Biochemical, genetic, and functional analyses of the phosphorylation sites on the Epstein-Barr virus-encoded oncogenic latent membrane protein LMP-1.
LMP-1 is the only Epstein-Barr virus-encoded latent protein known to have the properties of a transforming oncogene in rodent fibroblasts and the only latent protein, besides EBNA-1, detected in nasopharyngeal carcinoma and Hodgkin's lymphoma biopsies. LMP-1 is characterized by serine/threonine phosphorylation and rapid turnover (half-life, 2 to 3 h) due to specific proteolytic cleavage, which causes release of a phosphorylated C-terminal fragment (p25) into the cytoplasm. We used biochemical, functional, and mutational analyses to identify sites of phosphorylation. All of the phosphorylation sites detected lie in the C-terminal domain. In particular, we identified S-313 and T-324 as functionally important sites. Prevention of phosphorylation at S-313, by altering it to a glycine, prevented detectable phosphorylation of both LMP-1 and p25, indicating that it is a major site on both forms of the molecule. However, lack of detectable phosphorylation had no effect on p25 cleavage or on the ability of LMP-1 to transform Rat-1 fibroblasts. Alteration of S-313 to an aspartate resulted in a form of LMP-1 that was toxic to Rat-1 cells. Alteration of T-324 to a glycine residue had no detectable effect on the ability of LMP-1 to become serine phosphorylated or transform Rat-1 cells. Alteration of T-324 to a glutamate, however, inhibited all detectable phosphorylation and resulted in a form of LMP-1 that was unable to transform Rat-1 fibroblasts. These results are discussed in the context of a model in which LMP-1 function is modulated by phosphorylation and dephosphorylation at S-313 and T-324.
Moorthy
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RK
Department of Pathology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Thorley-Lawson
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DA
D000595:Amino Acid Sequence; D000818:Animals; D002472:Cell Transformation, Viral; D000090266:Chromobox Protein Homolog 5; D004252:DNA Mutational Analysis; D005347:Fibroblasts / Q000166:cytology; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D013912:Threonine / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*
C000715594:CBX5 protein, human; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D009856:Oncogene Proteins, Viral; D010446:Peptide Fragments; D010748:Phosphopeptides; D014763:Viral Matrix Proteins; D000090266:Chromobox Protein Homolog 5; D013912:Threonine; D012694:Serine