STAT1-cooperative DNA binding distinguishes type 1 from type 2 interferon signaling.

STAT1 is an indispensable component of a heterotrimer (ISGF3) and a STAT1 homodimer (GAF) that function as transcription regulators in type 1 and type 2 interferon signaling, respectively. To investigate the importance of STAT1-cooperative DNA binding, we generated gene-targeted mice expressing cooperativity-deficient STAT1 with alanine substituted for Phe77. Neither ISGF3 nor GAF bound DNA cooperatively in the STAT1F77A mouse strain, but type 1 and type 2 interferon responses were affected differently. Type 2 interferon-mediated transcription and antibacterial immunity essentially disappeared owing to defective promoter recruitment of GAF. In contrast, STAT1 recruitment to ISGF3 binding sites and type 1 interferon-dependent responses, including antiviral protection, remained intact. We conclude that STAT1 cooperativity is essential for its biological activity and underlies the cellular responses to type 2, but not type 1 interferon.

A family-wide assessment of latent STAT transcription factor interactions reveals divergent dimer repertoires.

The conversion of signal transducer and activator of transcription (STAT) proteins from latent to active transcription factors is central to cytokine signaling. Triggered by their signal-induced tyrosine phosphorylation, it is the assembly of a range of cytokine-specific STAT homo- and heterodimers that marks a key step in the transition of hitherto latent proteins to transcription activators. In contrast, the constitutive self-assembly of latent STATs and how it relates to the functioning of activated STATs is understood less well. To provide a more complete picture, we developed a co-localization-based assay and tested all 28 possible combinations of the seven unphosphorylated STAT (U-STAT) proteins in living cells. We identified five U-STAT homodimers-STAT1, STAT3, STAT4, STAT5A, and STAT5B-and two heterodimers-STAT1:STAT2 and STAT5A:STAT5B-and performed semi-quantitative assessments of the forces and characterizations of binding interfaces that support them. One STAT protein-STAT6-was found to be monomeric. This comprehensive analysis of latent STAT self-assembly lays bare considerable structural and functional diversity in the ways that link STAT dimerization before and after activation.

Evidence against a role for ß-arrestin1 in STAT1 dephosphorylation and the inhibition of interferon-γ signaling.

Signal transducer and activator of transcription 1 (STAT1) is activated by tyrosine phosphorylation upon interferon-γ (IFNγ) stimulation, which results in the expression of genes with antiproliferative and immunomodulatory functions. The inactivation of STAT1 occurs through tyrosine dephosphorylation by the tyrosine phosphatase TC45. It was proposed that recruitment of TC45 required the direct interaction of STAT1 with the scaffold protein ß-arrestin1, making ß-arrestin1 an essential negative regulator of STAT1 and IFNγ signaling (Mo et al., 2008). We tested the relevance of ß-arrestin1 for STAT1 activity. Our results do not confirm ß-arrestin1 as a STAT1-interacting protein. The STAT1 phosphorylation/dephosphorylation cycle was found to be unaffected by both the overexpression and the genetic deletion of ß-arrestin1. Accordingly, ß-arrestin1 did not inhibit STAT1 transcriptional activity or the induction of IFNγ target genes in response to IFNγ. Our data indicate that ß-arrestin1 is dispensable for STAT1 dephosphorylation and the termination of IFNγ signaling.

STAT2 Is a Pervasive Cytokine Regulator due to Its Inhibition of STAT1 in Multiple Signaling Pathways.

STAT2 is the quintessential transcription factor for type 1 interferons (IFNs), where it functions as a heterodimer with STAT1. However, the human and murine STAT2-deficient phenotypes suggest important additional and currently unidentified type 1 IFN-independent activities. Here, we show that STAT2 constitutively bound to STAT1, but not STAT3, via a conserved interface. While this interaction was irrelevant for type 1 interferon signaling and STAT1 activation, it precluded the nuclear translocation specifically of STAT1 in response to IFN-γ, interleukin-6 (IL-6), and IL-27. This is explained by the dimerization between activated STAT1 and unphosphorylated STAT2, whereby the semiphosphorylated dimers adopted a conformation incapable of importin-α binding. This, in turn, substantially attenuated cardinal IFN-γ responses, including MHC expression, senescence, and antiparasitic immunity, and shifted the transcriptional output of IL-27 from STAT1 to STAT3. Our results uncover STAT2 as a pervasive cytokine regulator due to its inhibition of STAT1 in multiple signaling pathways and provide an understanding of the type 1 interferon-independent activities of this protein.

SUMO conjugation of STAT1 protects cells from hyperresponsiveness to IFNγ.

The biologic effects of IFNγ are mediated by the transcription factor STAT1. The activity of STAT1 is inhibited by small ubiquitin-like modifier (SUMO) conjugation. This occurs both directly through decreasing STAT1 tyrosine phosphorylation and indirectly by facilitating STAT1 dephosphorylation consequential to increased STAT1 solubility because of suppressed paracrystal assembly. However, the physiologic implications of SUMO conjugation have remained unclear. Here, we used fibroblasts and bone marrow-derived macrophages (BMMs) from knockin mice expressing SUMO-free STAT1 to explore the consequences of STAT1 sumoylation for IFNγ signaling. Our experiments demonstrated buffer property of paracrystals for activated STAT1, such that SUMO-mediated paracrystal dispersal profoundly reduced phosphorylation of STAT1, which affected both the activating tyrosine 701 and the transcription-enhancing serine 727. Accordingly, the curtailed STAT1 activity in the nucleus caused by SUMO conjugation resulted in diminished transcription of IFNγ-responsive genes; and increased the IFNγ concentration more than 100-fold required to trigger lipopolysaccharide-induced cytotoxicity in bone marrow-derived macrophages. These experiments identify SUMO conjugation of STAT1 as a mechanism to permanently attenuate the IFNγ sensitivity of cells, which prevents hyperresponsiveness to this cytokine and its potentially self-destructive consequences. This sets the mode of SUMO-mediated inhibition apart from the other negative STAT regulators known to date.

Dimer formation and conformational flexibility ensure cytoplasmic stability and nuclear accumulation of Elk-1.

The ETS (E26) protein Elk-1 serves as a paradigm for mitogen-responsive transcription factors. It is multiply phosphorylated by mitogen-activated protein kinases (MAPKs), which it recruits into pre-initiation complexes on target gene promoters. However, events preparatory to Elk-1 phosphorylation are less well understood. Here, we identify two novel, functional elements in Elk-1 that determine its stability and nuclear accumulation. One element corresponds to a dimerization interface in the ETS domain and the second is a cryptic degron adjacent to the serum response factor (SRF)-interaction domain that marks dimerization-defective Elk-1 for rapid degradation by the ubiquitin-proteasome system. Dimerization appears to be crucial for Elk-1 stability only in the cytoplasm, as latent Elk-1 accumulates in the nucleus and interacts dynamically with DNA as a monomer. These findings define a novel role for the ETS domain of Elk-1 and demonstrate that nuclear accumulation of Elk-1 involves conformational flexibility prior to its phosphorylation by MAPKs.

Cytokine-induced paracrystals prolong the activity of signal transducers and activators of transcription (STAT) and provide a model for the regulation of protein solubility by small ubiquitin-like modifier (SUMO).

The biological effects of cytokines are mediated by STAT proteins, a family of dimeric transcription factors. In order to elicit transcriptional activity, the STATs require activation by phosphorylation of a single tyrosine residue. Our experiments revealed that fully tyrosine-phosphorylated STAT dimers polymerize via Tyr(P)-Src homology 2 domain interactions and assemble into paracrystalline arrays in the nucleus of cytokine-stimulated cells. Paracrystals are demonstrated to be dynamic reservoirs that protect STATs from dephosphorylation. Activated STAT3 forms such paracrystals in acute phase liver cells. Activated STAT1, in contrast, does not normally form paracrystals. By reversing the abilities of STAT1 and STAT3 to be sumoylated, we show that this is due to the unique ability of STAT1 among the STATs to conjugate to small ubiquitin-like modifier (SUMO). Sumoylation had one direct effect; it obstructed proximal tyrosine phosphorylation, which led to semiphosphorylated STAT dimers. These competed with their fully phosphorylated counterparts and interfered with their polymerization into paracrystals. Consequently, sumoylation, by preventing paracrystal formation, profoundly curtailed signal duration and reporter gene activation in response to cytokine stimulation of cells. The study thus identifies polymerization of activated STAT transcription factors as a positive regulatory mechanism in cytokine signaling. It provides a unifying explanation for the different subnuclear distributions of STAT transcription factors and reconciles the conflicting results as to the role of SUMO modification in STAT1 functioning. We present a generally applicable system in which protein solubility is maintained by a disproportionately small SUMO-modified fraction, whereby modification by SUMO partially prevents formation of polymerization interfaces, thus generating competitive polymerization inhibitors.

Green fluorescent protein-tagging reduces the nucleocytoplasmic shuttling specifically of unphosphorylated STAT1.

Fluorescence recovery after photobleaching (FRAP) and related techniques using green fluorescent protein (GFP)-tagged proteins are widely used to study the subcellular trafficking of proteins. It was concluded from these experiments that the cytokine-induced nuclear import of tyrosine-phosphorylated (activated) signal transducer and activator of transcription 1 (STAT1) was rapid, while the constitutive shuttling of unphosphorylated STAT1 was determined to be inefficient. However, unrelated experiments came to different conclusions concerning the constitutive translocation of STAT1. Because these discrepancies have not been resolved, it remained unclear whether or not unphosphorylated STAT1 is a relevant regulator of cytokine-dependent gene expression. This study was initiated to examine the influence of GFP-tagging on the nucleocytoplasmic shuttling of phosphorylated and unphosphorylated STAT1. In accordance with previous findings our results confirm the undisturbed rapid nuclear import of GFP-tagged activated STAT1. However, we reveal an inhibitory influence of GFP specifically on the constitutive nucleocytoplasmic cycling of the unphosphorylated protein. The decreased shuttling of unphosphorylated STAT1-GFP significantly reduced the activation level while nuclear accumulation was prolonged. Importantly, despite unimpaired nuclear import of activated STAT1 the transcription of a STAT1-dependent reporter gene was more than halved after GFP-tagging, which could be linked directly to reduced nucleocytoplasmic shuttling. In conclusion, it is demonstrated that GFP-based techniques considerably underestimate the actual shuttling rate of unphosphorylated native STAT1. The results confirm that the activation of STAT1 and hence its transcriptional activity is proportional to the nucleocytoplasmic shuttling rate of the unphosphorylated protein. Moreover, our data indicate that GFP-tagging may differently affect the mechanistically distinct translocation pathways of a shuttling protein.

A single residue modulates tyrosine dephosphorylation, oligomerization, and nuclear accumulation of stat transcription factors.

The NH(2) terminus of Stat proteins forms a versatile protein interaction domain that is believed to use discrete surfaces to mediate oligomerization and tyrosine dephosphorylation of Stat dimers. Here we show for Stat1 and Stat5a/b that these interfaces overlap and need to be reassigned to an unrelated region of the N-domain. Unexpectedly, our study showed for Stat1 that defective oligomerization of DNA-bound dimers was associated with prolonged interferon-induced nuclear accumulation. This uncoupling of DNA binding and nuclear retention was explained by the concomitant dephosphorylation deficiency that both Stat1 and Stat5a/b have in common and that for Stat1 was due to defective dephosphorylation by the phosphatase TC45. Furthermore, diminished N-domain-mediated oligomerization affected transcriptional activation by both Stat1 and Stat5a/b in a promoter-specific manner. DNA binding analysis indicated that oligomerization of Stats on DNA may be common, irrespective of the presence of multiple canonical binding sites. Accordingly, also transcription from promoters with only a single discernable gamma-activated sequence site was negatively effected by reduced tetramerization. Thus, these results indicate that defective oligomerization cannot generally be compensated for by enhanced tyrosine phosphorylation and prolonged nuclear accumulation. In addition, these data clarify the role of DNA binding in nuclear retention of Stat1.

Constitutive and IFN-gamma-induced nuclear import of STAT1 proceed through independent pathways.

STAT1 functions as both a constitutive transcriptional regulator and, in response to cytokine stimulation of cells, as an inducible tyrosine-phosphorylated transcription factor. Here, we identify and characterize a non-transferable nuclear targeting sequence in the STAT1 DNA-binding domain. This conserved signal is critical for the interferon-gamma (IFN-gamma)-induced nuclear import of phosphorylated STAT1 dimers and requires adjacent positively charged and hydrophobic residues for functioning. Additionally, the constitutive nucleocytoplasmic shuttling of STAT1 in the absence of IFN-gamma stimulation is revealed. Nuclear import and export of unphosphorylated STAT1 are demonstrated to be sensitive towards wheat germ agglutinin and to occur independently of the import receptor p97. Loss-of-function mutations of the dimer-specific import signal block nuclear entry of tyrosine-phosphorylated STAT1, which in turn also prevents induction of cytokine-inducible target genes. Nevertheless, nuclear import of unphosphorylated STAT1 continues and the STAT1-dependent constitutive expression of caspases and the tumor necrosis factor-alpha-mediated induction of apoptosis proceed unaltered. Thus, tyrosine-phosphorylated and unphosphorylated STAT1 molecules shuttle via independent pathways to distinct sets of target genes.