The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins

被引:137
作者
Zhang, BL
Zhang, YQ
Wang, ZX
Zheng, Y [1 ]
机构
[1] Univ Tennessee, Dept Biochem, Memphis, TN 38163 USA
[2] Acad Sinica, Inst Biophys, Beijing 100101, Peoples R China
关键词
D O I
10.1074/jbc.M001027200
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
The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg2+ cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rad, and RhoA. Differing from Ras and Rab proteins, which require Mg2+ for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg2+ similarly, with dissociation constants in the submicromolar concentration. The presence of Mg2+, however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides. The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg2+, and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg2+, suggesting that one role of GEF is to displace bound Mg2+ from the Rho proteins. The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg2+, indicating that the GEF-catalyzed nucleotide exchange involves a Mg2+-independent as well as a Mg2+-dependent mechanism. Although Mg2+ is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4-10-fold upon binding to Mg2+, and k(cat) values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg2+ was present. Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg2+ cofactor. These studies directly demonstrate a role of Mg2+ in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases, The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg2+, whereas RhoGAP utilizes the Mg2+ cofactor to achieve high catalytic efficiency and specificity.
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收藏
页码:25299 / 25307
页数:9
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共 47 条
[1]   Structure and mutagenesis of the Dbl homology domain [J].
Aghazadeh, B ;
Zhu, K ;
Kubiseski, TJ ;
Liu, GA ;
Pawson, T ;
Zheng, Y ;
Rosen, MK .
NATURE STRUCTURAL BIOLOGY, 1998, 5 (12) :1098-1107
[2]   A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the β-phosphate to destabilize GDP on ARF1 [J].
Béraud-Dufour, S ;
Robineau, S ;
Chardin, P ;
Paris, S ;
Chabre, M ;
Cherfils, J ;
Antonny, B .
EMBO JOURNAL, 1998, 17 (13) :3651-3659
[3]   PROTEINS REGULATING RAS AND ITS RELATIVES [J].
BOGUSKI, MS ;
MCCORMICK, F .
NATURE, 1993, 366 (6456) :643-654
[4]   REGULATION OF THE PHAGOCYTE RESPIRATORY BURST BY SMALL GTP-BINDING PROTEINS [J].
BOKOCH, GM .
TRENDS IN CELL BIOLOGY, 1995, 5 (03) :109-113
[5]   The structural basis of the activation of Ras by Sos [J].
Boriack-Sjodin, PA ;
Margarit, SM ;
Bar-Sagi, D ;
Kuriyan, J .
NATURE, 1998, 394 (6691) :337-343
[6]   G proteins - The arginine finger strikes again [J].
Bourne, HR .
NATURE, 1997, 389 (6652) :673-674
[7]   INTERACTIONS OF THE RAS-LIKE PROTEIN-P25RAB3A WITH MG2+ AND GUANINE-NUCLEOTIDES [J].
BURSTEIN, ES ;
MACARA, IG .
BIOCHEMICAL JOURNAL, 1992, 282 :387-392
[8]   The Dbl family of oncogenes [J].
Cerione, RA ;
Zheng, Y .
CURRENT OPINION IN CELL BIOLOGY, 1996, 8 (02) :216-222
[9]   GTPASE CASCADES CHOREOGRAPHING CELLULAR BEHAVIOR - MOVEMENT, MORPHOGENESIS, AND MORE [J].
CHANT, J ;
STOWERS, L .
CELL, 1995, 81 (01) :1-4
[10]   GEFs: structural basis for their activation of small GTP-binding proteins [J].
Cherfils, J ;
Chardin, P .
TRENDS IN BIOCHEMICAL SCIENCES, 1999, 24 (08) :306-311