Phosphatidylinositol-4-phosphate 5-kinase isoforms exhibit acyl chain selectivity for both substrate and lipid activator

Abstract

Phosphatidylinositol 4,5-bisphosphate is mostly produced in the cell by phosphatidylinositol-4-phosphate 5-kinases (PIP5K) and has a crucial role in numerous signaling events. Here we demonstrate that in vitro all three isoforms of PIP5K, α, β, and γ, discriminate among substrates with different acyl chains for both the substrates phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) although to different extents, with isoform γ being the most selective. Fully saturated dipalmitoyl-PtdIns4P was a poor substrate for all three isoforms, but both the 1-stearoyl-2-arachidonoyl and the 1-stearoyl-2-oleoyl forms of PtdIns4P were good substrates. V(max) was greater for the 1-stearoyl-2-arachidonoyl form compared with the 1-stearoyl-2-oleoyl form, although for PIP5Kβ the difference was small. For the α and γ isoforms, K(m) was much lower for 1-stearoyl-2-oleoyl PtdIns4P, making this lipid the better substrate of the two under most conditions. Activation of PIP5K by phosphatidic acid is also acyl chain-dependent. Species of phosphatidic acid with two unsaturated acyl chains are much better activators of PIP5K than those containing one saturated and one unsaturated acyl chain. PtdIns is a poor substrate for PIP5K, but it also shows acyl chain selectivity. Curiously, there is no acyl chain discrimination among species of phosphatidic acid in the activation of the phosphorylation of PtdIns. Together, our findings indicate that PIP5K isoforms α, β, and γ act selectively on substrates and activators with different acyl chains. This could be a tightly regulated mechanism of producing physiologically active unsaturated phosphatidylinositol 4,5-bisphosphate species in the cell.

FIGURE 1.

HA-PIP5K isoforms α, β, and γ show sensitivity to the acyl chain composition of PtdIns4P substrate. A–C, comparison of PIP5K activities with SA-, SO-, and DP-PtdIns4P at low substrate concentrations (total substrate concentration = 20 μm, equal to C_eff = 0.23 μm). The effective surface concentration (C_eff) of the substrate was calculated by multiplying the molar fraction of the substrate at the surface of the micelle by the total concentration of the substrate (28). D–F, comparison of PIP5K activities with SA-, and SO-PtdIns4P over a wide range of substrate concentrations (C_eff from 0.015 to 7.91 μm). Error bars, S.D.

FIGURE 2.

Activation of HA-PIP5K isoforms α, β, and γ by different PAs with SA-PtdIns4P (A–C), SO-PtdIns4P (D–F), and DP-PtdIns4P (G–I) as substrates. PIP5K enzymatic activity was measured with 10 μm (equal to C_eff = 0.06 μm) PtdIns4P and 50 μm (equal to C_eff = 1.42 μm) PA. Error bars, S.D.

FIGURE 3.

PIP5Kα has a strong preference for PtdIns4P as a substrate over PtdIns. PIP5K enzymatic activity was measured with either 20 μm SA-PtdIns4P, 20 μm SA-PtdIns (equal to C_eff = 0.23 μm), or 800 μm (equal to C_eff = 256 μm) SA-PtdIns. Error bars, S.D.

FIGURE 4.

HA-PIP5K isoforms α (A), β (B), and γ (C) show sensitivity to the acyl chain composition of PtdIns substrate. PIP5K enzymatic activity was measured with 700 μm (equal to C_eff = 204 μm) PtdIns. Error bars, S.D.

FIGURE 5.

HA-PIP5Kγ does not discriminate between different acyl chains of PA when either SA-PtdIns (A), SO-PtdIns (B), SL-PtdIns (C), or DL-PtdIns (D) is used as a substrate. PIP5K enzymatic activity was measured with 600 μm (equal to C_eff = 150 μm) PtdIns and 100 μm (equal to C_eff = 4.1 μm) PA. Error bars, S.D.

FIGURE 6.

Mutations L202I and L210I of c-Myc-PIP5Kα increase enzyme activation fold by DAPA. PIP5K enzymatic activity was measured with 10 μm (equal to C_eff = 0.06 μm) PtdIns4P and 50 μm (equal to C_eff = 1.42 μm) PA. Error bars, S.D.

FIGURE 7.

D322A mutant of FLAG-PIP5Kα is activated by DAPA. PIP5K enzymatic activity was measured with 30 μm (equal to C_eff = 0.5 μm) PtdIns4P and 60 μm (equal to C_eff = 2 μm) PA. Error bars, S.D.

FIGURE 8.

A and B, comparison of DAPA activation of PIP5Kα in a state of WT/WT homodimer, D322A/D322A homodimer, and WT/D322A heterodimer with either SO-PtdIns4P (A) or DP-PtdIns4P (B) used as substrate. C, comparison of enzyme activity ratios with SO- to DP-PtdIns4P used as substrates for PIP5Kα in a state of WT/WT homodimer, D322A/D322A homodimer, and WT/D322A heterodimer. Error bars, S.D.