ABSTRACT
Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for efficient conversion of primary human B lymphocytes to lymphoblastoid cell lines (LCLs) and for continued LCL growth. We used a transcomplementation assay in the context of LCLs transformed by an EBV with a conditional EBNA3C to identify the EBNA3C amino acids (aa) necessary for maintaining LCL growth. Surprisingly, we found that most EBNA3C aa were essential for continued LCL growth. Only EBNA3C mutants deleted for residues within aa 507-515, 516-620, 637-675, or 676-727 maintained full LCL growth, and EBNA3C mutants deleted for residues within aa 728-732 or 910-992 maintained slow LCL growth. In contrast, EBNA3C lacking aa 180-231, which mediate RBP-Jkappa association and are necessary for EBNA3C abrogation of EBNA2-induced transcription through RBP-Jkappa, could not support LCL growth. Furthermore, 2 EBNA3C alanine substitution mutants within aa 180-231, which were wild-type (wt) in abrogating EBNA2-mediated transcription through RBP-Jkappa, maintained LCL growth, and 2 alanine substitution mutants within aa 180-231, which were null in abrogating EBNA2-mediated transcription through RBP-Jkappa, did not maintain LCL growth. This indicates that EBNA3C regulation of transcription through RBP-Jkappa is critical to maintaining LCL growth. Several other EBNA3C functions also are critical for LCL growth, because EBNA3C mutants deleted for residues within aa 130-159, 251-506, or 733-909 were wt in abrogating transcription through RBP-Jkappa and expression level, but did not maintain LCL growth.
Subject(s)
Antigens, Viral/physiology , B-Lymphocytes/virology , Cell Transformation, Viral , Herpesvirus 4, Human/pathogenicity , Immunoglobulin J Recombination Signal Sequence-Binding Protein , Lymphoma, B-Cell/virology , Antigens, Viral/genetics , Epstein-Barr Virus Nuclear Antigens , Gene Expression Regulation , Genetic Complementation Test , Humans , Lymphoma, B-Cell/pathology , Mutagenesis, Site-Directed , Mutation , Transcription, Genetic , Viral ProteinsABSTRACT
Dok1 is an abundant Ras-GTPase-activating protein-associated tyrosine kinase substrate that negatively regulates cell growth and promotes migration. We now find that IkappaB kinase beta (IKKbeta) associated with and phosphorylated Dok1 in human epithelial cells and B lymphocytes. IKKbeta phosphorylation of Dok1 depended on Dok1 S(439), S(443), S(446), and S(450). Recombinant IKKbeta also phosphorylated Dok1 or Dok1 amino acids 430-481 in vitro. TNF-alpha, IL-1, gamma radiation, or IKKbeta overexpression phosphorylated Dok1 S(443), S(446), and S(450) in vivo, as detected with Dok1 phospho-S site-specific antisera. Moreover, Dok1 with S(439), S(443), S(446), and S(450) mutated to A was not phosphorylated by IKKbeta in vivo. Surprisingly, mutant Dok1 A(439), A(443), A(446), and A(450) differed from wild-type Dok1 in not inhibiting platelet-derived growth factor-induced extracellular signal-regulated kinase 1/2 phosphorylation or cell growth. Mutant Dok1 A(439), A(443), A(446), and A(450) also did not promote cell motility, whereas wild-type Dok1 promoted cell motility, and Dok1 E(439), E(443), E(446), and E(450) further enhanced cell motility. These data indicate that IKKbeta phosphorylates Dok1 S(439)S(443) and S(446)S(450) after TNF-alpha, IL-1, or gamma-radiation and implicate the critical Dok1 serines in Dok1 effects after tyrosine kinase activation.
