Identification of the amniotic fluid insulin-like growth
factor binding protein-1 phosphorylation sites and
propensity to proteolysis of the isoforms
Lorenzo Dolcini
1
, Alberto Sala
1
, Monica Campagnoli
1
, Sara Labo
`
1
, Maurizia Valli
1
, Livia Visai
1,2
,
Lorenzo Minchiotti
1
, Hugo L. Monaco
3
and Monica Galliano
1
1 Department of Biochemistry ‘A. Castellani’, University of Pavia, Italy
2 Center for Tissue Engineering (C. I. T), University of Pavia, Italy
3 Biocrystallography Laboratory, Department of Biotechnology, University of Verona, Italy
Introduction
The insulin-like growth factor binding proteins (IG-
FBP) are six homologous molecules (IGFBP-1–6) that
play critical roles in a wide variety of important physi-
ological processes. Upon binding they regulate the

from human normal amniotic fluid collected in the weeks 16–18, the intact
nonphosphorylated IGFBP-1 and five electrophoretically distinct phospho-
isoforms and have determined their in vivo phosphorylation state. The
unmodified protein was the most abundant component and mono-, bi-, tri-
and tetraphosphorylated forms were present in decreasing amounts. The
phosphorylation sites of IGFBP-1 were identified by liquid chromatogra-
phy–tandem mass spectrometry analysis of the peptides generated with
trypsin, chymotrypsin and Staphylococcus aureus V8 protease. Five serines
were found to be phosphorylated and, of these, four are localized in the
central, weakly conserved, region, at positions 95, 98, 101 and 119, whereas
one, Ser169, is in the C-terminal domain. The post-translational modifica-
tion predominantly involves the hydrophilic stretch of amino acids repre-
senting a potential PEST sequence (proline, glutamic acid, serine,
threonine) and our results show that the phosphorylation state influences
the propensity of IGFBP-1 to proteolysis.
Abbreviations
IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; LC-ESI-MS, liquid chromatography-electrospray ionization-
mass spectrometry.
FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS 6033
proteins are all secreted in the bloodstream, the func-
tional role of IGFBP-1 [5], IGFBP-3 [2] and IGFBP-5
[2] inside the cells has been well documented.
IGFBP-1, the subject of the present study, is predom-
inantly expressed in the liver and, in adult mammals,
the synthesis of the protein is upregulated in a number
of catabolic or stressful conditions, such as fasting and
diabetes [6]. Additionally, it has recently been shown
that a portion of intracellular IGFBP-1 localizes to
mitochondria, where it acts as a prosurvival factor and
protects the liver from apoptosis [5]. IGFBP-1 repre-

gestational periods. We have recently shown that nor-
mal amniotic fluid, collected in the weeks 16–18 of
gestation, contains the unmodified protein and five
electrophoretically distinct phosphoisoforms [20]. A
previous study has shown that IGFBP-1 expressed in
Chinese hamster ovary cells is phosphorylated at
Ser101, 119 and 169 [21,22] and a recent paper
described the modification of Ser98, which was
revealed in the highly phosphorylated protein isoforms
found in hypoxia-treated cells [23].
Post-translational modification influences the inter-
action of IGFBP-1 with IGF-I [21,22,24] and has been
associated with gestational and fetal abnormalities [24–
26]. However, neither the phosphorylation state of the
protein isolated from normal human amniotic fluid
nor the biological properties of the homogeneous
IGFBP-1 phosphoisoforms have been examined so far.
It has been shown that, in vitro, several metallo-
proteases recognize IGFBP-1 as a substrate [27] and a
modulating effect of phosphorylation on the backbone
cleavage of the protein has been recognized [8].
Recently, a specific IGFBP-1 protease activity has been
described in a patient with multiple myeloma and
identified as azurocidin [28].
Here we describe the purification on a preparative
scale of the six isoforms present in human normal amni-
otic fluid collected in the weeks 16–18. The availability
of sufficient amounts of the pure protein allowed a
detailed identification of the amino acid residues
involved in the IGFBP-1 post-translational modifica-

identical molecular mass of  30 kDa and that no
fragmentation of the protein had occurred during the
Phosphorylation of amniotic fluid human IGFBP-1 L. Dolcini et al.
6034 FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS
purification procedure. The peaks were pooled and
further purified by gel filtration chromatography. Six
homogeneous polypeptide chains with the same N-ter-
minal sequence, APWQCAPCSA, corresponding to
the first 10 residues of intact mature IGFBP-1, were
obtained. Nondenaturing gel electrophoresis (Fig. 2A)
and immunoblotting (Fig. 2B) showed that the electro-
phoretic mobilities of the IGFBP-1 isoforms increased,
but not progressively, as expected on the basis of the
anion exchange chromatography elution profile. Peak IV
migrated more anodically than the preceding chro-
matographic fraction and the last eluting fraction, VI,
displayed a very similar mobility to that of fraction V.
The six proteins were submitted to treatment with alka-
line phosphatase and, when analysed by isoelectric
focusing, all acquired the same isoelectric point of the
polypeptide chain eluting under peak I (data not shown).
This form thus represents the unmodified protein,
whereas the other fractions contain distinct phosphoiso-
forms. These results showed that the nonphosphorylated
protein, as well as the five distinct phosphoisoforms,
were obtained in homogeneous form by anion exchange
chromatography. Furthermore, the addition of the
chelating agent was effective in preventing proteolytic
cleavage, as only the intact proteins were obtained.
Mass determination of the IGFBP-1 isoforms

Q-Sepharose ion exchange chromatography. The elution was
carried out with a linear gradient from 0 to 100% using 6.25 m
M
Bistris-propane, pH 7.5 as buffer A and 6.25 mM Bistris-propane,
pH 9.5, 0.35
M NaCl as buffer B. The peaks I–VI, positive when
probed with anti-IGFBP-1 IgGs, were pooled and further purified by
gel filtration.
A
B
123456
123456
Fig. 2. Nondenaturing gel electrophoresis and western blot analysis
of IGFBP-1 isoforms. IGFBP-1 isoforms isolated by ion exchange
chromatography from peaks I–VI were (A) resolved on a 17% non-
denaturing polyacrylamide gel and stained with Coomassie Brilliant
Blue (lanes 1–6) and (B) following western blot were probed with
anti-IGFBP-1 IgGs (lanes 1–6).
Table 1. Molecular mass of the IGFBP-1 peaks isolated by anion
exchange chromatography.
IGFBP-1
isoforms
Measured
mass (Da)
Theoretical
mass (Da)
Dmass
(Da)
Peak I 25 251.2 25 252.4 +1.2
Peak II 25 331.1 25 332.4 +1.3

phosphopeptides is generally slightly lower than that
of their unmodified counterparts and only relatively
large fragments, between six and 25 amino acids, are
suitable for mass spectral analysis [32]. Therefore, to
enhance sequence coverage and ensure that each
phosphorylation site was contained in at least one
peptide of suitable size and hydrophobicity, the
IGFBP-1 isoforms were cleaved with three different
proteolytic enzymes. Reduction of disulfide bonds was
carried out before each enzymatic digestion and the
resulting peptide mixtures were immediately submitted
to LC-ESI-MS ⁄ MS analysis. The HPLC separation
1000
25+
24+
23+
22+
21+
20+
19+
18+
17+
16+
15+
14+
13+
25+
24+
23+
22+

19+
18+
17+
16+
15+
14+
13+
25+
26+
27+
24+
23+
22+
21+
20+
19+
18+
17+
16+
1200 1400
m/z
1600 1800 2000
25251 Da
25331 Da
25410 Da
25572 Da
25493 Da
E
D
C

PO
4
()98 for single charged ions and
)49 for doubly charged ions) dominated the fragmen-
tation pattern. A complete sequence coverage of the
protein was obtained (data not shown) and Table 2
lists the phosphorylated peptides observed. The V8
fragments spanning residues 93–100 (AGSPESPE)
from the unmodified protein, at m⁄ z 773.3, and from
the monophosphorylated one, at m ⁄ z 853.2, displayed
a mass difference of 80 Da, which is consistent with
the presence of one phosphate group. Fragmentation
in the ion trap showed that this peptide was phosphor-
ylated at Ser95 and, to a much lower extent, also at
Ser98. The presence of the fragment ion at m ⁄ z 755.3
is due to the loss of phosphoric acid (98 Da), the ion
at m ⁄ z 296.1 (b3+P) is consistent with the modifica-
tion of Ser95 and the ions at m ⁄ z 638.2, (y5+P) and
at m ⁄ z 442.2 (b5) are indicative of the modification at
Ser98 (Fig. 4B). Figure 4A shows the MS ⁄ MS spec-
trum obtained for the unmodified peptide. The two
peptides coelute and the doubly modified fragment was
not observed. The MS ⁄ MS spectra of the V8 fragment
93–103 (AGSPESPESTE) from the monophosphory-
lated isoform, at m ⁄ z 1172.1 (+80 Da shift) is shown
in Fig. 5A. The loss of phosphoric acid (98 Da) from
the precursor yielded a charged species at m⁄ z 1072.1
and the presence of a product ion at m ⁄ z 642.0
(y5+P) is consistent with phosphorylation at Ser101.
The fragment ions at m ⁄ z 562.2 and at m ⁄ z 649.0

83-108 +2P SDASAPHAAEAG
SPESPESTEITEEE 2787.1 2787.8 +0.7
93-100 +P AG
SPESPE 853.4 853.2 )0.2
93-103 +P AG
SPESPESTE 1169.4 1169.3 )0.1
93-103 +2P AG
SPESPESTE 1249.4 1249.2 )0.2
93-108 +P AGSPE
SPESTEITEEE 1770.7 1771.4 )0.3
100-111 +P
STEITEEELLD 1357.6 1357.6 0
109-121 +P LLDNFHLMAP
SEE 1594.7 1595.2 )0.5
109-122 +P LLDNFHLMAP
SEED 1708.7 1708.6 )0.1
112-121 +P NFHLMAP
SEE 1253.5 1253.9 +0.4
113-121 +P FHLMAP
SEE 1139.4 1139.9 +0.5
161-172 +P SLAKAQET
SGEE 1330.0 1330.3 +0.3
Tryptic peptides
165-175 +P AQET
SGEEISK 1258.5 1258.6 +0.1
165-183 +P AQET
SGEEISKFYLPNCNK 2237.0 2239.0 +2.0
L. Dolcini et al. Phosphorylation of amniotic fluid human IGFBP-1
FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS 6037
biphosphorylated fragment, Ser95 and Ser101 are

was revealed, and the phosphoacceptor sites Ser95,
Ser98, Ser119 and Ser169, previously described in
cultured cells [22,23], were confirmed in our natural
samples of human amniotic fluid IGFBP-1. In agree-
ment with previous data [22,35], we did not find
any evidence of threonine and tyrosine phosphor-
ylation. For this reason, and because it is well
Fig. 4. LC-MS ⁄ MS ion spectra of IGFBP-1
peptide 93–100. (A) MS ⁄ MS spectrum of
the singly charged 773.3 Da ion correspond-
ing to the nonphosphorylated AGSPESPE
fragment. (B) MS ⁄ MS spectrum of the
singly charged 853.2 Da ion corresponding
to the monophosphorylated AGSPESPE
sequence.
Phosphorylation of amniotic fluid human IGFBP-1 L. Dolcini et al.
6038 FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS
known that the phosphopeptides are not eluted
with high efficiency from RP-HPLC, a quantitative
analysis of each phosphorylation site could not be
performed.
Chromatographic separation of the V8 peptides
In order to decide whether a major phosphoacceptor
serine is present in the monophosphorylated protein, it
A
B
Fig. 5. LC-MS ⁄ MS ion spectra of IGFBP-1
peptide 93–103. (A) MS ⁄ MS spectrum of
the singly charged 1172.1 Da ion corre-
sponding to the monophosphorylated AGSP-

enzymatic cleavage. Furthermore, as no other evident
peak shift was appreciable, it can be argued that the
monophosphorylated IGFBP-1 form is predominantly
modified in the sequence spanning residues 93–103.
Proteolysis
In a previous paper [30], we described the isolation of
the C-terminal domain of IGFBP-1 and suggested that
the proteolytic cleavage could be ascribed to a specific
amniotic fluid metalloprotease. Therefore, prior to
examining the effect of phosphorylation on the proteo-
lytic process, we prefractionated the amniotic fluid
proteins by gel filtration and analysed the fractions
obtained for their IGFBP-1 protease activity. As shown
in Fig. 8A, unmodified IGFBP-1 incubated with the
fraction containing components with molecular mass
above 50 kDa, remained almost unchanged, whereas in
Fig. 7. HPLC elution profiles of the V8 peptides. Trace A was
obtained from the nonphosphorylated IGFBP-1 and trace B from
the monophosphorylated protein. The arrows indicate the major
differences between the two chromatograms (peaks 1 and 2).
A
B
C
Fig. 8. IGFBP-1 proteolysis. (A) Aliquots of the nonphosphorylated
protein (1 lgin20lLof20m
M Tris pH 7.5 containing 20 mM
CaCl
2
) were incubated overnight at 37 °C with 5 lL of the amniotic
fluid fractions containing proteins eluting ahead (lane 1) and after

bation was performed in the presence of 10 mm EDTA
(data not shown). The fraction containing the IGFBP-1
protease activity was then used to assess the dose
dependence of the proteolytic process. Figure 8B shows
that by increasing the amount added to the protein the
intensity of the 30 kDa band is progressively reduced.
The protease-containing fraction was then submitted to
zymography using gelatine and IGFBP-1 as substrates
in order to examine the specificity of the IGFBP-1
degrading activity. The gelatine substrate zymogram
showed the presence of several areas of lysis, represent-
ing gelatine-degrading proteinase activity in the sample.
On the contrary, only one area was evidenced in the
IGFBP-1 substrate zymogram (Fig. 8C). These results
indicate that a specific protease is involved in the prote-
olytic cleavage occurring in amniotic fluid. We then
examined the effect of phosphorylation on this process.
Each isoform was incubated with the proteinase-
containing fraction and the samples were then submit-
ted to western blot analysis, together with identical
aliquots of the untreated proteins (Fig. 9A). The
degradation process was evaluated on the basis of the
ratio between the integrated areas of the intact protein
in the treated and untreated samples. This value was
determined for each isoform and the results (Fig. 9B)
show that the monophosphorylated IGFBP-1 was
cleaved almost to the same extent as the unmodified
protein and that the susceptibility to proteolytic
degradation of the isoforms increased with the number
of phosphates linked to the polypeptide chains. The

2
) cartridge and only three
modified serines were located [36]. Enrichment meth-
ods are well suited when only minute amounts of the
phosphoprotein are available, but a detailed analysis
of phosphoptides demands relatively high quantities of
the homogeneous molecules. All the residues identified
in the present study were recognized by the protein
A
B
Fig. 9. Proteolysis of IGFBP-1 isoforms. (A) Aliquots of each
IGFBP-1 isoform (1 lgin20lLof20m
M Tris pH 7.5 containing
20 m
M CaCl
2
) were submitted to SDS electrophoresis and immuno-
blotting following incubation at 37 °C overnight with the addition of
a4lL aliquot of the amniotic fluid fraction containing IGFBP-1 pro-
tease activity. As a control, identical aliquots of the six isoforms
diluted to the same concentration with the buffer containing 20 m
M
CaCl
2
were incubated separately at 37 °C overnight and analysed.
For each isoform, indicated with the peak number, the left lane
contains the untreated protein and the right lane the sample treated
with the protease-containing fraction. (B) Histogram representation
of the susceptibility to proteolytic degradation of IGFBP-1 isoforms.
The y-axis shows the percentage of the ratio between the spot

data show that this phosphoacceptor site is also modi-
fied in protein purified from normal amniotic fluid.
The kinases responsible for in vivo phosphorylation of
IGFBP-1 are as yet unknown, but it has been shown
that IGFBP-1 is a substrate for several protein kinases
[31,35] and the residues adjacent to all the modified se-
rines form consensus sequences for post-translational
modification by casein kinase II.
A quantitative determination of the modification
occurring at each site could not be achieved because of
the poor ionization efficiency and fast degradation of
phosphopeptides. In particular, the detection of multi-
phosphorylated peptides is reduced owing to their high
propensity to adsorption to exposed surfaces [34]. Fur-
thermore, nonspecific and partial enzymatic cleavages
caused the presence of several fragments containing the
same phosphorylation site. However, the examination
of the peptides obtained following V8 proteolytic cleav-
age indicated that the monophosphorylated IGFBP-1
form is predominantly modified within the AGSPES-
PESTE sequence containing three of the five phosphor-
ylated residues identified in the present study. It is
interesting to note that this polypeptide sequence is
rich in proline, glutamic acid, serine and threonine, a
so-called PEST region, which is typical of rapidly
metabolized proteins. Briefly, the PEST hypothesis
suggests that protein regions containing a high local
concentration of the amino acids proline, glutamic acid,
serine, threonine, and, to a lesser extent, aspartic acid,
are suitable targets for proteolytic degradation [29].

metalloproteases, little is known about the susceptibil-
ity of IGFBP-1 to enzymatic cleavage. Because the
only proteolytic degradation of IGFBP-1 occurring
without the addition of endogenous reactants so far
described under normal conditions is that observed in
the amniotic fluid [41], we performed the assays using
a partially purified fraction of amniotic fluid displaying
IGFBP-1 specific protease activity. Our data clearly
show that the post-translational modification increases
the susceptibility to cleavage by the amniotic fluid pro-
tease and suggest that not only the degree, but also the
position of the phosphate groups influence the process.
As for the other IGFBPs, the proteolytic cleavage of
IGFBP-1 occurs within the central domain and we
have shown that the process generates a C-terminal
domain (residues 141–234) carrying a phosphate group
linked to Ser169 [30]. It is possible that the charge
repulsion between the phosphate groups in the mid-
region and the one located in the C-terminal portion
of the protein may cause a conformational change that
makes the cleavage site more exposed to the protease.
Thus, the post-translational modification might act as
Phosphorylation of amniotic fluid human IGFBP-1 L. Dolcini et al.
6042 FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS
a mechanism for increasing the bioavailability of IGFs
by enhancing the proteolytic cleavage as well as for
producing the C-terminal domain.
We have examined the amino acid sequences of the
other protein family members using the PESTfind
database and found that a high score region as a

poorly structured sequence composed of  65 amino
acids connecting the N- and the C-terminal domains.
The prevalent phosphoacceptor sites of the monopho-
sphorylated protein are located in a PEST region pres-
ent in proteins that are a target for phosphorylated
mediated degradation and our data show that this
post-translational modification increases the propensity
of IGFBP-1 to proteolytic cleavage.
Experimental procedures
Chemicals
Immobilon-P poly(vinylidene) difluoride transfer mem-
branes were obtained from Millipore (Bedford, MA, USA).
Sephadex G-100, Supedex-75 and Q-Sepharose resins, as
well as the enhanced chemiluminescence detection system
were purchased from Amersham Biosciences (Piscataway,
NY, USA). Gelatin, type A from porcine skin, and alkaline
phosphatase were purchased from Sigma (St Louis, MO,
USA). A MilliQ system (Millipore) was used for water
purification. Laboratory chemicals were purchased from
Sigma, unless otherwise specified.
Isolation of IGFBP-1 isoforms from human
amniotic fluid
Human amniotic fluid was obtained from discarded
amniocentesis samples collected in weeks 16–18, pooled
and stored frozen until used. EDTA (10 mm) was added
to avoid unwanted proteolysis. The fluid (2 L) was satu-
rated to 90% with ammonium sulfate and after centrifu-
gation at 10 000 g for 1 h, the pellet was dissolved in
50 mm Tris ⁄ HCl, pH 8.0, 150 mm NaCl, 10 mm EDTA,
dialysed against the same buffer and fractioned twice by

ously described [30] and antibody titres were assayed either
by ELISA or immunoblotting. The specific IgGs were puri-
fied by affinity chromatography on a Protein A-Sepharose
column according to the manufacturer’s recommendations
(Amersham Biosciences). For western blot analyses, after
electrophoresis, the proteins were transferred by electroblot-
ting to Immobilon-P poly(vinylidene difluoride) membranes
L. Dolcini et al. Phosphorylation of amniotic fluid human IGFBP-1
FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS 6043
using a Mini Protean II apparatus (Bio-Rad). The mem-
brane was blocked with 5% w ⁄ v skim milk and probed
with the mouse antiserum diluted 1 : 2000. Immunoreactive
spots were detected with horseradish peroxidase-conjugated
anti-mouse IgG and developed by the enhanced chemilumi-
nescence method.
LC-MS analysis of IGFBP-1 isoforms
The system used for LC-MS analyses was a Surveyor LC
coupled with an ion trap mass spectrometer LCQ (Thermo
Scientific, San Jose, CA, USA) equipped with an ESI
source and controlled by xcalibur software 1.3 (Thermo
Scientific). Experiments were carried out in positive ion
mode under constant instrumental conditions: source volt-
age 4.5 kV, capillary voltage )20 V, capillary temperature
210 °C, tube lens voltage )5 V. Mass spectra were acquired
in the mass range of 200–2000 m ⁄ z by scanning the
magnetic field in 200 ms. The purified isoforms, 20 lL out
of a 0.5 mgÆmL
)1
solution in 100 mm ammonium carbon-
ate, pH 8.5, were loaded onto a C18 column (Phenomenex

)1
with a gradient 2–60% B in
120 min, 60–98% B in 30 min and 98% B for 10 min. Sol-
vents consisted of water (A) and 60% acetonitrile (B) both
containing 0.1% trifluoroacetic acid. The eluent was analysed
online with an LCQ ion trap mass spectrometer (Thermo Sci-
entific) with the ESI ion source controlled by the xcalibur
software 1.4 (Thermo Scientific). Mass spectra were gener-
ated in positive ion mode under constant instrumental condi-
tions: source voltage 4.0 kV, capillary voltage 46 V, sheath
gas flow 40 (arbitrary units), auxiliary gas flow 10 (arbitrary
units), sweep gas flow 1 (arbitrary units), capillary tempera-
ture 250 °C, tube lens voltage )105 V. MS ⁄ MS spectra,
obtained by collision-induced dissociation in the linear ion
trap, were performed with an isolation width of 3 Da (m ⁄ z),
the activation amplitude was 35% of ejection radio frequency
amplitude, which corresponds to 1.58 V. MS ⁄ MS spectra
were interpreted manually with the assistance of the predic-
tion algorithm for peptide fragmentation proteinprospec-
tor [45] and were automatically analysed using peaks
studio, version 4.2 (Bioinformatic Solution Inc., Waterloo,
Canada).
RP-HPLC separation of the V8 peptides
Nonphosphorylated and monophosphorylated fractions
(1 mg) were digested with the same protocol reported above
and were loaded on to a Vydac C18 reverse phase column
(4.6 · 250 mm). Elution was carried out with a linear gradi-
ent from 0% to 100% solvent B in 100 min at a constant
flow rate of 1 mLÆmin
)1

gel casting (1 mgÆmL
)1
) and an aliquot of the amniotic fluid
fraction, diluted 1 : 1 in nonreducing sample buffer, was
electrophoresed. Only one lane, obtained with a spacer, was
made using this substrate, whereas the remaining part of
the cassette was filled with the resolving polyacrylamide gel
Phosphorylation of amniotic fluid human IGFBP-1 L. Dolcini et al.
6044 FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS
solution. Gelatine substrate zymography was carried out
using standard zymography methods, using gelatine at a
concentration of 1 mgÆmL
)1
[46].
Acknowledgements
We are grateful to Rossella Greco, Department of
General Biology and Medical Genetics, University of
Pavia for supplying the amniotic fluid; Patrizia Arcidi-
aco, Centro Grandi Strumenti, University of Pavia for
automated sequencing; and Angelo Gallanti, Depart-
ment of Biochemistry, University of Pavia for technical
assistance. This work was supported by a grant from
the Italian Ministry of Education and Scientific
Research (FIRB 2003).
References
1 Jones JI & Clemmons DR (1995) Insulin-like growth
factors and their binding proteins: biological actions.
Endocr Rev 16, 3–34.
2 Firth SM & Baxter RC (2002) Cellular actions of the
insulin growth factor binding proteins. Endocr Rev 23,

8 Gibson JM, Aplin JD, White A & Westwood M (2001)
Regulation of IGF bioavailability in pregnancy. Mol
Hum Reprod 7, 79–87.
9 Fowler DJ, Nicolaides KH & Miell JP (2000) Insulin-
like growth factor binding protein-1 (IGFBP-1): a
multifunctional role in the human female reproductive
tract. Hum Reprod 6, 495–504.
10 Tisi DK, Liu XJ, Wykes LJ, Skinner CD & Koski KG
(2005) Insulin-like growth factor II and binding proteins
1 and 3 from second trimester human amniotic fluid are
associated with infant birth weight. J Nutr 135,
1667–1672.
11 Irving JA & Lala PK (1995) Functional role of cell
surface integrins on human trophoblast cell migration:
regulation by TGF-beta, IGF-II, and IGFBP-1. Exp
Cell Res 217, 419–427.
12 Jones JI, Gockerman A, Busby WH Jr, Wright G &
Clemmons DR (1993) Insulin-like growth factor binding
protein 1 stimulates cell migration and binds to the
alpha 5 beta 1 integrin by means of its Arg-Gly-Asp
sequence. Proc Natl Acad Sci USA 90, 10553–10557.
13 Gockerman A, Prevette T, Jones JI & Clemmons DR
(1995) Insulin-like growth factor (IGF)-binding proteins
inhibit the smooth muscle cell migration responses to
IGF-I and IGF-II. Endocrinology 136, 4168–4173.
14 Gleeson LM, Chakraborty C, McKinnon T & Lala PK
(2001) Insulin-like growth factor-binding protein 1
stimulates human trophoblast migration by signaling
through alpha 5 beta 1 integrin via mitogen-activated
protein kinase pathway. J Clin Endocrinol Metab 86,

`
S & Galliano M (2007) Mapping the 5–50
fraction of human amniotic fluid by 2-DE and ESI-MS.
Proteomics Clin Appl 1, 167–175.
21 Jones JI, D’Ercole JA, Camacho-Hubner C & Clem-
mons DR (1991) Phosphorylation of insulin-like growth
factor (IGF)-binding protein 1 in cell culture and in
vivo: effects on affinity for IGF-I. Proc Natl Acad Sci
USA 88, 7481–7485.
22 Jones JI, Busby WH Jr, Wright G, Smith CE, Kimack
NM & Clemmons DR (1993) Identification of the sites
L. Dolcini et al. Phosphorylation of amniotic fluid human IGFBP-1
FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS 6045
of phosphorylation in insulin-like growth factor binding
protein-1. Regulation of its affinity by phosphorylation
of serine 101. J Biol Chem 268, 1125–1131.
23 Seferovic MD, Ali R, Kamei H, Liu S, Khosravi JM,
Nazarian S, Han VK, Duan C & Gupta MB (2009)
Hypoxia and leucine deprivation induce human insulin-
like growth factor binding protein-1 hyperphosphoryla-
tion and increase its biological activity. Endocrinology
159, 220–231.
24 Nissum M, Abuhehab M, Sukop U, Khosravi JM, Wild-
gruber R, Eckerskorn C, Han VKM & Gupta MB (2009)
Functional and complementary phosphorylation state
attributes of human insulin-like growth factor binding
protein-1 (IGFBP-1) isoforms resolved by free flow
electrophoresis. Mol Cell Proteomics 8, 1424–1435.
25 Bankowski E, Sobolewski K, Palka J & Jaworski S
(2004) Decreased expression of the insulin-like growth

domain of insulin-like growth factor-binding protein-1
isolated from human amniotic fluid. J Biol Chem 280,
29812–29819.
31 Blom N, Gammeltoft S & Brunak S (1999) Sequence-
and structure-based prediction of eukaryotic protein
phosphorylation sites. J Mol Biol 294, 1351–1362.
32 Gibson BW & Cohen P (1990) Liquid secondary
ion mass spectrometry of phosphorylated and
sulfated peptides and proteins. Methods Enzymol
193, 480–501.
33 Kim J, Camp DG II & Smith RD (2004) Improved
detection of multi-phosphorylated peptides in the
presence of phosphoric acid in liquid chromatography ⁄
mass spectrometry. J Mass Spectrom 39, 208–215.
34 Salih E (2005) Phosphoproteomics by mass spectro-
metry and classical protein chemistry approaches. Mass
Spectrom Rev 24, 828–846.
35 Frost RA & Tseng L (1991) Insulin-like growth factor-
binding protein-1 is phosphorylated by cultured human
endometrial stromal cells and multiple protein kinases
in vitro. J Biol Chem 266, 18082–18088.
36 Temporini C, Dolcini L, Abee A, Calleri E, Galliano
M, Caccialanza G & Massolini G (2008) Development
of an integrated chromatographic system for on-line
digestion and characterization of phosphorylated
proteins. J Chromatogr A 1183, 65–75.
37 Kabir-Salmani M, Shimizu Y, Sakai K & Iwashita M
(2005) Posttranslational modifications of decidual
IGFBP-1 by steroid hormones in vitro. Mol Hum
Reprod 11, 667–671.

45 Clauser KR, Baker PR & Burlingame AL (1999) Role
of accurate mass measurement (±10 ppm) in protein
identification strategies employing MS or MS ⁄ MS and
database searching. Anal Chem 71, 2871–2882.
46 Morodomi T, Ogata Y, Sasaguri Y, Morimatsu M &
Nagase H (1992) Purification and characterization of
matrix metalloproteinase 9 from U937 monocytic
leukaemia and HT1080 fibrosarcoma cells. Biochem J
285, 603–611.
Phosphorylation of amniotic fluid human IGFBP-1 L. Dolcini et al.
6046 FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS