Characterization of the Interactions of Estrogen Receptor and MNAR in the Activation of cSrc

Department of Molecular and Integrative Physiology, University of Illinois, Urbana-Champaign, Urbana, Illinois, United States
Molecular Endocrinology (Impact Factor: 4.02). 06/2004; 18(5):1096-108. DOI: 10.1210/me.2003-0335
Source: PubMed


In this study, we have evaluated the molecular mechanism of Src activation after its interaction with estrogen receptor alpha (ERalpha) and a newly identified scaffold protein, called MNAR (modulator of nongenomic activity of ER). Under basal condition, Src enzymatic activity is inhibited by intramolecular interactions. The enzyme can be activated by interaction between the SH2 domain of Src and phosphotyrosine-containing sequences and/or by interaction between the SH3 domain of Src and proteins containing PXXP motifs. Mutational analysis and functional evaluation of MNAR and the use of ERalpha and cSrc mutants revealed that MNAR interacts with Src's SH3 domain via its N-terminal PXXP motif. Mutation of this motif abolished both the MNAR-induced activation of Src and the stimulation of ER transcriptional activity. ER interacts with Src's SH2 domain using phosphotyrosine 537, and this complex was further stabilized by MNAR-ER interaction. Mapping studies reveal that both the A/B domain and Y537 of ERalpha are required for MNAR-induced activation of ER transcriptional activity. The region responsible for MNAR interaction with ER maps to two N-terminal LXXLL motifs of MNAR. Mutation of these motifs prevented ER-MNAR complex formation and eliminated activation of the Src/MAPK pathway. These data explicate how the coordinate interactions between MNAR, ER, and Src lead to Src activation. Our findings also demonstrate that MNAR is a scaffold protein that mediates ER-Src interaction and plays an important role in the integration of ER action in Src-mediated signaling.

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Available from: Frank Barletta, Apr 16, 2014
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    • "In epithelial, endothelial, and smooth muscle cells, Pelp1 was observed in cell nuclei along with ER-β [24]. In the present study, because Pelp1 was predominantly localized in the cytoplasm, Pelp1 expression is more likely to be cell-specific and, thereby, play a crucial role in estrogen signaling pathways on an individual cell basis rather than on the whole tissue [11,19,25,26]. Due to the documented role of Pelp1 in non-genomic estrogen and ER pathways involved in cell survival, apoptosis and, estrogen-induced osteo-, neuro-, and cardio-protection [27], Pelp1 may also participate in regulating these vital cell functions in BMSCs in individual cells, thereby contributing to different bone regulation and growth in different tissues of the body. "
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    ABSTRACT: Osteogenic induction and bone formation are heavily affected by environmental factors, including estrogen, estrogen receptors, and coregulatory proteins, such as the recently reported proline-, glutamic acid-, and leucine-rich protein 1(Pelp1). To investigate Pelp1 expression in rat bone mesenchymal stem cells (rBMSCs) during cell proliferation and osteogenic differentiation. rBMSCs were cultured in routine and osteogenic differentiation media. Cell proliferation was assessed at days 1, 3, 5, 7, 9, 11, 14, and 21. Pelp1 protein expression in the nucleus and cytoplasm were detected by immunocytochemical analysis. Real-time RT-PCR and western blot were used to detect mRNA and protein expressions of Pelp1, osteocalcin (OCN), and alkaline phosphatase (ALP). Over 21 days, rBMSCs in routine culture exhibited a 1-2 day lag phase and exponential growth from day 3 to 9, plateauing at day 9, and correlated with temporal mRNA expression of Pelp1, which almost reached baseline levels at day 21. In osteogenic induction cultures, Pelp1 mRNA levels rose at day 9 and steadily increased until day 21, reaching 6.8-fold greater value compared with day 1. Interestingly, Pelp1 mRNA expression in osteogenic cultures exhibited a trend similar to that of OCN expression. Pelp1 knockdown by siRNA transfection inhibited undifferentiated rBMSC proliferation, and bone markers OCN and ALP expressions in rBMSCs cultured in routine and osteogenic differentiation media. Pelp1 may be a key player in BMSCs proliferation and osteogenic differentiation, meriting further consideration as a target for development of therapies for pathological bone loss conditions, such as menopausal bone loss.
    PLoS ONE 12/2013; 8(10):e75477. DOI:10.1371/journal.pone.0075477 · 3.23 Impact Factor
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    • "The substitution of Y537 by a serine in the clone PC12-ER-Y537S evidences a decrease in cell viability and a return of cell viability equivalent to control one (Figure 2B). This PC12-ER-Y537S clone expresses a mutated form of ERα where the substitution of tyrosine 537 by a serine prevents the interaction of ERα with c-Src [27]. Moreover, the caspase activities in these cells and control ones are similar (Figure 2C). "
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    ABSTRACT: Many studies have reported proliferative, differentiating or protective effects of estradiol, notably through estrogen receptor alpha (ERα). On the contrary, the ligand-independent action of ERα is currently poorly documented notably in cell protection. The stable transfection of wild type, substituted or truncated form of ERα in PC12 cells (ERα negative cell line) lead the specific study of its ligand-independent action. Hence, we demonstrate here that, in the absence of E2, the expression of ERα prevents cells from apoptosis induced by serum deprivation. This protection is not due to an ERE-mediated transcription and does not require either AF-1 or AF-2 transactivation functions. It is afforded to the Y537 residue of ERα and activation of c-Src/Stat3 signaling pathway.
    PLoS ONE 06/2013; 8(6):e69081. DOI:10.1371/journal.pone.0069081 · 3.23 Impact Factor
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    • "Although several partners for extranuclear ERa have been described in different cell types, the most conserved partners are PI3K and the tyrosine kinase Src (Castoria et al, 2001; Simoncini et al, 2000; Song et al, 2005). Besides this core complex, several adaptor scaffold proteins such as p130 Cas and MNAR (modulator of non-genomic activity of the oestrogen receptor) have also been found to be part of the complex (Barletta et al, 2004; Cabodi et al, 2004; Shupnik, 2004). After oestrogenic stimulation, the rapid formation of the protein complex triggers the activation of downstream signalling cascades involving the Ras/MAPK and Akt pathways (Castoria et al, 2001; Hammes & Levin, 2007). "
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    ABSTRACT: Oestrogen receptors can mediate rapid activation of cytoplasmic signalling cascades by recruiting Src and PI3K. However, the involvement of this pathway in breast cancer remains poorly defined. We have previously shown that methylation of ERα is required for the formation of the ERα/Src/PI3K complex and that ERα is hypermethylated in a subset of breast cancers. Here, we used Proximity Ligation Assay to demonstrate that this complex is present in the cytoplasm of breast cancer cell lines as well as formalin-fixed, paraffin-embedded tumours. Of particular interest, the analysis of 175 breast tumours showed that overexpression of this complex in a subset of breast tumours correlates to the activation of the downstream effector Akt. Survival analysis revealed that high expression of this complex is an independent marker of poor prognosis and associated with reduced disease-free survival. Our data introduces the new concept that the rapid oestrogen pathway is operative in vivo. It also provides a rationale for patient stratification defined by the activation of this pathway and the identification of target therapies.
    EMBO Molecular Medicine 11/2012; 4(11). DOI:10.1002/emmm.201201615 · 8.67 Impact Factor
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