Inositol 1,3,4-Trisphosphate Acts in Vivo as a Specific Regulator of Cellular Signaling by Inositol 3,4,5,6-Tetrakisphosphate

Abstract

Ca2؉ -activated Cl ؊ channels are inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P 4 ) (Xie, W., Kaetzel, 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 stimulusdependent 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 [ 3 H]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 [ 3 H]Ins(3,4,5,6)P 4 levels increased 2-fold; there were no changes to levels of other 3 H-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 (IC 50 ‫؍‬ 0.17 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 (IC 50 ‫؍‬ 17 and >30 M, respectively). The non-physiological trisphosphates, D/LIns(1,2,4)P 3 , inhibited 1-kinase relatively potently (IC 50 ‫؍‬ 0.7 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.

Integration of Inositol Phosphate Signaling Pathways via Human ITPK1

“…The 3 10 4 helix also offers several extra interactions with the bound nucleotide that are not conserved in eITPK1. These include a bond from Ser-236 to the ADP-ribose o3 hydroxyl (2.7 Å, Fig.…”

“…hITPK1 Ser-232 makes a tight polar contact with the ␤-phosphate (2.5 Å) and also approaches the side chain of His-167 (3.4 Å). Note also that the helix 3 10 4 in hITPK1 aligns with a sequence in gmITPK4 that contains a proline residue (Fig. 4C), which would be expected to disrupt the intra-helix hydrogen bonding and geometry.…”

“…4B). The two crystal forms (Table 1) each contain two unique hITPK1 molecules in the asymmetric unit, and in each crystal form these molecules vary in the degree to which helix 3 10 4 encloses the nucleotide, indicating flexibility within this motif. In the conformation in which the nucleotide is most enveloped, the side chain of His-233 is able to reach hydrogen bonding distance to the AMPPNP ␥-phosphate, although the equivalent distance is 6 Å in the second molecule, beyond interacting range.…”

“…5). The hITPK1 inositol phosphate binding site is composed of residues from helices ␣1, ␣3, and 3 10 4, Arg-212 from sheet ␤11, and loop regions between ␤14-␣9 and ␤7-␤8 (Fig. 4, A and C).…”

“…Ins (3,4,5,6)P 4 is formed in a metabolic cycle with Ins(1,3,4,5,6)P 5 , whereas Ins(1,3,4)P 3 is formed by a kinase/phosphatase pathway as a consequence of receptordependent phospholipase C activity. The metabolic coupling of these two distinct pathways has been demonstrated in cells (10).…”