Multi-transmembrane protein K15 of Kaposi's sarcoma-associated herpesvirus targets Lyn kinase in the membrane raft and induces NFAT/AP1 activities.
Viral proteins of gamma-2 herpesviruses, such as LMP2A of Epstein Barr virus (EBV) and Tip of herpesvirus saimiri (HVS) dysregulate lymphocyte signaling by interacting with Src family kinases. K15 open reading frame of Kaposi's sarcoma associated herpesvirus (KSHV), located at the right end of the viral genome, encodes several splicing variants differing in numbers of transmembrane domains. Previously, we demonstrated that the cytoplasmic tail of the K15 protein interfered with B cell receptor signal transduction to cellular tyrosine phosphorylation and calcium mobilization. However, the detailed mechanism underlying this phenomenon was not understood. In the C-terminal cytoplasmic region of K15, putative binding domains for Src-SH2 and -SH3 were identified. In this study, we attempted to characterize these modular elements and cellular binding protein(s) by GST pull down and co-immunoprecipitation assays. These studies revealed that K15 interacted with the major B cell tyrosine kinase Lyn. In vitro kinase and transient co-expression assays showed that the expression of K15 protein resulted in activation of Lyn kinase activity. In addition, GST pull down assay suggested that the SH2 domain of Lyn alone was necessary for interaction with the C-terminal SH2B (YEEV) of K15, but the addition of Lyn SH3 to the SH2 domain increases the binding affinity to K15 protein. The data from luciferase assays indicate that K15 expression in BJAB cells induced NFAT and AP1 activities. The tyrosine residue in the C-terminal end of K15 required for the Lyn interaction appeared to be essential for NFAT/AP1 activation, highlighting the significance of the C-terminal SH2B of K15 as a modular element in interfering with B lymphocyte signaling through interaction with Lyn kinase.
Cho
Nam-Hyuk
NH
Department of Microbiology and Immunology, College of Medicine and Institute of Endemic Diseases, Seoul National University Medical Research Center and Bundang Hospital, Seoul 110-799, Korea.
Choi
Young-Ki
YK
Choi
Joong-Kook
JK
D002460:Cell Line; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D012514:Sarcoma, Kaposi / Q000821:virology; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D050778:NFATC Transcription Factors; D018808:Transcription Factor AP-1; D014764:Viral Proteins; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases