Mitotic phosphorylation activates hepatoma-derived growth factor as a mitogen

Department of Pediatrics, Cardiology Division, Johns Hopkins University, 600 N. Wolfe Street, Baltimore, MD 21287, USA.
BMC Cell Biology (Impact Factor: 2.34). 04/2011; 12(1):15. DOI: 10.1186/1471-2121-12-15
Source: PubMed


Hepatoma-derived growth factor (HDGF) is a nuclear protein that is a mitogen for a wide variety of cells. Mass spectrometry based methods have identified HDGF as a phosphoprotein without validation or a functional consequence of this post-translational modification.
We found that HDGF in primary mouse aortic vascular smooth muscle cells (VSMC) was phosphorylated. Wild type HDGF was phosphorylated in asynchronous cells and substitution of S103, S165 and S202 to alanine each demonstrated a decrease in HDGF phosphorylation. A phospho-S103 HDGF specific antibody was developed and demonstrated mitosis-specific phosphorylation. HDGF-S103A was not mitogenic and FACS analysis demonstrated a G2/M arrest in HDGF-S103A expressing cells, whereas cells expressing HDGF-S103D showed cell cycle progression. Nocodazole arrest increased S103 phosphorylation from 1.6% to 29% (P = 0.037).
Thus, HDGF is a phosphoprotein and phosphorylation of S103 is mitosis related and required for its function as a mitogen. We speculate that cell cycle regulated phosphorylation of HDGF may play an important role in vascular cell proliferation.


Available from: Monzur Rahman
Mitotic phosphorylation activates hepatoma-
derived growth factor as a mitogen
Allen D Everett
, Jun Yang
, Monzur Rahman
, Pratima Dulloor
and David L Brautigan
Background: Hepatoma-derived growth factor (HDGF) is a nuclear protein that is a mitogen for a wide variety of
cells. Mass spectrometry based methods have identified HDGF as a phosphoprotein without validation or a
functional consequence of this post-translational modification.
Results: We found that HDGF in primary mouse aortic vascular smooth muscle cells (VSMC) was phosphorylated.
Wild type HDGF was phosphorylated in asynchronous cells and substitution of S103, S165 and S202 to alanine
each demonstrated a decrease in HDGF phosphorylation. A phospho-S103 HDGF specific antibody was developed
and demonstrated mitosis-specific phosphorylation. HDGF-S103A was not mitogenic and FACS analysi s
demonstrated a G2/M arrest in HDGF-S103A expressing cells, whereas cells expressing HDGF-S103D showed cell
cycle progression. Nocodazole arrest increased S103 phosphorylation from 1.6% to 29% (P = 0.037).
Conclusions: Thus, HDGF is a phosphoprotein and phosphorylation of S103 is mitosis related and required for its
function as a mitogen. We speculate that cell cycle regulated phosphorylation of HDGF may play an important role
in vascular cell proliferation.
HDGF [GenBank: NM_004494] is a heparin binding pro-
tein originally isolated from conditioned media of a
human hepatoma (HuH-7) cell line. HDGF was subse-
quently shown to be a mitogen for many cell types with
nuclear localization necessary for its mitogenic activity
[1-6]. Expression of HDGF is developmentally regulated in
at least the renal, cardiovascular and pu lmonary systems
[1,3,7] and re-expressed at least in the both the lung [8]
and the arterial wall in response to injury [9], suggesting a
role in tissue repair. HDGF has also been identified at
least as an important prognostic marker in pathologic cell
growth, as it is overexpressed in a number of cancers with
expression linked to a poor outcome in lung, esophageal,
pancreatic and hepatic cancer [10-13].
Many nuclear proteins undergo post-translational modi-
fication to regulate their a ctivity. This is most clearly
demonstrated by the cell cycle regulatory cyclin and CDK
proteins which undergo both phosphorylation and depho-
sphorylation to reg ulate th ei r activity [ reviewed in [14]].
Previously we had shown by two-dimensional gel electro-
phoresis that HDGF in human melanoma cells has multi-
ple isoforms that migrated with the same mass in SDS but
had different pI [15], suggesting post-translational modifi-
cations, such as phosphorylation. In addition, in a proteo-
mic screen f or phosphoryla ted nucle ar protein s, HDGF
phosphorylated serines [16,17]. Whether HDGF is indeed
phosphorylated in vivo, and whether phosphorylation
affects HDGF function are all unknown.
In the present study, we detail that HDGF is indeed a
phosphoprotein, identify S103 as a significant phosphor-
ylation site and demonstra te that phosphorylation of
S103 plays a critical role in regulating HDGF mitogenic
Cell culture
HEK-293T, MDA-MB231 and COS-7 cells were obtained
from ATCC (Manassas, VA). Low passage mouse pri-
mary aortic vascular smooth muscle cells (VSMC) were
isolated as previously described [1] and all lines main-
tained in DMEM supplemented with 10% fetal bovine
serum (Gibco) at 37°C in 5% CO
. For proliferation
* Correspondence:
Department of Pediatrics, Cardiology Division, Johns Hopkins University,
600 N. Wolfe Street, Baltimore, 21287, USA
Full list of author information is available at the end of the article
Everett et al. BMC Cell Biology 2011, 12:15
© 2011 Everett e t al; licensee BioMed Central Ltd. This is an Open Access arti cle distributed under the terms of the Cr eative Commons
Attribution License ( censes/by/2.0), which permits unr estricted use , distribution, and reproduction in
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Page 1
experiments VSMC were serum starved for 36 hours,
then incubated overnight with BrdU (10 μM, R oche
Diagnostics, Indianapolis, IN). For cell cycle arrest stu-
dies, MDA-MB231 cells were seeded at 10
cells/ml in
6 well dishes containing a cover slip and DMEM with
10% serum. After 8 h cells were left in serum free (0.5%
serum) medi a for overnight. Next morning cells were re-
stimulated with 10% FCS. After 8 h cells were treated
with or without 200 nM nocodazole for next 16 h. Next
morning c ells were briefly washed with ice cold PBS and
fixed with 4% formaldehyde in DPBS.
Plasmids and transfections
Full length wild type rat HDGF was cloned in pK7-GFP
and pKH3 (vectors were gifts of Ian Macara, University of
Virginia) [4] and substitution of serine (S) 103, 165 and
202 to alanine (A) or aspartic acid (D) was done using
PCR (QuickChange Site Directed Mutagenesis, Strata-
gene). 1 × 10
HEK-293T, COS-7 or VSMC cells were
plated in 60 mm dishes and transfected the following day
with 4 ug of plasmid DNA using calcium phosphate (Pro-
Fection Mammalian Transfection System-Calcium Phos-
phate, Promega, WI) or FuGene (Roche Applied Science)
according to the manufacturers recommendations.
Fluorescent activated cell sorting
HEK-293T cells were transfected as above to express
cells were processed for cell cycle FACS analysis with
gating for no GFP and G FP after the method of Schmid
and Sakamoto [18] (Becton Dickinson FACSCalibur Dual
Laser) using ModFit LT softw are (Ve rity Softw are , Top-
sham, ME). Cell c ycle analysis was expressed as percent
in G1, G2 and S. Each FACS analysi s was perf ormed in
triplicate with the results pooled from 4-5 separate
Antibodies and immunoblotting
Anti-phospho-S103-HDGF was generated by Biosource
(Hopkington MA) using a synthetic phosphopeptide
corresponding to amino acids 95-107 of human HDGF
with an N-terminal cysteine (CVKASGYQS(pS)QKKS)
for coupling to keyhole limpet hemocyanin.
Western blot analysis was performed as previously
described [1,4,7]. Briefly, pho sphorylated proteins w ere
enriched from 4 × 10
COS-7 cells using the Phospho-
Protein Purification Kit (Qiagen, Valencia, CA) following
the manufacturers instructions. For immunoblot analysis,
COS-7 whole cell lysates and isolated proteins (20 μg)
were separated by 10% SDS-PAGE and transferred to
Trans-Blot Transfer Medium (Bio-Rad, Hercules, CA).
Blots were blocked in TBS-T (0.1% Tween, w/v) and 5%
bovine albumin (Roche) for one hour and probed with
either anti-phospho-S103-HDGF (1:500) or an ti-HDGF
(1:1000) in TBS-T for 1 ho ur at room temperature. After
washing with TBS-T membranes were incubated with an
anti-rabbit secondary antibody coupled to horseradish
peroxidase (1/30,000). After washing, the blots were
developed using enhanced chemiluminescence (GE
Immunocytochemical analysis was performed as previously
described [1,4,7]. Briefly, COS-7 cells grown on glass cover-
slips in six well plates were fixed in 4% buff ered parafor-
maldehyde for 30 minutes at room temperature then
washed with cold PBS. Separate coverslips were incubated
with the anti-HDGF (1:2000) or anti-phospho-S103-HDGF
(1:250). Control coverslips were incubated with no primary
antibody or preabsorbed primary antibody with 1 μgofthe
S103 phosphopeptide described above at the same concen-
tration as the primary antibody. For BrdU detection, cells
were fixed in 2% paraformaldehyde for 10 minutes at room
temperature, with BrdU detected using a mouse monoclo-
nal anti-BrdU antibody (6 ug/ml, Roche). Vector Red
(Vector Laboratories) was used as a fluorescent substrate
to identify specific HDGF or BrdU staining and DAPI as a
specific DNA counterstain. Images were acquired on a
Nikon Eclipse 400 microscope equipped with a MicroPubl-
isher digital ca mera (Qi maging, Burnaby, BC, Can ada)
and merged using Adobe Photoshop cs software (Adobe
Systems Inc., San Jose, CA). For nocodazole cell cycle
arrest studies, MDA-MB231 cells were immunostained for
anti-phospho-S103-HDGF and fluorescent microscope
acquired images analyzed by Nikon NIS-element software.
The total number of cells was counted by detecting size
and intensity of DAPI staining. The number of phospho-
S103 positive cells was identified as having at least 10 times
more intense staining than non-treated control cells. Cells
were counted from at least 3 different fields per coverslip
for each experiment with a total of 3 individual experi-
ments performed. Results were expressed as percent of
phospho-S103-HDGF positive nuclei analyzed using a
non-paired t-test with a P value of < 0.05 considered as
HDGF is a phosphoprotein
Previously we found multiple forms of HDGF by 2D gels
and suspected this was due to a post-translational modifi-
cation such as phosphorylation that could change the pI
of the protein. The NetPhosK 1. 0 computer algorithm
[19] using statistical ranking identified S103 as the most
likely candidate site for phosphorylation (Score 0.86).
In additio n, S165 and S202 were r ecently identified as
phosphorylation sites in HDGF by mass spectrometry of
HeLa nuclear and HT-29 cell extracts [16,17]. Sequence
comparisons confirmed that S103, S165 and S202 are
Everett et al. BMC Cell Biology 2011, 12:15
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conserved in mouse, rat and human HDGF. COS-7 cells
were transfected to express GFP fusions of wild type
HDGF or S103A, S165A or S202A substitution HDGF
polypeptides and metabolically radiolabeled with [
orthophosphate for 2 hours. The tagged proteins were
recovered by anti-GFP immu noprecipitation and as
shown in Figure 1, HDGF wt was phosphorylated and
phosphorylation of the S103A, S165A and S202A were
reduced relative to wild type (Figure 1) demonstrating
that all three of these serines were kinase substrates.
HDGF S103 is phosphorylated during mitosis
To further explore HDGF S103 phosphorylation we devel-
oped a phosp ho-S103- HDGF antibody. Western blotting
of enriched COS-7 phosphoproteins demonstrated detec-
tion of S103-HDGF phosphorylation (Figure 2). The phos-
pho-S103-HDGF antibody ident ified both the high and
low mass HDGF protein bands from the same COS-7 cell
lysate detected with a wild type HDGF antibody [1]. With
the phospho-S103 HDGF antibody, the high er mass
HDGF band was less distinct compared to the lower mass
band, but obvious with longer exposure. To map the
expression of S103-HDGF, we immunostained COS-7
cells with both total and phospho-S103-HDGF antibodies
(Figure 3A-C). Using the total HDGF antibody and COS-7
cell s, HD GF was highly expressed in 81.4% of nuclei and
with the phospho-S103-HDGF antibody, only 5.4% of cell
nuclei were immuno-positive (Figure 3A). Of interest, the
nuclei positive for phospho-S103-HDGF were all under-
going mitosis ( Figu re 3B), based on the condensed chro-
matin. Importantly preabsorbing the antibody with the
target phosphopeptide, or the absence of the primary anti-
body in the immunostaining reaction demonstrated that
staining was specific (Figure 3B). Further study (Figure 4)
revealed that phospho-S103-HDGF was only detected dur-
ing mitosis. Phospho-S103-HDGF was first detectable at
the time of nuclear condensation and breakdown of the
nuclear envelope, peaks at m etaphase with alignment of
chromosomes along the metaphase plate and disappears
with daughter cell separation in anaphase. This was also
evident when we cell cycle arrested cells with nocodazole
and stained those cells with the phospho-S103-HDGF
antibody (Figure 5). Nocodazole mitotic arrest significantly
increased the number of phospho-S103-HDGF positive
cells when compared to controls (29% vs 1.6%, P = 0.037,
Phosphorylation of S103 is necessary for HDGF mitogenic
HDGF is a potent mitogen for VSMC [1]. To examine
the role o f HDGF S103 phosphorylation in functio n as a
mitogen, mou se VSMC were transfected to express GFP-
HDGF S103A. Cells were serum starved for 24 hours
then pulse-labeled with BrdU. BrdU incorporati on (red)
into DNA (blue) by transfected (green) cells was detected
by fluorescent immunocytochemistry. As shown in
Figure 6, HDGF-S103A traffics normally to the nucleus,
Figure 1 HDGF is a phosphoprotein. Autoradiogram of HDGF
immunoprecipitated from COS-7 cells that transiently expressed
wild type rat GFP-HDGF (WT-HDGF) or S103A, S165A, or S202A-
HDGF substitution mutations after in vivo [
P] orthophosphate
labeling for two hours, and resolution by SDS-PAGE.
Figure 2 Phospho-S103-HDGF Immunoblotting.Westernblotof
COS-7 whole cell lysate (25 ug, Cell lysate), cell lysate PhosphoProtein
column flow through (25 ug) or PhosphoProtein column bound
proteins (25 ug, Po4 proteins, PhosphoProtein Purification Kit, see
Methods) using a specific anti-phospho-S103-HDGF (top panel, A) or
pan-anti-HDGF (bottom panel, B). Arrows = the high and low
molecular weight bands in the Po4 protein lane with both antibodies.
Everett et al. BMC Cell Biology 2011, 12:15
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Figure 3 Phospho-S103-HDGF expression in mitotic nuclei.(A)COS-7cellswereimmunostainedfor HDGF or phospho- S103-HDGF and
percentage positive cells determined. (B) Immunostaining of COS-7 cells (red) for total HDGF and middle panel for phospho-S103-HDGF. (C)
Staining specificity was proven by pre-absorbing the phospho-S103-HDGF antibody with the S103 phosphopeptide (Pre-Ab) or omission of the
phopho-S103 HDGF antibody (No primary). Cells were counterstained with DAPI to identify the nucleus and DNA.
Everett et al. BMC Cell Biology 2011, 12:15
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demon strating that S103 phosphorylation is not required
for nuclear translocation or retention. However as shown
in Figure 6, H DGF-S10 3A did not stimu late BrdU incor-
poration, showing it was not acting as a mitogen.
Phosphorylation of HDGF S103 is necessary for cell cycle
To test for a po ssible role o f phospho rylation in HDGF
function, S103 was mutated to an aspartic acid (S103D)
as a phospho-mimic residue. The effect of HDGF-
S103D on cell proliferation w as tested with asynchro-
nous HEK-293 cells transfected to express, GFP fused to
wild type HDGF, HDGF S103A or S103D and ce ll cycle
analysis performed by FACS o n the GFP positive cells
and the GFP negative cells from the same sample as a
control. As shown in F igure 7A, consiste nt with HDGF
being a growth factor, HDGF significa ntly decreased the
percentage of cells in G1 (43.3 ± 1.3% vs 49.6 + 3.0%,
P < 0.009) and increased the percentage in G2 (19.0 ±
1.8% vs 11.4 ± 1.1%, P < 0.004) as compared to G FP.
Unlike HDGF, HDGF S103A did not decrease the cell
population in G1 (43.3 ± 1.3% and 4 9.6 ± 3.0% respec-
tively), or increase the fraction of cells in S+G2 (56.7 ±
3.2 and 50.4 ± 3.0%, respectively). In contrast to HDGF
S103A, HDGF S103D significantly decreased the fraction
of cells in G1 (49.4 ± 3.0% and 33.4 ± 3.8%, respectively)
Figure 4 HDGF S103 is phosphorylated during M phase of the cell cycle. (A) Repre sentative immunostaining for phospho-S103 HDGF was
first detected in COS-7 cells that have undergone nuclear envelope breakdown and chromosomal condensation, (B) and in cells in metaphase
and early anaphase. (C and D) HDGF-S103 phosphorylation rapidly falls to undetectable levels as daughter cells separate in late anaphase and
telophase. Co-staining with DAPI demonstrates chromosomal DNA condensation in phospho-S103-HDGF immunostained cells (Merge).
Everett et al. BMC Cell Biology 2011, 12:15
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and increased the fraction of cells in the S+G2 phase
(50.4 ± 3.0% and 66.6 ± 3.8%, respectively). Therefore
expression of HDGF S103D, a phospho-mimic, was a
more potent mitogen than wild type HDGF and loss of
phosphorylation o n S103 abrogated HDGFs mitogenic
function. This effect on the cell cycle was limited to
transfected cells as non-transfected cells from the same
plate demonstrated normal cell cycle proportions
(Figure 7B). This demonstrates that the effects on the
cell cycle were not transfection artifacts.
HDG F is an abundant nuclear protein with activity as a
mitogen, in that it stimulates cell cycle progression. In
this study we demonstrate phosphorylation of S103 dur-
ing mitosis and show this phosphorylation is required
Figure 5 Phosphorylat ion of S103 HDGF is increased during mitot ic phase of cell division .A.MDA-MB231cellsweretreatedwithout
(control) or with nocodazole (200 nM) to arrest the cell cycle at mitosis and immunostained with the anti-phospho-S103-HDGF antibody and
DAPI. B. Graph representing percentage of phospho-S103-HDGF positive cells in control and nocodazole treated cells (n = 3; p = 0.037
calculated by unpaired two-tailed T test).
Everett et al. BMC Cell Biology 2011, 12:15
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Figure 6 HDGF-S103A is not a mitogen. HDGF-GFP-S103A was expressed in mouse VSMC for 24 hours and cells labeled with BrdU overnight.
Cells were immunostained for BrdU as a marker of DNA replication and observed for co-localization of GFP and BrdU. A = cells expressing
HDGF-GFP-S103A, B = no staining for BrdU, C = DAPI staining to show that HDGF-GFP-S103A nuclear targeting is not affected.
Figure 7 Phosphorylation of HDGF S103 regulates its ability to stimulate cell proliferation. A. HDGF-GFP, HDGF-GFP-S103A or HDGF-GFP-
S103D fusion proteins were expressed in HEK-293 cells for 24 hours and the cell cycle analyzed in GFP and non-GFP expressing cells by FACS. (n
= 4-5, with three replicates for each experiment.) B. Cell cycle analysis of cells expressing the HDGF fusions (GFP) vs non-GFP positive cells from
the same experiments in A. # = HDGF vs GFP, * = HDGF-S103A or D vs HDGF. * and # = P 0.05.
Everett et al. BMC Cell Biology 2011, 12:15
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for HDGF mitogenic activity. Our study of H DGF phos-
phorylation in vivo was suggested by the computer
search engine NetPhosK 1 [19] that matches amino acid
sequence to known protein kinase phosphorylation
motifs, with statistical ranking for significance. This type
of search engine is useful for identifying potential phos-
phorylation sites within a protein of interest. Separate
studies had identified HDGF as a phosphorylated
nuclear protein based on mass spectroscopy (MS)
[16,17] or by in vitro kinase assays [20]. These studies
indicated S132, S133, S165, T200 and S202 were phos-
phorylated in HeLa or HT-29 cells [16,17]. Our results
identify S103 as a new, previously unknown significant
HDGF phosphorylation site not previously identified by
MS. Because S103 is only phosphorylated during mito-
sis, based on immunostaining with a phospho-amino
acid specific antibody, this likely explains why pS103
was not found by MS in non-s ynchronized cells. This i s
supported by the relatively low levels of p S103-HDGF
we observed by immunoblotting whole cell extracts. It is
also unclear from these globa l MS studies whether the
peptide containing S103 was detected. We demonstrate
that S165 and S202 are also phosph orylated in vivo, but
at possibly lower level s in COS-7 cells relative to S103,
based on differences in radio labeling of the mutated
predicted to be a Cdk2 substrate based on sequence,
howev er mutation of S165 had no effect on the nuclear
targeting of HDGF or on its mitogenic activity [4,5].
Although the kinase for S103 is not known, Salvi et al
[20] have shown that HDGF can by phosphorylat ed in
vitro by casein kinase 2. It is not known whether S132/
133 are phosphoryl ated in vivo or whether S132/133
phosphorylation is functionally significant.
We found that phosphorylation of HDGF-S103 has a
significant effect on HDGF mitogenic activity. A substi-
tution mutation in HDGF to S103A to prevent phos-
phorylation nullified HDGF mitogenic activity, whereas
a S103D phospho-mimic mutation was constitutively
active, resulting in an increased mitogenic activity rela-
tive to wild type HDGF. This data would suggest that
one model of VSMC proliferation is t hat activation of
mitotic kinases results in p hosphorylation of S103-
HDGF, leading to increased cell proliferation. As the
impact of the S103 mutants on the cell cycle was much
more profound than the wild type protein, this would
suggest that HDGF mitogenic function is dependent on
phosphorylation and not just dependent on the amount
of HDGF present.
Although the mechanism of phospho-S103-HDGF
function during mitosis is unclear, it is of interest that
another HDGF family member LEDGF, demonstrates
metaphase chromatin binding, requiring cooperative
interaction of the PWWP and AT-hook domains.
Although HDGF does not contain AT-hook domains, it
does bind DNA directly requiring a large 36 bp recogni-
tion sequence and requires the PWWP domain for
DNA bindin g [21]. It is unclear how phosphorylation
regulates this process either to induce a conformational
change to increase binding or enhance binding with a
chromatin binding prot ein. The HDGF PWWP domain
was recent ly shown to dimerize on heparin and whether
phosphorylation plays a role in potentially regulating
HDGF dimerization on chromatin via the PWWP
domain is an area of active research.
It is of great in terest that a S282P mutation in the
DNA m ethyltransferase 3b (DNMT3b, also a PWWP
protein) gene results in the ICF syndrome (for immuno-
def iciency, centromeric instability, and facial anomalies)
[22]. This serine is 4 amino acids carboxy to the PWWP
domain in DNMT3b, and homologous to the location of
S103 in HDGF. The conservation of this serine in rela-
tion to the PWWP domain and its mutation associated
with a human disea se, strongly implicates these s erines
in the function of PWWP proteins.
HDGF is a mitotic phosphoprotein and phosphorylation
of S103 plays an important ro le in regulating the prolif-
eration of cells and the mitogenic function of HDGF.
Author details
Department of Pediatrics, Cardiology Division, Johns Hopkins University,
600 N. Wolfe Street, Baltimore, 21287, USA.
Center for Cell Signaling and
Department of Microbiology, University of Virginia, 1400 Jefferson Park
Avenue, Charlottesville, 22908, USA.
Authors contributions
ADE conceived the experiments and wrote the manuscript. JY made the
phospho HDGF mutants, generated the in vitro phosphorylation data and
drafted that experimental section. MR performed nocodazole and cell
sorting experiments and drafted the experimental results. PD performed cell
transfections and immunohistochemical analyses. DLB edited the draft and
contributed significantly to experimental design. All authors have read and
approved the final manuscript.
Received: 6 July 2009 Accepted: 13 April 2011 Published: 13 April 2011
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Cite this article as: Everett et al.: Mitotic phosphorylation activates
hepatoma-derived growth factor as a mitogen. BMC Cell Biology 2011
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Everett et al. BMC Cell Biology 2011, 12:15
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    • "Although HDGF was isolated from a hepatoma cell line, previous studies have shown that it plays important roles in the development and tissue repair of numerous normal organs, including the liver, kidneys, lungs and gut2728293031. In addition, Everett et al. [39] reported the functional involvement of HDGF in the development of and tissue repair in the cardiovascular system. "
    [Show abstract] [Hide abstract] ABSTRACT: The development of hepatocellular carcinoma (HCC) is an important complication of viral infection induced by hepatitis virus C, and our major research theme is to identify a new growth factor related to the progression of HCC. HDGF (hepatoma-derived growth factor) is a novel growth factor that belongs to a new gene family. HDGF was initially purified from the conditioned medium of a hepatoma cell line. HDGF promotes cellular proliferation as a DNA binding nuclear factor and a secreted protein acting via a receptor-mediated pathway. HDGF is a unique multi-functional protein that can function as a growth factor, angiogenic factor and anti-apoptotic factor and it participates in the development and progression of various malignant diseases. The expression level of HDGF may be an independent prognostic factor for predicting the disease-free and overall survival in patients with various malignancies, including HCC. Furthermore, the overexpression of HDGF promotes the proliferation of HCC cells, while a reduction in the HDGF expression inhibits the proliferation of HCC cells. This article provides an overview of the characteristics of HDGF and describes the potential role of HDGF as a growth-promoting factor for HCC.
    Full-text · Article · Jun 2015 · International Journal of Molecular Sciences
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    • "Among all reported activities, HRPs exhibit unique dual functions on both sides of the plasma membrane. Inside cells, HRPs have a transcriptional regulatory role in nuclear targeting , DNA binding and post-translational modification (Ikegame et al. 1999; Kishima et al. 2002; Singh et al. 2006; Yang and Everett 2007; Thakar et al. 2008; Bueno et al. 2010; Everett et al. 2011). Outside cells, HRPs were indentified to be a growth factor family. "
    [Show abstract] [Hide abstract] ABSTRACT: Hepatoma-derived growth factor (HDGF) recognizes cell surface heparan sulfate to promote its internalization though binding to its N-terminal HATH (homologous to amino terminus of HDGF) domain. HDGF-related proteins (HRPs) all have the HATH domain in their N terminus. In this study, we report on the commonality of heparin binding in all HRPs with a broad range of heparin-binding affinity: HRP-4 is the strongest binder, and the lens epithelium-derived growth factor shows a relatively weak binding, with binding affinities (KD) showing 30-fold difference in magnitude. With the HDGF HATH domain used as a model, residue K19 was the most critical basic residue in molecular recognition and protein internalization, and with its proximal proline-tryptophan-tryptophan-proline motif, coordinated a conformational change when binding to the heparin fragment. Other basic residues, K21, K61, K70, K72 and R79, confer added contribution in binding that the total ionic interaction from these residues represents more than 70% of the binding energy. Because the positive-charged residues are conserved in all HRP HATH domains, heparin binding outside of cells might be of equal importance for all HRPs in mediating downstream signaling; however, distinct effects and/or distribution might be associated with the varying affinities to heparin.
    Preview · Article · Jan 2012 · Glycobiology
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    [Show abstract] [Hide abstract] ABSTRACT: The prognosis of malignant melanoma is poor due to high incidence of metastasis, underscoring the demand for development of novel therapeutic strategies. Stress hormone pro-opiomelanocortin (POMC) is the precursor for several anti-inflammatory peptides that hold promise for management of cancer-related diseases. The present study evaluated the anti-metastatic potential and mechanism of POMC therapy for metastatic melanoma. Adenovirus-mediated POMC gene delivery potently inhibited the invasiveness of human and mouse melanoma cells. Moreover, after induction of lung metastasis, systemic POMC expression significantly reduced the foci formation and neovascularization in lungs. Mechanistic studies revealed POMC therapy inhibited the epithelial-mesenchymal transition (EMT) of melanoma cells by upregulation of E-cadherin and downregulation of vimentin and α-smooth muscle actin (α-SMA). In addition, microarray analysis unveiled POMC gene transfer reduced the mRNA level of multiple pro-metastatic factors including hepatoma-derived growth factor (HDGF). Cell culture and immunohistochemical studies further confirmed that POMC gene delivery significantly decreased the HDGF expression in melanoma cells and tissues. Despite of stimulating the invasion and EMT, exogenous HDGF supply only partially attenuated the POMC-mediated invasion inhibition and EMT change in melanoma cells. Finally, we delineated the contribution of melanocortins to POMC-induced inhibition of invasion, HDGF downregulation and E-cadherin upregulation. Together, these results indicate that HDGF downregulation participates in POMC-induced suppression of metastasis and EMT in melanoma.
    Full-text · Article · Mar 2013 · Molecular Cancer Therapeutics
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