To understand better the roles of ARD1 in cells, we prepared from ARD1-null mice mouse embryo fibroblast (MEF) lines stably transfected with plasmids for mifepristone (Mfp)-inducible expression of wild-type ARD1 protein (KO-WT), or ARD1 protein with inactivating mutations in E3 ligase domain (KO-E3), or containing persistently active GTP-bound (KO-GTP), or inactive GDP-bound (KO-GDP) GTPase domains. Inhibition of proteasomal proteases in mifepristone-induced KO-WT, KO-GDP, or KO-GTP MEFs resulted in accumulation of these ARD1 proteins, whereas KO-E3 accumulated without inhibitors. All data were consistent with the conclusion that ARD1 regulates its own steady state levels in cells by auto-ubiquitination. Based on reported growth factor receptor-cytohesin interactions, epidermal growth factor receptor (EGFR) was investigated in induced MEFs. Amounts of cell-surface and total EGFR were higher in KO-GDP and lower in KO-GTP than in KO-WT MEFs, with levels in both mutants greater (p =0.001) after proteasomal inhibition. Significant differences among MEF lines in content of TGFβreceptor III were similar to those in EGFR, albeit not as large. Differences in amounts of insulin receptor mirrored those in EGFR, but did not reach statistical significance. Overall, the capacity of ARD1 GTPase to cycle between active and inactive forms and its auto-ubiquitination both appear to be necessary for the appropriate turnover of EGFR and perhaps additional growth factor receptors.

Project Start
Project End
Budget Start
Budget End
Support Year
33
Fiscal Year
2011
Total Cost
$2,217,500
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
Zip Code
Chen, Pei-Wen; Jian, Xiaoying; Heissler, Sarah M et al. (2016) The Arf GTPase-activating Protein, ASAP1, Binds Nonmuscle Myosin 2A to Control Remodeling of the Actomyosin Network. J Biol Chem 291:7517-26
Li, Chun-Chun; Le, Kang; Kato, Jiro et al. (2016) Enhancement of ?-catenin activity by BIG1 plus BIG2 via Arf activation and cAMP signals. Proc Natl Acad Sci U S A 113:5946-51
Le, Kang; Li, Chun-Chun; Ye, Guan et al. (2013) Arf guanine nucleotide-exchange factors BIG1 and BIG2 regulate nonmuscle myosin IIA activity by anchoring myosin phosphatase complex. Proc Natl Acad Sci U S A 110:E3162-70
Lin, Sisi; Zhou, Chun; Neufeld, Edward et al. (2013) BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein modulates ATP-binding cassette transporter A-1 trafficking and function. Arterioscler Thromb Vasc Biol 33:e31-8
Shen, Xiaoyan; Li, Chun-Chun; Aponte, Angel M et al. (2012) Brefeldin A-inhibited ADP-ribosylation factor activator BIG2 regulates cell migration via integrin ?1 cycling and actin remodeling. Proc Natl Acad Sci U S A 109:14464-9
Le, Kang; Li, Ruifang; Xu, Suowen et al. (2012) PPAR? activation inhibits endothelin-1-induced cardiomyocyte hypertrophy by prevention of NFATc4 binding to GATA-4. Arch Biochem Biophys 518:71-8
Li, Chun-Chun; Kuo, Jean-Cheng; Waterman, Clare M et al. (2011) Effects of brefeldin A-inhibited guanine nucleotide-exchange (BIG) 1 and KANK1 proteins on cell polarity and directed migration during wound healing. Proc Natl Acad Sci U S A 108:19228-33
Meza-Carmen, Victor; Pacheco-Rodriguez, Gustavo; Kang, Gi Soo et al. (2011) Regulation of growth factor receptor degradation by ADP-ribosylation factor domain protein (ARD) 1. Proc Natl Acad Sci U S A 108:10454-9
Puxeddu, Ermanno; Uhart, Marina; Li, Chun-Chun et al. (2009) Interaction of phosphodiesterase 3A with brefeldin A-inhibited guanine nucleotide-exchange proteins BIG1 and BIG2 and effect on ARF1 activity. Proc Natl Acad Sci U S A 106:6158-63
Shen, Xiaoyan; Meza-Carmen, Victor; Puxeddu, Ermanno et al. (2008) Interaction of brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 and kinesin motor protein KIF21A. Proc Natl Acad Sci U S A 105:18788-93

Showing the most recent 10 out of 12 publications