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Molecular & Cellular Proteomics 6:1392-1405, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
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From the Cell Signalling Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia and the ¶ Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, Sydney, New South Wales 2065, Australia
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In the cellular environment, IGFBPs can modulate IGF access to the IGF-I receptor, either inhibiting or enhancing IGF-I receptor signaling, and can also act in ways that appear to be independent of their IGF binding activity. Paradoxically IGF-dependent actions include both the inhibition and the potentiation of IGF signaling, depending on the cellular context (2). For example, IGFBP-5 has been described as an inhibitor of the cell survival function of IGF in mammary gland (3) but as a potentiator of IGF action in stimulating osteoblast proliferation (4) in addition to its IGF-independent stimulatory action (5).
Post-translational modification of members of the IGFBP family is common. Several IGFBPs are secreted as phosphoproteins. In cell culture, IGFBP-3 shows constitutive phosphorylation of serine-containing motifs similar to consensus casein kinase 2 (CK2) phosphorylation sites. Serine phosphorylation of IGFBP-3 by CK2 in vitro affects its cell surface association and susceptibility to proteolysis but not its IGF binding affinity (6). In contrast, phosphorylation of IGFBP-3 by DNA-dependent protein kinase abolishes its IGF binding, increases its nuclear retention (7, 8), and has been reported to be essential for the apoptotic and growth-inhibitory effects of IGFBP-3 (8). The ability of IGFBP-1 to potentiate or inhibit IGF action appears to depend on its phosphorylation status. Highly phosphorylated IGFBP-1 has high binding affinity and is inhibitory to IGF action, whereas the hypophosphorylated protein has lower activity and potentiates IGF action (9). A single preliminary report over a decade ago suggested that IGFBP-5 is secreted as a phosphoprotein, but nothing is known about the phosphorylation sites, the protein kinases involved, or the effect of phosphorylation on IGFBP-5 function.
IGFBP-3 and IGFBP-4 have established N-glycosylation sites. N-Linked carbohydrate in IGFBP-3 is slightly inhibitory to cell surface association but has no other known function (10). Although the structure of N-linked oligosaccharides on IGFBP-4 has been described, their function is unknown (11). O-Linked glycosylation has been found in IGFBP-5 (12) and IGFBP-6 (13) and also has been noted in an early description of IGFBP-1 (14). Like the N-linked carbohydrate in IGFBP-3 (15), the O-glycosylation of IGFBP-6 is inhibitory to cell surface binding (13).
Part of the tertiary structure of IGFBP-5 has been described. Kalus et al. (16) characterized the N-terminal domain by NMR and revealed two disulfide bond pairs at Cys47 to Cys60 and Cys54 to Cys80. Potentially IGFBP-5 has nine disulfide bonds using all of the highly conserved cysteines of the IGFBP family. Ständker et al. (12) determined by Edman sequencing that IGFBP-5 was O-glycosylated at Thr152; however, the structure of the glycan is unknown. The central domain that contains the O-glycosylation is also the predicted location of phosphorylation sites because other IGFBP family members are phosphorylated in this domain. This has not been investigated for IGFBP-5.
We analyzed IGFBP-5 by mass spectrometry to determine the in vivo post-translational modifications. Using 32P-labeling of T47D cells and tryptic digestion of IGFBP-5, we identified two phosphorylation sites. We also determined the identity of the glycans at Thr152. By analyzing the intact protein we obtained quantitative data on the amount of phosphorylation and glycosylation of IGFBP-5, thereby providing information on the amount of protein in different pools of modified IGFBP-5. We also investigated CK2 as a candidate for the protein kinase that might phosphorylate the observed sites and explored the functional relevance of these modifications.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2-macroglobulin (5 µg/ml), leupeptin (0.5 µg/ml), and aprotinin (Trasylol, Bayer Pharmaceuticals, 1:20 dilution). Radioactive media were collected, and EDTA was added to a final concentration of 0.5 mg/ml. Initial experiments were performed in 12-place multiwells alongside non-radioactive replicate plates, and media were collected at various time points for analysis by radioimmunoassay (RIA) and Western ligand blot after precipitation using IGF-I-conjugated agarose. Kinase inhibitor studies were similarly performed, and resultant comparisons were made between control wells and those containing the CK2 inhibitor 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole (DRB) (Calbiochem) at 0.01 and 0.1 mm. For peptide mapping experiments, larger scale preparations of 32P-labeled IGFBP-5 were made in 420-cm2 flasks, and IGFBPs were semipurified from clarified medium by affinity chromatography using IGF-I-conjugated agarose. Bound IGFBPs were washed with 50 mm sodium phosphate, pH 6.5, and eluted using 1 m acetic acid. Radioactive fractions were pooled, lyophilized, and stored at –80 °C until used. Non-radiolabeled T47D cell-derived IGFBP-5 was purified from conditioned serum-free RPMI 1640 medium as described above. IGF-I affinity chromatography followed by reverse-phase HPLC yielded pure, intact IGFBP-5 free from IGFBP-2 and IGFBP-4 contamination as determined by mass spectrometry. Recombinant human IGFBP-5 (AdIGFBP-5) was expressed in 911 human retinoblastoma cells using an adenoviral vector as described previously (18).
Tryptic Digestion and HPLC—
Approximately 15 µg of purified 32P-labeled IGFBP-5 from T47D cells was prepared and purified by HPLC as described previously (19). The protein was dissolved in Laemmli sample buffer and heated to near boiling for 5 min. Reduced cysteines were alkylated by addition of acrylamide to a final concentration of 2% (v/v) for 45 min prior to SDS-PAGE. The gel was zinc-stained (20), and the IGFBP-5 band was excised and diced before being destained (20) and washed three times with 1 ml of water. The gel pieces were digested with 250 ng of trypsin in 75 µl of 50 mm ammonium bicarbonate for 16 h at 37 °C. A volume of acetonitrile equal to half of the total volume was added to the solution, and the tryptic peptides were extracted after vortexing for 15 min. A further extraction was obtained with 40 µl of 5% formic acid solution after 15 min of vortexing. The combined extract was dried down to 
4 µl. This solution was made up to 50 µl with 0.1% TFA and injected into a HPLC system (SMART system, GE Healthcare). The peptides were separated by reverse-phase chromatography using a 2.1 x 100-mm column (µRPC C2/C18, 3 µm, 120 Å, GE Healthcare) at 100 µl/min. The gradient was from 0 to 40% acetonitrile in 0.1% TFA in 25 min and then to 100% acetonitrile, 0.1% TFA in 10 min. Fractions (50 µl) were collected every 30 s, dried in a rotational vacuum concentrator (ALPHA-RVC IR, Martin Christ GmbH, Osterode, Germany), and dissolved in 5 µl of 10% acetonitrile solution. A 0.5-µl aliquot of each solution was spotted onto nitrocellulose and allowed to dry. The radioactive spots on the nitrocellulose were detected by a PhosphorImager (Storm 860, GE Healthcare) after exposure for 2 weeks.
Alkaline Phosphatase Treatment and Mass Spectrometry—
A 0.5-µl aliquot of each radioactive fraction was mixed with a 2-µl solution containing 50 mm ammonium bicarbonate, 2 mm magnesium chloride, and 0.3 units/µl Antarctic phosphatase (New England Biolabs) and incubated at 22 °C for 3 h. The phosphatase-treated sample was desalted using graphite or Poros R2 microcolumns as described previously (21). Graphite microcolumns were specifically used to trap hydrophilic peptides eluting in the first few fractions. The peptides were eluted from these columns with matrix solution (0.3% TFA, 60% acetonitrile aqueous solution containing 10 mg/ml -cyano-4-hydroxycinnamic acid and 10 mm ammonium dihydrogen phosphate) directly onto the MALDI plate. The phosphatase-treated sample was compared with an equal amount of untreated sample. The MALDI spots were analyzed using a Voyager DE-Pro MALDI mass spectrometer (Applied Biosystems, Boston, MA) in positive and negative linear and reflectron modes. External calibration was used. Phosphopeptides in the remaining sample were sequenced by tandem mass spectrometry using a QSTAR XL quadrupole-TOF mass spectrometer (Applied Biosystems/MDS Sciex) using the NanoSprayTM electrospray ion source (ESI-MSMS). Samples analyzed using the NanoSpray were concentrated using microcolumns and centrifuged into nanoelectrospray capillaries (Proxeon, Odense, Denmark) in 0.1% formic acid, 50% acetonitrile aqueous solution and were sprayed at 800–1,200 V. Argon was used as the collision gas. Tandem mass spectra of phosphopeptides were acquired in multiple channel acquisition mode for 5–15 min and were manually interpreted with the aid of Analyst 1.1 and BioAnalyst 1.1 (Applied Biosystems). Mass accuracy was between 20 and 60 ppm. No effort was made to distinguish between Gln/Lys and Leu/Ile. Phosphopeptides were distinguished from sulfated peptides (none found) by use of 32P-labeling and phosphatase treatment.
Intact IGFBP-5 protein, either AdIGFBP-5 or protein purified from T47D cell medium, was reconstituted in formic acid and diluted to 0.1% formic acid. It was then concentrated and desalted using a Poros R2 microcolumn to a final concentration of 20 µm and sprayed into the QSTAR XL mass spectrometer using a nanoelectrospray capillary in 0.5% formic acid, 50% acetonitrile aqueous solution. AdIGFBP-5 was dephosphorylated by incubation with 0.3 units/µl Antarctic phosphatase as above except that 10 mm magnesium chloride was used. Fresh phosphatase was added after 3 h, and the reaction was continued for another 3 h. The IGFBP-5 was purified from the Antarctic phosphatase by precipitation on IGF-I-agarose beads. A residual amount of bovine serum albumin from the precipitation was removed by loading it onto a Poros R3 microcolumn and eluting in steps of 10% acetonitrile. The purest IGFBP-5 fraction, which eluted at 30% acetonitrile, was concentrated to 20 µm and analyzed by ESI-MS. Mass spectra were deconvoluted using the Bayesian Protein Reconstruct tool of BioAnalyst 1.1. Mass spectra were first background subtracted using BioAnalyst 1.1 with a window width setting of m/z 4. A molecular mass spectrum in the range 25,000–35,000 Da was produced from the m/z range of 850–2,000 with a step mass of 0.5 Da, a signal-to-noise threshold of 1.5, and a minimum intensity of 0.01% for 20 iterations. We have reported the apex molecular mass. Each spectrum was acquired immediately after calibration with [Glu1]-fibrinopeptide B (Sigma-Aldrich). All molecular masses were reproducible to within ±1 Da under three repeated analyses.
Phosphorylation in Vitro with CK2—
A reaction volume of 40 µl was used to phosphorylate purified AdIGFBP-5. The reaction contained 3 µg of dephosphorylated IGFBP-5, 10 mm magnesium sulfate, 20 mm Tris, pH 7.4, 25 units of CK2 (Sigma-Aldrich), and 40 µm ATP with 10 µCi of [
-32P]ATP. This reaction was allowed to progress for 5 min. Another three lots of 3 µg of IGFBP-5 were phosphorylated with CK2 without 32P label for 30 min. These samples were also phosphorylated with extra magnesium (20 mm) and ATP (400 µm). After SDS-PAGE and tryptic digestion, prior to HPLC (see above), the three non-radioactive samples were added to the radioactive sample to boost the concentration of phosphopeptides, thereby improving the signal in the mass spectral analysis.
Binding Assays—
Non-phosphorylated and non-glycosylated samples for ligand binding comparisons were prepared by enzymatic treatment of AdIGFBP-5. Twenty micrograms of AdIGFBP-5 were dephosphorylated using Antarctic phosphatase as described above. Deglycosylated IGFBP-5 was achieved by incubation of AdIGFBP-5 (20 µg) with neuraminidase (50 milliunits) and O-glycosidase (10 milliunits) (Calbiochem) together overnight at room temperature in 50 mm sodium phosphate, pH 7.5, including the protease inhibitors 2-macroglobulin (5 µg/ml), leupeptin (0.5 µg/ml), and aprotinin (Trasylol, 1:20 dilution). Purification from the treatment enzymes was achieved using IGF-I affinity chromatography.
Heparin binding analysis was performed by manually loading IGFBP-5 (1 µg) onto a heparin sulfate Hi-Trap microcolumn (Amersham Biosciences) in 10 mm sodium phosphate, pH 7.5, 0.1% BSA. A linear 30-ml gradient (0–1 m NaCl in binding buffer) was then used to elute the bound IGFBP-5 at a flow rate of 0.5 ml/min. Fractions of 1 ml were collected from which 50 µl were assayed by RIA.
Binding of IGFBP-5 to IGF-I and IGF-II was performed by an adaptation of a method described previously (18). Briefly IGFBP-5 (0.025–5 ng) was incubated with 125I-IGF-I or 125I-IGF-II (20,000 cpm/100 µl) in a final volume of 250 µl of buffer containing 50 mm sodium phosphate, pH 6.5, 0.25% BSA for 2 h at 22 °C. Cold -globulin (0.25%, 250 µl) was added, and binary complexes were precipitated using polyethylene glycol 6000 (25%, 500 µl) followed by centrifugation. Ternary complex formation assays were performed using 125I-ALS (10,000 cpm/100 µl) to which IGFBP-5 (0.01–5 ng) was allowed to bind in the presence of either IGF-I (10 ng) or IGF-II (10 ng) in a final volume of 300 µl of buffer containing 50 mm sodium phosphate, pH 6.5, 0.25% BSA for 2 h at 22 °C. In-house IGFBP-5 antiserum (SFK Y.1, 5 µl) was added and incubated overnight at 4 °C, and complexes were precipitated using sheep anti-chicken -globulin (2 µl) followed by 1 ml of 60 g/liter polyethylene glycol 6000 in 0.15 m NaCl and centrifugation.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2:1 as determined by RIA (Fig. 1b). In a larger scale purification, 700 ml of culture medium, heavily conditioned by 5 x 108 T47D cells, contained 420 µg of IGFBP-5, determined by RIA, that after IGF-I affinity chromatography and reverse-phase HPLC gave a yield of 300 µg of pure IGFBP-5. Fig. 1c shows that after IGF-I affinity chromatography, reverse-phase HPLC clearly separated IGFBP-5 from IGFBP-2 and IGFBP-4. Peaks at 27 and 36 min were immunologically confirmed as IGFBP-5 and IGFBP-2, respectively. The peak at 29 min was confirmed as IGFBP-4 by N-terminal sequencing (data not shown).
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34-kDa band was excised and digested with trypsin. The tryptic digest was applied to a reverse-phase column, and peptides were collected in fractions. The radiation detected from each fraction was measured (Fig. 2b). Each radioactive fraction was subjected to Antarctic phosphatase treatment and compared with an untreated portion of the fraction using MALDI-TOF MS. The treated and untreated spectra were examined for signals that were a result of reductions in mass equal to multiples of 80 Da, demonstrating enzymatic dephosphorylation. The molecular mass of the signals identified as phosphopeptides by mass spectrometry were matched to theoretical molecular masses of IGFBP-5 tryptic peptides and are listed in Table I and indicated on Fig. 2b. Examples of the MALDI-TOF MS spectra are shown in Supplemental Fig. S1, a–h.
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Most of the phosphopeptides detected were from the same sequence of the IGFBP-5, 88EQVKIERDSREHEEPTTSEMAEETYSPKIFRPK120, with up to three missed trypsin cleavages (Fig. 2c). In assigning the 32P radioactive peaks, these phosphopeptides were found to contain the majority of radiation. Fractions 3 and 18–22 contained 88.5% of the total measured radiation (Fig. 2b). The remaining phosphopeptides from IGFBP-5 were from the C-terminal sequence 227YGMKLPGMEYVDGDFQCHTFDSSNVE252. The contribution of phospho-IGFBP-5-(227–252) to fraction 21 is probably negligible because it was detected as a very weak signal, whereas the contribution of 231LPGMEYVDGDFQCHTFDSSNVE252 in fractions 30 and 31 is small but significant. The exact contribution, in terms of radiation, was not possible to calculate because this phosphopeptide co-eluted with a contaminating phosphopeptide matching IGFBP-2-(100–142). However, there is sufficient resolution of the two phosphopeptides, in fractions 29, 30, and 31, to conclude that phospho-IGFBP-5-(231–252) contributed between 4.5 and 7.1% of the total radiation.
The isolation and detection of an IGFBP-2 phosphopeptide was a serendipitous finding. Most of the IGFBP-2 was excluded in the purification of IGFBP-5 (Fig. 1c), and a significant signal (>1%) for IGFBP-2 was undetectable in subsequent ESI-MS analysis of all the intact protein in our preparation (data not shown). Nevertheless a tryptic phosphopeptide was detected (Supplemental Fig. S1, g and h). Due to the abundance of important fragment ions, we localized a phosphorylation site for IGFBP-2. The phosphorylated serine was detected between b6 and b7 as a dehydroalanine residue (resulting from neutral loss of H3PO4 from a phosphoserine residue) and again as the intact phosphoserine residue between y363+ and y373+ (Fig. 3). Therefore, Ser106 is an in vivo phosphorylation site for IGFBP-2.
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Glycosylation Sites of IGFBP-5—
Signals for glycopeptides (showing neutral loss of carbohydrate) were detected in fractions 15 and 16 (Fig. 2b) when analyzed by MALDI-TOF MS. A base peak signal at m/z 1,255.6 was detected in the same spectra, matching the molecular mass of IGFBP-5-(145–156). This peptide was identified previously as containing an O-linked glycosylation site at Thr152 (12). However, in that study the constituents of the O-linked glycan were not determined or studied in any detail.
The glycopeptide fractions were combined and further analyzed using tandem MS (ESI-MSMS). Several related glycopeptides were detected as shown in Fig. 5a. Each glycopeptide was fragmented, and the carbohydrate constituents were determined. An example of a fragmentation product spectrum is shown in Fig. 5b. Because only molecular mass information was revealed, only the size of the carbohydrates, but not the structure of the glycan, could be determined. However, it was clear that the glycans were combinations of hexose (Hex), N-acetylhexosamines, (HexNAc), and N-acetylneuraminic acid (NeuAc, or sialic acid). Each glycopeptide contained some y and b type ions as well (Fig. 5b) that were a result of the fragmentation of the IGFBP-5-(145–156) sequence conjugated to the glycan. The non-modified signal was also fragmented confirming that the peptide was IGFBP-5-(145–156) (data not shown).
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Post-translational Modification of Intact IGFBP-5 in Vivo—
To quantify the extent of post-translational modification, we analyzed intact or full-length, rather than protease-treated, IGFBP-5. Analysis of purified human AdIGFBP-5 by ESI-MS confirmed the presence of the same phosphorylation sites, including phospho-Ser96, and confirmed similar glycopeptides (data not shown). Therefore a similar amount of post-translational modification of the intact protein was expected. The raw ESI-MS data from intact AdIGFBP-5 was converted to molecular masses (see "Experimental Procedures") and is shown in Fig. 6a. The mature chain of human IGFBP-5 has a molecular mass of 28,573 Da (Fig. 2c, Swiss-Prot accession number P24593 without the signal sequence, i.e. the first 20 amino acids), which would be reduced to 28,555 Da if all of the 18 cysteines were disulfide bonded as is believed to be the case for all IGFBPs. IGFBP-5 was found to be highly modified. We removed the contribution of phosphorylation by incubating IGFBP-5 with phosphatase (Fig. 6b). Signals at +1,027, +738, +444, +283, +160, and +80 Da were absent or greatly decreased after dephosphorylation. The molecular mass of the unmodified form of the protein was 28,556 Da prior to incubation with phosphatase and 28,564 Da after incubation. The difference in mass between these measurements is probably due to decreased preservation of disulfide bonds as the sample handling increased. Assuming the theoretical molecular mass is correct, then eight to nine disulfide bonds were preserved during the analysis of untreated IGFBP-5, and only four to five disulfide bonds were preserved after phosphatase treatment. The small signals at 15–18 and 30–36 Da larger than the five most prominent signals in the spectrum (Fig. 6a) were probably contributions from completely disulfide reduced and/or oxidized IGFBP-5. The phosphatase-treated sample was probably also affected by oxidation to a greater extent as shown by the presence of strong signals at 15 and 31 Da higher than the dominant signal (Fig. 6b).
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We also analyzed IGFBP-5 from T47D cells to determine whether endogenously expressed IGFBP-5 has a similar profile of post-translational modification. Fig. 6c shows the molecular mass spectrum of IGFBP-5 from T47D cells. This spectrum differs from the adenoviral vector-derived IGFBP-5 in that there are signals showing a loss of 113 Da in addition to the main signals of normal mass. A likely reason for this is the absence of Leu1 from the mature IGFBP-5 chain (Fig. 2c). Signals matching the molecular mass of 1LGSFVHCEPCDEK13 and 2GSFVHCEPCDEK13 were both detected by MALDI-TOF MS. This suggests some variability in the site of cleavage for the signal peptide from the IGFBP-5 precursor. It is notable that the IGFBP-5 with loss of 113 Da is not phosphorylated to the same extent as the full-length protein. Apart from the loss of 113 Da, the state of post-translational modification of the main signals is very similar to the AdIGFBP-5. In both signal strength and the extent of modification, there is generally similar phosphorylation and glycosylation. One clear exception is that the signal for monosialylated HexNAc + Hex (+656 Da) is slightly more intense in IGFBP-5 from T47D cells (Fig. 6c) than in AdIGFBP-5 (Fig. 6a).
Phosphorylation of IGFBP-5 in Vitro with Casein Kinase 2—
Because the phosphorylation sites at Ser96 and Ser248 are generally located near acidic residues, we sought to determine whether IGFBP-5 was a substrate for phosphorylation by the acidophilic CK2. The metabolic incorporation of [32P]phosphate into IGFBP-5 over 16 h in T47D breast cancer cells was significantly inhibited (p = 0.0075) by DRB, a CK2 inhibitor. At 0.1 mm, DRB reduced [32P]phosphate incorporation by 50% when densitometry analysis was normalized to its own immunostained internal control (Fig. 7, a and b). In vitro assays with 32P labeling also showed that IGFBP-5 could act as a substrate for CK2 before or after dephosphorylation with Antarctic phosphatase (Fig. 7c). The decreased 32P incorporation observed in Fig. 7c, lane 4, may be due to some residual phosphatase activity as a result of incomplete Antarctic phosphatase inactivation. Autophosphorylation of the CK2 catalytic subunit (44 kDa) and regulatory subunit (26 kDa), as seen in Fig. 7c, lane 5, is distinct from IGFBP-5 (30–32 kDa). We therefore phosphorylated phosphatase-treated IGFBP-5 with CK2 in vitro with [-32P]ATP, resolved the protein by SDS-PAGE, cut out and digested the band, separated the peptides by HPLC, and analyzed them by MALDI-TOF MS as above for the in vivo phosphorylation site analysis. The pattern of radioactive fractions collected from the HPLC was similar (Fig. 8a) with one main group and some low radiation fractions eluting late. However, an important exception was the lack of an early eluting fraction that should have contained phospho-IGFBP-5-(92–97).
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Functional Evaluation of IGFBP-5 Phosphorylation and Glycosylation—
To evaluate the functional consequences of IGFBP-5 post-translational modifications, the recombinant protein expressed using an adenoviral vector was dephosphorylated using Antarctic phosphatase, O-deglycosylated using a combination of O-deglycosidase and neuraminidase, and then repurified to remove the enzymes. Fig. 9a shows a Western immunoblot of the purified treated preparations. Dephosphorylation consistently resulted in more highly resolved IGFBP-5 variants after SDS-PAGE (Fig. 9a, lane 2), and deglycosylation produced a single IGFBP-5 band (Fig. 9a, lane 3). The Western ligand blot using 125I-IGF-I demonstrated no resultant major loss of ligand binding activity, and enzymatic treatment had no effect on immunological recognition as determined by RIA (data not shown). Because IGFBP-5 is known to associate with a variety of cell surface and matrix proteins, the effect of treatments on the interaction of IGFBP-5 with heparin (a typical sulfated glycosaminoglycan) was first evaluated. Fig. 9b shows that both enzymatic treatments enhanced the association of IGFBP-5 with heparin; that is, deglycosylated and dephosphorylated IGFBP-5 preparations were eluted from a heparin-agarose affinity column at a higher salt concentration than the untreated protein. Despite equivalent loading of the IGFBP-5 preparations onto the heparin column, the treated samples consistently displayed decreased recovery after elution, 50% compared with untreated IGFBP-5. A second peak at elution fraction 30 was also observed with dephosphorylated IGFBP-5, suggesting a second structural subspecies.
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We determined that the majority of secreted IGFBP-5 was phosphorylated (56%). Using 32P labeling it was evident that most of the phosphorylation was detected at Ser96, and it had the highest turnover of phosphate. Also diphosphorylation was uncommon (4%).
Tryptic peptides provided information on the type of glycans at a specific site. Analysis of the full-length protein confirmed the distribution of the types of glycans and also ruled out any significant amount of alternative post-translational modifications (e.g. acetylation or myristoylation). However, we clearly observed a variability in the site of cleavage of the signal peptide with IGFBP-5 from T47D cells with Leu1 (Leu21 of the IGFBP-5 precursor) being cleaved more often than not. Apart from this variability, there was generally little difference between adenoviral vector-derived IGFBP-5 and IGFBP-5 purified from T47D cells. The similar result for adenoviral vector-derived IGFBP-5 corroborates that we have correctly determined the in vivo phosphorylation and glycosylation state of IGFBP-5.
It should be noted that comparisons of mass spectral signal intensity are only valid when the molecules are alike. Small phosphopeptides are detected with different efficiency compared with the same peptides that lack a phosphate group (22). However, in the case of large phosphopeptides (>8,000 Da), the phosphate group makes no detectable difference (23). This is in agreement with observations that protein phosphorylation often has a much smaller than predicted effect on the pI of the protein (24) and thus its ability to be protonated (or the effect is smaller than the error in the measurement). No comparable studies have been made on the effect of glycosylation on detection efficiency, and therefore it must be noted that the mass spectral analysis of the intact IGFBP-5 may not be strictly quantitative. However, the amount of phosphorylation and glycosylation determined by ESI-MS was clearly in agreement with observed gel staining and 32P distribution (c.f. Fig. 6 and Table II with Fig. 2, a and b).
The major phosphorylation site at Ser96 of IGFBP-5 is at the beginning of the non-conserved central domain. Ser96 on IGFBP-5 shares the same proximity to the N-terminal domain as Ser101 on IGFBP-1 (25), Ser111 and Ser113 on IGFBP-3 (26), and Ser106 on IGFBP-2 (determined in this study). This proximity indicates a possible role in regulating the folding between the N-terminal domain and the central domain or even regulating IGF binding affinity as shown for IGFBP-1 (25). However, this has been shown not to be the case for IGF-I binding to IGFBP-3 (26), and we similarly saw no effect of IGFBP-5 dephosphorylation on IGF-I or IGF-II binding. Alternatively the phosphorylation may regulate binding to the short heparin-binding motif at residues 133–136 (Fig. 2c), although the glycosylation at Thr152 is much nearer to this motif. We found that both deglycosylation and dephosphorylation appeared to enhance the interaction between IGFBP-5 and heparin.
The minor phosphorylation site at Ser248 is five residues from the C terminus (Fig. 2c). It is therefore in a flexible position to simultaneously interact with any protein folds or binding partners near the C terminus. IGFBP-5 has many potential binding partners outside and within the cell, including fibronectin (27), retinoid X receptor- (28), Ras-association domain family 1 protein (29), four and a half LIM protein 2 (30), and others (2). A flexible C-terminal domain that can be phosphorylated may provide a regulatable docking site for these or other proteins. A C-terminal crystal structure of IGFBP-1 (41) is available for comparison with IGFBP-5, but the final residues after the last conserved cysteine are not clearly defined. In addition to the ligand binding comparisons performed in this study, mutagenesis and other phosphosite-specific studies will be required to advance the understanding of the roles of the two phosphorylation sites determined in this work.
Because both IGFBP-1 and IGFBP-3 are phosphorylated at central domain residues close to the cysteine-rich N-terminal domain, and the kinases involved were considered likely to be CK1, CK2, or a related enzyme (26, 31), we also considered whether CK2 might phosphorylate IGFBP-5. Ser96 was predicted as a potential CK2 phosphorylation site by the kinase specificity prediction program NetPhosK 1.0, and we observed that DRB, a specific inhibitor of CK2, reduced 32P incorporation into IGFBP-5 in cellular metabolic labeling experiments. Despite this, CK2 phosphorylated IGFBP-5 at Thr103 and Ser249 but failed to phosphorylate at Ser96 and Ser248 and is therefore not likely to be an in vivo protein kinase for IGFBP-5. However, there was some evidence that there may be more in vivo phosphorylation sites of very low abundance within the region 103–115. It remains to be determined whether these very low stoichiometry sites can have a significant effect on IGFBP-5 function. Ser96 and Ser248 share a slightly acidic context, DSXXXE, and may be phosphorylated by the same in vivo kinase, which also remains to be determined.
Variable glycosylation of IGFBP-5 may differentially affect its susceptibility to different proteases. IGFBP-5 has been reported to be cleaved C-terminally to residues 138 (32), 143 (33), 188 (12), and 169 (34, 35). This implies that, in various cell types, either N-terminal or C-terminal IGFBP-5 fragments could contain O-glycosylation at Thr152. The detection of both core 1 and core 2 type glycans at Thr152 indicates that there may be different pools of IGFBP-5 that have different functional glycosylation. This could occur if IGFBP-5 was exposed to different amounts of specific glycosyltransferases. For example, in normal breast, mucin O-glycans are largely core 2 type structures, whereas in breast carcinomas they are often smaller core 1 type structures (36). Differential glycosylation may affect cell association as reported for IGFBP-3 (15) and IGFBP-6 (13).
Variants of IGFBP-5 lacking either covalently linked phosphates or carbohydrates were prepared by treatment with Antarctic phosphatase or neuraminidase and O-glycosidase, respectively. Consistent with original data published by Conover and Kiefer (37), deglycosylation of adenoviral vector-derived IGFBP-5 resulted in a single band resolved after SDS-PAGE. Dephosphorylation of IGFBP-5 was determined to be complete by ESI-MS and produced more tightly resolved isoforms after SDS-PAGE and immunoblotting. Enzymatic treatments had no effect on immunorecognition and therefore allowed accurate quantitation of purified preparations by RIA. The ability of IGFBP-5 to interact with glycosaminoglycans, including heparin sulfate, has been documented previously (38). The enhanced retention of both deglycosylated and dephosphorylated IGFBP-5 preparations by heparin-agarose is consistent with an inhibitory effect of the negative charge carried by phosphates and carbohydrates, especially sialic acids, on the central or C-terminal domain heparin-binding motifs. This modulation of heparin binding affinity may similarly affect IGFBP-5 interactions with other glycosaminoglycan and other extracellular matrix components (39). Functional studies provided no evidence that IGFBP-5 phosphorylation and O-glycosylation can modulate IGF bioavailability at the levels of the binary complex with IGF-I or IGF-II or the ternary complex with ALS in the presence of either IGF-I or IGF-II. However, the dephosphorylated preparation did show a trend to lower ALS binding in the presence of either IGF-I or IGF-II; this is in contrast to the observed effect of phosphorylation on the binding of IGFBP-3 to ALS (6). Whether phosphorylation affects the molecular distribution of IGFBP-5 between binary and ternary complexes in the circulation, and perhaps influences the size redistribution observed in pregnancy serum (40), remains to be determined.
In conclusion, we demonstrated a major phosphorylation site in human IGFBP-5 at Ser96 and a minor site at Ser248. Although CK2 inhibition in T47D breast carcinoma cell culture inhibited IGFBP-5 phosphorylation, exposure of IGFBP-5 to CK2 in vitro led to phosphorylation at Thr103 (major site) and Ser249 (minor site), suggesting that CK2 is unlikely to be an important protein kinase for IGFBP-5 at least in these breast cancer cells. Whereas the full functional significance of the complex pattern of post-translational modification of IGFBP-5 and other IGFBPs remains to be established, this study has provided the first detailed demonstration of IGFBP-5 post-translational modifications and a firm biochemical basis on which to further investigate the biological consequences of phosphorylation and glycosylation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, May 11, 2007, DOI 10.1074/mcp.M700027-MCP200
1 The abbreviations used are: IGF, insulin-like growth factor; IGFBP, IGF-binding protein; ALS, acid-labile subunit; Hex, hexose; HexNAc, N-acetylhexosamine; NeuAc, N-acetylneuraminic acid; CK, casein kinase; RIA, radioimmunoassay; DRB, 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole; S/N, signal to noise. 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
Both authors contributed equally to this work.
|| To whom correspondence should be addressed: Kolling Inst. of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. Tel.: 61-2-9926-8486; Fax: 61-2-9926-8484; E-mail: robaxter{at}med.usyd.edu.au
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, 0 mm DRB; •, 0.01 mm DRB; 
, 0.1 mm DRB). c, IGFBP-5 is a substrate for CK2 in vitro. IGFBP-5 (untreated, lanes 1 and 2; Antarctic phosphatase (AP)-treated, lanes 3 and 4) was incubated with [
