A Novel, Phospholipase C-independent Pathway of Inositol 1,4,5-Trisphosphate Formation in Dictyostelium and Rat Liver

In an earlier study a mutant Dictyostelium cell-line (plc(-)) was constructed in which all phospholipase C activity was disrupted and nonfunctional, yet these cells had nearly normal Ins(1,4,5)P-3 levels (Drayer, A. L., Van Der Kaay, J., Mayr, G. W, Van Haastert, P. J. M. (1990) EMBO J. 13, 1601-1609). We have now investigated if these cells have a phospholipase C-independent de novo pathway of Ins(1,4,5)P-3 synthesis. We found that homogenates of plc(-) cells produce Ins(1,4,5)P-3 from endogenous precursors. The enzyme activities that performed these reactions were located in the particulate cell fraction, whereas the endogenous substrate was soluble and could be degraded by phytase. We tested various potential inositol polyphosphate precursors and found that the most efficient were Ins(1,3,4,5,6)P-3, Ins(1,3,4,5)P-4, and Ins(1,4,5,6)P-4. The utilization of Ins(1,3,4,5,6)P-5, which can be formed independently of phospholipase C by direct phosphorylation of inositol (Stephens, L. R. and Irvine, R. F. (1990) Nature 346, 580-582), provides Dictyostelium with an alternative and novel pathway of de novo Ins(1,4,5)P-3 synthesis. We further discovered that Ins(1,3,4,5,6)P-5 was converted to Ins(1,4,5)P-3 via both Ins(1,3,4,5)P-4 and Ins(1,4,5,6)P-4. In the absence of calcium no Ins(1,4,5)P-3 formation could be observed; half-maximal activity was observed at low micromolar calcium concentrations. These reaction steps could also be performed by a single enzyme purified from rat liver, namely, the multiple inositol polyphosphate phosphatase. These data indicate that organisms as diverse as rat and Dictyostelium possess enzyme activities capable of synthesizing the second messengers Ins(1,4,5)P-3 and Ins(1,3,4,5)P-4 via a novel phospholipase C-independent pathway.