Regulation of Estrogen related receptor alpha localization and signaling by the kinesin KIF17Nilsa M. Méndez1 and Geri E. Kreitzer2
1Industrial Biotechnology Department, University of Puerto Rico and 2Developmental and Cell Biology, Weill Cornell Medical College
Abstract Kinesins are motor proteins that transport a variety of cargos such
as organelles, vesicles, RNA, protein complexes and even viruses, to specific
destinations in the cell in a microtubule and ATP dependent manner. KIF17
is part of the kinesin family and it was found - by the methods of yeast two-
hybrid assay and immunoprecipitation - that one of the cargos that
interacts with is Estrogen related receptor alpha (ESRRA). ESRRA is an
orphan nuclear receptor structurally and functionally similar to the classic
estrogen receptors (ER) but its activity is independent of any known ligands,
like estrogen or estradiol. However, phosphorylation of ESRRA by EGF
signaling pathway can make ESRRA to change its conformation and enhance
DNA-binding. ESRRA activity is regulated in part by direct competition with
ERs and it must go to the nucleus in order to activate transcription of its
target genes. It has been previously reported that KIF17 is involved in
regulating CREM mediated transcription by interacting with and controlling
the intracellular localization of the transcriptional activator ACT in murine
male germ cells. Under the light of this precedent, we hypothesize that
KIF17 might be involved in regulating nuclear transport of ESRRA, and
hence, its 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 (nuclear vs cytoplasmic) in presence of EGF. We will
measure alterations of ESRRA localization and determine the
nuclear:cytoplasmic ratio by immunocytochemistry and 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
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
Materials and methodsCell 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-
GoalsWe want to determine the mechanism by which KIF17:
ReferencesMassarweh, 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
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 in cancer. Endocrine-Related Cancer (2006) 13 S25-S32
Results
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
mouse secondary antibody. DAPI was used as a nuclear stain.
Fluorescence microscopy―Nikon Eclipse TE2000-U microscope was used to take
the pictures and they were analyzed using MetaMorph version 6.3r1.
EGF treatment―MCF-7 and MDA-MB-231 cells were starved in DMEM Serum Free
Medium (Fatty Acid Free BSA) for 36 hours at 37oC and 5% CO2. Cells were treated
with 100 ng/mL EGF for the final 30 and 60 minutes of the incubation.
Phase contrast
Endogenous ESRRA DAPI
T 0 (c
ontr
ol)
T 30
(30
min
s, +
EGF)
T 60
(60
min
s, +
EGF)
Figure 5. Intracellular distribution of endogenous ESRRA in MCF-7 cells after EGF treatment
Summary Endogenous ESRRA (MCF-7 control) is mainly localized in the nucleus,
however after 30 minutes of treating the cells with EGF, ESRRA begins accumulating
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 ,
ESRRA is mostly nuclear (Figure 4) . Comparing both cell lines, ESRRA localization in
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
nuclear throughout the whole treatment. Our findings suggest that EGF might be
altering ESRRA intracellular localization. Overall, a complete understanding of the
mechanism by which EGF and KIF17 are involved in the subcellular translocation of
ESRRA is essential and may foster the development of new treatments for breast
cancer patients.
MCF-7 cells ER (+)
Phase contrast
Endogenous ESRRA DAPI
T 0
(con
trol
)
T 30
(30
min
s, +
EGF)
T 60
(60
min
s, +
EGF)
MDA-MB-231 cells ER (-)
Figure 6. Intracellular distribution of endogenous ESRRA in MDA-MB-231 cells after EGF treatment
Figure 2. KIF17 colocalizes with microtubules. GFP-KIF17-FL (red) and microtubules (green)
Figure 7. Over expression of GFP-ESRRA FL and RFP-KIF17 mut GE in MCF-7 cells
Abstract
IntroductionGoals
Materials and methods
Results
Summary
What’s next?
References
ACT
MT
NCREM
Figure 3. KIF17 binds to ACT and transports it into the nucleus, were
activates transcription of CREM dependent genes in murine male germ
cells . This is a novel regulatory function of KIF17.
ESRRA
MT
N
Figure 4. KIF17 might regulate ESRRA activity by controlling its transport
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.