Sebastian; Anstaett, Philipp; Camuña, Rafael C.; Bigler, Laurent; Gasser, Gilles; Schultz, Carsten; Wender, Paul A.; Jessen, Henning J.

doi:10.1038/ncomms10622

Cited by 83 publications

(94 citation statements)

References 57 publications

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“…Kinase and Phosphatase Assays-PPIP5K kinase activities were routinely measured at 37°C in 1-2-h incubations comprising 100 l of assay buffer containing 1 mM Na 2 EDTA, 50 mM KCl, 20 mM HEPES (pH 7.2), 7 mM MgCl 2 , 5 mM ATP, 0.5 mg/ml BSA, and either 1 M InsP 6 (Calbiochem) or 1 M chemically synthesized 5-InsP 7 (47). PPIP5K phosphatase activities were measured at 37°C in 30-min incubations comprising 100 l of assay buffer containing 1 mM Na 2 EDTA, 50 mM KCl, 20 mM HEPES (pH 7.2), 2 mM MgCl 2 , 0.5 mg/ml BSA, and either 1 or 0.05 M chemically synthesized 1-InsP 7 (48) or InsP 8 (30).…”

Section: Methodsmentioning

confidence: 99%

The Significance of the Bifunctional Kinase/Phosphatase Activities of Diphosphoinositol Pentakisphosphate Kinases (PPIP5Ks) for Coupling Inositol Pyrophosphate Cell Signaling to Cellular Phosphate Homeostasis

Nguyen

Hofer

et al. 2017

Journal of Biological Chemistry

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292

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Edited by Alex TokerPhosphate has multiple functions that direct the survival of all living organisms: in its organic form, P i is a component of genomic material, it serves as an energy currency, and it is ubiquitous in cell signaling. Thus, P i homeostasis is essential to life, but the mechanisms by which this occurs in humans and other metazoans are largely unknown (1, 2). Most of the previous work in this field of research has focused on yeast models (3-5). In particular, recent studies with Saccharomyces cerevisiae have revealed a new function in P i homeostasis for inositol pyrophosphates (5). The latter are soluble, intracellular signals that contain multiple phosphates and diphosphates; up to seven (InsP 7 ) 4 or eight (InsP 8 ) phosphates in total are crammed around a six-carbon inositol ring (see Refs. 6 -8 and Fig. 1). In S. cerevisiae, levels of one inositol pyrophosphate, 5-InsP 7 , track perturbations to P i homeostasis (5).This P i -sensing activity of 5-InsP 7 appears to reflect it being synthesized by a kinase class (kcs1 in yeast; IP6Ks in metazoans) that exhibits an unusually low affinity for ATP (9, 10). Consequently, cellular levels of 5-InsP 7 in yeast decrease in response to the drop in [ATP] that accompanies extracellular [P i ] depletion (5, 11). Furthermore, these ATP-driven changes in 5-InsP 7 levels appear to comprise a dynamic signaling response because 5-InsP 7 regulates proteins that maintain P i homeostasis through interactions with their SPX domains (5). However, it is not known to what extent this signaling response is applicable to metazoan cells, which lack orthologs of many of the yeast genes that function in P i sensing and P i homeostasis (2).

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Section: Methodsmentioning

confidence: 99%

The Significance of the Bifunctional Kinase/Phosphatase Activities of Diphosphoinositol Pentakisphosphate Kinases (PPIP5Ks) for Coupling Inositol Pyrophosphate Cell Signaling to Cellular Phosphate Homeostasis

Nguyen

Hofer

et al. 2017

Journal of Biological Chemistry

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292

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“…50 μl. Next, each sample was spiked with 1 nmol of InsP 6 (Calbiochem), and 1 nmol each of chemically synthesized 1-InsP 7 [22], 5-InsP 7 [23], and InsP 8 [24]. For some experiments (as indicated below), 20 nmol 5-InsP 7 were added.…”

Section: Methodsmentioning

confidence: 99%

Inositol Pyrophosphate Profiling of Two HCT116 Cell Lines Uncovers Variation in InsP8 Levels

Wilson

Jessen

et al. 2016

PLoS ONE

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The HCT116 cell line, which has a pseudo-diploid karotype, is a popular model in the fields of cancer cell biology, intestinal immunity, and inflammation. In the current study, we describe two batches of diverged HCT116 cells, which we designate as HCT116NIH and HCT116UCL. Using both gel electrophoresis and HPLC, we show that HCT116UCL cells contain 6-fold higher levels of InsP8 than HCT116NIH cells. This observation is significant because InsP8 is one of a group of molecules collectively known as ‘inositol pyrophosphates’ (PP-InsPs)—highly ‘energetic’ and conserved regulators of cellular and organismal metabolism. Variability in the cellular levels of InsP8 within divergent HCT116 cell lines could have impacted the phenotypic data obtained in previous studies. This difference in InsP8 levels is more remarkable for being specific; levels of other inositol phosphates, and notably InsP6 and 5-InsP7, are very similar in both HCT116NIH and HCT116UCL lines. We also developed a new HPLC procedure to record 1-InsP7 levels directly (for the first time in any mammalian cell line); 1-InsP7 comprised <2% of total InsP7 in HCT116NIH and HCT116UCL lines. The elevated levels of InsP8 in the HCT116UCL lines were not due to an increase in expression of the PP-InsP kinases (IP6Ks and PPIP5Ks), nor to a decrease in the capacity to dephosphorylate InsP8. We discuss how the divergent PP-InsP profiles of the newly-designated HCT116NIH and HCT116UCL lines should be considered an important research opportunity: future studies using these two lines may uncover new features that regulate InsP8 turnover, and may also yield new directions for studying InsP8 function.

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“…78 A recent study revealed that chemically synthesized 5-IP 7 released into mammalian cells is able to induce translocation of AKT from the plasma membrane to the cytoplasm. 79 It has been shown that AKT and other PH domains bind 5-IP 7 and IP 6 with higher affinity as compared with 1-IP 7 or IP 8 . 41 This study also suggested that PI(3,4,5)P 3 dependent recruitment of PPIP5K1 to the plasma membrane would deplete subplasmalemmal 5-IP 7 and IP 6 by using them as substrates, thus providing positive feedback to PI(3,4,5)P 3 binding by PH domains at the plasma membrane.…”

Section: Plasma Membranementioning

confidence: 99%

“…78,79,[126][127][128][129] Highly pure PP-IPs with β-phosphate moieties at specific carbon atoms have been synthesized by these groups, including non-hydrolysable analogues that are stable in cells and can bind target proteins but not pyrophosphorylate them. 47,78,130 A system for intracellular delivery and photouncaging of chemically synthesized PP-IPs has recently been developed, 79 which promises to open up new methods to study the functions of these molecules in different subcellular compartments. The use of IP 7 as an affinity reagent revealed two different classes of interacting proteins, depending on the absence or presence of the divalent cation Mg 2+ during the interaction, representing IP 7 binding or pyrophosphorylation targets respectively.…”

Section: Perspective On the Futurementioning

confidence: 99%

Inositol Pyrophosphates: Energetic, Omnipresent and Versatile Signalling Molecules

et al. 2017

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| Inositol pyrophosphates (PP-IPs) are a class of energy-rich signalling molecules found in all eukaryotic cells. These are derivatives of inositol that contain one or more diphosphate (or pyrophosphate) groups in addition to monophosphates. The more abundant and best studied PP-IPs are diphosphoinositol pentakisphosphate (IP 7 ) and bis-diphosphoinositol tetrakisphosphate (IP 8 ). These molecules can influence protein function by two mechanisms: binding and pyrophosphorylation. The former involves the specific interaction of a particular inositol pyrophosphate with a binding site on a protein, while the latter is a unique attribute of inositol pyrophosphates, wherein the β-phosphate moiety is transferred from a PP-IP to a pre-phosphorylated serine residue in a protein to generate pyrophosphoserine. Both these events can result in changes in the target protein's activity, localisation or its interaction with other partners. As a consequence of their ubiquitous presence in all eukaryotic organisms and all cell types examined till date, and their ability to modify protein function, PP-IPs have been found to participate in a wide range of metabolic, developmental, and signalling pathways.This review highlights many of the known functions of PP-IPs in the context of their temporal and spatial distribution in eukaryotic cells. The pathway of synthesis of inositol polyphosphates has been characterized in yeast, slime moulds, plants and animals. The simplest anabolic pathway, characterized in the yeast Saccharomyces cerevisiae (Fig. 1) (Fig. 1). Interestingly, the PPIP5Ks also possess a phosphatase domain which selectively cleaves the 1-position β-phosphate of 1-IP 7 and IP 8 , thus targeting the products of the kinase domain.35, 36 A recent study identified another yeast phosphatase, Siw14, that specifically targets the 5-position β-phosphate of PP-IPs 37 (Fig. 1). As PP-IPs are found in all eukaryotic organisms, they display many conserved and divergent 2, 131 Siw14, an inositol pyrophosphate phosphatase in yeast, preferentially cleaves the C5 β-phosphate on PP-IPs. 37 The yeast enzymes are depicted in purple, and mammalian enzymes are depicted in green and are bracketed. The undetermined inositol pyrophosphate structure is represented with an interrogation mark. Myoinositol contains five equatorial (parallel to the axis) and one axial (perpendicular to the axis) hydroxyl groups. Carbon atoms on the myo-inositol ring are numbered on the structures of PI(4,5)P 2 and IP 6 .

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Cellular delivery and photochemical release of a caged inositol-pyrophosphate induces PH-domain translocation in cellulo

Cited by 83 publications

References 57 publications

The Significance of the Bifunctional Kinase/Phosphatase Activities of Diphosphoinositol Pentakisphosphate Kinases (PPIP5Ks) for Coupling Inositol Pyrophosphate Cell Signaling to Cellular Phosphate Homeostasis

The Significance of the Bifunctional Kinase/Phosphatase Activities of Diphosphoinositol Pentakisphosphate Kinases (PPIP5Ks) for Coupling Inositol Pyrophosphate Cell Signaling to Cellular Phosphate Homeostasis

Inositol Pyrophosphate Profiling of Two HCT116 Cell Lines Uncovers Variation in InsP8 Levels

Inositol Pyrophosphates: Energetic, Omnipresent and Versatile Signalling Molecules

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