RESUMEN
The seven signal transducers of transcription (STATs) are cytokine-inducible modular transcription factors. They transmit the stimulation of cells with type I interferons (IFN-α/IFN-ß) and type II interferon (IFN-É£) into altered gene expression patterns. The N-terminal domain (NTD) of STAT1 is a surface for STAT1/STAT1 homodimer and STAT1/STAT2 heterodimer formation and allows the cooperative DNA binding of STAT1. We investigated whether the STAT1 NTD-mediated dimerization affected the IFN-induced tyrosine phosphorylation of STAT1, its nuclear translocation, STAT1-dependent gene expression, and IFN-dependent antiviral defense. We reconstituted human STAT1-negative and STAT2-negative fibrosarcoma cells with STAT1, NTD-mutated STAT1 (STAT1AA), STAT1 with a mutated DNA-binding domain (DBD), or STAT2. We treated these cells with IFN-α and IFN-É£ to assess differences between IFN-α-induced STAT1 homo- and heterodimers and IFN-É£-induced STAT1 homodimers. Our data demonstrate that IFNs induce the phosphorylation of STAT1 and STAT1AA at Y701 and their nuclear accumulation. We further reveal that STAT1AA can be phosphorylated in response to IFN-α in the absence of STAT2 and that IFN-É£-induced STAT1AA can activate gene expression directly. However, STAT1AA largely fails to bind STAT2 and to activate IFN-α-induced expression of endogenous antiviral STAT1/STAT2 target proteins. Congruent herewith, both an intact STAT1 NTD and STAT2 are indispensable to establish an antiviral state with IFN-α. These data provide new insights into the biological importance of the STAT1 NTD.
Asunto(s)
Interferón Tipo I/metabolismo , Interferón gamma/metabolismo , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/fisiología , Antivirales/metabolismo , Línea Celular , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Fosforilación/fisiología , Transporte de Proteínas/fisiología , Factor de Transcripción STAT2/metabolismoRESUMEN
Acetylation-dependent inactivation of STAT1 can be mimicked by the exchange of its lysine residues K410 and K413 to glutamine residues. STAT3 harbors non-acetylatable arginine moieties at the corresponding sites R414 and R417. It is unclear whether the mutation of these sites to glutamine residues antagonizes STAT3 activation. Here, we show that an arginine-glutamine-exchange at the STAT3 moieties R414 and R417 (R414Q and R417Q) reduces cytokine-dependent tyrosine phosphorylation of STAT3. This inhibitory effect can be partially rescued by phosphatase inhibition. In addition, the R414Q and R417Q mutations enhance the nuclear accumulation of unphosphorylated STAT3. STAT3 R414Q and STAT3 R417Q show a reduced response to cytokine stimulation emanating from the plasma membrane. Moreover, these STAT3 mutants have no direct inhibitory effect on the cytokine-induced activation of STAT1/STAT3-mediated gene expression. Since the mutations R414Q and R417Q reside within the STAT3 DNA binding domain (DBD), the STAT3 R414Q and R417Q mutants also lack intrinsic activity as transcription factors. Furthermore, in contrast to wild-type STAT3 they cannot compensate for a loss of STAT1 and they cannot promote STAT1/STAT3-dependent transcriptional activation. We further analyzed a STAT3 arginine-lysine-exchange mutant (R414K/R417K). This molecule mimics corresponding lysine residues found within the DBD of STAT1. Compared to wild-type STAT3, the STAT3 R414K/R417K mutant shows attenuated tyrosine phosphorylation and it is a less active transcription factor. In addition, STAT3 R414K/R417K is not activated by deacetylase inhibition. On the other hand, C-terminal acetylation of STAT3 is intact in STAT3 R414K/R417K. Our results suggest that the exchange of amino acid residues within the DBDs of STAT1/STAT3 affects their phosphorylation as well as their intracellular shuttling.
Asunto(s)
Arginina/metabolismo , ADN/metabolismo , Factor de Transcripción STAT3/metabolismo , Acetilación , Sustitución de Aminoácidos , Arginina/química , Línea Celular Tumoral , Histona Desacetilasas/química , Histona Desacetilasas/metabolismo , Humanos , Interferón-alfa/farmacología , Interleucina-6/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica , Estructura Terciaria de Proteína , Factor de Transcripción STAT3/genética , Transducción de Señal , Activación Transcripcional/efectos de los fármacosRESUMEN
The Janus tyrosine kinases JAK1-3 and tyrosine kinase-2 (TYK2) are frequently hyperactivated in tumors. In lung cancers JAK1 and JAK2 induce oncogenic signaling through STAT3. A putative role of TYK2 in these tumors has not been reported. Here, we show a previously not recognized TYK2-STAT3 signaling node in lung cancer cells. We reveal that the E3 ubiquitin ligase seven-in-absentia-2 (SIAH2) accelerates the proteasomal degradation of TYK2. This mechanism consequently suppresses the activation of STAT3. In agreement with these data the analysis of primary non-small-cell lung cancer (NSCLC) samples from three patient cohorts revealed that compared to lung adenocarcinoma (ADC), lung squamous cell carcinoma (SCC) show significantly higher levels of SIAH2 and reduced STAT3 phosphorylation levels. Thus, SIAH2 is a novel molecular marker for SCC. We further demonstrate that an activation of the oncologically relevant transcription factor p53 in lung cancer cells induces SIAH2, depletes TYK2, and abrogates the tyrosine phosphorylation of STAT1 and STAT3. This mechanism appears to be different from the inhibition of phosphorylated JAKs through the suppressor of cytokine signaling (SOCS) proteins. Our study may help to identify molecular mechanisms affecting lung carcinogenesis and potential therapeutic targets.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , TYK2 Quinasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular Tumoral , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Transducción de Señal/fisiología , Análisis de Matrices Tisulares , TransfecciónRESUMEN
Acetylation of signal transducer and activator of transcription (STAT) proteins has been recognized as a significant mechanism for the regulation of their cellular functions. Site-specific antibodies are available only for a minority of STATs. The detection of acetylated STATs by immunoprecipitation (IP) followed by western blot (WB) will be described in the following chapter. Defined conditions for cell lysis and IP will be elucidated on the basis of STAT1 acetylation.
Asunto(s)
Western Blotting/métodos , Inmunoprecipitación/métodos , Factores de Transcripción STAT/aislamiento & purificación , Factores de Transcripción STAT/metabolismo , Acetilación , Animales , Extractos Celulares , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Lisina/metabolismo , Ratones , Factores de Transcripción STAT/química , Factor de Transcripción STAT1/química , Factor de Transcripción STAT1/aislamiento & purificación , Factor de Transcripción STAT1/metabolismoRESUMEN
The cytokine-inducible transcription factors signal transducer and activator of transcription 5A and 5B (STAT5A and STAT5B) are important for the proper development of multicellular eukaryotes. Disturbed signaling cascades evoking uncontrolled expression of STAT5 target genes are associated with cancer and immunological failure. Here, we summarize how STAT5 acetylation is integrated into posttranslational modification networks within cells. Moreover, we focus on how inhibitors of deacetylases and tyrosine kinases can correct leukemogenic signaling nodes involving STAT5. Such small molecules can be exploited in the fight against neoplastic diseases and immunological disorders.
RESUMEN
Interferon-α (IFNα) has enormous potential for anti-proliferative and anti-viral treatments. However, clinical success is still hampered due to its limited bioavailability and thus, lack of sustained modulation of disease-relevant protective programs. Consequently, we here examined whether IFNα immobilized on nanoscale ferromagnetic R-Chitosan carriers is capable of inducing rapid and sustained activation of STAT1 signaling. We report the spontaneous formation of a stable nanoparticle-IFNα protein corona, which was exploited to generate IFNα-loaded spheres, obviating the need to specifically couple the cytokine to the nanoparticles (NPs). Notably, comprehensive experimental approaches ensure that formation of the IFNα NP-corona does not affect the biological activity of the cytokine, as STAT1 signaling was efficiently activated. Employing human prostate cancer and melanoma cell models, we found that the intensity and duration of STAT1 phosphorylation as well as the downstream activation of pathobiologically relevant genes were dose and particle dependent. In comparison with free IFNα, IFNα-loaded spheres resulted in a more sustained biologically relevant STAT1 activation, demonstrated also by conferring innate cellular immunity against vesicular stomatitis virus (VSV) infection. For one, our study demonstrates the advantages of biodegradable IFNα-coated R-Chitosan NPs for controlled cytokine release, and thereby improved therapy. Second, we reveal that the permanent presence of IFNα and not just the initial STAT1 phosphorylation ensures sustained IFNα-dependent signaling.
Asunto(s)
Antivirales/farmacología , Interferón-alfa/farmacología , Nanopartículas de Magnetita/química , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/efectos de los fármacos , Antivirales/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quitosano/química , Portadores de Fármacos/química , Humanos , Concentración de Iones de Hidrógeno , Inmunidad Innata/efectos de los fármacos , Interferón-alfa/química , Quinasas Janus/metabolismo , Fosforilación , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/metabolismo , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacosRESUMEN
A fascinating question of modern biology is how a limited number of signaling pathways generate biological diversity and crosstalk phenomena in vivo. Well-defined posttranslational modification patterns dictate the functions and interactions of proteins. The signal transducers and activators of transcription (STATs) are physiologically important cytokine-induced transcription factors. They are targeted by a multitude of posttranslational modifications that control and modulate signaling responses and gene expression. Beyond phosphorylation of serine and tyrosine residues, lysine acetylation has recently emerged as a critical modification regulating STAT functions. Interestingly, acetylation can determine STAT signaling codes by various molecular mechanisms, including the modulation of other posttranslational modifications. Here, we provide an overview on the acetylation of STATs and how this protein modification shapes cellular cytokine responses. We summarize recent advances in understanding the impact of STAT acetylation on cell growth, apoptosis, innate immunity, inflammation, and tumorigenesis. Furthermore, we discuss how STAT acetylation can be targeted by small molecules and we consider the possibility that additional molecules controlling STAT signaling are regulated by acetylation. Our review also summarizes evolutionary aspects and we show similarities between the acetylation-dependent control of STATs and other important molecules. We propose the concept that, similar to the 'histone code', distinct posttranslational modifications and their crosstalk orchestrate the functions and interactions of STAT proteins.
Asunto(s)
Factores de Transcripción STAT/metabolismo , Acetilación , Animales , Humanos , Transducción de SeñalRESUMEN
Head and neck squamous cell carcinomas (HNSCCs) are the sixth most common malignant neoplasm and more than 50% of patients succumb to this disease. HNSCCs are characterized by therapy resistance, which relies on the overexpression of anti-apoptotic proteins and on the aberrant regulation of the epidermal growth factor receptor (EGFR). As inherent and acquired resistance to therapy counteracts improvement of long-term survival, novel multi-targeting strategies triggering cancer cell death are urgently required. We investigated how induction of replicational stress by the ribonucleotide reductase inhibitor hydroxyurea (HU) combined with histone deacetylase inhibitors (HDACi) exerts anti-tumor activity. We treated HNSCC cell lines and freshly isolated tumor cells with HDACi, such as the clinically approved anti-epileptic drug valproic acid (VPA), in combination with HU. Our data demonstrate that at clinically achievable levels VPA/HU combinations efficiently block proliferation as well as clonogenic survival, and trigger apoptosis of HNSCC cells. In the presence of VPA/HU, such tumor cells increase expression of the pro-apoptotic BCL-2 family protein BIM, independent of wild-type p53 signaling and in the absence of increased expression of the p53 targets PUMA and BAX. The pro-apoptotic activity of BIM in HNSCCs was found critical for tumor cell death; ectopic overexpression of BIM induced HNSCC apoptosis and RNAi-mediated depletion of BIM protected HNSCC cells from VPA/HU. Also, significantly elevated BIM levels (p less than 0.01) were detectable in the apoptotic tumor centers versus proliferating tumor margins in HNSCC patients (n=31), underlining BIM's clinical relevance. Importantly, VPA/HU treatment additionally reduces expression and cell surface localization of EGFR. Accordingly, in a xenograft mouse model, VPA/HU efficiently blocked tumor growth (P less than 0.001) correlating with BIM induction and EGFR downregulation. We provide a molecular rationale for the potent anti-cancer activities of this drug combination. Our data suggest its exploitation as a potential strategy for the treatment of HNSCC and other tumor entities characterized by therapy resistance linked to dysregulated EGFR activation.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas Reguladoras de la Apoptosis/fisiología , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/tratamiento farmacológico , Inhibidores Enzimáticos/administración & dosificación , Genes erbB-1/efectos de los fármacos , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Proteínas de la Membrana/fisiología , Proteínas Proto-Oncogénicas/fisiología , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/patología , Inhibidores de Histona Desacetilasas/administración & dosificación , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Ribonucleótido Reductasas/antagonistas & inhibidores , Carcinoma de Células Escamosas de Cabeza y Cuello , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Signal transducer and activator of transcription 1 (STAT1) is important for innate and adaptive immunity. Histone deacetylase inhibitors (HDACi) antagonize unbalanced immune functions causing chronic inflammation and cancer. Phosphorylation and acetylation regulate STAT1 and different IFNs induce phosphorylated STAT1 homo-/heterodimers, e.g. IFNα activates several STATs whereas IFNγ only induces phosphorylated STAT1 homodimers. In transformed cells HDACi trigger STAT1 acetylation linked to dephosphorylation by the phosphatase TCP45. It is unclear whether acetylation differentially affects STAT1 activated by IFNα or IFNγ, and if cellular responses to both cytokines depend on a phosphatase-dependent inactivation of acetylated STAT1. Here, we report that HDACi counteract IFN-induced phosphorylation of a critical tyrosine residue in the STAT1 C-terminus in primary cells and hematopoietic cells. STAT1 mutants mimicking a functionally inactive DNA binding domain (DBD) reveal that the number of acetylation-mimicking sites in STAT1 determines whether STAT1 is recruited to response elements after stimulation with IFNγ. Furthermore, we show that IFNα-induced STAT1 heterodimers carrying STAT1 molecules mimicking acetylation bind cognate DNA and provide innate anti-viral immunity. IFNγ-induced acetylated STAT1 homodimers are though inactive, suggesting that heterodimerization and complex formation can rescue STAT1 lacking a functional DBD. Apparently, the type of cytokine determines how acetylation affects the nuclear entry and DNA binding of STAT1. Our data contribute to a better understanding of STAT1 regulation by acetylation.