RESUMO
We report the successful expression and detection of a phosphorylated form of human T cell tyrosine kinase, Lck, in Saccharomyes cerevisiae, which leads to growth suppression of the yeast cells. Expression of an inactive Lck mutant resulted in no phosphorylation and no growth suppression, indicating that cell growth inhibition by Lck is due to the activity of the kinase, consistent with the observed tyrosine-phosphorylation of the Lck and yeast host cell proteins. The addition of a known inhibitor of Lck to the cell culture resulted in recovery of cell growth expressing the active Lck, suggesting that the growth inhibition by lck gene expression can be used to screen inhibitors for the gene product. We have extended such approach to Tob, another potential therapeutic target.
Assuntos
Inibidores Enzimáticos/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Saccharomyces cerevisiae/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Dados de Sequência Molecular , Fosforilação , Pirimidinas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), whereas the closely related virus HTLV-2 has not been associated with such malignant conditions. HTLV-1 Tax1 oncoprotein transforms a rat fibroblast cell line (Rat-1) much more efficiently than does HTLV-2 Tax2. By using a differential display analysis, we isolated MAGI-3 as a Tax1-inducible gene in Rat-1 cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that Tax1 induced MAGI-3 in Rat-1 cells. MAGI-3 has multiple PDZ domains and interacted with Tax1 but not Tax2 in 293T cells. The interaction of Tax1 with MAGI-3 was dependent on a PDZ domain-binding motif, which is missing in Tax2. The interaction of Tax1 with MAGI-3 altered their respective subcellular localization, and moreover, the interaction correlated well with the high transforming activities of Tax1 in Rat-1 cells relative to Tax2. MAGI-3 mRNA and the allied MAGI-1, but not MAGI-2, were expressed in HTLV-1-infected T-cell lines. Our results suggest that the interaction of Tax1 and MAGI-3 alters their respective biological activities, which may play a role in transformation by Tax1 as well as in the pathogenesis of HTLV-1-associated diseases.