Littlepage Laurie E, Wu Hua, Andresson Thorkell, Deanehan Julia K, Amundadottir Laufey T, Ruderman Joan V
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2002 Nov 26;99(24):15440-5. doi: 10.1073/pnas.202606599. Epub 2002 Nov 6.
The activity of the kinase Aurora-A (Aur-A) peaks during mitosis and depends on phosphorylation by one or more unknown kinases. Mitotic phosphorylation sites were mapped by mass spec sequencing of recombinant Aur-A protein that had been activated by incubation in extracts of metaphase-arrested Xenopus eggs. Three sites were identified: serine 53 (Ser-53), threonine 295 (Thr-295), and serine 349 (Ser-349), which are equivalent to Ser-51, Thr-288, and Ser-342, respectively, in human Aur-A. To ask how phosphorylation of these residues might affect kinase activity, each was mutated to either alanine or aspartic acid, and the recombinant proteins were then tested for their ability to be activated by M phase extract. Mutation of Thr-295, which resides in the activation loop of the kinase, to either alanine or aspartic acid abolished activity. The S349A mutant had slightly reduced activity, indicating that phosphorylation is not required for activity. The S349D mutation completely blocked activation, suggesting that Ser-349 is important for either the structure or regulation of Aur-A. Finally, like human Aur-A, overexpression of Xenopus Aur-A transformed NIH 3T3 cells and led to tumors in nude mice. These results provide further evidence that Xenopus Aur-A is a functional ortholog of human Aur-A and, along with the recently described crystal structure of human Aur-A, should help in future studies of the mechanisms that regulate Aur-A activity during mitotic progression.
激酶Aurora-A（Aur-A）的活性在有丝分裂期间达到峰值，并且依赖于一种或多种未知激酶的磷酸化作用。通过对在中期阻断的非洲爪蟾卵提取物中孵育而被激活的重组Aur-A蛋白进行质谱测序，绘制了有丝分裂磷酸化位点。鉴定出三个位点：丝氨酸53（Ser-53）、苏氨酸295（Thr-295）和丝氨酸349（Ser-349），它们分别等同于人类Aur-A中的Ser-51、Thr-288和Ser-342。为了探究这些残基的磷酸化如何影响激酶活性，将每个位点突变为丙氨酸或天冬氨酸，然后测试重组蛋白被M期提取物激活的能力。位于激酶激活环中的Thr-295突变为丙氨酸或天冬氨酸会使活性丧失。S349A突变体的活性略有降低，表明活性并不需要磷酸化。S349D突变完全阻断了激活，这表明Ser-349对Aur-A的结构或调节很重要。最后，与人类Aur-A一样，非洲爪蟾Aur-A的过表达使NIH 3T3细胞发生转化，并在裸鼠中引发肿瘤。这些结果提供了进一步的证据，表明非洲爪蟾Aur-A是人类Aur-A的功能直系同源物，并且与最近描述的人类Aur-A晶体结构一起，应该有助于未来对有丝分裂进程中调节Aur-A活性机制的研究。
