Coccetti Paola, Tripodi Farida, Tedeschi Gabriella, Nonnis Simona, Marin Oriano, Fantinato Sonia, Cirulli Claudia, Vanoni Marco, Alberghina Lilia
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy.
Cell Cycle. 2008 May 15;7(10):1391-401. doi: 10.4161/cc.7.10.5825. Epub 2008 Feb 26.
The ubiquitin-conjugating enzyme Cdc34 was recently shown to be phosphorylated by CK2 on the C-terminal tail. Here we present novel findings indicating that in budding yeast CK2 phosphorylates Cdc34 within the N-terminal catalytic domain. Specifically, we show, by direct mass spectrometry analysis, that Cdc34 is phosphorylated in vitro and in vivo by CK2 on Ser130 and Ser167, and that the phosphoserines 130 and 167 are not present after CK2 inactivation in a cka1Deltacka2-8(ts) strain. CK2 phosphorylation of Ser130 and Ser167 strongly stimulates Cdc34 ubiquitin charging in vitro. The Cdc34(S130AS167A) mutant shows a basal ubiquitin charging activity which is indistinguishable from that of wild type but is not activated by CK2 phosphorylation and its expression fails to complement a cdc34-2(ts) yeast strain, supporting a model in which activation of Cdc34 involves CK2-mediated phosphorylation of its catalytic domain.
泛素结合酶Cdc34最近被证明在其C末端尾巴上被CK2磷酸化。在此，我们展示了新的发现，表明在芽殖酵母中，CK2在N末端催化结构域内磷酸化Cdc34。具体而言，我们通过直接质谱分析表明，在体外和体内，Cdc34在Ser130和Ser167位点被CK2磷酸化，并且在cka1Δcka2 - 8(ts)菌株中CK2失活后，磷酸化的丝氨酸130和167不存在。Ser130和Ser167的CK2磷酸化在体外强烈刺激Cdc34的泛素负载。Cdc34(S130A S167A)突变体显示出与野生型无法区分的基础泛素负载活性，但不能被CK2磷酸化激活，并且其表达不能互补cdc34 - 2(ts)酵母菌株，这支持了一种模型，即Cdc34的激活涉及CK2介导的其催化结构域的磷酸化。
