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Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia
Address all correspondence and requests for reprints to: Dr. Robert C. Baxter, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. E-mail: robaxter{at}med.usyd.edu.au
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGFBP-3 is a glycoprotein with two characteristic glycoforms of 40–45 kDa (16). In the circulation, it binds IGF-I or IGF-II in a ternary complex that also contains an 85-kDa glycoprotein, the acid-labile subunit (ALS) (2). Although the structural determinants on IGFBP-3 responsible for IGF binding are poorly defined, a cluster of basic residues near the carboxyl-terminus is known to be important for ALS binding (17), and among the five other IGFBPs, only IGFBP-5, which has a similar basic cluster, binds to ALS (18). The same basic domain is also involved in IGFBP-3 binding to glycosaminoglycans (19), plasminogen (20), and cell surfaces (17, 19) and is required for nuclear transport (15).
Consensus phosphorylation sites for a variety of serine-threonine kinases are present in the IGFBP-3 structure, mostly located in the central, nonconserved domain (21). The physiological roles of these kinases in modulating IGFBP-3 actions are poorly understood, although mitogen-activated protein (MAP) kinase has recently been implicated in the regulation of cellular sensitivity to IGFBP-3 (22). In cell culture, IGFBP-3 is secreted as a phosphoprotein, both by Chinese hamster ovary (CHO) cells transfected with a human IGFBP-3 complementary DNA (23) and by human skin fibroblasts (24). Mutagenesis studies in transfected CHO cells showed that conversion of serine residues at positions 111 and 113 of IGFBP-3 to alanine decreased [32P]phosphate incorporation by over 80%, suggesting that these were probably phospho-acceptor sites (23). As these serine residues are located within an acidic region (S111ES113EED) that matches consensus phosphorylation sequences for casein kinase 2 (CK2), this result was interpreted as indicating that an enzyme with specificity similar to that of CK2 is a major physiological IGFBP-3 kinase.
Accordingly, we investigated the effect of CK2 phosphorylation of plasma-derived IGFBP-3 on a range of its biological functions, including IGF binding, ternary complex formation, cell association, and regulation of DNA synthesis. As limited proteolysis is a well described mechanism for posttranslational modification of IGFBP-3 activity, the effect of phosphorylation on the susceptibility of IGFBP-3 to proteolytic degradation has also been evaluated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]ATP was obtained from NEN Life Science Products (Wilmington, DE). Endoglycosidase F (Endo-F)
was purchased from Roche Molecular Biochemicals (Mannheim,
Germany). Rabbit antihuman IGFBP-3 antisera R-30 and R-100, prepared in
this laboratory, have identical specificities.
IGFBP-3 phosphorylation
Typically, 10 µg IGFBP-3 were dissolved in 50 µl (final
volume) 20 mM Tris-HCl, 50 mM KCl, and 10
mM MgCl2 (pH 7.5), and ATP was added
to a concentration of 200 µM. The reaction was started
with the addition of 1 µl (500 U) CK2. Incubations were routinely
performed for 2 h at 30 C and were terminated by freezing at -80
C before final purification of the phospho-IGFBP-3 by reverse phase
HPLC. To monitor the reaction rate, similar incubations were set up,
with the further addition of 1 µCi
[-32P]ATP. Samples of 5 µl were removed at
various time points and added to 1 ml ice-cold trichloroacetic acid
(100 g/liter), and the radiolabeled IGFBP-3 was coprecipitated with 1
mg BSA, added as 100 µl of a 10 g/liter solution, and counted.
Phospho-IGFBP-3 was further purified to remove CK2 and other phosphorylation reagents, exactly as described for the final step of plasma IGFBP-3 purification (16), except that the 30-min acetonitrile gradient elution was preceded by a 10-min isocratic elution in 15% acetonitrile in 0.1% trifluoroacetic acid. Under these conditions, phospho-IGFBP-3 was eluted at 26–27 min. The concentrations of all IGFBP-3 preparations were determined by RIA (25). After phosphorylation under standard conditions, amino acid analysis (Australian Proteome Analysis Facility, Sydney, Australia) showed that the preparation contained 2.2 mol phosphoserine/mol IGFBP-3.
Immunoprecipitation of IGFBP-3
Radiolabeled IGFBP-3 was precipitated by IGFBP-3 antiserum R-30
(5 µl) and protein-A Sepharose (50 µl of a 50% suspension in PBS)
for 2 h at 22 C. Tubes were centrifuged, and the protein-A
Sepharose pellet was washed five times in 500 µl PBS containing 0.1%
Triton X-100.
Deglycosylation of IGFBP-3
For deglycosylation, radiolabeled IGFBP-3 was treated with
Endo-F. Approximately 1 µg phospho-IGFBP-3 was lyophilized and
dissolved in 15 µl distilled water with 1 g/liter BSA. Samples were
boiled for 5 min at 100 C, then 40 µl buffer containing 20
mM Tris buffer, 0.15 M NaCl, and Triton X-100
(0.5%), pH 6.5, were added. Endo-F (50 U) was added, and the reaction
mixture was incubated for 24 h at 37C. The reaction was terminated
by the addition of SDS-PAGE sample buffer.
SDS-PAGE, autoradiography, and Western blotting
SDS-PAGE was carried out as previously described (12). Samples
were heated at 100 C in sample buffer (15.5 mM Tris-HCl, 30
g/liter SDS, 10% glycerol, and 0.2 g/liter bromophenol blue at pH 6.8)
for 5 min, then applied to 10% gels. Separation was carried out over
15 h at 100 V. For autoradiography or phosphorimage analysis, gels
were stained in 1 g/liter Coomassie brilliant blue R-250 in 25%
isopropanol and 10% acetic acid, then destained in 25% methanol, 10%
acetic acid, and 3% glycerol for 2 h. Gels were dried and
analyzed by autoradiography using Hyperfilm-MP (Amersham Pharmacia Biotech, Aylesbury, UK) or by phosphorimaging using
ImageQuant software (Molecular Dynamics, Inc., Sunnyvale,
CA).
For immunoblot analysis of IGFBP-3, gels were equilibrated after electrophoresis in transfer buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol for 30 min, and proteins were transferred to Hybond-C extra nitrocellulose membrane (Amersham Pharmacia Biotech) at 250 mA over 1–2 h using an electrophoretic transfer unit (Pharmacia Biotech). After transfer, membranes were blocked with Tris-buffered saline (10 mM Tris and 150 mM NaCl, pH 7.4) containing 10 g/liter BSA for 3 h at 22 C, then probed with IGFBP-3 antiserum (R-100 at a final dilution of 1:5000) overnight at 22 C. Membranes were washed, then incubated with about 1 x 106 cpm [125I]protein A for an additional 1–2 h at 22 C. Membranes were analyzed by autoradiography or phosphorimaging.
Binding assays
Ligand binding and ternary complex formation assays were
conducted essentially as previously described (26). To measure binding
to IGF-I and -II, IGFBP-3 (0.1–25 ng) was incubated with
[125I]IGF-I or
[125I]IGF-II (10,000 cpm/100 µl) in a final
volume of 300 µl buffer containing 50 mM phosphate and
1% BSA, pH 6.5, for 2 h at 22 C. IGFBP-3 antiserum (R-30; 0.5
µl) was added for 1 h at 22 C, and complexes were precipitated
using goat antirabbit -globulin (2.5 µl), followed by 1 ml cold 60
g/liter polyethylene glycol 6000 in 0.15 M NaCl and
centrifugation. For ternary complex formation assays, IGFBP-3 was
incubated with IGF-I (10 ng/100 µl) and
[125I]ALS (10,000 cpm/100 µl) in a final
volume of 300 µl in 50 mM phosphate containing 10 g/liter
BSA at pH 6.5. Tubes were incubated for 2 h at 22 C, and IGFBP-3
antiserum (R-30; 0.5 µl) was added for 1 h at 22 C, then
complexes were precipitated as described above.
Cell binding of IGFBP-3
Cell binding of IGFBP-3 over 24 h was carried out in
confluent, serum-free cultures of simian virus 40-transformed
fibroblasts as previously described (12). For detection of bound
IGFBP-3, IGFBP-3 antibody R-30 was added at a 1:5,000 dilution for
incubation overnight at 22 C, followed by
[125I]protein A (20,000 cpm/well, diluted in SF
medium) for 4 h at 22 C. The radioactivity in cell lysates is
expressed as the total [125I]protein A
bound.
Proteolysis studies
The susceptibility of IGFBP-3 to proteases was tested in two
ways: by incubating 50 ng of each IGFBP-3 preparation at 37 C for
various times up to 30 h with 0.06 U plasmin (Sigma,
St. Louis, MO) in 50 µl 20 mM Tris-HCl and 150
mM NaCl, pH 7.5, or for various times up to 6 h with
50 µl conditioned medium from MCF-7 breast cancer cells, equilibrated
in 0.1 M sodium acetate, pH 5.5, as previously described
(27), and diluted to 100 µl in the same buffer. Increasing IGFBP-3
proteolysis was monitored as a loss of IGF-I binding, by incubating 0.5
ng proteolyzed IGFBP-3 with IGF-I tracer as described above under
Binding assays. IGFBP-3 (50 ng) incubated with the MCF-7
protease for 6 h at 37 C was also analyzed by SDS-PAGE and
immunoblotting as described above.
Thymidine incorporation
DNA synthesis was measured by incorporation of
[methyl-3H]thymidine (35
Ci/mmol; ICN Biomedicals, Inc., Costa Mesa, CA) into
neonatal foreskin fibroblasts and MCF-7 breast carcinoma cells as
previously described (28). Cells were grown to confluence in 24-well
multidishes (Nunc, Roskilde, Denmark) in RPMI 1640
medium containing 10% FCS and 10 µg/ml bovine insulin, then serum
deprived for 48 h in RPMI containing 1 g/liter BSA. Additions
(IGFBP-3 preparations and IGF-I) were made in serum-free medium. After
20 h, [methyl-3H]thymidine
(1 µCi/well) was added, incubations were continued for 4 h, then
cells were washed in 9 g/liter NaCl, fixed in methanol-acetic acid
(3:1) for 2–3 h at 4 C, and lysed in 5 g/liter SDS. Lysates were
counted in a liquid scintillation counter.
To examine IGFBP-3 potentiation of IGF-I-stimulated DNA synthesis (4), neonatal foreskin fibroblasts were plated in 48-well multidishes and grown in serum-containing medium to 50% confluence. Cells were serum deprived for 24 h, then medium containing IGFBP-3 (untreated and CK2 phosphorylated) was added, and incubations were continued for 24 h. Spent media were then removed, and IGF-I was added in fresh medium for a final 24-h incubation period. Thymidine incorporation was determined as described above during the final 4 h of this period.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]ATP
was included in the phosphorylation reaction established that under the
conditions described, IGFBP-3 phosphorylation was approaching
completion by 60 min (Fig. 1a
). A 2-h
incubation was routinely used. Under these conditions, some
incorporation of 32P into the 44-kDa 
-subunit
of CK2 was seen in the absence of IGFBP-3 (Fig. 1b, lane 1). When
IGFBP-3 was added during the reaction, radioactivity was predominantly
incorporated into the characteristic 40- to 45-kDa doublet band, with a
minor labeled band also seen near 30 kDa, corresponding to a
proteolyzed form of IGFBP-3 (Fig. 1b, lane 2). Both the doublet and the
30-kDa band were immunoprecipitable by a specific antihuman IGFBP-3
antiserum (Fig. 1b, lane 3). To test whether any of the incorporated
phosphate was present on carbohydrate,
[32P]phospho-IGFBP-3 was deglycosylated with
Endo F. As shown in Fig. 1c, this treatment reduced the apparent mol wt
of the IGFBP-3 glycosylated doublet to two major bands, probably
representing completely (
30 kDa) and incompletely (35 kDa)
deglycosylated protein, but did not reduce the intensity of the
radioactive signal, suggesting that little if any of the phosphate was
attached to carbohydrate. The minor bands at approximately 22 and 25
kDa are assumed to represent proteolyzed fragments of IGFBP-3.
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for an IGFBP-3 sample labeled with
32P, the peak of absorbance at 280 nm, eluting at
26–27 min (i.e. 16–17 min after commencing the linear
15–60% acetonitrile gradient) corresponded to the major peak of
incorporated phosphate, and when analyzed by SDS-PAGE and
autoradiography showed the expected doublet band typically seen for
IGFBP-3 (Fig. 2, inset). The peak emerging after 35 min,
containing a small amount of radioactivity, corresponds to the elution
time of CK2.
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compares the binding of radioiodinated
IGF-I or IGF-II to increasing concentrations of untreated or
phosphorylated IGFBP-3. Precipitation of complexes with an IGFBP-3
antiserum showed that phosphorylation had minimal effect on the binding
of either IGF-I or IGF-II by IGFBP-3. The binding of iodinated ALS to
the two preparations was similarly tested in the presence of unlabeled
IGF-I or IGF-II (Fig. 4, a and b). Dose
curves for phospho-IGFBP-3 were consistently shifted to the right,
indicating a decrease in ALS binding equivalent to approximately 50%
loss of potency for ternary complex formation. Competitive binding
curves performed with increasing concentrations of unlabeled ALS, as
shown in Fig. 4, c and d, similarly indicated a loss of ALS binding by
phospho-IGFBP-3 compared with the untreated preparation. Scatchard
analysis of these data showed no loss of binding affinity for ALS, but
an apparent loss of binding sites (not shown). This was not due to a
loss of immunoreactive or functional IGFBP-3 on phosphorylation, as
indicated by the unaltered IGF binding curves shown in Fig. 3.
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, the addition of untreated
IGFBP-3 at up to 500 ng/well resulted in a 5-fold increase in signal,
to 603 ± 28% of the control value. In contrast, addition of
CK2-phospho-IGFBP-3 caused only a 3-fold increase in signal, to
410 ± 19% of the control value at 500 ng/well. The inhibitory
effect of phosphorylation on IGFBP-3 cell association was highly
significant by ANOVA for repeated measures (P <
0.0001).
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shows that phospho-IGFBP-3
retained considerably more IGF-binding activity than the untreated
preparation over a 30-h incubation period in the presence of pure
plasmin, indicating that phosphorylation by CK2 leads to a relative
resistance to proteolytic degradation. A similar retention of
IGF-I-binding activity in the presence of the MCF-7 cell protease,
indicating resistance to the activity of this protease, was also seen
for the phosphorylated protein compared with the untreated preparation
(Fig. 6b). To confirm the results of the IGF-I binding assay, untreated
and phosphorylated IGFBP-3 were exposed to the MCF-7 cell protease
and analyzed by SDS-PAGE and immunoblotting. As shown in Fig. 6c, incubation of untreated IGFBP-3 with MCF-7 medium resulted in a
complete loss of immunoreactivity, whereas when phosphorylated IGFBP-3
was treated identically, a clear 40- to 45-kDa immunoreactive doublet
remained, again indicating that the phosphorylated protein was protease
resistant relative to the untreated preparation.
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, IGF-I (15 ng/ml) stimulated thymidine
incorporation 4-fold in both cell lines. Coincubation with increasing
doses of phosphorylated or control IGFBP-3 caused almost complete
reversal of the IGF-I effect, down to the baseline rate. However, there
was no difference between the effects of the two preparations in either
cell line.
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, this was not the case. If anything,
the phosphorylated preparation showed a slightly enhanced ability to
potentiate IGF-I-stimulated DNA synthesis, although the effect was
relatively small. The failure of phosphorylation to abrogate the
potentiating effect of IGFBP-3 suggests that mechanisms other than cell
association and partial proteolysis must be important in this
process.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We therefore sought to determine what effect phosphorylation by CK2 would have on a variety of IGFBP-3 functions. The untreated plasma-derived IGFBP-3 preparation contained less than 1 mol/mol phosphoserine and no detectable phosphothreonine, and at least four kinases (CK2, protein kinase A, protein kinase C, and MAP kinase) could catalyze [32P]phosphate incorporation into this material, evidence of significant unoccupied phosphoacceptor sites (not shown). After treatment with CK2, the phosphoserine content was 2.2 mol/mol, indicating, on the average, additional phosphorylation of close to two serine residues per molecule.
It had previously been reported that enzymatic dephosphorylation of secreted phospho-IGFBP-3 did not affect its IGF-I-binding activity (29), and our results are consistent with this, as no difference was seen between untreated and CK2-phosphorylated plasma-derived IGFBP-3 in their binding of IGF-I or IGF-II. Enzymatic dephosphorylation of phospho-IGFBP-3 has also been shown in a preliminary study to increase the apparent binding of ALS (32). In the present study we showed a decrease in ALS binding to the phosphorylated compared with the nonphosphorylated protein, consistent with the conclusion that the incorporation of phosphate into IGFBP-3 is inhibitory to ternary complex formation. As the immunoprecipitation method used to detect ternary IGFBP-3 complexes is identical to that used to detect binary complexes (which were unaffected by phosphorylation), we assume that this result is not an artifact due to altered immunoreactivity of phospho-IGFBP-3. ALS forms ternary complexes by binding to binary IGF-IGFBP-3 complexes (26). The interaction between ALS and these binary complexes is very sensitive to the ionic environment (33) and involves specific basic residues in the carboxyl-terminus of IGFBP-3 (17). An increase in the net negative charge of IGFBP-3 due to phosphorylation may therefore change the ionic environment sufficiently to diminish its interaction with ALS. This would only be of functional significance if circulating IGFBP-3 exists in different phosphorylation states or, at the cellular level, if the activity of secreted phospho-IGFBP-3 can be influenced by ALS in the extracellular environment.
IGFBP-3 is known to associate with the cell surface. We initially showed that heparin was able displace bound IGFBP-3, suggesting that the binding sites might be glycosaminoglycans (12); however, IGFBP-3 binding is unaffected when heparinase is used to remove glycosaminoglycans (34), indicating that this is not the case. Despite the putative identification of several proteins as IGFBP-3 receptors (35, 36), a signaling receptor remains to be identified, and there is no definitive proof that cell surface interaction is the primary step in a signaling pathway for IGFBP-3. Although the exact consequences of cell association of IGFBP-3 are unknown, structural studies have identified some of the determinants involved in the interaction. Truncation of the carboxyl-terminal domain of mammalian cell-derived human IGFBP-3 has been shown to abolish cell binding, and mutagenesis of five residues in the basic carboxyl-terminal domain (K228GRKR) to the corresponding residues of IGFBP-1 has the same effect (17). Synthetic peptides representing the basic domain sequence are also known to compete with IGFBP-3 for cell surface binding (19). In contrast to these observations, it has recently been reported that binding of an Escherichia coli-derived human IGFBP-3 preparation to Hs578T cells is partially displaced by peptides representing central domain regions of IGFBP-3 (37).
Our observation that phosphorylation of IGFBP-3, increasing its negative charge, inhibited its cell binding is consistent with the idea that positively charged residues of IGFBP-3 are essential for interaction with cell surface components. The extent of inhibition by CK2 phosphorylation was approximately 50%. In a previous study of 32P metabolically labeled IGFBP-3 produced by skin fibroblasts (24), we observed that [Leu24]IGF-I-(1–62) increased the amount of total IGFBP-3 in the cell medium without increasing the amount of phospho-IGFBP-3. As this IGF-I analog releases IGFBP-3 from the cell surface by a type I IGF-receptor-independent mechanism, this result was interpreted as indicating that the cell-bound IGFBP-3 released by the analog was in a nonphosphorylated form (24). The concept that extracellular phospho-IGFBP-3 preferentially localizes to the medium rather than the cell surface is supported by our observation that phosphorylation is inhibitory to cell association.
Limited proteolysis is well recognized as a mechanism for IGFBP regulation. In the case of IGFBP-3, there appear to be roles for both serum and tissue proteases. Circulating proteases in pregnancy, catabolic illness, and other conditions reduce the detectability of IGFBP-3 by ligand blotting (38), but their effects on the transport function of the protein are controversial, as in pregnancy IGFBP-3 appears fully proteolyzed, yet transports IGFs normally (39). At the cellular level, enzymes such as plasmin and thrombin (40, 41) can reduce IGFBP-3 affinity for its ligands, possibly releasing IGFs from the inhibitory effect of the IGFBP. This ability to increase IGF bioavailability in the cellular environment is believed to make an important contribution to the growth stimulatory activity of these proteases.
Some resistance to proteolysis by plasmin was observed even in the nonphospho-IGFBP-3 preparation, perhaps a result of a low level of phosphoserine detected before enzymatic phosphorylation. Interestingly, it appears that E. coli-derived IGFBP-3 may be fully proteolysed by plasmin (40), possibly reflecting its complete lack of glycosylation and phosphorylation. Phosphorylation by CK2 made IGFBP-3 relatively resistant to inactivation by either plasmin or a protease in MCF-7 breast cancer cell medium; it thus has the potential to decrease IGF-I bioavailability by preserving IGFBP-3 activity. In this way phosphorylation could effectively preserve the ability of IGFBP-3 to bind IGFs, even though no direct effect on binding was demonstrated.
As phosphorylation of IGFBP-3 affects cell surface association, it might be predicted to also affect the intracellular actions of IGFBP-3. However, in our study of two human cell lines (skin fibroblasts and MCF-7 breast cancer cells), no effect of either IGFBP-3 preparation was seen in the absence of added IGF-I. DNA synthesis, stimulated 4-fold by 15 ng/ml IGF-I in both cell types, was inhibited to baseline levels by the exogenous IGF-I preparations, but there was no difference between the phospho- and nonphospho forms, reflecting their similar binding of IGF-I.
CK2-phospho-IGFBP-3 binds less favorably to ALS and to the cell surface than the nonphosphoprotein, suggesting that it might preferentially localize in the extracellular environment, where its relative protection from proteolysis could allow it to function as an effective IGF regulator. However, the fact that no effect of IGFBP-3 phosphorylation on the inhibition of IGF-I-stimulated DNA synthesis was observed in two cell culture models suggests that neither proteolysis nor cell surface association has a major regulatory role in IGFBP-3 activity under these in vitro conditions. Cell binding and limited proteolysis of IGFBP-3 have also been associated with the ability of IGFBP-3 to potentiate IGF-I activity under some circumstances, involving the preincubation of cells with IGFBP-3 (4, 5). Phosphorylation, which decreases both cell binding and the susceptibility to proteolysis, might have been predicted to attenuate the ability of IGFBP-3 to potentiate IGF-I activity. However, a slight increase in the ability of phospho-IGFBP-3 to potentiate IGF-I-stimulated DNA synthesis was seen, again suggesting that other mechanisms must be more important in the potentiating effect of IGFBP-3.
These studies have demonstrated that IGFBP-3 phosphorylation by CK2 has the potential to alter its effects on cell function in several ways despite being without effect on IGF binding. However, modulation of IGFBP-3 activity in this way seems unlikely to be a dynamic method of regulating its cellular functions. If IGFBP-3 is secreted by cells with its CK2 phospho-acceptor sites already occupied, as implied by the results of Hoeck and Mukku (23), this phosphorylation would appear to be constitutive in nature and quite different from the rapid phosphorylation-dephosphorylation reactions characteristic of intracellular metabolic and signaling pathways. The possibility remains, however, that IGFBP-3 is subject to this more dynamic form of phosphorylation by enzymes other than CK2, as it contains consensus phospho-acceptor domains for a variety of kinases (21). Establishing whether these phosphorylation reactions actually occur and, if so, their biological consequences remains a fruitful area for further investigation.
Received June 16, 1999.
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) or
CK-2-phosphorylated (•) IGFBP-3. Bound radioactivity was
immunoprecipitated with anti-IGFBP-3 antiserum. Results are expressed
as the mean ± SEM from two experiments, each
performed in triplicate and normalized so that maximum binding is set
at 100%.
) or CK-2-phosphorylated
(•) IGFBP-3 on DNA synthesis stimulated by 15 ng/ml IGF-I in MCF-7
breast cancer cells (a) and neonatal human skin fibroblasts (b).
Thymidine incorporation is expressed relative to the rate in the
absence of IGF-I. Results are the mean ± SEM, pooled
for two experiments (a) or three experiments (b), each performed in
triplicate. In both cell lines, 200 ng/ml of either IGFBP-3 preparation
fully reversed the effect of IGF-I, with no difference between the
preparations.