Postdoctoral Research

My focus was on estrogen receptor (ER) phosphorylation and its effect on important receptor functions including, ligand, DNA and coactivator binding. Phosphorylation of ER and its coregulatory proteins has been implicated in drug resistance to antiestrogens and aromatase therapies in breast cancer.  Our studies examined the phosphorylation status of ER in two breast cancer cell lines with distinct physiological responses to breast cancer drug, tamoxifen. In parental MCF-7 breast cancer cells, ER phosphorylation is ligand-dependant and ligand-specific, where as in tamoxifen-resistant MCF-7 cells that have elevated levels of active kinases, both ligand dependency and ligand specificity is lost. We thus hypothesized that the modification status of the receptor (a) defines the alteration in receptor function and (b) presents a composite picture to the cell machinery dictating physiological response overcoming presence of antiestrogen and estrogen deprivation. To test our hypothesis, we analyzed:

Effect of ER phosphorylation on receptor function: 

 ER is phosphorylated at multiple sites on ERα namely, serine 102, 104, 118, 167, 263 and 305, and tyrosine 537 by various cellular kinases. Significantly, these sites fall in regions that are accessible and close to important docking sites (ligand, DNA and coregulatory proteins), and they result in modulation of ER function. To examine how phosphorylation events alter receptor function, we have utilized high throughput assays: Fluorescence polarization assay for ligand and DNA binding and Time Resolved Fluorescence Polarization (TRFRET) assay for coactivator recruitment to the DNA-bound full length ER in the presence of ligand. We observe for the first time that ER phosphorylation by MAPK decreases ER affinity for trans-hydroxy tamoxifen (TOT) and to the consensus response element containing DNA in the presence of TOT. Both, Src and PKA phosphorylations of the receptor increase affinity for E2 without modifying TOT binding, whereas AKT phosphorylation of the receptor increases affinity of ER for the specific DNA in presence of E2 and TOT. Using TRFRET assay, we observe increased affinity of the receptor for coactivator, SRC3, on ER phosphorylation by MAPK, AKT and PKA. In addition to affinity changes, these assays also indicate conformational differences between the studied receptor phosphorylations. Our study thus demonstrates that ER phosphorylation alters receptor functions effecting changes that could lead to an increase in E2 agonism and a decrease in TOT antagonistic activity, encountered in tamoxifen resistance in endocrine therapy of breast cancer.

Poster

Modification status of ER using middle down proteomics and mass spectrometry.

 Determination of modification patterns on ER is a challenging task involving a large 66 KDa protein and multiple modifications including phosphorylations and acetylations. To determine the modification state of the receptor, we are  utilizing middle down proteomics, employing limited proteolysis of ER to yield large fragments of ER encompassing regions of modification and analyzing them using mass spectrometry. Analysis of recombinant, full length phosphorylated ER digested with LysC enables detection of peptides that cover 90 % of the ER sequence and identify multiple phosphorylation events and calculate relative abundance ratios for each of the peptides. Surprisingly, our studies  detect a large number of phosphorylation events than predicted by Scansite or Netphos. We are currently analyzing these phosphorylation events by (a) limited proteolysis with other proteolytic agents, followed by phosphospecific and total protein staining (b) autoradiography  (c) sequencing limited proteolytic digests of phosphorylated ER using FTMS to identify the precise modification sites. Mapping of phosphorylation sites on the recombinant ER will set the stage for identification of modications in endogenous ER from parental and tamoxifen resistant MCF-7 cells. We believe that a comprehensive determination of the PTM code of ER would provide valuable information regarding estrogen action and the mechanism of hormone resistance that ultimately could be used to guide the optimum mode of hormone therapy in ER-positive breast cancer.

Poster

 We believe that a comprehensive determination of the PTM code of ER would provide valuable information regarding signaling milieu of the cell.  Integration of this information with phosphorylation-induced alterations in ER function would help understand mechanism of hormone resistance that ultimately could be used to guide the optimum mode of hormone therapy in ER-positive breast cancer.

Ph.D. Thesis: Interaction of the DNA-bound estrogen receptor(ER) with coregulatory proteins. 

  ER α and β are members of a large family of transcription factors that regulate gene expression in response to estrogen.  Although the role of ligand in altering coactivator recruitment and transcription has been well documented, the effect of DNA-binding on coregulator recruitment has not been addressed.  On binding to the estrogen response elements (EREs) in the target genes the ERs undergo DNA-induced changes in conformation.  This study investigates the role of DNA-induced receptor conformations on coregulator recruitment.  Here we have documented an ERE-dependent recruitment of coactivator proteins, amplified in breast cancer (AIB1) and transcription intermediary factor (TIF2) to ERα and ERβ.  In our efforts to identify novel interactions with the DNA-bound receptor, we have isolated several proteins that interact with the A2 ERE-bound ERα using DNA pull-down and agarose gel shift assays.  Identification of the components of these complexes reveal proteins involved in several cell processes including, transcription, DNA repair and replication, G-protein modulation, and scavengers of superoxide radicals.  A number of these proteins have already been implicated in ER-mediated transcription control where as several other identified proteins are players from divergent cellular pathways. To examine whether these proteins infact play role in ER-mediated transcription, we studied the role of DNA repair protein, 3-methyadenine DNA glycosylase (MPG) on ER action. Investigation of the association of ERα with the DNA repair protein, MPG reveals that this interaction modulates estrogen responsiveness and alters DNA repair.  Taken together, these studies demonstrate multiple roles ER plays at the promoter, including differential recruitment of coregulatory proteins and  integration of other cell process with  ER-mediated transcription.

 Modulation of coactivator recruitment by DNA-induced changes in ER conformation.
ER is a ligand-activated transcription factor that binds to EREs and regulates expression of target genes. Though the role of ligand in modulating receptor conformation and coactivator recruitment has been well documented, the effect of DNA-binding on coactivator recruitment has generally not been addressed.  Previous studies carried out in our laboratory demonstrated that ER conformation is modulated by individual ERE sequences.  In this study, we have examined the effect of DNA-induced receptor conformation on recruitment of coregulatory proteins.  The coactivator proteins steroid receptor coactivator 1 (SRC-1), glucocorticoid receptor interacting protein 1 (GRIP1 or its human homolog, transcriptional intermediary factor 2, TIF2) and amplified in breast cancer (AIB1) interact with the receptor through their receptor interaction domains (RIDs).  In gel shift studies, ERα bound to the vitellogenin A2, pS2, vitellogenin B1, and the oxytocin (OT) ERE and effectively recruited SRC-1 and GRIP1/TIF2 RIDs resulting in significant stabilization of ERα-DNA complex.  As the full-length coactivators may possess amino acid sequences that aid in recognizing differences in DNA-induced receptor conformations, the abilities of ERα and ERβ to bind to full-length coactivators present in nuclear extracts were examined.  We were able to detect differences in recruitment of TIF2, but not with AIB1 when ERα was bound to the A2 and the B1 EREs.  In addition, when ERβ was bound to the pS2 and the B1 ERE, it recruited significantly lower levels of TIF2 and AIB1 than the A2 ERE-bound receptor.  The OT ERE-bound ERβ recruited less AIB1 but similar levels of TIF2 compared to the A2 ERE-bound receptor.  Thus, we have documented the ability of individual EREs to influence the recruitment of known coactivators.  Taken together, these studies demonstrate that allosteric modulation of ER conformation by individual ERE sequences fosters the recruitment of different subpopulations of nuclear proteins and helps to explain how a single cell harboring numerous estrogen-responsive genes can differentially regulate expression of genes containing discrete ERE sequences.

 Interaction between ERα  and 3-methyladenine DNA glycosylase modulates transcription and DNA repair.
ERα initiates transcription of the target genes in conjunction with coregulatory proteins, chromatin modifiers and other signal transduction pathways.  We have identified a novel interaction between ERα and 3-methyladenine DNA glycosylase (MPG), a DNA repair protein, which alters estrogen responsiveness and modulates DNA repair.  Here we demonstrate that the ERα-MPG interaction is enhanced in the presence of 17 β-estradiol and A2 ERE-containing DNA.  The interaction between ERα and MPG is direct and occurs through the DNA binding domain, the hinge and the ligand binding domain of the receptor.  We also show interaction between endogenously expressed ERα and MPG from MCF-7 cells.  Interestingly, ERα stabilizes the binding of MPG with hypoxanthine-containing oligos and enhances MPG-catalyzed removal of hypoxanthine from the DNA.  Likewise, MPG dramatically stabilizes the interaction of the receptor with ERE-containing oligos.  Paradoxically, over expression of MPG decreases ERα-mediated activation of ERE-containing reporter plasmids in a dose-dependent manner.  The interaction between ERα-MPG modulates acetylation of both ERα and MPG.  Further, MPG interacts specifically with ER, thyroid receptor, peroxisomal activated factor γ and retinoic acid X receptors but not with the progesterone receptor.  Our studies suggests that recruitment of MPG to an ERE-containing promoter modulates ERα-mediated transcription and that this may also play a role in maintaining genome integrity by recruiting DNA repair proteins to actively transcribing DNA.

Interaction of DNA-bound ER with nuclear proteins.
ER regulates transcription of estrogen-responsive genes by binding to the promoter of target genes and recruiting coregulatory proteins as well as the transcription machinery.  Recent work indicates that the ligand and the DNA serve as allosteric modulators of receptor conformation altering recruitment of ERa-associated proteins. In our efforts to identify novel protein interactions with the DNA-bound receptor, we have isolated and identified nuclear proteins that interact with ERα.  We have also documented the formation of a large nuclear protein complex with the A2 ERE-bound ERα in agarose gel shift assays. Mass spectrometric analysis of the components of this ERα-associated complex revealed presence of several proteins involved in chromatin modification, transcription, G-protein modulation, redox regulation, and DNA repair.  Association of these proteins with the DNA-bound ERα may be playing a role in coordinating various cell processes with ERα-mediated transcription.  This study therefore suggests that the DNA-bound receptor fosters the recruitment of numerous proteins involved in multiple cell processes to estrogen-responsive genes and helps explain how cells can integrate various signal transduction pathways and coordinate a variety of celluar processes including transcription, DNA repair and replication.