Zhang Yan, Fillmore Rebecca A, Zimmer Warren E
Department of Systems Biology and Translational Medicine, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843-1114, USA.
Exp Biol Med (Maywood). 2008 Mar;233(3):297-309. doi: 10.3181/0709-RM-236.
Nkx3.1 is a member of the NK2 class of homeodomain proteins and is expressed in development, being an early marker of the sclerotome and prostate gland. It has been shown to be a critical factor for prostate differentiation and function. Previous studies suggested that Nkx3.1 interacts with Serum Response Factor (SRF) to transactivate the Smooth Muscle gamma-Actin (SMGA) promoter. In studies presented here, we examined the molecular mechanisms underlying the functional synergy of these factors upon SMGA transcription. We demonstrate that full length Nkx3.1 physically interacts with SRF in the absence of DNA and that these factors are able to co-associate in cellular context using a mammalian two-hybrid system. The segment of SRF responsible for Nkx3.1 interaction was mapped to a approximately 30 amino acid region (AAs 142-171) at the N-terminal segment of the MADS box. Two separate regions of Nkx3.1 were found to mediate interactions with SRF. Interestingly, recognized domains of NK2 proteins, namely the TN, homeodomain DNA binding segment, and the NK2-SD do not participate in SRF interactions. One of the Nkx3.1 SRF binding domains was mapped to the N-terminal of the protein consistent with recent studies of these proteins using NMR spectroscopy by Gelmann and colleagues (1). A second SRF binding region was mapped to amino acids C-terminal to the homeodomain. Structural predictions indicate that both of the SRF interacting segments are largely hydrophobic in character and beta-strand in structure. With co-transfection transcriptional analyses we found that interaction between SRF and Nkx3.1 as well as DNA binding by both factors was required for the observed transcriptional synergy. Thus our studies have identified novel protein-protein interacting domains within Nkx3.1 and SRF that operate in concert with their respective DNA binding domains to mediate functional transcriptional synergy of these factors to regulate SMGA gene activation.
Nkx3.1是同源结构域蛋白NK2家族的成员，在发育过程中表达，是生骨节和前列腺的早期标志物。它已被证明是前列腺分化和功能的关键因素。先前的研究表明，Nkx3.1与血清反应因子（SRF）相互作用以反式激活平滑肌γ-肌动蛋白（SMGA）启动子。在本文所呈现的研究中，我们研究了这些因子在SMGA转录时功能协同作用的分子机制。我们证明全长Nkx3.1在没有DNA的情况下与SRF发生物理相互作用，并且这些因子能够在细胞环境中使用哺乳动物双杂交系统共同结合。负责与Nkx3.1相互作用的SRF片段被定位到MADS盒N端的大约30个氨基酸区域（第142 - 171位氨基酸）。发现Nkx3.1的两个不同区域介导与SRF的相互作用。有趣的是，NK2蛋白的公认结构域，即TN、同源结构域DNA结合片段和NK2-SD不参与与SRF的相互作用。与Nkx3.1的SRF结合结构域之一被定位到该蛋白的N端，这与Gelmann及其同事最近使用核磁共振光谱对这些蛋白的研究结果一致（1）。第二个SRF结合区域被定位到同源结构域C端的氨基酸。结构预测表明，两个与SRF相互作用的片段在很大程度上具有疏水性且结构为β链。通过共转染转录分析，我们发现SRF与Nkx3.1之间的相互作用以及两个因子的DNA结合对于观察到的转录协同作用是必需的。因此，我们的研究在Nkx3.1和SRF中鉴定出了新的蛋白质-蛋白质相互作用结构域，它们与其各自的DNA结合结构域协同作用，介导这些因子的功能性转录协同作用以调节SMGA基因激活。
