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CtBP1/BARS is an activator of phospholipase D1 necessary for agonist-induced macropinocytosis.

Haga Y, Miwa N, Jahangeer S, Okada T, Nakamura S - EMBO J. (2009)

Bottom Line: Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis.Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems.The present results shed light on the molecular basis of how the 'fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe, Japan.

ABSTRACT
Vesicular trafficking such as macropinocytosis is a dynamic process that requires coordinated interactions between specialized proteins and lipids. A recent report suggests the involvement of CtBP1/BARS in epidermal growth factor (EGF)-induced macropinocytosis. Detailed mechanisms as to how lipid remodelling is regulated during macropinocytosis are still undefined. Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis. EGF-induced macropinocytosis was specifically blocked by 1-butanol but not by 2-butanol. In addition, stimulation of cells by serum or EGF resulted in the association of CtBP1/BARS with PLD1. Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems. The present results shed light on the molecular basis of how the 'fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis.

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Effect of PLD downregulation by subtype-specific siRNAs on EGF-induced macropinocytosis. (A) COS7 cells were transfected with control or PLD subtype-specific siRNAs and cultured for 30 h. Cells were then metabolically labelled with radioactive lysophosphatidylcholine for 18 h and assayed for PLD in the presence of 0.3% 1-butanol. PtdBut, phosphatidylbutanol. (B) Transfected cells as in (A) were serum-starved for 1 h and stimulated with 100 ng/ml EGF in the presence of tetramethylrhodamine-labelled dextran for 8 min, washed, fixed and analysed for macropinocytosis by confocal microscopy. Bars, 10 μm. (C) Quantification of macropinocytosing cells treated as in (B). Data are means±s.e. from at least four independent experiments carried out in triplicate. *P<0.05 and **P<0.01 compared with control siRNA.
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f2: Effect of PLD downregulation by subtype-specific siRNAs on EGF-induced macropinocytosis. (A) COS7 cells were transfected with control or PLD subtype-specific siRNAs and cultured for 30 h. Cells were then metabolically labelled with radioactive lysophosphatidylcholine for 18 h and assayed for PLD in the presence of 0.3% 1-butanol. PtdBut, phosphatidylbutanol. (B) Transfected cells as in (A) were serum-starved for 1 h and stimulated with 100 ng/ml EGF in the presence of tetramethylrhodamine-labelled dextran for 8 min, washed, fixed and analysed for macropinocytosis by confocal microscopy. Bars, 10 μm. (C) Quantification of macropinocytosing cells treated as in (B). Data are means±s.e. from at least four independent experiments carried out in triplicate. *P<0.05 and **P<0.01 compared with control siRNA.

Mentions: Fluid-phase macropinocytosis was analysed in COS7 cells. Macropinocytosis was inactive under basal conditions and was markedly enhanced upon stimulation by EGF (Figure 1A and B) as reported earlier (Hewlett et al, 1994). As membrane lipid remodelling is essential for dynamic vesicular trafficking including macropinocytosis, we hypothesized that membrane lipid-metabolizing enzyme(s) must be involved during EGF-induced macropinocytosis. Among various candidates for such enzymes, we focused on PLD because this enzyme is known to be involved in various vesicular trafficking steps and to be activated by various growth factors, including EGF (Billah and Anthes, 1990). To assess the possibility of PLD involvement in EGF-induced macropinocytosis, the effect of 1-butanol, which is known to inhibit PLD-mediated processes by facilitating the PLD-specific transphosphatidylation reaction at the expense of PtdOH production was studied. Importantly, EGF-induced macropinocytosis was completely blocked by 1-butanol (Figure 1A and B). However, 2-butanol, which is unable to facilitate transphosphatidylation by PLD, had no effect on macropinocytosis, indicating that the 1-butanol effect was a PLD-specific and non-cytotoxic one and that PLD-catalysed PtdOH formation may be necessary for EGF-induced macropinocytosis. To assess further the physiological importance of PLD in macropinocytosis, endogenous PLD was specifically downregulated by RNA interference and then the agonist-induced macropinocytosis was evaluated. Both PLD1 and PLD2 isoforms are expressed in COS7 cells with a higher ratio of PLD1/PLD2 when compared with other cell lines (Mitchell et al, 2003). Phorbol 12-myristate 13-acetate-induced PLD activity was downregulated by almost 50% in COS7 cells transfected either with PLD1- or PLD2-siRNA (small interfering RNA) compared with control siRNA-treated cells (Figure 2A), confirming that both siRNAs worked properly in this system as reported earlier (Du et al, 2004; Sonoda et al, 2007). These PLD isoform-downregulated cells showed lower macropinocytotic activity compared with cells treated with control siRNA, whereas PLD1-downregulated cells exhibited stronger inhibition than PLD2-downregulated cells (Figure 2B and C). The inhibition of EGF-induced macropinocytosis caused either by PLD1 or PLD2 knockdown was rescued by the co-expression of the respective rat PLD isoforms, whereas the expression of a lipase inactive mutant, lipase-negative (LN)-PLD1 or LN-PLD2, did not restore macropinocytotic activity (Figure 3A). In these rescue experiments, actin dynamics in living COS7 cells were monitored using the actin-binding domain of filamin fused to RFP (ABD-filamin–RFP) (Figure 3B and C). This marker enabled visualization of early stages of EGF-induced macropinocytosis. GFP–PLD1 accumulated in the membrane-ruffled area of the cells that invaginated inward to form cup-shaped structures (Figure 3B, see arrows). It is interesting to note that transfected LN-PLD1 also accumulated in the membrane-ruffled area but failed in subsequent closure of invaginated and cup-shaped structures (Figure 3C, see arrows), suggesting that PLD1 activity is necessary for early stages of macropinocytosis. Expression of PLD2 also rescued the EGF-induced macropinocytosis in PLD2-knockdown cells. In contrast to PLD1, however, PLD2 accumulation surrounding macropinosomes was observed at relatively later stages of macropinocytosis (20 min after EGF treatment) in COS7 cells transiently expressing GFP–PLD2 (see Supplementary Figure 1).


CtBP1/BARS is an activator of phospholipase D1 necessary for agonist-induced macropinocytosis.

Haga Y, Miwa N, Jahangeer S, Okada T, Nakamura S - EMBO J. (2009)

Effect of PLD downregulation by subtype-specific siRNAs on EGF-induced macropinocytosis. (A) COS7 cells were transfected with control or PLD subtype-specific siRNAs and cultured for 30 h. Cells were then metabolically labelled with radioactive lysophosphatidylcholine for 18 h and assayed for PLD in the presence of 0.3% 1-butanol. PtdBut, phosphatidylbutanol. (B) Transfected cells as in (A) were serum-starved for 1 h and stimulated with 100 ng/ml EGF in the presence of tetramethylrhodamine-labelled dextran for 8 min, washed, fixed and analysed for macropinocytosis by confocal microscopy. Bars, 10 μm. (C) Quantification of macropinocytosing cells treated as in (B). Data are means±s.e. from at least four independent experiments carried out in triplicate. *P<0.05 and **P<0.01 compared with control siRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f2: Effect of PLD downregulation by subtype-specific siRNAs on EGF-induced macropinocytosis. (A) COS7 cells were transfected with control or PLD subtype-specific siRNAs and cultured for 30 h. Cells were then metabolically labelled with radioactive lysophosphatidylcholine for 18 h and assayed for PLD in the presence of 0.3% 1-butanol. PtdBut, phosphatidylbutanol. (B) Transfected cells as in (A) were serum-starved for 1 h and stimulated with 100 ng/ml EGF in the presence of tetramethylrhodamine-labelled dextran for 8 min, washed, fixed and analysed for macropinocytosis by confocal microscopy. Bars, 10 μm. (C) Quantification of macropinocytosing cells treated as in (B). Data are means±s.e. from at least four independent experiments carried out in triplicate. *P<0.05 and **P<0.01 compared with control siRNA.
Mentions: Fluid-phase macropinocytosis was analysed in COS7 cells. Macropinocytosis was inactive under basal conditions and was markedly enhanced upon stimulation by EGF (Figure 1A and B) as reported earlier (Hewlett et al, 1994). As membrane lipid remodelling is essential for dynamic vesicular trafficking including macropinocytosis, we hypothesized that membrane lipid-metabolizing enzyme(s) must be involved during EGF-induced macropinocytosis. Among various candidates for such enzymes, we focused on PLD because this enzyme is known to be involved in various vesicular trafficking steps and to be activated by various growth factors, including EGF (Billah and Anthes, 1990). To assess the possibility of PLD involvement in EGF-induced macropinocytosis, the effect of 1-butanol, which is known to inhibit PLD-mediated processes by facilitating the PLD-specific transphosphatidylation reaction at the expense of PtdOH production was studied. Importantly, EGF-induced macropinocytosis was completely blocked by 1-butanol (Figure 1A and B). However, 2-butanol, which is unable to facilitate transphosphatidylation by PLD, had no effect on macropinocytosis, indicating that the 1-butanol effect was a PLD-specific and non-cytotoxic one and that PLD-catalysed PtdOH formation may be necessary for EGF-induced macropinocytosis. To assess further the physiological importance of PLD in macropinocytosis, endogenous PLD was specifically downregulated by RNA interference and then the agonist-induced macropinocytosis was evaluated. Both PLD1 and PLD2 isoforms are expressed in COS7 cells with a higher ratio of PLD1/PLD2 when compared with other cell lines (Mitchell et al, 2003). Phorbol 12-myristate 13-acetate-induced PLD activity was downregulated by almost 50% in COS7 cells transfected either with PLD1- or PLD2-siRNA (small interfering RNA) compared with control siRNA-treated cells (Figure 2A), confirming that both siRNAs worked properly in this system as reported earlier (Du et al, 2004; Sonoda et al, 2007). These PLD isoform-downregulated cells showed lower macropinocytotic activity compared with cells treated with control siRNA, whereas PLD1-downregulated cells exhibited stronger inhibition than PLD2-downregulated cells (Figure 2B and C). The inhibition of EGF-induced macropinocytosis caused either by PLD1 or PLD2 knockdown was rescued by the co-expression of the respective rat PLD isoforms, whereas the expression of a lipase inactive mutant, lipase-negative (LN)-PLD1 or LN-PLD2, did not restore macropinocytotic activity (Figure 3A). In these rescue experiments, actin dynamics in living COS7 cells were monitored using the actin-binding domain of filamin fused to RFP (ABD-filamin–RFP) (Figure 3B and C). This marker enabled visualization of early stages of EGF-induced macropinocytosis. GFP–PLD1 accumulated in the membrane-ruffled area of the cells that invaginated inward to form cup-shaped structures (Figure 3B, see arrows). It is interesting to note that transfected LN-PLD1 also accumulated in the membrane-ruffled area but failed in subsequent closure of invaginated and cup-shaped structures (Figure 3C, see arrows), suggesting that PLD1 activity is necessary for early stages of macropinocytosis. Expression of PLD2 also rescued the EGF-induced macropinocytosis in PLD2-knockdown cells. In contrast to PLD1, however, PLD2 accumulation surrounding macropinosomes was observed at relatively later stages of macropinocytosis (20 min after EGF treatment) in COS7 cells transiently expressing GFP–PLD2 (see Supplementary Figure 1).

Bottom Line: Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis.Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems.The present results shed light on the molecular basis of how the 'fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe, Japan.

ABSTRACT
Vesicular trafficking such as macropinocytosis is a dynamic process that requires coordinated interactions between specialized proteins and lipids. A recent report suggests the involvement of CtBP1/BARS in epidermal growth factor (EGF)-induced macropinocytosis. Detailed mechanisms as to how lipid remodelling is regulated during macropinocytosis are still undefined. Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis. EGF-induced macropinocytosis was specifically blocked by 1-butanol but not by 2-butanol. In addition, stimulation of cells by serum or EGF resulted in the association of CtBP1/BARS with PLD1. Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems. The present results shed light on the molecular basis of how the 'fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis.

Show MeSH
Related in: MedlinePlus