Signal transducing adaptor protein‐2 (STAP‐2) is a recently identified adaptor protein, that contains pleckstrin and Src homology 2 (SH2)‐like domains as well as a YXXQ motif in its C‐terminal region. Our previous studies have demonstrated that STAP‐2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, STAP‐2 was found to positively regulate LPS/TLR4‐mediated signals in macrophages. Disruption of STAP‐2 resulted in impaired LPS/TLR4‐induced cytokine production and NF‐κB activation. Conversely, overexpression of STAP‐2 enhanced these LPS/TLR4‐induced biological activities. STAP‐2, particularly its SH2‐like domain, bound to both MyD88 and IKK‐α/β, but not TRAF6 or IRAK1, and formed a functional complex composed of MyD88‐STAP‐2‐IKK‐α/β. These interactions augmented MyD88‐ and/or IKK‐α/β‐dependent signals, leading to enhancement of the NF‐κB activity. These results demonstrate that STAP‐2 may constitute an alternative LPS/TLR4 pathway for NF‐κB activation instead of the TRAF6‐IRAK1pathway.
Tyrosine kinase-2 (Tyk2), a member of the Jak family of kinases, mediates the signals triggered by various cytokines, including type I IFNs, IL-12, and IL-23. In the current study, we investigated the in vivo involvement of Tyk2 in several IL-12/Th1– and IL-23/Th17–mediated models of experimental diseases, including methylated BSA injection-induced footpad thickness, imiquimod-induced psoriasis-like skin inflammation, and dextran sulfate sodium- or 2,4,6-trinitrobenzene sulfonic acid-induced colitis. In these disease models, Tyk2 deficiency influenced the phenotypes in immunity and/or inflammation. Our findings demonstrate a somewhat broader contribution of Tyk2 to immune systems than previously expected and suggest that Tyk2 may represent an important candidate for drug development by targeting both the IL-12/Th1 and IL-23/Th17 axes.
Smads proteins play a key role in the intracellular signaling of the transforming growth factor (TGF)- family of growth factors, which exhibits a diverse set of cellular responses, including cell proliferation and differentiation. In particular, Smad7 acts as an antagonist of TGF- signaling, which could determine the intensity or duration of its signaling cascade. In this study we identified a protein inhibitor of activated STAT (signal transducers and activators of transcription), PIASy, as a novel interaction partner of Smad7 by yeast two-hybrid screening using the MH2 domain of Smad7 as bait. The association of Smad7 and PIASy was confirmed using co-expressed tagged proteins in 293T cells. Moreover, we found that other Smads including Smad3 also associated with PIASy through its MH2 domain, and PIASy suppressed TGF--mediated activation of Smad3. PIASy also stimulated the sumoylation of Smad3 in vivo. Furthermore, endogenous PIASy expression was induced by TGF- in Hep3B cells. These findings provide the first evidence that a PIAS family protein, PIASy, associates with Smads and involves the regulation of TGF- signaling using the negative feedback loop.
Signal transducers and activators of transcription (STATs) mediate cell proliferation, differentiation and survival in immune responses, hematopoiesis, neurogenesis and other biological processes. For example, STAT3 has been reported to be constitutively activated in numerous cancer cells. To clarify the molecular mechanisms underlying the STAT activation, we performed yeast two-hybrid screening and identified KAP1/TIF1b as a novel STATbinding partner. KAP1 is a universal corepressor protein for the Kruppel-associated box zinc-finger protein superfamily of transcriptional repressors. We found endogenous KAP1 associated with endogenous STAT3 in vivo. Importantly, small-interfering RNA-mediated reduction of KAP1 expression enhanced interleukin (IL)-6-induced STAT3-dependent transcription and gene expression. Furthermore, reduction of KAP1 expression resulted in the marked accumulation of STAT3 phosphorylated on Ser727 in the nucleus, a modification that regulates its transcriptional activation. These results indicate that KAP1 may serve as a transcriptional regulator of the IL-6/STAT3 signaling pathway.
Daxx has been shown to play an essential role in type I IFN-αβ-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify how Daxx regulates B cell development, we examined Daxx interacting partners by yeast two-hybrid screening. DNA methyltransferase 1 (DNMT1)-associated protein (DMAP1) was identified and demonstrated to interact with Daxx. The interaction regions in both proteins were mapped, and the cellular localization of the interaction was examined. Both Daxx and DMAP1 formed a complex with DNMT1 and colocalized in the nucleus. DMAP1 enhanced Daxx-mediated repression of glucocorticoid receptor transcriptional activity. Furthermore, Daxx protected protein degradation of DMAP1 in vivo. These results provide the novel molecular link between Daxx and DNMT1, which establishes a repressive transcription complex in the nucleus.
Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin homology- and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, we find that STAP-2-deficient splenocytes or T cells exhibit enhanced cell adhesion to fibronectin after PMA treatment, and that STAP-2-deficient T cells contain the increased protein contents of focal adhesion kinase (FAK). Furthermore, overexpression of STAP-2 induces a dramatic decrease in the protein contents of FAK and integrin-mediated T cell adhesion to fibronectin in Jurkat T cells via the degradation of FAK. Regarding the mechanism for this effect, we found that STAP-2 associates with FAK and enhances its degradation, proteasome inhibitors block FAK degradation, and STAP-2 recruits an endogenous E3 ubiquitin ligase, Cbl, to FAK. These results reveal a novel regulation mechanism for integrin-mediated signaling in T cells via STAP-2, which directly interacts with and degrades FAK.
Granuloma formation is an important host defense mechanism against intracellular bacteria; however, uncontrolled granulomatous inflammation is pathologic. T helper 17 (TH17) cells are thought to have a pathogenic role in autoimmune and inflammatory diseases, including in granulomas. Here, we report that the PDZ-LIM domain protein PDLIM2 inhibited TH17 cell development and granulomatous responses by acting as a nuclear ubiquitin E3 ligase that targeted signal transducer and activator of transcription 3 (STAT3), a transcription factor critical for the commitment of naïve CD4+ T cells to the TH17 lineage. PDLIM2 promoted the polyubiquitination and proteasomal degradation of STAT3, thereby disrupting STAT3-mediated gene activation. Deficiency in PDLIM2 resulted in the accumulation of STAT3 in the nucleus, enhanced the extent of TH17 cell differentiation, and exacerbated granuloma formation. This study delineates an essential role for PDLIM2 in inhibiting TH17 cell-mediated inflammatory responses by suppressing STAT3 signaling and provides a potential therapeutic target for the treatment of autoimmune diseases.
Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains, as well as a YXXQ motif in its C-terminal region. Our previous studies revealed that STAP-2 regulates integrin-mediated T cell adhesion. In the present study, we find that STAP-2 expression affects Jurkat T cell migration after stromal cell-derived factor-1α (SDF-1α)-treatment. Furthermore, STAP-2-deficient T cells exhibit reduced cell migration after SDF-1α-treatment. Importantly, overexpression of STAP-2 in Jurkat T cells induces activation of small guanine triphosphatases, such as Rac1 and Cdc42. Regarding the mechanism for this effect, we found that STAP-2 associates with Vav1, the guanine-nucleotide exchanging factor for Rac1, and enhances downstream Vav1/Rac1 signaling. These results reveal a novel STAP-2-mediated mechanism for the regulation of SDF-1α-induced chemotaxis of T cells via activation of Vav1/Rac1 signaling.
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