PIP4Ks Suppress Insulin Signaling through a Catalytic-Independent Mechanism

Abstract

Insulin stimulates the conversion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) to phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P₃), which mediates downstream cellular responses. PI(4,5)P₂ is produced by phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and by phosphatidylinositol-5-phosphate 4-kinases (PIP4Ks). Here, we show that the loss of PIP4Ks (PIP4K2A, PIP4K2B, and PIP4K2C) in vitro results in a paradoxical increase in PI(4,5)P₂ and a concomitant increase in insulin-stimulated production of PI(3,4,5)P₃. The reintroduction of either wild-type or kinase-dead mutants of the PIP4Ks restored cellular PI(4,5)P₂ levels and insulin stimulation of the PI3K pathway, suggesting a catalytic-independent role of PIP4Ks in regulating PI(4,5)P₂ levels. These effects are explained by an increase in PIP5K activity upon the deletion of PIP4Ks, which normally suppresses PIP5K activity through a direct binding interaction mediated by the N-terminal motif VMLΦPDD of PIP4K. Our work uncovers an allosteric function of PIP4Ks in suppressing PIP5K-mediated PI(4,5)P₂ synthesis and insulin-dependent conversion to PI(3,4,5)P₃ and suggests that the pharmacological depletion of PIP4K enzymes could represent a strategy for enhancing insulin signaling.

Keywords: Akt; PI(3,4,5)P(3); PI(4,5)P(2); PI3K; PI5P4K; PIP4K; PIP5K; RTK; insulin; signaling.

Figure 1.. 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.

Figure 2.. Identification of a Catalytic-Independent Function of PIP4K

(A and B) Quantification of PI(4)P and PI(4,5)P₂ levels by HPLC upon knockdown of one or multiple isoforms of PIP4K in 293T cells (A) or HeLa cells (B) using tandem shRNA constructs. Hairpin constructs are indicated on the × axis, with descriptions in Table S2. (C and D) Western blot validation of rescue cell line panel with reconstitution of kinase-active or kinase-dead PIP4K2A^KD or kinase-active PIP4K2C into HeLa TKD cells (C) or 293T TKO cells (D) at near-endogenous levels. Tagged 3xHA-PIP4K proteins are larger than the endogenous protein. A faint non-specific band is visible running slightly faster than the endogenous protein. (E–J) Quantification by HPLC of PI(4,5)P₂ in HeLa TKD rescue cell lines (E) and 293T TKO rescue cell lines (F). Quantification of PI(4)P in HeLa TKD rescue cell lines (G) and 293T TKO rescue cell lines (H). Quantification of PI(5)P in HeLa TKD rescue cell lines (I) and 293T TKO rescue cell lines (J). Significance calculated using ANOVA with Holm-Sidak multiple comparisons to TKD or TKO cell lines. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Data are represented as means ± SEMs. n = 3.

Figure 3.. 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-PIP4K2C^VD, 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 PIP4K2C^VD. (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 PIP4K2C^VD. (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 PIP4K2C^VD. 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.

Figure 4.. Structural Role of PIP4K in Regulating PIP5K and the PI3K Pathway Is Distinct from Its Catalytic Role in Autophagy

(A) Insulin stimulation of PI(3,4,5)P₃ synthesis by PI3K in 293T rescue cell lines, quantified by HPLC. Cells were serum starved for 20 h, then stimulated with 50 ng/mL insulin for 5 min. (B) Insulin stimulation of Akt activation in HeLa rescue cell line panel. Cells were serum starved for 12 h, then stimulated with 250 ng/mL insulin for 10min. Western blot band intensities were measured by Li-Cor and are indicated under the relevant band. (C) Quantification of insulin-stimulated increase in pAkt-473in HeLa rescue cell line panel, normalized to total Akt. n = 3. (D and E) Immunofluorescence detection of lysosomal marker Lamp1 in the 293T rescue cell line panel shows dependence on catalytic activity to rescue increased lysosomal accumulation. Representative images are shown (D), and full quantification is detailed (E). (F) Quantification of TFEB target gene expression by qRT-PCR in 293T rescue cell line panel. Significance calculated using ANOVA with Holm-Sidak multiple comparisons to control cell line. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and n.s., not significant.