SMG1 and CDK12 Link ΔNp63α Phosphorylation to RNA Surveillance in Keratinocytes

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 T₃₅₇/S₃₅₈ and S₃₆₈ was dependent on SMG1, whereas CDK12 increased the phosphorylation of ΔNp63α at S₆₆/S₆₈ and S₃₀₁. 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.