FIGURE 6.

Effect of ITPK-induced Ins(1,3,4,5)P₄ generation on activity of Ins(1,4,5)P₃ 5-PPT in H1299 cells. A, uninhibited 5-PPT activity in control and ITPKA kd cells. Ins(1,4,5)P₃-5-PPT activity was analyzed in cell lysates from H1299 control and H1299 kd cells under inactive kinase conditions where ATP and the ATP reconstitution system were omitted from the assay buffer. Shown are the averages of four determinations. B, inhibitory effect of Ins(1,3,4,5)P₄ on Ins(1,4,5)P₃-5-PPT activity during Ins(1,4,5)P₃ transients generated with total cellular protein extracts. Ins(1,4,5)P₃-5-PPT activity was analyzed in cell lysates from control and ITPKA kd cells under active kinase conditions with and without Ca²⁺/CaM activation. From a total of eight different transients of Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄ versus time, an inhibition curve of Ins(1,4,5)P₃-5-PPT activity by Ins(1,3,4,5)P₄ was derived at a constant Ins(1,4,5)P₃ of 4.75 μm. C, transients of Ins(1,4,5)P₃ are strongly prolonged by high total cellular ITPK activity. Transients of Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄ obtained with cell extracts from control and ITPKA kd cells under active kinase conditions with and without Ca²⁺/CaM activation of ITPK are plotted against time. Data show one representative series of experiments out of three performed with different cell extracts.