Department of Molecular Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
J Proteome Res. 2021 Dec 3;20(12):5347-5358. doi: 10.1021/acs.jproteome.1c00427. Epub 2021 Nov 11.
The tumor suppressor p53-like protein p63 is required for self-renewal of epidermal tissues. Loss of p63 or exposure to ultraviolet (UV) irradiation triggers terminal differentiation in keratinocytes. However, it remains unclear how p63 diverts epidermal cells from proliferation to terminal differentiation, thereby contributing to successful tissue self-renewal. Here, we used bottom-up proteomics to identify the proteome at the chromatin in normal human epidermal keratinocytes following UV irradiation and p63 depletion. We found that loss of p63 increased DNA damage and that UV irradiation recruited the cyclin-dependent kinase CDK12 and the serine/threonine protein kinase SMG1 to chromatin only in the presence of p63. A post-translational modification analysis of ΔNp63α with mass spectrometry revealed that phosphorylation of T/S and S was dependent on SMG1, whereas CDK12 increased the phosphorylation of ΔNp63α at S/S and S. Indirect phosphorylation of ΔNp63α in the presence of SMG1 enabled ΔNp63α to bind to the tumor suppressor p53-specific DNA recognition sequence, whereas CDK12 rendered ΔNp63α less responsive to UV irradiation and was not required for specific DNA binding. CDK12 and SMG1 are known to regulate the transcription and splicing of RNAs and the decay of nonsense RNAs, respectively, and a subset of p63-specific protein-protein interactions at the chromatin also linked p63 to RNA transcription and decay. We observed that in the absence of p63, UV irradiation resulted in more ORF1p. ORF1p is the first protein product of the intronless non-LTR retrotransposon LINE-1, indicating a derailed surveillance of RNA processing and/or translation. Our results suggest that p63 phosphorylation and transcriptional activation might correspond to altered RNA processing and/or translation to protect proliferating keratinocytes from increased genotoxic stress.
肿瘤抑制因子 p53 样蛋白 p63 对于表皮组织的自我更新是必需的。p63 的缺失或暴露于紫外线 (UV) 辐射会触发角质形成细胞的终末分化。然而，p63 如何将表皮细胞从增殖转向终末分化，从而促进成功的组织自我更新，目前仍不清楚。在这里，我们使用自上而下的蛋白质组学方法，在正常的人类表皮角质形成细胞中，在 UV 照射和 p63 缺失后，鉴定了染色质上的蛋白质组。我们发现，p63 的缺失增加了 DNA 损伤，并且只有在存在 p63 的情况下，UV 照射才能招募细胞周期蛋白依赖性激酶 CDK12 和丝氨酸/苏氨酸蛋白激酶 SMG1 到染色质上。通过质谱对 ΔNp63α 的翻译后修饰分析表明，T/S 和 S 的磷酸化依赖于 SMG1，而 CDK12 增加了 ΔNp63α 在 S/S 和 S 上的磷酸化。在 SMG1 的存在下，ΔNp63α 的间接磷酸化使 ΔNp63α 能够结合肿瘤抑制因子 p53 特异性的 DNA 识别序列，而 CDK12 使 ΔNp63α 对 UV 照射的反应性降低，并且不是特异性 DNA 结合所必需的。CDK12 和 SMG1 分别被认为调节 RNA 的转录和剪接以及无意义 RNA 的降解，染色质上的一组 p63 特异性蛋白质-蛋白质相互作用也将 p63 与 RNA 转录和降解联系起来。我们观察到，在没有 p63 的情况下，UV 照射会导致更多的 ORF1p。ORF1p 是无内含子非 LTR 逆转录转座子 LINE-1 的第一个蛋白质产物，表明 RNA 加工和/或翻译的监测失控。我们的结果表明，p63 的磷酸化和转录激活可能对应于改变的 RNA 加工和/或翻译，以保护增殖的角质形成细胞免受增加的遗传毒性应激。
