Cholesterol depletion results in site-specific increases in epidermal growth factor receptor phosphorylation due to membrane level effects. Studies with cholesterol enantiomers

Abstract

In A431 cells, depletion of cholesterol with methyl-beta-cyclodextrin induced an increase in both basal and epidermal growth factor (EGF)-stimulated EGF receptor phosphorylation. This increase in phosphorylation was site-specific, with significant increases occurring at Tyr845, Tyr992, and Tyr1173, but only minor changes at Tyr1045 and Tyr1068. The elevated level of receptor phosphorylation was associated with an increase in the intrinsic kinase activity of the EGF receptor kinase, possibly as a result of the cyclodextrin-induced enhancement of the phosphorylation of Tyr845, a site in the kinase activation loop known to be phosphorylated by pp60src. Cholesterol and its enantiomer (ent-cholesterol) were used to investigate the molecular basis for the modulation of EGF receptor function by cholesterol. Natural cholesterol (nat-cholesterol) was oxidized substantially more rapidly than ent-cholesterol by cholesterol oxidase, a protein that contains a specific binding site for the sterol. By contrast, the ability of nat- and ent-cholesterol to interact with sphingomyelins and phosphatidylcholine and to induce lipid condensation in a monolayer system was the same. These data suggest that, whereas cholesterol-protein interactions may be sensitive to the absolute configuration of the sterol, sterol-lipid interactions are not. nat- and ent-cholesterol were tested for their ability to physically reconstitute lipid rafts following depletion of cholesterol. nat- and ent-cholesterol reversed to the same extent the enhanced phosphorylation of the EGF receptor that occurred following removal of cholesterol. Furthermore, the enantiomers showed similar abilities to reconstitute lipid rafts in cyclodextrin-treated cells. These data suggest that cholesterol most likely affects EGF receptor function because of its physical effects on membrane properties, not through direct enantioselective interactions with the receptor.

Fig.1

Structures of nat-cholesterol and its unnatural enantiomer, ent-cholesterol.

Fig.2. Effect of cholesterol depletion on EGF receptor phosphorylation

A, A431 cells were treated without or with the indicated concentrations of methyl-β-cyclodextrin for 30 min to remove cholesterol. Cells were lysed and analyzed for basal receptor phosphorylation by Western blotting with anti-phosphotyrosine antibody PY20. Results were quantitated by densitometry. B, cells were treated without (control (CON)) or with 7.5 mM methyl-β-cyclodextrin (CDX) for 30 min and were then stimulated with the indicated doses of EGF for 5 min. Cells were lysed and analyzed as described for A. Results from a representative experiment were quantified by densitometry and subjected to nonlinear curve fitting using GraphPAD Prism.

Fig.3. Effect of cholesterol depletion on ¹²⁵I-EGF binding

A431 cells were treated without (●) or with (○) 5 mM methyl-β-cyclodextrin (CDX) and assayed for ¹²⁵I-EGF binding as described under “Experimental Procedures.” Points represent the mean of triplicate determinations.

Fig.4. Effect of cholesterol on site-specific tyrosine phosphorylation of the EGF receptor

A431 cells were treated without (control (CON)) or with 7.5 mM methyl-β-cyclodextrin (CDX) to remove cholesterol and then stimulated with the indicated doses of EGF for 5 min. Cells were lysed and analyzed for receptor phosphorylation by Western blotting with antibodies that recognize phosphorylated tyrosines 845, 992, 1045, 1068, and 1173 on the EGF receptor. Results were quantified by densitometry and subjected to nonlinear curve fitting using GraphPAD Prism. Results from a representative experiment are shown.

Fig. 5. Oxidation of nat- and ent-cholesterol by cholesterol Oxidase

Equal amounts of nat-cholesterol (■) and ent-cholesterol (□) were subjected to oxidation by cholesterol oxidase as described under “Experimental Procedures.” Samples were incubated for the indicated times at 37 °C and analyzed for absorbance at 600 nm.

Fig.6. Condensation of lipids by nat- and ent-cholesterol in monolayers

Surface pressure versus average molecular area behavior was measured as described under “Experimental Procedures” for pure nat- or ent-cholesterol or mixtures of these enantiomers with sphingomyelin. ▽ and ▼, isotherms for pure nat- and ent-cholesterol, respectively; ○ and •, isotherms for 30 mol % nat- and ent-cholesterol, respectively, mixed with 70 mol % egg sphingomyelin; □ and ■, isotherms for 30 mol % nat- and ent-cholesterol, respectively, mixed with 70 mol % N-stearoylsphingomyelin (the naturally occurring form of this lipid); ◇ and ◆, isotherms for 1:1 mixtures of nat- and ent-cholesterol, respectively, with palmitoyloleoylphosphatidylcholine. Each trace represents the average of three or more experimental isotherms. The standard errors are shown. mNm, milli-Newton meter.

Fig.7. Effect of cholesterol repletion on phosphorylation of the EGF receptor

A, A431 cells were treated without (control (CON)) or with 7.5 mM methyl-β-cyclodextrin (CDX) to remove cholesterol. Some cultures were subsequently repleted with cholesterol by incubation with either nat-cholesterol (NAT) or ent-cholesterol (ENT) in complex with methyl-β-cyclodextrin. Concentrations given refer to the amount of cholesterol added. Cells were lysed and analyzed for receptor phosphorylation by Western blotting with anti-phosphotyrosine antibody PY20. Results were quantified by densitometry. B, cells were treated as described for A, but prior to lysis, cells were stimulated with 5 nM EGF for 5 min.

Fig.8. Effect of cholesterol repletion on phosphorylation of the EGF receptor

Chinese hamster ovary cells expressing EGF receptors were treated without (control (CON)) or with 7.5 mM methyl-β-cyclodextrin (CDX) to remove cholesterol. Some cultures were subsequently repleted with cholesterol by incubation with either natcholesterol (Nat-Chol) or ent-cholesterol (Ent-Chol) in complex with methyl-β-cyclodextrin. Concentrations given refer to the amount of cholesterol added. Cells were then stimulated with 25 nM EGF for 5 min, lysed, and analyzed for receptor phosphorylation by Western blotting with anti-phosphotyrosine antibody PY20. Results were quantified by densitometry.

Fig.9. Effect of cholesterol depletion and repletion on dephosphorylation of the EGF receptor

A431 cells were treated without (control (CON)) or with 7.5 mM methyl-β-cyclodextrin (CDX) to remove cholesterol. Some cultures were subsequently repleted by incubation for 30 min with 0.2 mM nat-cholesterol (NAT) or ent-cholesterol (ENT) in complex with methyl-β-cyclodextrin. Cells were stimulated with 1.25 nM EGF for 5 min and washed to remove residual EGF as described under “Experimental Procedures.” Cells were incubated in DMEM/BSA for the indicated times and then analyzed for EGF receptor phosphorylation by Western blotting with anti-phosphotyrosine antibody PY20. Results from a representative experiment were quantified by densitometry.

Fig. 10. Effect of repletion with nat- or ent-cholesterol on in vitro autophosphorylation of the EGF receptor

A431 cells were treated without (control (CON)) or with 10 mM methyl-β-cyclodextrin (CDX). Cholesterol-depleted cells were subsequently repleted by incubation for 30 min with 0.2 mM nat-cholesterol (NAT) or ent-cholesterol (ENT) in complex with methyl-β-cyclodextrin. Membranes were prepared, and aliquots were subjected to in vitro autophosphorylation assays as described under “Experimental Procedures.” Results from a representative experiment were quantified by densitometry.

Fig. 11. Reconstitution of lipid rafts by nat- and ent-cholesterol

A431 cells were treated without (control (CON)) or with methyl-β-cyclodextrin (CDX) for 30 min. Some methyl-β-cyclodextrin-treated cultures were subsequently repleted with cholesterol by incubation for 30 min with 0.2 mM nat-cholesterol (NAT) or ent-cholesterol (ENT) in complex with methyl-β-cyclodextrin. Lipid rafts were prepared as described under “Experimental Procedures.” Equal aliquots of fractions from the sucrose density gradient were analyzed by SDS-PAGE and Western blotting for the EGF receptor or flotillin.