A molecular basis for inositol polyphosphate synthesis in Drosophila melanogaster.
Metabolism of inositol 1,4,5-trisphosphate (I(1,4,5)P3) results in the production of diverse arrays of inositol polyphosphates (IPs), such as IP4, IP5, IP6) and PP-IP5. Insights into their synthesis in metazoans are reported here through molecular studies in the fruit fly, Drosophila melanogaster. Two I(1,4,5)P3 kinase gene products are implicated in initiating catabolism of these important IP regulators. We find dmIpk2 is a nucleocytoplasmic 6-/3-kinase that converts I(1,4,5)P3 to I(1,3,4,5,6)P5, and harbors 5-kinase activity toward I(1,3,4,6)P4, and dmIP3K is a 3-kinase that converts I(1,4,5)P3 to I(1,3,4,5)P4. To assess their relative roles in the cellular production of IPs we utilized complementation analysis, RNA interference, and overexpression studies. Heterologous expression of dmIpk2, but not dmIP3K, in ipk2 mutant yeast recapitulates phospholipase C-dependent cellular synthesis of IP6. Knockdown of dmIpk2 in Drosophila S2 cells and transgenic flies results in a significant reduction of IP6 levels; whereas depletion of dmIP3K, either alpha or beta isoforms or both, does not decrease IP6 synthesis but instead increases its production, possibly by expanding I(1,4,5)P3 pools. Similarly, knockdown of an I(1,4,5)P3 5-phosphatase results in significant increase in dmIpk2/dmIpk1-dependent IP6 synthesis. IP6 production depends on the I(1,3,4,5,6)P5 2-kinase activity of dmIpk1 and is increased in transgenic flies overexpressing dmIpk2. Our studies reveal that phosphatase and kinase regulation of I(1,4,5)P3 metabolic pools directly impinge on higher IP synthesis, and that the major route of IP6 synthesis depends on the activities of dmIpk2 and dmIpk1, but not dmIP3K, thereby challenging the role of IP3K in the genesis of higher IP messengers.
Department of Pharmacology, Howard Hughes Medical Institute, Duke University Medical Center, DUMC 3813, Durham, North Carolina 27710, USA.
Andrew M
AM
Seeds
Joshua C
JC
Sandquist
Eric P
EP
Spana
John D
JD
York
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D029721:Drosophila Proteins / Q000737:chemistry*; D004331:Drosophila melanogaster / Q000378:metabolism; D020224:Expressed Sequence Tags; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis* / Q000378:metabolism; D007295:Inositol Phosphates / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008957:Models, Genetic; D008969:Molecular Sequence Data; D005810:Multigene Family; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry*; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012469:Salivary Glands / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors; D019076:Transgenes
D029721:Drosophila Proteins; D007295:Inositol Phosphates; D011994:Recombinant Proteins; D015544:Inositol 1,4,5-Trisphosphate; D011494:Protein Kinases; C494447:Ipk1 protein, Drosophila; C494446:Ipk2 protein, Drosophila; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase