Phosphorylation of inositol 1,4,5-trisphosphate analogues by 3-kinase and dephosphorylation of inositol 1,3,4,5-tetrakisphosphate analogues by 5-phosphatase.
A series of 32P-labeled D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] analogues was enzymically prepared from the corresponding D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] analogues using recombinant rat brain Ins(1,4,5)P3 3-kinase and [gamma-32P]ATP. Ins(1,4,5)P3 analogues with bulky groups at the 2-OH position, substitutions of phosphates by thiophosphates and D-6-deoxy-myo-Ins(1,4,5)P3 were tested. Using [3H]Ins(1,4,5)P3 and ATP gamma S, a [3H]Ins(1,3,4,5)P4 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)P4 analogues were purified and their degradation by type-I Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase was compared with the degradation of Ins(1,3,4,5)P4. 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)P4.
Van Dijken
P
P
Department of Biochemistry, University of Groningen, The Netherlands.
Lammers
A A
AA
Ozaki
S
S
Potter
B V
BV
Erneux
C
C
Van Haastert
P J
PJ
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology; D015544:Inositol 1,4,5-Trisphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism*; D000072181:Inositol Polyphosphate 5-Phosphatases; D015394:Molecular Structure; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007295:Inositol Phosphates; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; C081655:inositol-1,3,4,5-tetrakisphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C066722:myo-inositol D-3 hydroxykinase; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases