Epstein-Barr virus LMP1 activates EGFR, STAT3, and ERK through effects on PKCdelta

Abstract

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that infects more than 90% of the world's adult population and is linked to multiple malignancies, including Burkitt lymphoma, Hodgkin disease, and nasopharyngeal carcinoma (NPC). The EBV oncoprotein LMP1 induces transcription of the epidermal growth factor receptor (EGFR), which is expressed at high levels in NPC. EGFR transcription is induced by LMP1 through a p50 NFκB1-Bcl-3 complex, and Bcl-3 is induced by LMP1-mediated activation of STAT3. This study reveals that LMP1, through its carboxyl-terminal activation domain 1 (LMP1-CTAR1), activates both STAT3 and EGFR in a serum-independent manner with constitutive serine phosphorylation of STAT3. Upon treatment with EGF, the LMP1-CTAR1-induced EGFR was additionally phosphorylated and STAT3 became phosphorylated on tyrosine, concomitant with upregulation of a subset of STAT3 target genes. The kinase responsible for LMP1-CTAR1-mediated serine phosphorylation of STAT3 was identified to be PKCδ using specific RNAi, a dominant negative PKCδ, and the PKCδ inhibitor rottlerin. Interestingly, inhibition of PKCδ also inhibited constitutive phosphorylation of EGFR and LMP1-CTAR1-induced phosphorylation of ERK. Inhibition of PKCδ blocked LMP1-CTAR1-mediated transformation of Rat-1 cells, likely through the inhibition of ERK activation. These findings indicate that LMP1 activates multiple distinct signaling pathways and suggest that PKCδ functions as a master regulator of EGFR, STAT3, and ERK activation by LMP1-CTAR1.

Fig. 1.

LMP1-CTAR1-mediated activation of EGFR and STAT3. (A) C33A cells stably transduced with pBabe or LMP1-CTAR1 were maintained in DMEM with 10% FBS. LMP1-CTAR1 cells were then serum starved for 24 h and treated with DMSO (lane 3), EGF (500 ng/ml) for 10 min (lane 4), EGF/AG1478 (inhibitor of EGFR tyrosine kinase, 100 μM) for 10 min (lane 5), EGF for 30 min (lane 6), or EGF/AG1478 for 30 min (lane 7). Whole-cell protein lysates were prepared for Western blot analysis. Activation of EGFR was detected by using antibody against tyrosine-phosphorylated EGFR. The solid arrow points at LMP1-CTAR1-activated EGFR, and the dashed arrow points at the shifted EGFR after EGF treatment. GAPDH was detected as a loading control, and HA-specific antibody was used to detect expression of LMP1-CTAR1. Data are representative of results of three independent experiments. (B) Total RNA was extracted from serum-starved LMP1-CTAR1 cells treated with DMSO, EGF for 10 min, or EGF for 30 min, and quantitative real-time RT-PCR was performed with primers against STAT3 target genes. Results from QRT-PCR were normalized to GAPDH, and expressions of STAT3 targets in pBabe control cells were set to 1. Results were graphed as mean averages with standard errors of the mean and were computed from triplicate results of three or more separate experiments. Statistical significance was evaluated using a computerized, paired two-tailed Student t test. Differences were considered significant at P < 0.05.

Fig. 2.

LMP1 effects on EGFR ligands and phosphorylation. (A) Total RNA was extracted from serum-starved pBabe control or LMP1-CTAR1-expressing C33A cells, and QRT-PCR was performed with primers against potential EGFR ligands. Results from QRT-PCR were normalized to GAPDH, and levels of EGFR ligands in pBabe control cells were set to 1. Results were graphed as mean averages with standard errors of the mean and were computed from triplicate results of four separate experiments. Statistical significance was evaluated using a computerized, paired two-tailed Student t test. Differences were considered significant at P < 0.05. (B) Serum-starved pBabe control and LMP1-CTAR1-expressing C33A cells were treated with either DMSO (Mock), EGF 500 ng/ml, or EGF plus 100 μM AG1478 for 10 min before the whole-cell lysates were subject to Western blot analysis using antibodies against total EGFR, activated (tyrosine-phosphorylated) EGFR, or specific phosphorylation of EGFR (Tyr 992, 1045, or 1068). Expression of GAPDH was measured as the loading control. The solid arrow indicates LMP1-CTAR1-activated EGFR, and the dashed arrow indicates the EGF-induced band-shift of EGFR. Images shown here are representative of results of three independent experiments.

Fig. 3.

LMP1-CTAR1-induced serine phosphorylation of STAT3 is inhibited with PKCδ inhibitor rottlerin. (A) C33A cells stably transduced with pBabe or LMP1-CTAR1 were maintained in DMEM with 10% FBS. The media were replaced with serum-free medium for 24 h, and both groups of cells were treated with DMSO for 5 h (lanes 1 and 2). LMP1-CTAR1 cells were also treated with the PKCδ inhibitor (50 or 40 μM rottlerin, lanes 3 and 4), PKCα/β inhibitor (5 nM Gö6976, lane 5), MEK/ERK inhibitor (10 μM U0126, lane 6), and PI3K inhibitor (25 μM LY294002, lane 7) for 5 h. Band intensities were determined using ImageJ software, normalized to GAPDH levels, and represented relative to the pBabe control level. Results were graphed as mean averages with standard errors of the mean and were computed from triplicate results of three or more separate experiments. (B) pBabe and LMP1-CTAR1 cells were treated with rottlerin for 5 h. Whole-cell protein lysates were prepared for Western blot analysis. HSC70 was detected as a loading control.

Fig. 4.

LMP1-CTAR1-induced serine phosphorylation of STAT3 is dependent on PKCδ. C33A cells stably transduced with pBabe or LMP1-CTAR1 were transfected with pSUPER.PKCdelta.RNAi (RNAi) or PKCδ-dominant negative (DN) for 24 h in serum free media. Whole-cell lysates were prepared and separated by SDS-PAGE for immunoblot analysis. HSC70 was used as a loading control. Band intensities were determined using ImageJ software, normalized to HSC70 (or EGFR for p-EGFR) levels, and represented relative to the control level. Band intensities were determined using ImageJ software, normalized to HSC70 levels, and represented relative to the pBabe control level. Results were graphed as mean averages with standard errors of the mean and were computed from triplicate results of three separate experiments.

Fig. 5.

Rottlerin inhibits LMP1-CTAR1-induced focus formation. (A) Rat-1 cells were transduced with pBabe and LMP1-CTAR1 (1–220) at three different dilutions, maintained for 10 days, stained with crystal violet, and observed for focus formation. The effect of PKCδ inhibition was determined using 1 μM rottlerin. (B) Whole-cell protein lysates were prepared from a duplicate of the focus formation experiment described for panel A. (C) p-Babe or LMP1-CTAR1 Rat-1 cells were treated for 5 h with DMSO (lane 1) or rottlerin (5 or 40 μM). Western blot analysis on cell lysates separated by SDS-PAGE was performed using antibodies against total ERK and phosphorylated ERK. GAPDH or HSC70 were detected as loading controls. Data shown are representative of results of three independent experiments.

Fig. 6.

PKCδ is required for LMP1-induced loss of contact inhibition. (A) Rat-1 cells stably expressing pBabe, LMP1, pBabe.PKCδ-DN, or LMP1.PKCδ-DN were grown for 10 to 14 days. The medium was replaced every 2 days with fresh DMEM. Cells were stained with crystal violet and observed for focus formation at ×10 magnification with phase contrast. (B) Confluent dishes of pBabe, LMP1, pBabe.PKCδ-DN, and LMP1.PKCδ-DN Rat-1 cells were harvested, and cell lysates were analyzed by immunoblot analysis for p-ERK levels, with HSC70 detected as a loading control.

Fig. 7.

Model of LMP1 activation of EGFR, STAT3, and ERK. LMP1-CTAR1 induces intrinsic activation/phosphorylation of EGFR, which can be further activated by EGF to induce STAT3 tyrosine phosphorylation and the expression of STAT3 target genes (black arrows). LMP1-CTAR1 induces PKCδ-dependent serine phosphorylation of STAT3 and Bcl-3 upregulation (gray arrows). The PKCδ inhibitor, rottlerin, and PKCδ DN inhibit serine phosphorylation of STAT3, activation of EGFR, and activation of MEK/ERK (white arrows). The MEK/ERK inhibitor U0126 blocks LMP1-CTAR1-mediated phosphorylation of ERK as well as transformation of Rat-1 cells, indicating that PKCδ contributes to LMP1's transforming ability by functioning upstream of MEK.