Dephosphorylation of phosphotyrosine on STAT1 dimers requires extensive spatial reorientation of the monomers facilitated by the N-terminal domain - PubMed (original) (raw)

Dephosphorylation of phosphotyrosine on STAT1 dimers requires extensive spatial reorientation of the monomers facilitated by the N-terminal domain

Claudia Mertens et al. Genes Dev. 2006.

Abstract

We report experiments that infer a radical reorientation of tyrosine-phosphorylated parallel STAT1 dimers to an antiparallel form. Such a change in structure allows easy access to a phosphatase. With differentially epitope-tagged molecules, we show that the two monomers of a dimer remain together during dephosphorylation although they most likely undergo spatial reorientation. Extensive single amino acid mutagenesis within crystallographically established domains, manipulation of amino acids in an unstructured tether that connects the N-terminal domain (ND) to the core of the protein, and the demonstration that overexpressed ND can facilitate dephosphorylation of a core molecule lacking an ND all support this model: When the tyrosine-phosphorylated STAT1 disengages from DNA, the ND dimerizes and somehow assists in freeing the reciprocal pY-SH2 binding between the monomers of the dimer while ND ND dimerization persists. The core of the monomers rotate allowing reciprocal association of the coiled:coil and DNA-binding domains to present pY at the two ends of an antiparallel dimer for ready dephosphorylation.

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Figures

Figure 1.

Figure 1.

Schematic of salient features of STAT1 structure. The domains are N-terminal domain (ND), coiled:coil domain (CC), DNA-binding domain (DBD), linker domain (L), and SH2 domain (SH2). The ND is shown tied to the CC through a flexible tether (not to scale), and the residues C-terminal to the –SH2 include the Y701, which is phosphorylated (red dot) when the molecule is activated. The C-terminal region is also flexible as indicated by the wavy black line. At the bottom of the figure are diagrams of the parallel and antiparallel structures supported by crystallographic results (Chen et al. 1998; Mao et al. 2005). Notable is the F172 residue that is important in the antiparallel structure.

Figure 2.

Figure 2.

Monomers of a STAT1 phosphodimer remain associated during dephosphorylation. (A,B) Purification of STAT1-Flag:STAT1-Myc phosphodimers. 293T cells were cotransfected with expression constructs for Flag-tagged STAT1 and Myc-tagged STAT1. After IFN-γ induction, whole-cell extracts were subjected to a three-step affinity-purification scheme (see Materials and Methods). Material was evaluated by EMSA with a M67 probe before purification (A) and after purification (B) using anti-Flag and anti-Myc antibodies for shifting STAT1–DNA complexes. (C,D) Continued association of STAT1 monomers in a dimer after in vitro dephosphorylation with GST-TC45. Purified doubly tagged STAT1 phosphodimers alone (C,D, lanes 1,3) or as mixture with purified nontagged phospho STAT1β (C,D, lanes 2,4) were incubated for 60 min with purified tyrosine phosphatase TC45. (Top panels) Tyrosine phosphorylation of STAT1 proteins was monitored before and after TC45 treatment by EMSA. (Bottom panels) The presence of Flag-Myc heterodimers before and after dephosphorylation was assayed by pull-down with Ni-NTA agarose and immunoblot detection of coimmunoprecipitated proteins with anti-Flag antibodies. Note that in C approximately equal amounts of doubly tagged STAT1 (lane 2, upper band) and nontagged STAT1β (lane 2, lower band) were used, whereas in D 10-fold as much nontagged STAT1β was included to assay for a possible dissociation and reassociation of tagged dimers.

Figure 3.

Figure 3.

Persistent phosphorylation of STAT1 tether mutants. (A) Amino acid sequence of STAT1 tether and tether mutants. (B) Dephosphorylation of STAT1 and STAT1 mutants. U3A cells that lack STAT1 were left untransfected (U3A) or transfected for 24 h with vectors encoding wild-type STAT1 (WT), STAT1 lacking the first 135 amino acids (Δ135), or other mutant STAT1 proteins: F172W, Δ17T, Δ12T, Δ7T; as indicated in A. Parallel samples of cells were treated or not treated with IFN-γ for 30 min, and IFN-γ for 30 min followed by the tyrosine kinase inhibitor staurosporine for an additional 45 min. Total cell extracts were prepared and Western blotted with anti-pY STAT1 (top) and anti-STAT1 antibodies (bottom). (C) Time course of dephosphorylation in staurosporine-treated cells. U3A cells were complemented with wild-type STAT1 or tether mutants 12TCR (12 central amino acids reversed, shown in red in A) or Δ12TC (12 central amino acids deleted). Expressed STAT1 proteins were IFN-γ activated for 30 min followed by treatment with staurosporine for the indicated times (30, 60, 90, 120 min). Immunoblot analysis of whole-cell extracts as in B. (D) Same assay as in B with wild-type STAT1 or mutants Δ12T and MetH-T in which residues 121–141 of STAT1 are replaced with 21 amino acids of human methionine synthase (shown in green in A).

Figure 4.

Figure 4.

Expression of free STAT1-ND but not STAT5b-ND rescues the phenotype of mutant Δ135. (A) Coexpression of Δ135 with STAT1 ND constructs in U3A cells. Cells were transfected with indicated combinations of Δ135 and empty vector, Δ135 and HA-tagged wild-type ND, or Δ135 and HA-tagged ND-F77/L78A, which prevents ND dimerization. After IFN-γ activation followed by staurosporine treatment for indicated times (in minutes), total cell extracts were subjected to immunoblot analysis. (Bottom panel) Expression of NDs was detected with anti-HA antibodies. (B) Coexpression of Δ135 with ND of STAT1 and STAT5b. Preparation of samples as in A with staurosporine treatment for 45 min. Experiments were carried out in duplicate.

Figure 5.

Figure 5.

Mutations of the pocket for residue F172 in the DBD cause persistent phosphorylation. (A) Shown is the crystallographically determined structure of amino acids in DBD of STAT1 that form a pocket in which the F172 residues of the CCDs interact. (B,C) STAT1 proteins with indicated mutations that were designed to disrupt the CCD/DBD dimer interface were expressed in U3A cells. After activation with IFN-γ for 30 min and staurosporine treatment for 45 min, total cell extracts were analyzed by immunoblotting with anti-pY STAT1 (top) and anti-STAT1 antibodies (bottom). (D) DNA binding of activated pocket mutants. Total cell extracts prepared as in B and C were subjected to EMSA analysis with an M67 probe.

Figure 6.

Figure 6.

Nuclear entry of persistently phosphorylated STAT1 mutants. (A) U3A cells transfected with expression constructs for wild-type STAT1 and mutants Δ135 and Q340A were treated with IFN-γ for 30 min and staurosporine for 45 min. Whole-cell extracts (WCE) and nuclear extracts (NE) were prepared in parallel from identical samples by dividing cells evenly before lysate preparation. Samples were subjected to immunoblotting with antibodies to pY STAT1 and STAT1. (B) Flow of activated STAT1 proteins upon staurosporine addition. U3A cells expressing wild-type STAT1 or mutant proteins Δ135 and Q340A were treated with IFN-γ for 30 min followed by staurosporine treatment for the indicated times (in minutes). Cytoplasmic (Cy) and nuclear extracts (NE) were analyzed by immunoblotting.

Figure 7.

Figure 7.

Model for STAT1 dephosphorylation using crystal structures of ND and core of STAT1. The domains and icons for STAT1 are given in Figure 1. Note that the red dot at Y701 indicates phosphorylation. First consider the parallel phosphorylated dimer (pSTAT dimer) bound to DNA. Disengagement from DNA is shown in the second panel; flexibility of the free pSTAT dimer allows contact between NDs because they are on an ∼60 Å tether (Chen et al. 1998). The ND, possibly by contacting the body of the pSTAT, induces pY–SH2 disengagement. The dimer still held together by the ND/ND interface allows rotation of the monomers so that CC/DBD interaction can occur, presenting the pY at 701 for dephosphorylation. Exit to the cytoplasm of the antiparallel nonphosphorylated dimer then occurs. In the cytoplasm, nonphosphorylated STAT1 is shown as either a monomer or dimer and can likely be tyrosine-phosphorylated in either mode. STAT4 may be obligatorily a dimer in order to be phosphorylated at the receptor (Ota et al. 2004).

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