Abstract
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.
Highlights
Interleukin-6 (IL-6) and ␥-interferon (IFN␥) activate an overlapping set of genes via the Jak/STAT pathway
We examined by electrophoretic mobility shift assays (EMSAs) the inhibitory effects of several synthetic peptides containing each one of the six tyrosine residues of the intracellular domain of gp130 in a nonphosphorylated and a phosphorylated form (Fig. 1A)
The phosphopeptide YPRHE which has the same sequence as YP767 except for a point mutation Gln 3 Glu at position ϩ3 showed almost no competition indicating that the glutamine in position ϩ3 from tyrosine 767 is important for acute phase response factor (APRF) activation
Summary
(Received for publication, October 25, 1995, and in revised form, February 20, 1996). Two members of the STAT family of transcription factors, namely APRF (or STAT3) and STAT1 are rapidly phosphorylated on tyrosine in a number of cell types upon IL-6 stimulation, a process for which Jak kinase was found to be essential (12, 19 –21) They homo- or heterodimerize and are translocated into the nucleus where they bind to specific enhancers within promoter regions of acute phase protein genes [22, 23]. Stahl et al [24] have recently demonstrated that any of the last four carboxyl-terminal tyrosine-containing motifs within the cytoplasmic domain of gp130 is sufficient for the activation of APRF They proposed the amino acid sequence YXXQ as an APRF binding consensus sequence. The identification of a novel phosphotyrosine motif within the cytoplasmic domain of gp130 important for STAT1 activation strongly suggests that STAT1 transiently associates with gp130 independent of APRF
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