Fig 2: PIP4K Inhibits PIP5K Activity(A) Changes in PIP5K activity in 293T cells. Cell pellets were normalized to cell number and sonicated in the presence of excess PI(4)P. Radioactive ATP32 was added for kinase reaction. Lipids were extracted and quantified by TLC, with confirmation by HPLC after deacylation.(B–D) Quantification of in vitro kinase assay measuring conversion of PI(4)P to radiolabeled PI(4,5)P2 by PIP5K1A. PIP5K1A activity is inhibited in vitro by addition of 2- to 10-fold molar excess of PIP4K2A, PIP4K2B, or PIP4K2C (B). Once denatured, PIP4K2C no longer inhibits PIP5K1A (C). In the presence of 0.1% Triton X-100, PIP4K2A no longer inhibits PIP5K1A activity (D).(E) Western blot validation of near-endogenous level expression of 3xHA-PIP5K1A levels in 293T control and TKO cells. Tagged 3xHA-PIP5K1A is slightly larger than the endogenous protein.(F) Pull-down using anti-HA beads from 293T WT cells expressing empty 3xHA vector (empty) or 3xHA-PIP5K1A (PIP5K1A). Western blot of input (first two lanes) and immunoprecipitation of endogenous PIP4K2A with PIP5K1A (second two lanes).(G and H) 293T lysates were incubated with beads pre-incubated with either GST or GST-PIP4K2C, used as bait. Pull-downs analyzed by western blot strongly to detect the enrichment of PIP5K1A and PIP5K1C(G). Analysis of flow-through reflects the extent to which lysate is depleted of PIP5K1A and PIP5K1C by bait (H).(I) Sequence homology of N-terminal region in PIP4K. Mutated residues of PIP4K2C aa69–75 are indicated in red.(J) 293T lysates were incubated with beads conjugated to GST-PIP4K2C or GST-PIP4K2CVD, used as bait. Bead pull-downs were analyzed by western blot for the presence of PIP5K1A, which is able to bind WT PIP4K2C but not PIP4K2CVD.(K) Quantification of in vitro kinase assay measuring the conversion of PI(4)P to radiolabeled PI(4,5)P2 by PIP5K1A, which is inhibited by PIP4K2C but not PIP4K2CVD.(L and M) Quantification by HPLC of PI(4)P (L) and PI(4,5)P2 (M) in HEK293T TKO cell lines rescued with PIP4K2C and PIP4K2CVD.Significance calculated using ANOVA with Holm-Sidak multiple comparisons to control cell line. **p < 0.01, ****p < 0.0001, and n.s., not significant. Data are represented as means ± SEMs. n = 3.

Fig 3: Validation of Tools to Eliminate All Three PIP4K Isoforms Reveals Paradoxical Increase in PI(4,5)P2(A) Western blots showing the efficiency of knockdown of PIP4K isoforms in HeLa cells. Cell line notation and their descriptions are listed in Table S2.(B) Western blots showing 293T clones with CRISPR-mediated knockout of PIP4K2A, PIP4K2B, and PIP4K2C, with cell line descriptions in Table S2. Parental cell line is in the first lane, and the CRISPR clonal cell line with intact PIP4K is designed as WT. A faint non-specific band that runs slightly faster than PIP4Ks is just visible.(C and D) Quantification by HPLC of PI(5)P, which increases in cells with the loss of PIP4K, shown in HeLa TKD cells (C) as well as 293T TKO cells (D).(E) Knock down of PIP4K enhances insulin signaling in HeLa cells, as shown by western blot. Quantification by Li-Cor indicated the respective bands below.(F) Immunofluorescence detection of lysosomal marker Lamp1 showing increased lysosomal accumulation in 293T TKO versus control cells. Representative images shown.(G and H) PI(4,5)P2 increases in cells with loss of PIP4K, shown in HeLa TKD cells (G) and 293T TKO cells (H), measured by HPLC.(I and J) PI(4)P decreases in cells with loss of PIP4K, shown in HeLa TKD cells (I) and 293T TKO cells (J), measured by HPLC.(K) 293T TKO cells have higher levels of PI(3,4,5)P3, as measured by HPLC.Significance calculated using ANOVA with Holm-Sidak multiple comparisons to control cell line with intact PIP4K. **p < 0.01, ***p < 0.001, and ****p < 0.0001. Data are represented as means ± SEMs. n = 3.

Fig 4: Expression of PIP4K2A, PIP4K2B, and PIP4K2C in normal and malignant hematopoietic cells. (A) Graphical legend for the myeloid differentiation hierarchy illustrating the analyzed cell subpopulations used in the analysis (https://mindthegraph.com/ (accessed on 2 August 2023)). Abbreviations: HSC, hematopoietic stem cells; CMP, common myeloid progenitor; GMP, granulocyte macrophage progenitor; MEP, megakaryocyte/erythrocyte progenitor; ERY, erythrocytes; MEGA, megakaryocytes; META, metamyelocytes; NEU, neutrophils; EOS, eosinophils; BASO, basophils; and MONO, monocytes. (B–D) Gene expression profile of PIP4K2A (probe 205570_at), PIP4K2B (probe 201080_at), and PIP4K2C (probe 218942_at) in myeloid cell subpopulations (GSE24759). The p-values and cell lineages are indicated in the graphs: * p < 0.05 cell lineage vs. HSC1, # p < 0.01 cell lineage vs. HSC2; ANOVA and Bonferroni post hoc test. (E) Schematic representation of PIP4K2A, PIP4K2B, and PIP4K2C expression in the different molecular subtypes of AML obtained from the BloodSpot Database (https://servers.binf.ku.dk/bloodspot/ (accessed on 2 August 2023)). (F) PIP4K2A, PIP4K2B, and PIP4K2C mRNA levels in normal CD34+ cells and AML cell lines. ACTB and HPRT1 were used as the reference genes, and CD34+ cells were used as a calibrator. (G) Western blot analysis of the PIP4K2A, PIP4K2B, and PIP4K2C levels in the total cell extracts from CD34+ cells and AML cell lines; the membranes were re-probed with the antibody for the detection of actin and developed with the SuperSignal™ West Dura Extended Duration Substrate system using a G: BOX Chemi XX6 gel document system.