Nucleus-associated phosphorylation of Ins(1,4,5)P3 to InsP6 in Dictyostelium

Although many cells contain large amounts of InsP(6), its metabolism and function is still largely unknown. In Dictyostelium lysates, the formation of InsP(6) by sequential phosphorylation of inositol via Ins(3,4,6)P-3 has been described [Stevens and Irvine (1990) Nature (London) 346, 580-583]; the second messenger Ins(1,4,5)P, was excluded as a potential substrate or intermediate for InsP(3) formation. However, we observed that mutant cells labelled in vivo with [H-3]inositol showed altered labelling of both [H-3]Ins(1,4, 5)P-3 and [H-3]InsP(6). In this report we demonstrate that Ins(1,4,5)P-3 is converted into InsP(6) in vitro by nucleus-associated enzymes, in addition to the previously described stepwise phosphorylation of inositol to InsP(6) that occurs in the cytosol. HPLC analysis indicates that Ins(1,4,5)P-3 is converted into InsP(6) via sequential phosphorylation at the 3-, 6- and 2-positions. Ins[P-32]P-6, isolated from cells briefly labelled with [P-32]P-i, was analysed using Paramecium phytase, which removes the phosphates of InsP(6) in a specific sequence. The 6-position contained significantly more P-32 radioactivity than the 4- or 5-positions, indicating that the 6-position is phosphorylated after the other two positions. The results from these in vivo and in vitro experiments demonstrate a metabolic route involving the phosphorylation of Ins(1,4,5)P-3 via Ins(1,3,4,5)P-4 and Ins(1,3,4,5,6)P-5 to InsP(6) in a nucleus-associated fraction of Dictyostelium cells.