Fig. 4.
Perturbation of Pikfyve activity suggests that the PI5P derives from PI(3,5)P2. (A) Inhibition of Pikfyve in mouse primary fibroblasts by 1.6 μM YM201636 for the times specified results in a rapid depletion of PI(3,5)P2 and PI5P (n = 4). (B) Stimulation of mouse fibroblasts by refeeding with normal growth medium, insulin, and transferrin following 1-h starvation in HBSS with Mg2+ and Ca2+ reveals that within 2.5 min all lipids, with the exception of PI5P, increased and plateaued at a new steady-state level. PI5P plateaued at its new steady-state level at 10 min (n = 3). (C) Expression of human PIKFYVE in wild-type yeast resulted in a large elevation in PI(3,5)P2, a small increase in PI5P, and a corresponding reduction in PI3P. (D) The sum of PI3P, PI(3,5)P2, and PI5P was the same in wild-type yeast with PIKFYVE overexpression and with vector control (n = 3). This result suggests that PI5P levels are dependent on PI3P via dephosphorylation of PI(3,5)P2.