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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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Activation of PLD1 by CtBP1/BARS in a purified recombinant protein system. PLD1 (A) and PLD2 (B) activities were measured as a function of time in the absence or presence of 10 nM CtBP1/BARS using mixed phospholipid vesicles. PLD1 activity was reconstituted with various combinations of 1 nM CtBP1/BARS and 50 nM ARF1 in the absence or presence of 100 μM guanine nucleotides (C) or with various combinations of 1 nM CtBP1/BARS, 1 nM CtBP1/BARS(S147D), 4 nM ARF6, 300 nM RhoA and 100 nM PKCα (D). Enzymatic reactions proceeded for 20 min and PtdEtOH formation was measured. Data presented are means±s.e. of at least six independent experiments carried out in triplicate. *P<0.05 compared with no addition of activators. PtdEtOH, phosphatidylethanol.
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f9: Activation of PLD1 by CtBP1/BARS in a purified recombinant protein system. PLD1 (A) and PLD2 (B) activities were measured as a function of time in the absence or presence of 10 nM CtBP1/BARS using mixed phospholipid vesicles. PLD1 activity was reconstituted with various combinations of 1 nM CtBP1/BARS and 50 nM ARF1 in the absence or presence of 100 μM guanine nucleotides (C) or with various combinations of 1 nM CtBP1/BARS, 1 nM CtBP1/BARS(S147D), 4 nM ARF6, 300 nM RhoA and 100 nM PKCα (D). Enzymatic reactions proceeded for 20 min and PtdEtOH formation was measured. Data presented are means±s.e. of at least six independent experiments carried out in triplicate. *P<0.05 compared with no addition of activators. PtdEtOH, phosphatidylethanol.

Mentions: Next, the ability of CtBP1/BARS to activate PLD was tested in a purified in vitro system. In contrast to bacterial and plant PLDs, mammalian enzyme is latent in its activity in a purified system and requires several cofactors such as phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) (Brown et al, 1993) and phosphatidylethanolamine (Nakamura et al, 1996) to express high activity. For this reason, the effect of CtBP1/BARS on PLD activity was measured in mixed phospholipid vesicles (Brown et al, 1993). Importantly, CtBP1/BARS doubled PLD1 activity (Figure 9A). PLD2 activity was unaffected by CtBP1/BARS (Figure 9B). These results are consistent with the observation that association of CtBP1/BARS with PLD1 but not PLD2 increased after serum stimulation of COS7 cells (Figure 7). The activation of PLD1 by CtBP1/BARS was further characterized by using various combinations of cofactors. ARF1, a small G protein implicated in macropinocytosis (Cohen et al, 2007) and a well-characterized subtype known to activate PLD, was also tested for its ability to stimulate PLD in concert with CtBP1/BARS. When both CtBP1/BARS and ARF1 were added in the PLD assay, synergistic activation was observed in a manner dependent on GTPγS or GTP but not on GDP (Figure 9C), suggesting that CtBP1/BARS synergizes with the active form of ARF1. As ARF6 is also considered to be an important regulator for macropinocytosis (Donaldson et al, 2009) along with the observation of ARF6 accumulation surrounding the newly formed macropinosomes (see Supplementary Figure 2), the ability of this ARF subtype was also assessed for PLD activation with other known PLD activators, RhoA (Bowman et al, 1993; Malcolm et al, 1994) and protein kinase Cα (PKCα) (Conricode et al, 1992; Singer et al, 1996). ARF6- and RhoA-supported PLD activity was further stimulated by CtBP1/BARS to 2.8- and 2.1-fold, respectively, although further stimulation of PKCα-supported PLD activity was not further enhanced by CtBP1/BARS (Figure 9D). Interestingly, simultaneous addition of all these proteins caused a robust activation of PLD1, suggesting that CtBP1/BARS causes a synergistic activation of PLD1 with other activators and that CtBP1/BARS is acting on a site of PLD1 distinct from the sites for previously reported PLD activators. Furthermore, it has recently been reported that during EGF-induced macropinocytosis, CtBP1/BARS undergoes p21-activated kinase-dependent phosphorylation at Ser-147, which is important for translocation to the macropinocytic cup and its surrounding membranes and subsequent fission of the macropinocytic cup (Liberali et al, 2008). To explain these phenomena through PLD-mediated signalling, we prepared the phosphorylation mimicking mutant of CtBP1/BARS, CtBP1/BARS (S147D) and tested its ability to stimulate PLD activity. As expected, this mutant showed about two-fold higher capacity to stimulate PLD1 as compared with wild-type CtBP1/BARS (Figure 9D).


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)

Activation of PLD1 by CtBP1/BARS in a purified recombinant protein system. PLD1 (A) and PLD2 (B) activities were measured as a function of time in the absence or presence of 10 nM CtBP1/BARS using mixed phospholipid vesicles. PLD1 activity was reconstituted with various combinations of 1 nM CtBP1/BARS and 50 nM ARF1 in the absence or presence of 100 μM guanine nucleotides (C) or with various combinations of 1 nM CtBP1/BARS, 1 nM CtBP1/BARS(S147D), 4 nM ARF6, 300 nM RhoA and 100 nM PKCα (D). Enzymatic reactions proceeded for 20 min and PtdEtOH formation was measured. Data presented are means±s.e. of at least six independent experiments carried out in triplicate. *P<0.05 compared with no addition of activators. PtdEtOH, phosphatidylethanol.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f9: Activation of PLD1 by CtBP1/BARS in a purified recombinant protein system. PLD1 (A) and PLD2 (B) activities were measured as a function of time in the absence or presence of 10 nM CtBP1/BARS using mixed phospholipid vesicles. PLD1 activity was reconstituted with various combinations of 1 nM CtBP1/BARS and 50 nM ARF1 in the absence or presence of 100 μM guanine nucleotides (C) or with various combinations of 1 nM CtBP1/BARS, 1 nM CtBP1/BARS(S147D), 4 nM ARF6, 300 nM RhoA and 100 nM PKCα (D). Enzymatic reactions proceeded for 20 min and PtdEtOH formation was measured. Data presented are means±s.e. of at least six independent experiments carried out in triplicate. *P<0.05 compared with no addition of activators. PtdEtOH, phosphatidylethanol.
Mentions: Next, the ability of CtBP1/BARS to activate PLD was tested in a purified in vitro system. In contrast to bacterial and plant PLDs, mammalian enzyme is latent in its activity in a purified system and requires several cofactors such as phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) (Brown et al, 1993) and phosphatidylethanolamine (Nakamura et al, 1996) to express high activity. For this reason, the effect of CtBP1/BARS on PLD activity was measured in mixed phospholipid vesicles (Brown et al, 1993). Importantly, CtBP1/BARS doubled PLD1 activity (Figure 9A). PLD2 activity was unaffected by CtBP1/BARS (Figure 9B). These results are consistent with the observation that association of CtBP1/BARS with PLD1 but not PLD2 increased after serum stimulation of COS7 cells (Figure 7). The activation of PLD1 by CtBP1/BARS was further characterized by using various combinations of cofactors. ARF1, a small G protein implicated in macropinocytosis (Cohen et al, 2007) and a well-characterized subtype known to activate PLD, was also tested for its ability to stimulate PLD in concert with CtBP1/BARS. When both CtBP1/BARS and ARF1 were added in the PLD assay, synergistic activation was observed in a manner dependent on GTPγS or GTP but not on GDP (Figure 9C), suggesting that CtBP1/BARS synergizes with the active form of ARF1. As ARF6 is also considered to be an important regulator for macropinocytosis (Donaldson et al, 2009) along with the observation of ARF6 accumulation surrounding the newly formed macropinosomes (see Supplementary Figure 2), the ability of this ARF subtype was also assessed for PLD activation with other known PLD activators, RhoA (Bowman et al, 1993; Malcolm et al, 1994) and protein kinase Cα (PKCα) (Conricode et al, 1992; Singer et al, 1996). ARF6- and RhoA-supported PLD activity was further stimulated by CtBP1/BARS to 2.8- and 2.1-fold, respectively, although further stimulation of PKCα-supported PLD activity was not further enhanced by CtBP1/BARS (Figure 9D). Interestingly, simultaneous addition of all these proteins caused a robust activation of PLD1, suggesting that CtBP1/BARS causes a synergistic activation of PLD1 with other activators and that CtBP1/BARS is acting on a site of PLD1 distinct from the sites for previously reported PLD activators. Furthermore, it has recently been reported that during EGF-induced macropinocytosis, CtBP1/BARS undergoes p21-activated kinase-dependent phosphorylation at Ser-147, which is important for translocation to the macropinocytic cup and its surrounding membranes and subsequent fission of the macropinocytic cup (Liberali et al, 2008). To explain these phenomena through PLD-mediated signalling, we prepared the phosphorylation mimicking mutant of CtBP1/BARS, CtBP1/BARS (S147D) and tested its ability to stimulate PLD activity. As expected, this mutant showed about two-fold higher capacity to stimulate PLD1 as compared with wild-type CtBP1/BARS (Figure 9D).

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