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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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Involvement of both PLD1 and CtBP1/BARS in EGF-induced macropinocytosis. COS7 cells were transiently co-transfected with expression vectors encoding GFP–PLD1 or CFP–CtBP1/BARS and cultured for 2 days. Cells were serum-starved for 1 h and stimulated with 100 ng/ml EGF in the presence of tetramethylrhodamine-labelled dextran and analysed for macropinocytosis by confocal microscopy. Representative frames of time-lapse images for GFP–PLD1 (green), CFP–CtBP1/BARS (grey) and tetramethylrhodamine-labelled dextran (red) and merged signals are shown. Arrows and arrowheads indicate newly forming macropinosomes. Bars, 10 μm.
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f8: Involvement of both PLD1 and CtBP1/BARS in EGF-induced macropinocytosis. COS7 cells were transiently co-transfected with expression vectors encoding GFP–PLD1 or CFP–CtBP1/BARS and cultured for 2 days. Cells were serum-starved for 1 h and stimulated with 100 ng/ml EGF in the presence of tetramethylrhodamine-labelled dextran and analysed for macropinocytosis by confocal microscopy. Representative frames of time-lapse images for GFP–PLD1 (green), CFP–CtBP1/BARS (grey) and tetramethylrhodamine-labelled dextran (red) and merged signals are shown. Arrows and arrowheads indicate newly forming macropinosomes. Bars, 10 μm.

Mentions: To demonstrate more clearly that EGF-induced association of PLD1 and CtBP1/BARS is implicated in the formation of macropinosomes, we monitored the dynamics of these proteins during macropinocytosis in living cells. COS7 cells transiently expressing both GFP–PLD1 and CFP–CtBP1/BARS were stimulated with EGF and the dynamics of these fluorescent proteins during macropinocytosis were visualized by time-lapse confocal microscopy (Figure 8). GFP–PLD1 distributed predominantly in the cytoplasm in a reticular pattern, whereas CFP–CtBP1/BARS was localized in the cytoplasm in a diffuse pattern. On stimulation with EGF, both proteins gathered around newly macropinocytosing areas in a concurrent manner, that is, these two proteins gathered almost simultaneously around macropinocytosing cups (Figure 8, see arrows and arrowheads). These results suggest functional roles for both proteins in the induction of macropinocytosis.


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)

Involvement of both PLD1 and CtBP1/BARS in EGF-induced macropinocytosis. COS7 cells were transiently co-transfected with expression vectors encoding GFP–PLD1 or CFP–CtBP1/BARS and cultured for 2 days. Cells were serum-starved for 1 h and stimulated with 100 ng/ml EGF in the presence of tetramethylrhodamine-labelled dextran and analysed for macropinocytosis by confocal microscopy. Representative frames of time-lapse images for GFP–PLD1 (green), CFP–CtBP1/BARS (grey) and tetramethylrhodamine-labelled dextran (red) and merged signals are shown. Arrows and arrowheads indicate newly forming macropinosomes. Bars, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664659&req=5

f8: Involvement of both PLD1 and CtBP1/BARS in EGF-induced macropinocytosis. COS7 cells were transiently co-transfected with expression vectors encoding GFP–PLD1 or CFP–CtBP1/BARS and cultured for 2 days. Cells were serum-starved for 1 h and stimulated with 100 ng/ml EGF in the presence of tetramethylrhodamine-labelled dextran and analysed for macropinocytosis by confocal microscopy. Representative frames of time-lapse images for GFP–PLD1 (green), CFP–CtBP1/BARS (grey) and tetramethylrhodamine-labelled dextran (red) and merged signals are shown. Arrows and arrowheads indicate newly forming macropinosomes. Bars, 10 μm.
Mentions: To demonstrate more clearly that EGF-induced association of PLD1 and CtBP1/BARS is implicated in the formation of macropinosomes, we monitored the dynamics of these proteins during macropinocytosis in living cells. COS7 cells transiently expressing both GFP–PLD1 and CFP–CtBP1/BARS were stimulated with EGF and the dynamics of these fluorescent proteins during macropinocytosis were visualized by time-lapse confocal microscopy (Figure 8). GFP–PLD1 distributed predominantly in the cytoplasm in a reticular pattern, whereas CFP–CtBP1/BARS was localized in the cytoplasm in a diffuse pattern. On stimulation with EGF, both proteins gathered around newly macropinocytosing areas in a concurrent manner, that is, these two proteins gathered almost simultaneously around macropinocytosing cups (Figure 8, see arrows and arrowheads). These results suggest functional roles for both proteins in the induction of macropinocytosis.

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