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Comparative Study
. 2000 Oct;20(19):7132-9.
doi: 10.1128/MCB.20.19.7132-7139.2000.

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

T Zhang  1 , W H Kee, K T Seow, W Fung, X Cao
Affiliations
Comparative Study

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

T Zhang et al. Mol Cell Biol. 2000 Oct.

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 alpha-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 alpha-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.

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Figures

FIG. 1
FIG. 1
Schematic diagrams of the structural domains and deletion mutants of Stat3. α1, α2, α3, and α4 represent the α-helices in the coiled-coil region.
FIG. 2
FIG. 2
Tyr phosphorylation of Stat3 deletion mutants stimulated with EGF or IL-6. (A) COS-1 cells were transfected with control (C) or Stat3 expression plasmids as labeled and either left untreated or treated with EGF for 15 min. The cell lysates were prepared and subjected to Western blot analysis using antibody against an anti-phospho-Tyr-705-Stat3 (pY705ST3), as indicated in the upper panel. The membrane was stripped and reprobed with an anti-Stat3 antibody (lower panel). (B) Transfected HepG2 cells were either left untreated (−) or treated with IL-6 (+) for 15 min. Cell lysates were immunoprecipitated (IP) with FLAG antibody, and the immunoprecipitates were subjected to Western blot analysis using the anti-pY705ST3 antibody (upper panel). The blots were stripped and reprobed with Stat3 antibody (lower panel).
FIG. 3
FIG. 3
Deletion of helix α1 or α2 abolishes tyrosine phosphorylation of Stat3 in response to EGF or IL-6. COS-1 (A) or HepG2 (B) cells were transfected with the control plasmid (C), full-length Stat3 (FL), or the mutants in which either helix α1 (ΔH1) or α2 (ΔH2) was deleted and either left untreated (−) or treated with EGF or IL-6 (+). The tyrosine phosphorylation and expression of Stat3 were monitored by Western blot analysis (A) and immunoprecipitation/blotting analysis (B,) as described in the legend to Fig. 1.
FIG. 4
FIG. 4
Effect of the coiled-coil domain of Stat3 on its binding to the phosphorylated peptide derived from IL-6 receptor subunit gp130. (A) Diagram of the structure and Tyr phosphorylation sites of human gp130. Y1 to Y6 represent tyrosine residues 683, 759, 767, 814, 905, and 915 on the gp130 cytoplasmic tail, respectively. TM, transmembrane domain. a.a., amino acids. (B) COS-1 cells were transfected with the Stat3 plasmids as labeled on top of the upper panel and lysed after 48 h. The biotinylated peptides (pY2, SSTVQ-YPO4−-STVVHS; pY3, VVHSG-YPO4−-RHQVPS; and Y3, VVHSGYRHQVPS) derived from gp130 were incubated with streptavidin-Sepharose. The beads were washed and then incubated with aliquots of lysates. The complexes were washed, fractionated on SDS-PAGE, and immunoblotted with anti-Stat3 antibody (upper panel). The cell lysates were subjected to Western blot analysis with anti-Stat3 antibody to monitor the expression of the various Stat3 proteins in transfected cells (lower panel). (C) The blot used in panel B was stripped and reprobed with anti-SHP-2 antibody. Binding of the endogenous SHP-2 from cells transfected with full-length Stat3 to its docking site pY2 is shown.
FIG. 5
FIG. 5
Point mutant Asp170 of Stat3 diminishes its peptide-binding activity and the tyrosine phosphorylation stimulated by EGF. (A) Sequence alignment of helix α1 in the coiled-coil domain of STATs. The highly conserved hydrophilic residues are boxed and indicated at the bottom. (B) COS-1 cells were transfected with point mutants of Stat3 as labeled on top of the upper panel. The peptide-binding experiments were performed as described in the legend to Fig. 4. The expression of the mutant Stat3 proteins is examined in Western blot analysis in the lower panel. (C) COS-1 cells were transfected with control vector or Stat3 plasmids as labeled on top of the upper panel and induced by EGF for 15 min. The cell lysates were subjected to Western blot analysis with antibodies against phospho-Tyr-705-Stat3 (upper panel) or Stat3 (lower panel).
FIG. 6
FIG. 6
Phosphorylation and DNA binding of the helix α1 deletion mutant in vitro. Baculovirus-expressed full-length Stat3 (b-ST3-FL) and Stat3 with deletions of the N-domain and helix α1 (b-ST3-ΔN1H) were purified from Sf9 cells and incubated with Src kinase. The reaction mixture was divided so that each part contained 0.5 μg of Stat3 proteins and subjected to either Western blot analysis with the antibodies indicated (A) or EMSA using [32P]hSIE as a probe (B). FP, free probe.
FIG. 7
FIG. 7
Direct interaction of Stat3 and the gp130-derived phosphopeptide. Baculovirus-expressed full-length Stat3 (b-ST3-FL) and the deletion mutants (b-ST3-ΔN1H and b-ST3-ΔN4H) were purified from Sf9 cells, and 0.5 μg of each protein was subjected to peptide binding with either pY3 or Y3 as described in the legend to Fig. 4 (upper panel) or Western blot analysis (lower panel).
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