I-KAPPA-B/MAD-3 MASKS THE NUCLEAR-LOCALIZATION SIGNAL OF NF-KAPPA-B P65 AND REQUIRES THE TRANSACTIVATION DOMAIN TO INHIBIT NF-KAPPA-B P65 DNA-BINDING
I-KAPPA-B/MAD-3 MASKS THE NUCLEAR-LOCALIZATION SIGNAL OF NF-KAPPA-B P65 AND REQUIRES THE TRANSACTIVATION DOMAIN TO INHIBIT NF-KAPPA-B P65 DNA-BINDING
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DOI：
10.1091/mbc.3.12.1339
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发表时间：
1992-12-01
影响因子：
3.3
通讯作者：
BALLARD, DW
中科院分区：
文献类型：
--
作者：
GANCHI, PA;SUN, SC;BALLARD, DW
NF-κ B转录因子复合物的活性核形式由两个DNA结合亚基NF-κ B p65和NF-κ B p50组成，这两个亚基与v-rel癌基因产物具有广泛的N-末端序列同源性。NF-κ B p65亚基在该复合物中提供反式激活活性，并作为NF-κ B细胞质抑制剂（称为IkappaB）的细胞内受体。相比之下，单独的NF-κ B p50不能刺激κ B定向转录，并且基于先前的体外研究，NF-κ B p50不受IkappaB直接调节。为了研究NF-κ B和IkappaB/MAD-3之间关键调节相互作用的分子基础，鉴定了一系列人NF-κ B p65突变体，其功能分离DNA结合、IkappaB介导的抑制和IkappaB诱导的该转录因子的核排斥。用这些NF-κ B p65突变体进行的体内表达研究的结果显示如下：1）IkappaB/MAD-3完全抑制人T淋巴细胞中通过人免疫缺陷病毒1型kappaB增强子介导的NF-κ B p65依赖性转录激活，2）IkappaB/MAD 3与NF-κ B p65的结合足以将NF-κ B p65从细胞核重新靶向细胞质，3）NF-κ B p65的Rel同源结构域中存在的功能性核定位信号的选择性缺失破坏了其接合IkappaB/MAD-3的能力，和4）NF-κ B p65的独特C-末端减弱了其自身的核定位，并含有IkappaB介导的NF-κ B p65 DNA结合活性抑制所需的序列。总之，这些发现表明核定位信号和NF-κ B p65的反式激活结构域构成了一个二分系统，该系统在IkappaB/MAD-3的抑制功能中起关键作用。出乎意料的是，我们的体内研究也表明，IkappaB/MAD-3直接结合NF-κ B p50。这种相互作用是功能性的，因为它导致NF-κ B p50从细胞核重新靶向细胞质。然而，没有观察到DNA结合活性的损失，这可能反映了与NF-κ B p65中存在的独特C-末端结构域不同的独特C-末端结构域。
The active nuclear form of the NF-kappaB transcription factor complex is composed of two DNA binding subunits, NF-kappaB p65 and NF-kappaB p50, both of which share extensive N-terminal sequence homology with the v-rel oncogene product. The NF-kappaB p65 subunit provides the transactivation activity in this complex and serves as an intracellular receptor for a cytoplasmic inhibitor of NF-kappaB, termed IkappaB. In contrast, NF-kappaB p50 alone fails to stimulate kappaB-directed transcription, and based on prior in vitro studies, is not directly regulated by IkappaB. To investigate the molecular basis for the critical regulatory interaction between NF-kappaB and IkappaB/MAD-3, a series of human NF-kappaB p65 mutants was identified that functionally segregated DNA binding, IkappaB-mediated inhibition, and IkappaB-induced nuclear exclusion of this transcription factor. Results from in vivo expression studies performed with these NF-kappaB p65 mutants revealed the following: 1) IkappaB/MAD-3 completely inhibits NF-kappaB p65-dependent transcriptional activation mediated through the human immunodeficiency virus type 1 kappaB enhancer in human T lymphocytes, 2) the binding of IkappaB/MAD3 to NF-kappaB p65 is sufficient to retarget NF-kappaB p65 from the nucleus to the cytoplasm, 3) selective deletion of the functional nuclear localization signal present in the Rel homology domain of NF-kappaB p65 disrupts its ability to engage IkappaB/MAD-3, and 4) the unique C-terminus of NF-kappaB p65 attenuates its own nuclear localization and contains sequences that are required for IkappaB-mediated inhibition of NF-kappaB p65 DNA binding activity. Together, these findings suggest that the nuclear localization signal and transactivation domain of NF-kappaB p65 constitute a bipartite system that is critically involved in the inhibitory function Of IkappaB/MAD-3. Unexpectedly, our in vivo studies also demonstrate that IkappaB/MAD-3 binds directly to NF-kappaB p50. This interaction is functional as it leads to retargeting of NF-kappaB p50 from the nucleus to the cytoplasm. However, no loss of DNA binding activity is observed, presumably reflecting the unique C-terminal domain that is distinct from that present in NF-kappaB p65.