Sumoylation of Smad3 stimulates its nuclear export during PIASy-mediated suppression of TGF-beta signaling.

Sma- and MAD-related protein 3 (Smad3) plays crucial roles in the transforming growth factor-beta (TGF-beta)-mediated signaling pathway, which produce a variety of cellular responses, including cell proliferation and differentiation. In our previous study, we demonstrated that protein inhibitor of activated STATy (PIASy) suppresses TGF-beta signaling by interacting with and sumoylating Smad3. In the present study, we examined the molecular mechanisms of Smad3 sumoylation during PIASy-mediated suppression of TGF-beta signaling. We found that small-interfering RNA-mediated reduction of endogenous PIASy expression enhanced TGF-beta-induced gene expression. Importantly, coexpression of Smad3 with PIASy and SUMO1 affected the DNA-binding activity of Smad3. Furthermore, coexpression of Smad3 with PIASy and SUMO1 stimulated the nuclear export of Smad3. Finally, fluorescence resonance energy transfer analyses revealed that Smad3 interacted with SUMO1 in the cytoplasm. These results suggest that PIASy regulates TGF-beta/Smad3-mediated signaling by stimulating sumoylation and nuclear export of Smad3.

BART is essential for nuclear retention of STAT3.

Signal transducers and activators of transcription (STATs) mediate cell proliferation, differentiation and survival in immune responses, hematopoiesis, neurogenesis and other biological processes. STAT3, for example, is involved in the epithelial-mesenchymal transition during gastrulation, organogenesis, wound healing and cancer progression. STAT activity is regulated by a variety of mechanisms, including nuclear translocation. To clarify the molecular mechanisms underlying the regulation of STAT activity, we performed yeast two-hybrid screening. Here, we identified binder of ADP-ribosylation factor-like two (BART) as a novel STAT-binding partner. Importantly, we showed that BART is essential for the transcriptional activity and nuclear retention of STAT3. Furthermore, an effector of BART, ADP-ribosylation factor-like 2 (ARL2) was also involved in nuclear retention of STAT3. These results indicate that BART plays an essential role in the nuclear retention of STAT3 through interaction with ARL2.

Modulation of TLR4 signaling by a novel adaptor protein signal-transducing adaptor protein-2 in macrophages.

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 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-kappaB activation. Conversely, overexpression of STAP-2 enhanced these LPS/TLR4-induced biological activities. STAP-2, particularly its Src homology 2-like domain, bound to both MyD88 and IkappaB kinase (IKK)-alphabeta, but not TNFR-associated factor 6 or IL-1R-associated kinase 1, and formed a functional complex composed of MyD88-STAP-2-IKK-alphabeta. These interactions augmented MyD88- and/or IKK-alphabeta-dependent signals, leading to enhancement of the NF-kappaB activity. These results demonstrate that STAP-2 may constitute an alternative LPS/TLR4 pathway for NF-kappaB activation instead of the TNFR-associated factor 6-IL-1R-associated kinase 1 pathway.

Phosphorylation of threonine-265 in Zipper-interacting protein kinase plays an important role in its activity and is induced by IL-6 family cytokines.

Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase that has been implicated in cell death and transcriptional regulation, but its mechanism of regulation remains unknown. Here, we identified threonine-265 (Thr265) in ZIPK as a major autophosphorylation site. Mutational analyses revealed that autophosphorylation of Thr265 were essential for its full catalytic activity toward an exogenous substrate as well as for cell death induction. Furthermore, leukemia inhibitory factor (LIF) stimulated Thr265 phosphorylation of ZIPK, thereby leading to phosphorylation and activation of signal transducer and activator of transcription (STAT3). Taken together, our findings demonstrate that ZIPK is positively regulated through Thr265 phosphorylation and that this phosphorylation is essential for its function.

Physical and functional interactions between STAT3 and ZIP kinase.

Signal transducer and activator of transcription 3 (STAT3) is a latent cytoplasmic transcription factor that can be activated by cytokines and growth factors. It plays important roles in cell growth, apoptosis and cell transformation, and is constitutively active in a variety of tumor cells. In this study, we provide evidence that zipper-interacting protein kinase (ZIPK) interacts physically with STAT3. ZIPK specifically interacted with STAT3, and did not bind to STAT1, STAT4, STAT5a, STAT5b or STAT6. ZIPK phosphorylated STAT3 on serine 727 (Ser727) and enhanced STAT3 transcriptional activity. Small interfering RNA-mediated reduction of ZIPK expression decreased leukemia inhibitory factor (LIF)- and IL-6-induced STAT3-dependent transcription. Furthermore, LIF- and IL-6-mediated STAT3 activation stimulated ZIPK activity. Taken together, our data suggest that ZIPK interacts with STAT3 within the nucleus to regulate the transcriptional activity of STAT3 via phosphorylation of Ser727.

Nuclear retention of STAT3 through the coiled-coil domain regulates its activity.

Signal transducer and activator of transcription 3 (STAT3), which mediates biological actions in many physiological processes, is activated by cytokines and growth factors via specific tyrosine phosphorylation, dimerization, and nuclear translocation. However, the mechanism involved in its nuclear translocation remains unclear. A previous study demonstrated that STAT3 with Arg-214/215 mutations in the coiled-coil domain (R214A/R215A; STAT3 RA) failed to undergo nuclear translocation. Here, we re-examined the nature of the STAT3 RA mutant and found that it showed higher and more extensive tyrosine-phosphorylation as well as much higher STAT3 transcriptional activity in response to stimuli. Furthermore, STAT3 RA showed nuclear translocation and faster nuclear export than wild-type STAT3 after stimulation. Moreover, nuclear retention of STAT3 RA by a chromosomal region maintenance 1 (CRM1) inhibitor, leptomycin B, decreased the enhanced STAT3 activation by stimuli. These data demonstrate that Arg-214/215 are involved in CRM1-mediated STAT3 nuclear export and the regulation of STAT3 activity.

Roles for lysine residues of the MH2 domain of Smad3 in transforming growth factor-beta signaling.

Sma and MAD-related protein 3 (Smad3) plays a key role in the intracellular signaling of the transforming growth factor-beta (TGF-beta) family of growth factors, which exhibits a diverse set of cellular responses, including cell proliferation and differentiation. Smad3 has the N-terminal Mad homology (MH) 1 and the C-terminal MH2 domains. MH2 domain is essential for the TGF-beta-induced transcriptional activation, because the MH2 domain of Smad3 is involved in the interactions with several transcriptional cofactors as well as the type I TGF-beta receptor (TbetaR-I). In this study, we examined the roles for four lysine residues (Lys-333, Lys-341, Lys-378, and Lys-409) in the Smad3 MH2 domain. Mutation of the lysine (K)-378 to arginine (R) (K378R) abolished the interaction with TbetaR-I, phosphorylation, transcriptional activation by an active TbetaR-I. The K341R mutant also failed to stimulate TGF-beta-induced transcription by resting in the cytoplasm. However, the K409R mutant showed a higher transcriptional activity by stronger interactions with co-activators, such as p300/CBP. Furthermore, both the K341R and K378R mutants act as dominant-negative inhibitors in the TGF-beta-induced target genes of endogenous TGF-beta signal. Thus, the lysine residues of Smad3 MH2 domain play important roles in the transcriptional regulation of TGF-beta signals through TbetaR-I.

The RING domain of PIASy is involved in the suppression of bone morphogenetic protein-signaling pathway.

Bone morphogenetic proteins (BMPs) play central roles in differentiation, development, and physiologic tissue remodeling. Recently, we have demonstrated that a protein inhibitor of activated STAT, PIASy, suppresses TGF-beta signaling by interacting with Sma and MAD-related protein 3 (Smad3). In this study, we examined a PIASy-dependent inhibitory effect on BMP signaling. PIASy expression was induced by BMP-2 stimulation and suppressed BMP-2-dependent Smad activity in hepatoma cells. Furthermore, BMP-2-regulated Smads directly bound to PIASy. We also demonstrated that the RING domain of PIASy played an important role in PIASy-mediated suppression of Smad activity. We here provide evidence that the inhibitory action of PIASy on BMP-regulated Smad activity was due to direct physical interactions between Smads and PIASy through its RING domain.

Cross-talk between endocrine-disrupting chemicals and cytokine signaling through estrogen receptors.

STAT3 mainly acts as a signal transducer of IL-6 family cytokines and transcriptionally activates specific target genes. STAT3 has also been demonstrated to mediate cellular transformation and is found in numerous cancers. Endocrine-disrupting chemicals (EDCs) are a diverse group of chemicals that bind to estrogen receptors (ERs), mimic estrogenic actions, and may have adverse effects on human health. In our previous study, we demonstrated that estrogens suppressed the STAT3-mediated transcription activity through ERs. In this study, we examined the effects of EDCs on STAT3-mediated signaling through ERs. Surprisingly, some of EDCs enhanced STAT3-mediated transcription activity through ERs. This finding strongly suggests that EDCs may play an important role in the endocrine functions by mimicking cytokine activity by stimulating STAT3 actions through ERs.

Physical and functional interactions between Daxx and DNA methyltransferase 1-associated protein, DMAP1.

Daxx has been shown to play an essential role in type I IFN-alphabeta-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.

Involvement of NF-kappaB in TGF-beta-mediated suppression of IL-4 signaling.

Control of immune response requires the coordinated integration of both stimulatory and inhibitory factors. Therefore, the cross-talk of different signaling pathways is critical in providing an integrated cellular response to multiple external signals. Both interleukin-4 (IL-4) and transforming growth factor (TGF-beta) are pleiotropic cytokines and play critical roles in controlling immune responses. For example, IL-4 mediates important pro-inflammatory functions in asthma including induction of the IgE isotype switch and expression of vascular cell adhesion molecules. Whereas, TGF-beta is secreted from B, T, and dendritic cells as well as macrophages, and negatively regulates their proliferation, differentiation, and activation by other cytokines. In this study, we examined the effect of TGF-beta on IL-4 signaling using B cells as well as embryonic kidney cells. TGF-beta inhibited IL-4-induced IgG1 production and gene expression of germline epsilon transcripts in B cells. In embryonic kidney cells, TGF-beta signals suppressed IL-4-induced transcription, when we monitored using germline epsilon promoter DNA. Furthermore, activation of NF-kappaB resulted in a resistance to TGF-beta-mediated suppression of IL-4 signaling. These results indicate that TGF-beta-mediated regulation of IL-4 signaling may act by targeting NF-kappaB signaling.

XB51 isoforms mediate Alzheimer's beta-amyloid peptide production by X11L (X11-like protein)-dependent and -independent mechanisms.

XB51 (derived from X11-like binding protein of clone number 51) was isolated by yeast two-hybrid cDNA screening using the N-terminal domain of X11L (X11-like protein) as a bait. X11L is a neuron-specific adaptor protein that is known to down-regulate APP (beta-amyloid precursor protein) metabolism by associating with the cytoplasmic domain of APP, but the detailed mechanisms are still unknown. Thus the X11L-associated protein XB51 is believed to regulate APP metabolism by modifying X11L function through its interaction with X11L. Here we report that the hXB51 (human XB51 ) gene can yield two transcripts, one with exon 9 spliced out (resulting in the hXB51beta isoform) and the other containing exon 9 (yielding the hXB51alpha isoform). hXB51alpha binds to X11L to form a tripartite complex composed of hXB51alpha, X11L and APP. Complex-formation results in blocking X11L's suppression of Abeta (beta-amyloid) generation from APP. hXB51beta associates with X11L and inhibits its interaction with APP. However, hXB51beta suppresses Abeta generation and secretion in an X11L-independent manner. Thus the hXB51 isoforms regulate Abeta generation differently, either enhancing it by modifying the association of X11L with APP or suppressing it in an X11L-independent manner. These observations advance our understanding of the molecular mechanisms regulating intracellular Abeta production and the pathogenesis of Alzheimer's disease.

Regulation of transforming growth factor-beta signaling by protein inhibitor of activated STAT, PIASy through Smad3.

Smads proteins play a key role in the intracellular signaling of the transforming growth factor (TGF)-beta 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-beta 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-beta-mediated activation of Smad3. PIASy also stimulated the sumoylation of Smad3 in vivo. Furthermore, endogenous PIASy expression was induced by TGF-beta in Hep3B cells. These findings provide the first evidence that a PIAS family protein, PIASy, associates with Smads and involves the regulation of TGF-beta signaling using the negative feedback loop.

Molecular interactions between STAT3 and protein inhibitor of activated STAT3, and androgen receptor.

STAT3 mainly acts as a signal transducer of IL-6 family cytokines and transcriptionally activates specific target genes. The recently discovered protein inhibitor of activated STAT3 (PIAS3) binds directly to STAT3 and blocks transcriptional activation. In our previous report, we demonstrated that PIAS3 directly interacted with androgen receptor (AR) and affected AR-mediated gene activation. Furthermore, we also showed that AR associated with STAT3 and enhanced its activity. Here, we examined molecular interactions between STAT3, PIAS3, and AR to underline the mechanism of how they regulate each other. AR activation overcame the inhibitory effect on STAT3-mediated transcription by PIAS3. Co-immunoprecipitation experiments revealed that an active form of AR relieved STAT3 from STAT3-PIAS3 complex formation. These results indicate that AR and PIAS3 regulate the STAT3-mediated transcriptional activity by their physical protein-protein competition on STAT3.