Insufficient p65 phosphorylation at S536 specifically contributes to the lack of NF-κB activation and transformation in resistant JB6 cells
Insufficient p65 phosphorylation at S536 specifically contributes to the lack of NF-κB activation and transformation in resistant JB6 cells
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DOI：
10.1093/carcin/bgh198
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发表时间：
2004-10-01
期刊：
影响因子：
4.7
通讯作者：
Colburn, NH
中科院分区：
文献类型：
--
作者：
Hu, J;Nakano, H;Colburn, NH
TNF-α诱导的JB 6小鼠表皮细胞转化需要NF-κ B活化。p65的活化缺陷导致转化抗性（P-）细胞中NF-κ B活化的缺乏。我们假设，差异NF-κ B激活涉及差异p65磷酸化所产生的酶活性差异。在这里，我们表明，TNF-α诱导更大的ERK依赖性p65磷酸化在S536转化敏感（P+）细胞比P-细胞。我们的研究结果证实，有限的ERK含量导致低IkappaB激酶（IKK β）水平，进而导致P-细胞中TNF-α刺激后S536处p65磷酸化不足。P65在S536处的磷酸化似乎在TNF-α诱导的p65 DNA结合和p300向p65复合物的募集以及与HDAC 1和3结合的p65的释放中起作用。在S536处阻断p65磷酸化，而不是在S276或S529处阻断，消除p65反式激活活性。在转化抗性P细胞中p65而不是p65磷酸化突变体（S536 A）的过表达使得这些细胞对TNF-α诱导的转化敏感。p65磷酸化的过表达模拟P-细胞中的p65-S536 D或p65-S536 E，并且还挽救转化反应。这些发现提供了直接证据，即在JB 6转化模型中，TNF-α诱导的NF-κ B活化需要S536处的p65磷酸化。在P-细胞中观察到的NF-κ B活化的缺乏可归因于S536上p65磷酸化水平不足，这是由IKK β不足引起的，而IKK β不足又由ERK不足引起。因此，p65在S536处的磷酸化为癌症预防提供了潜在的分子靶点。
NF-kappaB activation is required for TNF-alpha-induced transformation of JB6 mouse epidermal cells. Deficient activation of p65 contributes to the lack of NF-kappaB activation in transformation-resistant (P-) cells. We hypothesized that the differential NF-kappaB activation involves differential p65 phosphorylation arising from enzyme activity differences. Here we show that TNF-alpha induces greater ERK-dependent p65 phosphorylation at S536 in transformation sensitive (P+) cells than in P- cells. Our results establish that limited ERK content contributes to a low IkappaB kinase (IKKbeta) level, in turn resulting in insufficient p65 phosphorylation at S536 upon TNF-alpha stimulation in P- cells. Phosphorylation of p65 at S536 appears to play a role in TNF-alpha-induced p65 DNA binding and recruitment of p300 to the p65 complex as well as in release of p65 bound to HDAC1 and 3. Blocking p65 phosphorylation at S536, but not at S276 or S529, abolishes p65 transactivational activity. Over-expression of p65 but not p65 phosphorylation mutant (S536A) in transformation-resistant P- cells renders these cells sensitive to TNF-alpha-induced transformation. Over-expression of p65 phosphorylation mimics p65-S536D or p65-S536E in P- cells and also rescues the transformation response. These findings provide direct evidence that phosphorylation of p65 at S536 is required for TNF-alpha-induced NF-kappaB activation in the JB6 transformation model. The lack of NF-kappaB activation seen in P- cells can be attributed to an insufficient level of p65 phosphorylation on S536 that arises from insufficient IKKbeta that in turn arises from insufficient ERK. Thus, p65 phosphorylation at S536 offers a potential molecular target for cancer prevention.