Phosphatidylinositol 4-phosphate is a major source of GPCR-stimulated phosphoinositide production

Abstract

Phospholipase C (PLC) enzymes hydrolyze the plasma membrane (PM) lipid phosphatidylinositol 4,5-bisphosphate (PI4,5P₂) to generate the second messengers inositol trisphosphate (IP₃) and diacylglycerol (DAG) in response to receptor activation in almost all mammalian cells. We previously found that stimulation of G protein-coupled receptors (GPCRs) in cardiac cells leads to the PLC-dependent hydrolysis of phosphatidylinositol 4-phosphate (PI4P) at the Golgi, a process required for the activation of nuclear protein kinase D (PKD) during cardiac hypertrophy. We hypothesized that GPCR-stimulated PLC activation leading to direct PI4P hydrolysis may be a general mechanism for DAG production. We measured GPCR activation-dependent changes in PM and Golgi PI4P pools in various cells using GFP-based detection of PI4P. Stimulation with various agonists caused a time-dependent reduction in PI4P-associated, but not PI4,5P₂-associated, fluorescence at the Golgi and PM. Targeted depletion of PI4,5P₂ from the PM before GPCR stimulation had no effect on the depletion of PM or Golgi PI4P, total inositol phosphate (IP) production, or PKD activation. In contrast, acute depletion of PI4P specifically at the PM completely blocked the GPCR-dependent production of IPs and activation of PKD but did not change the abundance of PI4,5P₂ Acute depletion of Golgi PI4P had no effect on these processes. These data suggest that most of the PM PI4,5P₂ pool is not involved in GPCR-stimulated phosphoinositide hydrolysis and that PI4P at the PM is responsible for the bulk of receptor-stimulated phosphoinositide hydrolysis and DAG production.

Fig. 1.. Receptor-stimulated Golgi PI4P hydrolysis occurs in MEFs, PANC-1 cells, RASMs, and HEY cells.

(A to D) Cells were transduced with adenovirus expressing FAPP-PH-GFP. After 16 hours, the cells were serum-starved for 4 hours and then imaged. Full confocal stacks were collected at each time point to ensure that the confocal plane was maintained during the experiment. (A) Left: Representative images of MEFs expressing FAPP-PH-GFP before and after stimulation with 100 nM ET-1. Right: Time course of the changes in fluorescence of n = 4 cells for each treatment. (B) Left: Representative images of RASM cells expressing FAPP-PH-GFP before and after stimulation with 100 nM ET-1. Right: Time course of the changes in fluorescence of n = 3 cells for each treatment. (C) Time course of PI4P hydrolysis in untreated PANC-1 cells and PANC-1 cells treated with 100 nM NT. Data are combined from n = 6 cells for each treatment. (D) Time course of PI4P hydrolysis in untreated HEY cells and HEY cells treated with 1% FBS. Data are combined from n = 4 cells for each treatment. All traces are means ± SEM. Each time point on the curves for treated cells was compared to those of the control curves and was analyzed by one-tailed unpaired t test. *P < 0.05 and **P < 0.01; ns, not significant (P > 0.05).

Fig. 2.. NT stimulates PI4P depletion even after removal of the bulk of PI4,5P₂.

(A and B) PANC-1 cells were transduced with adenoviruses expressing the FKBP–5-phosphatase system and GFP-2×P4M and were imaged by epifluorescence microscopy. Cells were treated with dimethyl sulfoxide (DMSO) (vehicle) or RAPA for 15 min before being incubated with vehicle or 100 nM NT (at the times indicated by the arrow) for 25 min. (A) Representative image time courses were produced with the ImageJ montage setting with higher fluorescence represented in black. (B) Compiled data for at least 12 cells from at least three independent experiments. All traces are means ± SEM. All curves were compared using two-way analysis of variance (ANOVA) with multiple comparisons within each row. Comparisons between control (Ctrl) and RAPA treatments were not statistically significant at any time point. P values represent Ctrl compared to NT-treated (red) and RAPA compared to RAPA NT-treated (purple). *P < 0.05, **P < 0.01, *** P < 0.001, and ****P < 0.0001.

Fig. 3.. ET-1 stimulates PI4P depletion even after removal of the bulk of PI4,5P₂.

(A and B) MEFs were transduced with adenoviruses expressing the FKBP–5-phosphatase system and GFP-P4M and then were imaged by TIRF microscopy. Cells were treated with 1 μM RAPA or DMSO (vehicle) for 20 min, which was followed by the addition of 100 nM ET-1 or vehicle at the time indicated by the arrow. (A) Representative images of MEFs before and after treatment with 100 nM ET-1 with DMSO vehicle or 1 μM RAPA. (B) Time courses of PI4P depletion for combined experiments with six or seven cells analyzed for each condition in each of three independent experiments. All traces are means ± SEM. All curves are compared by two-way ANOVA with multiple comparisons within each row. P values represent RAPA, Ctrl compared to DMSO, ET-1–treated (blue) and RAPA compared to RAPA, ET-1–treated (red). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 4.. NT-1 does not affect the amount of steady-state PM PI4,5P₂ and causes only transient IP₃ production.

(A) PANC-1 cells were transfected with plasmid encoding the GFP-PLCδ-PH domain, treated with vehicle or 100 nM NT, and visualized by epifluorescence microscopy. Representative GFP images are shown. Time is seconds after beginning imaging. NT was added after 100 s. (B) Regions of interest containing GFP-PLCδ-PH fluorescence were identified and quantitated over time with ImageJ software. (C) Regions of interest containing Tubby-GFP fluorescence were identified and quantitated over time with ImageJ. Data in (B) and (C) are compiled from 10 cells in at least three independent experiments. All traces are means ± SEM. All curves were compared by two-way ANOVA with multiple comparisons within each row. **P < 0.01; values at all other time points were not statistically significant.

Fig. 5.. NT-1 stimulates in the rapid accumulation of IP₂ and the generation of very low amounts of IP₃.

(A) Scheme showing the potential paths to total IP production. The reagents listed in blue were used to manipulate PI4P abundance throughout the study. (B) A representative chromatogram from an experiment in which MEFs were labeled with [³H]myo-inositol before being stimulated with 100 nM ET-1 for 30 min, and then IP species were extracted and fractionated by ion exchange chromatography as described in Materials and Methods. CPM, counts per minute. (C) Quantitation of the relative contributions of each of the indicated IP species to the total IPs produced by MEFs in response to stimulation with ET-1. Data are compiled from three separate experiments performed as described for (B). Data are means ± SEM. (D) Time course of the production of the indicated IP species after stimulation with 100 nM ET-1. Data are from duplicate measurements and are representative of three independent experiments.

Fig. 6.. PI4P depletion at the PM blocks total IP production.

(A to C) Analysis of total IP production in vehicle- or ET-1–treated MEFs (left) and in vehicle- or NT-treated PANC-1 cells (right) after global PI4P depletion with PAO (A), after PM PI4P depletion with A1 (B), and after Golgi PI4P depletion with PIK93. The indicated cells were serum-starved and labeled with [³H]inositol for 24 hours and then were pretreated for 15 min with 10 μM PAO, 100 nM A1, or 300 nM PIK93 before being treated for 30 min with vehicle, 100 nM ET-1, or 100 nM NT in the presence of LiCl. The results are expressed in counts per minute (left) or fold over unstimulated cells (right). Data are means ± SD and were analyzed by one-way ANOVA with Tukey’s post-test. **P < 0.01 and ****P < 0.0001.

Fig. 7.. PI4P depletion at the PM inhibits PKD activation.

(A) The indicated cells were treated with 10 μM PAO for 15 min to deplete global PI4P, which was followed by the addition of agonist for 30 min (100 nM ET-1 or 100 nM NT). The cells were then analyzed by Western blotting with antibodies against total PKD and pPKD (Ser⁹¹⁶). (B) MEFs were treated with 100 nM A1 or 300 nM PIK93 for 15 min, which was followed by the addition of 100 nM ET-1 for 30 min. The cells were then analyzed by Western blotting with antibodies against total PKD and pPKD (Ser⁹¹⁶). (C) PANC-1 cells were treated with A1 or PIK93 for 15 min, which was followed by the addition of 100 nM NT for 30 min. The cells were then analyzed by Western blotting with antibodies against total PKD and pPKD (Ser⁹¹⁶). Western blots in all panels are representative of three independent experiments. Graphs in each panel show the abundance of pPKD relative to that of total PKD from three independent experiments. Data are means ± SEM and were analyzed by one-way ANOVA with Tukey’s post-test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 8.. PI4,5P₂ depletion inhibits neither total IP production nor PKD activation.

Cells were transfected (HEK) or transduced (PANC-1, MEF, and NRVM) with an adenovirus expressing the FKBP–5-phosphatase (5P) system as described in fig. S2 or yellow fluorescent protein (YFP) (YF). (A) Cells were treated with 1 μM RAPA for 20 min, which was followed by stimulation with the indicated agonists [100 nM NT, 100 nM ET-1, or 2 μM adenosine triphosphate (ATP)]. Total IPs were analyzed as described in Fig. 6. Data are means ± SEM of three independent experiments. (B) Cells were treated with 1 μM RAPA for 20 min, which was followed by stimulation with the indicated agonists (100 nM ET-1 and 100 nM NT). PKD activation was analyzed by Western blotting as described in Fig. 7. Western blots are representative of three independent experiments. (C) Grouped data from MEFs from three experiments are shown as means ± SEM and were analyzed by one-way ANOVA with Tukey’s post-test. *P < 0.05.