The non-catalytic carboxyl-terminal domain of ARFGAP1 regulates actin cytoskeleton reorganization by antagonizing the activation of Rac1

Ka Yu Siu, Mei Kuen Yu, Xinggang Wu, Min Zong, Michael G. Roth, Hsiao Chang Chan, Sidney Yu

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Background: The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton. Principal Findings: As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci. Conclusions: ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein.

Original languageEnglish (US)
Article numbere18458
JournalPLoS One
Volume6
Issue number4
DOIs
StatePublished - 2011

Fingerprint

microfilaments
Actin Cytoskeleton
Actins
Chemical activation
actin
cells
GTPase-activating proteins
GTPase-Activating Proteins
Membranes
phosphatidylinositol kinases
Blister
Phosphatidylinositols
G-proteins
catalytic activity
GTP-Binding Proteins
cell movement
Plating
active sites
traffic
Polymerization

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

The non-catalytic carboxyl-terminal domain of ARFGAP1 regulates actin cytoskeleton reorganization by antagonizing the activation of Rac1. / Siu, Ka Yu; Yu, Mei Kuen; Wu, Xinggang; Zong, Min; Roth, Michael G.; Chan, Hsiao Chang; Yu, Sidney.

In: PLoS One, Vol. 6, No. 4, e18458, 2011.

Research output: Contribution to journalArticle

Siu, Ka Yu ; Yu, Mei Kuen ; Wu, Xinggang ; Zong, Min ; Roth, Michael G. ; Chan, Hsiao Chang ; Yu, Sidney. / The non-catalytic carboxyl-terminal domain of ARFGAP1 regulates actin cytoskeleton reorganization by antagonizing the activation of Rac1. In: PLoS One. 2011 ; Vol. 6, No. 4.
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abstract = "Background: The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton. Principal Findings: As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci. Conclusions: ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein.",
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T1 - The non-catalytic carboxyl-terminal domain of ARFGAP1 regulates actin cytoskeleton reorganization by antagonizing the activation of Rac1

AU - Siu, Ka Yu

AU - Yu, Mei Kuen

AU - Wu, Xinggang

AU - Zong, Min

AU - Roth, Michael G.

AU - Chan, Hsiao Chang

AU - Yu, Sidney

PY - 2011

Y1 - 2011

N2 - Background: The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton. Principal Findings: As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci. Conclusions: ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein.

AB - Background: The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton. Principal Findings: As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci. Conclusions: ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein.

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