Transcriptional coactivators such as p300 act as crucial elements in the eukaryotic gene regulation network. These proteins bind to various transcription factors which recruit them to specific gene regions whose chromatin structure subsequently is remodeled. Previously, we have shown that C/EBPβ recruits p300 by interacting with the E1A‐binding site of the coactivator. We now show that C/EBPβ not only binds to p300 but also triggers massive phosphorylation of p300. This novel activity of C/EBPβ is dependent on the E1A‐binding region of p300 as well as on several subdomains of C/EBPβ, all of which are involved in the p300–C/EBPβ interaction. We have identified several sites of C/EBPβ‐inducible phosphorylation within the C‐terminal domain of p300. Mutation of these sites substantially impairs the activity of p300 as a coactivator of C/EBPβ. Interestingly, phosphorylation of p300 is also triggered by other C/EBP family members as well as by various other transcription factors that interact with the E1A‐binding domain of p300, suggesting that this novel phosphorylation mechanism may be of general relevance.

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 Stat3␤. 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.Acute-phase response factor (APRF) was originally identified and characterized by us as a transcription factor rapidly activated in response to interleukin-6 (IL-6) and IL-6-related cytokines (36,37). APRF was then demonstrated to be a member of the signal transducer and transcription (STAT) factor family and is now called Stat3 (1,40). In addition to IL-6-type cytokines, i.e., IL-11, oncostatin M, leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotrophin-1, other cytokines and hormones have been shown to activate APRF (Stat3). Among them are alpha interferon, gamma interferon (in murine cells), IL-2, IL-5, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), CSF-1, growth hormone, prolactin, epidermal growth factor, and others (2-5, 7, 12, 18, 22, 25, 26). As is true for other STAT family members, inactive Stat3 is a cytoplasmic protein that associates with the intracellular parts of cytokine receptors upon ligand binding (20). The factors recruited in this way are tyrosine phosphorylated, most probably by the receptor-associated JAK protein tyrosine kinases (20). The STAT factors then dissociate from the receptor to form dimers (31). Subsequently, STAT dimers translocate to the nucleus, where they bind to regulatory DNA elements of target genes.The specificity of STAT factor activation and function is controlled at various levels. Tyrosine motifs within the cytokine receptor cytoplasmic parts that are phosphorylated after...

The cytokine interleukin-6 (IL-6) rapidly activates a latent cytoplasmic transcription factor, acute-phase response factor (APRF), by tyrosine phosphorylation. Activation and DNA binding of APRF are inhibited by inhibitors of protein tyrosine kinases but not serinelthreonine kinases. However, immediateearly gene induction by IL-6 and, as we show here, stimulaton of the promoters of the genes for az-macroglobulin, Jun-B, and intercellular adhesion molecule-I (ICAM-I) are blocked by the serinelthreonine kinase inhibitor 1-17. We now show that IL-6 triggers a delayed phosphorylation of APRF at serine resudues which can be reversed in vitro by protein phosphatase 2A and is also inhibited by H7. Therefore, APRF serine phosphorylation is likely to represent a crucial event in IL-6 signal transduction leading to target gene induction.

Several growth factors exert their mitogenic effect through the intrinsic tyrosine kinase activity located at the intracellular domain of their receptors [1]. The common event in the signal transduction of these receptors is the formation of a multimeric complex comprising the phosphotyrosine domains of the receptor and SH2/SH3 domains of effector or adapter proteins [2][3][4]. The phosphorylated platelet derived growth factor (PDGF) receptor appears to directly interact with SH2 domains in phosphatidylinositol (PI) 3-kinase or other proteins [5,6]. Similarly, phosphorylated epidermal growth factor (EGF)-receptors have been shown to form a complex including the SH2/SH3 adapter Grb-2 and the GTP exchange factor Sos-1 which subsequently activates Ras [7]. In