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-␣/␤) 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-␣ stimulation compared with wild-type STAT2. Therefore, this mutation reveals that a prolonged response to IFN-␣ could account for one difference between tumor cell lines that undergo IFN-␣-induced apoptosis compared with those that display an antiproliferative response but do not die.

Identification of STAT2 Serine 287 as a Novel Regulatory Phosphorylation Site in Type I Interferon-induced Cellular Responses

“…EMSA-Five g of nuclear protein was mixed with 32 P-labeled double-stranded oligonucleotide probe corresponding to the IFN-␥ activated sequence (GAS) of the Fc␥RI gene or an ISRE sequence derived from the promoter of the ISG15 gene as described previously (20,21). Rabbit anti-STAT2 (C-20) and rabbit anti-STAT1 (E-23) were used for supershifting STAT2 and STAT1, respectively.…”

“…The transcriptional activity of STAT2 can also be modulated by a stretch of amino acid residues mapped to the SH2 domain of STAT2. Our laboratory identified a novel motif consisting of proline 630, tyrosine 631, threonine 632, and lysine 633 that we termed PYTK and is conserved in STAT1 and STAT3 (20,21). A point mutation of Pro-630 to leucine surfaced under IFN-␣ selection pressure and was found to abrogate IFN-␣ induced apoptosis, whereas mutation of Y631 to phenylalanine had the opposite effect as it promoted the apoptotic effects of IFN-␣.…”

Mutations leading to constitutive active gp130/JAK1/STAT3 pathway

“…The fourth mutation, Y640F, was in the PYTK motif conserved in STAT1, STAT2, and STAT3. Interestingly, substitution of the corresponding Y631 to phenylalanine in STAT2 promotes type I IFN signaling [29,30]. The three other mutations found in IHCA were distributed throughout the protein: the altered leucine-78, which disturbs latent dimer formation [31,32]; glutamate-166, which is part of helix alpha 1 involved in the interaction with gp130 [33]; and aspartate-502, which is located in the alpha-helical ''connector'' domain.…”

Wedelolactone, a Naturally Occurring Coumestan, Enhances Interferon-γ Signaling through Inhibiting STAT1 Protein Dephosphorylation

“…It has been shown that certain genotoxic agents could sensitize cells in response to very low doses of either interferon-␣ or ␥ through activation of STAT1 (48). Enhanced STAT1 phosphorylation caused by a mutation in the SH2 domain of STAT2 promoted type I IFNinduced apoptosis (49). Therefore, increased STAT1 phosphorylation appears to be a valid strategy to induce the cell death of tumor cells, and wedelolactone is a unique chemical modulator TCPTP (gene name PTPN2) was originally cloned from a human peripheral T cell cDNA library (50).…”