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.