Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 2011 Nov 18;286(46):40354-64. doi: 10.1074/jbc.M111.258707. Epub 2011 Sep 27.
The zinc finger transcription factor Krüppel-like factor 5 (KLF5) is regulated posttranslationally. We identified SMAD ubiquitination regulatory factor 2 (SMURF2), an E3 ubiquitin ligase, as an interacting protein of KLF5 by yeast two-hybrid screen, coimmunoprecipitation, and indirect immunofluorescence studies. The SMURF2-interacting domains in KLF5 were mapped to its carboxyl terminus, including the PY motif of KLF5 and its zinc finger DNA-binding domain. KLF5 protein levels were reduced significantly upon overexpression of SMURF2 but not catalytically inactive SMURF2-C716A mutant or SMURF1. SMURF2 alone reduced the protein stability of KLF5 as shown by cycloheximide chase assay, indicating that SMURF2 specifically destabilizes KLF5. In contrast, KLF5(1-165), a KLF5 amino-terminal construct that lacks the PY motif and DNA binding domain, was not degraded by SMURF2. The degradation of KLF5 by SMURF2 was blocked by the proteasome inhibitor MG132, and SMURF2 efficiently ubiquitinated both overexpressed and endogenous KLF5. In contrast, knocking down SMURF2 by siRNAs significantly enhanced KLF5 protein levels, reduced ubiquitination of KLF5, and increased the expression of cyclin D1 and PDGF-A, two established KLF5 target genes. In consistence, SMURF2, but not the E3 ligase mutant SMURF2-C716A, significantly inhibited the transcriptional activity of KLF5, as demonstrated by dual luciferase assay using the PDGF-A promoter, and suppressed the ability of KLF5 to stimulate cell proliferation as measured by BrdU incorporation. Hence, SMURF2 is a novel E3 ubiquitin ligase for KLF5 and negatively regulates KLF5 by targeting it for proteasomal degradation.
锌指转录因子 Krüppel 样因子 5（KLF5）受翻译后调控。我们通过酵母双杂交筛选、共免疫沉淀和间接免疫荧光研究，发现 SMAD 泛素化调节因子 2（SMURF2）是 KLF5 的相互作用蛋白。KLF5 与 SMURF2 的相互作用结构域定位于其羧基末端，包括 KLF5 的 PY 基序及其锌指 DNA 结合结构域。过表达 SMURF2 可显著降低 KLF5 蛋白水平，但过表达无催化活性的 SMURF2-C716A 突变体或 SMURF1 则没有这种作用。SMURF2 本身通过环己酰亚胺追踪实验降低 KLF5 的蛋白稳定性，表明 SMURF2 特异性地使 KLF5 不稳定。相比之下，KLF5(1-165)，一种缺乏 PY 基序和 DNA 结合结构域的 KLF5 氨基末端构建体，不受 SMURF2 降解。SMURF2 可有效泛素化过表达和内源性 KLF5，且其对 KLF5 的降解可被蛋白酶体抑制剂 MG132 阻断。相反，siRNA 敲低 SMURF2 可显著提高 KLF5 蛋白水平，降低 KLF5 的泛素化，并增加两个已确立的 KLF5 靶基因 cyclin D1 和 PDGF-A 的表达。与预期一致，SMURF2（而非 E3 连接酶突变体 SMURF2-C716A）可显著抑制 PDGF-A 启动子的双荧光素酶报告基因检测中的 KLF5 转录活性，并抑制 KLF5 刺激细胞增殖的能力（通过 BrdU 掺入来衡量）。因此，SMURF2 是 KLF5 的一种新型 E3 泛素连接酶，通过靶向 KLF5 进行蛋白酶体降解来负调控 KLF5。
