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Phosphorylation of Rat Insulin-like Growth Factor Binding Protein-1 Does Not Affect its Biological Properties
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 357, No. 1, September 1, pp. 101–110, 1998 Article No. BB980797
Phosphorylation of Rat Insulin-like Growth Factor Binding Protein-1 Does Not Affect its Biological Properties Beverly Peterkofsky,1 Anna Gosiewska,2 Shirley Wilson, and Yeon-Ran Kim3 Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20982-4255
Received April 15, 1998, and in revised form June 11, 1998
Insulin-like growth factors (IGFs) I and II stimulate growth and expression of specific genes through binding to cell membrane receptors. IGF binding proteins also bind IGF-I with higher affinity than the receptor. They are found in the circulation and tissues and can modulate IGF actions. Human IGFBP-1 is phosphorylated on serine residues, which increases its affinity for IGF-I. An acidic, presumably phosphorylated, form of human IGFBP-1 inhibits IGF-I-stimulated DNA synthesis in cultured cells, while a less acidic, unphosphorylated form potentiates this function. Phosphorylation of human IGFBP-3, however, does not affect its affinity for IGF-I. Previously we found that multiple forms of rat IGFBP-1 are obtained by anion-exchange chromatography, raising the possibility that it also is phosphorylated, which led us to examine its properties. Phosphopeptide analysis of 32P-labeled, immunoprecipitated rat IGFBP-1 synthesized by H-4-II-EC3 rat hepatoma cells indicated that it is phosphorylated on two sites that were deduced to be ser107 and ser132 in the central nonconserved domain. Dephosphorylation of purified phosphorylated rat IGFBP-1 did not affect its affinity for IGF-I or its specific binding activity, and the dephosphorylated form inhibited DNA synthesis in 3T3 cells. Incubation of cells labeled with radioactive proline in the presence of monensin and brefeldin A, which inhibit secretion at different sites, led to intracellular accumulation of the least phosphorylated form of rat IGFBP-1, but prevented further phosphorylation. The results suggested that phosphorylation occurs at two sites in cells, the cis-Golgi and the trans-Golgi network. In summary, these studies have shown that rat IGFBP-1 is phosphorylated on
two sites by reactions that occur in different secretory organelles and that similar to human IGFBP-3, but unlike human IGFBP-1, phosphorylation does not affect its affinity for IGF-I. © 1998 Academic Press Key Words: insulin-like growth factor; insulin-like growth factor binding proteins; phosphorylation; ligand affinity; DNA synthesis.
Insulin-like growth factors (IGFs)4- I and II stimulate growth and expression of specific genes through binding to cell membrane receptors (1). In vivo, circulating IGFs are bound in a complex with an acid-labile subunit and insulin-like growth factor binding protein (IGFBP)-3 (1). There are six other IGFBPs (1–3), and normally their levels in the circulation are quite low, but they may be increased by pathological conditions (1). Most of the IGFBPs are expressed mainly in liver, but they also are produced to varying extents in other tissues and in cells in culture (1, 2). While the primary function of IGFBP-3 appears to be as a carrier of IGFs in the circulation, several of the IGFBPs, including free IGFBP-3, inhibit IGF-I action in cell cultures (1), and this may be the major role of the noncarrier IGFBPs. Serum from fasted or vitamin C-deficient (scorbutic) guinea pigs inhibit DNA, collagen, and proteoglycan synthesis in cultured connective tissue cells (4, 5), and inhibition is due mainly to IGFBP-1, and to a lesser extent IGFBP-2 (6), which are induced during these nutritional deficiencies (6, 7). The affinity of IGFBP-1 for IGF-I is greater than the affinity of the cellular 4
1 To whom correspondence should be addressed. Fax: (301) 4023095. E-mail: [email protected]. 2 Present address: Johnson and Johnson, Wound Healing Technology Resource Center, Skillman, NJ 08558. 3 Present address: Department of Microbiology, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea.
0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Abbreviations used: CK1, casein kinase 1; CK2, casein kinase 2; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; PMSF, phenylmethylsulfonyl fluoride; TBS, 0.11 M NaCl/0.05 M Tris–HCl, pH 7.4; RER, rough endoplasmic reticulum; TGN, trans Golgi network; CHO, Chinese hamster ovary; QA, a less acidic form of IGF-1 binding activity peak; QB, a more acidic form of IGF-1 binding activity peak; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; G-CK, Golgi-casein kinase. 101
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receptor (6), allowing IGFBP-1 to compete favorably for the common ligand. IGFBP-1 purified from human amniotic fluid separates into two forms on cation exchange chromatography; a more acidic form that inhibits and a less acidic form that potentiates DNA synthesis stimulated by IGF-I in several different primary cell cultures (8, 9). Human IGFBP-1 produced by endometral stromal (10) and HepG2 cells (11), and present in amniotic fluid (11, 12), occurs as a mixture of phosphorylated forms and the unphosphorylated form. Human IGFBP-1 expressed in transfected CHO cells is phosphorylated on serine residues 101, 119, and 169, and when phosphorylation is prevented by mutation of residue 101, the affinity for IGF-I is substantially decreased, although the phosphorylated and mutant forms were not tested for inhibition or potentiation (13). A phosphorylated form of human IGFBP-1 has been purified from normal plasma, and it also has a higher affinity for IGF-I than its dephosphorylated form, although the affinity for IGF-II is unaffected (14). It has been proposed that the acidic form of IGFBP-1 in amniotic fluid consists of the most highly phosphorylated form which, with its higher affinity for IGF-I, would be a more potent inhibitor than the less phosphorylated form (11). It has been reported, however, that the less acidic form of human amniotic fluid IGFBP-1 does not potentiate, but rather inhibits DNA synthesis in cartilage cultures (15), while in fetal skin fibroblasts, both the unphosphorylated and phosphorylated forms potentiate DNA synthesis (12). In addition, phosphorylation of two serines in human IGFBP-3 does not affect its affinity for IGF-I (16). Thus, the function of phosphorylation in IGFBPs remains unclear. Several of our previous observations suggest that phosphorylation may not affect the inhibitory function of guinea pig or rat IGFBP-1. Normal guinea pig serum contains a high level of alkaline phosphatase activity (17) and very low levels of IGFBP-1 (4 –7), while the reverse is true in scorbutic guinea pig serum (17, 4 –7). This situation suggests that the phosphorylation state of IGFBP-1 in scorbutic guinea pig serum would be high, which might explain its inhibitory activity. The inhibitory activity of IGFBP-1 in scorbutic guinea pig serum, however, is not eliminated when cells are cultured in mixtures of normal and scorbutic serum (4, 5), conditions that should cause dephosphorylation. Like human IGFBP-1, the rat protein yields several species that elute at different salt concentrations during anion exchange chromatography, which suggests that it also may be phosphorylated (6). Addition of the most acidic form of purified rat IGFBP-1 to cells cultured in normal guinea pig serum, conditions that should cause dephosphorylation, results in inhibition of collagen and DNA synthesis (6). Rat IGFBP-1 contains several potential phosphorylation sites (2) similar to those in human
IGFBP-1, so we determined whether the multiple forms of rat IGFBP-1 obtained from anion exchange chromatography could be accounted for by phosphorylation. We found that rat IGFP-1 was phosphorylated at two sites, and so we further determined whether phosphorylation occurred in more than one subcellular compartment during secretion and also whether phosphorylated and dephosphorylated forms differed with respect to their affinity for IGF-I and their ability to inhibit DNA synthesis. EXPERIMENTAL PROCEDURES Materials. Rat hepatoma H-4-II-EC3 (H-35) cells (ATCC 8065) were obtained from the American Type Culture Collection (Rockville, MD). Dexamethasone, monensin, brefeldin A, and HPLC-pure trypsin were purchased from Sigma (St. Louis, MO). Minimal essential medium without phosphate was purchased from ICN Biochemical Inc. (Costa Mesa, CA). [14C]Proline was obtained from Moravek Biochemicals Inc. (Brea, CA), 32P-labeled inorganic phosphate was from NEN (Boston, MA), and [125I]IGF-I was from Amersham (Arlington Heights, IL). Servalyt ampholytes (40% solutions) were obtained from Crescent Chemical Co. (Hauppauge, NY). Econo-Pac Q cartridges were obtained from Bio-Rad (Hercules, CA). Escherichia coli alkaline phosphatase, 0.4 units/ml in 2.6 M ammonium sulfate, was purchased from United States Biochemical Corp. (Cleveland, OH). Endoproteinase Asp-N was purchased from Calbiochem (San Diego, CA). Gel electrophoresis. Electrophoresis was performed in a Novex minigel apparatus. For SDS–PAGE, 10% gels were used as described previously (6). Isoelectric focusing gel electrophoresis was carried out with 5.5% polyacrylamide gels containing 10% glycerol, 2.4% ampholytes with pH ranges of 5– 8 or 3–10, with or without 4 M urea. Sample buffer consisted of 15% glycerol, 2.4% ampholytes, and 1% Triton X-100. Separated proteins were transferred to an Immobilon P membrane for ligand blotting with [125I]IGF-I, as described below, or when samples were labeled with [14C]proline, fluorograms were prepared by fixing gels in 50% methanol, briefly soaking them in FluoroHance, and then drying them under vacuum, as described previously (18). The dried gels were exposed to Kodak X-OMAT film. Purification of rat IGFBP-1. Purification was carried out as described in detail previously (6). In brief, the conditioned medium from dexamethasone-treated H-35 rat hepatoma cells was fractionated with ammonium sulfate (50% saturated), and the precipitated fraction containing IGFBP-1 was passed through an S200 HR column. Fractions containing IGF-I binding activity were pooled and further fractionated by anion exchange chromatography on an Econo-Pac Q cartridge. Several peaks of IGF-I binding activity were obtained, and they were collected into two pools, a less acidic form (QA), and a more acidic form (QB). Analysis of phosphorylated and dephosphorylated purified IGFBP-1. For analysis of [125I]IGF-I binding affinity, SDS–PAGE, and isoelectric focusing gel electrophoresis, QA or QB forms of purified IGFBP-1 (26 ng) were incubated for 30 min at 37°C with or without 0.16 units of alkaline phosphatase in a final volume of 10 ml containing 6 ml of TBS/0.1% bovine serum albumin, 0.1 M ammonium sulfate, 0.1% Triton X-100, and 0.5 mM PMSF. To measure [125I]IGF-I binding affinity, the reaction was stopped by the addition of 140 ml of ice-cold 0.1 M Tris–HCl, pH 7.6, containing 0.1% bovine serum albumin. Ten-microliter portions of diluted control and alkaline phosphatase-treated samples were assayed for binding of [125I]IGF-I, with competition by unlabeled IGF-I (0 –1 nM), as described previously (19). For isoelectric focusing gel electrophoresis or SDS–PAGE, 10 ml of the appropriate 23 sample buffer was added to the 10 ml reaction mixture, the samples were electrophoresed, and
PHOSPHORYLATION OF RAT INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-1 ligand blots were prepared using [125I]IGF-I as a probe, as described previously (6). In some experiments, samples of the QB form of purified rat IGFBP-1 (32– 65 mg) that were either untreated, or that had been incubated without or with 0.02 units/ml alkaline phosphatase, were rechromatographed on a Q cartridge by the same procedure used for the purification (6). Portions of fractions from these columns were analyzed by one or more of the procedures described above and also tested in the DNA synthesis assay described below. DNA synthesis assay. DNA synthesis was measured in quiescent BALB 3T3-714 cells, as described previously (6), with 100 ml of basal medium that contained EGF, PDGF, and IGF-I at 10 ng/ml each, which are saturating concentrations, and 0.8 mCi of [3H]thymidine. Purified rat IGFBP-1 samples were added at the time of [3H]thymidine addition. The amount of IGFBP-1 added was determined based on the binding activity of fractions compared to the specific activity (binding units/ng) of untreated purified rat IGFBP-1 (QB). Labeling of H-35 cells with radioactive precursors. H-35 cells were grown as described previously (6), except that cultures were seeded at 1.8 3 106 cells in 6 ml of MEM-5-PIE medium in 60-mm cell culture dishes. On day 3, when the cells were nearly confluent, dexamethasone (5 3 1027 M) was added to the medium, and incubation was continued for 3 h. Then the medium was removed, and cell layers were washed twice with serum-free medium containing dexamethasone, but without phosphate (MEM-0). One milliliter of MEM-0 containing dexamethasone was added, along with 10 mCi of [14C]proline or 100 mCi of 32PO2 4 , and cells were incubated for 4 h at 37°C. In some experiments, monensin and brefeldin A were added at the time of labeling with [14C]proline at concentrations specified in legends to figures. After the labeling period, the dishes were placed on ice and the culture medium was removed. The cell layer was rinsed with 1 ml per dish of TBS, and the rinse was combined with the medium. The cells were scraped off in 1 ml of TBS, they were collected by centrifugation at 500g for 5 min, and the supernatant was pooled with the medium. The cell pellet was suspended in 0.5 ml of TBS, the suspension was sonicated, and the sonicate was centrifuged at 43,000g for 10 min to obtain the supernatant. For [14C]proline-labeled samples, duplicate dishes were used for each experimental condition, and the cell sonicate and medium fractions from the two dishes were combined and concentrated to a final volume of 200 ml with a Savant Speed Vac. For 32P-labeled samples, the medium fractions from six dishes were pooled and concentrated. Immunoprecipitation. For [14C]proline-labeled samples, equivalent portions of cell and medium concentrates, each in duplicate, were used. For 32P-labeled samples, only portions of the medium concentrate were used. Samples (50 ml) were incubated for 16 h at 4°C in a total volume of 70 ml containing 0.01 M EDTA, pH 7, 0.05% Tween 20, 0.3 mg/ml bovine serum albumin, 0.01 M Tris–HCl, pH 7.6, and antibody to rat IGFBP-1 (6) at a final dilution of 1:175. Immune complexes were isolated with Protein A-Sepharose beads. For analysis by SDS–PAGE under nonreducing conditions, the beads were resuspended in sample buffer and boiled for 5 min. The suspension was cooled to room temperature, electrophoresed on a 10% gel, and a fluorogram was prepared, as described above. The relative amount of IGFBP-1 on fluorograms was quantitated by densitometry using the NIH Image program. For isoelectric focusing gel electrophoresis, the immune complex bound to protein A-Sepharose was suspended in 200 ml of 0.36 N acetic acid (pH 3.0) to dissociate the complex, and then acetic acid was removed by evaporation under vacuum. The dissociated complex was incubated at 37°C for 30 min in a total volume of 10 ml containing 5 ml of TBS; 0.1% Triton X-100; 0.5 mM phenylmethylsulfonyl fluoride, 0.08 M ammonium sulfate, with or without 0.12 units of E. coli alkaline phosphatase. The reaction mixture was cooled to room temperature, 10 ml of 23 isoelectric focusing sample buffer was added, and the samples were electrophoresed on an isoelectric focusing denaturing gel containing 4 M urea, as described above.
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FIG. 1. Anion exchange chromatography step in the purification of rat IGFBP-1. Rat IGFBP-1 was isolated from H-35 cells and purified as described under Experimental Procedures. The fractionation step on a Q cartridge is shown. Fractions were analyzed for binding of [125I]IGF-I (F) and for protein (E). Fractions 19 –23 (QA) and 26 –31 (QB) were pooled. Recovery of binding activity for four runs averaged 82%.
Phosphopeptide mapping. The procedures of Boyle et al. (20) were followed for elution of proteins from gel strips, proteolytic cleavage, and two-dimensional phosphopeptide mapping. Briefly, 32 P-labeled proteins immunoprecipitated from the medium fraction were separated by SDS–PAGE on a 10% gel, the gel was dried under vacuum at 60°C for 2 h, and it was exposed to Kodak X-OMAT film. Using the autoradiogram as a template, the band of radiolabeled IGFBP-1 was excised from the gel, the strip was homogenized in 50 mM NH4CO3, and the proteins were denatured by adding b-mercaptoethanol (0.5%), and SDS (1%) and boiling for 5 min. The eluted proteins were precipitated with trichloroacetic acid and oxidized with performic acid prior to incubation with endoproteinase Asp-N (1 mg/ml) and/or trypsin (1 mg/ml) in 50 mM NH4CO3, pH 8.0, twice for 18 –24 h at 37°C. Two-dimensional mapping of the phosphopeptides was carried out on thin-layer cellulose plates. In the first dimension, electrophoresis was performed in pH 1.9 buffer consisting of formic acid/acetic acid/H2O (1/3.1/35.9), at 22 mA constant current and 40°C for 50 min. In the second dimension, ascending chromatography was performed with 1-butanol/pyridine/acetic acid/H2O (5/2.3/1/4). The radiolabeled peptides were visualized using a PhosphoImager.
RESULTS
Properties of Rat IGFBP-1 Fractionated by Ion-Exchange Chromatography Rat IGFBP-1 was isolated from H-35 cells and purified by a series of steps including precipitation at 50% saturated ammonium sulfate, gel filtration, and anionexchange chromatography on a Q cartridge, as described previously (6). Several peaks of [125I]IGF-I binding activity were detected by anion-exchange chromatography, and fractions were pooled as QA, which included two less acidic forms, and QB, a more acidic form that appeared relatively homogeneous (Fig. 1). The two pools were analyzed by SDS–PAGE followed
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FIG. 2. SDS–PAGE of Q cartridge-purified fractions. (A) Silver staining of QA and QB pools electrophoresed on a 10% gel. (B) The QB pool (26 ng) was immunoprecipitated with 1 ml (lanes 2 and 3) or 4 ml (lanes 4 and 5) of antibody prepared against QA. Samples were incubated without (2) or with (1) alkaline phosphatase (APase), or not incubated at all (NI), and then electrophoresed on a 10% gel. IGFBP activity was detected by ligand blotting with [125I]IGF-I.
by silver staining. For both pools, single bands were observed that migrated as 31- to 32-kDa proteins, but QB (Fig. 2A, lanes 4 – 6) appeared to migrate slightly slower and as a more diffuse band than QA (Fig. 2A, lanes 1–3). This observation suggested that, like the human protein (10, 11), rat IGFBP-1 may be phosphorylated. Immunoprecipitated QB showed a slight increase in migration on SDS–PAGE after alkaline phosphatase treatment (Fig. 2B, lanes 3 and 5), supporting this conclusion. Treated and untreated rat IGFBP-1 were equally well precipitated, which established that both phosphorylated and unphosphorylated forms react with the antibody, which was raised against the less acidic QA form. Further evidence for phosphorylation of rat IGFBP-1 was obtained by isoelectric focusing gel electrophoresis. On a gel with a pH 3–10 gradient, the QA pool of IGFBP-1 consisted of two bands (Fig. 3A, lane 1). QB also gave rise to two bands (Fig. 3A, lane 3), of which the upper one migrated similarly to the lower QA band. Alkaline phosphatase converted all of the Q proteins to a less acidic form (Fig. 3A, lanes 2 and 4) that presumably is unphosphorylated IGFBP-1. When the QB fraction was analyzed on a pH 5– 8 gradient, two very acidic forms were observed (Fig. 3B, lane 1). After incubation in buffer alone (Fig. 3B, lane 2), most of QB remained highly phosphorylated, with some partial dephosphorylation. Alkaline phosphatase converted all of the forms to a single unphosphorylated form (Fig. 3B, lane 3). Control and alkaline phosphatase-treated samples of rat IGFBP-1 (QB) also were analyzed by rechromatographing them on a Q cartridge (Fig. 4) under the same conditions that were used for the initial purification. Most of the IGF-I binding activity in the control sample
still was found in the fractions eluting at the end of the 0.15 M NaCl elution step (pool II), with a peak in fractions 24 and 25. Analysis of this peak by isoelectric focusing gel electrophoresis showed that it migrated like the original highly phosphorylated QB fraction (data not shown). About 30% of the activity was present in the less acidic heterogeneous fractions comprising pool I. Isoelectric focusing analysis demonstrated that this pool consisted of partially phosphorylated forms, so although some dephosphorylation of QB occurred during chromatography, none of these species was completely dephosphorylated (data not shown). After alkaline phosphatase treatment, most of the IGF-I binding activity shifted to a peak in fractions 15 and 16. Isoelectric focusing gel electrophoresis analysis of these fractions showed that it consisted mainly of a band at the same position as the completely unphosphosphorylated form obtained by alkaline phosphatase treatment of the original QB sample (data not shown). Analysis of Phosphorylation Sites by Phosphopeptide Mapping The sites of phosphorylation in human IGFBP-1 have been found at ser101, 119, and 169, as shown in Fig. 5A. These sites resemble CK2 phosphorylation
FIG. 3. Isoelectric focusing gel electrophoresis of purified fractions QA and QB. Samples were incubated without (2) or with (1) alkaline phosphatase (APase), or not incubated at all (NI), and then electrophoresed on nondenaturing isoelectric focusing gels using ampholytes with a gradient of pH 3–10 (A) or 5– 8 (B). Ligand blotting with [125I]IGF-I was used to detect IGFBP activity. The positions of carbonic anhydrase marker (pI 5.4/5.9) are shown on both sides of the figure. On the left, the top arrow indicates the unphosphorylated form in pool A (lane 1), the middle arrow indicates a partially phosphorylated form in pools A and B (lanes 1 and 3), and the bottom arrow indicates a more phosphorylated form in pool B (lane 3). On the right, the bottom arrow indicates the most phosphorylated form in pool B (lanes 1 and 2), and the top two arrows indicate the positions of partially dephosphorylated forms (lane 2).
PHOSPHORYLATION OF RAT INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-1
FIG. 4. Rechromatography of rat IGFBP-1 (QB) on a Q cartridge. The QB form of purified rat IGFBP-1 was incubated without (F, 32 mg) or with (E, 65 mg) 0.02 units/ml of alkaline phosphatase. The [125I]IGF-I binding activities of IGFBP-1 after incubation without or with alkaline phosphatase were 17 and 18 binding units/ng, respectively. Samples were dialyzed against starting buffer and chromatographed on a Q cartridge. The [125I]IGF-I binding activity of fractions was measured and is plotted as the percentage of the activity applied to the column. For some experiments, aliquots of fractions were combined to obtain phosphorylated and unphosphorylated pools I, II, and III, as indicated above the profiles.
sites (21, 22), with acidic amino acids in position 12 and/or 13 on the C-terminal side of the serine. Sites 101 and 119 are located in the nonconserved intermediate domain, while site 169 is located within the C-terminal domain, which is highly conserved between species (2) and contains cysteine residues that participate in intramolecular disulfide bonds (23). The rat IGFBP-1 sequence has been determined (2), which allowed us to identify several serine residues as potential phosphorylation sites, based on a comparison with the human IGFBP-1 phosphorylation sites (Fig. 5A). The site at ser107, SEDE, is not conserved in the human sequence, but it is a sequence in which serine is frequently phosphorylated in other secreted proteins (21, 22). The site at ser132 in rat IGFBP-1 (SRED) is very similar to the ser119 site in the human protein (SEED), except for an arg residue in place of a glu. The sequence at ser114 in the rat protein was not considered equivalent to the human ser101 site because it contains a pro residue in the 11 position, and neither of the two ser-pro-glu sequences preceding ser101 in human IGFBP-1 are phosphorylated (13). Similarly, thr118 in a TEE sequence was not considered a potential site, since the analogous site at thr105 (TEEE) in the human protein is not phosphorylated (13), even with an additional glu residue. Ser96 in the rat protein lacks an acidic residue at the 11 and 12 positions, but it does contain two glu residues at 13 and 14. The other
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fourteen serine and nine threonine residues in rat IGFBP-2 were not in motifs that are found phosphorylated in secreted proteins (21, 22), and thus they were not considered as potential sites. Two-dimensional phosphopeptide mapping was carried out on rat IGFBP-1 that was labeled with 32PO2 4 in hepatoma cells treated with dexamethasone. Immunoprecipitated IGFBP-1 was purified by SDS–PAGE, and it was cleaved with either trypsin or endoproteinase Asp-N or by sequential treatment with both enzymes. Results of the phosphopeptide analysis showed that initial trypsin digestion reproducibly yielded a single 32 P-labeled peptide (Fig. 6A). As outlined in Fig. 5B, this could have resulted only if ser96 (T77–102), but not ser107 and ser132 (T86 –133), were phosphorylated or alternatively, if ser107 and/or ser132 were phosphorylated but not ser96. After additional digestion with endoproteinase Asp-N, the single peptide gave rise to two overlapping radioactive peptides (Fig. 6C). Since peptide T77–102 which contains ser96 would not be cleaved by Asp-N (Fig. 5B), the results strongly
FIG. 5. Potential phosphorylation sites and predicted phosphopeptides for rat IGFBP-1. (A) A comparison of the sequences containing three serine phosphorylation sites reported for human IGFBP-1 (lower sequence, S101, S119, and S169) and three potential sites in rat IGFBP-1 (S96, S107, and S132). The potential or actual serine residues phosphorylated, and adjacent acidic amino acid residues, are underlined. The alanine residue at position 182 in rat IGFBP-1 that occurs in place of ser169 in the human protein is in bold. (B) Predicted radioactive peptides that would be obtained from 32Plabeled rat IGFBP-1 cleaved by trypsin (T) or endoproteinase Asp-N (AN) or by sequential digestion with both (T/AN or AN/T). The peptides are numbered with their inclusive sequences. The potential phosphorylated serine residues are in bold.
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FIG. 7. Inhibition of IGFBP-1 secretion by brefeldin A. Brefeldin A (BFA) was added to dexamethasone-treated H-35 cells at the same time as [14C]proline. IGFBP-1 was immunoprecipitated from the cell and medium fractions with antiserum to rat IGFBP-1, and it was separated by SDS–PAGE on a 10% gel. The concentrations of brefeldin A used were (mg/ml): none (lanes 3 and 4), 2.5 (lanes 5 and 6), 5.0 (lanes 7 and 8), 10.0 (lanes 9 and 10). Aliquots of the samples without brefeldin A treatment served as minus antibody controls (lane 2). Lane 1 contained carbonic anhydrase as a molecular mass marker (30 kDa).
Subcellular Localization of Phosphorylation
FIG. 6. Two-dimensional phosphopeptide analysis of rat IGFBP-1. Dexamethasone-treated H-35 cells were labeled with 32PO2 4 , and IGFBP-1 secreted into the medium was immunoprecipitated. IGFBP-1 was purified by SDS–PAGE, extracted from the gel, and digested with proteases. The released phosphopeptides were analyzed by electrophoresis (1) and chromatography (2), as described under Experimental Procedures. Digestion was performed with either trypsin (A), endoproteinase Asp-N (B), or sequentially with trypsin/Asp-N (C), or Asp-N/trypsin (D). The vertical arrows indicate the point of application, the double horizontal arrows indicate two distinct peptides produced by Asp-N digestion, and the single horizontal arrows indicate the position of two overlapping peptides produced by sequential digestion.
suggested that both ser107 and ser132 were phosphorylated, but not ser96. Initial digestion with endoproteinase Asp-N yielded two radioactive peptides (Fig. 6B). Since the trypsin results indicated that ser96 was not a phosphorylation site, formation of two Asp-N peptides implied that ser107 in AN1-108 and ser132 in AN124-134 were phosphorylated, as outlined in Fig. 5B. Further digestion of the Asp-N-derived peptides with trypsin yielded the same pattern (Fig. 6D) as that obtained by the reverse digestion process (Fig. 6C). These results established that rat IGFBP-1 is phosphorylated at two sites, which were deduced to be ser107 and ser132.
Since two phosphosphorylation reactions occur during the secretion of rat IGFBP-1, it was of interest to determine whether they were accomplished in the same or separate subcellular sites. To investigate this question, cells were labeled with [14C]proline in the presence of either monensin, which blocks secretion between the cis- and medial-Golgi compartments (24), or brefeldin A, which blocks secretion prior to the TGN by disrupting the Golgi apparatus (25). Radioactive rat IGFBP-1 in the cell and medium fractions was immunopreciptated and analyzed on SDS–PAGE. The time course of labeling and secretion was examined and maximum secretion occurred after 4 h (data not shown). Dose-response experiments were performed in a similar manner after labeling cells for 4 h. Brefeldin A at 10 mg/ml (Fig. 7) inhibited secretion by 97%, while 0.2 mM monensin (Fig. 8) inhibited secretion by 95%,
FIG. 8. Inhibition of IGFBP-1 secretion by monensin. Procedures used were those described in the legend to Fig. 7, except that monensin was added to cell cultures. The concentrations used were (mM): none (lanes 1 and 2), 0.05 (lanes 3 and 4) ; 0.1 (lanes 5 and 6); 0.2 (lanes 7 and 8).
PHOSPHORYLATION OF RAT INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-1
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These results suggest that one phosphorylation step occurs prior to both the brefeldin A and monensin block, placing it in the RER or cis-Golgi compartment. The other phosphorylation must occur in the TGN or beyond, since brefeldin A blocks passage to the TGN. Effect of Phosphorylation of Rat IGFBP-1 on its Affinity for IGF-I
FIG. 9. Denaturing isoelectric focusing gel electrophoresis of [14C]proline-labeled IGFBP-1. H-35 cells were labeled with [14C]proline for 4 h in the absence or presence of inhibitors, and IGFBP-1 was immuprecipitated, as described in the legends to Figs. 7 and 8. Immune complexes were incubated with (1) or without (2) alkaline phosphatase (APase), and phosphorylated and unphosphorylated forms were separated on a denaturing isoelectric focusing gel as described under Experimental Procedures. Cultures either had no additions (NA, lanes 1 and 2), or were treated with 10 mg/ml brefeldin A (BFA, lanes 3 and 4) or 1 mM monensin (Mon, lanes 5 and 6). The positions of three phosphorylated forms (1–3) and the unphosphorylated form (UP) are indicated on the left.
and IGFBP-1 accumulated intracellularly. In the untreated control cultures (Fig. 7, lanes 3 and 4; Fig. 8, lanes 1 and 2) the amounts of radioactive IGFBP-1 secreted were 91 and 85% of the total, respectively. In order to determine whether the accumulated intracellular IGFBP-1 in the inhibited cultures was phosphorylated or not, the [14C]proline-labeled immunopreciptates were also analyzed by isoelectric focusing gel electrophoresis after dissociating the immune complex. Samples were incubated without or with alkaline phosphatase prior to electrophoresis to identify phosphorylated forms. Electrophoresis was carried out on a denaturing gel in order to prevent reassociation of the complex, and the migration of phosphorylated forms differed somewhat from that observed in the nondenatured gels described above. In the untreated sample (Fig. 9, lane 1), most of the radioactive IGFBP-1 was in the medium and, as with the purified protein, it consisted of several phosphorylated forms. The protein in band 1 was slightly more acidic than the unphosphorylated form obtained by alkaline phosphatase treatment (Fig. 9, lane 2) and probably contained a single phosphorylation site, while the protein in band 3 was the most acidic form, which suggested that it contained both phosphorylated sites. The small amount of band 2 may result from dephosphorylation of the band 3 protein to yield the second monophosphorylated protein. In cells treated with brefeldin A or monensin, IGFBP-1 that accumulated intracellularly contained mainly the band 1 form (Fig. 9, Cell, lanes 3 and 5).
The QB form of purified rat IGFBP-1 was dephosphorylated with alkaline phosphatase and the affinity for [125I]IGF-I was measured either directly or after the samples were rechromatographed on a Q cartridge. Controls consisted of QB that was either unincubated or incubated under the same conditions as for the dephosphorylation, except that alkaline phosphatase was not present. The results of a number of experiments showed that there was no significant difference in the affinity of the phosphorylated and unphosphorylated forms (Fig. 10) or for QA (data not shown). Scatchard plots of the data indicated that the Ka of all forms was 1.1 3 1010 M21. In addition, the specific activity of IGFBP-1 for IGF-I binding (binding units/ng protein) did not change after alkaline phosphatase treatment (see the legend to Fig. 4).
FIG. 10. IGF-I affinity competition with phosphorylated and dephosphorylated rat IGFBP-1. Samples were analyzed in duplicate for [125I]IGF-I binding activity in the presence of varying amounts of unlabeled IGF-I, as indicated. Results are presented as a percentage of the maximum binding without unlabeled ligand. For the phosphorylated samples (2), five separate analyses were performed. One sample was the original, unincubated QB analyzed directly. Four samples were QB incubated without alkaline phosphatase; two of these were analyzed as such, while two were rechromatographed as described in the legend to Fig. 4, and a pool of the most highly phosphorylated fractions (24 1 25) was used. For the dephosphorylated samples (1 APase), four separate analyses were performed: two were incubated with alkaline phosphatase and analyzed directly, while two were incubated with alkaline phosphatase and rechromatographed on a Q cartridge, and a pool of the completely dephosphorylated fractions (15 1 16) was analyzed.
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Inhibition of DNA Synthesis by Phosphorylated and Unphosphorylated Forms of Rat IGFBP-1 We previously found that the QB form of purified rat IGFBP-1 inhibited DNA synthesis in quiescent 3T3 cells (6). In this system, cells are incubated with saturating concentrations of PDGF and EGF, so that DNA synthesis is dependent only on the addition of IGF-I. To determine whether the dephosphorylated form would inhibit DNA synthesis, control or dephosphorylated QB was rechromatographed on a Q cartridge and pools I, II, and III of both fractionations were tested (Fig. 11). Unphosphorylated and phosphorylated forms of rat IGFBP-1 inhibited DNA synthesis almost identically, with 50% inhibition at approximately 120 ng/ml. Even when PDGF and EGF were omitted, so that the stimulation of DNA synthesis by IGF-I alone was much reduced, all of the forms of rat IGFBP-1 still inhibited. Potentiation with unphosphorylated human IGFBP-1 has been observed with 20 –100 ng/ml (8, 9, 12). DISCUSSION
The major observations of this study were that purified rat IGFBP-1 is phosphorylated on two sites, compared to three in human IGFBP-1, that these phosphorylations occur in two different subcellular compartments of the secretory pathway, and that unlike human IGFBP-1, the phosphorylation state of rat IGFBP-1 did not affect its affinity for IGF-I. The specific activity for binding also was not affected. In addition, the dephosphorylated form of rat IGFBP-1 inhibited DNA synthesis in cultured cells, contrary to previous findings for human IGFBP-1 (1, 8, 9). It has been proposed that the lower affinity of the unphosphorylated human protein is the basis for the ability of the human protein to potentiate, rather than inhibit DNA synthesisis (1). In some cases, potentiation is dependent on the addition of a factor in platelet-poor plasma (26), but it also occurs in the absence of this factor (12). The plasma factor may not affect the innate ability of rat IGFBP-1 to act as an inhibitor, however, since its unphosphorylated form does not have a lower affinity than the phosphorylated form. It has been pointed out previously (27) that, although the affinities of the dephosphorylated and phosphorylated forms of human IGFBP-1 for IGF-I differ, both are relatively high compared to the affinity of the receptor, so inhibition of IGF-I function may be the major role of all IGFBPs. The affinity for both forms of rat IGFBP-1, which are identical, are approximately an order of magnitude greater than that of cellular receptors for IGF-I (6). The two phosphorylated sites in rat IGFBP-1 were deduced to be serine residues 107 and 132, based on a comparison of the rat sequence with the known phosphorylation sites in human IGFBP-1 and -3 and on the
FIG. 11. Inhibition of DNA synthesis by dephosphorylated and phosphorylated rat IGFBP-1. DNA synthesis was measured in the absence, or in the presence, of varying amounts of rat IGFBP-1. Samples were pooled fractions from the I, II, and III regions of rechromatographed IGFBP-1 (QB) that were either phosphorylated (2, F) or dephosphorylated with alkaline phosphatase (1APase, E), as described in the legend to Fig. 4. Results are plotted as the percentage inhibition of the [3H]thymidine incorporation obtained in the absence of IGFBP-1.
pattern of phosphopeptides obtained after cleavage with trypsin and/or endoproteinase Asp-N. As discussed above under Results, there are a number of other serine and threonine residues in IGFBP-1, but they are not within motifs generally found as phosphorylated in secreted proteins (28, 29). The ser107 site is not conserved in the human sequence, while the ser132 site is similar to the human ser119 site. Both phosphorylation sites are in the nonconserved central domain while in human IGFBP-1 two sites are in the nonconserved domain, and one (ser169) is in the conserved C-terminal domain. The two serines phosphor-
PHOSPHORYLATION OF RAT INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-1
ylated in IGFBP-3 also are in the nonconserved central domain (16). From results of experiments which used the secretion inhibitors monensin and brefeldin A, it was concluded that one of the phosphorylation steps occurs in the RER or cis-Golgi and the other in the TGN. This conclusion is based on the fact that both inhibitors allowed formation of the least acidic, phosphorylated form of rat IGFBP-1 (band 1), but prevented further phosphorylation to band 3. Multiple cellular sites for serine phosphorylation in other proteins have been observed. ChromograninB and secretogranin are phosphorylated on serine early in the secretory pathway, probably in the medial- or trans-golgi (30). Phosphorylation of connexin43, an intercellular gap junction protein, is presumed to occur in the RER or Golgi apparatus (31). In contrast, serine phosphorylation in progastrin occurs late, in either the TGN or the secretory granules (32). Brefeldin A does not inhibit phosphorylation of b- and g-caseins but it does inhibit phosphosphorylation of a-casein, suggesting that the former are phosphorylated in the Golgi apparatus, while the latter is phosphorylated in the TGN (33). Our results extend previous observations by demonstrating that a single protein may be phosphorylated at different steps in the secretion pathway, and they also imply that two different kinases are involved. CK1 and CK2 are ubiquitous enzymes that phosphorylate casein in vitro, and apparent substrates for CK2 include both nuclear and cytoplasmic proteins (21, 22, 34). Another type of kinase, designated as Golgicasein kinase (G-CK), has been purified from a Golgienriched fraction of mammary gland (35) and liver (29) and is thought to be responsible for the phosphorylation of secreted proteins such as casein. Only a small percentage of total CK2 activity in the liver was detected in the Golgi fraction (29). The biochemical properties and substrate requirements of G-CK clearly differentiate it from CK1 and CK2. Threonine essentially is not phosphorylated by G-CK, and a peptide containing a motif ser-X-glu/ser(P)-X, an in vivo casein phosphorylation site, is a specific substrate for this enzyme, as it is not phosphorylated by either CK1 or CK2 (29, 35). For CK2, the preferred sequence is X-ser-glu/aspasp/glu-glu/asp, but an acidic residue at 13 appears to be critical (21, 22). Current evidence supports the idea that G-CK is responsible for phosphorylation of most serine residues in secreted proteins, while CK2 may play a minor role. Phosphorylation of apolipoproteins is detected using Golgi, but not RER, vesicles from liver in vitro, and CK2 is not involved (36). An enzyme purified from an RER and Golgi vesicle-enriched fraction from osteosarcoma cells was identified as CK2 on the basis of its immunogenicity and biochemical properties, and it phosphorylates the acidic bone proteins osteopontin
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and phosphoryn in vitro (37). Of the 28 phosphorylated sites in osteopontin, however, all but one of them are on serine, and G-CK rather than CK2 is probably responsible because almost all of the phosphoserines are located in ser-X-glu/ser(P)-X motifs. Two kinases that phosphorylate human IGFBP-1 in vitro were extracted from HepG2 cells, but it was not determined whether they originate in subcellular organelles (38). One of the enzymes exhibits properties similar to those of CK2, while the other enzyme does not appear to be either CK1 or CK2, raising the possibility that it is G-CK. In human IGFBP-3, mutation of aspartic acid to alanine at the 13 position in the SEED motif did not affect phosphorylation of the serine residue, while mutation of the two glutamic acid residues at 11 and 12 to alanine abolished it (16). Since an acidic residue at the 13 position is critical for CK2 activity, while a glu residue is required at 12 for G-CK actiivity, it is very likely that G-CK acts at this site. This site is identical to the ser119 site in human IGFBP-1 and similar to the SRED site in rat IGFBP-1, which suggests that G-CK is involved in their phosphorylation. Ser107 in the SEDE motif of rat IGFBP-1 is more likely to be phosphorylated by CK2, since the aspartic acid in the 12 residue makes this sequence a poor substrate for G-CK (29, 35). Since phosphorylation of rat IGFBP-1 and human IGFBP-3 does not affect their affinity for IGF-I, unlike human IGFBP-1, this raises the question of what the function of phosphorylation is in these IGFBPs. Although the effect of ser/thr phosphorylation on the activity of numerous enzymes and transcription factors is well documented (21, 22, 34), relatively little is known about the function of phophoserines in secreted proteins. The most well established example is casein, where the phosphates on serine are required for calcium binding and subsequent aggregation for micelle formation in milk (33). Phosphorylation of osteopontin can determine whether the protein will bind to the cell membrane or to fibronectin in the extracellular matrix (39), and it also may be important in bone mineralization (40). Early phosphorylation of connexin43 may prevent misfolding or degradation of the protein before its oligomerization in the Golgi apparatus (31). Based on our isoelectric focusing gel results, inhibition of phosphorylation by brefeldin A and monensin in hepatoma cells did not cause an appreciable decrease in total rat IGFBP-1, suggesting that the function of phosphorylation is not related to intracellular degradation in this case. The present studies have ruled out some possible functions for phosphorylation of IGFBPs, and have provided new information on the intracellular site of phosphorylation of rat IGFBP-1 and some insight into the kinases that may be responsible.
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ACKNOWLEDGMENTS We thank Drs. S. G. Rhee and D. Y. Jhon (NHLBI) for their generous assistance with the phosphopeptide analyses.
REFERENCES 1. Jones, J. I., and Clemmons, D. R. (1995) Endocr. Rev. 16, 3–34. 2. Shimasaki, S., and Ling, N. (1991) Prog. Growth Factor Res. 3, 243–266. 3. Wilson, E. M., Oh, Y., and Rosenfeld, R. (1997) J. Clin. Endocrinol. Metab. 87, 1301–1303. 4. Palka, J., Bird, T. A., Oyamada, I., and Peterkofsky, B. (1989) Growth Factors 1, 147–156. 5. Oyamada, I., Palka, J., Schalk, E. M., Takeda, K., and Peterkofsky, B. (1990) Arch. Biochem. Biophys. 276, 85–93. 6. Peterkofsky, B., Gosiewska, A., Kipp, D. E., Shah, V., and Wilson, S. (1994) Growth Factors 10, 229 –241. 7. Gosiewska, A., Wilson, S., Kwon, D., and Peterkofsky, B. (1994) Endocrinology 134, 1329 –1339. 8. Busby, W. H., Jr., Klapper, D. G., and Clemmons, D. R. (1988) J. Biol. Chem. 263, 14203–14210. 9. Elgin, R. G., Busby, W. H., Jr., and Clemmons, D. R. (1987) Proc. Natl. Acad. Sci. USA 84, 3254 –3258. 10. Frost, R. A., and Tseng, L. (1991) J. Biol. Chem. 266, 18082– 18088. 11. Jones, J. I., D’Ercole, J., Camacho-Hubner, C., and Clemmons, D. R. (1991) Proc. Natl. Acad. Sci. USA 88, 7481–7485. 12. Koistinen, R., Angervo, M., Leinonen, P., Hakala, T, and Seppa¨la¨, M. (1993) Clin. Chim. Acta 215, 189 –199. 13. Jones, J. I., Busby, W. H., Jr., Wright, G., Smith, C. E., Kimack, N. M., and Clemmons, D. R. (1993) J. Biol. Chem. 268, 1125– 1131. 14. Westwood, M., Gibson, J. M., and White, A. (1997) Endocrinology 138, 1130 –1136. 15. Burch, W. M., Correa, J., Shively, J. E., and Powell, D. R. (1990) J. Clin. Endocrinol. Metab. 70, 173–180. 16. Hoeck, W. G., and Mukku, V. R. (1994) J. Cell. Biochem. 56, 262–273. 17. Mahmoodian, F., Gosiewska, A., and Peterkofsky, B. (1996) Arch. Biochem. Biophys. 336, 86 –96. 18. Peterkofsky, B., Haralson, M. A., Dimari, S. J., and Miller, E. J. (1995) in Extracellular Matrix. A Practical Approach (Haralson, M. A., and Hassell, J. R., Eds.), pp. 31–71, Oxford University Press, New York.
19. Palka, J., and Peterkofsky, B. (1988) Anal. Biochem. 175, 442– 449. 20. Boyle, W. J., van der Geer, P., and Hunter, T. (1991) Methods Enzymol. 201, 110 –149. 21. Tuazon, P. T., and Traugh, J. A. (1991) in Advances in Second Messenger and Phosphoprotein Research (Greengard, P., and Robison, G.A., Eds.), Vol. 23, pp. 123–164, Raven Press, New York. 22. Songyang, Z., Lu, K. P., Kwon, Y. T., Tsai, L. H., Filhol, O., Cochet, C., Brickey, D. A., Soderling, T. R., Bartleson, C., Graves, D. J., DeMaggio, A. J., Hoekstra, M. F., Blenis, J., Hunter, T., and Cantley, L. C. (1996) Mol. Cell. Biol. 16, 6486 – 6493. 23. Brinkman, A., Korleve, D. F., Schuller, A. G. P., Zwarthoff, E. C., and Drop, S. L. S. (1991) FEBS Lett. 291, 264 –268. 24. Tartakoff, A. M. (1983) Methods Enzymol. 98, 47–59. 25. Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz, J. (1992) J. Cell Biol. 116,1071–1080. 26. Clemmons, D. R., and Gardner, L. I. (1990) J. Cell. Physiol. 145, 129 –135. 27. Lee, P. D. K., Giudice, L. C., Conover, C. A., and Powell, D. R. (1997) Proc. Soc. Exp. Biol. Med. 216, 319 –357. 28. Sorensen, E. S., Hojrup, P., and Petersen, T. E. (1995) Protein Sci. 4, 2040 –2049. 29. Lasa, M., Marin, O., and Pinna, L. A. (1997) Eur. J. Biochem. 243, 719 –725. 30. Rosa, P., Mantovani, S., Rosbach, R., and Huttner, W. B. (1992) J. Biol. Chem. 267, 12227–12232. 31. Laird, D. W., Castillo, M., and Kasprzak, L. (1995) J. Cell Biol. 131, 1193–1203. 32. Varro, A., Henry, J., Vaillant, C., and Dockray, G. J. (1994) J. Biol. Chem. 269, 20764 –20770. 33. Turner, M. D., Handel, S. E., Wilde, C. F., and Burgoyne, R. D. (1993) J. Cell Sci. 106, 1221–1226. 34. Allende, J. E., and Allende, C. C. (1995) FASEB J. 9, 313–323. 35. Lasa-Benito, M., Marin, O., Meggio, F., and Pinna, L. A. (1996) FEBS Lett. 382, 149 –152. 36. Swift, L. L. (1996) J. Biol. Chem. 271, 31491–31495. 37. Wu, C. B., Shiizu, Y., Ng., A., and Pan, Y. M. (1996) Connect. Tissue Res. 34, 23–32. 38. Ankrapp, S. P., Jones, J. I., and Clemmons, D. R. (1996) J. Cell. Biochem. 60, 387–389. 39. Nemir, M., DeVouge, M. W., and Mukherjee, B. B. (1989) J. Biol. Chem. 264, 18202–18208. 40. Sfeir, C., and Veis, A. (1995) J. Bone Miner. Res. 10, 607– 615.
Vol. 357, No. 1, September 1, pp. 101–110, 1998 Article No. BB980797
Phosphorylation of Rat Insulin-like Growth Factor Binding Protein-1 Does Not Affect its Biological Properties Beverly Peterkofsky,1 Anna Gosiewska,2 Shirley Wilson, and Yeon-Ran Kim3 Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20982-4255
Received April 15, 1998, and in revised form June 11, 1998
Insulin-like growth factors (IGFs) I and II stimulate growth and expression of specific genes through binding to cell membrane receptors. IGF binding proteins also bind IGF-I with higher affinity than the receptor. They are found in the circulation and tissues and can modulate IGF actions. Human IGFBP-1 is phosphorylated on serine residues, which increases its affinity for IGF-I. An acidic, presumably phosphorylated, form of human IGFBP-1 inhibits IGF-I-stimulated DNA synthesis in cultured cells, while a less acidic, unphosphorylated form potentiates this function. Phosphorylation of human IGFBP-3, however, does not affect its affinity for IGF-I. Previously we found that multiple forms of rat IGFBP-1 are obtained by anion-exchange chromatography, raising the possibility that it also is phosphorylated, which led us to examine its properties. Phosphopeptide analysis of 32P-labeled, immunoprecipitated rat IGFBP-1 synthesized by H-4-II-EC3 rat hepatoma cells indicated that it is phosphorylated on two sites that were deduced to be ser107 and ser132 in the central nonconserved domain. Dephosphorylation of purified phosphorylated rat IGFBP-1 did not affect its affinity for IGF-I or its specific binding activity, and the dephosphorylated form inhibited DNA synthesis in 3T3 cells. Incubation of cells labeled with radioactive proline in the presence of monensin and brefeldin A, which inhibit secretion at different sites, led to intracellular accumulation of the least phosphorylated form of rat IGFBP-1, but prevented further phosphorylation. The results suggested that phosphorylation occurs at two sites in cells, the cis-Golgi and the trans-Golgi network. In summary, these studies have shown that rat IGFBP-1 is phosphorylated on
two sites by reactions that occur in different secretory organelles and that similar to human IGFBP-3, but unlike human IGFBP-1, phosphorylation does not affect its affinity for IGF-I. © 1998 Academic Press Key Words: insulin-like growth factor; insulin-like growth factor binding proteins; phosphorylation; ligand affinity; DNA synthesis.
Insulin-like growth factors (IGFs)4- I and II stimulate growth and expression of specific genes through binding to cell membrane receptors (1). In vivo, circulating IGFs are bound in a complex with an acid-labile subunit and insulin-like growth factor binding protein (IGFBP)-3 (1). There are six other IGFBPs (1–3), and normally their levels in the circulation are quite low, but they may be increased by pathological conditions (1). Most of the IGFBPs are expressed mainly in liver, but they also are produced to varying extents in other tissues and in cells in culture (1, 2). While the primary function of IGFBP-3 appears to be as a carrier of IGFs in the circulation, several of the IGFBPs, including free IGFBP-3, inhibit IGF-I action in cell cultures (1), and this may be the major role of the noncarrier IGFBPs. Serum from fasted or vitamin C-deficient (scorbutic) guinea pigs inhibit DNA, collagen, and proteoglycan synthesis in cultured connective tissue cells (4, 5), and inhibition is due mainly to IGFBP-1, and to a lesser extent IGFBP-2 (6), which are induced during these nutritional deficiencies (6, 7). The affinity of IGFBP-1 for IGF-I is greater than the affinity of the cellular 4
1 To whom correspondence should be addressed. Fax: (301) 4023095. E-mail: [email protected]. 2 Present address: Johnson and Johnson, Wound Healing Technology Resource Center, Skillman, NJ 08558. 3 Present address: Department of Microbiology, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea.
0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Abbreviations used: CK1, casein kinase 1; CK2, casein kinase 2; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; PMSF, phenylmethylsulfonyl fluoride; TBS, 0.11 M NaCl/0.05 M Tris–HCl, pH 7.4; RER, rough endoplasmic reticulum; TGN, trans Golgi network; CHO, Chinese hamster ovary; QA, a less acidic form of IGF-1 binding activity peak; QB, a more acidic form of IGF-1 binding activity peak; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; G-CK, Golgi-casein kinase. 101
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receptor (6), allowing IGFBP-1 to compete favorably for the common ligand. IGFBP-1 purified from human amniotic fluid separates into two forms on cation exchange chromatography; a more acidic form that inhibits and a less acidic form that potentiates DNA synthesis stimulated by IGF-I in several different primary cell cultures (8, 9). Human IGFBP-1 produced by endometral stromal (10) and HepG2 cells (11), and present in amniotic fluid (11, 12), occurs as a mixture of phosphorylated forms and the unphosphorylated form. Human IGFBP-1 expressed in transfected CHO cells is phosphorylated on serine residues 101, 119, and 169, and when phosphorylation is prevented by mutation of residue 101, the affinity for IGF-I is substantially decreased, although the phosphorylated and mutant forms were not tested for inhibition or potentiation (13). A phosphorylated form of human IGFBP-1 has been purified from normal plasma, and it also has a higher affinity for IGF-I than its dephosphorylated form, although the affinity for IGF-II is unaffected (14). It has been proposed that the acidic form of IGFBP-1 in amniotic fluid consists of the most highly phosphorylated form which, with its higher affinity for IGF-I, would be a more potent inhibitor than the less phosphorylated form (11). It has been reported, however, that the less acidic form of human amniotic fluid IGFBP-1 does not potentiate, but rather inhibits DNA synthesis in cartilage cultures (15), while in fetal skin fibroblasts, both the unphosphorylated and phosphorylated forms potentiate DNA synthesis (12). In addition, phosphorylation of two serines in human IGFBP-3 does not affect its affinity for IGF-I (16). Thus, the function of phosphorylation in IGFBPs remains unclear. Several of our previous observations suggest that phosphorylation may not affect the inhibitory function of guinea pig or rat IGFBP-1. Normal guinea pig serum contains a high level of alkaline phosphatase activity (17) and very low levels of IGFBP-1 (4 –7), while the reverse is true in scorbutic guinea pig serum (17, 4 –7). This situation suggests that the phosphorylation state of IGFBP-1 in scorbutic guinea pig serum would be high, which might explain its inhibitory activity. The inhibitory activity of IGFBP-1 in scorbutic guinea pig serum, however, is not eliminated when cells are cultured in mixtures of normal and scorbutic serum (4, 5), conditions that should cause dephosphorylation. Like human IGFBP-1, the rat protein yields several species that elute at different salt concentrations during anion exchange chromatography, which suggests that it also may be phosphorylated (6). Addition of the most acidic form of purified rat IGFBP-1 to cells cultured in normal guinea pig serum, conditions that should cause dephosphorylation, results in inhibition of collagen and DNA synthesis (6). Rat IGFBP-1 contains several potential phosphorylation sites (2) similar to those in human
IGFBP-1, so we determined whether the multiple forms of rat IGFBP-1 obtained from anion exchange chromatography could be accounted for by phosphorylation. We found that rat IGFP-1 was phosphorylated at two sites, and so we further determined whether phosphorylation occurred in more than one subcellular compartment during secretion and also whether phosphorylated and dephosphorylated forms differed with respect to their affinity for IGF-I and their ability to inhibit DNA synthesis. EXPERIMENTAL PROCEDURES Materials. Rat hepatoma H-4-II-EC3 (H-35) cells (ATCC 8065) were obtained from the American Type Culture Collection (Rockville, MD). Dexamethasone, monensin, brefeldin A, and HPLC-pure trypsin were purchased from Sigma (St. Louis, MO). Minimal essential medium without phosphate was purchased from ICN Biochemical Inc. (Costa Mesa, CA). [14C]Proline was obtained from Moravek Biochemicals Inc. (Brea, CA), 32P-labeled inorganic phosphate was from NEN (Boston, MA), and [125I]IGF-I was from Amersham (Arlington Heights, IL). Servalyt ampholytes (40% solutions) were obtained from Crescent Chemical Co. (Hauppauge, NY). Econo-Pac Q cartridges were obtained from Bio-Rad (Hercules, CA). Escherichia coli alkaline phosphatase, 0.4 units/ml in 2.6 M ammonium sulfate, was purchased from United States Biochemical Corp. (Cleveland, OH). Endoproteinase Asp-N was purchased from Calbiochem (San Diego, CA). Gel electrophoresis. Electrophoresis was performed in a Novex minigel apparatus. For SDS–PAGE, 10% gels were used as described previously (6). Isoelectric focusing gel electrophoresis was carried out with 5.5% polyacrylamide gels containing 10% glycerol, 2.4% ampholytes with pH ranges of 5– 8 or 3–10, with or without 4 M urea. Sample buffer consisted of 15% glycerol, 2.4% ampholytes, and 1% Triton X-100. Separated proteins were transferred to an Immobilon P membrane for ligand blotting with [125I]IGF-I, as described below, or when samples were labeled with [14C]proline, fluorograms were prepared by fixing gels in 50% methanol, briefly soaking them in FluoroHance, and then drying them under vacuum, as described previously (18). The dried gels were exposed to Kodak X-OMAT film. Purification of rat IGFBP-1. Purification was carried out as described in detail previously (6). In brief, the conditioned medium from dexamethasone-treated H-35 rat hepatoma cells was fractionated with ammonium sulfate (50% saturated), and the precipitated fraction containing IGFBP-1 was passed through an S200 HR column. Fractions containing IGF-I binding activity were pooled and further fractionated by anion exchange chromatography on an Econo-Pac Q cartridge. Several peaks of IGF-I binding activity were obtained, and they were collected into two pools, a less acidic form (QA), and a more acidic form (QB). Analysis of phosphorylated and dephosphorylated purified IGFBP-1. For analysis of [125I]IGF-I binding affinity, SDS–PAGE, and isoelectric focusing gel electrophoresis, QA or QB forms of purified IGFBP-1 (26 ng) were incubated for 30 min at 37°C with or without 0.16 units of alkaline phosphatase in a final volume of 10 ml containing 6 ml of TBS/0.1% bovine serum albumin, 0.1 M ammonium sulfate, 0.1% Triton X-100, and 0.5 mM PMSF. To measure [125I]IGF-I binding affinity, the reaction was stopped by the addition of 140 ml of ice-cold 0.1 M Tris–HCl, pH 7.6, containing 0.1% bovine serum albumin. Ten-microliter portions of diluted control and alkaline phosphatase-treated samples were assayed for binding of [125I]IGF-I, with competition by unlabeled IGF-I (0 –1 nM), as described previously (19). For isoelectric focusing gel electrophoresis or SDS–PAGE, 10 ml of the appropriate 23 sample buffer was added to the 10 ml reaction mixture, the samples were electrophoresed, and
PHOSPHORYLATION OF RAT INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-1 ligand blots were prepared using [125I]IGF-I as a probe, as described previously (6). In some experiments, samples of the QB form of purified rat IGFBP-1 (32– 65 mg) that were either untreated, or that had been incubated without or with 0.02 units/ml alkaline phosphatase, were rechromatographed on a Q cartridge by the same procedure used for the purification (6). Portions of fractions from these columns were analyzed by one or more of the procedures described above and also tested in the DNA synthesis assay described below. DNA synthesis assay. DNA synthesis was measured in quiescent BALB 3T3-714 cells, as described previously (6), with 100 ml of basal medium that contained EGF, PDGF, and IGF-I at 10 ng/ml each, which are saturating concentrations, and 0.8 mCi of [3H]thymidine. Purified rat IGFBP-1 samples were added at the time of [3H]thymidine addition. The amount of IGFBP-1 added was determined based on the binding activity of fractions compared to the specific activity (binding units/ng) of untreated purified rat IGFBP-1 (QB). Labeling of H-35 cells with radioactive precursors. H-35 cells were grown as described previously (6), except that cultures were seeded at 1.8 3 106 cells in 6 ml of MEM-5-PIE medium in 60-mm cell culture dishes. On day 3, when the cells were nearly confluent, dexamethasone (5 3 1027 M) was added to the medium, and incubation was continued for 3 h. Then the medium was removed, and cell layers were washed twice with serum-free medium containing dexamethasone, but without phosphate (MEM-0). One milliliter of MEM-0 containing dexamethasone was added, along with 10 mCi of [14C]proline or 100 mCi of 32PO2 4 , and cells were incubated for 4 h at 37°C. In some experiments, monensin and brefeldin A were added at the time of labeling with [14C]proline at concentrations specified in legends to figures. After the labeling period, the dishes were placed on ice and the culture medium was removed. The cell layer was rinsed with 1 ml per dish of TBS, and the rinse was combined with the medium. The cells were scraped off in 1 ml of TBS, they were collected by centrifugation at 500g for 5 min, and the supernatant was pooled with the medium. The cell pellet was suspended in 0.5 ml of TBS, the suspension was sonicated, and the sonicate was centrifuged at 43,000g for 10 min to obtain the supernatant. For [14C]proline-labeled samples, duplicate dishes were used for each experimental condition, and the cell sonicate and medium fractions from the two dishes were combined and concentrated to a final volume of 200 ml with a Savant Speed Vac. For 32P-labeled samples, the medium fractions from six dishes were pooled and concentrated. Immunoprecipitation. For [14C]proline-labeled samples, equivalent portions of cell and medium concentrates, each in duplicate, were used. For 32P-labeled samples, only portions of the medium concentrate were used. Samples (50 ml) were incubated for 16 h at 4°C in a total volume of 70 ml containing 0.01 M EDTA, pH 7, 0.05% Tween 20, 0.3 mg/ml bovine serum albumin, 0.01 M Tris–HCl, pH 7.6, and antibody to rat IGFBP-1 (6) at a final dilution of 1:175. Immune complexes were isolated with Protein A-Sepharose beads. For analysis by SDS–PAGE under nonreducing conditions, the beads were resuspended in sample buffer and boiled for 5 min. The suspension was cooled to room temperature, electrophoresed on a 10% gel, and a fluorogram was prepared, as described above. The relative amount of IGFBP-1 on fluorograms was quantitated by densitometry using the NIH Image program. For isoelectric focusing gel electrophoresis, the immune complex bound to protein A-Sepharose was suspended in 200 ml of 0.36 N acetic acid (pH 3.0) to dissociate the complex, and then acetic acid was removed by evaporation under vacuum. The dissociated complex was incubated at 37°C for 30 min in a total volume of 10 ml containing 5 ml of TBS; 0.1% Triton X-100; 0.5 mM phenylmethylsulfonyl fluoride, 0.08 M ammonium sulfate, with or without 0.12 units of E. coli alkaline phosphatase. The reaction mixture was cooled to room temperature, 10 ml of 23 isoelectric focusing sample buffer was added, and the samples were electrophoresed on an isoelectric focusing denaturing gel containing 4 M urea, as described above.
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FIG. 1. Anion exchange chromatography step in the purification of rat IGFBP-1. Rat IGFBP-1 was isolated from H-35 cells and purified as described under Experimental Procedures. The fractionation step on a Q cartridge is shown. Fractions were analyzed for binding of [125I]IGF-I (F) and for protein (E). Fractions 19 –23 (QA) and 26 –31 (QB) were pooled. Recovery of binding activity for four runs averaged 82%.
Phosphopeptide mapping. The procedures of Boyle et al. (20) were followed for elution of proteins from gel strips, proteolytic cleavage, and two-dimensional phosphopeptide mapping. Briefly, 32 P-labeled proteins immunoprecipitated from the medium fraction were separated by SDS–PAGE on a 10% gel, the gel was dried under vacuum at 60°C for 2 h, and it was exposed to Kodak X-OMAT film. Using the autoradiogram as a template, the band of radiolabeled IGFBP-1 was excised from the gel, the strip was homogenized in 50 mM NH4CO3, and the proteins were denatured by adding b-mercaptoethanol (0.5%), and SDS (1%) and boiling for 5 min. The eluted proteins were precipitated with trichloroacetic acid and oxidized with performic acid prior to incubation with endoproteinase Asp-N (1 mg/ml) and/or trypsin (1 mg/ml) in 50 mM NH4CO3, pH 8.0, twice for 18 –24 h at 37°C. Two-dimensional mapping of the phosphopeptides was carried out on thin-layer cellulose plates. In the first dimension, electrophoresis was performed in pH 1.9 buffer consisting of formic acid/acetic acid/H2O (1/3.1/35.9), at 22 mA constant current and 40°C for 50 min. In the second dimension, ascending chromatography was performed with 1-butanol/pyridine/acetic acid/H2O (5/2.3/1/4). The radiolabeled peptides were visualized using a PhosphoImager.
RESULTS
Properties of Rat IGFBP-1 Fractionated by Ion-Exchange Chromatography Rat IGFBP-1 was isolated from H-35 cells and purified by a series of steps including precipitation at 50% saturated ammonium sulfate, gel filtration, and anionexchange chromatography on a Q cartridge, as described previously (6). Several peaks of [125I]IGF-I binding activity were detected by anion-exchange chromatography, and fractions were pooled as QA, which included two less acidic forms, and QB, a more acidic form that appeared relatively homogeneous (Fig. 1). The two pools were analyzed by SDS–PAGE followed
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FIG. 2. SDS–PAGE of Q cartridge-purified fractions. (A) Silver staining of QA and QB pools electrophoresed on a 10% gel. (B) The QB pool (26 ng) was immunoprecipitated with 1 ml (lanes 2 and 3) or 4 ml (lanes 4 and 5) of antibody prepared against QA. Samples were incubated without (2) or with (1) alkaline phosphatase (APase), or not incubated at all (NI), and then electrophoresed on a 10% gel. IGFBP activity was detected by ligand blotting with [125I]IGF-I.
by silver staining. For both pools, single bands were observed that migrated as 31- to 32-kDa proteins, but QB (Fig. 2A, lanes 4 – 6) appeared to migrate slightly slower and as a more diffuse band than QA (Fig. 2A, lanes 1–3). This observation suggested that, like the human protein (10, 11), rat IGFBP-1 may be phosphorylated. Immunoprecipitated QB showed a slight increase in migration on SDS–PAGE after alkaline phosphatase treatment (Fig. 2B, lanes 3 and 5), supporting this conclusion. Treated and untreated rat IGFBP-1 were equally well precipitated, which established that both phosphorylated and unphosphorylated forms react with the antibody, which was raised against the less acidic QA form. Further evidence for phosphorylation of rat IGFBP-1 was obtained by isoelectric focusing gel electrophoresis. On a gel with a pH 3–10 gradient, the QA pool of IGFBP-1 consisted of two bands (Fig. 3A, lane 1). QB also gave rise to two bands (Fig. 3A, lane 3), of which the upper one migrated similarly to the lower QA band. Alkaline phosphatase converted all of the Q proteins to a less acidic form (Fig. 3A, lanes 2 and 4) that presumably is unphosphorylated IGFBP-1. When the QB fraction was analyzed on a pH 5– 8 gradient, two very acidic forms were observed (Fig. 3B, lane 1). After incubation in buffer alone (Fig. 3B, lane 2), most of QB remained highly phosphorylated, with some partial dephosphorylation. Alkaline phosphatase converted all of the forms to a single unphosphorylated form (Fig. 3B, lane 3). Control and alkaline phosphatase-treated samples of rat IGFBP-1 (QB) also were analyzed by rechromatographing them on a Q cartridge (Fig. 4) under the same conditions that were used for the initial purification. Most of the IGF-I binding activity in the control sample
still was found in the fractions eluting at the end of the 0.15 M NaCl elution step (pool II), with a peak in fractions 24 and 25. Analysis of this peak by isoelectric focusing gel electrophoresis showed that it migrated like the original highly phosphorylated QB fraction (data not shown). About 30% of the activity was present in the less acidic heterogeneous fractions comprising pool I. Isoelectric focusing analysis demonstrated that this pool consisted of partially phosphorylated forms, so although some dephosphorylation of QB occurred during chromatography, none of these species was completely dephosphorylated (data not shown). After alkaline phosphatase treatment, most of the IGF-I binding activity shifted to a peak in fractions 15 and 16. Isoelectric focusing gel electrophoresis analysis of these fractions showed that it consisted mainly of a band at the same position as the completely unphosphosphorylated form obtained by alkaline phosphatase treatment of the original QB sample (data not shown). Analysis of Phosphorylation Sites by Phosphopeptide Mapping The sites of phosphorylation in human IGFBP-1 have been found at ser101, 119, and 169, as shown in Fig. 5A. These sites resemble CK2 phosphorylation
FIG. 3. Isoelectric focusing gel electrophoresis of purified fractions QA and QB. Samples were incubated without (2) or with (1) alkaline phosphatase (APase), or not incubated at all (NI), and then electrophoresed on nondenaturing isoelectric focusing gels using ampholytes with a gradient of pH 3–10 (A) or 5– 8 (B). Ligand blotting with [125I]IGF-I was used to detect IGFBP activity. The positions of carbonic anhydrase marker (pI 5.4/5.9) are shown on both sides of the figure. On the left, the top arrow indicates the unphosphorylated form in pool A (lane 1), the middle arrow indicates a partially phosphorylated form in pools A and B (lanes 1 and 3), and the bottom arrow indicates a more phosphorylated form in pool B (lane 3). On the right, the bottom arrow indicates the most phosphorylated form in pool B (lanes 1 and 2), and the top two arrows indicate the positions of partially dephosphorylated forms (lane 2).
PHOSPHORYLATION OF RAT INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-1
FIG. 4. Rechromatography of rat IGFBP-1 (QB) on a Q cartridge. The QB form of purified rat IGFBP-1 was incubated without (F, 32 mg) or with (E, 65 mg) 0.02 units/ml of alkaline phosphatase. The [125I]IGF-I binding activities of IGFBP-1 after incubation without or with alkaline phosphatase were 17 and 18 binding units/ng, respectively. Samples were dialyzed against starting buffer and chromatographed on a Q cartridge. The [125I]IGF-I binding activity of fractions was measured and is plotted as the percentage of the activity applied to the column. For some experiments, aliquots of fractions were combined to obtain phosphorylated and unphosphorylated pools I, II, and III, as indicated above the profiles.
sites (21, 22), with acidic amino acids in position 12 and/or 13 on the C-terminal side of the serine. Sites 101 and 119 are located in the nonconserved intermediate domain, while site 169 is located within the C-terminal domain, which is highly conserved between species (2) and contains cysteine residues that participate in intramolecular disulfide bonds (23). The rat IGFBP-1 sequence has been determined (2), which allowed us to identify several serine residues as potential phosphorylation sites, based on a comparison with the human IGFBP-1 phosphorylation sites (Fig. 5A). The site at ser107, SEDE, is not conserved in the human sequence, but it is a sequence in which serine is frequently phosphorylated in other secreted proteins (21, 22). The site at ser132 in rat IGFBP-1 (SRED) is very similar to the ser119 site in the human protein (SEED), except for an arg residue in place of a glu. The sequence at ser114 in the rat protein was not considered equivalent to the human ser101 site because it contains a pro residue in the 11 position, and neither of the two ser-pro-glu sequences preceding ser101 in human IGFBP-1 are phosphorylated (13). Similarly, thr118 in a TEE sequence was not considered a potential site, since the analogous site at thr105 (TEEE) in the human protein is not phosphorylated (13), even with an additional glu residue. Ser96 in the rat protein lacks an acidic residue at the 11 and 12 positions, but it does contain two glu residues at 13 and 14. The other
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fourteen serine and nine threonine residues in rat IGFBP-2 were not in motifs that are found phosphorylated in secreted proteins (21, 22), and thus they were not considered as potential sites. Two-dimensional phosphopeptide mapping was carried out on rat IGFBP-1 that was labeled with 32PO2 4 in hepatoma cells treated with dexamethasone. Immunoprecipitated IGFBP-1 was purified by SDS–PAGE, and it was cleaved with either trypsin or endoproteinase Asp-N or by sequential treatment with both enzymes. Results of the phosphopeptide analysis showed that initial trypsin digestion reproducibly yielded a single 32 P-labeled peptide (Fig. 6A). As outlined in Fig. 5B, this could have resulted only if ser96 (T77–102), but not ser107 and ser132 (T86 –133), were phosphorylated or alternatively, if ser107 and/or ser132 were phosphorylated but not ser96. After additional digestion with endoproteinase Asp-N, the single peptide gave rise to two overlapping radioactive peptides (Fig. 6C). Since peptide T77–102 which contains ser96 would not be cleaved by Asp-N (Fig. 5B), the results strongly
FIG. 5. Potential phosphorylation sites and predicted phosphopeptides for rat IGFBP-1. (A) A comparison of the sequences containing three serine phosphorylation sites reported for human IGFBP-1 (lower sequence, S101, S119, and S169) and three potential sites in rat IGFBP-1 (S96, S107, and S132). The potential or actual serine residues phosphorylated, and adjacent acidic amino acid residues, are underlined. The alanine residue at position 182 in rat IGFBP-1 that occurs in place of ser169 in the human protein is in bold. (B) Predicted radioactive peptides that would be obtained from 32Plabeled rat IGFBP-1 cleaved by trypsin (T) or endoproteinase Asp-N (AN) or by sequential digestion with both (T/AN or AN/T). The peptides are numbered with their inclusive sequences. The potential phosphorylated serine residues are in bold.
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FIG. 7. Inhibition of IGFBP-1 secretion by brefeldin A. Brefeldin A (BFA) was added to dexamethasone-treated H-35 cells at the same time as [14C]proline. IGFBP-1 was immunoprecipitated from the cell and medium fractions with antiserum to rat IGFBP-1, and it was separated by SDS–PAGE on a 10% gel. The concentrations of brefeldin A used were (mg/ml): none (lanes 3 and 4), 2.5 (lanes 5 and 6), 5.0 (lanes 7 and 8), 10.0 (lanes 9 and 10). Aliquots of the samples without brefeldin A treatment served as minus antibody controls (lane 2). Lane 1 contained carbonic anhydrase as a molecular mass marker (30 kDa).
Subcellular Localization of Phosphorylation
FIG. 6. Two-dimensional phosphopeptide analysis of rat IGFBP-1. Dexamethasone-treated H-35 cells were labeled with 32PO2 4 , and IGFBP-1 secreted into the medium was immunoprecipitated. IGFBP-1 was purified by SDS–PAGE, extracted from the gel, and digested with proteases. The released phosphopeptides were analyzed by electrophoresis (1) and chromatography (2), as described under Experimental Procedures. Digestion was performed with either trypsin (A), endoproteinase Asp-N (B), or sequentially with trypsin/Asp-N (C), or Asp-N/trypsin (D). The vertical arrows indicate the point of application, the double horizontal arrows indicate two distinct peptides produced by Asp-N digestion, and the single horizontal arrows indicate the position of two overlapping peptides produced by sequential digestion.
suggested that both ser107 and ser132 were phosphorylated, but not ser96. Initial digestion with endoproteinase Asp-N yielded two radioactive peptides (Fig. 6B). Since the trypsin results indicated that ser96 was not a phosphorylation site, formation of two Asp-N peptides implied that ser107 in AN1-108 and ser132 in AN124-134 were phosphorylated, as outlined in Fig. 5B. Further digestion of the Asp-N-derived peptides with trypsin yielded the same pattern (Fig. 6D) as that obtained by the reverse digestion process (Fig. 6C). These results established that rat IGFBP-1 is phosphorylated at two sites, which were deduced to be ser107 and ser132.
Since two phosphosphorylation reactions occur during the secretion of rat IGFBP-1, it was of interest to determine whether they were accomplished in the same or separate subcellular sites. To investigate this question, cells were labeled with [14C]proline in the presence of either monensin, which blocks secretion between the cis- and medial-Golgi compartments (24), or brefeldin A, which blocks secretion prior to the TGN by disrupting the Golgi apparatus (25). Radioactive rat IGFBP-1 in the cell and medium fractions was immunopreciptated and analyzed on SDS–PAGE. The time course of labeling and secretion was examined and maximum secretion occurred after 4 h (data not shown). Dose-response experiments were performed in a similar manner after labeling cells for 4 h. Brefeldin A at 10 mg/ml (Fig. 7) inhibited secretion by 97%, while 0.2 mM monensin (Fig. 8) inhibited secretion by 95%,
FIG. 8. Inhibition of IGFBP-1 secretion by monensin. Procedures used were those described in the legend to Fig. 7, except that monensin was added to cell cultures. The concentrations used were (mM): none (lanes 1 and 2), 0.05 (lanes 3 and 4) ; 0.1 (lanes 5 and 6); 0.2 (lanes 7 and 8).
PHOSPHORYLATION OF RAT INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-1
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These results suggest that one phosphorylation step occurs prior to both the brefeldin A and monensin block, placing it in the RER or cis-Golgi compartment. The other phosphorylation must occur in the TGN or beyond, since brefeldin A blocks passage to the TGN. Effect of Phosphorylation of Rat IGFBP-1 on its Affinity for IGF-I
FIG. 9. Denaturing isoelectric focusing gel electrophoresis of [14C]proline-labeled IGFBP-1. H-35 cells were labeled with [14C]proline for 4 h in the absence or presence of inhibitors, and IGFBP-1 was immuprecipitated, as described in the legends to Figs. 7 and 8. Immune complexes were incubated with (1) or without (2) alkaline phosphatase (APase), and phosphorylated and unphosphorylated forms were separated on a denaturing isoelectric focusing gel as described under Experimental Procedures. Cultures either had no additions (NA, lanes 1 and 2), or were treated with 10 mg/ml brefeldin A (BFA, lanes 3 and 4) or 1 mM monensin (Mon, lanes 5 and 6). The positions of three phosphorylated forms (1–3) and the unphosphorylated form (UP) are indicated on the left.
and IGFBP-1 accumulated intracellularly. In the untreated control cultures (Fig. 7, lanes 3 and 4; Fig. 8, lanes 1 and 2) the amounts of radioactive IGFBP-1 secreted were 91 and 85% of the total, respectively. In order to determine whether the accumulated intracellular IGFBP-1 in the inhibited cultures was phosphorylated or not, the [14C]proline-labeled immunopreciptates were also analyzed by isoelectric focusing gel electrophoresis after dissociating the immune complex. Samples were incubated without or with alkaline phosphatase prior to electrophoresis to identify phosphorylated forms. Electrophoresis was carried out on a denaturing gel in order to prevent reassociation of the complex, and the migration of phosphorylated forms differed somewhat from that observed in the nondenatured gels described above. In the untreated sample (Fig. 9, lane 1), most of the radioactive IGFBP-1 was in the medium and, as with the purified protein, it consisted of several phosphorylated forms. The protein in band 1 was slightly more acidic than the unphosphorylated form obtained by alkaline phosphatase treatment (Fig. 9, lane 2) and probably contained a single phosphorylation site, while the protein in band 3 was the most acidic form, which suggested that it contained both phosphorylated sites. The small amount of band 2 may result from dephosphorylation of the band 3 protein to yield the second monophosphorylated protein. In cells treated with brefeldin A or monensin, IGFBP-1 that accumulated intracellularly contained mainly the band 1 form (Fig. 9, Cell, lanes 3 and 5).
The QB form of purified rat IGFBP-1 was dephosphorylated with alkaline phosphatase and the affinity for [125I]IGF-I was measured either directly or after the samples were rechromatographed on a Q cartridge. Controls consisted of QB that was either unincubated or incubated under the same conditions as for the dephosphorylation, except that alkaline phosphatase was not present. The results of a number of experiments showed that there was no significant difference in the affinity of the phosphorylated and unphosphorylated forms (Fig. 10) or for QA (data not shown). Scatchard plots of the data indicated that the Ka of all forms was 1.1 3 1010 M21. In addition, the specific activity of IGFBP-1 for IGF-I binding (binding units/ng protein) did not change after alkaline phosphatase treatment (see the legend to Fig. 4).
FIG. 10. IGF-I affinity competition with phosphorylated and dephosphorylated rat IGFBP-1. Samples were analyzed in duplicate for [125I]IGF-I binding activity in the presence of varying amounts of unlabeled IGF-I, as indicated. Results are presented as a percentage of the maximum binding without unlabeled ligand. For the phosphorylated samples (2), five separate analyses were performed. One sample was the original, unincubated QB analyzed directly. Four samples were QB incubated without alkaline phosphatase; two of these were analyzed as such, while two were rechromatographed as described in the legend to Fig. 4, and a pool of the most highly phosphorylated fractions (24 1 25) was used. For the dephosphorylated samples (1 APase), four separate analyses were performed: two were incubated with alkaline phosphatase and analyzed directly, while two were incubated with alkaline phosphatase and rechromatographed on a Q cartridge, and a pool of the completely dephosphorylated fractions (15 1 16) was analyzed.
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Inhibition of DNA Synthesis by Phosphorylated and Unphosphorylated Forms of Rat IGFBP-1 We previously found that the QB form of purified rat IGFBP-1 inhibited DNA synthesis in quiescent 3T3 cells (6). In this system, cells are incubated with saturating concentrations of PDGF and EGF, so that DNA synthesis is dependent only on the addition of IGF-I. To determine whether the dephosphorylated form would inhibit DNA synthesis, control or dephosphorylated QB was rechromatographed on a Q cartridge and pools I, II, and III of both fractionations were tested (Fig. 11). Unphosphorylated and phosphorylated forms of rat IGFBP-1 inhibited DNA synthesis almost identically, with 50% inhibition at approximately 120 ng/ml. Even when PDGF and EGF were omitted, so that the stimulation of DNA synthesis by IGF-I alone was much reduced, all of the forms of rat IGFBP-1 still inhibited. Potentiation with unphosphorylated human IGFBP-1 has been observed with 20 –100 ng/ml (8, 9, 12). DISCUSSION
The major observations of this study were that purified rat IGFBP-1 is phosphorylated on two sites, compared to three in human IGFBP-1, that these phosphorylations occur in two different subcellular compartments of the secretory pathway, and that unlike human IGFBP-1, the phosphorylation state of rat IGFBP-1 did not affect its affinity for IGF-I. The specific activity for binding also was not affected. In addition, the dephosphorylated form of rat IGFBP-1 inhibited DNA synthesis in cultured cells, contrary to previous findings for human IGFBP-1 (1, 8, 9). It has been proposed that the lower affinity of the unphosphorylated human protein is the basis for the ability of the human protein to potentiate, rather than inhibit DNA synthesisis (1). In some cases, potentiation is dependent on the addition of a factor in platelet-poor plasma (26), but it also occurs in the absence of this factor (12). The plasma factor may not affect the innate ability of rat IGFBP-1 to act as an inhibitor, however, since its unphosphorylated form does not have a lower affinity than the phosphorylated form. It has been pointed out previously (27) that, although the affinities of the dephosphorylated and phosphorylated forms of human IGFBP-1 for IGF-I differ, both are relatively high compared to the affinity of the receptor, so inhibition of IGF-I function may be the major role of all IGFBPs. The affinity for both forms of rat IGFBP-1, which are identical, are approximately an order of magnitude greater than that of cellular receptors for IGF-I (6). The two phosphorylated sites in rat IGFBP-1 were deduced to be serine residues 107 and 132, based on a comparison of the rat sequence with the known phosphorylation sites in human IGFBP-1 and -3 and on the
FIG. 11. Inhibition of DNA synthesis by dephosphorylated and phosphorylated rat IGFBP-1. DNA synthesis was measured in the absence, or in the presence, of varying amounts of rat IGFBP-1. Samples were pooled fractions from the I, II, and III regions of rechromatographed IGFBP-1 (QB) that were either phosphorylated (2, F) or dephosphorylated with alkaline phosphatase (1APase, E), as described in the legend to Fig. 4. Results are plotted as the percentage inhibition of the [3H]thymidine incorporation obtained in the absence of IGFBP-1.
pattern of phosphopeptides obtained after cleavage with trypsin and/or endoproteinase Asp-N. As discussed above under Results, there are a number of other serine and threonine residues in IGFBP-1, but they are not within motifs generally found as phosphorylated in secreted proteins (28, 29). The ser107 site is not conserved in the human sequence, while the ser132 site is similar to the human ser119 site. Both phosphorylation sites are in the nonconserved central domain while in human IGFBP-1 two sites are in the nonconserved domain, and one (ser169) is in the conserved C-terminal domain. The two serines phosphor-
PHOSPHORYLATION OF RAT INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-1
ylated in IGFBP-3 also are in the nonconserved central domain (16). From results of experiments which used the secretion inhibitors monensin and brefeldin A, it was concluded that one of the phosphorylation steps occurs in the RER or cis-Golgi and the other in the TGN. This conclusion is based on the fact that both inhibitors allowed formation of the least acidic, phosphorylated form of rat IGFBP-1 (band 1), but prevented further phosphorylation to band 3. Multiple cellular sites for serine phosphorylation in other proteins have been observed. ChromograninB and secretogranin are phosphorylated on serine early in the secretory pathway, probably in the medial- or trans-golgi (30). Phosphorylation of connexin43, an intercellular gap junction protein, is presumed to occur in the RER or Golgi apparatus (31). In contrast, serine phosphorylation in progastrin occurs late, in either the TGN or the secretory granules (32). Brefeldin A does not inhibit phosphorylation of b- and g-caseins but it does inhibit phosphosphorylation of a-casein, suggesting that the former are phosphorylated in the Golgi apparatus, while the latter is phosphorylated in the TGN (33). Our results extend previous observations by demonstrating that a single protein may be phosphorylated at different steps in the secretion pathway, and they also imply that two different kinases are involved. CK1 and CK2 are ubiquitous enzymes that phosphorylate casein in vitro, and apparent substrates for CK2 include both nuclear and cytoplasmic proteins (21, 22, 34). Another type of kinase, designated as Golgicasein kinase (G-CK), has been purified from a Golgienriched fraction of mammary gland (35) and liver (29) and is thought to be responsible for the phosphorylation of secreted proteins such as casein. Only a small percentage of total CK2 activity in the liver was detected in the Golgi fraction (29). The biochemical properties and substrate requirements of G-CK clearly differentiate it from CK1 and CK2. Threonine essentially is not phosphorylated by G-CK, and a peptide containing a motif ser-X-glu/ser(P)-X, an in vivo casein phosphorylation site, is a specific substrate for this enzyme, as it is not phosphorylated by either CK1 or CK2 (29, 35). For CK2, the preferred sequence is X-ser-glu/aspasp/glu-glu/asp, but an acidic residue at 13 appears to be critical (21, 22). Current evidence supports the idea that G-CK is responsible for phosphorylation of most serine residues in secreted proteins, while CK2 may play a minor role. Phosphorylation of apolipoproteins is detected using Golgi, but not RER, vesicles from liver in vitro, and CK2 is not involved (36). An enzyme purified from an RER and Golgi vesicle-enriched fraction from osteosarcoma cells was identified as CK2 on the basis of its immunogenicity and biochemical properties, and it phosphorylates the acidic bone proteins osteopontin
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and phosphoryn in vitro (37). Of the 28 phosphorylated sites in osteopontin, however, all but one of them are on serine, and G-CK rather than CK2 is probably responsible because almost all of the phosphoserines are located in ser-X-glu/ser(P)-X motifs. Two kinases that phosphorylate human IGFBP-1 in vitro were extracted from HepG2 cells, but it was not determined whether they originate in subcellular organelles (38). One of the enzymes exhibits properties similar to those of CK2, while the other enzyme does not appear to be either CK1 or CK2, raising the possibility that it is G-CK. In human IGFBP-3, mutation of aspartic acid to alanine at the 13 position in the SEED motif did not affect phosphorylation of the serine residue, while mutation of the two glutamic acid residues at 11 and 12 to alanine abolished it (16). Since an acidic residue at the 13 position is critical for CK2 activity, while a glu residue is required at 12 for G-CK actiivity, it is very likely that G-CK acts at this site. This site is identical to the ser119 site in human IGFBP-1 and similar to the SRED site in rat IGFBP-1, which suggests that G-CK is involved in their phosphorylation. Ser107 in the SEDE motif of rat IGFBP-1 is more likely to be phosphorylated by CK2, since the aspartic acid in the 12 residue makes this sequence a poor substrate for G-CK (29, 35). Since phosphorylation of rat IGFBP-1 and human IGFBP-3 does not affect their affinity for IGF-I, unlike human IGFBP-1, this raises the question of what the function of phosphorylation is in these IGFBPs. Although the effect of ser/thr phosphorylation on the activity of numerous enzymes and transcription factors is well documented (21, 22, 34), relatively little is known about the function of phophoserines in secreted proteins. The most well established example is casein, where the phosphates on serine are required for calcium binding and subsequent aggregation for micelle formation in milk (33). Phosphorylation of osteopontin can determine whether the protein will bind to the cell membrane or to fibronectin in the extracellular matrix (39), and it also may be important in bone mineralization (40). Early phosphorylation of connexin43 may prevent misfolding or degradation of the protein before its oligomerization in the Golgi apparatus (31). Based on our isoelectric focusing gel results, inhibition of phosphorylation by brefeldin A and monensin in hepatoma cells did not cause an appreciable decrease in total rat IGFBP-1, suggesting that the function of phosphorylation is not related to intracellular degradation in this case. The present studies have ruled out some possible functions for phosphorylation of IGFBPs, and have provided new information on the intracellular site of phosphorylation of rat IGFBP-1 and some insight into the kinases that may be responsible.
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ACKNOWLEDGMENTS We thank Drs. S. G. Rhee and D. Y. Jhon (NHLBI) for their generous assistance with the phosphopeptide analyses.
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