PHOSPHORYLATION OF INOSITOL 1,4,5-TRISPHOSPHATE ANALOGS BY 3-KINASE AND DEPHOSPHORYLATION OF INOSITOL 1,3,4,5-TETRAKISPHOSPHATE ANALOGS BY 5-PHOSPHATASE

A series of P-32-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 [gamma-P-32]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, were tested. Using [H-3]Ins(1,4,5)P-3 and ATP gamma S, a [H-3]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, 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.