Open AccessPhosphorylation of Inositol 1,4,5–Trisphosphate Analogues by 3‐Kinase and Dephosphorylation of Inositol 1,3,4,5‐Tetrakisphosphate Analogues by 5‐Phosphatase
Author(s) -
Duken Peter,
Lammers Aleida A.,
Ozaki Shoichiro,
Potter Barry V. L.,
Erneux Christophe,
Haastert Peter J. M.
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb20081.x
Subject(s) - dephosphorylation , thiophosphate , inositol , phosphatase , phosphorylation , chemistry , inositol phosphate , phosphate , kinase , stereochemistry , biochemistry , receptor , organic chemistry
A series of 32 P‐labeled d ‐ myo ‐inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5) P 4 ] analogues was enzymically prepared from the corresponding d ‐ myo –inositol 1,4,5‐trisphosphate [Ins(1,4,5) P 3 ] analogues using recombinant rat brain Ins(1,4,5)P 3 3‐kinase and [γ‐ 32 P]ATP. Ins(1,4,5) P 3 analogues with bulky groups at the 2‐OH position, substitutions of phosphates by thiophosphates and d ‐6‐deoxy‐ myo ‐Ins(1,4,5) P 3 were tested. Using [ 3 H]Ins(1,4,5) P 3 and ATPγS, a [ 3 H]Ins(1,3,4,5) P 4 analogue with a thiophosphate at the D‐3 position was prepared. The D‐4 and/or D‐5 phosphate group seemed to be important for 3‐kinase activity, while the OH group at position 6 was not crucial. The addition of bulky groups at the 2‐OH position did not prevent phosphorylation. The labeled Ins(1,3,4,5) P 4 analogues were purified and their degradation by type‐I Ins(1,4,5) P 3 /Ins(1,3,4,5) P 4 5‐phosphatase was compared with the degradation of Ins(1,3,4,5) P 4 . Substitution of the phosphate group at positions 1 or 3 by a thiophosphate, or the addition of bulky groups at the 2‐OH position did not prevent degradation. d ‐6–Deoxy‐ myo ‐inositol 1,3,4,5‐tetrakisphosphate could not be degraded by the 5‐phosphatase, indicating the importance of the 6‐OH group for 5‐phosphatase action. d ‐6‐Deoxy‐ myo –inositol 1,3,4,5‐tetrakisphosphate could be an important tool in elucidating the cellular functions of Ins(1,3,4,5)P 4