PIP5Ks interact with anionic phospholipids. (a) Colocalization of PIP5K isoforms with the negative surface charge marker, R-Pre. (b) RAW cells were cotransfected with a kinase-deficient GFP–PIP5K-β and RFP-PH–PLC-δ without or with a construct encoding the 5-phosphatase domain of synaptojanin2 targeted to the membrane by attachment of a C-terminal CaaX box (Sj2-CaaX). (c) Quantification of the membrane association of GFP–PIP5K-β, kinase-deficient GFP–PIP5K-β, and RFP-PH–PLC-δ in the presence or absence of Sj2-CaaX. Data are means ± SEM (n ≥ 25); *, P < 0.001. (d) The amino acid sequence of wild-type (WT) PIP5K-α from residues 410–464. The residues replaced in mutants A and B are highlighted in bold type with asterisks. (e) RAW cells were transiently cotransfected with GFP chimeras of with either wild-type PIP5K-α (left), mutant A (middle), or mutant B and PM-RFP (right). The distribution of the fluorescent proteins was analyzed by spinning-disc confocal microscopy, and representative images acquired near the middle of the cell are illustrated. Bars, 3 µm.