A role for rat inositol polyphosphate kinases rIPK2 and rIPK1 in inositol pentakisphosphate and inositol hexakisphosphate production in rat-1 cells.
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.
Fujii
Makoto
M
Departments of Pharmacology and Cancer Biology and of Biochemistry, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.
York
John D
JD
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003260:Contact Inhibition; D020869:Gene Expression Profiling; D005816:Genetic Complementation Test; D007295:Inositol Phosphates / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010833:Phytic Acid / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats
D007295:Inositol Phosphates; D034741:RNA, Small Interfering; D010833:Phytic Acid; C496699:Ipmk protein, rat; C496698:Ippk protein, rat; D017853:Phosphotransferases (Alcohol Group Acceptor); D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase