Ribonucleic acid polymerase II binding subunit 3 (Rpb3), a potential nuclear target of insulin-like growth factor binding protein-3
Ribonucleic acid polymerase II binding subunit 3 (Rpb3), a potential nuclear target of insulin-like growth factor binding protein-3
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DOI：
10.1210/en.2005-1269
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发表时间：
2006-05-01
期刊：
影响因子：
4.8
通讯作者：
Rodgers, BD
中科院分区：
文献类型：
--
作者：
Oufattole, M;Lin, SWJ;Rodgers, BD
胰岛素样生长因子结合蛋白（IGFBP）-3具有内在的抗增殖和促凋亡的功能，是独立的IGF结合，并可能涉及核定位。我们确定，外源性IGFBP-3迅速易位到成肌细胞核和22个残基的肽含有金属结合结构域（MBD）和核定位序列（NLS）可以类似地指导嵌合GFP成肌细胞核。此外，非IGF结合IGFBP-3突变体抑制成肌细胞增殖而不刺激凋亡。这些结果表明，IGFBP-3抑制肌肉细胞生长的IGF-独立的方式，可能是其快速核定位的影响。因此，我们通过使用酵母双杂交试验和两个N端缺失突变体作为诱饵：BP 3/231（231个残基，L 61至K291）和BP 3/111（K181-K291）筛选大鼠L 6成肌细胞cDNA文库来鉴定IGFBP-3相互作用蛋白。先前已知与IGFBP-3相互作用的蛋白质以及几种新的蛋白质被鉴定，包括RNA聚合酶II结合亚基3（Rpb 3）。随后使用不同的IGFBP-3缺失突变体鉴定Rpb 3结合所需的结构域，并将其定位于MBD/NLS表位。Rpb 3/IGFBP-3的结合被证实通过免疫共沉淀试验与特定的抗血清，而一个NLS突变体IGFBP-3不与Rpb 3，这表明一个功能性NLS是必需的。Rpb 3促进聚合酶复合物向特定转录因子的募集，并且对于许多基因的反式激活是必需的。它与IGFBP-3的结合为IGFBP-3在基因转录的直接调节中提供了功能性作用。
IGF-binding protein (IGFBP)-3 has intrinsic antiproliferative and proapoptotic functions that are independent of IGF binding and may involve nuclear localization. We determined that exogenous IGFBP-3 rapidly translocates to myoblast nuclei and that a 22-residue peptide containing the metal binding domain (MBD) and nuclear localization sequence (NLS) can similarly direct chimeric GFP into myoblast nuclei. Furthermore, a non-IGF-binding IGFBP-3 mutant inhibited myoblast proliferation without stimulating apoptosis. These results suggest that IGFBP-3 inhibits muscle cell growth in an IGF-independent manner that may be influenced by its rapid nuclear localization. We therefore identified IGFBP-3 interacting proteins by screening a rat L6 myoblast cDNA library using the yeast two-hybrid assay and two N-terminal deletion mutants as bait: BP3/231 (231 residues, L61 to K291) and BP3/111 (K181-K291). Proteins previously known to interact with IGFBP-3 as well as several novel proteins were identified, including RNA polymerase II binding subunit 3 (Rpb3). The domain necessary for Rpb3 binding was subsequently identified using different IGFBP-3 deletion mutants and was localized to the MBD/NLS epitope. Rpb3/IGFBP-3 binding was confirmed by coimmunoprecipitation assays with specific antisera, whereas a NLS mutant IGFBP-3 did not associate with Rpb3, suggesting that a functional NLS is required. Rpb3 facilitates recruitment of the polymerase complex to specific transcription factors and is necessary for the transactivation of many genes. Its association with IGFBP-3 provides a functional role for IGFBP-3 in the direct modulation of gene transcription.