In GFP with high risk HPV-18E6 fusion protein expressed 293T and MCF-7 cells, the endogenous wild-type p53 could be transiently phosphorylated at multiple sites

Abstract

Background: Infected cells recognize viral replication as a DNA damage stress and elicit the host surveillance mechanism to anti-virus infection. Modulation of the activity of tumor suppressor p53 is a key event in the replication of many viruses. They could manipulate p53 function through phosphorylation modification for their own purpose. But there is rarely research about p53 phosphorylation status in the context of HPV-E6. Therefore, we investigated whether p53 could be phosphorylated by HPV-E6.

Methods: We used a mammalian green fluorescence protein (GFP) expression system to express HPV-18E6 with GFP fusion proteins (GFP-18E6) in wild-type (wt) p53 cell lines, such as 293T and MCF-7 cells to trace the traffic and subcellular location of E6 protein. By immunofluorescence technique and immunoblotting, we determined the positive phosphorylated sites of p53 and observed the distribution of phosphorylated p53 in the context of GFP-18E6.

Results: GFP-18E6 was predominantly located in nuclei of wt p53 cell lines, and it could induce transient phosphorylation of p53 at multiple sites, such as Ser15, Ser20, and Ser392. All the three sites of phosphorylated p53s were localized in nuclei together with GFP-18E6.

Conclusion: In GFP with high risk HPV-18E6 fusion protein expressed 293T and MCF-7 cells, the endogenous wt p53 could be transiently phosphorylated at multiple sites.

Figure 1

GFP-18E6 is predominantly located in nuclei. Representative photographs of 293T and MCF-7 cells at 21 h after transfection with GFP and GFP-18E6 expression plasmid. The green fluorescence is emitted by the cells transfected with pGFP and pGFP-18E6 respectively. The red is DAPI stained nuclei. Scale bar = 8 μm. The photographs are examined at 400× magnification by fluorescence microscope.

Figure 2

Data analysis of GFP-18E6 level in 293T and MCF-7 cells. The expression level of GFP and GFP-18E6 are examined by fluorescence intensity dynamically. One hundred cells are examined for each plasmid from 20 random fields. The N/(N+C) indicates fluorescence intensity ratio of GFP fusion protein in nuclei (N) to it in both nuclei and cytoplasm (N+C).

Figure 3

HPV-18E6 promotes multiple sites phosphorylation of p53 along with up-regulation of ATM and Chk2. The phosphorylated responses appear obviously at three sites: Ser¹⁵, Ser²⁰, and Ser³⁹² of p53 along with the up-regulation of ATM and Chk2 at 24 h in GFP-18E6 expressing 293T and MCF-7 cells. IgG is an irrelative antibody used as the negative control. Data are normalized to β-actin and representative of three independent western blot analyses.

Figure 4

Localization and expression level of phosphorylated p53 proteins. (A) In 293T and MCF-7 cells, the phosphorylated p53s are located in nuclei together with GFP-18E6. Green fluorescence indicates the protein of GFP, and GFP-18E6 expressed by the transfected cells, Red fluorescence indicates phosphorylated p53 proteins, which are labelled with phosphorylated anti-p53 antibodies plus anti-rabbit-Cy3 secondary antibody. The photographs are examined at 400× magnification by confocal microscopy. Scale bar = 8 μm. The results shown are representative of three independent experiments. (B) Level of phosphorylated p53 in the context of GFP-18E6 from 12 h to 72 h. The data of phosphorylated p53s level are examined by fluorescence intensity. 100 cells are examined for each phosphorylated site of p53 from 20× random fields.