NUCLEUS-ASSOCIATED PHOSPHORYLATION OF INS(1,4,5)P-3 TO INSP(6) IN DICTYOSTELIUM

Although many cells contain large amounts of InsP(6), its metabolism a nd function is still largely unknown. In Dictyostelium lysates, the fo rmation 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 po tential substrate or intermediate for InsP(3) formation. However, we o bserved that mutant cells labelled in vivo with [H-3]inositol showed a ltered 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 de scribed 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-positi ons. 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 significan tly more P-32 radioactivity than the 4- or 5-positions, indicating tha t the 6-position is phosphorylated after the other two positions. The results from these in vivo and in vitro experiments demonstrate a meta bolic 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 frac tion of Dictyostelium cells.