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

Research output: Contribution to journalArticle

43 Citations (Scopus)

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
Externally publishedYes

Fingerprint

Cell signaling
Phosphotransferases
Inhibitory Concentration 50
Type C Phospholipases
Inositol Phosphates
Second Messenger Systems
Inositol
Metabolites
inositol 1,3,4-trisphosphate
inositol-3,4,5,6-tetrakisphosphate
Aorta
Esters
Chemical activation
Switches

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Inositol 1,3,4-trisphosphate acts in vivo as a specific regulator of cellular signaling by inositol 3,4,5,6-tetrakisphosphate. / Yang, Xiaonian; Rudolf, Marco; Carew, Mark A.; Yoshida, Masako; Nerreter, Volkmar; Riley, Andrew M.; Chung, Sung Kee; Bruzik, Karol S.; Potter, Barry V L; Schultz, Carsten; Shears, Stephen B.

In: Journal of Biological Chemistry, Vol. 274, No. 27, 02.07.1999, p. 18973-18980.

Research output: Contribution to journalArticle

Yang, X, Rudolf, M, Carew, MA, Yoshida, M, Nerreter, V, Riley, AM, Chung, SK, Bruzik, KS, Potter, BVL, Schultz, C & Shears, SB 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, vol. 274, no. 27, pp. 18973-18980. https://doi.org/10.1074/jbc.274.27.18973
Yang, Xiaonian ; Rudolf, Marco ; Carew, Mark A. ; Yoshida, Masako ; Nerreter, Volkmar ; Riley, Andrew M. ; Chung, Sung Kee ; Bruzik, Karol S. ; Potter, Barry V L ; Schultz, Carsten ; Shears, Stephen B. / Inositol 1,3,4-trisphosphate acts in vivo as a specific regulator of cellular signaling by inositol 3,4,5,6-tetrakisphosphate. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 27. pp. 18973-18980.
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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.",
author = "Xiaonian Yang and Marco Rudolf and Carew, {Mark A.} and Masako Yoshida and Volkmar Nerreter and Riley, {Andrew M.} and Chung, {Sung Kee} and Bruzik, {Karol S.} and Potter, {Barry V L} and Carsten Schultz and Shears, {Stephen B.}",
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TY - JOUR

T1 - Inositol 1,3,4-trisphosphate acts in vivo as a specific regulator of cellular signaling by inositol 3,4,5,6-tetrakisphosphate

AU - Yang, Xiaonian

AU - Rudolf, Marco

AU - Carew, Mark A.

AU - Yoshida, Masako

AU - Nerreter, Volkmar

AU - Riley, Andrew M.

AU - Chung, Sung Kee

AU - Bruzik, Karol S.

AU - Potter, Barry V L

AU - Schultz, Carsten

AU - Shears, Stephen B.

PY - 1999/7/2

Y1 - 1999/7/2

N2 - 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.

AB - 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.

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