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Serine 28 phosphorylation of NRIF3 confers its co-activator function for estrogen receptor-alpha transactivation.

ABSTRACT: NRIF3 is an estrogen-inducible nuclear receptor coregulator that stimulates estrogen receptor-alpha (ERalpha) transactivation functions and associates with the endogenous ER and its target gene promoter. p21-activated protein kinase 1 (Pak1) phosphorylates ERalpha at Ser305 and this modification is important in ERalpha transactivation function. Although ERalpha transactivation functions are regulated by co-activator activity of NRIF3, it remains unclear whether Pak1 could impact ER functions via a posttranslational modification of NRIF3. Here, we report that Pak1 phosphorylates NRIF3 at Serine28 and that NRIF3 binds to Pak1 in vitro and in vivo. We found that NRIF3 phosphorylation, co-activator activity and association with ERalpha increased following Pak1 phosphorylation of NRIF3's Ser28 and that activated ERalpha-Ser305 and NRIF3-Ser28 cooperatively support transactivation of ERalpha. NRIF3 expression increased significantly in cells with inducible Pak1 expression. We found that NRIF3 and ERalpha interaction, subcellular localization and ERalpha transactivation activity all increased in cells expressing the Pak1 phosphorylation-mimicking mutant NRIF3-Ser28Glu. Consistently, the NRIF3-Ser28Glu mutant exhibited an enhanced recruitment to the endogenous ER target genes and increased expression following estrogen stimulation. Finally, breast cancer cells with stable overexpression of NRIF3 showed increased proliferation and enhanced anchorage-independent growth. These findings suggest that NRIF3-Ser28 is a physiologic target of Pak1 signaling and contributes to the enhanced NRIF3 co-activator activity, leading to coordinated potentiation of ERalpha transactivation, its target gene expression and estrogen responsiveness of breast cancer cells.

SUBMITTER: Talukder AH

PROVIDER: S-EPMC3621709 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Serine 28 phosphorylation of NRIF3 confers its co-activator function for estrogen receptor-alpha transactivation.

Talukder A H AH Li D-Q DQ Manavathi B B Kumar R R

Oncogene 20080602 39

NRIF3 is an estrogen-inducible nuclear receptor coregulator that stimulates estrogen receptor-alpha (ERalpha) transactivation functions and associates with the endogenous ER and its target gene promoter. p21-activated protein kinase 1 (Pak1) phosphorylates ERalpha at Ser305 and this modification is important in ERalpha transactivation function. Although ERalpha transactivation functions are regulated by co-activator activity of NRIF3, it remains unclear whether Pak1 could impact ER functions via ...[more]

PMID: 18521086

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Project description:BACKGROUND: The placenta-specific 1 (PLAC1) gene encodes a membrane-associated protein which is selectively expressed in the placental syncytiotrophoblast and in murine fetal tissues during embryonic development. In contrast to its transcriptional repression in all other adult normal tissues, PLAC1 is frequently activated and highly expressed in a variety of human cancers, in particular breast cancer, where it associates with estrogen receptor ? (ER?) positivity. In a previous study, we showed that ER?-signaling in breast cancer cells transactivates PLAC1 expression in a non-classical pathway. As the members of the p160/nuclear receptor co-activator (NCOA) family, NCOA1, NCOA2 and NCOA3 are known to be overexpressed in breast cancer and essentially involved in estrogen-mediated cancer cell proliferation we asked if these proteins are involved in the ER?-mediated transactivation of PLAC1 in breast cancer cells. METHODS: Applying quantitative real-time RT-PCR (qRT-PCR), Western Blot analysis and chromatin immunoprecipitation, we analyzed the involvement of NCOA1, NCOA2, NCOA3 in the ER?-mediated transactivation of PLAC1 in the breast cancer cell lines MCF-7 and SK-BR-3. RNAi-mediated silencing of NCOA3, qRT-PCR, Western blot analysis and ER? activation assays were used to examine the role of NCOA3 in the ER?-mediated regulation of PLAC1 in further detail. Transcript expression of NCOA3 and PLAC1 in 48 human breast cancer samples was examined by qRT-PCR and statistical analysis was performed using Student's t-test. RESULTS: We detected selective recruitment of NCOA3 but not NCOA1 or NCOA2 to the PLAC1 promoter only in ER?-positive MCF-7 cells but not in ER?-negative SK-BR-3 breast cancer cells. In addition, we demonstrate that silencing of NCOA3 results in a remarkable decrease of PLAC1 expression levels in MCF-7 cells which cannot be restored by treatment with estradiol (E?). Moreover, significant higher transcript levels of PLAC1 were found only in ER?-positive human breast cancer samples which also show a NCOA3 overexpression. CONCLUSIONS: In this study, we identified NCOA3 as a selective co-activator of ER?-mediated transactivation of PLAC1 in MCF-7 breast cancer cells. Our data introduce PLAC1 as novel target gene of NCOA3 in breast cancer, supporting the important role of both factors in breast cancer biology.

Project description:BackgroundEstrogen receptor α (ERα) has been suggested to regulate anti-inflammatory signaling in brain microglia, the only resident immune cells in the brain. ERα conserves the phosphorylation motif at Ser216 within the DNA binding domain. Previously, Ser216 was found to be phosphorylated in neutrophils infiltrating into the mouse uterus and to enable ERα to regulate migration. Given the implication of this phosphorylation in immune regulation, ERα was examined in mouse microglia to determine if Ser216 is phosphorylated and regulates microglia's inflammation. It was found that Ser216 was constitutively phosphorylated in microglia and demonstrated that in the absence of phosphorylated ERα in ERα KI brains microglia inflamed, confirming that phosphorylation confers ERα with anti-inflammatory capability. ERα KI mice were obese and weakened motor ability.MethodsMixed glia cells were prepared from brains of 2-days-old neonates and cultured to mature and isolate microglia. An antibody against an anti-phospho-S216 peptide of ERα (αP-S216) was used to detect phosphorylated ERα in double immunofluorescence staining with ERα antibodies and a microglia maker Iba-1 antibody. A knock-in (KI) mouse line bearing the phosphorylation-blocked ERα S216A mutation (ERα KI) was generated to examine inflammation-regulating functions of phosphorylated ERα in microglia. RT-PCR, antibody array, ELISA and FACS assays were employed to measure expressions of pro- or anti-inflammatory cytokines at their mRNA and protein levels. Rotarod tests were performed to examine motor connection ability.ResultsDouble immune staining of mixed glia cells showed that ERα is phosphorylated at Ser216 in microglia, but not astrocytes. Immunohistochemistry with an anti-Iba-1 antibody showed that microglia cells were swollen and shortened branches in the substantial nigra (SN) of ERα KI brains, indicating the spontaneous activation of microglia as observed with those of lipopolysaccharide (LPS)-treated ERα WT brains. Pro-inflammatory cytokines were up-regulated in the brain of ERα KI brains as well as cultured microglia, whereas anti-inflammatory cytokines were down-regulated. FACS analysis showed that the number of IL-6 producing and apoptotic microglia increased in those prepared from ERα KI brains. Times of ERα KI mice on rod were shortened in Rotarod tests.ConclusionsBlocking of Ser216 phosphorylation aggravated microglia activation and inflammation of mouse brain, thus confirming that phosphorylated ERα exerts anti-inflammatory functions. ERα KI mice enable us to further investigate the mechanism by which phosphorylated ERα regulates brain immunity and inflammation and brain diseases. Video abstract.

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Serine 28 phosphorylation of NRIF3 confers its co-activator function for estrogen receptor-alpha transactivation.

Publications

Serine 28 phosphorylation of NRIF3 confers its co-activator function for estrogen receptor-alpha transactivation.

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