Importance of Radioactive Labelling to Elucidate Inositol Polyphosphate Signalling

Abstract

Inositol polyphosphates, in their water-soluble or lipid-bound forms, represent a large and multifaceted family of signalling molecules. Some inositol polyphosphates are well recognised as defining important signal transduction pathways, as in the case of the calcium release factor Ins(1,4,5)P₃, generated by receptor activation-induced hydrolysis of the lipid PtdIns(4,5)P₂ by phospholipase C. The birth of inositol polyphosphate research would not have occurred without the use of radioactive phosphate tracers that enabled the discovery of the "PI response". Radioactive labels, mainly of phosphorus but also carbon and hydrogen (tritium), have been instrumental in the development of this research field and the establishment of the inositol polyphosphates as one of the most important networks of regulatory molecules present in eukaryotic cells. Advancements in microscopy and mass spectrometry and the development of colorimetric assays have facilitated inositol polyphosphate research, but have not eliminated the need for radioactive experimental approaches. In fact, such experiments have become easier with the cloning of the inositol polyphosphate kinases, enabling the systematic labelling of specific positions of the inositol ring with radioactive phosphate. This approach has been valuable for elucidating their metabolic pathways and identifying specific and novel functions for inositol polyphosphates. For example, the synthesis of radiolabelled inositol pyrophosphates has allowed the discovery of a new protein post-translational modification. Therefore, radioactive tracers have played and will continue to play an important role in dissecting the many complex aspects of inositol polyphosphate physiology. In this review we aim to highlight the historical importance of radioactivity in inositol polyphosphate research, as well as its modern usage.

Keywords: Inositol; Metabolism; Phosphate; Pyrophosphates; Radioactivity.

Fig. 1

Schematic representation of the inositol cycle. Inositol acquired from the extracellular space is incorporated into lipids by the action of the phosphatidylinositol synthase (PI-synthase). The conversion of PtdIns (PI), first to PtdIns(4)P (PIP) and then to PtdIns(4,5)P₂ (PIP₂), generates the substrate for phospholipase C (PLC, boxed in red). Once receptor activation occurs, PLC generates two second messengers: the (plasma) membrane-resident diacylglycerol (DAG) and the calcium (Ca²⁺) release factor Ins(1,4,5)P₃ (IP₃). The latter is converted back to inositol via two dephosphorisation steps, closing the cycle. The inositol cycle is particularly active in stimulated mammalian cells. The Hokin “PI response” [8, 9] measures the [³²P] taken up by the cell and its conversion to [³²P] γATP, with the subsequent radioactive phosphorylation of DAG to phosphatidic acid (PA). PA is then reattached to inositol, creating radioactive PI. For graphical reasons, inositol is abbreviated here as “I” instead of “Ins”, and phosphatidylinositol as “PI” instead of “PtdIns”

Fig. 2

myo-Inositol structure and its radiolabelled derivatives. While nine stereoisomeric configurations of inositol are possible, the structure of myo-inositol is depicted in (a), referred to in the review simply as inositol, since it is by far the most common and biologically relevant form of inositol. The modern D-numbering system for inositols is counterclockwise as viewed from above and assigns the single axial hydroxyl group of myo-inositol to the carbon in position 2, while the other five hydroxyls are equatorial. myo-Inositol possesses an axis of symmetry through carbons 2 and 5 (dashed line), making positions 1,3 and 4,6 enantiomeric. The most common commercially available tritium-labelled inositol (b) possess the [³H] radiolabelled in position 1 and/or 2, while in [¹⁴C]inositol the radiolabel is uniformly distributed (c)

Fig. 3

Inositol polyphosphates synthetic pathway. The synthesis of higher phosphorylated inositol polyphosphates begins with the synthesis of Ins(1,4,5)P₃. Saccharomyces cerevisiae uses only phospholipase C (PLC) hydrolysis of the lipid PI(4,5)P₂ to synthesise Ins(1,4,5)P₃ [28], whereas Dictyostelium discoideum utilises the cytosolic route, of which the enzymology is not fully elucidated (dashed line) [110]. Ins(1,4,5)P₃ is metabolised by ITPKA, B, or C to synthesise Ins(1,3,4,5)P₃, which is acted on by the 5-phosphatase (grey line) to generate the Ins(1,3,4)P₃ converted by ITPK1 to Ins(1,3,4,5,6)P₅. However, this isomer of InsP₅ can also be directly generated by IPMK from Ins(1,4,5)P₃. InsP₅ is converted to InsP₆ by the IP₅-2Kinase IPPK. Phosphorylation of InsP₆ by the IP6Ks generates the inositol pyrophosphate InsP₇, specifically the depicted isomer 5PP-InsP₅, which is further acted on by PPIP5K1,2 to IP₈, specifically to 1,5(PP)₂-IP₄. The IP6K enzymes can also use Ins(1,3,4,5,6)P₅ as a substrate, generating the inositol pyrophosphate PP-IP₄. In this figure, for visual reasons, inositol is abbreviated as “I” instead of “Ins”, and phosphatidylinositol as “PI” instead “PtdIns”. Kinases catalysing each step are indicated in red (human) and blue (S. cerevisiae)

Fig. 4

Schematic synthesis of radiolabelled InsP₅. The most abundant inositol pentakisphosphate isomer, Ins(1,3,4,5,6)P₅, is the common end product of two multikinases, IPMK and ITPK1, using different starting inositol triphosphates (see Fig. 3). Therefore, using these two enzymes and different species of InsP₃ and InsP₄, it is possible to generate InsP₅ labelled in different positions of the inositol ring. The top reaction illustrates the ability of Entamoeba histolytica ITPK1 (EhITPK1) to phosphorylate position 1 [34, 35], enabling specific synthesis of 1[³²P]Ins(1,3,4,5,6)P₅. Conversely, the bottom reaction uses the mammalian IPMK, a 3,6 kinase. Recombinant IPMK can thus be used to generate radiolabelled 3,6[³²P]Ins(1,3,4,5,6)P₅ [88]. Different atoms are colour-coded as follows: carbon black circle; oxygen green circle; phosphate yellow circle; radioactive phosphate red circle

Fig. 5

Schematic synthesis of radiolabelled 5[³²P]βInsP₇. Two different InsP₇ isomers can be easily and rapidly synthesised biochemically, using recombinant IP6K1 or the kinase domain of PPIP5K1 (or its yeast counterpart Vip1). Using radiolabelled [³²P]γATP and InsP₆ as substrate, IP6K1 generates 5[³²P]βInsP₇ (top), thus transferring the radioactive [³²P] from ATP to the phosphorylated position 5 of InsP₆. PPIP5K instead generates the isomer 1[³²P]βInsP₇ (bottom). Different atoms are colour-coded as follows: carbon black circle; oxygen green circle; phosphate yellow circle; radioactive phosphate red circle