Macrophage migration inhibitory factor (MIF) is an important mediator that plays a central role in the control of the host immune and inflammatory response. To investigate the molecular mechanism of MIF action, we have used the yeast two-hybrid system and identified PAG, a thiol-specific antioxidant protein, as an interacting partner of MIF. Association of MIF with PAG was found in 293T cells transiently expressing MIF and PAG. The use of PAG mutants (C52S, C71S, and C173S) revealed that this association was significantly affected by C173S, but not C52S and C71S, indicating that a disulfide involving Cys 173 of PAG is responsible for the formation of MIF⅐PAG complex. In addition, the interaction was highly dependent on the reducing conditions such as dithiothreitol or -mercaptoethanol but not in the presence of H 2 O 2 . Analysis of the activities of the interacting proteins showed that the D-dopachrome tautomerase activity of MIF was decreased in a dose-dependent manner by coexpression of wild-type PAG, C52S, and C71S, whereas C173S was almost ineffective, suggesting that the direct interaction may be involved in the control of D-dopachrome tautomerase activity of MIF. Moreover, MIF has been shown to bind to PAG and it also inhibits the antioxidant activity of PAG.
Using the yeast two-hybrid assay and in vivo binding assay, we investigated whether B-myb oncogene products (Bmyb) can associate with each other. Specificity tests of the yeast two-hybrid system showed a self-association of B-myb proteins in yeast. Cotransfection experiments demonstrated that B-myb proteins form a complex in vivo. Deletion analysis revealed that this binding was sufficiently mediated by the carboxy-terminal conserved region of B-myb. In addition, the B-myb selfassociation is directly dependent on the amount of expressed Bmyb in cells and slightly increased by the dephosphorylation state. These results suggested that B-myb could form a complex and influence its transcriptional activity.z 1999 Federation of European Biochemical Societies.
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