Implications of an antiparallel dimeric structure of nonphosphorylated STAT1 for the activation-inactivation cycle

Abstract

IFN-gamma treatment of cells leads to tyrosine phosphorylation of signal transducer and activator of transcription (STAT) 1 followed by dimerization through a reciprocal Src homology 2-phosphotyrosine interaction near the -COOH end of each monomer, forming a parallel structure that accumulates in the nucleus to drive transcription. Prompt dephosphorylation and return to the cytoplasm completes the activation-inactivation cycle. Nonphosphorylated STATs dimerize, and a previously described interface between N-terminal domain (ND) dimers has been implicated in this dimerization. A new crystal structure of nonphosphorylated STAT1 containing the ND dimer has two possible configurations for the body of STAT1, one of which is antiparallel. In this antiparallel structure, the Src homology 2 domains are at opposite ends of the dimer, with the coiled:coil domain of one monomer interacting reciprocally with the DNA-binding domain of its partner. Here, we find that mutations in either the coiled:coil/DNA-binding domain interface or the ND dimer interface block dimerization of nonphosphorylated molecules and cause a resistance to dephosphorylation in vivo and resistance to a tyrosine phosphatase in vitro. We conclude that a parallel STAT1 phosphodimer not bound to DNA most likely undergoes a conformational rearrangement (parallel to antiparallel) to present the phosphotyrosine efficiently for dephosphorylation.

Fig. 1.

Coimmunoprecipitation of nonphosphorylated differentially tagged STAT1 proteins. (A) Sites of domain boundaries, point mutations, and epitope tagging are indicated. (B) Diagram of known STAT1 structures (2, 3). The DNA (blue coil)-bound tyrosine-phosphorylated structure is labeled parallel; in this structure, the NDs cannot interact (2). The two antiparallel diagrams are from ref. and are discussed in the Introduction. (C) 293T cells were transfected to express the indicated epitope-tagged proteins: F172W, single mutant; F77A/L78A, double mutant; F172W/F77A/L78A, triple mutant. Cell extracts were immunoprecipitated with anti-Flag antibody (IP:Flag) or anti-Myc antibody (IP:Myc). After precipitation and epitope release, SDS/PAGE and immunoblotting (IB) with anti-Myc or anti-Flag antibody was carried out.

Fig. 4.

Possible scenarios for STAT1 inactivation and predicted phenotypes for the F172W, F77A, and L78A mutants.

Fig. 2.

Effects of various STAT1 mutations on IFN-γ activation and subsequent inactivation. (A) Time course (min) of IFN-γ activation of cells expressing STAT1α or mutants assayed by EMSA. (B) Time course (hr) of activation of STAT1 and mutants assayed by EMSA. (C) Persistent tyrosine phosphorylation of mutant STAT1. U3A cells were transiently transfected (or not) with the indicated expression vectors and treated (or not) with IFN-γ for 0.5 or 6 h. Western blotting was with anti-pY STAT1 antibody or STAT1 antibody. (D) Failure to bind DNA, accomplished with the N460A mutation, does not affect the prolonged phosphorylation phenotype. Reconstituted U3A cells were treated with IFN-γ for 0.5 or 5 h. A portion of the extracts was subjected to EMSA (Left) whereas the remainder was analyzed for tyrosine phosphorylation by Western blotting with anti-pY STAT1 antibody (Right). Anti-STAT1 Western blots show the total expressed protein levels.

Fig. 3.

Dephosphorylation resistance of the STAT1 mutants. (A) Effect of the tyrosine kinase inhibitor staurosporine. After a 30-min induction with IFN-γ, U3A cells containing indicated STAT1 protein were treated as indicated with staurosporine and extracts were subjected to EMSA or pY Western blotting. The F172W (B) and F77A/L78A (C) mutants resist in vitro dephosphorylation. Purified tyrosine-phosphorylated proteins were incubated with increasing amounts of GST-TC45, and tyrosine phosphorylation was assayed by Western blotting with anti-pY STAT1 antibody. The anti-STAT1 Western blots show the total expressed protein levels.