Cross-talk between remodeling and de novo pathways maintains phospholipid balance through ubiquitination

Abstract

Phosphatidylcholine (PtdCho), the major phospholipid of animal membranes, is generated by its remodeling and de novo synthesis. Overexpression of the remodeling enzyme, LPCAT1 (acyl-CoA:lysophosphatidylcholine acyltransferase) in epithelia decreased de novo PtdCho synthesis without significantly altering cellular PtdCho mass. Overexpression of LPCAT1 increased degradation of CPT1 (cholinephosphotransferase), a resident Golgi enzyme that catalyzes the terminal step for de novo PtdCho synthesis. CPT1 degradation involved its multiubiquitination and processing via the lysosomal pathway. CPT1 mutants harboring arginine substitutions at multiple carboxyl-terminal lysines exhibited proteolytic resistance to effects of LPCAT1 overexpression in cells and restored de novo PtdCho synthesis. Thus, cross-talk between phospholipid remodeling and de novo pathways involves ubiquitin-lysosomal processing of a key molecular target that mechanistically provides homeostatic control of cellular PtdCho content.

FIGURE 1.

Pulmonary expression of LPCAT1 (A), LPCAT2 (B), and LPCAT3 (C). Primary murine lung type II cells, alveolar macrophages, and fibroblasts were isolated, and total cellular RNA was reverse transcribed to cDNA for subsequent qRT-PCR. The mRNA levels of each LPCAT were measured and normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA. The data represent cells isolated from at least 5 mice/group. *, p < 0.05 versus type II cells.

FIGURE 2.

LPCAT1 expression regulates surfactant metabolism. MLE cells were nucleofected with an LPCAT1 plasmid or an empty (E) vector. After 24 h, cells were collected and processed for LPCAT1 mRNA (measured relative to 18 S mRNA) by qRT-PCR (A), LPCAT1 and β-actin immunoblotting (B, inset), or enzyme activity (B), [¹⁴C]LysoPtdCho incorporation into surfactant DPPtdCho (C), or cellular mass of PtdCho, phosphatidylinositol (PI), and phosphatidylglycerol (PG) (D). B–D were normalized for cellular protein. E, surfactant secretion was measured by [³H]palmitic acid labeling of cells and activities retrieved in medium. Data are expressed as cpm medium/ml/total cellular DPPtdCho phospholipid phosphorus/mg of protein. Results are mean ± S.E. from at least three individual experiments except for PtdCho mass (two experiments) and phosphatidylinositol and phosphatidylglycerol mass (n = 1). *, p < 0.05 versus empty construct.

FIGURE 3.

LPCAT1 expression inhibits the denovo (CDP-choline) pathway. LPCAT1 was expressed in cells and processed for [³H]choline incorporation into PtdCho as a measure of de novo synthesis (A), CK activity (B), CCT activity (C), and CPT activity (D). E and F, primary rat type II cells were isolated, cultured, and infected with LPCAT1-V5 packaged in lentivirus prior to assays for [³H]choline incorporation into PtdCho (E), V5 and LPCAT1 immunoblotting (F, inset), and CPT1 activity (F). G and H, cells were left untreated or treated with KGF (20 ng/ml), and 24 h post-treatment, cells were harvested and processed for LPCAT immunoblotting and activity (G) and CPT activities (H). Results are mean ± S.E. from at least three individual experiments except for LPCAT activity (G) (two experiments). *, p < 0.05 versus empty construct.

FIGURE 4.

LPCAT1 expression promotes CPT degradation. A, MLE cells were nucleofected with empty, CPT1, or CEPT1 cDNA vectors, and CPT activity was assayed. B, cells were transfected with empty (E), LPCAT1 (L), or CPT1 (C) or in combination with empty or LPCAT1 plasmids, and after 16 h, CPT1, LPCAT1, and β-actin levels were detected by immunoblotting. C, experiments similar to B were performed, except CEPT1 was overexpressed instead of CPT1. D, cells were transfected with empty (E) or LPCAT1 expression vectors, and relative mRNA levels of CPT1, CEPT1, and CCT in cells were assayed using qRT-PCR. E, empty (E), LPCAT1 (L), or CPT1 (C) plasmids were transfected in cells alone or in combination, and after 12 h, cells were exposed to CHM with or without either NH₄Cl or lactacystin (Lac). After 18 h, cells were harvested and processed for immunoblotting for V5-CPT1, LPCAT1, and β-actin protein levels. The lower graphs in B, C, and E show densitometric values of bands from respective immunoblots. Data in each panel (A–E) represent three individual experiments (*, p < 0.05 versus empty group).

FIGURE 5.

CPT1 is localized to Golgi but degraded within the lysosomes. MLE cells were transfected with CFP-CPT1, βGT-YFP, and/or LPCAT1. A (top), cells were fixed and stained using a β-1,3-Gal-T2 (αβ-GT) primary antibody and an Alexa568-labeled goat anti-rabbit secondary antibody. To-Pro-3 was used to visualize nuclei. Bottom, cells were co-transfected with CFP-CPT1 and βGT-YFP and processed for imaging. White bar, 20 nm. B, transfected cells were exposed to NH₄Cl for 24 h as indicated. Cells were allowed to recover (without NH₄Cl) for 2 h prior to incubation with LysoTracker dye for 1 h. Cells were then fixed and visualized using a confocal microscope. The arrows in merged panels represent co-localization with LysoTracker.

FIGURE 6.

CPT1 is ubiquitinated. A, cells were co-transfected with ubiquitin plasmid, FLAG-luciferase (Luc), and FLAG-CPT1 with or without NH₄Cl (25 mm) for 24 h. Total cell lysates were processed for FLAG and β-actin immunoblotting. The graph on the right represents densitometric analysis of immunoblots. B, cells were nucleofected with FLAG-CPT1, HA-ubiquitin, or CPT1-V5his, with or without or LPCAT1, incubated for 18 h, and treated with NH₄Cl for 24 h using methods described previously. Cell lysates were loaded onto SDS-PAGE (Input) or incubated with HA antibody/protein A/G matrix, eluted, and visualized by FLAG immunoblotting (top and bottom). NS, nonspecific bands; arrows denote higher migrating CPT1-UB conjugates. C, cells were nucleofected with CPT1-V5his or CCT-V5, and 18 h after transfection, cells were treated with NH₄Cl for 24 h. Cells were harvested and incubated with Ni²⁺ resin at 4 °C for 4 h, and protein was eluted and visualized using SDS-PAGE and V5 immunoblotting. D, schematic of candidate ubiquitin acceptor sites targeted for generation of multiple Lys → Arg CPT1-V5his mutants. The CPT1_K4R mutant harbors mutations at Lys²⁵⁴, Lys²⁸², Lys²⁸³, and Lys²⁹², and the CPT1_K6R construct also harbors substitutions at Lys³⁰⁷ and Lys³¹¹. E (left), CPT1, CPT1_K4R, CPT1_K6R, and luciferase-V5 (Luc) plasmids were transfected into cells; after 18 h, cells were exposed to NH₄Cl for 24 h and harvested. Cell lysates were loaded onto SDS-PAGE (Input) or incubated with UbiQapture-Q kit matrix (4 °C for 4 h), eluted, and visualized by V5 immunoblotting (bottom). The arrows indicate slower migrating bands that exhibit differential levels of intensities between recombinant CPT1 and mutant constructs. Right, untransfected cells, CCT, and CPT1 alone or co-transfected with LPCAT1 lysates were processed using UbiQapture-Q matrix and visualized. All panels were exposed extensively to detect ubiquitinated products except for the far right panel. Results are representative of at least three individual experiments. F and G, cells were co-transfected with CPT1 or CPT1_K6R plasmid with either an empty (E) or LPCAT1 plasmid and exposed to CHM. Cells from each group were harvested at times after CHM treatment and processed for V5 immunoblotting (left) and densitometric analysis (right) of autoradiograms. The starting (0 h) time point was normalized to equal 1 for each test group. Best fit lines are a result of one-phase exponential decay line fitting performed by Prism graphing software. Data in F and G represent two individual experiments. H and I, cells were transfected with CFP-CPT1_K6R alone or in combination with βGT-YFP or LPCAT1. F, cells were co-transfected with CFP-CPT1_K6R and βGT-YFP, fixed, and visualized. The arrows indicate co-localization of CFP-CPT1_K6R with βGT-YFP. G, transfected cells were exposed to NH₄Cl treatment for 24 h. Cells were allowed to recover (no NH₄Cl) for 2 h prior to incubation with LysoTracker dye for 1 h. Cells were then fixed and visualized by confocal microscopy.

FIGURE 7.

CPT1 ubiquitin fusion proteins target the lysosome. MLE cells were transfected with CFP-CPT1, CFP-CPT1–1×Ub, CFP-CPT1–2×Ub, CFP-CPT1–3×Ub, or CFP-CPT1–4×Ub. After 18 h, cells were incubated with LysoTracker dye for 1 h. Cells were then fixed and visualized using confocal microscopy.

FIGURE 8.

CPT1 Lys → Arg mutants are resistant to LPCAT1-induced degradation. A, MLE cells were untransfected or transfected with CPT1-V5his, CPT1_K4R, or CPT1_K6R constructs alone or in combination with LPCAT1 and processed for LPCAT1, V5 (CPT1), and β-actin immunoblotting (A), CPT activities (B), or [³H]choline incorporation into PtdCho (C). Results are means ± S.E. from at least three individual experiments. *, p < 0.05 versus paired controls.