A Mutation in the SH2 domain of STAT2 prolongs tyrosine phosphorylation of STAT1 and promotes type I IFN-induced apoptosis

Abstract

Type I interferons (IFN-alpha/beta) induce apoptosis in certain tumor cell lines but not others. Here we describe a mutation in STAT2 that confers an apoptotic effect in tumor cells in response to type I IFNs. This mutation was introduced in a conserved motif, PYTK, located in the STAT SH2 domain, which is shared by STAT1, STAT2, and STAT3. To test whether the tyrosine in this motif might be phosphorylated and affect signaling, Y631 of STAT2 was mutated to phenylalanine (Y631F). Although it was determined that Y631 was not phosphorylated, the Y631F mutation conferred sustained signaling and induction of IFN-stimulated genes. This prolonged IFN response was associated with sustained tyrosine phosphorylation of STAT1 and STAT2 and their mutual association as heterodimers, which resulted from resistance to dephosphorylation by the nuclear tyrosine phosphatase TcPTP. Finally, cells bearing the Y631F mutation in STAT2 underwent apoptosis after IFN-alpha stimulation compared with wild-type STAT2. Therefore, this mutation reveals that a prolonged response to IFN-alpha could account for one difference between tumor cell lines that undergo IFN-alpha-induced apoptosis compared with those that display an antiproliferative response but do not die.

Figure 1.

The PYTK motif within the SH2 domain of STAT1, STAT2 and STAT3. (A) The conserved motif PYTK (boxed) is conserved in STAT1, STAT2 and STAT3 (B) Domains of human STAT2. The position of PYTK motif located within the SH2 domain is marked. C-C indicates coil-coil domain; DBD, DNA-binding domain; SH2, Src-homology-2, and TAD, transactivation domain. (C) Panel of U6A cells expressing STAT2 with single amino acid substitutions were stimulated with or without IFN-α (3000 U/ml) or IFN-γ (10 ng/ml) and (%) growth inhibition was measured on day 3 by MTS assay. Results are shown as mean ± SD. Lower panel shows immunoblot analysis with anti-STAT2 and anti-actin antibodies. STAT2 expression levels were normalized against actin using densitometry and compared relative to U6A WT STAT2 expression. This is a representative experiment of three independent experiments.

Figure 2.

STAT2 Y631F stimulates the induction of apoptosis by type I IFNs. (A) U6A (STAT2−/−) cells or U6A reconstituted with wild-type STAT2 or STAT2 Y631F were stimulated with either 500 or 3000 U/ml IFN-α and growth inhibition was measured on day 3. (B) Same as in A except cells were stimulated with 3000 U/ml IFN-α and dually stained with Annexin V (x-axis) and propidium iodide (y-axis) and analyzed by flow cytometry. (C) Confocal microscopy of U6A cells reconstituted with wild-type STAT2 (top row) or STAT2 Y631F (bottom row) stimulated with or without IFN-α (3000 U/ml), IFN-β (3000 U/ml), or IFN-γ (10 ng/ml). (D) Same as in (C) except cells were pretreated for 2 h with the caspase inhibitor ZVAD (50 μM) before treatment with IFN-α (top row). Light field microscopy of cells shows morphological changes of apoptosis (bottom row). These experiments were repeated at least three times with similar results. Data are shown as mean ± SD.

Figure 3.

STAT2 Y631F prolongs STAT1 activation. Nuclear extracts were prepared from cells incubated with or without IFN-α for the indicated times. (A) Nuclear extracts were resolved by SDS-PAGE and immunoblot analysis was performed with antibodies against phosphoSTAT1-Y701 and phosphoSTAT2-Y690. Immunoblots were reprobed with RPA70 antibody to control for both equal loading of protein and purity of the nuclear extracts (lower panels). (B) Subcellular distribution of STAT1 and STAT2 was analyzed by confocal immunofluorescence microscopy. Cells were permeabilized and stained with anti-STAT1 (magenta), anti-Flag (STAT2, green), and counterstained with DAPI. STAT1 and STAT2 overlay is depicted as white. (C) Nuclear extracts were immunoprecipitated with anti-STAT2. Immunoblots were probed with anti-phospho-STAT1 (top panel) anti-phospho-STAT2 (middle panel) or anti-STAT2 (bottom panel) antibodies to ensure equal levels of immunoprecipitated proteins. Immunoprecipitation with IgG or beads alone were used as nonspecific negative controls (D) Nuclear extracts were assayed by EMSA for formation of ISGF3 and STAT1 homodimers with an ISRE and GRR probe, respectively.

Figure 4.

STAT2 Y631F augments ISGF3-mediated gene expression. Total RNA was isolated and qRT-PCR was performed in triplicate wells using specific primers to the indicated genes. 18S primers were included as an internal control to normalize for equal amount of cDNA. This is a representative experiment of three that were performed.

Figure 5.

STAT2 Y631F inhibits STAT1 tyrosine dephosphorylation. (A) Cells were stimulated with IFN-α (1000 U/ml) for 30 min followed by pulse chase with 500 ng/ml staurosporine (STS) for the indicated times. Whole-cell extracts were prepared and immunoblots were probed with an antibody against phosphoSTAT1-Y701. Membrane was reprobed for total STAT1 (bottom panel) to ensure equal loading of protein. (B) Same as in A except, STAT1 was immunoprecipitated from nuclear extracts and then incubated with increasing amounts of the protein tyrosine phosphatase TcPTP. IgG immunoprecipitates are shown as irrelevant negative controls.