Evidence of autophosphorylation in Txk: Y91 is an autophosphorylation site.
We have previously shown that Txk, a member of Tec family tyrosine kinase, is expressed in Th1 and ThO cells and directly contributes to gene transcription of Th1-related proteins, including interferon (IFN)-gamma, through nuclear translocation in response to mitogenic stimuli. Btk, another member of Tec family tyrosine kinase, has been shown to have a Src family tyrosine kinase-dependent transphosphorylation site and an autophosphorylation site. However, little is known about the phosphorylation mechanism of Txk, except that 420 tyrosine residue was identified as the transphosphorylation site. In this study, we found that Txk autophosphorylated itself by using an in vitro kinase assay. To elucidate the role of phosphorylation in Txk function, we studied IFN-gamma secretion by Jurkat T cells expressing mutant Txk proteins. While transfection with the wild-type Txk resulted in increased IFN-gamma production, the function was abrogated by disruption of the ATP biding site, which is presumably involved in the autophosphorylation mechanism. The results suggest that phosphorylated Txk is an active form to promote IFN-gamma synthesis. The 91 tyrosine residue of Txk is deduced to be an autophosphorylation site by comparing its structure with Btk. In Jurkat cells transfected with Txk Y91A, IFN-gamma production was decreased in comparison with the wild-type Txk transfected Jurkat cells. These data suggest that phosphorylation of the 91 tyrosine residue in Txk plays a positive regulatory role in Txk function.
Kashiwakura
Jun-Ichi
J
Department of Biochemistry, Hoshi University, Tokyo, Japan.
Suzuki
Noboru
N
Takeno
Mitsuhiro
M
Itoh
Saotomo
S
Oku
Teruaki
T
Sakane
Tsuyoshi
T
Nakajin
Shizuo
S
Toyoshima
Satoshi
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006706:Homeostasis / Q000502:physiology; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics; D019169:Jurkat Cells; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D014158:Transcription, Genetic; D015854:Up-Regulation / Q000502:physiology
D017931:DNA Primers; D007371:Interferon-gamma; D000255:Adenosine Triphosphate; C089848:Tec protein-tyrosine kinase; D011505:Protein-Tyrosine Kinases