The Coiled-Coil Domain of Stat3 Is Essential for Its SH2 Domain-Mediated Receptor Binding and Subsequent Activation Induced by Epidermal Growth Factor and Interleukin-6

Abstract

STAT proteins are a family of latent transcription factors that mediate the response to various cytokines and growth factors. Upon stimulation by cytokines, STAT proteins are recruited to the receptors via their SH2 domains, phosphorylated on a specific tyrosine, dimerized, and translocated into the nucleus, where they bind specific DNA sequences and activate the target gene transcription. STATs share highly conserved structures, including an N-domain, a coiled-coil domain, a DNA-binding domain, a linker domain, and an SH2 domain. To investigate the role of the coiled-coil domain, we performed a systematic deletion analysis of the N-domain and each of the ␣-helices and mutagenesis of conserved residues in the coiled-coil region of Stat3. Our results indicate that the coiled-coil domain is essential for Stat3 recruitment to the receptor and the subsequent tyrosine phosphorylation and tyrosine phosphorylation-dependent activities, such as dimer formation, nuclear translocation, and DNA binding, stimulated by epidermal growth factor (EGF) or interleukin-6 (IL-6). Single mutation of Asp170 or, to a lesser extent, Lys177 in ␣-helix 1 diminishes both receptor binding and tyrosine phosphorylation. Furthermore, the Asp170 mutant retains its ability to bind to DNA when phosphorylated on Tyr705 by Src kinase in vitro, implying a functional SH2 domain. Finally, we demonstrate a direct binding of Stat3 to the receptor. Taken together, our data reveal a novel role for the coiled-coil domain that regulates the early events in Stat3 activation and function.Growth factors and cytokines regulate cell growth and differentiation by triggering various intracellular signaling pathways that lead to gene expression. JAK-STAT is a key pathway that mediates cellular responses to a variety of cytokines (reviewed in references 11, 20, and 30). As the name suggests, STATs (signal transducers and activators of transcription) represent a family of proteins with dual functions that transduce the signal in the cytoplasm and activate gene expression in the nucleus. Seven STATs with different functions have been identified so far in mammalian cells, and over 40 different polypeptides are known to activate one or more STATs (reviewed in reference 10). Among them, Stat3 was identified both as an acute-phase response factor activated by interleukin-6 (IL-6) in mouse liver and by homology to Stat1 (2, 46). Stat3 is also activated by other members of the IL-6 family, such as IL-11, ciliary neurotrophic factor, oncostatin M, and leukemia-inhibitory factor, which share the common transducing gp130 receptor subunit (2, 25, 33). IL-6, as a pleiotropic cytokine, exhibits various functions in immune response, hematopoiesis, and neuronal differentiation (37).The model of activation of Stat3 by IL-6 has been established. Binding of IL-6 to its receptor gp80 (subunit ␣) induces homodimerization of gp130 (subunit ␤) and phosphorylation of the gp130-associated Janus kinases (JAKs). JAKs phosphorylate the tyrosine residues on gp130 that serve as docking sites fo...

A Novel Sequence in the Coiled-coil Domain of Stat3 Essential for Its Nuclear Translocation

“…In this model, the monomeric Stat3 protein forms an intramolecular interaction between the coiled-coil domain and the C-terminal domains that facilitates the recruitment of Stat3 to the cytokine receptor via its SH2 domain in response to cytokine stimulation (35,49). In this report, we have identified Arg-214/215 in the coiledcoil domain as the potential NLS element for its nuclear translocation.…”

The role of Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8 regulator of transcription activation (RTA) in control of gene expression

“…RTA mutants that fail to stimulate STATactivated reporter gene expression also fail to induce STAT dimerization. The cytokine IL-6, and others, are known to provide an activation signal for STAT3 and to promote its translocation into the nucleus (Smith and Crompton, 1998;Schumann et al, 1999;Zhang et al, 2000). Therefore, in the presence of IL-6 and other mediators, a significant proportion of STAT3 will be in the nucleus, thereby increasing the probability of interactions with RTA.…”

The LIM/Homeodomain Protein Islet1 Recruits Janus Tyrosine Kinases and Signal Transducer and Activator of Transcription 3 and Stimulates Their Activities

“…This is in agreement to the results of EMSA described above. To further verify this point, we examined the effect of Isl1 on the Stat3 mutants, which harbor distinct point mutations in the tyrosine phosphorylation site (Y705F), SH2 domain (R609L), or DNA binding domain (R414/417A), that lack transcriptional activity (Zhang et al, 2000;Ma et al, 2003). The wildtype and the mutant Stat3 were transfected into COS-1 cells with the luciferase reporter gene m67-hSIE-luc in the absence or presence of Isl1.…”

“…The expression vectors for the murine Jak1 and the dominant negative form of Jak1 (K907E) were gifts from J. Ihle (Saint Jude Children's Research Hospital, Memphis, TN). The various Stat3 deletion mutations, the glutathione Stransferase (GST)-Stat3 fusion protein, and the Stat1 and Stat5 constructs have been described previously (Zhang et al, 2000;Lim and Cao, 2001;Lufei et al, 2003;Ma et al, 2003). All PCR products were checked by sequencing.…”

“…Nuclear extracts were prepared and used in DNA binding assays as described previously (Zhang et al, 2000;Ma et al, 2003). Briefly, 10 g of nuclear extract was used in the DNA binding assays with 32 P m67-hSIE, the high-affinity binding site of Stat3 as a radiolabeled probe.…”