Opposed regulation of type I IFN-induced STAT3 and ISGF3 transcriptional activities by histone deacetylases (HDACS) 1 and 2.

The antiviral and antiproliferative responses mediated by type I interferons (IFNs) depend on JAK/STAT signaling and ISGF3 (STAT1:STAT2:IRF9)-dependent transcription. In addition, type I IFNs stimulate STAT3 activation in many cell types, an event generally associated with cell cycle progression, survival, and proliferation. To gather more insight into this functionally contradictive phenomenon, we studied the regulation of STAT3 transcriptional activity upon type I IFN treatment. We show that IFNα2 stimulation strongly induces STAT3 phosphorylation, nuclear translocation, and promoter binding, yet the activation of transcription of a STAT3-dependent reporter and endogenous genes, such as SOCS3 and c-FOS, is impaired. Simultaneous treatment with IFNα2 and trichostatin A, as well as combined HDAC1/HDAC2 silencing, restores STAT3-dependent reporter gene and endogenous gene expression, strongly suggesting that HDAC1 and HDAC2 are directly involved in repressing IFNα2-activated STAT3. Of note, single silencing of only one of the two HDACs does not lead to enhanced STAT3 activity, supporting a functional redundancy between these two enzymes. In sharp contrast, HDAC1 and HDAC2 activities are required for ISGF3-dependent gene expression. We conclude that HDAC1 and HDAC2 differentially modulate STAT activity in response to IFNα2: while they are required for the induction of ISGF3-responsive genes, they impair the transcription of STAT3-dependent genes.

The many faces of the SOCS box.

The suppressors of cytokine signalling (SOCS) box is a structural domain found at the C-terminus of over 70 human proteins. It is usually coupled to a protein interaction module such as an SH2 domain in case of SOCS proteins, a family of modulators of cytokine signaling. The SOCS box participates in the formation of E3 ligase complexes, marking activated cytokine receptor complexes for proteasomal degradation. A similar mechanism was recently uncovered for controlling SOCS activity itself, since SOCS2 was found to enhance the turnover of other SOCS proteins. The SOCS box can also add unique features to individual SOCS proteins: it can function as an adaptor domain as was demonstrated for SOCS3, or as a modulator of substrate binding in case of CIS. In this review we discuss these multiple roles of the SOCS box, which emerges as a versatile module controlling cytokine signaling via multiple mechanisms.

HyperISGylation of Old World monkey ISG15 in human cells.

BACKGROUND: ISG15 is an Ubiquitin-like protein, highly induced by Type I Interferons. Upon the cooperative activity of specific Ubiquitinating enzymes, ISG15 can be conjugated to its substrates. Increasing evidence points to a role for protein ISGylation in anti-viral and anti-tumoral defense. PRINCIPAL FINDINGS: We identified ISG15 from Old World Monkeys (OWm) as a hyper-efficient protein modifier. Western blot analysis visualized more efficient conjugation of OWmISG15 relative to HuISG15 in human (Hu), monkey and mouse (Mo) cell-lines. Moreover, the substrates of OWmISG15 identified upon Tandem Affinity Purification followed by LC-MS/MS identification largely outnumbered these of HuISG15 itself. Several Ubiquitin-Conjugating enzymes were identified as novel ISGylated substrates. Introduction of a N89D mutation in HuISG15 improved its ISGylation capacity, and additional Q31K/T33A/D133N mutations yielded a HuISG15 variant with an ISGylation efficiency comparable to OWmISG15. Homology modeling and structural superposition situate N89 in the interaction interface with the Activating enzyme. Analysis of the UbE1L residues in this interface revealed a striking homology between OWmUbE1L and HuUbE1, the Activating enzyme of Ubiquitin. In line with this observation, we found efficient activation of AgmISG15, but not HuISG15 or MoISG15, by HuUbE1, thus providing a likely explanation for OWm hyperISGylation. CONCLUSIONS: This study discloses the poor conjugation competence of HuISG15 compared to OWmISG15 and maps the critical determinants for efficient conjugation. HyperISGylation may greatly assist ISGylation studies and may enhance its function as positive regulator of Interferon-related immune responses or as anti-tumoral modulator.

Elongin B/C recruitment regulates substrate binding by CIS.

SOCS proteins play a major role in the regulation of cytokine signaling. They are recruited to activated receptors and can suppress signaling by different mechanisms including targeting of the receptor complex for proteasomal degradation. The activity of SOCS proteins is regulated at different levels including transcriptional control and posttranslational modification. We describe here a novel regulatory mechanism for CIS, one of the members of this protein family. A CIS mutant deficient in recruitment of the Elongin B/C complex completely failed to suppress STAT5 activation. This deficiency was not caused by altered turnover of CIS but by loss of cytokine receptor interaction. Intriguingly, no such effect was seen for binding to MyD88. The interaction between CIS and the Elongin B/C complex, which depends on the levels of uncomplexed Elongin B/C, was easily disrupted. This regulatory mechanism may be unique for CIS, as similar mutations in SOCS1, -2, -3, -6, and -7 had no functional impact. Our findings indicate that the SOCS box not only plays a role in the formation of E3 ligase complexes but, at least for CIS, can also regulate the binding modus of SOCS box-containing proteins.

Review: Negative regulation of leptin receptor signalling.

Leptin was discovered as an adipostat, regulating body weight by balancing food intake and energy expenditure. Recently, leptin emerged as a pleiotropic cytokine. It plays a substantial role in a wide spectrum of other functions including immune regulation, bone formation and fertility. Leptin signalling is under tight control. Aberrations of this stringent control system may be implicated in a variety of pathologies. Here, we review the various mechanisms that control cellular leptin receptor signalling.

Functional cross-modulation between SOCS proteins can stimulate cytokine signaling.

SOCS (suppressors of cytokine signaling) proteins are negative regulators of cytokine signaling that function primarily at the receptor level. Remarkably, in vitro and in vivo observations revealed both inhibitory and stimulatory effects of SOCS2 on growth hormone signaling, suggesting an additional regulatory level. In this study, we examined the possibility of direct cross-modulation between SOCS proteins and found that SOCS2 could interfere with the inhibitory actions of other SOCS proteins in growth hormone, interferon, and leptin signaling. This SOCS2 effect was SOCS box-dependent, required recruitment of the elongin BC complex, and coincided with degradation of target SOCS proteins. Detailed mammalian protein-protein interaction trap (MAPPIT) analysis indicated that SOCS2 can interact with all members of the SOCS family. SOCS2 may thus function as a molecular bridge between a ubiquitin-protein isopeptide ligase complex and SOCS proteins, targeting them for proteasomal turnover. We furthermore extended these observations to SOCS6 and SOCS7. Our findings point to a unique regulatory role for SOCS2, SOCS6, and SOCS7 within the SOCS family and provide an explanation for the unexpected phenotypes observed in SOCS2 and SOCS6 transgenic mice.

A complex interaction pattern of CIS and SOCS2 with the leptin receptor.

Hypothalamic leptin receptor signalling plays a central role in weight regulation by controlling fat storage and energy expenditure. In addition, leptin also has direct effects on peripheral cell types involved in regulation of diverse body functions including immune response, bone formation and reproduction. Previous studies have demonstrated the important role of SOCS3 (suppressor of cytokine signalling 3) in leptin physiology. Here, we show that CIS (cytokine-inducible SH2 protein) and SOCS2 can also interact with the leptin receptor. Using MAPPIT (mammalian protein-protein interaction trap), a cytokine receptor-based two-hybrid method operating in intact cells, we show specific binding of CIS with the conserved Y985 and Y1077 motifs in the cytosolic domain of the leptin receptor. SOCS2 only interacts with the Y1077 motif, but with higher binding affinity and can interfere with CIS and STAT5a prey recruitment at this site. Furthermore, although SOCS2 does not associate with Y985 of the leptin receptor, we find that SOCS2 can block interaction of CIS with this position. This unexpected interference can be explained by the direct binding of SOCS2 on the CIS SOCS box, whereby elongin B/C recruitment is crucial to suppress CIS activity.