Phosphorylation of STAT2 on serine-734 negatively regulates the IFN-α-induced antiviral response.

Serine phosphorylation of STAT proteins is an important post-translational modification event that, in addition to tyrosine phosphorylation, is required for strong transcriptional activity. However, we recently showed that phosphorylation of STAT2 on S287 induced by type I interferons (IFN-α and IFN-ß), evoked the opposite effect. S287-STAT2 phosphorylation inhibited the biological effects of IFN-α. We now report the identification and characterization of S734 on the C-terminal transactivation domain of STAT2 as a new phosphorylation site that can be induced by type I IFNs. IFN-α-induced S734-STAT2 phosphorylation displayed different kinetics to that of tyrosine phosphorylation. S734-STAT2 phosphorylation was dependent on STAT2 tyrosine phosphorylation and JAK1 kinase activity. Mutation of S734-STAT2 to alanine (S734A) enhanced IFN-α-driven antiviral responses compared to those driven by wild-type STAT2. Furthermore, DNA microarray analysis demonstrated that a small subset of type I IFN stimulated genes (ISGs) was induced more by IFNα in cells expressing S734A-STAT2 when compared to wild-type STAT2. Taken together, these studies identify phosphorylation of S734-STAT2 as a new regulatory mechanism that negatively controls the type I IFN-antiviral response by limiting the expression of a select subset of antiviral ISGs.

Identification of STAT2 serine 287 as a novel regulatory phosphorylation site in type I interferon-induced cellular responses.

STAT2 is a positive modulator of the transcriptional response to type I interferons (IFNs). STAT2 acquires transcriptional function by becoming tyrosine phosphorylated and imported to the nucleus following type I IFN receptor activation. Although most STAT proteins become dually phosphorylated on specific tyrosine and serine residues to acquire full transcriptional activity, no serine phosphorylation site in STAT2 has been reported. To find novel phosphorylation sites, mass spectrometry of immunoprecipitated STAT2 was used to identify several phosphorylated residues. Of these, substitution of serine 287 with alanine (S287A) generated a gain-of-function mutant that enhanced the biological effects of IFN-α. S287A-STAT2 increased cell growth inhibition, prolonged protection against vesicular stomatitis virus infection and enhanced transcriptional responses following exposure of cells to IFN-α. In contrast, a phosphomimetic STAT2 mutant (S287D) produced a loss-of-function protein that weakly activated IFN-induced ISGs. Our mechanistic studies suggest that S287A-STAT2 likely mediates its gain-of-function effects by prolonging STAT2/STAT1 dimer activation and retaining it in transcriptionally active complexes with chromatin. Altogether, we have uncovered that in response to type I IFN, STAT2 is serine phosphorylated in the coiled-coil domain that when phosphorylated can negatively regulate the biological activities of type I IFNs.

STAT2 phosphorylation and signaling.

STAT2 is an essential transcription factor in type I IFN mediated anti-viral and anti-proliferative signaling. STAT2 function is regulated by tyrosine phosphorylation, which is the trigger for STAT-dimerization, subsequent nuclear translocation, and transcriptional activation of IFN stimulated genes. Evidence of additional STAT2 phosphorylation sites has emerged as well as novel roles for STAT2 separate from the classical ISGF3-signaling. This review aims to summarize knowledge of phosphorylation-mediated STAT2-regulation and future avenues of related STAT2 research.

The role of signal transducer and activator of transcription-2 in the interferon response.

The signal transducer and activator of transcription-2 (STAT2) was discovered as a cellular component of the DNA binding complex known as interferon (IFN) stimulated gene factor-3. Numerous studies have confirmed that STAT2 operates as a positive regulator in the transcriptional activation response elicited by IFNs. In this article, we highlight the progress made in elucidating the pivotal role of STAT2 in driving the expression of IFN-induced genes, innate antiviral immunity, apoptosis, and cancer. A better understanding of the functional role of STAT2 in the IFN response and how STAT2 is regulated will uncover new clues to its role in diseases.

Interferon-lambda as a potential therapeutic agent in cancer treatment.

The discovery that type I interferon (IFN-alpha/beta) inhibited tumor cell growth was welcomed initially with great excitement as it rapidly became a U.S. Food and Drug Administration-approved drug to treat several forms of cancer. In time, this enthusiasm diminished as severe toxicity associated with IFN-alpha administration, resistance to the therapy, or less than optimal responses became evident in cancer patients, thus restricting its clinical use and reducing its potential as an anticancer drug. The recent discovery of a third type of IFN [IFN-lambda/interleukin (IL)-29/IL-28], which shares the same biological properties of type I IFNs, opens the door for evaluating the therapeutic potential of IFN-lambda as it uses a distinct receptor complex whose expression, unlike type I IFN receptors, is restricted to cells of specific lineage. It is unclear whether the mechanism by which type III IFNs restrict tumor cell proliferation is different or the same from the one utilized by type I IFN. Nevertheless, accumulating evidence as described in this review suggests that, in contrast to IFN-alpha therapy, IFN-lambda therapy could be less toxic and suitable for certain types of malignancies as not all cells are responsive to this cytokine.

Resistance to IFN-alpha-induced apoptosis is linked to a loss of STAT2.

Type I IFNs (IFN-alpha/beta) are pleitropic cytokines widely used in the treatment of certain malignancies, hepatitis B and C, and multiple sclerosis. IFN resistance is a challenging clinical problem to overcome. Hence, understanding the molecular mechanism by which IFN immunotherapy ceases to be effective is of translational importance. In this study, we report that continuous IFN-alpha stimulation of the human Jurkat variant H123 led to resistance to type I IFN-induced apoptosis due to a loss of signal transducers and activators of transcription 2 (STAT2) expression. The apoptotic effects of IFN-alpha were hampered as STAT2-deficient cells were defective in activating the mitochondrial-dependent death pathway and ISGF3-mediated gene activation. Reconstitution of STAT2 restored the apoptotic effects of IFN-alpha as measured by the loss of mitochondrial membrane potential, cytochrome c release from mitochondria, caspase activation, and ultimately cell death. Nuclear localization of STAT2 was a critical event as retention of tyrosine-phosphorylated STAT2 in the cytosol was not sufficient to activate apoptosis. Furthermore, silencing STAT2 gene expression in Saos2 and A375S.2 tumor cell lines significantly reduced the apoptotic capacity of IFN-alpha. Altogether, we show that STAT2 is a critical mediator in the activation of type I IFN-induced apoptosis. More importantly, defects in the expression or nuclear localization of STAT2 could lessen the efficacy of type I IFN immunotherapy.