Huang Yi-Wei, Surka Mark C, Reynaud Denis, Pace-Asciak Cecil, Trimble William S
Program in Cell Biology, Hospital for Sick Children, Toronto, ON, Canada.
FEBS J. 2006 Jul;273(14):3248-60. doi: 10.1111/j.1742-4658.2006.05333.x.
Septins are a family of conserved proteins that are essential for cytokinesis in a wide range of organisms including fungi, Drosophila and mammals. In budding yeast, where they were first discovered, they are thought to form a filamentous ring at the bridge between the mother and bud cells. What regulates the assembly and function of septins, however, has remained obscure. All septins share a highly conserved domain related to those found in small GTPases, and septins have been shown to bind and hydrolyze GTP, although the properties of this domain and the relationship between polymerization and GTP binding/hydrolysis is unclear. Here we show that human septin 2 is phosphorylated in vivo at Ser218 by casein kinase II. In addition, we show that recombinant septin 2 binds guanine nucleotides with a Kd of 0.28 microm for GTPgammaS and 1.75 microm for GDP. It has a slow exchange rate of 7 x 10(-5) s(-1) for GTPgammaS and 5 x 10(-4) s(-1) for GDP, and an apparent kcat value of 2.7 x 10(-4) s(-1), similar to those of the Ras superfamily of GTPases. Interestingly, the nucleotide binding affinity appears to be altered by phosphorylation at Ser218. Finally, we show that a single septin protein can form homotypic filaments in vitro, whether bound to GDP or GTP.
Septins是一类保守的蛋白质，对于包括真菌、果蝇和哺乳动物在内的多种生物体的胞质分裂至关重要。在首次发现它们的芽殖酵母中，它们被认为在母细胞和芽细胞之间的桥处形成丝状环。然而，是什么调节Septins的组装和功能仍不清楚。所有Septins都共享一个与小GTP酶中发现的结构域高度保守的结构域，并且已证明Septins能结合并水解GTP，尽管该结构域的特性以及聚合与GTP结合/水解之间的关系尚不清楚。在这里，我们表明人Septin 2在体内被酪蛋白激酶II磷酸化于Ser218位点。此外，我们表明重组Septin 2结合鸟嘌呤核苷酸，对GTPγS的Kd为0.28微摩尔，对GDP的Kd为1.75微摩尔。它对GTPγS的交换速率为7×10⁻⁵ s⁻¹，对GDP的交换速率为5×10⁻⁴ s⁻¹，表观kcat值为2.7×10⁻⁴ s⁻¹，与GTP酶的Ras超家族相似。有趣的是，核苷酸结合亲和力似乎因Ser218位点的磷酸化而改变。最后，我们表明单个Septin蛋白在体外无论结合GDP还是GTP都能形成同型丝。
