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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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Serum induces association of CtBP1/BARS with native PLD. After serum starvation for 16 h, COS7 cells were stimulated with 100 ng/ml EGF or 10% fetal calf serum for 30 min. Cells were lysed and immunoprecipitated with anti-CtBP1/BARS antibody. Pulled down PLD-associated with CtBP1/BARS was assayed for PtdBut production in the absence or presence of 50 nM ARF. Data presented are means±s.e. of six independent experiments carried out in triplicate. *P<0.05 compared with no addition of agonists.
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f6: Serum induces association of CtBP1/BARS with native PLD. After serum starvation for 16 h, COS7 cells were stimulated with 100 ng/ml EGF or 10% fetal calf serum for 30 min. Cells were lysed and immunoprecipitated with anti-CtBP1/BARS antibody. Pulled down PLD-associated with CtBP1/BARS was assayed for PtdBut production in the absence or presence of 50 nM ARF. Data presented are means±s.e. of six independent experiments carried out in triplicate. *P<0.05 compared with no addition of agonists.

Mentions: It is well known that PLD is activated by a variety of growth factors and cytokines, including EGF and serum, in various types of mammalian cells. The observation that both CtBP1/BARS and PLD were required for EGF-induced macropinocytosis prompted us to assess the causal relationship between the two proteins. First, we studied the effect of CtBP1/BARS on agonist-stimulated PLD activation. When COS7 cells were stimulated with EGF, PLD was activated as assessed by the accumulation of PtdBut in the presence of 1-butanol in culture medium (Figure 5). Serum also induced PLD activation as reported earlier (Billah and Anthes, 1990). Importantly, when cellular CtBP1/BARS was downregulated by CtBP1/BARS-targeted gene silencing, EGF- or serum-induced activation of PLD was strongly attenuated, suggesting that CtBP1/BARS is required for the agonist-induced PLD activation. To gain further insight into the molecular mechanisms underlying the involvement of CtBP1/BARS in agonist-induced PLD activation, we analysed the interaction between CtBP1/BARS and PLD before and after agonist stimulation. Endogenous CtBP1/BARS was immunoprecipitated from COS7 cell lysates prepared from the cells treated with or without EGF or serum and immunoprecipitated beads were subjected to PLD assay. EGF or serum stimulation of COS7 cells caused increase in PLD activity pulled down with CtBP1/BARS-containing beads (Figure 6), indicating that EGF or serum stimulation of cells facilitated the association of the two endogenous proteins in intact cells. In mammals, there are two PLD isoforms, PLD1 and PLD2. To identify the PLD isoform involved in this interaction, COS7 cells were transiently transfected with either PLD1 or PLD2 and endogenous CtBP1/BARS was immunoprecipitated using anti-CtBP1/BARS antibody and associated PLD isoform was identified by immunoblot analysis. In the cells expressing PLD1, CtBP1/BARS was associated with this isoform in a serum-dependent manner (Figure 7A and B). This association was diminished again under low serum conditions (data not shown). These results suggest that PLD1 has the capacity to associate with CtBP1/BARS in a manner reversibly regulated by serum. On the other hand, PLD2 showed relatively higher association with CtBP1/BARS under the basal conditions and the association changed little even under high serum conditions (Figure 7C and D). These results suggest that agonists such as serum may induce CtBP1/BARS association with PLD1 under physiological conditions as seen in Figure 6. To support this notion in an endogenous cell system, PLD activity pulled down by anti-CtBP1/BARS antibody in cell lysates prepared from serum-stimulated cells was stimulated by the small G-protein ARF1 (Figure 6), an activity characteristic of PLD1 (Exton, 1999).


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)

Serum induces association of CtBP1/BARS with native PLD. After serum starvation for 16 h, COS7 cells were stimulated with 100 ng/ml EGF or 10% fetal calf serum for 30 min. Cells were lysed and immunoprecipitated with anti-CtBP1/BARS antibody. Pulled down PLD-associated with CtBP1/BARS was assayed for PtdBut production in the absence or presence of 50 nM ARF. Data presented are means±s.e. of six independent experiments carried out in triplicate. *P<0.05 compared with no addition of agonists.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f6: Serum induces association of CtBP1/BARS with native PLD. After serum starvation for 16 h, COS7 cells were stimulated with 100 ng/ml EGF or 10% fetal calf serum for 30 min. Cells were lysed and immunoprecipitated with anti-CtBP1/BARS antibody. Pulled down PLD-associated with CtBP1/BARS was assayed for PtdBut production in the absence or presence of 50 nM ARF. Data presented are means±s.e. of six independent experiments carried out in triplicate. *P<0.05 compared with no addition of agonists.
Mentions: It is well known that PLD is activated by a variety of growth factors and cytokines, including EGF and serum, in various types of mammalian cells. The observation that both CtBP1/BARS and PLD were required for EGF-induced macropinocytosis prompted us to assess the causal relationship between the two proteins. First, we studied the effect of CtBP1/BARS on agonist-stimulated PLD activation. When COS7 cells were stimulated with EGF, PLD was activated as assessed by the accumulation of PtdBut in the presence of 1-butanol in culture medium (Figure 5). Serum also induced PLD activation as reported earlier (Billah and Anthes, 1990). Importantly, when cellular CtBP1/BARS was downregulated by CtBP1/BARS-targeted gene silencing, EGF- or serum-induced activation of PLD was strongly attenuated, suggesting that CtBP1/BARS is required for the agonist-induced PLD activation. To gain further insight into the molecular mechanisms underlying the involvement of CtBP1/BARS in agonist-induced PLD activation, we analysed the interaction between CtBP1/BARS and PLD before and after agonist stimulation. Endogenous CtBP1/BARS was immunoprecipitated from COS7 cell lysates prepared from the cells treated with or without EGF or serum and immunoprecipitated beads were subjected to PLD assay. EGF or serum stimulation of COS7 cells caused increase in PLD activity pulled down with CtBP1/BARS-containing beads (Figure 6), indicating that EGF or serum stimulation of cells facilitated the association of the two endogenous proteins in intact cells. In mammals, there are two PLD isoforms, PLD1 and PLD2. To identify the PLD isoform involved in this interaction, COS7 cells were transiently transfected with either PLD1 or PLD2 and endogenous CtBP1/BARS was immunoprecipitated using anti-CtBP1/BARS antibody and associated PLD isoform was identified by immunoblot analysis. In the cells expressing PLD1, CtBP1/BARS was associated with this isoform in a serum-dependent manner (Figure 7A and B). This association was diminished again under low serum conditions (data not shown). These results suggest that PLD1 has the capacity to associate with CtBP1/BARS in a manner reversibly regulated by serum. On the other hand, PLD2 showed relatively higher association with CtBP1/BARS under the basal conditions and the association changed little even under high serum conditions (Figure 7C and D). These results suggest that agonists such as serum may induce CtBP1/BARS association with PLD1 under physiological conditions as seen in Figure 6. To support this notion in an endogenous cell system, PLD activity pulled down by anti-CtBP1/BARS antibody in cell lysates prepared from serum-stimulated cells was stimulated by the small G-protein ARF1 (Figure 6), an activity characteristic of PLD1 (Exton, 1999).

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