代写dissertation HOPS: a novel cAMP-dependent shuttling protein involved in protein synthesis regulation
Maria Agnese
Introduction
The liver has the capacity to restore damaged or lost liver cellmass by cell proliferation (Bucher, 1963). The process of liverregeneration has been studied extensively in a rat model using70% partial hepatectomy (PH), first described by Higginsand Anderson (Higgins and Anderson, 1931). After PH the
remaining differentiated liver cells re-enter the cell cyclethrough transition from the quiescent G0 phase to the G1 phase(priming phase) followed by DNA synthesis and G2-M phases.The initial rounds of cell division are synchronous and the firstDNA replication occurs within 24 or 38 hours after surgery in
rat and mouse, respectively (Fausto, 2000). After one or tworeplication rounds the hepatocytes return to the quiescent state.The regeneration process is completed within 10 to 12 days
(Bucher, 1963). Hormones, growth factors, cytokines and theircoupled signal transduction pathways have been implicated ingoverning hepatocyte proliferation, but the precise orchestrationof these factors is still poorly understood (Cressman et al., 1996;Yamada et al., 1997; Michalopoulos and DeFrances, 1997;
Fausto et al., 1995; Fausto, 2001). The increase of cellularcAMP, observed in the first hours after PH, is followed by amodification of protein kinase A expression (Ekanger et al.,
1989; Diehl and Rai, 1996; Servillo et al., 2001; Servillo et al.,
2002). A direct role for cAMP-responsive transcription inproliferation after PH has been established (Della Fazia et al.,
1997). It has been shown that inducible cAMP early repressor(ICER) expression is strongly induced upon PH (Servillo et al.,
1997) and that cAMP responsive element modulator (CREM)plays a critical role in the proliferative process through analysisof liver regeneration in CREM knockout mice (Servillo et al.,1998). Lack of CREM results in reduced proliferation andderegulation of the residual hepatocyte cell cycle. Studies on
CREM knockout mice revealed delayed expression of variouscyclin genes (Servillo et al., 1998).Hepatocytes are resting cells in G0 phase that after PH gothrough G1 phase and progress to the cell cycle. The process
that allows hepatocytes to pass from G0 to G1 phase has beendescribed as priming. Growth factors such as hepatocytegrowth factor (HGF), transforming growth factor-α (TGF-α)and epidermal growth factor (EGF) act primarily onhepatocytes in liver regeneration (Fausto, 2000). Manyresearchers have shown that liver regeneration is a multistepprocess and that priming is necessary for hepatocyteproliferation. HGF, TGF-α and EGF alter gene expression inresidual hepatocytes and in the subsequent proliferative steps.
Following priming, two major events occur in residualhepatocytes after PH, activation of several genes in response
to proliferation and restoration of the quiescent state ofhepatocytes by activated genes.
To describe the mechanisms involved in the proliferativeresponse, we focussed on the molecular changes occurring
during liver regeneration following PH throughcharacterization of novel genes activated in residual
hepatocytes. A cDNA library was constructed with mRNAsderived from residual hepatocytes at different times following
PH. We performed a rat regenerating liver cDNA library
3185
The liver has the ability to autonomously regulate growth
and mass. Following partial hepatectomy, hormones,
growth factors, cytokines and their coupled signal
transduction pathways have been implicated in hepatocyte
proliferation. To understand the mechanisms responsible
for the proliferative response, we studied liver regeneration
by characterization of novel genes that are activated in
residual hepatocytes. A regenerating liver cDNA library
screening was performed with cDNA-subtracted probes
derived from regenerating and normal liver. Here, we
describe the biology of Hops (for hepatocyte odd protein
shuttling). HOPS is a novel shuttling protein that contains
an ubiquitin-like domain, a putative NES and a prolinerich
region. HOPS is rapidly exported from the nucleus and
is overexpressed during liver regeneration. Evidence shows
that cAMP governs HOPS export in hepatocytes of normal
and regenerating liver and is mediated via CRM-1. We
demonstrate that HOPS binds to elongation factor eEF-1A
and interferes in protein synthesis. HOPS overexpression
in H-35-hepatoma and 3T3-NIH cells strongly reduces
proliferation.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/118/14/3185/DC1
Key words: shuttling protein, proliferation, liver, eEF-1A, hepatoma
cells, liver regeneration
Summary
HOPS: a novel cAMP-dependent shuttling protein
involved in protein synthesis regulation
Maria Agnese Della Fazia, Marilena Castelli, Daniela Bartoli, Stefania Pieroni, Valentina Pettirossi,
Danilo Piobbico, Mariapia Viola-Magni and Giuseppe Servillo*
Department of Clinical and Experimental Medicine, University of Perugia, Policlinico Monteluce, 06122 Perugia, Italy
*Author for correspondence (e-mail: [email protected])
Accepted 21 April 2005
Journal of Cell Science 118, 3185-3194 Published by The Company of Biologists 2005
doi:10.1242/jcs.02452
Research Article
Journal of Cell Science
3186
screening with cDNA-subtracted probes derived from rat
regenerating liver cDNAs (2-18 hours after PH) and rat normal
liver mRNAs. Screening allowed us to isolate up to 40 genes.
All isolated genes were upregulated in the liver after PH and
in hepatoma cells. One of these novel genes expressed in liver
regeneration has been characterized previously (Della Fazia et
al., 2002). This gene Lal-1 is involved during liver regeneration
and in the proliferative process. At present our attention has
been drawn to another of these genes that we named hepatocyte
odd protein shuttling (Hops).
In this paper, we demonstrate that HOPS is a novel shuttling
protein, which via CRM-1 (Fornerod et al., 1997), actively
directs proliferation of cells controlling protein synthesis.
Evidence is provided that cAMP governs HOPS export in
hepatocytes of normal and regenerating liver. Following
PH, HOPS is rapidly exported from the nucleus and is
overexpressed during liver regeneration. It has been established
that HOPS binds to elongation factor EF-1A and interferes
with protein synthesis. Overexpression of HOPS in H-35
hepatoma cells and 3T3-NIH cells strongly reduces cell
proliferation.
Materials and Methods
Cell cultures
H-35 rat hepatoma and COS-1 cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal calf
serum (FCS; GIBCO Invitrogen) (Taub et al., 1987). NIH-3T3 cells
were cultured in DMEM supplemented with BCS (GIBCO
Invitrogen). The Phoenix cells were cultured in DMEM supplemented
with FBS (GIBCO Invitrogen).
Animals
Experiments were performed on 3-month-old male animals: Sprague-
Dawley rats and SVJ-129 mice. The animals were purchased from
Harlan-Nossan and received human care according to NIH guidelines.
Animals were maintained in a 12 hours :12 hours light:dark cycle with
food and water ad libitum. Liver resection was performed between 8
a.m. and 12 a.m. removing about 70% of the liver mass (Higgins and
Anderson, 1931). As control, sham operation by transverse abdominal
incision followed by digital manipulation of the liver was performed.
Rats were sacrificed at 2, 5, 8, 12, 18, 24, 48 and 72 hours after PH.
Mice were sacrificed at 15, 30 minutes, 1, 2, 5, 8, 12, 18, 36, 38, 48,
60 and 72 hours after PH. Four animals per experimental group were
used for each time point and livers were pooled prior to analysis.
RNA analysis
Total RNA extraction was performed and analyzed by northern blot
analysis as described previously (Sambrook et al., 1989; Della Fazia
et al., 1992).
Cell cycle synchronization and stable clones cells
H-35 cell cycle was synchronized at the G0/G1 boundary in serum-free
medium and in G1/S phases by the double thymidine method. Briefly,
H-35 were cultured in the absence of serum, arrested for 72 hours and
released in fresh culture medium in the presence of serum. The time
point at which H-35 received serum after starvation was considered
time 0. The double thymidine method to arrest the cells at the G1/S
boundary was performed as described previously (Crosio et al., 2002).
The time point, corresponding to the G1/S transition, was considered
time 0. The percentage of H-35 cells, labeled with propidium iodide,
was determined at different phases of the cell cycle by flow cytometry
(FACS analysis; Becton Dickinson FACStar Plus flow cytometer).
H-35 stable cell clones overexpressing HOPS were generated by
transfection with pcDNA3 vector containing full-length Hops and by
selection in culture medium with geneticin (0.4 mg/ml). H-35 stable
cell clones generated by transfection with empty pcDNA3 vector were
used as control. Thymidine incorporation in H-35 cells and H-35
stable cell clones was performed as described previously (Della Fazia
et al., 2001).
Retroviral vector production and cell infection
pBabe-puro, MuLV-based retroviral vector, was used to transduce the
Hops gene (Morgenstern et al., 1990). Wild-type Hops cDNA was
inserted into pBabe-puro vector to produce pBabe-Hops.
Phoenix cells were plated at 1.5106 per plate 2 days before
transfection. For each transfection, 2 μg of pBabe-puro or pBabe-
Hops plasmid were used. Viral supernatants were concentrated and
collected 48 hours after transfection according to Nolan laboratory
protocol (www.stanford.edu/group/nolan/tutorials). Culture
supernatants containing retroviral vectors were then added to NIH-
3T3 with polybrene (8 μg/ml) (Sigma). Cells were cultured for 24
hours and selected in the same medium containing puromycin (2
μg/ml) for 4 days. Resistant cells (1104) were plated and counted
every 2 days for 10 days after selection.
Hops gene isolation
Hops gene isolation from a regenerating liver library was performed
using a subtracted probes procedure. A number of positive clones
were isolated and tested by northern blot analysis of the time course
of RNA extracted at different times following PH. Selected cDNAs
overexpressed during liver regeneration were isolated and sequenced.
Hops DNA sequence and putative protein prediction procedures have
been previously described (Della Fazia et al., 2002).
Antibody production
/Polyclonal anti-HOPS was generated in our laboratory by immunizing
rabbits with KHL-coupled peptide (H T T E S T D P L P Q S S G T
T T P A Q P S E) corresponding to N-terminal sequence (aa 32-54)
of the mouse HOPS protein. The serum of two rabbits was collected
and immunopurified on a column Sulfo-Link coupling gel (Pierce)
where the specific peptide had been immobilized. To test the
specificity of the antibody, a quenching test was performed with
different amounts of specific peptide in western blot and in
immunohistochemistry analyses (Della Fazia et al., 2002).
Western analyses
Protein extracts were resolved by standard SDS-polyacrylamide gel
electrophoresis from total liver and H-35 hepatoma rat cells. Liver and
cells were minced immediately in RIPA buffer. Each sample (50 μg)
was separated by gel electrophoresis and blotted onto a nitrocellulose
membrane (Schleicher and Schuell). The blots were incubated with
rabbit anti-HOPS polyclonal antibody and with rabbit anti-CREB
polyclonal antibody (Cell Signaling Technology) the signals were
detected using an ECL kit (Amersham Pharmacia Biotech).
Histological and immunofluorescence analyses
The livers were embedded into OCT compound for cryosectioning.
Sections (7 μm) were cut and mounted on slides. For histological
analysis, sections were stained with Hematoxylin-Eosin. Slides were
blocked with 3% BSA and then incubated with specific primary
polyclonal anti-HOPS antibody. After three washes in PBS, slides
were incubated with anti-rabbit Cy-3-conjugated secondary antibody
Journal of Cell Science 118 (14)
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HOPS: a novel shuttling protein 3187
in 3% BSA. DAPI was added at the final concentration of 10 ng/ml.
Images were captured with a Zeiss Axioplan fluorescence microscope
controlled by a Spot-2 cooled camera (Diagnostic Instruments) with
a 40 objective lens.
H-35 leptomycin B treatment
H-35 hepatoma cells were pretreated for 30 minutes with 10 ng/ml of
cycloheximide (CHX; Sigma). The cells were then treated for an
additional 6 hours with 20 ng/ml LMB. After CHX treatment PBS
was added to the control cell population. At the end of treatment the
cells were fixed in 4% paraformaldehyde and hybridized with specific
anti-HOPS antibody overnight. The cells were examined using a
fluorescence microscope with a standard filter for red fluorescence.
Six random fields of cells stained for HOPS in the presence or absence
of LMB were counted. The image was captured with a Zeiss Axioplan
fluorescence microscope controlled by Spot-2 cooled camera
(Diagnostic Instruments). Images were saved as TIFF-files.
Two-hybrid screening and analysis
Hops full-length cDNA was cloned into yeast expression
vector pGBKT7. The pGBKT7-Hops plasmid was
transformed into yeast strain AH109. Two-hybrid
screening was carried out according to the manufacturer’s
protocol (Clontech) using a VP-16 DNA activation
domain fusion library in an E9.5-12.5 mouse embryo
cDNA library in the vector pASV3 (Le Douarin et al.,
1995). The transformants were plated onto appropriate
selective medium supplemented with 25 mM 3-aminotriazole.
β-gal assays were performed on isolated clones
and carried out in Y190 yeast strain. The results reported
are in Miller units and are the means of triplicate
measurements performed using three distinct
transformations. The plasmids extracted by lysing cells
with acid-washed beads were electroporated in E. coli
bacterial strain HB101 and then plated onto M9 (–Leu)
plates (Vojtek et al., 1993). The isolated clones were
sequenced using the Sanger method.
Immunoprecipitation
H-35 cells were harvested, washed and resuspended in
lysis buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 2%
Triton X-100) supplemented with protease inhibitor
cocktail and 1 mM phenylmethanesulfonyl fluoride
(PMSF). Immunoprecipitation was carried out as
described (Bardoni et al., 1999) using the anti-eEF-1A
(Upstate Biotech). The proteins bound to the beads were
separated by electrophoresis on 10-12% SDS-PAGE and
visualized by immunoblot using the anti-HOPS antibody.
At the same time, the cell lysate was coimmunoprecipitated
using anti-HOPS and detected with
anti-eEF-1A.
HOPS recombinant protein: production and
purification in bacteria
The HOPS gene was cloned in pET-14Tb expression
vector (Novagen). The resulting gene was expressed in
BL21 (DE3) cells (Novagen) and after induction with 0.5
mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for
24 hours at 25°C the recombinant protein was purified
under denaturing conditions using His-Bind affinity
chromatography. Expression and purification were
carried out according to the manufacturer’s protocol
(Novagen).
In vitro translation inhibition assay
An aliquot (1 μg) of luciferase cDNA (Promega) was added to 100 μl
of a rabbit reticulocyte lysate in vitro translation reaction (Promega)
in the presence of HOPS or GST purified recombinant proteins at
different concentrations: 60, 120, 240, 360 and 420 nM. The reaction
mixture was incubated at 30°C for 2 hours. Newly synthesized 35Slabeled
luciferase protein was analyzed by 10% SDS-PAGE and
results were quantified using densitometry analysis with the Scion
Image 4.0 program. Western blot analysis was performed using antieEF-
1A as control.
Results
HOPS is a novel shuttling protein
We have cloned and characterized a novel gene named Hops.
A cDNA library was constructed with mRNA isolated from
residual hepatocytes at different times after the G0/G1 to S
Fig. 1. The Hops gene. (A) Northern blot analysis of Hops mRNA extracted
from rat at different times following PH (h PH). Normal liver (NL) was used as a
control. rRNA of the same samples were used as a loading control. (B) Protein
sequence of HOPS. The ubiquitin-like domain (102-175) is underlined; the
proline-rich domain (176-183) is in bold; the putative NES is boxed.
(C) Schematic representation of HOPS. The characteristic domains of the amino
acid sequence are indicated. (D) Western blot analysis of HOPS during liver
regeneration in mouse using a specific antibody. NL and liver after PH (minutes
and hours PH). Anti-CREB was used as a loading control.
Journal of Cell Science
3188
phase transition following PH.
Screening of the rat regenerating liver
cDNA library was performed using a
subtracted probe derived from mRNA
of normal liver and cDNA of
regenerating liver that enabled us to
isolate up to 40 novel genes. All
isolated genes were tested by
northern blot analysis to verify
expression at different times
following PH. Our interest was
directed to one of the isolated
genes overexpressed during liver
regeneration, Hops. The HOPS
nucleotide sequence and the deduced
amino acid sequence were
determined. Northern blot analysis
showed that Hops mRNA was
expressed in rat regenerating liver
and the transcript length was
estimated. Hops mRNA expression,
almost undetectable in normal liver,
presented a specific band of 1.5 kb
corresponding in size to the Hops
transcript. Liver mRNAs were
evaluated at different times during
liver regeneration and in shamoperated
rats. Furthermore, induction
of gene expression was detected in
the first hours of liver regeneration
after PH (Fig. 1A). No change in
Hops gene expression from shamoperated
animal mRNA was detected
(data not shown). HOPS protein is
composed of an ubiquitin-like
domain (102-175 aa), a proline rich
region (176-183 aa) and three leucine
rich α-helixes: 13-32, 190-212, 197-
209 (Fig. 1B,C). The rat, mouse and
human HOPS genes were cloned
and the nucleotide sequences have
been submitted to the NCBI
database under accession numbers
AY603378, AY603379 and
AY603380, respectively. The rat and
mouse HOPS amino acid sequences
showed 96% identity and 97% similarity. Human HOPS
showed 90% identity with respect to mouse and rat (data not
shown). The amino acid identity in the C-terminal of HOPS in
the three species was about 97% (data not shown). Rat Hops
spans over 1381 bp with a +1 ATG sequence at 132 bp showing
an ORF of 735 bp (Fig. 1C).
HOPS expression is modulated during liver regeneration
To investigate HOPS expression and validate the results
obtained in differential screening, we analyzed HOPS
expression by western blot and immunolocalization analyses in
mouse normal liver and regenerating liver. The specific anti-
HOPS antibody was raised in rabbit against a peptide localized
in the N terminus of HOPS. Transfection of CMV-Hops in
COS-1 cells detected two specific bands of different molecular
masses: 27 kDa corresponding to full-length Hops and an
additional band of 24 kDa (see Fig. S1A in supplementary
material). Western blot analysis performed on mouse normal
liver showed an appreciable single band of 27 kDa
corresponding to the translation of full-length Hops cDNA.
During mouse liver regeneration HOPS expression gradually
increased with a peak at 48 hours after PH in concurrence with
the first mitotic wave of residual hepatocytes. In the following
hours protein expression progressively decreased. The
additional band of different molecular mass, 24 kDa, was
detected 8 hours after PH (Fig. 1D). This additional band was
associated with post-translation modifications that affected
HOPS at the C-terminal of the protein. A fusion protein, HOPSGFP,
expressed in cells, with GFP placed at the C terminus of
Journal of Cell Science 118 (14)
Fig. 2. Immunohistological analysis of HOPS in normal (NL) and regenerating liver at different
times following PH (30 minutes to 72 hours) using (left column) anti-HOPS (original
magnification 400 and inset, higher magnification, 1000). (Middle column) DAPI staining of
the same slide. (Right column) Merged images of anti-HOPS and DAPI. Scale bars: 20 μm.
Journal of Cell Science
HOPS: a novel shuttling protein 3189
HOPS, showed release of GFP from HOPS. Western blot
analysis with anti-GFP revealed a specific cleavage in the C
terminus of HOPS (Fig. S1 in supplementary material).
HOPS is differently localized in normal and regenerating
hepatocytes
Following western blot analysis where overexpression
of HOPS was detected after PH, we tested HOPS
immunolocalization in histological specimens of normal and
regenerating mouse liver. In normal hepatocytes, HOPS was
localized mainly in the nucleus with a small amount detectable
in the cytoplasm (Fig. 2). At 30 minutes after PH in the residual
hepatocytes, HOPS migrated to the cytoplasm. This
phenomenon was observed until 8 hours after PH. At 12 hours
the shuttling protein returned progressively to the nucleus and
at 48 hours, in concurrence with the first mitotic wave of the
residual hepatocytes, HOPS was strongly expressed in the
nucleus. At 72 hours after PH, in synchronization with a
second mild wave of proliferation, a slow migration pattern of
HOPS was detected in cytoplasm (Fig. 2).
Different HOPS expression and intracellular localization
in proliferating cells
Following the results obtained in vivo in liver regeneration,
the role of HOPS in H-35 rat hepatoma cells was examined.
HOPS western blot analysis in H-35 proliferating cells showed
two distinct bands, at 27 and 24 kDa (Fig. 3A). To investigate
HOPS expression at different stages of the cell cycle, H-35
cells were synchronized using the double thymidine arrest
technique. As demonstrated by flow cytometry, highly
synchronized cells were obtained. Thymidine inhibited DNA
synthesis and arrested cells on the G1/S border. At the end of
the double thymidine arrest (time 0), 90-92% of hepatoma
cells arrested in G1/S phase (Fig. 3A).
Protein extracts from H-35 synchronized cells were
collected at different times after release from the double
thymidine block. HOPS was strongly expressed at time 0 in
relation to the arrest of the cell cycle of hepatoma cells and
down-regulated rapidly after release (Fig. 3A). Similar results
were obtained by blocking the cell cycle of H-35 cells in G0/G1
by serum deprivation. During cell starvation the progressive
increase of HOPS expression was analyzed. When the cell
cycle was arrested, at the end of 72 hours of serum deprivation,
HOPS was overexpressed in the starved cells. Addition of
serum to the culture medium down-regulated HOPS expression
in the cells (Fig. 3A).
In light of these results, we investigated a possible change
of HOPS localization in relation to the cell cycle. The
progressive starvation of H-35 cells, following serum
deprivation, allowed us to study the redistribution of HOPS
localization during cell cycle arrest. HOPS was diffused
in the nucleus and cytoplasm of wild-type hepatoma cells.
Fig. 3. HOPS expression in
H-35 hepatoma cells.
(A) Western blot analysis of
HOPS in (Top) H-35 hepatoma
cells arrested by double
thymidine excess. Time 0: the
cell at the end of treatment; 2-
12 h: hours after addition of
serum and release from cell
cycle arrest. (Bottom) Western
blot analysis of HOPS at
different times after serum
deprivation in H-35 hepatoma
cells. 0-72 h: hours of serum
deprivation; +1, +2 h: hours
after addition of serum to cells.
(B) An aliquot of H-35 cells
was treated with propidium
iodide and assayed by
FACScan analysis to evaluate
the cell cycle. (Top) H-35 cells
not treated; (middle) time 0:
H-35 cells following treatment
with double thymidine;
(bottom) 72 sd: H-35 cells
starving for 72 hours following
serum deprivation (sd).
(C) Immunolocalization of
HOPS in H-35 cells at
different times (12-72 hours)
after serum deprivation (12-72
hours) and 2 hours after
addition of serum (+2 hours).
Scale bars: 20 μm.
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3190
The protein progressively migrated to the nucleus 12 hours
after serum deprivation. In the following hours, cells
progressively stopped proliferating and HOPS accumulated
in the nuclei. At 72 hours after serum deprivation, when
almost 90% of the cells were in G0/G1 phase (Fig. 3B) and
HOPS was overexpressed, the protein was localized mainly
in the nucleus. Addition of serum to the culture medium
caused a rapid increase in cell proliferation, as demonstrated
by flow cytometry, and redistribution of HOPS in the cells
(Fig. 3C).
HOPS is a shuttling protein
Rapid HOPS export from the nucleus to the cytoplasm in
proliferating hepatocytes raised the question of whether there
are agents that could affect shuttling. During liver regeneration
different factors act on residual hepatocytes to modify
expression and induce proliferation. In particular, our attention
focused on two factors that act rapidly on residual hepatocytes
following PH, EGF and cAMP. The effects of EGF and cAMP
on HOPS shuttling in the liver were analyzed. In EGF-treated
mice a small amount of HOPS migrated into the cytoplasm of
hepatocytes at 30 minutes after treatment and almost all protein
returned to the nucleus after 60 minutes. At 90 minutes HOPS
was detected again in the nucleus (data not shown). In cAMPtreated
mice the shuttling protein was detected in the nucleus
at 15 and 30 minutes after treatment and migrated in part into
the cytoplasm. At 60 minutes HOPS was exported completely
into the cytoplasm. At 90 and 120 minutes HOPS returned
progressively to the nucleus, showing a distribution pattern
similar to normal hepatocytes (Fig. 4).
Analysis of the HOPS amino acid sequence revealed the
motif LACLLVLALA in the N-terminal region, a typical
nuclear export signal (NES) region, present in proteins that are
exported from the nucleus to the
cytoplasm via CRM-1 (Fig. 1B and Fig.
5A) (Fornerod et al., 1997; Fukuda et al.,
1997; Macara, 2001).
To assess the export of HOPS via CRM-
1, we investigated HOPS localization in H-
35 hepatoma cells following treatment
with leptomycin B (LMB), a specific
inhibitor of nuclear export mediated by
leucine-rich NES (Wolff et al., 1997).
Nuclear export inhibition revealed
enrichment of HOPS in the nucleus
compared with the control, demonstrating
that HOPS export acts via CRM-1 (Fig.
5B,C).
Binding specificity between HOPS
and eEF-1A
To gain further insight into the role of
HOPS and its shuttling function,
screenings were performed using the twohybrid
system in yeast; HOPS was used as
bait to identify proteins that specifically
bind it. Positive yeast clones were isolated
from a cDNA library of total E9.5-12.5
embryos in selected medium during the
two-hybrid system screening and all were
positive for β-galactosidase activity. In
addition, there was no β-galactosidase
activity in the yeast strain transformed
with the empty vector (pASV3; Fig. 6A).
Our attention was drawn to one of the
isolated clones that is present in more
than 10% of all positive yeast clones
characterized. The clone displays high β-
galactosidase activity and its sequence
corresponds to eukaryotic elongation
factor-1A (eEF-1A) (Fig. 6B).
Immunoprecipitation studies conducted in
endogenous protein on H-35 hepatoma cell
lines and in liver confirmed the native
interaction of HOPS with eEF-1A. Protein
extracts were immunoprecipitated using
Journal of Cell Science 118 (14)
Fig. 4. HOPS localization in liver following cAMP induction. (Left colum) Localization of
HOPS (anti-HOPS) in normal liver and at 15, 30, 60, 90 and 120 minutes following
intraperitoneal injection of cAMP. (Middle column) DAPI staining of the same slides at
different times following cAMP injection. (Right column) Merged images of anti-HOPS
and DAPI. Scale bars: 20 μm.
Journal of Cell Science
HOPS: a novel shuttling protein 3191
polyclonal anti-HOPS followed by immunoblotting with
the monoclonal anti-eEF-1A antibody (Fig. 6C). Similarly,
immunoprecipitated protein extracts were tested with
monoclonal anti-eIF2A antibody as control (Fig. 6C). The
results confirmed the in vivo interaction between HOPS and
eEF-1A.
High HOPS levels inhibit in vitro translation
Following immunoprecipitation studies on binding specificity
between HOPS and eEF-1A, the possibility of a significant role
for this interaction was analyzed. Because eEF-1A is essential
in protein synthesis in peptide chain elongation, in vitro protein
synthesis levels were evaluated in the presence of different
HOPS recombinant protein concentrations. In vitro
transcription and translation experiments were performed
using luciferase cDNA as the reporter gene. The amount of
luciferase protein was evaluated in the presence and absence
of HOPS recombinant protein. HOPS recombinant protein was
added to the in vitro translation (Fig. 7A) at different
concentrations (60, 120, 240, 360 and 420 nM). There was a
slight increase in protein synthesis when 60 nM of recombinant
HOPS was added while 120 nM had no effect. Surprisingly,
recombinant HOPS at 240 nM reduced protein synthesis to
almost 50% (Fig. 7A). No synthesis of luciferase was detected
Fig. 5. Effect of leptomycin B (LMB) on HOPS shuttling.
(A) Comparison of NES sequence in HOPS and other proteins.
(B) Immunohistological staining (anti-HOPS) of H-35 hepatoma
cells left untreated (–LMB) or treated with LMB (+LMB) resulted in
accumulation of HOPS in nucleus in treated cells. DAPI: DAPI
staining of the same slides; merge: merged images of anti-HOPS and
DAPI. (C) Quantification of HOPS staining in the nucleus and
cytoplasm in the presence or absence of LMB. Data are
representative of five separate experiments. Standard errors of the
mean are indicated by error bars. Scale bars: 20 μm.
Fig. 6. Isolation of HOPS binding protein by the yeast two-hybrid
system. HOPS full-length protein was used as bait. (A) Growth of
transformants co-expressing HOPS and eEF-1A on selective
medium. HOPS, HOPS-Gal4-DBD; eEF-1A, the clone isolated
from the screening of the library; Gal4DBD, VP-16-AD and lamin
are negative controls. Individual Trp+ and Leu+ transformants were
plated on selective medium (Trp– Leu–) with histidine and
adenosine (HIS+/ADE+) or without histidine and adenosine
(HIS–/ADE–). (B) β-galactosidase
assay in selected colonies of yeast
expressing HOPS and eEF-1A. β-
galactosidase units are expressed
in Miller units.
(C) Coimmunoprecipitation of
HOPS-EF-1A complex from H-35
hepatoma cells. (Top) Western
blot analysis of H-35 protein
extract immuno-precipitated with
preimmune and anti-HOPS sera
(IP). H-35, protein extract as
control. Detection was performed
with anti-EF-1A.
(Bottom) Western blot with antieIF-
2a as control.
Journal of Cell Science
3192
using 360 and 420 nM (Fig. 7A,B). Analogous experiments
performed with the same concentration of GST, used as protein
control, had no significant effects on the synthesis of luciferase
(Fig. 7B). In reticulate lysates with different HOPS
concentrations the expression of eEF-1A was tested by western
blot analysis. No differences in eEF-1A expression were
detected in all samples examined (Fig. 7A).
HOPS and cell proliferation
The increased level of HOPS expression detected in H-35 cells
induced to arrest proliferation or in residual hepatocytes
following PH suggest a possible involvement of HOPS in
proliferation. To test this hypothesis, H-35 stable cells
overexpressing HOPS were generated. Stable cell cultures
were analyzed by cytofluorimetric analysis and [3H]thymidine
incorporation was evaluated. The study was performed on four
different stable clones. The results showed that HOPS
overexpression blocked H-35 cell growth. Colony growth assay
showed that [3H]thymidine incorporation was drastically
reduced in H-35 stable clones overexpressing HOPS with
respect to H-35 stable clones used as control. In H-35 stable
cells overexpressing HOPS the percentage of [3H]thymidine
incorporation showed a reduction of about of 65% (Fig. 7C)
with respect to controls. In H-35 stable clones HOPS is
localized in the nucleus and cytoplasm (Fig. S2 in
supplementary material). These results implicate the
involvement of HOPS during cell proliferation. To further
verify this hypothesis, experiments were performed in NIH-
3T3 proliferating cells and the HOPS anti-proliferative effect
was quantified. NIH-3T3 cells were infected with pBabe-puro
or pBabe-Hops. A growth cell selection by puromycin was
carried out for 5 days in both types of infected cells. The results
indicate that HOPS inhibits proliferation in NIH-3T3 cells
infected with pBabe-Hops. After puromycin selection the
number of cells at day 2 decreased in both types of infected
cells. In the following days, a stronger proliferation of cells
infected with pBabe-puro was observed than in cells infected
with pBabe-Hops. The number of the cells switched from
approximately 58104 with pBabe-puro to about 22104 with
pBabe-Hops, showing a reduction of almost 65% (Fig. 7D).
Discussion
In this paper, the role played during cell proliferation of a
newly identified gene, Hops, isolated in regenerating liver,
is described. Evidence is provided that HOPS is a
nucleocytoplasmic shuttling protein that contributes to the
control of cell proliferation by regulating protein synthesis. In
liver regeneration, early after PH, many factors such as
hormones and growth factors act on residual hepatocytes that
rearrange gene expression and protein synthesis. Following
PH, the residual hepatocytes begin to proliferate in synchrony.
Recent studies speculate that a crucial decision regarding
growth and proliferation arrest must be taken in the cell after
cell division. In fact, studies indicate that external and internal
factors act on the cell directing quiescence or proliferation
(Malumbres and Barbacid, 2001).
In liver regeneration many factors act on residual
hepatocytes to organize the reconstitution of the original liver
Journal of Cell Science 118 (14)
Fig. 7. Inhibition of
translation by HOPS in
vitro and reduction of
proliferation in vivo.
(A) Luciferase cDNA was
translated in an in vitro
translation system with
and without (C: control)
different concentrations of
purified recombinant
HOPS protein (60-420
nM). Similar experiments
were performed with or
without purified
recombinant GST protein.
eEF-1A expression was
analyzed by western
blotting on reticulate
lysates with different
HOPS concentrations.
(B) The synthesized
luciferase was quantified
by densitometric analysis
after gel electrophoresis.
The average results from
three experiments are
shown (in arbitrary units).
Black bars: the amount of
luciferase after the addition of different concentrations of HOPS; gray bars: the amount of luciferase after the addition of different
concentrations of GST. (C) Percentage thymidine incorporation by stable H-35 stable clones (C1-C3) overexpressing HOPS with respect to H-
35 control. (D) Number of puro selected NIH-3T3 cells at different days after infection. The gray line shows the number of control cells
infected with plasmid Puro; the black line shows the number of cells infected with plasmid Puro containing HOPS.
Journal of Cell Science
HOPS: a novel shuttling protein 3193
mass. After PH, change occur in stable hepatocytes at many
levels (protein synthesis, energy requirement, proliferation)
arranging their cell program on the basis of actual needs. It has
been demonstrated that the growth factors EGF, TGF-α and
HGF work as priming factors on the residual hepatocytes in
the first hours after PH, and increased levels in cAMP
concentration have been detected in the first hours after PH.
We observed in vivo that the increased level of cAMP allows
HOPS to export in proliferating hepatocytes following PH or
in normal hepatocytes after cAMP intraperitoneal injection in
mice. Our results show that the rapid export of HOPS from the
nucleus to cytoplasm is ascribed to high levels of cAMP in the
cells. The return of HOPS to the hepatocyte nucleus, 90
minutes after cAMP injection compared with regenerating
hepatocytes where HOPS returns 12 hours after PH, would
suggest that HOPS stimulated by cAMP migrates into the
cytoplasm, but proliferation of regenerating liver retains HOPS
in the cytoplasm.
The compartmentalization of proteins and their regulation in
export and import mechanisms from the nucleus to cytoplasm
is an important system of control in cell functions. The
presence of a NES domain in the HOPS sequence and specific
protein accumulation in hepatoma cell nucleus after treatment
with LMB indicates an involvement of CRM-1 in nuclear
export of HOPS protein. Based on these data we speculate that
CRM-1 is responsible for the nuclear export of HOPS and in
turn regulates HOPS cytoplasmic functions.
The identification of eEF-1A as a molecular partner binding
HOPS in liver and in hepatoma cells shows a specific
functional interaction between the two proteins. eEF-1Α plays
a key role in protein synthesis and controls the first step of
elongation of the growing peptide. In the cells, eEF-1A is
located predominantly in the cytoplasm. Recent studies
showed that eEF-1Α is actively exported from the nucleus to
keep the nuclear eEF-1Α concentration down to 1/100 with
respect to the cytoplasmic concentration, preventing eventual
nuclear translation (Bohnsack et al., 2002; Calado et al., 2002).
During cell proliferation, translation machinery of protein
synthesis rapidly increases and protein synthesis factors,
ribosomes and regulating factors guarantee a rapid processing
of transcripts (Thomas, 2000; Ruggero and Pandolfi, 2003).
In the first hours following PH, in the period preceding cell
mitosis, the residual hepatocytes are hypertrophic and protein
synthesis is strongly activated. Our findings suggest a
molecular mechanism in which, during residual hepatocyte
proliferation after PH, HOPS shuttles from the nucleus to
cytoplasm playing a pivotal role in the control of protein
synthesis by eEF-1Α activity regulation (Fig. 8). These
assumptions are supported by results of in vitro translation
assay in which the addition of recombinant HOPS regulates
protein synthesis.
During liver regeneration HOPS rapidly migrates from the
nucleus to the cytoplasm 12 hours after PH and the shuttling
protein returns to the nucleus where it is overexpressed until 72
hours. In hepatoma cells, the cell cycle is arrested by starvation
and HOPS is progressively overexpressed and accumulates in
the nucleus. Upon analysis the two phenomena may appear
incongruous. HOPS is overexpressed in proliferating cells, such
as residual hepatocytes in liver regeneration, and in hepatoma
cells induced to arrest proliferation. After PH the residual
hepatocytes are programmed to perform one or two rounds of
the cell cycle, unlike hepatoma cells that lack the capacity to
control the proliferative process. It could be that proliferative
signals act, on residual hepatocytes in the first hours following
PH in vivo, and signals from the serum act on hepatoma cells
allowing HOPS to shuttle from the nucleus to cytoplasm. The
absence of these signals, in the late phases of liver regeneration
or in serum-deprived cells, induces HOPS migration into the
nucleus and its overexpression. These events precede the
proliferative arrest in residual hepatocytes after a round of
proliferation as well as in starved hepatoma cells. This
hypothesis is supported by experiments performed in the H-35
hepatoma cells. H-35 stable clones overexpressing HOPS have
a strong reduction in thymidine uptake and a delay in growth.
Furthermore, our data confirm that HOPS overexpression in
NIH-3T3 cells is able to strongly reduce cell proliferation.
These findings suggest that cAMP and/or other proliferative
signals act on HOPS to regulate shuttling and expression. We
believe that HOPS is a protein that finely regulates cell
proliferation at the level of protein synthesis. HOPS exerts its
role by affecting eEF-1A in cytoplasm thus regulating cell
proliferation.
Recently it has been suggested that a key role is played by
translation factors and protein synthesis in the transformation
and regulation of cell proliferation (Caraglia et al., 2000;
Ruggero and Pandolfi, 2003). Alterations in eEF-1A
expression correlate with cancer and potential metastatic
activity of mammary adenocarcinoma (Edmonds et al., 1996).
Furthermore, the oncogene PTI1 (prostate tumor inducing
gene), a hybrid molecule containing a truncated form eEF-1A,
appears to play an important role in prostate cancer
(Gopalkrishnan et al., 1999).
The implication of translation factors in protein synthesis in
cancer cells may facilitate identification of novel therapeutic
agents that act on protein synthesis.
The authors thank Stefano Brancorsini, Emira Ayroldi, Eileen
Mahoney Zannetti and all the members of the Servillo laboratory for
help, fruitful discussion and for critical reading of the manuscript, and
Silvano Pagnotta and Maria Luisa Alunni for their excellent technical
assistance. This work was supported by the Associazione Italiana per
la Ricerca sul Cancro (AIRC) and Ministero dell’Università e
Fig. 8. Schematic representation of HOPS function. Cell
proliferation signals and factors that increased cAMP allow export of
HOPS via CRM-1. HOPS binds eEF-1A in the cytoplasm and then
returns to the nucleus.
Journal of Cell Science
3194
della Ricerca Scientifica 2003-2004, Cofin prot. 2003063402_004,
Italy.
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Journal of Cell Science