Ulrike Hemmann

MUTATIONAL ANALYSIS OF ACUTE-PHASE RESPONSE FACTOR/STAT3 ACTIVATION AND DIMERIZATION

Signal transducer and transcription (STAT) factors are activated by tyrosine phosphorylation in response to a variety of cytokines, growth factors, and hormones. Tyrosine phosphorylation triggers dimerization and nuclear translocation of these transcription factors. In this study, the functional role of carboxy-terminal portions of the STAT family member acute-phase response factor/Stat3 in activation, dimerization, and transactivating potential was analyzed. We demonstrate that truncation of 55 carboxy-terminal amino acids causes constitutive activation of Stat3 in COS-7 cells, as is known for the Stat3 isoform Stat3beta. By the use of deletion and point mutants, it is shown that both carboxy- and amino-terminal portions of Stat3 are involved in this phenomenon. Dimerization of Stat3 was blocked by point mutations affecting residues both in the vicinity of the tyrosine phosphorylation site (Y705) and more distant from this site, suggesting that multiple interactions are involved in dimer formation. Furthermore, by reporter gene assays we demonstrate that carboxy-terminally truncated Stat3 proteins are incapable of transactivating an interleukin-6-responsive promoter in COS-7 cells. In HepG2 hepatoma cells, however, these truncated Stat3 forms transmit signals from the interleukin-6 signal transducer gp130 equally well as does full-length Stat3. We conclude that, dependent on the cell type, different mechanisms allow Stat3 to regulate target gene transcription either with or without involvement of its putative carboxy-terminal transactivation domain.

Characterization and Binding Specificity of the Monomeric STAT3-SH2 Domain

Signal transducers and activators of transcription (STATs) are important mediators of cytokine signal transduction. STAT factors are recruited to phosphotyrosine-containing motifs of activated receptor chains via their SH2 domains. The subsequent tyrosine phosphorylation of the STATs leads to their dissociation from the receptor, dimerization, and translocation to the nucleus. Here we describe the expression, purification, and refolding of the STAT3-SH2 domain. Proper folding of the isolated protein was proven by circular dichroism and fluorescence spectroscopy. The STAT3-SH2 domain undergoes a conformational change upon dimerization. Using an enzyme-linked immunosorbent assay we demonstrate that the monomeric domain binds to specific phosphotyrosine peptides. The specificity of binding to phosphotyrosine peptides was assayed with the tyrosine motif encompassing Tyr705 of STAT3 and with all tyrosine motifs present in the cytoplasmic tail of the signal transducer gp130.

A complex of the soluble interleukin-6 receptor and interleukin-6 is internalized via the signal transducer gp130

In human body fluids a soluble form of the interleukin‐6 receptor (sIL‐6R) has been found which together with interleukin‐6 (IL‐6) acts agonistically on cells expressing the signal transducer gp130. The means by which the sIL‐6R is removed from the circulation is unknown. Here, we show that a complex of 125I‐labelled recombinant sIL‐6R and IL‐6 is internalized by MDCK cells stably transfected with gp130 and by human hepatoma cells HepG2 that endogenously express the IL‐6R and gp130. We further show that most of the internalized sIL‐6R is degraded within lysosomes. Our studies suggest that cells expressing gp130 are capable of endocytosing an IL‐6/sIL6R complex, thereby removing both from the circulation.

Endocytosis of interleukin-6—soluble interleukin-6 receptor complex and its intralysosomal degradation

Binding and internalization of interleukin-6—soluble interleukin-6 receptor complex by MDCK and MDCK-gp130 (transfected with gp 130 signal transductor) cells are studied. Binding of labeled complex depends on the concentration of interleukin-6; an effective internalization of the complex is shown. Binding and endocytosis of the complex are demonstrated in human hepatoma cells expressing interleukin-6 receptor and gp 130. These processes depend on the concentration of interleukin-6. The inhibitors of lysosomal functions ammonium chloride, monensin, and leupeptin suppress intralysosomal degradation of the complex, which confirms the important role of intralysosomal cleavage of the complex.