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Regulation of Estrogen related receptor alpha localization and signaling by the kinesin KIF17 Nilsa M. Méndez1 and Geri E. Kreitzer2 1Industrial Biotechnology Department, University of Puerto Rico and 2Developmental and Cell Biology, Weill Cornell Medical College ACT Abstract Abstract Kinesins are motor proteins that transport a variety of cargos such dehyde 2% and permeabilized with -20oC methanol for 15 secs. Endo- ESRRA was stained using anti human ESRRA mouse monoclonal primary antibody and Cy5 anti as organelles, vesicles, RNA, protein complexes and even viruses, to specific N destinations in the cell in a microtubule and ATP dependent manner. KIF17 CREM mouse secondary antibody. DAPI was used as a nuclear stain. MT the pictures and they were analyzed using MetaMorph version 6.3r1. Medium (Fatty Acid Free BSA) for 36 hours at 37oC and 5% CO2. Cells were treated ESRRA is an orphan with 100 ng/mL EGF for the final 30 and 60 minutes of the incubation. nuclear receptor structurally and functionally similar to the classic estrogen estrogen or estradiol. However, phosphorylation of ESRRA by EGF signaling pathway can make ESRRA to change its conformation and enhance DNAbinding. ESRRA activity is regulated in part by direct competition with ERs Figure 3. KIF17 binds to ACT and transports it into the nucleus, were Phase contrast cells . This is a novel regulatory function of KIF17. Endogenous ESRRA MCF-7 (ER positive) cells starts in the nucleus and after the treatment, the distribution becomes both nuclear and cytoplasmic, while MDA-MB-231 is mostly altering ESRRA intracellular localization. Overall, a complete understanding of the MCF-7 cells ER (+) activates transcription of CREM dependent genes in murine male germ ESRRA is mostly nuclear (Figure 4) . Comparing both cell lines, ESRRA localization in nuclear throughout the whole treatment. Our findings suggest that EGF might be Results Results receptors (ER) but its activity is independent of any known ligands, like in the outer periphery of the nucleus. After 60 minutes of treatment, ESRRA is localized within the nucleus and the cytoplasm in MCF-7 cells. In MDA-MB-231 , EGF treatment―MCF-7 and MDA-MB-231 cells were starved in DMEM Serum Free hybrid assay and immunoprecipitation - that one of the cargos that interacts Endogenous ESRRA (MCF-7 control) is mainly localized in the nucleus, however after 30 minutes of treating the cells with EGF, ESRRA begins accumulating Fluorescence microscopy―Nikon Eclipse TE2000-U microscope was used to take is part of the kinesin family and it was found - by the methods of yeast two- with is Estrogen related receptor alpha (ESRRA). Summary Summary mechanism by which EGF and KIF17 are involved in the subcellular translocation of DAPI ESRRA is essential and may foster the development of new treatments for breast cancer patients. genes. It has been previously reported that KIF17 is involved in regulating T0 (control) and it must go to the nucleus in order to activate transcription of its target ESRRA CREM mediated transcription by interacting with and controlling the What’s next? germ cells. Under the light of this precedent, we hypothesize that KIF17 might be involved in regulating nuclear transport of ESRRA, and hence, its N MT activity. Our goal is to reveal the functional significance of KIF17-ESRRA interaction in breast cancer cells and we will do this by first, determining the mechanisms by which KIF17 controls the intracellular distribution of ESRRA T30 (30 mins, +EGF) intracellular localization of the transcriptional activator ACT in murine male Measure nuclear: cytoplasmic ratio to see if there’s a significant change in ESRRA distribution after EGF treatment fluorescence microscopy. Knowing the mechanisms underlying ESRRA regulation is important because its activity is related to aggressive tumor behavior and poor prognoses in breast cancer patients. Introduction Introduction Figure 1. Kinesin structure. Kinesins are motor proteins that transport a variety of cargos. The N-term is the motor domain that binds to MT, the coiled-coil domain regulates homodimerization and the C-term is the cargo binding domain Figure 2. KIF17 colocalizes with microtubules. GFP-KIF17-FL (red) and microtubules (green) into the nucleus. ESRRA is an orphan nuclear receptor that is constitutively active and does not bind to any known physiological ligand. Even though ESRRA is widely expressed in normal tissues, studies have shown that ESRRA is an unfavorable biomarker for breast cancer. Goals We want to determine the mechanism by which KIF17: Figure 5. Intracellular distribution of endogenous ESRRA in MCF-7 cells after EGF treatment MDA-MB-231 cells ER (-) Phase contrast 1. controls the intracellular distribution of ESRRA (nuclear vs cytoplasmic) 2. can modulate transcription of ESRRA target genes 3. regulates interactions of transcriptional coactivators with ESRRA Materials Materials and and methods methods Cell lines―the cells used were MCF-7 and MDA-MB-231. These are mammary epithelial cells Estrogen receptor positive and negative, respectively. Cell culture―all cells were grown in 10 cm dishes with DMEM medium supplemented with 10% Fetal Bovine Serum (10% FBS) and 20mM HEPES, incubated at 37oC and 5% CO2. Fixation and immunocytochemistry― all cells were rinsed with Phosphate buffered saline (PBS) with Ca2+ and Mg2+, fixed during 2 minutes in paraformal- Endogenous ESRRA DAPI T0 (control) and Figure 7. Over expression of GFP-ESRRA FL and RFP-KIF17 mut GE in MCF-7 cells References Massarweh, S. and Schiff, R. Resistance to endocrine therapy in breast cancer: exploiting estrogen receptor/growth factor signaling crosstalk. Endocrine-Related Cancer (2006) 13, S15-S24 Ciguere, V. and Barry, J. Epidermal Growth Factor-induced signaling in breast cancer cells results in selective target gene activation by orphan nuclear receptor T60 (60 mins, +EGF) immunocytochemistry Figure 4. KIF17 might regulate ESRRA activity by controlling its transport T30 (30 mins, +EGF) ESRRA localization and determine the nuclear:cytoplasmic ratio by T60 (60 mins, +EGF) (nuclear vs cytoplasmic) in presence of EGF. We will measure alterations of Look at up/down regulation of transcription of ESRRA target genes (+/- EGF, +/KIF17) Do live, time-lapse imaging of the movement of ESRRA into the nucleus after EGF treatment (+/KIF17 GE or tail domain) Estrogen-Related α. Cancer Res 2005; 65: (14) 6120-6129 Kotaja, N., Macho, B. and Sassone-Corsi, P. Microtubule-independent and Protein Kinase A-mediated function of KIF17b controls the intracellular transport of activator of CREM in testis (ACT). J. Biol. Chem. 280, 31739-31745 Stein, R.A. and McDonnell, D.P. Estrogen-related receptor α as a therapeutic target Figure 6. Intracellular distribution of endogenous ESRRA in MDA-MB-231 cells after EGF treatment in cancer. Endocrine-Related Cancer (2006) 13 S25-S32