Xn. Yang et al., Inositol 1,3,4-trisphosphate acts in vivo as a specific regulator of cellular signaling by inositol 3,4,5,6-tetrakisphosphate, J BIOL CHEM, 274(27), 1999, pp. 18973-18980
Ca2+-activated Cl- channels are inhibited by inositol 3,4,5,6-tetrakisphosp
hate (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 activati
on of phospholipase C (PLC). In this study, we show that inositol 1,3,4-tri
sphosphate (Ins(1,3,4)P-3) is the specific signal that ties increased cellu
lar levels of Ins(3,4,5,6)P-4 to changes in PLC activity. We first demonstr
ated that Ins(1,3,4)P-3 inhibited Ins(3,4,5,6)P-4 1-kinase activity that wa
s 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-lab
eled AR4-2J cells with cell permeant and non-radiolabeled 2,5,6-tri-O-butyr
yl-myo-inositol 1,3,4-trisphosphate-hexakis(acetoxymethyl) ester. This trea
tment 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 othe
r H-3-labeled inositol phosphates. This experiment provides the first direc
t 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 compar
ed with Ins(1,3,4)P-3 itself (IC50 = 0.17 mu M). This result shows that dep
hosphorylation of Ins(1,3,4)P-3 in vivo is an efficient mechanism to "switc
h-off" the cellular regulation of Ins(3,4,5,6)P-4 levels that comes from In
s(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 an
d >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 th
e idea that Ins(1,3,4)P-3 acts in an important signaling cascade.