Dept. Biochemistry, Eötvös Loránd University, Budapest, Hungary.
Biochem Biophys Res Commun. 2011 Oct 28;414(3):493-8. doi: 10.1016/j.bbrc.2011.09.093. Epub 2011 Sep 24.
LC8 dynein light chain (now termed DYNLL1 and DYNLL2 in mammals), a dimeric 89 amino acid protein, is a component of the dynein multi-protein complex. However a substantial amount of DYNLL1 is not associated to microtubules and it can thus interact with dozens of cellular and viral proteins that display well-defined, short linear motifs. Using DYNLL1 as bait in a yeast two-hybrid screen of a human heart library we identified ATMIN, an ATM kinase-interacting protein, as a DYNLL1-binding partner. Interestingly, ATMIN displays at least 18 SQ/TQ motifs in its sequence and DYNLL1 is known to bind to proteins with KXTQT motifs. Using pepscan and yeast two-hybrid techniques we show that DYNLL1 binds to multiple SQ/TQ motifs present in the carboxy-terminal domain of ATMIN. Recombinant expression and purification of the DYNLL1-binding region of ATMIN allowed us to obtain a polypeptide with an apparent molecular mass in gel filtration close to 400 kDa that could bind to DYNLL1 in vitro. The NMR data-driven modelled complexes of DYNLL1 with two selected ATMIN peptides revealed a similar mode of binding to that observed between DYNLL1 and other peptide targets. Remarkably, co-expression of mCherry-DYNLL1 and GFP-ATMIN mutually affected intracellular protein localization. In GFP-ATMIN expressing-cells DNA damage induced efficiently nuclear foci formation, which was partly impeded by the presence of mCherry-DYNLL1. Thus, our results imply a potential cellular interference between DYNLL1 and ATMIN functions.
LC8 动力蛋白轻链（现在在哺乳动物中称为 DYNLL1 和 DYNLL2），是一种二聚体 89 个氨基酸的蛋白质，是动力蛋白多蛋白复合物的组成部分。然而，大量的 DYNLL1 与微管没有关联，因此可以与数十种具有明确短线性基序的细胞和病毒蛋白相互作用。我们用人心脏文库作为诱饵，在酵母双杂交筛选中使用 DYNLL1 鉴定出 ATMIN，一种 ATM 激酶相互作用蛋白，作为 DYNLL1 的结合伴侣。有趣的是，ATMIN 在其序列中至少显示 18 个 SQ/TQ 基序，而 DYNLL1 已知与具有 KXTQT 基序的蛋白质结合。使用 pepscan 和酵母双杂交技术，我们表明 DYNLL1 结合到 ATMIN 羧基末端结构域中存在的多个 SQ/TQ 基序。重组表达和纯化 ATMIN 的 DYNLL1 结合区使我们能够获得在凝胶过滤中表观分子量接近 400 kDa 的多肽，该多肽可在体外与 DYNLL1 结合。基于 NMR 数据的 DYNLL1 与两个选定的 ATMIN 肽的模型复合物揭示了与在 DYNLL1 与其他肽靶标之间观察到的相似的结合模式。值得注意的是，mCherry-DYNLL1 和 GFP-ATMIN 的共表达相互影响细胞内蛋白质定位。在 GFP-ATMIN 表达细胞中，DNA 损伤有效地诱导核焦点形成，而 mCherry-DYNLL1 的存在部分阻碍了焦点形成。因此，我们的结果表明 DYNLL1 和 ATMIN 功能之间存在潜在的细胞干扰。
