The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, P.O.B. 653, Be'er-Sheva 84105, Israel; The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, P.O.B. 653, Be'er-Sheva 84105, Israel.
Department of Chemical Engineering, Ben-Gurion University of the Negev, P.O.B. 653, Be'er-Sheva 84105, Israel.
J Mol Biol. 2018 Oct 19;430(21):4359-4368. doi: 10.1016/j.jmb.2018.08.028. Epub 2018 Sep 4.
Signaling via lysine methylation by protein lysine methyltransferases (PKMTs), has been linked to diverse biological and disease processes. The mono-methyltransferase SETD6 (SET-domain-containing protein 6) is a member of the PKMT family and was previously shown to regulate essential cellular processes such as the NF-κB, WNT and the oxidative stress pathways. However, on the biochemical level, little is known about the enzymatic mode of action of SETD6. Here we provide evidence that SETD6 forms high-molecular-weight structures. Specifically, we demonstrate that SETD6 monomeric, dimeric and trimeric forms are stabilized by the methyl donor, S-adenosyl-l-methionine. We then show that SETD6 has auto-methylation activity at K39 and K179, which serves as the major auto-methylation sites with a moderate auto-methylation activity toward K372. A point mutation at K179 but not at K39 and K372, located at the SET domain of SETD6, impaired SETD6 ability to form a trimer, strongly implying a link between the auto-methylation and the oligomerization state. Finally, by radioactive in vitro methylation experiments and biochemical kinetics analysis, we show that the auto-methylation at K39 and K179 increases the catalytic rate of SETD6. Collectively, our data support a model by which SETD6 auto-methylation and self-interaction positively regulate its enzymatic activity in vitro and may suggest that other PKMTs are regulated in the same manner.
蛋白赖氨酸甲基转移酶（PKMTs）通过赖氨酸甲基化进行信号转导，与多种生物和疾病过程有关。单甲基转移酶 SETD6（SET 结构域蛋白 6）是 PKMT 家族的成员，先前已被证明可调节 NF-κB、WNT 和氧化应激途径等重要的细胞过程。然而，在生化水平上，对 SETD6 的酶促作用模式知之甚少。在这里，我们提供了 SETD6 形成高分子量结构的证据。具体来说，我们证明 SETD6 单体、二聚体和三聚体形式通过甲基供体 S-腺苷甲硫氨酸稳定。然后我们表明，SETD6 在 K39 和 K179 处具有自动甲基化活性，这是主要的自动甲基化位点，对 K372 具有中等的自动甲基化活性。位于 SETD6 的 SET 结构域中的 K179 点突变，但不是 K39 和 K372 点突变，会损害 SETD6 形成三聚体的能力，这强烈暗示自动甲基化和寡聚状态之间存在联系。最后，通过放射性体外甲基化实验和生化动力学分析，我们表明 K39 和 K179 的自动甲基化增加了 SETD6 的催化速率。总的来说，我们的数据支持这样一种模型，即 SETD6 的自动甲基化和自我相互作用正向调节其体外的酶活性，并且可能表明其他 PKMT 以相同的方式受到调节。
