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Signal transducer and activator of transcription 5 (STAT5) is a transcription factor that activates prolactin (PRL)-dependent gene expression in the mammary gland. For the activation of its target genes, STAT5 recruits coactivators like p300 and the CREB-binding protein (CBP). In this study we analyzed the function of p300/CBP-associated members of the p160/SRC/NCoA-family in STAT5-mediated transactivation of β-casein expression. We found that only one of them, NCoA-1, acts as a coactivator for both STAT5a and STAT5b. The two coactivators p300/CBP and NCoA-1 cooperatively enhance STAT5a-mediated transactivation. For NCoA-1-dependent coactivation of STAT5, both the activation domain 1 and the amino-terminal bHLH/PAS domain are required. The amino-terminal region mediates the interaction with STAT5a in cells. A motif of three amino acids in an α-helical region of the STAT5a-transactivation domain is essential for the binding of NCoA-1 and for the transcriptional activity of STAT5a. Moreover we observed that NCoA-1 is involved in the synergistic action of the glucocorticoid receptor and STAT5a on the β-casein promoter. These findings support a model in which STAT5, in concert with the glucocorticoid receptor, recruits a multifunctional coactivator complex to initiate the PRL-dependent transcription.
Signal transducer and activator of transcription 6 (STAT6) is a transcription factor that is activated by interleukin-4 (IL-4)-induced tyrosine phosphorylation and mediates most of the IL-4-induced gene expression. Transcriptional activation by STAT6 requires the interaction with coactivators like p300 and the CREB-binding protein (CBP). In this study we have investigated the function of the CBP-associated members of the p160/steroid receptor coactivator family in the transcriptional activation by STAT6. We found that only one of them, NCoA-1, acts as a coactivator for STAT6 and interacts directly with the transactivation domain of STAT6. The N-terminal part of NCoA-1 interacts with the far C-terminal part of the STAT6 transactivation domain but does not interact with the other members of the STAT family. This domain of NCoA-1 has a strong inhibitory effect on STAT6-mediated transactivation when overexpressed in cells, illustrating the importance of NCoA-1 for STAT6-mediated transactivation. In addition, we showed that both coactivators CBP and NCoA-1 bind independently to specific regions within the STAT6 transactivation domain. Our results suggest that multiple contacts between NCoA-1, CBP, and STAT6 are required for transcriptional activation. These findings provide new mechanistic insights into how STAT6 can recruit coactivators required for IL-4-dependent transactivation.
Signal transducer and activator of transcription 6 (STAT6) regulates transcriptional activation in response to interleukin-4 (IL-4)-induced tyrosine phosphorylation by direct interaction with coactivators. The CREB-binding protein and the nuclear coactivator 1 (NCoA-1), a member of the p160/steroid receptor coactivator family, bind independently to specific regions of STAT6 and act as coactivators. In this study we show that an LXXLL motif in the STAT6 transactivation domain mediates the interaction with NCoA-1. Peptides representing this motif as well as antibodies generated against this motif inhibited STAT6/NCoA-1 interaction in glutathione S-transferase pulldown assays. Peptides derived from the STAT6 transactivation domain adjacent to the LXXLL motif as well as antibodies against these peptides showed no inhibitory effect. Mutagenesis of the LXXLL motif eliminated the STAT6/NCoA-1 interaction in vitro and in vivo, supporting the specific role of this motif in NCoA-1 binding. Importantly, mutagenesis of the STAT-LXXLL motif strongly diminished the IL-4-regulated activation of the endogenous STAT6 target gene eotaxin-3. Taken together, these results indicate that the STAT6-LXXLL-binding motif mediates the interaction with NCoA-1 in transcriptional activation and represents a new potential drug target for the inhibition of the STAT6 transactivation function in allergic diseases.
Transcriptional activation involves the ordered recruitment of coactivators via direct interactions between distinct binding domains and recognition motifs. The p160/SRC/NCoA coactivator family comprises three members (NCoA-1, -2 and -3), which are organized in multiprotein coactivator complexes. We had identified the PAS-B domain of NCoA-1 as an LXXLL motif binding domain. Here we show that NCoA family members are able to interact with other full-length NCoA proteins via their PAS-B domain and they specifically interact with the CBP-interaction domain (CID/AD1) of NCoA-1. Peptide competition, binding experiments and mutagenesis of LXXLL motifs point at distinct binding motif specificities of the NCoA PAS-B domains. NMR studies of different NCoA-1-PAS-B/LXXLL peptide complexes revealed similar although not identical binding sites for the CID/AD1 and STAT6 transactivation domain LXXLL motifs. In mechanistic studies, we found that overexpression of the PAS-B domain is able to disturb the binding of NCoA-1 to CBP in cells and that a CID/AD1 peptide competes with STAT6 for NCoA-1 in vitro. Moreover, the expression of an endogenous androgen receptor target gene is affected by the overexpression of the NCoA-1 or NCoA-3 PAS-B domains. Our study discloses a new, complementary mechanism for the current model of coactivator recruitment to target gene promoters.
Objective: Establishment of an immunocompetent mouse model representing the typical progressive stages observed in malignant human gliomas for the in vivo evaluation of novel target-specific regimens.
Methods: Isolated clones from tumours that arose spontaneously in GFAP-v-src transgenic mice were used to develop a transplantable brain tumour model in syngeneic B6C3F1 mice. STAT3 protein was knocked down by infection of tumour cells with replication-defective lentivirus encoding STAT3-siRNA. Apoptosis is designed to be induced by soluble recombinant TRAIL + chemical Bcl-2/Bcl-xL inhibitors.
Results: Striatal implantation of 105 mouse tumour cells resulted in the robust development of microscopically (2 – 3 mm) infiltrating malignant gliomas. Immunohistochemically, the gliomas displayed the astroglial marker GFAP and the oncogenic form of STAT3 (Tyr-705-phosphorylated) which is found in many malignancies including gliomas. Phosphorylated STAT3 was particularly prominent in the nucleus but was also found at the plasma membrane of peripherally infiltrating glioma cells. To evaluate the role of STAT3 in tumour progression, we stably expressed siRNA against STAT3 in several murine glioma cell lines. The effect of STAT3 depletion on proliferation, invasion and survival will be first assessed in vitro and subsequently after transplantation in vivo. Upstream and downstream components of the STAT3 signalling pathway as well as possible non-specific side effects of STAT3-siRNA expression after lentiviral infection will be examined, too.
Conclusions: Its high rate of engraftment, its similarity to the malignant glioma of origin, and its rapid locally invasive growth should make this murine model useful in testing novel therapies for malignant gliomas.