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Abstract
Over 30 inositol polyphosphates are known to exist in mammalian cells; however, the majority of them have uncharacterized functions. In this study we investigated the molecular basis of synthesis of highly phosphorylated inositol polyphosphates (such as inositol tetrakisphosphate, inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6)) in rat cells. We report that heterologous expression of rat inositol polyphosphate kinases rIPK2, a dual specificity inositol trisphosphate/inositol tetrakisphosphate kinase, and rIPK1, an IP5 2-kinase, were sufficient to recapitulate IP6 synthesis from inositol 1,4,5-trisphosphate in mutant yeast cells. Overexpression of rIPK2 in Rat-1 cells increased inositol 1,3,4,5,6-pentakisphosphate (I(1,3,4,5,6)P5) levels about 2-3-fold compared with control. Likewise in Rat-1 cells, overexpression of rIPK1 was capable of completely converting I(1,3,4,5,6)P5 to IP6. Simultaneous overexpression of both rIPK2 and rIPK1 in Rat-1 cells increased both IP5 and IP6 levels. To reduce IPK2 activity in Rat-1 cells, we introduced vector-based short interference RNA against rIPK2. Cells harboring the short interference RNA had a 90% reduction of mRNA levels and a 75% decrease of I(1,3,4,5,6)P5. These data confirm the involvement of IPK2 and IPK1 in the conversion of inositol 1,4,5-trisphosphate to IP6 in rat cells. Furthermore these data suggest that rIPK2 and rIPK1 act as key determining steps in production of IP5 and IP6, respectively. The ability to modulate the intracellular inositol polyphosphate levels by altering IPK2 and IPK1 expression in rat cells will provide powerful tools to study the roles of I(1,3,4,5,6)P5 and IP6 in cell signaling.
Highlights
Most of the over 30 inositol polyphosphates present in mammalian cells have unknown physiological functions [1,2,3]
We report that heterologous expression of rat inositol polyphosphate kinases rat ortholog of IPK2 (rIPK2), a dual specificity inositol trisphosphate/inositol tetrakisphosphate kinase, and rat ortholog of IPK1 (rIPK1), an IP5 2-kinase, were sufficient to recapitulate IP6 synthesis from inositol 1,4,5-trisphosphate in mutant yeast cells
To elucidate the molecular basis for synthesis of these important messengers we established cell lines in which inositol polyphosphate levels were altered through overexpression or RNA interference (RNAi) of inositol polyphosphate kinases
Summary
I(1,4,5)P3, inositol 1,4,5-trisphosphate; rIPK2, rat inositol polyphosphate kinase 2; rIPK1, rat inositol polyphosphate kinase 1; HPLC, high pressure liquid chromatography; IP, inositol polyphosphate; I(1,3,4)P3, inositol 1,3,4-trisphosphate; I(1,4,5,6)P4, inosiparticipates in intracellular Ca2ϩ mobilization [4] and serves as a precursor of highly phosphorylated inositol polyphosphates such as inositol tetrakisphosphate (IP4), inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6) [1,2,3]. Most of the data mentioned above were determined in vitro or in yeast, and evidence in mammalian cells tol 1,4,5,6-tetrakisphosphate; I(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; I(1,3,4,6)P4, inositol 1,3,4,6-tetrakisphosphate; I(1,3,4,5,6)P5, inositol 1,3,4,5,6-pentakisphosphate; IP3, inositol trisphosphate; IP4, inositol tetrakisphosphate; IP5, inositol pentakisphosphate; IP6, inositol hexakisphosphate; RNAi, RNA interference; siRNA, short interference RNA; FBS, fetal bovine serum; RT, reverse transcription; GFP, green fluorescent protein; GST, glutathione S-transferase; At, A. thaliana; Sc, S. cerevisiae; dm, D. melanogaster; I(1,4)P2, inositol 1,4-bisphosphate; I(1)P, inositol 1-phosphate. The ability to modulate the intracellular inositol polyphosphate levels shown here by altering IPK2 and IPK1 expression in rat cells will provide powerful tools to study the roles of I(1,3,4,5,6)P5 and IP6 in eukaryotic cell signaling
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