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.