LPS Impairs Phospholipid Synthesis by Triggering β-Transducin Repeat-containing Protein (β-TrCP)-mediated Polyubiquitination and Degradation of the Surfactant Enzyme Acyl-CoA:Lysophosphatidylcholine Acyltransferase I (LPCAT1)

Abstract

with Arg abrogated LPCAT1 polyubiquitination. LPS profoundly reduced immunoreactive LPCAT1 levels and impaired lung surfactant mechanics, effects that were overcome by siRNA to ␤-TrCP and GSK-3␤ or LPCAT1 gene transfer, respectively. Thus, LPS appears to destabilize the LPCAT1 protein by GSK-3␤-mediated phosphorylation within a canonical phosphodegron for ␤-TrCP docking and site-specific ubiquitination. LPCAT1 is the first lipogenic substrate for ␤-TrCP, and the results suggest that modulation of the GSK-3␤-SCF␤ TrCP E3 ligase effector pathway might be a unique strategy to optimize dipalmitoylphosphatidylcholine levels in sepsis.

Acyl-CoA:Lysophosphatidylcholine Acyltransferase I (Lpcat1) Catalyzes Histone Protein O-Palmitoylation to Regulate mRNA Synthesis

“…2 cells were maintained with HITES medium supplemented with 10% FBS in a 37°C incubator and 5% CO 2 as described previously (22). Human embryonic kidney (HEK) 293FT cells were cultured in modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin, and 2 mM L-glutamine.…”

“…The washed beads were mixed with SDS-PAGE loading dye prior to SDS-PAGE and immunoblot analysis. Immunoblotting was performed as described previously (22). Microsomes were isolated as described previously (22).…”

“…[ 14 C]Palmitoyl-CoA was dried and solubilized in 5ϫ palmitoylation buffer by sonication (final working concentration 65 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 12.5 mM fatty acid-free BSA, and 2 mM EDTA) as we described previously (22). Each reaction contained 1 g of histone H4 recombinant protein, 2 Ci of [ 14 C]palmitoyl-CoA, and 10 ng of either recombinant Lpcat1, heat-inactivated Lpcat1, or SPTLC2.…”

Extracellular Signal-regulated Kinase (ERK) Regulates Cortactin Ubiquitination and Degradation in Lung Epithelial Cells

“…For example, ␤-Trcp targets phosphorylated IB (48) and phosphorylated ␤-catenin (49). We have shown that Lpcat1 is serine-phosphorylated by GSK3␤, and subsequently the phosphoenzyme is targeted by ␤-Trcp for disposal (36). Serine phosphorylation of cortactin on Ser 405 and Ser 418 by ERK has been well demonstrated (21,23,24); however, its link to cortactin stability has not been investigated.…”

“…␤-Trcp targets phosphorylated IB for its degradation (48), a protein also regulated by LPS. Our prior work demonstrates that ␤-Trcp also targets acyl-coA:lysophosphatidylcholine acyltransferase 1 (Lpcat1), a phospholipid-remodeling enzyme, for disposal in lung epithelial cells (36). Hence, these observations suggest that SCF ␤-Trcp appears to exert diverse effects on fundamental processes within lung epithelium.…”