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Phosphatidic acid plays a regulatory role in clathrin-mediated endocytosis.

Antonescu CN, Danuser G, Schmid SL - Mol. Biol. Cell (2010)

Bottom Line: We examined the effect of altering cellular PA levels on CME by manipulating the activities and/or levels of either phospholipase D (PLD1 and PLD2) or diacylglycerol kinase (DGK), two enzyme classes involved in PA production.DGK inhibition resulted in a dramatic reduction of cellular PA, measured directly using an enzyme-coupled reaction, which resulted in a decreased rate of EGFR internalization measured biochemically.Consistent with opposite effects on cellular PA levels, PLD inhibition had opposite effects on EGFR internalization and CCP dynamics, compared with DGK inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA.

ABSTRACT
Clathrin-mediated endocytosis (CME) is the main route of internalization of receptor-ligand complexes. Relatively little is known about the role of specific lipids in CME, in particular that of phosphatidic acid (PA). We examined the effect of altering cellular PA levels on CME by manipulating the activities and/or levels of either phospholipase D (PLD1 and PLD2) or diacylglycerol kinase (DGK), two enzyme classes involved in PA production. DGK inhibition resulted in a dramatic reduction of cellular PA, measured directly using an enzyme-coupled reaction, which resulted in a decreased rate of EGFR internalization measured biochemically. This corresponded to a decreased rate of clathrin-coated pit (CCP) initiation and increased lifetimes of productive CCPs, as determined by quantitative live-cell total internal reflection fluorescence microscopy. Unexpectedly, PLD inhibition caused an increase in cellular PA, suggesting that PLD activity negatively regulates PA synthesis by other more productive pathways. Consistent with opposite effects on cellular PA levels, PLD inhibition had opposite effects on EGFR internalization and CCP dynamics, compared with DGK inhibition. Importantly, the constitutive internalization of transferrin receptors was unaffected by either treatment. These findings demonstrate that PA plays a regulatory rather than obligatory role in CME and differentially regulates ligand-stimulated CME of EGFR.

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EGF treatment does not affect the reduction of PA levels due to inhibition of DGK. BSC-1 cells were treated with either (A) the PLD inhibitor FIPI (750 nM) or (B) the DGK inhibitor R59949 (30 μM) or left untreated (0.1% DMSO vehicle, control) for 20 min as indicated and then stimulated or not with EGF, as indicated for 5 min. Cells were then immediately subject to extraction of lipids and determination of cellular PA content. Shown are the means ± SE of six independent experiments. *p < 0.05.
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Figure 6: EGF treatment does not affect the reduction of PA levels due to inhibition of DGK. BSC-1 cells were treated with either (A) the PLD inhibitor FIPI (750 nM) or (B) the DGK inhibitor R59949 (30 μM) or left untreated (0.1% DMSO vehicle, control) for 20 min as indicated and then stimulated or not with EGF, as indicated for 5 min. Cells were then immediately subject to extraction of lipids and determination of cellular PA content. Shown are the means ± SE of six independent experiments. *p < 0.05.

Mentions: EGF stimulation has been reported to increase both PLD (Lee et al., 2009a) and DGK (Cai et al., 2009) activities. Therefore, it remained possible that EGF stimulation changes the contribution of each enzyme to cellular PA levels, thereby accounting for the differential effects of these perturbations on EGF uptake. However, in cells stimulated for 5 min with either 2 or 20 ng/ml EGF, we did not observe a statistically significant increase in total PA levels (Figure 6). Moreover, the effects of FIPI and R59949 on PA levels were unchanged when cells were stimulated with EGF. Thus, although EGF stimulation may result in an increased activity of PLD and/or certain DGKs, this appears to contribute a negligible amount of PA relative to that produced constitutively, largely through class I DGKs. Hence, regardless of EGF stimulation, PLD has a minor and negative contribution toward total cellular PA levels, whereas DGK type I are the major contributors to total cellular PA levels.


Phosphatidic acid plays a regulatory role in clathrin-mediated endocytosis.

Antonescu CN, Danuser G, Schmid SL - Mol. Biol. Cell (2010)

EGF treatment does not affect the reduction of PA levels due to inhibition of DGK. BSC-1 cells were treated with either (A) the PLD inhibitor FIPI (750 nM) or (B) the DGK inhibitor R59949 (30 μM) or left untreated (0.1% DMSO vehicle, control) for 20 min as indicated and then stimulated or not with EGF, as indicated for 5 min. Cells were then immediately subject to extraction of lipids and determination of cellular PA content. Shown are the means ± SE of six independent experiments. *p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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Figure 6: EGF treatment does not affect the reduction of PA levels due to inhibition of DGK. BSC-1 cells were treated with either (A) the PLD inhibitor FIPI (750 nM) or (B) the DGK inhibitor R59949 (30 μM) or left untreated (0.1% DMSO vehicle, control) for 20 min as indicated and then stimulated or not with EGF, as indicated for 5 min. Cells were then immediately subject to extraction of lipids and determination of cellular PA content. Shown are the means ± SE of six independent experiments. *p < 0.05.
Mentions: EGF stimulation has been reported to increase both PLD (Lee et al., 2009a) and DGK (Cai et al., 2009) activities. Therefore, it remained possible that EGF stimulation changes the contribution of each enzyme to cellular PA levels, thereby accounting for the differential effects of these perturbations on EGF uptake. However, in cells stimulated for 5 min with either 2 or 20 ng/ml EGF, we did not observe a statistically significant increase in total PA levels (Figure 6). Moreover, the effects of FIPI and R59949 on PA levels were unchanged when cells were stimulated with EGF. Thus, although EGF stimulation may result in an increased activity of PLD and/or certain DGKs, this appears to contribute a negligible amount of PA relative to that produced constitutively, largely through class I DGKs. Hence, regardless of EGF stimulation, PLD has a minor and negative contribution toward total cellular PA levels, whereas DGK type I are the major contributors to total cellular PA levels.

Bottom Line: We examined the effect of altering cellular PA levels on CME by manipulating the activities and/or levels of either phospholipase D (PLD1 and PLD2) or diacylglycerol kinase (DGK), two enzyme classes involved in PA production.DGK inhibition resulted in a dramatic reduction of cellular PA, measured directly using an enzyme-coupled reaction, which resulted in a decreased rate of EGFR internalization measured biochemically.Consistent with opposite effects on cellular PA levels, PLD inhibition had opposite effects on EGFR internalization and CCP dynamics, compared with DGK inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA.

ABSTRACT
Clathrin-mediated endocytosis (CME) is the main route of internalization of receptor-ligand complexes. Relatively little is known about the role of specific lipids in CME, in particular that of phosphatidic acid (PA). We examined the effect of altering cellular PA levels on CME by manipulating the activities and/or levels of either phospholipase D (PLD1 and PLD2) or diacylglycerol kinase (DGK), two enzyme classes involved in PA production. DGK inhibition resulted in a dramatic reduction of cellular PA, measured directly using an enzyme-coupled reaction, which resulted in a decreased rate of EGFR internalization measured biochemically. This corresponded to a decreased rate of clathrin-coated pit (CCP) initiation and increased lifetimes of productive CCPs, as determined by quantitative live-cell total internal reflection fluorescence microscopy. Unexpectedly, PLD inhibition caused an increase in cellular PA, suggesting that PLD activity negatively regulates PA synthesis by other more productive pathways. Consistent with opposite effects on cellular PA levels, PLD inhibition had opposite effects on EGFR internalization and CCP dynamics, compared with DGK inhibition. Importantly, the constitutive internalization of transferrin receptors was unaffected by either treatment. These findings demonstrate that PA plays a regulatory rather than obligatory role in CME and differentially regulates ligand-stimulated CME of EGFR.

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