Regulation of p90RSK phosphorylation by SARS-CoV infection in Vero E6 cells

Abstract

The 90 kDa ribosomal S6 kinases (p90RSKs) are a family of broadly expressed serine/threonine kinases with two kinase domains activated by extracellular signal-regulated protein kinase in response to many growth factors. Our recent study demonstrated that severe acute respiratory syndrome (SARS)-coronavirus (CoV) infection of monkey kidney Vero E6 cells induces phosphorylation and dephosphorylation of signaling pathways, resulting in apoptosis. In the present study, we investigated the phosphorylation status of p90RSK, which is a well-known substrate of these signaling pathways, in SARS-CoV-infected cells. Vero E6 mainly expressed p90RSK1 and showed weak expression of p90RSK2. In the absence of viral infection, Ser221 in the N-terminal kinase domain was phosphorylated constitutively, whereas both Thr573 in the C-terminal kinase domain and Ser380 between the two kinase domains were not phosphorylated in confluent cells. Ser380, which has been reported to be involved in autophosphorylation by activation of the C-terminal kinase domain, was phosphorylated in confluent SARS-CoV-infected cells, and this phosphorylation was inhibited by , which is an inhibitor of p38 mitogen-activated protein kinases (MAPK). Phosphorylation of Thr573 was not upregulated in SARS-CoV-infected cells. Thus, in virus-infected cells, phosphorylation of Thr573 was not necessary to induce phosphorylation of Ser380. On the other hand, Both Thr573 and Ser380 were phosphorylated by treatment with epidermal growth factor (EGF) in the absence of p38 MAPK activation. Ser220 was constitutively phosphorylated despite infection. These results indicated that phosphorylation status of p90RSK by SARS-CoV infection is different from that by stimulation of EGF. This is the first detailed report regarding regulation of p90RSK phosphorylation by virus infection.

Figure 1

Phosphorylation of signaling pathways in SARS‐CoV‐infected cells. Vero and Vero E6 cells were prepared at confluence in 24‐well plates and the cells were infected with SARS‐CoV at 10 m.o.i. Protein samples were obtained at 17, 24, and 44 h.p.i. The protein of Vero E6 cells at 44 h.p.i. could not be obtained due to strong morphological changes caused by apoptosis. Western blotting analyses were performed to examine signaling pathways (A) and apoptotic marker proteins (B).

Figure 2

p90RSK family expressed in Vero E6 cells. Vero E6 cells were prepared at densities of 1 × 10⁶ and 0.6 × 10⁵ in 6‐well plates. HeLa cells, a clonal cell line for another study, were used as controls. Both K562 and Jurkat cell lysates were obtained from Clontech Laboratories Inc. Western blotting analysis was performed using the same amounts of protein. According to the antibody product data sheets, anti‐human p90RSK1 monoclonal antibody (Epitomics) does not cross‐react with other RSK family members. Anti‐human and mouse p90RSK2 antibody (Zymed) does not react with overexpressed p90RSK1, 3, or 4. Anti‐human p90RSK3 antibody (Zymed) does not react with overexpressed p90RSK1, 2, or 4. Anti‐human p90RSK antibody (Zymed) does not react with p90RSK1, 2, or 3. Anti‐p90RSK1/2/3 antibody (Cell Signaling) detects endogenous levels of RSK1, RSK2, and RSK3 proteins. In Vero E6 cells, the band of RSK1 was stronger than that of RSK2 as described in the text. The weak bands were enhanced using Adobe Photoshop.

Figure 3

Phosphorylation of p90RSK Ser221 in Vero E6 cells. (A) Vero E6 cells were prepared at densities of 10, 5, 2.5, 1.25, 0.6, and 0.3 × 10⁵ cells in DMEM containing 5% FBS per well in 6‐well plates. Proteins were obtained from these cells after 24 h, and Western blotting was performed using anti‐phospho p90RSK (Ser221). (B) The confluency of Vero E6 cells used in this study is shown. (C) 2 × 10³ cells in DMEM containing 0.2% and 5% FBS were prepared in 96‐well plates. After 4 days, cell number was counted using a WST‐1 cell proliferation assay kit. (D) 0.25 and 10 × 10⁵ cells in DMEM containing various concentrations of FBS were prepared in 6‐well plates. Western blotting was performed using proteins obtained after 24 h.

Figure 4

Phosphorylation of p90RSK Thr573 and Ser380 in Vero E6 cells. Confluent Vero E6 cells in 24‐well plates were treated with EGF. Western blotting analysis was performed using proteins obtained at 0, 1, 5, and 10 min (A). (B) Vero E6 cells were prepared at 10, 5, 2.5, 1.25, 0.6, and 0.3 × 10⁵ cells in 6‐well plates. Proteins were obtained from these cells after 24 h, and Western blotting was performed using anti‐phospho p90RSK (Thr573 and Ser380). The proteins used in (B) were the same as those in Fig. 3A, and equal amount of proteins were blotted.

Figure 5

Phosphorylation of p90RSK in SARS‐CoV‐infected Vero E6 cells. (A) 1 × 10⁶ cells in 6‐well plates were prepared (100% confluency). The cells were infected with SARS‐CoV at 50 m.o.i. Western blotting analysis was performed using proteins obtained at 16 and 24 h.p.i. (B) One hour after viral inoculation, cells were treated with http://SB203580 (20 μM). Proteins were obtained at 24 h.p.i. for Western blotting analysis. Mock‐infected cells were treated with DMSO as a control.