Yohe Marielle E, Rossman Kent L, Gardner Olivia S, Karnoub Antoine E, Snyder Jason T, Gershburg Svetlana, Graves Lee M, Der Channing J, Sondek John
Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599-7295, USA.
J Biol Chem. 2007 May 4;282(18):13813-23. doi: 10.1074/jbc.M700185200. Epub 2007 Mar 2.
Dbl-related oncoproteins are guanine nucleotide exchange factors specific for Rho-family GTPases and typically possess tandem Dbl homology (DH) and pleckstrin homology domains that act in concert to catalyze exchange. Because the ability of many Dbl-family proteins to catalyze exchange is constitutively activated by truncations N-terminal to their DH domains, it has been proposed that the activity of Dbl-family proteins is regulated by auto-inhibition. However, the exact mechanisms of regulation of Dbl-family proteins remain poorly understood. Here we show that the Dbl-family protein, Tim, is auto-inhibited by a short, helical motif immediately N-terminal to its DH domain, which directly occludes the catalytic surface of the DH domain to prevent GTPase activation. Similar to the distantly related Vav isozymes, auto-inhibition of Tim is relieved by truncation, mutation, or phosphorylation of the auto-inhibitory helix. A peptide comprising the helical motif inhibits the exchange activity of Tim in vitro. Furthermore, substitutions within the most highly conserved surface of the DH domain designed to disrupt interactions with the auto-inhibitory helix also activate the exchange process.
与Dbl相关的癌蛋白是Rho家族GTP酶特异的鸟嘌呤核苷酸交换因子，通常拥有串联的Dbl同源（DH）结构域和普列克底物蛋白同源结构域，二者协同作用以催化交换反应。由于许多Dbl家族蛋白催化交换的能力被其DH结构域N端的截短所组成性激活，因此有人提出Dbl家族蛋白的活性受自身抑制调节。然而，Dbl家族蛋白的确切调节机制仍知之甚少。在此我们表明，Dbl家族蛋白Tim被其DH结构域紧邻N端的一个短螺旋基序自身抑制，该基序直接封闭DH结构域的催化表面以防止GTP酶激活。与远亲的Vav同工酶类似，Tim的自身抑制通过自身抑制螺旋的截短、突变或磷酸化而解除。包含该螺旋基序的肽在体外抑制Tim的交换活性。此外，在DH结构域最保守表面进行的旨在破坏与自身抑制螺旋相互作用的替换也激活了交换过程。
