HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.
Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg(176) to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334) was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.
Division of Genome Science and Microbiology, Department of Pathological Sciences, School of Medicine, Faculty of Medical Sciences, University of Fukui, Eiheiji, Japan.
Kenji
K
Nakashima
Kenji
K
Takeuchi
Kazuyasu
K
Chihara
Tomoko
T
Horiguchi
Xuedong
X
Sun
Lin
L
Deng
Ikuo
I
Shoji
Hak
H
Hotta
Kiyonao
K
Sada
D001402:B-Lymphocytes / Q000201:enzymology*; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers; D006801:Humans; D008024:Ligands; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D051076:Proto-Oncogene Proteins c-fyn / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D018909:src Homology Domains; D012324:RNA-Dependent RNA Polymerase
D017931:DNA Primers; D008024:Ligands; D051076:Proto-Oncogene Proteins c-fyn; D017361:Viral Nonstructural Proteins; C494957:FYN protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase