The Pleckstrin Homology (PH) Domain of the Arf Exchange Factor Brag2 Is an Allosteric Binding Site

被引:28
|
作者
Jian, Xiaoying [1 ]
Gruschus, James M. [2 ]
Sztul, Elizabeth [3 ]
Randazzo, Paul A. [1 ]
机构
[1] NCI, Lab Cellular & Mol Biol, Ctr Canc Res, Bethesda, MD 20892 USA
[2] NHLBI, Lab Mol Biophys, NIH, Bethesda, MD 20892 USA
[3] Univ Alabama Birmingham, Dept Cell Dev & Integrat Biol, Birmingham, AL 35294 USA
基金
美国国家卫生研究院; 美国国家科学基金会;
关键词
ADP-RIBOSYLATION FACTOR; GUANINE-NUCLEOTIDE-EXCHANGE; AMINO-TERMINUS; ACTIN CYTOSKELETON; MYRISTOYLATED ARF1; KINETIC MECHANISM; STRUCTURAL BASIS; CELL-ADHESION; FACTOR ARNO; G-PROTEINS;
D O I
10.1074/jbc.M112.368084
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Brag2, a Sec7 domain (sec7d)-containing guanine nucleotide exchange factor, regulates cell adhesion and tumor cell invasion. Brag2 catalyzes nucleotide exchange, converting Arf.GDP to Arf.GTP. Brag2 contains a pleckstrin homology (PH) domain, and its nucleotide exchange activity is stimulated by phosphatidylinositol 4,5-bisphosphate (PIP2). Here we determined kinetic parameters for Brag2 and examined the basis for regulation by phosphoinositides. Using myristoylated Arf1.GDP as a substrate, the k(cat) was 1.8 +/- 0.1/s as determined by single turnover kinetics, and the K-m was 0.20 +/- 0.07 mu M as determined by substrate saturation kinetics. PIP2 decreased the K-m and increased the k(cat) of the reaction. The effect of PIP2 required the PH domain of Brag2 and the N terminus of Arf and was largely independent of Arf myristoylation. Structural analysis indicated that the linker between the sec7d and the PH domain in Brag2 may directly contact Arf. In support, we found that a Brag2 fragment containing the sec7d and the linker was more active than sec7d alone. We conclude that Brag2 is allosterically regulated by PIP2 binding to the PH domain and that activity depends on the interdomain linker. Thus, the PH domain and the interdomain linker of Brag2 may be targets for selectively regulating the activity of Brag2.
引用
收藏
页码:24273 / 24283
页数:11
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