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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U2 - 10.1074/jbc.274.27.18973
DO - 10.1074/jbc.274.27.18973
M3 - Article
C2 - 10383396
AN - SCOPUS:0033516581
SN - 0021-9258
VL - 274
SP - 18973
EP - 18980
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 27
ER -