Late cornified envelope 1C (LCE1C), a transcriptional target of TAp63 phosphorylated at T46/T281, interacts with PRMT5

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.

Figure 1

TAp63 is phosphorylated at T46 and T281 by GAK in vitro. (a) GST-p63^p40/p51 protein shows a phosphorylated band (horizontal arrow) in the presence of GST-tagged GAK-k (tilted arrow), the kinase domain of GAK. (b) A schematic representation of TAp63 structure and TC1–TC5 fragments denoting the sites of functional domains and amino acid numbers. TA, transactivation; DB, DNA binding; OD, oligomerization; SAM, C-terminal sterile alpha motif. (c) Radio-autograph of SDS-PAGE analysis after in vitro kinase assays of full-size and TC1–TC5 fragments of TAp63 in the absence (no kinase) or presence of GAK-k (horizontal arrow). White arrows denote the bands for full-size and TC1–TC5 fragments of TAp63 as stained by Simply Blue, whereas white arrowheads indicate the phosphorylated bands in the radio-autograph. Horizontal arrows denote the band for auto-phosphorylation of GAK-k. (d) Radio-autograph showing that TC1-2 (30–50) and TC3-1 (273–307) fragments, but not TC1-1 (1–29), TC1-3 (51–100), or TC3-2 (306–354) fragments, of TAp63 are the phosphorylation substrates of GAK-k. Black arrowheads or black arrows denote the bands for phosphorylated TAp63 fragments or GAK-k, respectively. NS, no substrate. (e) A schematic representation of TC1-2 and TC3-1 fragments of TAp63 that were identified as the phosphorylated substrates by GAK-k. Location of S and T residues that are possible phosphorylation targets of GAK-k are shown, together with their alanine (A) substitutes that are used as the GST-tagged substrates (green triangles). (f) Radio-autograph showing that the band intensities for T46A and T281A fragments were weakened (red arrowheads) due to T/A substitutions. Black arrowheads denote the phosphorylated bands for TC1-T39A, TC1-T44A, TC3-285A, and TC3-297A fragments. Horizontal black arrows denote the bands for GAK-k.

Figure 2

LCE1C is a transcriptional target of TAp63. (a) Wb shows that expression of Myc-TAp63 proteins drastically increased from Dox (−) to Dox (+) conditions in Tet-ON inducible Myc-TAp63-WT, Myc-TAp63-T46A/T281A (AA), or Myc-TAp63-T46D/T281D (DD) U2OS cell lines. Vec means Myc-vector alone. The amount of α-tubulin was monitored as a loading control. (b) List of the nine genes arranged in the descending order of averaged fold change values of signal intensity, obtained by DNA microarray analysis. (c) Scatter plot of the DNA microarray data for average values from Dox (−) versus Dox (+) conditions in Tet-ON inducible Myc-TAp63-WT cells. The y-axis and x-axis show log₂[hybridization signal intensity] of microarray data from Dox (+) and Dox (−), respectively. Notable genes with high fold change values are plotted. (d,e) Bar graphs of qRT-PCR data show relative mRNA levels in Tet-ON inducible Myc-vector, Myc-TAp63-WT, Myc-TAp63-T46A/T281A (AA), or Myc-TAp63-T46D/T281D (DD) U2OS cell lines in Dox (−) and Dox (+) conditions for the denoted genes (i–iii). Red and black arrows highlight the notable alterations of the mRNA levels. The y-axis values are magnified in (e). (f) A schematic representation of the GAK_TAp63-T46/T281_LCE1C axis.

Figure 3

Luciferase reporter assays using the promoter region of the LCE1C gene. (a) A schematic representation of the promoter region (~2000 bp) of LCE1C. Green box indicates the location of the luciferase gene. (b) Bar graph to indicate the promoter activity of #1–4 promoter regions (arbitrary units). Promoter activity of SV40 promoter was measured as a positive control. Promoter activities using no TAp63 DNA (NT) or vector alone (Vec) in the reaction mixture were also measured as negative controls. (c) Bar graph to examine the alteration of the promoter activity of LCE1C-promoter #1 by addition of Myc-TAp63-WT, Myc-TAp63-T46A/T281A (AA), or Myc-TAp63-T46D/T281D (DD) plasmids into the reaction mixture. Promoter activities using no TAp63 DNA (NT) or vector alone (Vec) in the reaction mixture were also measured as negative controls (NC). Addition of SV40 promoter was examined as a positive control (PC). N.S. indicates no significant difference. (d,e) Wb to confirm the exogenous expression of Myc-TAp63 in luciferase assays. α-tubulin was detected as a loading control. (f) A schematic representation of the action of the GAK_TAp63-T46/T281_LCE1C axis (i), which was destroyed in the Myc-TAp63-T46A/T281A (AA) expressing cells (ii).

Figure 4

LCE1C is comprised of oligomers and interacts with PRMT5. (a) Dot blot analysis of the homemade anti-LCE1C antibody in which the denoted amount of antigen peptide (TPKCPPKCPTPKCPP) was dotted on the Wb filter. (b) Wb on the cell extracts from 293T cells expressing FLAG-vector alone or FLAG-LCE1C. Putative bands highlighted by red font (1, 2, 3, and 4) indicate monomer, dimer, trimer, or tetramer forms of LCE1C proteins. (c) A line graph shows a linear distribution of migration distance in SDS-PAGE used for Wb and molecular weight of LCE1C. Red arrowheads correspond to the band for indicated oligomers. (d) Co-IP on extract of 293T cells expressing FLAG-LCE1C shows an interaction of FLAG-LCE1C tetramer with PRMT5 (red arrowhead). (e) Co-IP on extract of 293T cells expressing Myc-LCE1C and/or FLAG-PRMT5 shows an interaction of FLAG-LCE1C tetramer with FLAG-PRMT5 (red arrow). LE; long exposure onto X-ray film. SE; short exposure onto X-ray film. Red arrow indicates a band for putative LCE1C tetramer.

Figure 5

Subcellular localization of Myc-LCE1C and FLAG-PRMT5 in U2OS cells expressing these proteins. Pink arrows denote FLAG-PRMT5 signals showing nuclear dots. Scale bar, 30 µm.

Figure 6

MS analysis identified importin α as one of the association partners of LCE1C. (a) Cell extracts prepared from logarithmically growing 293T cells expressing the FLAG-vector or FLAG-LCE1C were immunoprecipitated with an anti-FLAG antibody and the precipitates were applied to SDS-PAGE. After silver-staining, a strong band was detected only in 293T cells expressing FLAG-LCE1C; the gel fragment highlighted by a red rectangle was subjected to LC-MS/MS analysis. (b) A list of genes that were identified as association partners of LCE1C. Molecular weight and the score for each candidate protein are shown. The score signifies the “Mascot probability based scoring” that is used to judge whether a result is significant or not; the larger the number, the higher the probability that the detected peptide is derived from the denoted protein in the database. (c) Amino acid sequence of LCE1C protein. Candidate for NLS (RRRR) is highlighted by red font. (d) Co-IP analysis using the extract of 293T cells expressing the FLAG-vector or FLAG-LCE1C, which was immunoprecipitated with an anti-FLAG antibody. The precipitates were probed with anti-importin α or anti-FLAG antibodies. Red arrowhead indicates a band that shows an interaction between FLAG-LCE1C and importin α in vivo. Black arrow or green arrowhead indicates a band for monomer or dimer forms of FLAG-LCE1C proteins, respectively. (e) A schematic representation of the action of the GAK_TAp63-T46/T281_LCE1C axis, which is followed by interaction of LCE1C with importin α or PRMT5.