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 correspondin g D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3] analogues using r ecombinant 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, substitutio ns 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. Th e D-4 and/or D-5 phosphate group seemed to be important for 3-kinase a ctivity, while the OH group at position 6 was not crucial. The additio n 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 degrada tion by type-I Ins(1,4,5)P-3/ Ins(1,3,4,5)P-4 5-phosphatase was compar ed with the degradation of Ins(1,3,4,5)P-4. Substitution of the phosph ate group at positions 1 or 3 by a thiophosphate, or the addition of b ulky groups at the 2-OH position did not prevent degradation. D-6-Deox y-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-phosp hatase 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.