MOLECULAR AND CELLULAR BIOLOGY, July 2011, p. 2683–2695
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 31, No. 13
The Ras Signaling Inhibitor LOX-PP Interacts with Hsp70 and c-Raf
To Reduce Erk Activation and Transformed Phenotype of Breast
Seiichi Sato,1Philip C. Trackman,2Joni M. Ma ¨ki,3Johanna Myllyharju,3
Kathrin H. Kirsch,4† and Gail E. Sonenshein1†*
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 021111; Division of Oral Biology,
Boston University Goldman School of Dental Medicine, Boston, Massachusetts 021182; Oulu Center for Cell-Matrix Research,
Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu,
FIN-90014 Oulu, Finland3; and Department of Biochemistry, Boston University School of
Medicine, Boston, Massachusetts 021184
Received 29 September 2010/Returned for modification 5 December 2010/Accepted 15 April 2011
The lysyl oxidase gene (LOX) inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its
activity was mapped to the 162-amino-acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein.
LOX-PP inhibits Erk signaling, motility, and tumor formation in a breast cancer xenograft model; however, its
mechanism of action is largely unknown. Here, a copurification-mass spectrometry approach was taken using
ectopically expressed LOX-PP in HEK293T cells and the heat shock/chaperone protein Hsp70 identified.
Hsp70 interaction with LOX-PP was confirmed using coimmunoprecipitation of intracellularly and bacterially
expressed and endogenous proteins. The interaction was mapped to the Hsp70 peptide-binding domain and to
LOX-PP amino acids 26 to 100. LOX-PP association reduced Hsp70 chaperone activities of protein refolding
and survival after heat shock. LOX-PP interacted with the Hsp70 chaperoned protein c-Raf. With the use of
ectopic expression of LOX-PP wild-type and deletion proteins, small interfering RNA (siRNA) knockdown, and
Lox?/?mouse embryo fibroblasts, LOX-PP interaction with c-Raf was shown to decrease downstream activa-
tion of MEK and NF-?B, migration, and anchorage-independent growth and reduce its mitochondrial local-
ization. Thus, the interaction of LOX-PP with Hsp70 and c-Raf inhibits a critical intermediate in Ras-induced
MEK signaling and plays an important role in the function of this tumor suppressor.
Lysyl oxidase (LOX) (protein-6-oxidase; EC 18.104.22.168) is the
key extracellular enzyme that controls collagen and elastin
cross-linking, which is required for the biosynthesis of func-
tional extracellular matrices. The LOX gene was isolated as the
Ras recision gene (rrg) with the ability to inhibit the transform-
ing activity of the H-Ras oncogene in NIH 3T3 fibroblasts (10,
35). Reduced LOX expression has been reported to occur in
many carcinomas (5, 21, 23, 33, 60, 63, 73). Ectopic LOX gene
expression in gastric cancer cells resulted in reduced tumor
formation in nude mice (33). Lysyl oxidase is synthesized and
secreted as a 50-kDa inactive proenzyme (Pro-LOX), which is
processed by proteolytic cleavage to a functional 32-kDa en-
zyme (LOX) and an 18-kDa propeptide (LOX-PP). Expression
of Pro-LOX in Ras-transformed NIH 3T3 fibroblasts inhibited
the activities of the Erk1/2 and Akt kinases and transcription
factor NF-?B (30). Subsequently, LOX-PP was identified as
the inhibitor of Ras signaling and transformed phenotype in
NIH 3T3 fibroblasts (51), NF639 breast cancer cells (44), and
H1299 lung cancer and PANC-1 pancreatic cancer cells, with
mutant RAS and TP53 genes (73). In NF639 cells, driven by
Her-2/neu which signals via Ras, LOX-PP expression de-
creased Her-2/neu-mediated signaling and Erk activation in
exponentially growing cells or following serum stimulation
(44). LOX-PP also reduced mesenchymal phenotype in vitro,
as judged by induction of epithelial and reduction of mesen-
chymal markers and invasive colony formation in Matrigel and
tumor xenograft formation in a nude mouse model (44). Fur-
thermore, LOX-PP attenuated fibronectin-stimulated integrin
signaling and migration in breast cancer cells (75). Together,
these studies suggest a role for LOX-PP in inhibiting the in-
vasive phenotype of carcinomas, but the exact mechanisms by
which it functions have not been elucidated.
Hsp70s are a family of stress response proteins that act as
molecular chaperones that prevent protein aggregation and
also refold denatured or unfolded proteins via their ATPase
activity, catalyzed by ATP-hydrolysis (22, 32). The human
Hsp70 family includes at least 8 distinct genes that code
for Hsp70 isoforms, located on several different chromo-
somes. Hsp70 family proteins are structurally conserved, and
include an ATPase domain in the amino-terminal region, a
peptide binding domain in the carboxy-terminal region, and an
acidic motif (EEVD) in the extreme carboxy-terminal region.
Hsp70 family proteins localize to the nucleus, mitochondria,
endoplasmic reticulum, Golgi apparatus, cytosol, and extracel-
lular cell surface. The two major cytoplasmic isoforms are
Hsc70 and Hsp70/Hsp72, which is also termed Hsp70. Gener-
ally, Hsc70 is abundantly and ubiquitously expressed in nontu-
mor tissues, whereas Hsp70/Hsp72 expression is induced by
various signals. Constitutively expressed Hsp70/Hsp72 proteins
* Corresponding author. Mailing address: Department of Biochem-
istry, Tufts University School of Medicine, 150 Harrison Avenue, Bos-
ton, MA 02111. Phone: (617) 636-4091. Fax: (617) 636-2049. E-mail:
† K.H.K. and G.E.S. contributed equally to the design and develop-
ment of this work and therefore share senior authorship.
?Published ahead of print on 2 May 2011.
have been detected in several tumors. In breast cancer, Hsp70
overexpression has been associated with shorter disease-free
survival, a more invasive phenotype, and poorer prognosis (9).
The exact roles of the Hsp70/Hsp72 family in cancer are not
fully understood; however, these proteins can contribute to
tumor cell survival and tumorigenesis via multiple antiapop-
totic functions and through their role as a cochaperone for
Hsp90 (47, 66, 76). Interestingly, it is reported that dual silenc-
ing of Hsc70 and Hsp70 induces tumor specific apoptosis (57).
In the present study, we identified Hsp70 protein as a novel
binding partner of LOX-PP, mapped the domains of interac-
tion, and demonstrated LOX-PP can also associate with c-Raf,
an Hsp70 client. Functionally, the interaction with LOX-PP
reduced the chaperone and survival functions of Hsp70 upon
stress and decreased the activation of c-Raf that promotes a
more transformed phenotype of breast cancer cells.
MATERIALS AND METHODS
Plasmid construction. For construction of N-terminally glutathione S-trans-
ferase (GST)-tagged LOX-PP and its deletion mutants, the cDNA encoding
amino acids (aa) 1 to 162 (full length), 1 to 115 (?C1), 1 to 100 (?C2), 1 to 77
(?C3), 1 to 61 (?C4), 1 to 25 (?C5), 26 to 162 (?N1), 62 to 162 (?N2), 78 to 162
(?N3), 101 to 162 (?N4), 116 to 162 (?N5), and 26 to 100 (M1) and deletions of
aa 26 to 61 (?M1), 26 to 77 (?M2), and 26 to 100 (?M3) of LOX-PP was
amplified from full-length cDNA (44) and inserted into the BamHI/ClaI site of
pEBG-GST mammalian expression vector, a generous gift from Bruce Mayer
(University of Connecticut Health Center, Farmington, CT). For construction of
C-terminally GST-tagged LOX-PP, the cDNAs encoding GST and LOX-PP
were amplified and inserted into pcDNA3.1(?) (Invitrogen, Carlsbad, CA). For
construction of C-terminally V5/His-tagged proteins, the cDNAs encoding LOX-
PP, LOX, or Pro-LOX were inserted into pcDNA4/V5-His vector (Invitrogen).
For preparation of recombinant rat LOX-PP, recombinant LOX-PP (rLOX-
PP)–myc-His protein, in which the signal peptide of Pro-LOX was replaced with
the one from osteonectin (BM-40) in the pcDNA4/TO/myc-His vector, was used
as described previously (70). pCXbsr-Hsp70 and pAD-c-Raf were kindly supplied
by Michael Sherman and Vladimir Gabai (Boston University School of Medi-
cine, Boston, MA) and Linda Van Aelst (Cold Spring Harbor Laboratories, Cold
Spring Harbor, NY), respectively. The cDNA encoding the Hsp70 wild type
(WT) was inserted into pEBG, pGEX4T-3 (GE Healthcare, Uppsala, Sweden),
and pFLAG-CMV-2 (Sigma-Aldrich, St. Louis, MO). The cDNAs encoding
Hsp70 ?SmaI and ?BglII were digested with SmaI and BglII, respectively, as
described by Milarski and Morimoto (43), and inserted into pFLAG-CMV2. The
cDNA encoding c-Raf WT was subcloned into pEGFP-C1 (Clontech, Palo Alto,
CA) and pGEX4T-3 vector. The cDNA encoding LOX-PP WT and ?M3 was
subcloned into pGEX4T-3 vector. All constructs with deletions were generated
by PCR and verified by DNA sequencing.
Cell culture and treatment conditions. ER?-positive MCF-7 and ZR-75 and
ER?-negative Hs578T breast cancer cells, which contain a mutated constitutively
active H-Ras, were purchased from the American Type Culture Collection
(ATCC; Manassas, VA). Human embryonic kidney HEK293T cells and Bosc23
cells were obtained from the ATCC. Cells were maintained in Dulbecco’s min-
imal essential medium (DMEM) supplemented with 10% heat-inactivated fetal
bovine serum (FBS), 2 mM glutamine, and penicillin-streptomycin, as recom-
mended by the ATCC. Mouse embryo fibroblasts (MEFs) from Lox?/?or
control C57BL/6 mice were prepared as described previously (39) and cultured
in high-glucose DMEM supplemented with 20% FBS in DMEM with L-glu-
tamine, nonessential amino acids, bicarbonate, and antibiotics. T-Rex293 cells
were cultured as described previously (70). NIH 3T3 cells were kindly provided
by Amitha Palamakumbura (Boston University Goldman School of Dental Med-
icine, Boston, MA) and cultured as described previously (30). The NF639 cell
line, kindly provided by P. Leder (Harvard Medical School, Boston, MA) was
derived from a mammary gland tumor in a mammary tumor virus (MMTV)-
ERBB2 transgenic mouse and cultured as described previously (18). For heat
shock treatment, cells were transfected with GST-tagged WT or ?M3 LOX-PP
or GST using Lipofectamine 2000 (Invitrogen). After 24 h, cells were submerged
in a 45°C water bath for 10 min and allowed to recover at 37°C for 24 h. Cells
were then assayed using 0.4% trypan blue uptake (Invitrogen) for the percentage
of cell death. Assays were performed three independent times, each in duplicate,
and the means ? standard deviations (SD) are presented. The RNA duplexes
used for targeting mouse LOX (oligonucleotide A, 5?-TAGGGCGGATGTCA
GAGACTA-3?; oligonucleotide B, 5?-AACGATCCTTTCAAATTATAA-3?)
were purchased from Qiagen (Germantown, MD) and transfected at a final
concentration of 20 nM using Lipofectamine RNAiMAX (Invitrogen).
Antibodies and immunoblot analysis. Antibodies against Hsp70 (SPA-810),
Hsp70/Hsc70 (SPA-820), and Hsp90 (SPA-830) were purchased from Stressgen
(Victoria, BC, Canada). Antibodies against ?-tubulin (DM1A), ?-tubulin (TUB
2.1), ?-tubulin (GTU-88), ?-actin (AC-15), and FLAG (M2) were from Sigma-
Aldrich. Antibodies against Erk1/2 (no. 9102), p-Erk1/2 (phospho-Thr202/
Tyr204; no. 9101), Akt (no. 9272), MEK1/2 (L38C12; no. 4694), p-MEK1/2
(phopho-Ser217/221; no. 9121), and EGFR (no. 2232) were purchased from Cell
Signaling (Danvers, MA). Antibodies from Santa Cruz Biotechnology (Santa
Cruz, CA) included anti-GST (B-14) and anti-B-Raf (F-7). Antibodies against
c-Raf (clone 53) and Apaf-1 (A92820) were from BD Transduction (Franklin
Lakes, NJ). Monoclonal antibodies against V5 (R960-25) and COX-1 (COX 111)
and polyclonal antibody against green fluorescent protein (GFP) (A-6455) were
from Invitrogen. Rabbit polyclonal antibody against V5 (E14) from Delta Bio-
Labs (Gilroy, CA) was used for immunoprecipitation and immunofluorescence
microscopy. Antibody against His tag (120-003-812) was from Macs Miltenyi
Biotec (Germany). Rabbit polyclonal antibodies against LOX-PP were either
prepared as described previously (27) or purchased from Novus Biologicals
(NBP1-30327) (Littleton, CO) and react with mouse or rat or human or rat,
For preparation of whole-cell lysates, cells were solubilized in phosphate-
buffered saline (PBS) with 0.5% SDS, 1 mM Na3VO4, and 10 mM NaF. Lysates
were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF)
membranes, and subjected to immunoblotting using the appropriate primary
antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies
(goat anti-mouse IgG-HRP [Santa Cruz; no. sc-2005] or anti-rabbit IgG-HRP
[Bio-Rad; no. 170-6515]), as described previously (30).
Affinity isolation of LOX-PP-interacting proteins. HEK293T cells plated on
six 35-mm dishes were transfected with 2 ?g of pEBG or pEBG-mLOX-PP
expression plasmid per dish using Lipofectamine 2000 reagent according to the
manufacturer’s protocol. After 24 h, cells were lysed in 300 ?l of buffer A (25
mM HEPES-KOH [pH 7.2], 150 mM KCl, 2 mM EDTA, 1 mM phenylmethyl-
sulfonyl fluoride (PMSF), 1 mM dithiothreitol, 0.5 ?g/ml leupeptin, 2 ?M pep-
statin A, 1 ?g/ml aprotinin, and 1% Triton X-100) per dish. The lysates were
centrifuged in a microcentrifuge for 10 min at 13,000 rpm at 4°C to remove
insoluble material. The supernatant (about 2 mg) was incubated with 20 ?l
glutathione-Sepharose 4B (GE Healthcare) for 2 h at 4°C. The resin was washed
four times with lysis buffer, and proteins were eluted with SDS-PAGE loading
buffer. Following separation by 10% SDS-PAGE, proteins were visualized by
Coomassie blue staining and bands corresponding to 70 kDa and 52 kDa were
isolated. In-gel proteolytic digestion and mass spectrometry (liquid chromatog-
raphy-tandem mass spectrometry [LC-MS-MS]) was performed by the Taplin
Biological Mass Spectrometry Facility (Boston, MA).
Effects of ATP on the association of Hsp70 and LOX-PP. HEK293T cells,
plated on 35-mm dishes, were transfected with the indicated expression plasmids.
After 24 h, cell lysates were prepared in 300 ?l of lysis buffer A or in the same
buffer with 1 mM ATP and 2 mM MgCl2but without EDTA. GST pulldown
assays were performed as described above. The beads were washed with lysis
buffer, and the precipitated proteins were detected by immunoblotting.
Luciferase refolding assay. The cDNA of cytoplasm-localized luciferase (Luc-
Cyto) in which the C-terminal peroxisomal localization signal has been destroyed
by a Leu-Val mutation of the amino acid at position 550 was made by site-direct
mutagenesis as described by Michels et al. (42). HEK293T cells were transfected
with cytomegalovirus (CMV) promoter-driven Luc-Cyto expression plasmid and
GST-tagged WT or ?M3 LOX-PP or GST using Lipofectamine 2000 according
to the manufacturer’s protocol. After 24 h, cultures were treated with 20 ?g/ml
cycloheximide for 30 min at 37°C. To denature the expressed luciferase protein,
cells were incubated at 45°C for 30 min in a water bath. The cells were then
incubated for the indicated periods of time at 37°C to allow for refolding of the
luciferase and lysed, and protein concentrations measured using a protein assay
kit (Bio-Rad). The luciferase activities in equal amounts of protein lysates were
measured using the luciferase assay system (Promega, Madison, WI) according
to the manufacturer’s protocol.
Preparation of recombinant proteins. GST, GST–LOX-PP WT and ?M3,
GST-Hsp70, and GST–c-Raf were expressed in Escherichia coli BL21(DE3)
pLysS (Invitrogen). The bacteria were grown in Luria broth at 30°C to an A600of
0.5, and protein expression was induced with 0.1 mM isopropyl ?-D-thiogalacto-
side. At 3 h for GST, GST–LOX-PP WT and ?M3, and GST-Hsp70 or 1 h for
GST–c-Raf postinduction, the bacteria were harvested by centrifugation
2684SATO ET AL.MOL. CELL. BIOL.
(3,700 ? g for 10 min at 4°C), resuspended in lysis buffer (1% Triton X-100, 50
mM Tris, 150 mM NaCl, 1 mM EDTA, and 1 mM PMSF [pH 8.0]), and lysed by
sonication. The lysate was centrifuged at 13,000 rpm for 10 min. The supernatant
was loaded on to a glutathione-Sepharose 4B column, washed extensively with
lysis buffer containing 300 mM NaCl and 0.1% Triton X-100, and eluted with
phosphate buffer (36 mM Na2HPO4and 14 mM NaH2PO4[pH 7.2]) containing
100 mM NaCl, 0.1% Triton X-100, and 30 mM glutathione. Purified rLOX-PP–
myc-His protein, with the BM-40 signal peptide (70), and GST fusion proteins
(0.5 ?M) were used in binding assays conducted in phosphate buffer containing
100 mM NaCl and 0.1% Triton X-100. Purified binding partners, at the indicated
concentrations, were incubated for 3 h at 4°C. Glutathione-Sepharose 4B was
then added and allowed to incubate for 1.5 h at 4°C with gentle rotation. The
beads were sedimented in a microcentrifuge. After extensive washing, the pro-
teins bound to the beads were solubilized in sample buffer and subjected to
SDS-PAGE followed by immunoblotting.
Immunoprecipitation analysis. NF639, ZR-75, or NIH 3T3 cells were lysed
with buffer A, as described above. Either rabbit anti-LOX-PP (reference 27 or
NBP1-30327 [Novus Biologicals]), mouse anti-Hsp70/Hsc70, or rabbit anti-V5
tag (2 ?g) was added to 500 ?g cell lysate, followed by overnight incubation at
4°C. Protein G-Sepharose beads (Invitrogen) were then added to the mixture,
followed by incubation at 4°C for 2 h with gentle shaking. The beads were washed
four times with buffer A. The immune complexes were eluted from the Sephar-
ose beads with SDS-PAGE sample buffer and the precipitated proteins analyzed
by Western blotting.
Migration, invasion, colony, and NF-?B activity assays. Suspensions of 1 ?
104NF639 cells or MEFs were layered, in triplicate, in the upper compartments
of a Transwell (Costar, Cambridge, MA) on a 6.5-mm-diameter polycarbonate
filter (8-?m pore size) and incubated at 37°C for the indicated times. Migration
of the cells to the lower side of the filter was evaluated by staining with crystal
violet and quantified by spectrometric determination at A570as described previ-
ously (75). Assays were performed three times independently, each time in
triplicate, and the means ? SD are presented. Suspensions of 1 ? 104NF639
cells were layered in the upper compartment of a Transwell on a 6.5-mm-
diameter polycarbonate filter (8-?m pore size) precoated with 10 ?g of Matrigel
and incubated at 37°C for 6 h. Migration of cells was quantified as described
above for cell migration. Soft agar assays (30) and NF-?B element-driven lucif-
erase assays (73) were performed as we have described previously.
Immunofluorescence microscopy. NF639 cells plated on coverslips were trans-
fected with small interfering RNA (siRNA) for 48 h. Cells were incubated for 30
min in 250 nM MitoTracker (Invitrogen), washed with PBS, and fixed with
methanol at ?20°C for 5 min. Following washing with PBS, cells were blocked by
incubation in 2% (wt/vol) bovine serum albumin (A3059; Sigma) in PBS (buffer
1) and incubated for 1 h in primary antibody diluted in buffer 1. Following
washing with PBS, the coverslips were incubated for 1 h in appropriate secondary
antibodies conjugated with Alexa Fluor 488 (Invitrogen) diluted in buffer 1.
Subsequently, the coverslips were mounted in Slowfade Gold antifade reagent
(Invitrogen) with Hoechst 33342 (Invitrogen) and observed under a Nikon
Eclipse E400 microscope. The images are representative of two independent
experiments performed in duplicate.
Isolation of mitochondrial membranes. NF639 cells, cultured on 100-mm
dishes and transfected with 20 nM control siRNA or siLOX oligonucleotide B
RNA for 48 h or wild-type or Lox?/?MEFs cultured on 150-mm dishes, were
harvested with a cell scraper, followed by centrifugation. Further steps were
carried out on ice or at 4°C. Cells were washed with 1? PBS and then homog-
enized with 20 strokes in 3 volumes of homogenization buffer (10 mM Tris-HCl,
pH 7.5, 0.25 M sucrose, 5 mM MgCl2, 10 mM KCl, and 1 mM PMSF) in a
stainless steel homogenizer. After centrifugation at 800 ? g for 10 min, the
postnuclear supernatant was centrifuged at 3,000 ? g for 10 min and the resulting
mitochondrial membrane preparation washed once with homogenization buffer.
Mass spectrometry identifies Hsp70 as a LOX-PP associ-
ated protein. To identify proteins that can associate with LOX-
PP, the murine propeptide was cloned into the mammalian
pEBG vector, which expresses inserted cDNAs as GST fusion
proteins (41). HEK293T cells were transfected with pEBG-
mLOX-PP LOX-PP expression plasmid or a pEBG empty-
vector (EV) control. After 24 h, extracts were prepared and
incubated with glutathione-Sepharose 4B resin and bound pro-
teins washed extensively and eluted with SDS-PAGE loading
buffer. Following 10% SDS-PAGE, proteins were visualized by
Coomassie blue staining (Fig. 1A). Bands of approximately 70
kDa and 52 kDa were seen in GST–LOX-PP precipitates and
not in the control GST lane. These were excised and subjected
to in-gel proteolytic digestion and mass spectrometry (LC-MS-
MS). The bands were identified as the 70-kDa heat shock
protein (Hsp70) and ?/?-tubulin, respectively (Table 1). To
confirm the mass spectrometry analysis, the GST–LOX-PP and
GST proteins were purified from HEK293T cells in a small-
scale GST pulldown assay and subjected to Western blotting
for Hsp70, for ?-tubulin, ?-tubulin, and ?-tubulin, and for
?-actin and GST as loading controls. Hsp70 specifically copu-
rified with GST–LOX-PP, as did all three of the tubulin pro-
teins (Fig. 1B), confirming the associations identified by mass
spectrometry. Hsp90, which frequently functions as a cochap-
erone with Hsp70, was somewhat unexpectedly not detected in
the proteins that purified with LOX-PP on the resin column as
visualized by Coomassie blue staining in Fig. 1A. Consistently,
when we tested directly for its presence by immunoblotting,
Hsp90 was not detectable among the proteins that coprecipi-
tated with GST–LOX-PP (Fig. 1B). The association of ectop-
ically expressed LOX-PP with endogenous Hsp70 proteins was
also readily seen in several breast cancer cell lines, including
ZR-75 and Hs578T (Fig. 1C) and MCF-7 (not shown), follow-
ing transient GST–LOX-PP-versus-GST expression. Similarly,
endogenous LOX-PP and Hsp70 proteins were found to co-
immunoprecipitate in extracts from ZR-75 cells (Fig. 1D),
which express low levels of LOX-PP, and from NIH 3T3 (Fig.
1E) and NF639 cells (see Fig. 6B).
Next, we asked whether Hsp70 can interact with LOX-PP
using purified preparations of myc-His-tagged recombinant rat
rLOX-PP (Fig. 2A) and GST-Hsp70 protein (Fig. 2B). GST-
Hsp70 protein brought down recombinant LOX-PP, confirm-
ing the direct association between these two proteins (Fig. 2C).
Furthermore, LOX-PP was coprecipitated with GST-Hsp70 in
HEK293T cells (Fig. 2D). Interestingly, ?-tubulin was not de-
tected in the copurified proteins, indicating that tubulin is not
an intermediate in the Hsp70–LOX-PP protein complex (Fig.
2D). The association with ?-tubulin is consistent with our pre-
vious observations in differentiating MC3T3-E1 cells, where
LOX-PP was found to interact with the microtubule network
(19). Thus, LOX-PP associates with Hsp70 and ?/?/?-tubulin.
Furthermore, our findings suggest that LOX-PP is a client of
Hsp70 but not Hsp90.
Interaction of LOX-PP with Hsp70 is inhibited upon ATP
binding. Previous investigations of the Hsp70 family have
shown that ATP binding induces a conformational change and
that the interaction of Hsp70 proteins with their binding part-
ners is frequently sensitive to the nucleotide-bound state (20).
Thus, we next tested whether the interaction of LOX-PP with
Hsp70 is ATP dependent. In the presence of 1 mM ATP, the
interaction of LOX-PP and Hsp70, but not ?-tubulin, was
almost completely eliminated (Fig. 2E). These findings suggest
a conformational dependence for the interaction with Hsp70
and regulation through an ATP-binding/hydrolysis cycle.
Mapping the domains of Hsp70 mediating interaction with
LOX-PP. Hsp70 has both ATPase and peptide binding do-
mains (22, 32). To map the regions interacting with LOX-PP,
deletion mutants of these domains in Hsp70 were constructed
VOL. 31, 2011 LOX-PP INTERACTION INHIBITS Hsp70 AND c-Raf FUNCTIONS2685
as described by Milarski and Morimoto (43) (Fig. 3A). Hsp70
?BglII lacks amino acids 121 to 427 in the BglII-BglII frag-
ment, which codes for the ATPase domain, and Hsp70 ?SmaI
lacks amino acids 439 to 617 in the SmaI-SmaI fragment, which
codes for the peptide binding domain. These Hsp70 constructs
were coexpressed with GST–LOX-PP or GST, as a control, in
HEK293T cells. Following pulldown of the GST proteins,
bound proteins were identified by Western blotting using a
Flag antibody (Fig. 3B, top panel). GST–LOX-PP brought
down Hsp70 ?BglII as well as Hsp70 WT but not Hsp70
?SmaI (Fig. 3B, GST pulldown lanes). GST failed to bring
down any proteins, as expected. Furthermore, analysis of ali-
quots of each of the original lysates confirmed essentially equal
loading of expressed proteins and efficient pulldown of all of
the GST proteins (Fig. 3B, lower panel). Thus, the peptide
binding domain of Hsp70 mediates interaction with LOX-PP.
Mapping the domains of LOX-PP mediating interaction
with Hsp70. Structural prediction and circular dichroism anal-
yses of the propeptide region of LOX-PP indicated that it
assembles as an intrinsically disordered protein (IDP) (44, 70),
which occurs in proteins that take on structures once assem-
bled in a complex. To begin to map the region(s) mediating
binding with Hsp70, a series of progressive C-terminal (?C1 to
?C5) and N-terminal (?N1 to ?N5) deletion mutants of
LOX-PP were constructed (Fig. 4A). Expression of the de-
letion proteins was confirmed following transfection into
HEK293T cells (Fig. 4B, bottom panel). GST or GST-tagged
proteins were pulled down with glutathione-Sepharose 4B
beads, and the binding of Hsp70 and ?-tubulin assessed by
Western blotting. Hsp70 binding was reduced when the region
between aa 78 and 100 was deleted from the C terminus or
when aa 26 to 62 were removed from the N terminus (Fig. 4B,
top panel). To further map the LOX-PP-interacting domain,
additional deletion constructs within the region of aa 25 to aa
101 were prepared and analyzed (Fig. 4C). While ?M1 and
?M2 retained a low level of interaction with Hsp70 and ?-tu-
bulin, the ?M3 LOX-PP variant, with a deletion of aa 26 to
100, failed to interact with either protein (Fig. 4D and data not
shown). Finally, to test the ability of this region for interaction,
the M1 construct, which expresses a peptide consisting of this
FIG. 1. Identification of Hsp70/72 and tubulin as LOX-PP-interacting proteins. (A) HEK293T cells were transfected with vectors expressing
either GST or GST–LOX-PP. Proteins were precipitated with glutathione-Sepharose 4B beads and resolved by SDS-PAGE, and gels were stained
with Coomassie brilliant blue R-250. The positions of the GST and GST–LOX-PP bands are indicated. The bands at approximately 52 kDa and
70 kDa were excised, subjected to in-gel digestion, and then analyzed by LC-MS-MS and identified as ?/?-tubulin (?/?-tub.) and Hsp70/72,
respectively (Table 1). Stars represent bands present in both lanes, presumably nonspecific associated proteins. Positions of molecular mass
markers are given in the left lane. (B) Lysates of HEK293T cells expressing either GST or GST–LOX-PP plated on a 35-mm dish were purified
as described for panel A using a GST pulldown assay and subjected to Western blotting with the indicated antibodies. For estimation of the
amounts of expressed proteins, 4% of each of the lysates was separated and immunoblotted (Input). (C) ZR-75 (upper panels) and Hs578T (lower
panels) breast cancer cells were transfected with expression plasmids for GST or GST–LOX-PP and the resulting lysates subjected to GST
pulldown and Western blotting as described for panel B. (D) Triton X-100 extracts of ZR-75 cells were immunoprecipitated (IP) with rabbit
anti-IgG or Novus LOX-PP antibodies, as indicated. The precipitated proteins were analyzed by Western blotting with antibodies against Hsp70
and LOX-PP. As the band of precipitated LOX-PP migrated close to that of rabbit IgG light chain, protein A-conjugated HRP was used as a
secondary “antibody” to detect immunoprecipitated LOX-PP. (E) Triton X-100 extracts of NIH 3T3 fibroblasts were immunoprecipitated with
rabbit IgG or LOX-PP antibodies (upper panels) or mouse IgG or Hsp70 antibodies (lower panels) and subjected to Western blotting with the
indicated antibodies as described for panel D.
TABLE 1. Summary of proteins identified by mass spectrometrya
70 P11142 Heat shock 70-kDa
protein 8 (HSC70)
Heat shock 70-kDa
protein 1 (Hsp70)
30 255/646 (39.5)
P0810724 164/641 (25.6)
aA summary of the analysis of the results of mass spectrometry of the two
bands excised as described for Fig. 1A is shown.
2686 SATO ET AL.MOL. CELL. BIOL.
region (aa 26 to aa 100), was prepared. The M1 peptide inter-
acted with both Hsp70 and ?-tubulin (Fig. 4E). Thus, the
domain of LOX-PP that interacts with Hsp70 and ?-tubulin is
mapping internally to aa 26 to aa 100.
LOX-PP reduces Hsp70-mediated protein folding and cell
survival. To elucidate the functional effects of LOX-PP on
Hsp70, we first measured whether LOX-PP alters Hsp70 ex-
pression. No change in either its level or its rate of decay was
seen (not shown). Next, the effects of LOX-PP on the ability of
Hsp70 to promote correct refolding were assessed using a
luciferase protein refolding assay. A cytoplasm-localized lucif-
erase (Luc-Cyto), in which the C-terminal peroxisomal local-
ization signal has been destroyed as described in Materials and
Methods, was employed. Transfected Luc-Cyto was inacti-
vated by incubating HEK293T–EV, HEK293T–LOX-PP
WT, or HEK293T–LOX-PP ?M3 cells at 45°C for 30 min.
After a subsequent 1-, 2-, or 3-h recovery period at 37°C, lucifer-
ase activity was assayed using a luminometer. A decreased ability
to promote luciferase refolding was seen over the time course
in the HEK293T–LOX-PP WT versus HEK293T-EV cells
(Fig. 5A). In contrast, the LOX-PP-?M3 deletion, which can-
not interact with Hsp70, had no effect on luciferase refolding
compared to what was found for the cells with the EV DNA.
Thus, LOX-PP decreases the ability of Hsp70 to promote pro-
tein folding, and this requires the region of association.
As another measure of the effects of LOX-PP on Hsp70
function, the ability of Hsp70 to protect cells from heat shock-
induced cell death was assessed. Specifically, we tested whether
survival of ZR-75 breast cancer cells is affected by expression
of either the LOX-PP WT or the ?M3 mutant following heat
shock at 45°C for 10 min and recovery for 24 h. Cell death was
measured by uptake of trypan blue (Fig. 5B). While only ?5%
of ZR-75 cells died with EV DNA, an approximately 5-fold
increase in cell death was seen upon expression of LOX-PP
WT protein. In contrast, the LOX-PP-?M3 mutant had no
detectable effect on survival of heat-shocked ZR-75 cells (Fig.
5B, left panels). Western blotting was performed for GST and
?-tubulin, which confirmed efficient synthesis of the GST pro-
teins and equal loading, respectively. Similarly, NF639 breast
cancer cells were found to be substantially more sensitive to
heat-shock induced cell death following ectopic expression of
LOX-PP WT protein but not LOX-PP-?M3 mutant (Fig. 5B,
right panels). Thus, LOX-PP enhanced the sensitivity of ZR-75
and NF639 breast cancer cells to heat shock. Together, these
studies indicate that LOX-PP has the ability to decrease the
chaperone function of Hsp70. The inability of the ?M3 mutant
to either prevent refolding or induce cell death suggests that
these effects are mediated by interaction of Hsp70 with the
LOX-PP associates with c-Raf, an Hsp70-interacting pro-
tein. Hsp70 has been found to interact with a diverse set of
proteins in mediating its various functions. To begin to address
the functional role of the LOX-PP interaction with Hsp70, we
tested for association of LOX-PP with Hsp70-interacting pro-
FIG. 2. The interaction between LOX-PP and Hsp70 is direct and is reduced in an ATP-dependent manner. (A) T-Rex293 cells were
transfected with a pcDNA4/TO/LOX-PP/myc-His vector expressing rLOX-PP–myc-His protein. rLOX-PP–myc-His (LOX-PP–myc-His) protein
was purified on a nickel affinity column, a sample (1 ?g) was subjected to SDS-PAGE, and the gel was stained with Coomassie blue. (B) E.
coli-expressed GST and GST-tagged Hsp70 (G-Hsp70) proteins were purified on glutathione-Sepharose 4B beads, and 1 ?g of each protein was
resolved by gel electrophoresis. Left panel, Coomassie blue-stained gel; right panel, Western blotting (WB) with a GST antibody. (C) rLOX-PP–
myc-His (0.5 ?M), shown in panel A, was incubated with GST (0.5 ?M) or GST-Hsp70 (G-Hsp70; 0.5 ?M) for 3 h, following incubation with
glutathione-Sepharose 4B beads for 90 min. The precipitated proteins and a sample of the input were analyzed by immunoblotting with antibodies
against His tag and GST. Molecular mass markers are indicated on the left. (D) HEK293T cells were transfected with expression plasmids for GST
or GST-Hsp70 (G-Hsp70) with LOX-PP-V5, and GST pulldown assays were performed as described for Fig. 1B and subjected to Western blotting
with the indicated antibodies. (E) HEK293T cells were transfected with GST, N-terminal GST-tagged LOX-PP (G–LOX-PP), or C-terminal
GST-tagged LOX-PP (LOX-PP-G). After 24 h, cell lysates were subjected to GST pulldown with glutathione-Sepharose 4B beads in the presence
(?) or absence (?) of 1 mM ATP. The coprecipitated proteins were examined by Western blotting with antibodies against Hsp70, ?-tubulin
(?-tub.), and GST. For estimation of the amounts of expressed proteins, 4% of each of the lysates was separated and immunoblotted (Input).
VOL. 31, 2011LOX-PP INTERACTION INHIBITS Hsp70 AND c-Raf FUNCTIONS 2687
teins that are mediators of signaling pathways known to be
affected by LOX-PP in breast cancer cells (Fig. 6A). Notably
GST–LOX-PP brought down c-Raf and Apaf-1 in addition to
Hsp70, whereas no association was detected with the Hsp70-
interacting proteins Akt, B-Raf, epidermal growth factor
receptor (EGFR), Erk1/2, and MEK1/2 (Fig. 6A) and cyto-
chrome c, JNK1, Src, or poly(ADP-ribose) polymerase
(PARP) (data not shown). Interestingly, we have noted that
ectopic LOX-PP expression reduced Erk1/2 activation by the
Ras/Raf pathway in NF639 breast cancer cells, which are
driven by a Her-2/neu to Ras signaling cascade, and in Ras-
transformed NIH 3T3 fibroblasts (30, 44). Thus, we sought to
test the ability of endogenous c-Raf and LOX-PP to associate
in these lines. Triton X-100-soluble lysates from NF639 and
NIH 3T3 cells were immunoprecipitated with an antibody
against either LOX-PP or Hsp70 or its isogenically matched
antibody, rabbit IgG or mouse IgG, respectively (Fig. 6B and
C). The antibody against LOX-PP brought down Hsp70 and
c-Raf; interestingly, in both lines, the pulldown of c-Raf
appears somewhat more effective than that of Hsp70 (Fig.
6B and C). The LOX-PP antibody precipitated the precur-
sor Pro-LOX, which contains the peptide on its amino-
terminal domain, and the processed LOX-PP peptide. The
antibody against Hsp70 brought down both LOX-PP and Pro-
LOX as well as c-Raf, consistent with interaction via the pro-
peptide domain. Notably, the LOX-PP produced extracellu-
larly by proteolytic cleavage of Pro-LOX, which was detected
at only low levels in the cell extracts, as judged by the input
samples, was quite readily detected in association with Hsp70
presumably in the intracellular compartment. To further assess
the domains of interaction, NF639 cells were transfected with
vectors expressing either LOX-PP, Pro-LOX, which contains
LOX-PP and the LOX enzyme domains, or the LOX enzyme
alone. The interaction with c-Raf was observed with LOX-PP
and Pro-LOX but not the enzyme (Fig. 6D). Thus, LOX-PP
associated quite effectively with c-Raf, as well as Hsp70, in
these breast cancer cells, which is consistent with our previous
observations that ectopic LOX-PP expression in the NF639
and NIH 3T3 cells inhibits the activity of Erk1/2 kinases (30,
44), which are downstream of c-Raf.
We next tested for direct interaction of LOX-PP and c-Raf,
using a GST-tagged protein (Fig. 6E, inset). Recombinant
LOX-PP–myc-His was incubated with either GST or GST–c-
Raf for 3 h, and then GST-tagged and associated proteins were
purified using glutathione-Sepharose 4B beads. The protein
bound to the resin was visualized by immunoblotting with a His
tag antibody or Coomassie blue staining (Fig. 6E, upper or
lower panel, respectively). A band at the position of full-length
LOX-PP was seen with GST-Raf but not GST alone, indicating
that LOX-PP can associate directly with c-Raf, although this
interaction was weaker than expected based on the coimmu-
noprecipitation seen in cell extracts as described above (Fig.
6B and C). This might be due to the lack of a modification(s)
on the bacterially expressed c-Raf necessary to promote its
direct interaction with the propeptide or the possibility that
Hsp70 might stabilize the interaction of c-Raf and LOX-PP.
Interaction with LOX-PP reduces Raf signaling via Erk.
The finding that LOX-PP associates with c-Raf led us to fur-
ther assess the effects of the propeptide on signaling down-
stream of this kinase. To test whether LOX-PP reduces acti-
vation of MEK1/2 by c-Raf, plasmids expressing GFP-tagged
c-Raf or control GFP were transfected into HEK293T cells in
the absence or presence of GST–LOX-PP or GST (Fig. 7A).
Resulting cell lysates from GST–LOX-PP- and c-Raf-express-
ing cells displayed reduced MEK1/2 activity compared to those
from the GST alone, consistent with inhibition of c-Raf activity
by LOX-PP. The ability of LOX-PP to inhibit Erk1/2 activity in
two additional breast cancer lines, Hs578T and ZR-75, was
assessed next. Ectopic LOX-PP expression reduced Erk1/2 ac-
tivity in both lines without affecting total Erk1/2 levels (Fig.
7B). Next, we tested whether the region comprising aa 26 to
100 of LOX-PP is necessary for its interaction with c-Raf as
well as with Hsp70. Lysates were prepared from HEK293T
cells ectopically expressing either GST, GST–LOX-PP WT,
GST–LOX-PP ?M3 (?26-100), or GST–LOX-PP M1 (26-100)
and subjected to glutathione-Sepharose 4B beads. Binding of
c-Raf to full-length LOX-PP and to LOX-PP M1 containing
only aa 26 to 100 was readily detected, whereas no binding was
seen with the GST–LOX-PP ?M3 protein, which is missing
this region, or with the GST control protein (Fig. 7C). The
mutants were next compared to WT LOX-PP for their effects
on activation of MEK1/2 by c-Raf, as described above and
shown in Fig. 7A. The GST–LOX-PP ?M3 protein was unable
to reduce c-Raf activity, as judged by levels of phospho-MEK
measured, whereas the WT and M1 proteins were effective.
FIG. 3. Mapping of the binding site for LOX-PP on Hsp70. (A) A
schematic representation of the Hsp70 mutants used in this study is
shown. Full-length (WT) or deletion mutant Hsp70 cDNAs (?BglII
and ?SmaI) were inserted into the pFLAG-CMV-2 vector. Positions
of the ATPase and peptide binding (bdg.) domains and the acidic motif
EEVD are as indicated. (B) GST or GST–LOX-PP was coexpressed
with full-length FLAG-Hsp70 (WT) or mutant FLAG-Hsp70 ?BglII
or FLAG-Hsp70 ?SmaI in HEK293T cells. GST proteins were pre-
cipitated with glutathione-Sepharose 4B beads and bound proteins
subjected to Western blotting with anti-FLAG and anti-GST antibod-
ies. For estimation of the amounts of expressed proteins, 4% of each
of the lysates was separated and immunoblotted (Input panels).
2688 SATO ET AL.MOL. CELL. BIOL.
The data from three independent experiments were quantified
using ImageJ software and the average level (? SD) relative to
the level for EV, set at 100%, for the LOX-PP ?M3 protein
was 108% ? 13%, whereas values of 74% ? 7% and 63% ?
10% were obtained for the LOX-PP WT and M1 proteins,
respectively (data not shown).
To test whether the interaction with c-Raf and Hsp70 is
necessary for the observed inhibition of the transformed phe-
notype by LOX-PP (44, 75), the effects of LOX-PP WT versus
?M3 deletion on anchorage-independent growth and migra-
tion of NF639 cells were compared. Purified bacterially ex-
pressed GST, full-length GST–LOX-PP WT, or GST–LOX-PP
?M3 proteins (Fig. 7D, bottom panel) were tested for their
effect on soft agar colony formation by NF639 cells. While
LOX-PP WT protein robustly inhibited growth of NF639 cells
in soft agar, the GST–LOX-PP ?M3 protein had no effect
compared to the GST protein alone (Fig. 7D). Migration as-
says were performed 48 h after ectopic expression of GST,
full-length GST–LOX-PP WT, or GST–LOX-PP ?M3 in
NF639 breast cancer cells (Fig. 7E). Consistent with our pre-
vious studies, an ?40% reduction was seen with expression of
full-length LOX-PP. In contrast, no reduction in NF639 cell
migration was seen with the LOX-PP ?M3 mutant (Fig. 7E).
Thus, the domain comprising aa 26 to 100, which mediates the
interaction of LOX-PP with c-Raf, is required for inhibition of
c-Raf activity and reduction in the transformed phenotype by
LOX gene knockdown or knockout induces a more trans-
formed phenotype. To determine the effects of knockdown of
the endogenous LOX-PP protein on Erk activation, two siLOX
RNAs (oligonucleotide A or oligonucleotide B) and a scram-
bled control siRNA were employed. NF639 and NIH 3T3 cells
were transfected with the siRNA at 20 nM (final concentra-
tion) and cultures incubated for 48 h. Media and isolated
whole cell extracts (WCEs) were subjected to immunoblot
analysis for LOX proteins using an antibody that recognizes
FIG. 4. LOX-PP binding to Hsp70 maps to aa 26 to 100 of LOX-PP. (A) Schematic representation of the initial LOX-PP mutants used in this
study is shown. SP, signal peptide. (B) HEK293T cells were transfected with plasmids expressing the deletion mutants indicated in panel A. GST
constructs were pulled down with glutathione-Sepharose 4B beads, and the bound proteins were detected by Western blotting with antibodies
against Hsp70, ?-tubulin (?-tub.), and GST (upper panels). For estimation of the amounts of expressed proteins, 4% of each of the lysates was
separated and immunoblotted (Input). (C) Schematic representation of additional deletion constructs of LOX-PP is shown. (D and E) HEK293T
cells were transfected with EV or plasmids expressing WT, ?M1, ?M2, and ?M3 (D) or EV, WT, ?M3, and M1 (E). GST constructs were pulled
down and bound proteins detected by Western blotting as described for panel B. (Input) Ten percent of each of the lysates was separated and
VOL. 31, 2011LOX-PP INTERACTION INHIBITS Hsp70 AND c-Raf FUNCTIONS 2689
the propeptide domain (Fig. 8A). The data confirmed the
ability of both siLOX RNAs to effectively knock down secreted
and intracellular LOX-PP and its precursor Pro-LOX, as ex-
pected. Notably, the decreases in LOX-PP levels were accom-
panied by substantial increases in active phospho-Erk in both
NF639 and NIH 3T3 cells, whereas no changes were observed
in total Erk1/2 (Fig. 8A). We next tested the effects of LOX
knockdown on the ability of NF639 cells to migrate. A robust
increase in the ability of NF639 cells to migrate was noted
upon treatment with the two siLOX RNAs compared to the
level for the control siRNA, with oligonucleotide B being
somewhat more effective (Fig. 8B, left panel). Next the effects
of the siRNAs were tested on invasion by NF639 cells. The
ability of NF639 cells to invade through Matrigel was signifi-
cantly increased upon treatment with the two siLOX RNAs
compared to the level for the control siRNA, with oligonucle-
otide B causing a larger increase (Fig. 8B, right panel).
MEFs were derived from mice null for the LOX gene, which
die perinatally (38), and from WT C57BL/6 mice and assayed
for their ability to migrate. MEFs from Lox?/?mice displayed
increased migration compared to those from the wild-type
mice (Fig. 8C). Previously, we demonstrated that LOX-PP
inhibits NF-?B activity in Ras-transformed NIH 3T3 cells (30).
Consistently, a 4.28 ? 0.94-fold increase in the activity of an
NF-?B element-driven reporter was observed in Lox?/?MEFs
compared to the level for MEFs from wild-type mice (not
shown). Thus, decreased LOX gene expression is accompanied
by a more transformed phenotype, as judged by increases in
Erk and NF-?B activities and in the ability of cells to migrate
and invade through Matrigel.
LOX gene knockdown leads to a more mitochondrial local-
ization of c-Raf. Rapp and coworkers have observed that ac-
tivation of its downstream targets Erk1/2 by c-Raf is accompa-
nied by a more mitochondrial localization of c-Raf (15). This
led us to test the effects of LOX gene knockdown on c-Raf
localization to the mitochondria. NF639 cells were treated with
20 nM (final concentration) of siLOX RNA oligonucleotide B
or with scrambled control siRNA. After 48 h of incubation,
mitochondrial proteins were isolated and subjected to immu-
noblotting for c-Raf (Fig. 9A, upper panel). Knockdown of
LOX-PP was associated with an increased localization of c-Raf
to the mitochondria. Western blotting for the mitochondrial
protein COX-1 confirmed equal loading. Similarly, the amount
of c-Raf localized in the mitochondrial fraction from the
Lox?/?MEFs was substantially higher than that from MEFs
from wild-type C57BL/6 mice (Fig. 9A, lower panel). Mito-
tracker and immunofluorescence analysis were next used to
visualize how the localization of c-Raf to the mitochondria was
FIG. 5. LOX-PP reduces Hsp70 chaperone and cell survival functions. (A) HEK293T cells were transfected with a cytoplasm-localized firefly
luciferase expression plasmid and either GST–LOX-PP WT (WT), GST–LOX-PP-?M3 (?M3), or GST (EV) using Lipofectamine 2000. After
24 h, 20 ?g/ml cycloheximide was added to prevent further protein synthesis. The cultures were incubated for 30 min at 37°C and then shifted to
45°C for 30 min to denature the luciferase protein. The cells were then incubated at 37°C to recover the folding of luciferase. At the indicated time
points, the cells were lysed, protein concentrations were measured, and luciferase activities of equal amounts of protein were determined using a
luciferase assay system (upper panels). (Lower panels) Western blotting was performed for GST and ?-tubulin, which confirmed efficient synthesis
of the GST proteins and equal loading, respectively. (B) ZR-75 and NF639 cells (left and right panels, respectively) were transfected with vectors
expressing GST–LOX-PP WT (WT), GST–LOX-PP-?M3 (?M3), or GST (EV). After 24 h, cultures were incubated for 10 min at 45°C and then
returned to 37°C for 24 h. (Upper panels) ZR-75 and NF639 cells were stained with 0.4% trypan blue and positive (dead) cells counted. The results
from 3 individual experiments (means ? SD) are shown. (Lower panels) WCEs were subjected to immunoblotting for GST and ?-tubulin, as
described for panel A.
2690SATO ET AL.MOL. CELL. BIOL.
altered by knockdown of LOX-PP in NF639 cells (Fig. 9B). In
cells treated with control siRNA, c-Raf displayed both a cyto-
plasmic and a mitochondrial localization. A substantial in-
crease in c-Raf staining in the mitochondria was detected with
siLOX RNA oligonucleotide B treatment of NF639 cells (Fig.
9B). Furthermore, the mitochondria appeared more clustered
and perinuclear than filamentous, reminiscent of the changes
observed with induction of a constitutive active c-Raf protein
in NIH 3T3 cells by Rapp and coworkers (15). Similar data
were obtained with the WT and Lox?/?MEFs (data not
shown). Thus, LOX-PP appears to reduce the localization of
c-Raf to the mitochondria and to decrease Erk signaling.
Here, a novel mechanism of action of the Ras inhibitor
protein LOX-PP that occurs via its association with Hsp70 and
c-Raf, leading to reduced Hsp70 function and c-Raf-mediated
signaling and a transformed phenotype in breast cancer cells,
was elucidated. Hsp70 was identified as a binding partner of
LOX-PP using copurification-mass spectrometry, and this in-
teraction was shown to reduce Hsp70 functional ability to pro-
mote refolding and survival from heat shock-induced death.
The domains of interaction were mapped to the peptide bind-
ing region of Hsp70 and to aa 26 to aa 100 of LOX-PP. Testing
of the known Hsp70 clients revealed that LOX-PP is also
associated with c-Raf. This identification of c-Raf is consistent
with previous observations showing that LOX-PP reduces sig-
naling downstream of c-Raf in breast, lung, pancreatic, and
prostate cancer cells, including Erk1/2 activation in growing
cells or upon stimulation with serum or basic fibroblast growth
factor (bFGF) (30, 44, 52, 73) and migration and the trans-
formed phenotype (30, 44, 45, 73). Furthermore, we now show
that knockdown or knockout of LOX-PP has the inverse ef-
fects, i.e., leads to increased cell migration and invasion, and
Erk1/2 and NF-?B activities. Knockdown of LOX-PP levels
also led to increased localization of c-Raf to the mitochondria,
which correlated with the activation of Erk1/2. These findings
are consistent with the report by Rapp and coworkers (15)
showing that active c-Raf localizes to the mitochondria and
that this localization plays an important role in activation of
Erk1/2. Thus, LOX-PP forms complexes comprising Hsp70
and c-Raf, which reduces the signaling mediated by these two
factors, which have been shown to contribute to the progres-
sion of breast and other cancer cells (2, 4, 49, 72). Overall, our
findings suggest the potential use of this peptide in treatment
of cancers driven by signaling via these pathways.
LOX-PP appears to associate directly with Hsp70 in breast
cancer cells, as judged by in vitro binding assays and coimmu-
noprecipitation analysis. Elevated Hsp70 has been detected in
many cancers, including breast cancers (6, 48), and the expres-
FIG. 6. Hsp70-interacting protein c-Raf associates with LOX-PP. (A) HEK293T cells were transfected with expression plasmids for GST or
GST–LOX-PP (G–LOX-PP) and expressed proteins purified on glutathione-Sepharose 4B beads. Bound proteins were analyzed by Western
blotting for the presence of Hsp70 and proteins known to interact with Hsp70, including c-Raf, Apaf-1, Akt, B-Raf, EGFR, Erk1/2, and MEK1/2,
and for GST as the control (pulldown). For estimation of the amounts of expressed proteins, 4% of each of the lysates was immunoblotted (Input).
(B and C) Triton X-100 extracts of NF639 cells (B) and NIH 3T3 fibroblasts (C) were immunoprecipitated with the antibodies indicated at the
top. The precipitated proteins were analyzed by Western blotting with antibodies against Hsp70, c-Raf, and LOX-PP. As the band of precipitated
LOX-PP and Pro-LOX migrated close to that of rabbit IgG light and heavy chains, respectively, protein A-conjugated HRP was used as a secondary
“antibody” to detect immunoprecipitated LOX-PP and precursor Pro-LOX. (D) NF639 cells were transfected with expression plasmid for empty
vector (EV) or V5-tagged LOX-PP, LOX, or Pro-LOX proteins. Triton X-100 extracts were immunoprecipitated with V5 antibodies and the
precipitated proteins detected with antibodies against the c-Raf or V5 tag. (E) rLOX-PP–myc-His (0.5 ?M) was incubated with GST (0.5 ?M) or
GST–c-Raf (G-c-Raf; 0.5 ?M) for 3 h and then with glutathione-Sepharose 4B beads for 90 min. The proteins bound to the resin were separated
by SDS-PAGE and visualized by immunoblotting with His tag antibody (upper panel) or Coomassie brilliant blue staining (lower panel). For
estimation of the amount of rLOX-PP–myc-His protein present, 5% of the mixture was separated and immunoblotted (Input). (Inset) Coomassie
blue-stained gel of bacterial expressed and purified ?100-kDa GST–c-Raf (1 ?g) is shown. Stars denote putative products of degradation or
incomplete synthesis. The same molecular weight markers were used, and their positions are indicated on the left.
VOL. 31, 2011LOX-PP INTERACTION INHIBITS Hsp70 AND c-Raf FUNCTIONS 2691
sion level of Hsp70 correlates with metastasis, resistance to
anticancer drugs, and poor prognosis in many human cancers
(reviewed in reference 8). Hsp70 prevents caspase-dependent
and -independent cell death triggered by apoptotic stimuli,
such as heat shock, tumor necrosis factor, serum withdrawal,
and chemotherapeutic agents. Furthermore, Hsp70 has been
shown to downregulate Jun N-terminal protein kinase (JNK),
p38 mitogen-activated protein kinase (MAPK), and caspase
and to stabilize lysosomal membrane integrity (14). Notably,
the association with LOX-PP functionally reduced the ability
of Hsp70 to promote correct folding of luciferase protein and
resulted in substantially increased cell death upon heat shock
of breast cancer cells. These findings lead us to hypothesize
that the interaction with LOX-PP compromises Hsp70 func-
tions essential for growth and survival of tumor cells.
Hsp70 family proteins are highly conserved and have three
regions mediating interaction with various proteins, including
an ATPase domain in the N-terminal region, a peptide binding
domain in the C-terminal region, and an acidic motif (EEVD)
at the C terminus. The peptide binding domain of Hsp70 has
been reported to bind to PKC?II, p53, Rictor, apoptosis-in-
ducing factor (AIF), JNK1, TRAF2, TRAF6, Ku70, and MstI
(7, 11, 16, 29, 37, 40, 54, 58, 59). Meanwhile, Hsp70 interacts
with Hip, Bag-1, Bax, hYVH1, PARP-1, CD40, and Ask1 via its
ATPase domain (3, 17, 25, 26, 36, 53, 64) and with Hop
(Hsp70/Hsp90-organizing protein) and CHIP via the EEVD
motif (1, 12). In this study, mutational analysis demonstrated
that the peptide binding domain of Hsp70 is necessary for the
interaction with LOX-PP. Interestingly LOX-PP does not in-
teract with the common Hsp70 cochaperone Hsp90 in either
breast cancer or HEK293T cells. While it is somewhat rarer to
be a sole client of Hsp70, other examples include Rb, CD40,
and TRAF6 (3, 7, 24, 28). Importantly, LOX-PP interacted
with c-Raf, an Hsp70-associating protein (65), to inhibit the
signaling via Erk1/2 that promotes a migratory phenotype in
breast cancer cells. It should be noted that we did not observe
interaction of LOX-PP with MEK1/2, Erk1/2 or B-Raf kinase,
or H-Ras (unpublished results). These results suggest that our
previous data showing that LOX-PP reduces Ras-mediated
Erk1/2 activation (30, 44, 45, 73) is due to the ability of
LOX-PP to reduce the activity of its upstream kinase c-Raf.
Since the LOX gene was originally identified as the Ras reci-
sion gene with ability to suppress Ras-mediated transformation
(10, 35), an active LOX enzyme which is lacking the propep-
tide region was found to promote an invasive phenotype in
breast cancer cells (13, 55, 56). These paradoxical results can
be explained, in part, by our findings that LOX-PP, but not the
LOX enzyme, can interact with c-Raf and reduce its activity.
Previous structure prediction studies of LOX-PP using
DISOPRED, GlobPlot and DisProt, and circular dichroism
(CD) analysis have indicated that the propeptide assembles as
an intrinsically disordered protein (44, 70). Here, the domains
of LOX-PP mediating binding with Hsp70 were mapped to aa
26 to 100. This sequence contains a proline-rich region be-
tween aa 26 and 34 and an arginine-rich region between aa 62
and 72. While prolines within a peptide sequence disrupt or-
dered secondary structures, proline-rich sequences are impor-
tant for mediating interactions with several protein-protein
interaction domains, including the Src homology 3 (SH3), Ena/
VASP homology 1 (EVH1), and WW domains (74). Indeed, in
FIG. 7. The region comprising aa 26 to 100 of LOX-PP that inter-
acts with Hsp70 is necessary for inhibition of c-Raf kinase and the
transformed phenotype. (A) HEK293T cells were transfected with the
plasmids expressing GFP-tagged c-Raf (Raf) or the GFP (G) control
in the presence of GST–LOX-PP (PP) or the GST (G) control, as
indicated. Cell lysates (20 ?g) were analyzed for the effects on the
activity of the c-Raf downstream mediator MEK1/2 by Western blot-
ting using anti-phospho-MEK1/2 (Ser-217/221) and for total MEK1/2
as well as for GFP, GST, and ?-actin as loading controls. (B) Hs578T
and ZR-75 cells were transfected with plasmids expressing V5-tagged
LOX-PP or EV DNA. Samples of WCEs (20 ?g protein) were sub-
jected to immunoblotting using antibodies against phospho-Erk1/2
(Thr-202/Tyr-204), total Erk1/2, the V5 tag, and ?-actin. (C) HEK293T
cells were transfected with the expression plasmids for the indicated
GST-tagged LOX-PP proteins (WT, ?M3, or M1) or GST alone (EV).
Proteins were purified using glutathione-Sepharose 4B beads and sub-
jected to Western blotting for c-Raf and GST. For estimation of the
amounts of expressed proteins, 4% of each of the lysates (Input) was
immunoblotted. (D) Bacterially expressed GST, GST–LOX-PP WT,
or GST–LOX-PP ?M3 proteins were purified (bottom panel) and
their effects on the ability of NF639 cells to form colonies after 2
weeks of growth in soft agar assessed. Plates were photographed at
?4 magnification. (E) GST (EV), GST–LOX-PP WT, or GST–
LOX-PP ?M3 was ectopically expressed in NF639 breast cancer
cells. After 48 h, cells were subjected to a migration assay for 16 h
in triplicate, and cells that migrated to the lower side of the filter
were stained with crystal violet and quantified by spectrometric
determination at A570. Averages ? SD are given. P values were
calculated using Student’s t test.*, P ? 0.01.
2692 SATO ET AL.MOL. CELL. BIOL.
more recent LOX-PP copurification-mass spectrometry analy-
sis of ZR-75 human breast cancer cells, an adaptor protein
containing an SH3 domain was identified as a novel LOX-PP-
interacting protein (S. Sato, unpublished observations), and
work is in progress to test for their association. Arginine is
known to be an electrically positively charged amino acid and
prefers to be on the outside of the proteins. Consistently,
hydropathy plot analysis predicts that this region of LOX-PP is
on the surface. Lastly, the carboxy-terminally truncated construct
of LOX-PP appeared to exhibit stronger binding to Hsp70 and
tubulin than full-length LOX-PP. This finding suggests that aa
116 to 162 in the carboxy-terminal region may contain a domain
that suppresses the interaction with Hsp70 and tubulin. Further
study is required to address this possibility.
Previous work implicated the carboxy-terminal region of
LOX-PP in productive secretion of Pro-LOX. Specifically, Ka-
gan and coworkers (31) established that the amino terminus of
the propeptide region of Pro-LOX contains a signal sequence
essential for normal secretion. Sommer and Mecham and their
collaborators found that Pro-LOX mutants that lack aa 23 to
157 were not secreted (62, 67). These findings suggest that the
carboxy-terminal region of the propeptide plays a critical role
in Pro-LOX secretion in addition to the signal peptide. To test
this possibility, we analyzed the localization of WT and M1
LOX-PP in breast cancer cells by immunofluorescence analy-
sis. While WT LOX-PP localized to the Golgi apparatus in
Hs578T cells, as judged by colocalization with Golgi marker
protein, consistent with previous work using osteoblasts (19),
FIG. 8. Knockdown of LOX gene expression enhances Erk signaling and cell migration and invasion. (A) NF639 and NIH 3T3 cells were
transfected with 20 nM (each) siLOX RNAs oligonucleotide A or oligonucleotide B or scrambled control siRNA for 48 h. Samples of medium (20
?l) and WCEs (20 ?g) were subjected to immunoblot analysis for LOX using an antibody that recognizes the propeptide domain, pErk1/2, total
Erk1/2, and ?-actin. (B) NF639 cells were transfected as described for panel A. (Left panel) After 48 h, cells were subjected to a migration assay
for 6 h in triplicate as described for Fig. 7E. Averages ? SD are given. P values were calculated using Student’s t test.*, P ? 0.01. (Right panel)
After 48 h, cells were subjected to an invasion assay for 6 h in triplicate as described in Materials and Methods. Averages ? SD are given. P values
were calculated using Student’s t test.*, P ? 0.01. (C) WT or Lox?/?MEFs were subjected to a migration assay for 16 h in triplicate and the data
quantified as described for Fig. 7E. Averages ? SD are given. P values were calculated using Student’s t test.*, P ? 0.01.
FIG. 9. Knockdown of LOX gene expression enhances targeting of c-Raf to the mitochondria. (A) NF639 cells treated with control siRNA or
siLOX oligonucleotide B RNA for 48 h (upper panels). The mitochondrial fractions (10 ?g) were subjected to 10% SDS-PAGE and analyzed by
immunoblotting for c-Raf and COX-1. The mitochondrial fraction (10 ?g) of WT or Lox?/?MEFs was subjected to immunoblotting for c-Raf
and COX-1, as described above (lower panels). (B) NF639 cells were transfected as described for panel A. After 48 h, cells were incubated
with 250 nM Mitotracker for 30 min, fixed with methanol, and processed for immunofluorescence analysis as described in Materials and
Methods. Bars: 5 ?m.
VOL. 31, 2011 LOX-PP INTERACTION INHIBITS Hsp70 AND c-Raf FUNCTIONS2693
the M1 LOX-PP mutant localized throughout the cytosol and
failed to associate with the Golgi apparatus (data not shown).
Taken together, these findings suggest that the c-Raf suppres-
sor domain of LOX-PP maps to the region of aa 26 to 100 and
that the carboxy-terminal region is required for proper lo-
calization and correct processing. In this regard, it is known
that glycosylation occurs within the Golgi apparatus during
secretion (50), and it has been reported that LOX-PP is
posttranslationally modified by N-glycosylation and O-gly-
cosylation (68, 70).
Here, tubulins were identified as additional binding proteins
of LOX-PP in breast cancer and HEK293T cells. Notably,
tubulin was not an intermediate protein for the association of
Hsp70 with LOX-PP. This finding suggests that the tubulin–
LOX-PP and Hsp70–LOX-PP complexes are independent of
each other. Interestingly, the colocalization and association of
LOX-PP with tubulin in the microtubules of differentiated
osteoblasts were observed previously using confocal micros-
copy and overlay binding analysis (19). The microtubule net-
work plays important roles in the control of cell cycle progres-
sion, cell movement, vesicle transport, and signal transduction
(34, 69, 71). It will be important to determine the effects of
LOX-PP association on these diverse functions.
Ectopic LOX-PP expression was seen to enhance cell death
of breast cancer cells upon heat shock. Recently, we reported
that LOX-PP sensitizes pancreatic and breast cancer cells to
doxorubicin-induced apoptosis, which is caspase dependent
(46). Interestingly, we observed that Apaf-1 was also copre-
cipitated with LOX-PP. Hsp70 has been shown to block Apaf-
1/cytochrome c-mediated caspase activation (61), suggesting
that LOX-PP may promote apoptosis via overriding the inhi-
bition of Apaf-1 protease activity mediated by its interaction
with Hsp70 protein. In summary, LOX-PP has been shown to
interact with Hsp70 and c-Raf and thereby to reduce chaper-
one function and signaling via Erk1/2 that promote survival
and a more transformed phenotype in breast cancer cells. It
will be important to test whether this association can be ex-
tended to other cancers driven by Ras signaling.
We gratefully acknowledge Bruce Mayer, Linda Van Aelst, Michael
Sherman, and Vladimir Gabai for providing cloned DNAs and Phil
Leder and Amitha Palamakumbura for the NF639 and NIH 3T3 cell
These studies were supported by Public Health Service grants CA-
082742, CA-129129, and CA-143108 from the National Cancer Insti-
tute, Academy of Finland grant 202469, and the Sigrid Juse ´lius Foun-
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