Inositol 1,3,4-trisphosphate acts in vivo as a specific regulator of cellular signaling by inositol 3,4,5,6-tetrakisphosphate

Ca2+-activated Cl- channels are inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P-4) (Xie, W. Haetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears, S. B., and Nelson, D. J. (1996) J. Biol. Chem. 271, 14092-14097), a novel second messenger that is formed after stimulus-dependent activation of phospholipase C (PLC). In this study, we show that inositol 1,3,4-trisphosphate (Ins(1,3,4)P-3) is the specific signal that ties increased cellular levels of Ins(3,4,5,6)P-4 to changes in PLC activity. We first demonstrated that Ins(1,3,4)P-3 inhibited Ins(3,4,5,6)P-4 1-kinase activity that was either (i) in lysates of AR4-2J pancreatoma cells or (ii) purified 22,500-fold (yield = 13%) from bovine aorta. Next, we incubated [H-3]inositol-labeled AR4-2J cells with cell permeant and non-radiolabeled 2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate-hexakis(acetoxymethyl) ester. This treatment increased cellular levels of Ins(1,3,4)P-3 2.7-fold, while [H-3]Ins(3,4,5,6)P-4 levels increased a-fold; there were no changes to levels of other H-3-labeled inositol phosphates. This experiment provides the first direct evidence that levels of Ins(3,4,5,6)P-4 are regulated by Ins(1,3,4)P-3 in vivo, independently of Ins(1,3,4)P-3 being metabolized to Ins(3,4,5,6)P-4. In addition, we found that the Ins(1,3,4)P-3 metabolites, namely Ins(1,3)P-2 and Ins(3,4)P-2, were >100-fold weaker inhibitors of the 1-kinase compared with Ins(1,3,4)P-3 itself (IC50 = 0.17 mu M). This result shows that dephosphorylation of Ins(1,3,4)P-3 in vivo is an efficient mechanism to "switch-off" the cellular regulation of Ins(3,4,5,6)P-4 levels that comes from Ins(1,3,4)P-3-mediated inhibition of the 1-kinase. We also found that Ins(1,3,6)P-3 and Ins(1,4,6)P-3 were poor inhibitors of the 1-kinase (IC50 = 17 and >30 mu M, respectively). The non-physiological trisphosphates, D/L-Ins(1,2,4)P-3, inhibited 1-kinase relatively potently (IC50 = 0.7 mu M), thereby suggesting a new strategy for the rational design of therapeutically useful kinase inhibitors. Overall, our data provide new information to support the idea that Ins(1,3,4)P-3 acts in an important signaling cascade.