PI(3,4,5)P3 potentiates phospholipase C-beta activity

Abstract

Phospholipase C-beta (PLC-beta) isozymes are key effectors in G protein-coupled signaling pathways. Previously, we showed that PLC-beta1 and PLC-beta3 bound immobilized PIP(3). In this study, PIP(3) was found to potentiate Ca(2+)-stimulated PLC-beta activities using an in vitro reconstitution assay. LY294002, a specific PI 3-kinase inhibitor, significantly inhibited 10 min of agonist-stimulated total IP accumulation. Both LY294002 and wortmannin inhibited 90 sec of agonist-stimulated IP(3) accumulation in intact cells. Moreover, transfected p110CAAX, a constitutively activated PI 3-kinase catalytic subunit, increased 90 sec of oxytocin-stimulated IP(3) accumulation. Receptor-ligand binding assays indicated that LY294002 did not affect G protein-coupled receptors directly, suggesting a physiological role for PIP(3) in directly potentiating PLC-beta activity. When coexpressed with p110CAAX, fluorescence-tagged PLC-beta3 was increasingly localized to the plasma membrane. Additional observations suggest that the PH domain of PLC-beta is not important for p110CAAX-induced membrane association.

Figure 1. PIP₃ increases Ca²⁺ stimulated, but not Gα₁₁ or Gβγ stimulated, PLC-β activity in vitro

Reconstitution PIP₂ hydrolysis assay was performed as described in materials and methods. A, Stimulation of PLC-β3 (35 ng) activity by 50 ng Gα₁₁ or GTPγS-activated Gα₁₁ (Gα₁₁ + GTPγS) was quantitated in the presence of 15 μM PI(4,5)P₂ (white bar) or 15 μM PI(4,5)P₂ plus 5 μM PIP₃ (gray bar). B, Stimulation of PLC-β3 (35 ng) activity by 0.15 μM free Ca²⁺ (Ca²⁺ buffer) or 60 ng Gβ₁γ₂ (Gβγ) was quantitated in the presence of 15 μM PI(4,5)P₂ (white bar) or 15 μM PI(4,5)P₂ plus 5 μM PIP₃ (gray bar). C, Stimulation of PLC-β3 (35 ng) activity by 0.15 μM free Ca²⁺ (Ca²⁺ buffer) or 60 ng Gβ₁γ₂ (Gβγ) was quantitated in the presence of 15 μM PI(4,5)P₂ (white bar) or 15 μM PI(4,5)P₂ plus 5 μM PI(3,5)P₂ (gray bar). D, Ca²⁺-stimulated PLC-β1 (12 ng), PLC-β2 (40 ng) or PLC-β3 (35 ng) activity was quantitated in the presence of 15 μM PI(4,5)P₂ (white bar) or 15 μM PI(4,5)P₂ plus 5 μM PIP₃ (gray bar). Results are mean ± S.D for triplicate determination and are representative of two experiments. ** indicates p < 0.01 and *** indicates p < 0.001 compared with controls.

Figure 2. LY294002 inhibits agonist-stimulated IPn accumulation in whole cells

A, 1321N1 cells were pre-treated with 0.1% DMSO vehicle (white bars) or two concentrations of LY294002, 10 μM (grey bars) or 50 μM (black bars), for 30 minutes followed by stimulation with 1 mM Carbachol (Carbachol) or DMEM vehicle (Basal) for 10 minutes. Cells were lysed and total inositol phosphates (IP) were extracted and quantitated as described in materials and methods. B, Transfected HEK 293 cells stably expressing human oxytocin receptor were pre-treated with 0.1% DMSO vehicle (white bars) or 50 μM LY294002 (grey bars) for 30 minutes followed by stimulation with 100 nM oxcytocin (OT) or DMEM vehicle (Basal) for 10 minutes. Cells were lysed and total inositol phosphates (IP) were quantitated. Results are mean ± S.D for triplicate determination and *** indicates p < 0.001 compared with DMSO controls. C, 1321N1 cells were pretreated with 0.1% DMSO vehicle (DM, lanes 1, 3 and 4) or 50 μM LY294002 (LY, lanes 2, 5 and 6) in Kreb's solution for 30 min followed by stimulation with 1mM carbachol for 10 minutes (Carb, lanes 3-6). Cell lysates were separated by SDS-PAGE and phospho-Akt (top panel) or β-actin (bottom panel) was detected by Western blot with selective antibodies.

Figure 3. PI 3-kinase inhibitors inhibit agonist-stimulated IP₃ accumulation in whole cells

A, 1321N1 cells were pre-treated with 0.1% DMSO vehicle (white bars) or 50 μM LY294002 (grey bars) for 30 minutes followed by stimulation with 1 mM Carbachol or DMEM vehicle (Basal) for 90 seconds. Cells were lysed and total inositol trisphosphate (IP₃) was extracted and quantitated as described materials and methods. B, Transfected HEK 293 cells stably expressing human oxytocin receptor were pre-treated with 0.1% DMSO vehicle (white bars) or 50 μM LY294002 (grey bars) for 30 minutes followed by stimulation with 100 nM oxcytocin (OT) or DMEM vehicle (Basal) for 90 seconds. Cells were lysed and total inositol trisphosphate (IP₃) was quantitated. C, Transfected HEK 293 cells stably expressing human oxytocin receptor were pre-treated with 0.05% DMSO vehicle (white bars) or 0.5 μM wortmannin (grey bars) for 30 minutes followed by stimulation with 100 nM oxcytocin (OT) or DMEM vehicle (Basal) for 90 seconds. Cells were lysed and total inositol trisphosphate (IP₃) was quantitated. Results are mean ± S.D for triplicates. *** indicates p < 0.001 and ** indicates p < 0.01 compared with DMSO controls.

Figure 4. p110CAAX, constitutively activated PI 3-kinase, potentiates agonist-stimulated IP₃ accumulation in whole cells

A, HEK 293 cells stably expressing human oxytocin receptor were transfected with vehicle control (Mock) or p110CAAX (PI3K*) for 48 hours followed by stimulation with 100 nM oxcytocin (OT) or DMEM vehicle (Basal) for 90 seconds. Cells were lysed and total inositol trisphosphate (IP₃) was quantitated. Results are mean ± S.D for triplicate determination and * indicates p < 0.05 compared with DMSO controls. B, HEK 293 cells were transfected with vehicle control (Mock, lanes 1 and 2) or p110CAAX (PI3K*, lanes 3 and 4) for 48 hours and cell lysates were separated by SDS-PAGE. Phospho-Akt (top panel) or β-actin (bottom panel) was detected by Western blot with selective antibodies.

Figure 5. p110CAAX, constitutively activated PI 3-kinase, increases PLC-β3 membrane association

A, HEK 293 cells were transiently transfected with GFP tagged Akt-PH domain (Akt-PH) or GFP tagged Akt-PH R25C mutant (Akt-PH (R25C)). B, HEK 293 cells were co-transfected with GFP tagged PLC-β3 plus mock vector (β3+Mock) or GFP tagged PLC-β3 plus p110CAAX (β3+PI3K*). After transfected 48 hours, cells were fixed and GFP fluorescence was detected at 488 nm by confocal microscopy. Scale bar is 5 μm.

Figure 6. PLC-β PH domain localization to cytosol is not affected by p110CAAX-over-expression

HEK 293 cells were transiently transfected with GFP tagged PLC-β1 PH domain (PLC-β1 PH) or GFP tagged PLC-β3 PH domain (PLC-β3 PH). After transfected 48 hours, cells were fixed and GFP fluorescence was detected at 488 nm by confocal microscopy. Scale bar is 2 μm.