LPS impairs phospholipid synthesis by triggering beta-transducin repeat-containing protein (beta-TrCP)-mediated polyubiquitination and degradation of the surfactant enzyme acyl-CoA:lysophosphatidylcholine acyltransferase I (LPCAT1)

Abstract

Acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a relatively newly described and yet indispensable enzyme needed for generation of the bioactive surfactant phospholipid, dipalmitoylphosphatidylcholine (DPPtdCho). Here, we show that lipopolysaccharide (LPS) causes LPCAT1 degradation using the Skp1-Cullin-F-box ubiquitin E3 ligase component, β-transducin repeat-containing protein (β-TrCP), that polyubiquitinates LPCAT1, thereby targeting the enzyme for proteasomal degradation. LPCAT1 was identified as a phosphoenzyme as Ser(178) within a phosphodegron was identified as a putative molecular recognition site for glycogen synthase kinase-3β (GSK-3β) phosphorylation that recruits β-TrCP docking within the enzyme. β-TrCP ubiquitinates LPCAT1 at an acceptor site (Lys(221)), as substitution of Lys(221) with Arg abrogated LPCAT1 polyubiquitination. LPS profoundly reduced immunoreactive LPCAT1 levels and impaired lung surfactant mechanics, effects that were overcome by siRNA to β-TrCP and GSK-3β or LPCAT1 gene transfer, respectively. Thus, LPS appears to destabilize the LPCAT1 protein by GSK-3β-mediated phosphorylation within a canonical phosphodegron for β-TrCP docking and site-specific ubiquitination. LPCAT1 is the first lipogenic substrate for β-TrCP, and the results suggest that modulation of the GSK-3β-SCFβ(TrCP) E3 ligase effector pathway might be a unique strategy to optimize dipalmitoylphosphatidylcholine levels in sepsis.

FIGURE 1.

LPCAT1 is degraded by the ubiquitin-proteasomal pathway. A, MLE cells during exponential growth were serum-starved and treated with 10 μg/ml cycloheximide (CHX) in a time course. Cells were lysed and subjected to LPCAT1 and β-actin immunoblotting (IB). B, densitometry results of the immunoblotting were plotted for half-life analyses using PRISM software. C, LPCAT1 was overexpressed in cells for 18 h, and cells were treated and analyzed as in A. D–F, cells were serum-starved and treated with MG132 (20 μg/ml) (D and E) or leupeptin (20 μg/ml) (F) in a time course and analyzed by LPCAT1 and β-actin immunoblotting. Data in E show levels of LPCAT1 accumulation over time. G, V5-LPCAT1 was overexpressed in cells prior to treatment with or without MG132 (20 μg/ml) for 24 h. Cells were lysed in the presence of 1 μg/ml ubiquitin aldehyde and subjected to LPCAT1 immunoblot analyses. Asterisk represents endogenous LPCAT1. Multiple polyubiquitin (polyUb) bands were detected. H, HA-tagged ubiquitin and FLAG or V5-tagged LPCAT1 were co-expressed in cells. Cell lysates were immunoprecipitated (IP) with HA antibody, and the precipitates were analyzed by V5 immunoblotting. Each panel is representative of three to six independent experiments, with the exception of E and F (n = 1).

FIGURE 2.

SCF^β-TrCP ubiquitinates LPCAT1. A, MLE cells were cultured alone (nontransfected cells (NT)) or overexpressed with candidate V5-tagged SCF E3 ubiquitin ligase subunits. Cell lysates were subjected to V5, LPCAT1, and β-actin immunoblotting (IB). B, cells were nucleofected with pcDNA3.1/His/V5/Lpcat1 plasmid or co-nucleofected with pcDNA3.1/His/V5/β-TrCP plasmids for 18 h. Cell lysates were subjected to V5 immunoblotting (B) or LPCAT1 activity (C) analysis. *, p < 0.05 by analysis of variance. D, cell lysates were immunoprecipitated (IP) (10% input) with LPCAT1 antibody and the precipitates analyzed by β-TrCP immunoblotting. E, in vitro ubiquitination assays were conducted (“Experimental Procedures.”). Left panel shows the input of SCF-box subunit protein components, and right panel shows ubiquitination reactions using these components in the presence (+) or absence (−) of β-TrCP in the reaction mixtures. Products were run on SDS-PAGE and immunoblotted using LPCAT1 antibody. The bands below ∼45 kDa represent nonspecific proteins. F, β-TrCP-specific siRNA oligos were nucleofected in MLE cells for 48 h. Cells were treated with 10 μg/ml cycloheximide for times as indicated. Cell lysates were subjected to immunoblotting. Each panel is representative of three independent experiments, with the exception of E (n = 2). PolyUb, polyubiquitin.

FIGURE 3.

A conserved β-TrCP-docking site resides in LPCAT1. A, alignment of the amino acid sequence of a putative β-TrCP recognition degron within LPCAT1 and the known consensus sequence. B, MLE cells were nucleofected with wild type (WT) and serine-mutated LPCAT1 plasmids, respectively, for 24 h in the presence or absence of MG132. Cell lysates were analyzed by LPCAT1 immunoblotting (IB). Plasmids encode WT, and two point mutants (S178A and S182A) and a double mutant S178A/S182A within LPCAT1. C, cells were nucleofected with wild type or individual serine-mutated LPCAT1 plasmids. Nucleofected cells were treated with MG132 in a time course analysis, and cell lysates were subjected to LPCAT1 immunoblotting. D, wild type or serine-mutated LPCAT1 were overexpressed in cells. Cell lysates were subjected to V5 antibody immunoprecipitation (IP) followed by β-TrCP immunoblotting. Each panel is representative of three independent experiments. E, co-localization of LPCAT1 and β-TrCP. Cells were co-transfected with LPCAT1-CFP and β-TrCP-YFP (2 μg of plasmid/chamber), and LPCAT1 and β-TrCP localization was detected at the single cell level using a combination laser-scanning microscope system.

FIGURE 4.

GSK-3β phosphorylates LPCAT1. A, MLE cells were nucleofected with GSK-3β-specific siRNA or control RNA oligos followed by cycloheximide (CHX) treatment. Cell lysates were subjected to LPCAT1, GSK-3β, and β-actin immunoblotting (IB). B, wild type (GSK-3βwt), hyperactive (GSK-3βhyper), and kinase-dead (GSK-3βkd) GSK-3β plasmids and wild type or serine-mutated LPCAT1 plasmids were co-nucleofected in MLE cells for 16 h. Cell lysates were analyzed by immunoblotting as indicated. C, LPCAT1 in vitro phosphorylation assays were performed (see under “Experimental Procedures”). V5-tagged wild type and serine-mutated LPCAT1 were overexpressed in cells, and LPCAT1 was isolated with LPCAT1 antibody and protein A/G-agarose beads. Lower panel shows the input of LPCAT1 that was visualized by V5 immunoblotting. D, V5-tagged wild type and Ser¹⁷⁸ and/or Ser¹⁸²-mutated LPCAT1 plasmids were co-overexpressed with or without GSK-3β siRNA in MLE cells, and cell lysates were subjected to V5 immunoprecipitation followed by immunoblotting using phosphoserine antibodies to detect phosphorylated LPACT1 (pLPCAT1). Immunoblots were also probed with antibodies for LPCAT1 and GSK-3β. Cell lysates (10% input) were used for immunoblotting analyses as indicated. A and D are representative of five and three independent experiments, respectively, and the other panels are duplicate experiments.

FIGURE 5.

Lys²²¹ within LPCAT1 is a ubiquitin acceptor site for SCF^β-TrCP. A, MLE cells were nucleofected with wild type and lysine-mutated LPCAT1 plasmids for 16 h followed by MG132 treatment for another 12 h. Cell lysates were analyzed by immunoblotting (IB). B and C, nucleofected cells were treated with MG132 in a time course, and cell lysates were subjected to LPCAT1 immunoblotting (B), and the densitometry of the immunoblotting results was plotted (C). D, HA-tagged β-TrCP and V5-tagged wild type or lysine-mutated LPCAT1 plasmids were co-expressed in cells, and HA immunoprecipitation (IP) was performed followed by V5 immunoblotting. Each panel is representative of three independent experiments except D (n = 2).

FIGURE 6.

LPS triggers LPCAT1 degradation via the ubiquitin proteasome. A, MLE cells were maintained in serum-free medium and exposed to different concentrations of LPS in the presence or absence of MG132 for 24 h. Cell lysates were analyzed by LPCAT1 and β-actin immunoblotting (IB). B, cells were exposed to LPS (10 μg/ml) in the presence or absence of MG132, and cells were collected in a time course for LPCAT1 immunoblotting. C and D, cells were transfected with control RNA, β-TrCP siRNA, or GSK-3β siRNA oligos for 48 h followed by treatment with cycloheximide (CHX) (10 μg/ml) with or without LPS (10 μg/ml) for another 12 h as indicated. Cell lysates were processed for LPCAT1, β-TrCP, or GSK-3β immunoblotting. Each panel is representative of three independent experiments.

FIGURE 7.

LPS down-regulates LPCAT1 in a mouse model. A–F, mice were given vector alone or a lentivirus encoding LPCAT1 (10⁸ plaque-forming units/mouse) for 96 h prior to intratracheal inoculation with LPS (5 mg/kg) for 24 h. Animals were then mechanically ventilated, and lung resistance (B), compliance (C), and elastance (lung stiffness, D) were measured and pressure-volume loops (E) generated. Each group contained 4–6 mice. Mice lung tissues were also homogenized and sonicated in cell lysis buffer followed by LPCAT1 immunoblotting (IB) analysis (A). *, p < 0.05 versus groups as indicated by analysis of variance. F, PtdCho levels in lung lavage were assayed in groups.