Interleukin-6 (IL-6) and gamma-interferon (IFNgamma) activate an overlapping set of genes via the Jak/STAT pathway. However, at least in human cells, a differential activation of STAT transcription factors was observed: IL-6 activates both acute phase response factor (APRF)/STAT3 and STAT1, whereas IFNgamma leads only to STAT1 activation. All STATs cloned so far contain SH2 domains. Since all cytokine receptors using the Jak/STAT pathway were found to be tyrosine-phosphorylated after ligand binding, it has been proposed that specific phosphotyrosine modules within the cytoplasmic domain of the receptor chains recruit different STAT factors. We have analyzed by mutational studies and by phosphopeptide competition assays which of the tyrosine modules of the IL-6 signal transducer gp130 are capable of recruiting either APRF or STAT1. We found that two of the four tyrosine modules that are important for APRF activation also activate STAT1. For these modules, we propose the new consensus sequence YXPQ. We further present evidence that STAT1 is activated independently from APRF suggesting that gp130 contains multiple independent STAT binding sites. We compare the APRF and STAT1 activation motifs of gp130 with the STAT1 activation motif of the IFNgamma receptor and demonstrate that the specificity of activation can be changed from APRF to STAT1 and vice versa by only two point mutations within a tyrosine module. These data strongly support the concept that the activation of a specific STAT is determined mainly by the phosphotyrosine module. The significance of these findings for other receptor systems is discussed.
Distinct yet overlapping sets of STAT transcription factors are activated by different cytokines. One example is the differential activation of acute phase response factor (APRF, also called Stat3) and Stat1 by interleukin 6 and interferon-␥. Interleukin 6 activates both factors while, at least in human cells, interferon-␥ recruits only Stat1. Stat1 activation by interferon-␥ is mediated through a cytosolic tyrosine motif, Y440, of the interferon-␥ receptor. In an accompanying paper (Gerhartz, C., Heesel, B., Sasse, J., Hemmann, U., Landgraf, C., Schneider-Mergener, J., Horn, F., Heinrich, P. C., and Graeve, L. (1996) J. Biol. Chem. 271, 12991-12998), we demonstrated that two tyrosine motifs within the cytoplasmic part of the interleukin 6 signal transducer gp130 specifically mediate APRF activation while two others can recruit both APRF and Stat1. By expressing a series of Stat1/APRF domain swap mutants in COS-7 cells, we now determined which domains of Stat1 and APRF are involved in the specific recognition of phosphotyrosine motifs. Our data demonstrate that the SH2 domain is the sole determinant of specific STAT factor recruitment. Furthermore, the SH2 domain of Stat1 is able to recognize two unrelated types of phosphotyrosine motifs, one represented by the interferon-␥ receptor Y440DKPH peptide, and the other by two gp130 YXPQ motifs. By molecular modeling, we propose three-dimensional model structures of the Stat1 and APRF SH2 domains which allow us to explain the different binding preferences of these factors and to predict amino acids crucial for specific peptide recognition.Most interleukins, colony-stimulating factors, and interferons bind to plasma membrane receptors which are members of the hematopoietic receptor superfamily (1). These cytokines regulate cellular functions and gene expression via various intracellular signaling cascades of which the so-called JAK-STAT 1 pathway has recently attracted considerable attention (2). This pathway has first been established for interferon (IFN) signaling. The transcription factors Stat1␣, Stat1, and Stat2, formerly known as p91, p84, and p113 components, respectively, of the IFN-stimulated gene factor-3 complex were shown to be activated by tyrosine phosphorylation in response to IFN␣ (3) and Stat1 also by IFN␥ (4, 5). Subsequent to their phosphorylation, STAT factors homo-or heterodimerize, translocate to the nucleus, and bind to regulatory DNA elements of target genes. STAT factors contain putative SH3 and SH2 domains in their carboxyl-terminal parts as well as potential leucine zipper-like ␣-helical structures toward their amino termini (6). The SH2 domains seem to be involved in both the activation process and the dimerization of the STATs (7). A centrally located portion of Stat1 has recently been demonstrated to represent its DNA-binding domain (8). Tyrosine phosphorylation of STATs is most likely catalyzed by members of the JAK family of protein-tyrosine kinases (9). To date, four members of that family, Jak1, Jak2, Jak3, and Tyk2, have been cloned,...
cDNAs coding for the two receptor subunits of the interleukin-6 receptor have been stably expressed in Madine Darby canine kidney (MDCK) cells. The fate of the IL-6 binding protein (IL-6R) and of the signal transducing protein gpl30 was studied independently. Both proteins were proteolytically cleaved from cells metabolically labeled with [3"S]methionine/cysteine leading to the release of soluble receptor proteins of 55 kDa and 100 kDa, respectively. In contrast to the shedding of the ILdR gp130 was inefficiently released from the cells and the process was not significantly stimulated by the phorbolester PMA. In addition we show that the soluble forms of the IL-6R and gp130 released by transfected cells can form a ternary complexe with interleukin-6 indicating that such complexes also may occur in vivo.
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