Proteomic identification and functional characterization of a novel ARF6 GTPase-activating protein, ACAP4.
ARF6 GTPase is a conserved regulator of membrane trafficking and actin-based cytoskeleton dynamics at the leading edge of migrating cells. A key determinant of ARF6 function is the lifetime of the GTP-bound active state, which is orchestrated by GTPase-activating protein (GAP) and GTP-GDP exchanging factor. However, very little is known about the molecular mechanisms underlying ARF6-mediated cell migration. To systematically analyze proteins that regulate ARF6 activity during cell migration, we performed a proteomic analysis of proteins selectively bound to active ARF6 using mass spectrometry and identified a novel ARF6-specific GAP, ACAP4. ACAP4 encodes 903 amino acids and contains two coiled coils, one pleckstrin homology domain, one GAP motif, and two ankyrin repeats. Our biochemical characterization demonstrated that ACAP4 has a phosphatidylinositol 4,5-bisphosphate-dependent GAP activity specific for ARF6. The co-localization of ACAP4 with ARF6 occurred in ruffling membranes formed upon AIF(4) and epidermal growth factor stimulation. ACAP4 overexpression limited the recruitment of ARF6 to the membrane ruffles in the absence of epidermal growth factor stimulation. Expression of GTP hydrolysis-resistant ARF6(Q67L) resulted in accumulations of ACAP4 and ARF6 in the cytoplasmic membrane, suggesting that GTP hydrolysis is required for the ARF6-dependent membrane remodeling. Significantly the depletion of ACAP4 by small interfering RNA or inhibition of ARF6 GTP hydrolysis by overexpressing GAP-deficient ACAP4 suppressed ARF6-dependent cell migration in wound healing, demonstrating the importance of ACAP4 in cell migration. Thus, our study sheds new light on the biological function of ARF6-mediated cell migration.
Fang
Zhiyou
Z
Laboratory of Cellular Dynamics, Hefei National Laboratory for Physical Sciences at Microscale, Hefei 230027, China.
Miao
Yong
Y
Ding
Xia
X
Deng
Hui
H
Liu
Siqi
S
Wang
Fengsong
F
Zhou
Rihong
R
Watson
Charles
C
Fu
Chuanhai
C
Hu
Qicong
Q
Lillard
James W
JW
Powell
Michael
M
Chen
Yong
Y
Forte
John G
JG
Yao
Xuebiao
X
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D017607:Aluminum Compounds / Q000737:chemistry; D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology*; D003599:Cytoskeleton / Q000502:physiology; D004815:Epidermal Growth Factor / Q000378:metabolism; D005459:Fluorides / Q000737:chemistry; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D011485:Protein Binding; D040901:Proteomics
D000089982:ADP-Ribosylation Factor 6; D000199:Actins; D017607:Aluminum Compounds; D020690:GTPase-Activating Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; C050992:tetrafluoroaluminate; D004815:Epidermal Growth Factor; C488555:ASAP3 protein, human; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D005459:Fluorides