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

Xiaonian Yang, Marco Rudolf, Mark A. Carew, Masako Yoshida, Volkmar Nerreter, Andrew M. Riley, Sung Kee Chung, Karol S. Bruzik, Barry V.L. Potter, Carsten Schultz, Stephen B. Shears

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43 Scopus citations

Abstract

Ca2+-activated Cl- channels are inhibited by inositol 3,4,5,6- tetrakisphosphate (Ins(3,4,5,6)P4) (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 stimulus- dependent activation of phospholipase C (PLC). In this study, we show that inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) is the specific signal that ties increased cellular levels of Ins(3,4,5,6)P4 to changes in PLC activity. We first demonstrated that Ins(1,3,4)P3 inhibited Ins(3,4,5,6)P4 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 [3H]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)P3 2.7-fold, while [3H]Ins(3,4,5,6)P4 levels increased 2-fold; there were no changes to levels of other all-labeled inositol phosphates. This experiment provides the first direct evidence that levels of Ins(3,4,5,6)P4 are regulated by Ins(1,3,4)P3 in vivo, independently of Ins(1,3,4)P3 being metabolized to Ins(3,4,5,6)P4. In addition, we found that the Ins(1,3,4)P3 metabolites, namely Ins(1,3)P2 and Ins(3,4)P2, were > 100-fold weaker inhibitors of the 1-kinase compared with Ins(1,3,4)P3 itself (IC50 = 0.17 μM). This result shows that dephosphorylation of Ins(1,3,4)P3 in vivo is an efficient mechanism to 'switch-off' the cellular regulation of Ins(3,4,5,6)P4 levels that comes from Ins(1,3,4)P3-mediated inhibition of the 1 -kinase. We also found that Ins(1,3,6)P3 and Ins(1,4,6)P3 were poor inhibitors of the 1- kinase (IC50 = 17 and >30 μM, respectively). The non-physiological trisphosphates, D/L-Ins(1,2,4)P3, inhibited 1-kinase relatively potently (IC50 = 0.7 μM), 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)P3 acts in an important signaling cascade.

Original languageEnglish (US)
Pages (from-to)18973-18980
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number27
DOIs
StatePublished - Jul 2 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Yang, X., Rudolf, M., Carew, M. A., Yoshida, M., Nerreter, V., Riley, A. M., Chung, S. K., Bruzik, K. S., Potter, B. V. L., Schultz, C., & Shears, S. B. (1999). Inositol 1,3,4-trisphosphate acts in vivo as a specific regulator of cellular signaling by inositol 3,4,5,6-tetrakisphosphate. Journal of Biological Chemistry, 274(27), 18973-18980. https://doi.org/10.1074/jbc.274.27.18973