Cell cycle stage-specific phosphorylation of the Epstein-Barr virus immortalization protein EBNA-LP

EBNA-LP is a viral nuclear oncoprotein implicated in the immortalization of B lymphocytes by Epstein-Barr virus. An analysis of EBNA-LP migration on polyacrylamide gels was performed with protein derived from the X50-7 lymphoblastoid cell line blocked by hydroxyurea or aphidicolin at the G(1)/S phase of the cell cycle or by nocodazole at the G(2)/M phase. More slowly migrating species of EBNA-LP were detected in G(2)/M phase-arrested cell extracts. Release from nocodazole G(2)/M block or treatment with phosphatase caused the more slowly migrating species of EBNA-LP to disappear. Analyses of (PO43-)-P-32-labeled EBNA-LP protein immunoprecipitated from the drug-synchronized cells showed that phosphorylated EBNA-LP was present throughout the cell cycle but that phosphorylation increased in G(2) and was maximal at G(2). Phosphoamino acid analysis revealed that all phosphorylation was on serine residues only. The ability of EBNA-LP to be phosphorylated by p34(cdc2) kinase and casein kinase II exclusively on serines implicates these enzymes as being potentially involved in EBNA-LP phosphorylation.