Epstein-Barr Virus Latent Membrane Protein 2A Regulates c-Jun Protein through Extracellular Signal-Regulated Kinase

Abstract

Epstein-Barr virus (EBV

Syk Tyrosine Kinase Mediates Epstein-Barr Virus Latent Membrane Protein 2A-induced Cell Migration in Epithelial Cells

“…Twenty g of extracted protein was denatured at 100°C for 5 min and resolved on SDS-8% polyacrylamide gels. The proteins were electrotransferred onto a polyvinylidene fluoride (Immobilon-P; Millipore, Bedford, MA), and the blot was blocked in washing buffer containing 4% skim milk and then incubated with anti-phosphotyrosine antibody (4G10; Upstate Biotechnology, Inc.), anti-Syk antibody (4D10, Santa Cruz Biotechnology), anti-phospho-Syk antibody (New England Biolabs, Beverly, MA), anti-LMP2A antibody (22), anti-HA antibody (Babco, Richmond, CA), or anti-actin antibody (Sigma) for 1 h. After washing with Tris-buffered saline containing 0.05% Tween 20, the blot was incubated with peroxidase-labeled goat anti-mouse or goat anti-rabbit antibody (The Jackson Laboratory, West Grove, PA). Finally, bands were visualized using the Renaissance kit (PerkinElmer Life Sciences).…”

“…Immunoprecipitation-Nonidet P-40-solubilized cell lysates (125 g) were precleared with protein A-Sepharose beads (Amersham Biosciences) for 2 h at 4°C, and samples were incubated overnight with 1 g of anti-phosphotyrosine antibody (4G10), rabbit anti-Syk antibody (N-19, Santa Cruz Biotechnology), anti-LMP2A antibody (22), mouse immunoglobulin (Dako, Glostrup, Denmark), or rabbit immunoglobulin (Dako) at 4°C. The immune complexes were then precipitated by protein A-Sepharose at 4°C for 2 h and washed four times with PBS or 1% Nonidet P-40 buffer.…”

“…Plasmid Construction-pRc/CMV-LMP2A is an LMP2A expression plasmid driven by the cytomegalovirus promoter (22). Wild type LMP2A plasmid and plasmids with deletions in LMP2A coding sequences for amino acids 21-64 (⌬21-64), 21-85 (⌬21-85), and 80 -112 (⌬80 -112) or point mutation of tyrosine codons 31 (Y31F), 74 (Y74F), 85 (Y85F), 101 (Y101F), and 112 (Y112F) to phenylalanine were described by Fruehling et al (26).…”

“…To determine whether LMP2A can regulate endogenous Syk in epithelial cells, the tyrosine phosphorylation status of Syk was examined by immunoprecipitation and Western blotting. Anti-Syk, anti-phosphotyrosine antibodies, or immunoglobulin controls were used in immunoprecipitation experiments with LMP2A-expressing stable clones or transient transfectants (22). The immunoprecipitated proteins were then analyzed by Western blotting with anti-phosphotyrosine and anti-Syk antibodies.…”

“…LMP2A can induce cell migration and harbors an ITAM, the interaction motif of LMP2A with Syk in B cells (22,25). Thus, Syk is a prime candidate for mediating LMP2A-induced cell motility in epithelial cells.…”