Abstract
p63, a transcriptional factor that belongs to the p53 family, regulates epidermal differentiation, stemness, cell death, tumorigenesis, metastasis, and senescence. However, its molecular mechanism remains elusive. We report here that TAp63 phosphorylated at T46/T281 specifically upregulates the late cornified envelope 1C (LCE1C) gene that is essential at a relatively late stage of epithelial development. We identified these phosphorylation sites during a search for the targets of Cyclin G-associated kinase (GAK) in vitro. LCE1C was drastically upregulated by doxycycline-dependent expression of Myc-TAp63 wild-type protein. Luciferase reporter assays using the promoter region of the LCE1C gene confirmed that the phosphorylations of TAp63-T46/T281 contributed to full transcriptional activation of the LCE1C gene. LCE1C interacted with protein arginine methyltransferase 5 (PRMT5) and translocated it from the nucleus to the cytoplasm. Mass spectrometry and co-immunoprecipitation identified importin-α as one of the association partners of LCE1C. In summary, we propose that the GAK_TAp63-pT46/pT281_LCE1C axis plays an important role in preventing the nuclear function of PRMT5.
Highlights
As a member of the p53/p63/p73 family, p63 is a transcriptional regulator that is involved in epidermal differentiation, stemness, cell death, tumorigenesis, metastasis, and senescence1. p63 can individually induce cell cycle arrest and collaborates with other members, including p53, p73 and other splicing isoforms of p63, to induce apoptosis in response to DNA damage[2,3,4]
QRT-PCR revealed that the mRNA level of late cornified envelope 1C (LCE1C) in U2OS cells expressing TAp63-WT was dramatically increased in a Dox-dependent manner (Fig. 2d,e), suggesting that the transcription of LCE1C is dependent on the amount of TAp63 protein
Luciferase reporter assays using the promoter region of the LCE1C gene revealed that transcription of LCE1C was markedly induced in WT-expressing cells to quantitative reverse transcription-polymerase chain reaction (qRT-PCR), but the LCE1C promoter activity was apparently decreased in AA-expressing cells (Fig. 3)
Summary
As a member of the p53/p63/p73 family, p63 is a transcriptional regulator that is involved in epidermal differentiation, stemness, cell death, tumorigenesis, metastasis, and senescence1. p63 can individually induce cell cycle arrest and collaborates with other members, including p53, p73 and other splicing isoforms (for example, γ isoform) of p63, to induce apoptosis in response to DNA damage[2,3,4]. As a member of the p53/p63/p73 family, p63 is a transcriptional regulator that is involved in epidermal differentiation, stemness, cell death, tumorigenesis, metastasis, and senescence. ΔNp63 (DN) isoform lacking the N-terminal transactivation domain of p63 is commonly expressed and acts as a dominant negative inhibitor of p63 function with gain of oncogenic properties[6]. It is traditionally accepted that TAp63 has tumor-suppressive activities while ΔNp63 has oncogenic activities, this model does not necessarily apply to all functions of p63 in cancer and normal tissue developments. Novel kinase targets of GAK in the nucleus are expected to be identified to elucidate the role of GAK in cancer cells. DNA microarray analysis identified LCE1C as a novel transcriptional target of TAp63, and the mRNA level of LCE1C was higher in TAp63-expressing cells than in empty vector-expressing cells. We propose that the GAK_TAp63-pT46/pT281_LCE1C axis constitutes a novel function of TAp63, partially explaining the phenotype of p63-null mice, and this axis may contribute to suppression of the tumorigenesis through PRMT5
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