Cytokines are multifunctional mediators that classically modulate immune activity by receptor-mediated pathways. Macrophage migration inhibitory factor (MIF) is a cytokine that has a critical role in several inflammatory conditions but that also has endocrine and enzymatic functions. The molecular targets of MIF action have so far remained unclear. Here we show that MIF specifically interacts with an intracellular protein, Jab1, which is a coactivator of AP-1 transcription that also promotes degradation of the cyclin-dependent kinase inhibitor p27Kip1 (ref. 10). MIF colocalizes with Jab1 in the cytosol, and both endogenous and exogenously added MIF following endocytosis bind Jab1. MIF inhibits Jab1- and stimulus-enhanced AP-1 activity, but does not interfere with the induction of the transcription factor NFkappaB. Jab1 activates c-Jun amino-terminal kinase (JNK) activity and enhances endogenous phospho-c-Jun levels, and MIF inhibits these effects. MIF also antagonizes Jab1-dependent cell-cycle regulation by increasing p27Kip1 expression through stabilization of p27Kip1 protein. Consequently, Jab1-mediated rescue of fibroblasts from growth arrest is blocked by MIF. Amino acids 50-65 and Cys 60 of MIF are important for Jab1 binding and modulation. We conclude that MIF may act broadly to negatively regulate Jab1-controlled pathways and that the MIF-Jab1 interaction may provide a molecular basis for key activities of MIF.

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The molecular mechanism of action of MIF, a cytokine that plays a critical role in the host immune and inflammatory response, has not yet been identified. We recently demonstrated that MIF is an enzyme that exhibits oxidoreductase activity by a cysteine thiol-mediated mechanism. Here we further investigated this function by examining the reduction of insulin disulfides by wild-type human MIF (wtMIF) using various substrates, namely glutathione (GSH), dihydrolipoamide, Lcysteine, L L-mercaptoethanol and dithiothreitol. The activity of wtMIF was compared to that of the relevant cysteine mutants of MIF and to two carboxy-truncated mutants. Only GSH and dihydrolipoamide were found to serve as reductants, whereas the other substrates were not utilized by MIF. Reduction of insulin disulfides by MIF was closely dependent on the presence of the Cys SU -Ala-Leu-Cys TH (CALC) motif-forming cysteines C57 and C60, whereas C81 was not involved (activities : 51 þ 13%, 14 þ 5%, and 70 þ 12% of wtMIF, respectively, and 20 þ 3% for the double mutant C57S/C60S). Confirming the notion that the activity of MIF was dependent on the CALC motif in the central region of the MIF sequence, the C-terminal deletion mutants MIF(1^105) and MIF(1^110) were found to be fully active. The favored use of GSH and dihydrolipoamide indicated that MIF may be involved in the regulation of cellular redox processes and was supported further by the finding that MIF expression by the cell lines COS-1 and RAW 264.7 was significantly induced upon treatment with the oxidant hydrogen peroxide.z 1998 Federation of European Biochemical Societies.

Macrophage migration inhibitory factor (MIF) displays both cytokine and enzyme activities, but its molecular mode of action is still unclear. MIF contains three cysteine residues and we showed recently that the conserved Cys57-AlaLeu-Cys60 (CALC) motif is critical for the oxidoreductase and macrophage-activating activities of MIF. Here we probed further the role of this catalytic centre by expression, purification, and characterization of the cysteine3serine mutants Cys60Ser, Cys57Ser/Cys60Ser, and Cys81Ser of human MIF and of mutants Ala58Gly/ Leu59Pro and Ala58Gly/Leu59His, containing a thioredoxin (Trx)-like and protein disulphide isomerase (PDI)-like dipeptide, respectively. The catalytic centre mutants formed inclusion bodies and the resultant mutant proteins Cys57Ser/Cys60Ser, Ala58Gly/Leu59Pro, and Als58Gly/Leu59His were only soluble in organic solvent or 6 m GdmHCl when reconstituted at concentrations above 1 mg´mL 21 . This made it necessary to devise new purification methods. By contrast, mutant Cys81Ser was soluble. Effects of pH, solvent, and ionic strength conditions on the conformation of the mutants were analysed by far-UV CD spectropolarimetry and mutant stability was examined by denaturant-induced unfolding. The mutants, except for mutant Cys81Ser, showed a close conformational similarity to wild-type (wt) MIF, and stabilization of the mutants was due mainly to acid pH conditions. Intramolecular disulphide bond formation at the CALC region was confirmed by near-UV CD of mutant Cys60Ser. Mutant Cys81Ser was not involved in disulphide bond formation, yet had decreased stability. Analysis in the oxidoreductase and a MIF-specific cytokine assay revealed that only substitution of the active site residues led to inactivation of MIF. Mutant Cys60Ser had no enzyme and markedly reduced cytokine activity, whereas mutant Cys81Ser was active in both tests. The Trx-like variant showed significant enzyme activity but was less active than wtMIF; PDI-like MIF was enzymatically inactive. However, both variants had full cytokine activity. Together with the low but nonzero cytokine activity of mutant Cys60Ser, this indicated that the cytokine activity of MIF may not be tightly regulated by redox effects or that a distinguishable receptor mechanism exists. This study provides evidence for a role of the CALC motif in the oxidoreductase and cytokine activities of MIF, and suggests that Cys81 could mediate conformational effects. Availability and characterization of the mutants should greatly aid in the further elucidation of the mechanism of action of the unusual cytokine MIF.

The structure of the cytokine MIF has been investigated by X-ray crystallography, NMR, and biochemical methods with conflicting results regarding the structural and functional oligomerization state of this protein. Determination of the oligomeric state(s) is important for understanding more precisely the molecular mechanism of MIF action. To address this issue, we performed cross-linking of human and mouse MIF and selected mutants by various methods and analyzed the oligomerization by SDS-PAGE and gel filtration. MIF was found to form a mixture of monomeric, dimeric, and trimeric states at physiological concentrations, with the monomer and dimer representing the major species. Similar results were obtained when the carboxy-truncated mutants MIF(1^104) and MIF(1^109) were examined, indicating that the C-terminus of MIF is not critical for trimer stabilization. Cross-linking analysis of the isosteric CysCSer mutants C56S and C80S of human MIF resulted in a similar oligomer distribution, whereas substitution of Cys SW led to a significant reduction in the dimeric and trimeric forms, indicating that the hydrophobic region around Cys SW is important for the oligomerization of MIF. Together, our data argue that physiological MIF solutions contain a mixture of monomers, dimers, and trimers.z 1998 Federation of European Biochemical Societies.

Macrophage migration inhibitory factor (MIF) is a cytokine with broad regulatory functions in innate immunity. MIF belongs to the few cytokines displaying catalytic activities, i.e. MIF has a Pro2-dependent tautomerase and a Cys-Ala-Leu-Cys (CALC) cysteine-based thiol-protein oxidoreductase activity. Previous studies have addressed the roles of the catalytic site residues and the C-terminus. The two activities have not been directly compared. Here we report on the N-terminal mutational analysis and minimization of MIF and on a dissection of the two catalytic activities by comparing mutants P2AMIF, D4MIF, D5MIF, D6MIF, D7MIF, D8MIF, and D10MIF with the cysteine mutants of MIF. As N-terminal deletion was predicted to interfere with protein structure due to disruption of the central b sheet, it was surprising that deletion of up to six N-terminal residues resulted in normally expressed proteins with wild-type conformation. Strikingly, such mutants exhibited full MIF-specific immunologic activity. While mutation of Pro2 eliminated tautomerase activity, the CALC cysteine residues had no influence on this activity. However, mutant C81SMIF, which otherwise has full biologic activity, only had 32% tautomerase activity. Deletion of four N-terminal residues did not interfere with insulin reduction by MIF. By contrast, reduction of 2-hydroxyethyldisulfide (HED) was markedly affected by N-terminal manipulation, with P2AMIF and D2MIF exhibiting 40% activity, and D4MIF completely failing to reduce HED. This study constitutes the first comparison of the two catalytic activities of MIF and should assist in understanding the molecular links between the catalytic and immunologic activities of this cytokine and in providing guidelines for N-terminal protein minimization.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine for which a receptor has not been identified. That MIF has intracellular functions has been suggested by its enzymatic activity and constitutive expression profile. The discovery of functional MIF-c-Jun activation domain binding protein 1 (JAB1) binding has confirmed this notion and indicated that nonreceptor-based signaling mechanisms are important for MIF function. Here, we have generated and tested several biologically active labeled MIF derivatives to further define target protein binding by MIF and its cellular uptake characteristics. (35)S-MIF, biotinylated MIF, and fluoresceinated MIF were demonstrated to exhibit full biologic activity. Neither by applying a standard iodinated MIF preparation nor by using the biologically active (35)S-MIF derivative in receptor-binding studies were we able to measure any receptor-binding activity on numerous cells, confirming that uptake of MIF into target cells and MIF signaling can occur by receptor-independent pathways. When MIF derivatives were applied in cellular uptake studies, MIF was found to be endocytosed into both immune and nonimmune cells and targeted to the cytosol and lysosomes. The entry of MIF was temperature and energy dependent and was inhibited by monodansylcadaverine but not by ouabain. Endocytosed biotin-MIF bound JAB1 not only in macrophages, as shown previously, but also in nonimmune cells. A tagged MIF construct, MIF-enhanced green fluorescent protein (EGFP), was shown to be a valuable tool, as EGFP constructs of critical MIF cysteine mutants exhibited identical cellular localization properties to those of wild-type MIF (wtMIF). Our results indicate that MIF membrane receptors are not widely expressed, if at all, and suggest that the cellular uptake of MIF occurs by nonreceptor-mediated endocytosis rather than penetration. All the derivatives investigated, except for iodinated MIF, represent valuable tools for further MIF target protein and cellular studies.

Carboxy-truncated mutants of human MIF (MIF(1-104) and MIF(1-109)) were used in structure activity studies. CD spectroscopy revealed an overall structural similarity between the mutants and MIF. Denaturant-induced unfolding demonstrated that the C-terminus contributed significantly to the conformational stability of MIF. This appears to be due to the formation of two C-terminal ~-strands. The mutants were enzymatically active, exhibiting half of the enzymatic redox activity of MIF. However, immunological analysis showed that deletion of both 5 and 10 C-terminal residues resulted in loss of the macrophage activating.properties of MIF, providing functional evidence that the C-terminus is important for immunological activity and trimer formation. A more detailed study of the C-terminus may assist in identifying the molecular basis for the immunological and enzymatic activities of MIF.