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Endocrinology, doi:10.1210/en.2003-0089
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Endocrinology Vol. 144, No. 9 4042-4050
Copyright © 2003 by The Endocrine Society

Phosphorylation of Insulin-Like Growth Factor (IGF) Binding Protein-3 by Breast Cancer Cell Membranes Enhances IGF-I Binding

Suresh Mishra and Liam J. Murphy

Departments of Internal Medicine and Physiology, University of Manitoba, Winnipeg, Canada, R3E 0W3

Address all correspondence and requests for reprints to: Liam J. Murphy, Room 843, John Buhler Research Centre, University of Manitoba 715 McDermot Avenue, Winnipeg, Canada, MB R3E 3P4. E-mail: ljmurph{at}cc.umanitoba.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cross-linking of nonglycosylated biotinylated IGF binding protein (IGFBP)-3 to T-47D cell membranes identifies complexes with Mr of 32, 50, 70, and 100 kDa. Nonbiotinylated glycosylated IGFBP-3 competed for binding to each of these sites. The 32-kDa band approximated the size of intact nonglycosylated IGFBP-3, but its abundance was enhanced by cross-linking, and it had a more acidic isoelectric point on isoelectric focusing, suggesting that it had undergone phosphorylation. Immobilized IGFBP-3 was phosphorylated in the presence of 32P-{gamma}ATP by both T-47D cell membranes and by intact cells treated with phenylarsine oxide to inhibit internalization. MCF-7 and COS-1 cells were also able to bind and phosphorylated IGFBP-3. IGF-I inhibited both IGFBP-3 binding to membranes and phosphorylation. However, incubation of T-47D cells with IGFBP-3 enhanced binding of 125I-IGF-I to the cell monolayer indicating that membrane bound IGFBP-3 was able to bind IGF-I. Immobilized IGFBP-3 when phosphorylated by T-47D membranes bound significantly more 125I-IGF-I than nonphosphorylated IGFBP-3. Treatment with alkaline phosphatase significantly reduced 125I-IGF-I binding to phosphorylated immobilized IGFBP-3 and also reduced 125I-IGF-I to T-47D cell monolayers preincubated with IGFBP-3. Phosphorylation of IGFBP-3 by T-47D membranes was partially blocked by inhibitors of both protein kinase A and C. These data demonstrate that binding of IGFBP-3 to breast cancer membranes is accompanied by phosphorylation at the plasma membrane and that both processes are inhibited by IGF-I. However, once phosphorylated the ability of IGFBP-3 to bind IGF-I is enhanced, resulting in increased association of the IGF-I with the cell membrane.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF BINDING PROTEIN (IGFBP)-3 is the most abundant of the IGFBPs in the circulation and functions to transport and modulate the actions of IGF-I and -II. In addition to these IGF-dependent effects, emerging evidence suggests that IGFBP-3 also functions in an IGF-independent manner to stimulate apoptosis and inhibit cellular proliferation of various cell lines including human breast cancer cells (1, 2, 3). These IGF-independent effects are clearly demonstrable with mutant IGFBP-3 and IGFBP-3 fragments that have minimal affinity for IGF-I (4, 5) and with cell lines devoid of IGF-I receptors (1, 6).

The recent interest in the IGF-independent effects of IGFBP-3 has led to an investigation of IGFBP-3 binding sites on plasma membranes. It has been assumed that these IGF-independent effects of IGFBP-3 are mediated via specific cell surface binding sites, although this remains to be proven. IGFBP-3 binding sites may also facilitate delivery of IGF-I to the cell membrane and therefore enhance rather than inhibit the IGF-I effect.

IGFBP-3 interacts via a cluster of basic residues at the C terminus with a variety of proteins including the acid labile subunit (7), glycosaminoglycans (8), and plasminogen (9). This same region of the molecule is also involved in binding to plasma membrane binding sites (10), although the central domain is also considered to be important (11).

The interaction of IGFBP-3 with a variety of membrane proteins has been demonstrated using cross-linking techniques (12, 13, 14). With the exception of the type V TGF-ß receptor (15), the identity of these membrane proteins is not known. Mutant IGFBP-3 molecules that lack the ability to bind IGF-I retain their ability to bind plasma membranes (4) and IGF-I has been reported to inhibit the binding of IGFBP-3 to cell membranes (13, 16). In a similar fashion, IGFBP-3 is able to inhibit binding of IGF-I to the type 1 IGF receptor (17). These observations are of interest because IGF-I has proliferative and antiapoptotic actions (17), whereas IGFBP-3 promotes apoptosis and inhibits proliferation in various cell lines (1, 2, 3).

As part of our ongoing studies to understand the mechanisms that mediate the IGF-independent growth inhibitory effects of IGFBP-3, we examined the interaction of IGFBP-3 with T-47D cell membranes. Here we report the novel finding that IGFBP-3 is phosphorylated at the plasma membrane by a process that is inhibited by IGF-I and does not require internalization. However, after phosphorylated by the membrane kinase, the binding of IGF-I to phosphorylated IGFBP-3 is enhanced.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and reagents
T-47D, MCF-7, and COS-1 cells were obtained from the American Type Tissue Culture (Manassas, VA). Cell culture reagents were from Life Technologies, Inc., Life Sciences (Burlington, Ontario, Canada). Glycosylated and nonglycosylated IGFBP-3 were obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Protein kinase inhibitors, H-89, (N-[2-(P-bromocinnamylamino)ethyl-5-iso-quinolinesulfonamide), PD 98059 (2'-amino-3'-methoxyflavone), H-7 (1-[5-isoquinolinesulfonyl)-2-methylpiperazone and genistein, were obtained from Calbiochem (Hornby, Ontario, Canada). All other reagents were obtained from Sigma-Aldrich Canada (Oakville, Ontario, Canada) unless otherwise stated.

Biotinylation of IGFBP-3
Nonglycosylated Escherichia coli-derived IGFBP-3 was dissolved in 500 µl PBS at a concentration of 1 µg/µl and incubated with 7.5 µl p-biotinoyl-aminocaproic acid-N-hydroxy-succinamide ester (Roche Molecular Biochemicals, Mannheim, Germany) for 2 h at room temperature. At the end of incubation, free biotin ester was separated on a Sephadex G-25 column preequilibrated with 5 ml blocking solution. The protein concentration was measured using Bradford protein assay (Bio-Rad Laboratories Inc., Mississauga, Canada). Biotinylation of IGFBP-3 had no effect on binding of IGFBP-3 to membranes or binding of 125I-IGF-I to IGFBP-3. The biotinylated samples were stored at -80 C until used.

Cross-linking of biotinylated IGFBP-3
Solubilized cell membranes were prepared by using membrane preparation kit and protocol according to the manufacturer’s instructions (Pierce, Rockford, IL). Two micrograms of biotinylated-IGFBP-3 was added to 200 µl of solubilized cell membrane and incubated at 4 C for 1 h. All experiments were undertaken in the presence of the protease inhibitor, aprotinin (2 µg/ml), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1 mM EDTA. At the end of incubation, disucinimidyl suberate (DSS, 10 µg/ml) was added and incubated for another 15–20 min. Finally, IGFBP-3 cross-linked membrane proteins were precipitated with streptavidin-agarose, washed three times and boiled in sodium dodecyl sulfate sample buffer, and analyzed on an 11% SDS-PAGE gel. Proteins were transferred to the nitrocellulose membrane. Membranes were blocked in 5% milk washed in TBST [10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20 (pH 8.0)] and incubated with streptavidin-horseradish peroxidase (HRP) conjugate diluted 1:3000 for 1 h at room temperature. ECL was used to analyze the membrane. For whole cell cross-linking studies, T-47D, MCF-7, or COS-1 cells were grown on a 35-mm culture dish. After 90% confluence, cells were washed three times with ice-cold PBS and finally incubated with biotinylated IGFBP-3 (200 ng/ml) in PBS for 1 h at 4 C. At the end of incubation, DSS (10 µg/ml) was added and the incubation continued for another 15–20 min. Cells were washed three times with ice-cold PBS and recovered with the help of cell scraper in ice cold PBS containing aprotinin (2 µg/ml), 1 mM PMSF, and 0.1 mM EDTA. Samples were centrifuged, supernatant discarded, and pellet (washed twice in ice-cold PBS) boiled in loading buffer and electrophoresed on 11% gel. Separated proteins were transferred to nitrocellulose membrane and the IGFBP-3 cross-linked proteins were detected either using streptavidin-HRP as described above or using anti-IGFBP-3 antibodies. Antibody AF675 directed against the whole molecule was obtained from R&D Systems (Minneapolis, MN). Antibodies SC 6002 and SC 9028, directed against the C terminus and internal domain residues 113-210, respectively, were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibody 06-108 directed against the whole molecule was obtained from Upstate Biotechnology Inc. (Lake Placid, NY). To investigate whether the IGF-I/IGFBP-3 complex binds to the cell membrane, 125I-IGF-I (0.5 µCi) and IGFBP-3 (0.5 µg) incubated in phosphate buffer for 2 h at room temperature and subsequently cross-linked with DSS (10 µg/ml). The resulting 125I-IGF-I/IGFBP-3 complex was used for membrane cross-linking to T-47D cell monolayer as described above.

Gel electrophoresis
A Mini-PROTEAN II tube cell (Bio-Rad, Hercules, CA) was used for two-dimensional gel electrophoresis of the IGFBP-3 cross-linked proteins. Isoelectric focusing was done at 400 V for 6 h. After first dimension run was complete, gels were extruded from the capillaries and overlaid on 10% SDS-PAGE for second dimension run. Subsequently, proteins were transferred to nitrocellulose membrane, incubated with streptavidin-HRP, and detected with ECL.

Phosphorylation of IGFBP-3
Polystyrene tubes were coated with streptavidin and blocked with BSA (Sigma Chemical Co., St. Louis, MO), washed with saline, and stored at -20 C until used. Biotinylated IGFBP-3 was incubated in streptavidin-coated tubes for 2 h on ice. At the end of incubation excess, unbound IGFBP-3 were removed. The tubes were placed on ice and the phosphorylation reaction cocktail containing [32P]-{gamma}-ATP [60 µCi/ml in Tris-buffered saline (pH 7.5)] was added. Finally, membrane or intact cells (~1 x 106), were added to the tube and incubated at 30 C for 30 min. At the end of incubation, reaction mixture was aspirated and SDS-PAGE sample buffer added to each tube, boiled for 7–10 min, and analyzed on 11% gel. Subsequently, gels were transferred to nitrocellulose membrane and the membranes were processed for autoradiography or for ECL detection as described above. For these experiments, cells were grown in 100-mm tissue culture dishes and harvested using trypsin and incubated in complete media to neutralize the trypsin and allow for recovery. Subsequently, the cells were washed three times in PBS using centrifugation at 850 x g before use. A second method was also used to examine phosphorylation of IGFBP-3 by intact cell monolayers. Biotinylated IGFBP-3 in the phosphorylation reaction cocktail was added to washed cell monolayers in 24-well plates and incubated at 37 C for 5–10 min. At the end of incubation, the reaction mix was aspirated and radiolabeled biotinylated IGFBP-3 was detected by SDS-PAGE and autoradiography. In some experiments, phosphorylated IGFBP-3 was dephosphorylated by incubating with 5 U of alkaline phosphatase for 1 h at room temperature. In some whole cell experiments, phenylarsine oxide was added in a final concentration of 10 µM to inhibit internalization.

Binding of 125I-IGF-I to phosphorylated and nonphosphorylated IGFBP-3
Biotinylated IGFBP-3 was immobilized on the streptavidin-coated tubes and either used directly or phosphorylated using T-47D cell membranes in the presence of radiolabeled or unlabelled ATP (100 µM) as above. In some experiments, nonbiotinylated glycosylated IGFBP-3 was immobilized on tubes by simple adsorption. After phosphorylation, the reaction mixture was aspirated, the tubes were washed and 125I-IGF-I (50,000 cpm, Perkin-Elmer Life Sciences Inc., Boston, MA) in PBS containing 0.002% BSA and 0.01 M glycine (pH 7.4) was added to each tube. After incubation at 22 C for 2 h, the unbound 125I-IGF-I was aspirated, the tubes were washed and the radioactivity in each tube was determined. To verify that 125I-IGF-I bound to the immobilized IGFBP-3 rather than to other residual membrane proteins, DSS (10 µg/ml) was added to the tubes at the completion of the incubation with 125I-IGF-I and cross-linked radioactive proteins analyzed by SDS-PAGE and autoradiography. In some experiments to determine whether membrane protein inhibited binding of IGF-I to the IGFBP-3, varying amounts of solubilized membrane protein was added to the reaction mix containing 125I-IGF-I and immobilized phosphorylated or nonphosphorylated IGFBP-3.

Effects of protein kinase inhibitors on phosphorylation of IGFBP-3
Solubilized T-47D membranes were preincubated with the protein kinase inhibitors, H-89 at 50 and 100 nM, PD 98059 at 0.5 and 1 µM, genistein at 5 and 10 µM, and H-7 at 5 and 10 µM for 1 h on ice. Subsequently, the ability of the membrane preparation to phosphorylate immobilized IGFBP-3 was examined.

Statistical analysis
Data are expressed as the mean ± SEM. Student’s t test was used for single comparisons. For determining statistical differences between multiple groups, an ANOVA with repeated measures followed by Dunnett’s t test was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of IGFBP-3 binding sites on T-47D cell membranes
Biotinylated IGFBP-3 was incubated with T-47D cells and subsequently cross-linked using DSS. Three major bands were identified. These had an Mr of approximately 32, 70, and 100 kDa, with the 100-kDa species being the least abundant (Fig. 1AGo). Western blot analysis using a variety of antibodies directed against IGFBP-3 indicated that the approximately 32-, 70-, and 100-kDa bands contained immunoreactive IGFBP-3 (data not shown). Interestingly, the 32-kDa band, which had a similar apparent molecular mass to the IGFBP-3 standard, was also increased by cross-linking. When solubilized membranes rather than whole cells were used, a similar pattern of IGFBP-3 binding was observed (Fig. 1BGo). A coordinate increase in the intensity of each of the bands were observed when cells were incubated for varying time periods before cross-linking (Fig. 1CGo). The three bands were also apparent when IGFBP-3 was cross-linked to membranes from MCF-7 cells (data not shown).



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  		FIG. 1. Cross-linking with DSS of biotinylated IGFBP-3 to T-47D cell monolayers and solubilized membranes. Biotinylated IGFBP-3 was incubated with intact cells for 1 h at 4 C (A) or solubilized membranes (B) before cross-linking with DSS (+). After SDS-PAGE, the biotinylated complexes were identified using streptavidin-HRP. The position of the 100-, 70-, and 32-kDa complexes are indicated by the arrowheads. C, Biotinylated IGFBP-3 was incubated with solubilized membranes for variable times before cross-linking with DSS.

 
IGF-I inhibits binding of IGFBP-3 to plasma membranes
When 125I-IGF-I was preincubated with biotinylated IGFBP-3 before incubating with the cell monolayer, no radiolabel was associated with the cell membrane after cross-linking (Fig. 2AGo, lower panel) despite the detection IGFBP-3 in the 32- and 70-kDa bands using streptavidin-HRP (Fig. 2AGo, upper panel). In this particular experiment, the 100-kDa band was not seen. We considered the possibilities that the 125I-IGF-I/IGFBP-3 complex had a reduced ability to bind to the membrane or that binding of the 125I-IGF-I/IGFBP-3 complex to membranes resulted in the unloading of 125I-IGF-I bound to IGFBP-3. To address these possibilities, we investigated whether 125I-IGF-I cross-linked to IGFBP-3 was able to bind to the cell membranes. Cross-linking 125I-IGF-I to IGFBP-3 reduced the loss of labeled when the complex was analyzed by SDS-PAGE (lane 2, Fig. 2BGo). However, when cross-linked 125I-IGF-I/IGFBP-3 was incubated with cell monolayers, only a small amount of radioactivity was bound to the plasma membranes (lane 4, Fig. 2BGo). We interpreted these data to indicate that 125I-IGF-I/IGFBP-3 complex had a reduced affinity for the IGFBP-3 binding sites on the cell membrane compared with IGFBP-3 itself. Furthermore, incubation of the biotinylated IGFBP-3 and T-47D cell membrane, together with increasing amounts of unlabeled IGF-I, reduced the amount of biotinylated IGFBP-3 cross-linked to the membranes, indicating that IGF-I was able to inhibit IGFBP-3 binding to membrane presumably by enhancing formation of the binary complex (Fig. 2CGo).



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  		FIG. 2. Reduced affinity of the binary complex for binding to T-47D solubilized membranes and monolayers. A, Radiolabeled IGF-I was preincubated with biotinylated IGFBP-3 and then incubated the membranes and cross-linked with DSS. Streptavidin-agarose was used to precipitated the IGFBP-3 and associated proteins. The upper part of panel A shows biotinylated IGFBP-3 detected by ECL whereas the lower part shows the corresponding autoradiogram. B, 125I-IGF-I/IGFBP-3 binary complex that had (+), or had not been cross-linked (-, lanes 1 and 2), was analyzed by SDS-PAGE and autoradiography. Cross-linked 125I-IGF-I/IGFBP-3 binary complex was incubated with cell monolayers and further cross-linked to the monolayer (lanes 3 and 4). C, Biotinylated IGFBP-3 was incubated with cell monolayers in the presence of up to 100-fold molar excess of IGF-I. After cross-linking and analysis on SDS-PAGE, biotinylated IGFBP-3 was detected by ECL. Incubations were carried out at 4 C for 1 h as described in Materials and Methods.

 
Phosphorylation of IGFBP-3 by plasma membranes
Because the 32-kDa band identified in the cross-linking experiments appeared to migrate slightly faster than the native nonglycosylated biotinylated IGFBP-3 (Fig. 2AGo, upper panel) and was more abundant in experiments where DSS was used (Fig. 1Go), we speculated that it represented IGFBP-3, which had been cross-linked to the membrane and biochemically modified rather than simply loosely bound IGFBP-3. We used two-dimensional gel electrophoresis to investigate the nature of this band. Biotinylated IGFBP-3 immobilized on streptavidin-coated tubes was used to exclude the potential confounding factor of endogenously expressed IGFBP-3. Isoelectric focusing revealed that the isoelectric point (pI) of the 32 kDa-band was more acidic compared with the biotinylated IGFBP-3 standard (Fig. 3Go). We hypothesized that this change in pI was due to phosphorylation, and we demonstrated that dephosphorylation with alkaline phosphatase returned the pI of the 32-kDa band to that of the IGFBP-3 standard (Fig. 3Go, lower panel).



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  		FIG. 3. Two-dimensional gel electrophoresis analysis of IGFBP-3 cross-linked to solubilized T-47D cell membranes. Biotinylated IGFBP-3 was incubated with solubilized T-47D cell membranes as described in Materials and Methods. After cross-linking and analysis on SDS-PAGE, biotinylated IGFBP-3 was detected by ECL. The migration pattern of the biotinylated-IGFBP-3 standard is shown in the upper panel. In the center panel, the position of the cross-linked IGFBP-3-membrane protein complexes are shown. In the lower panel, the cross-linked membrane preparation was treated with alkaline phosphatase before analysis.

 
When cell membranes were incubated with biotinylated IGFBP-3 immobilized on streptavidin-coated tubes in the presence of 32P-{gamma}-ATP, the 32-kDa band was radiolabeled (Fig. 4AGo, lower panel). Phosphorylation of 70 and 100 kDa was also seen on some occasions, even in the absence of cross-linking (Fig. 4BGo). In addition, intact T-47D cells were able to phosphorylate immobilized IGFBP-3 (Fig. 4BGo). The presence of phenylarsine oxide, an inhibitor of internalization, had no effect on the ability of the cells to phosphorylate immobilized IGFBP-3. In four separate experiments where we quantified the results by densitometry, we compared phosphorylation of IGFBP-3 by phenylarsine oxide (PAO)-treated cells with untreated cells the phosphorylation of IGFBP-3 by PAO-treated cells was 99.7 ± 10.1% of untreated cells. Conditioned medium from T-47D cells was not able to phosphorylate immobilized IGFBP-3. Phosphorylation of immobilized IGFBP-3 by T-47D membranes was inhibited by the presence of IGF-I (Fig. 4CGo). We demonstrated that MCF-7 and COS-1 cell membranes and intact cells also phosphorylated IGFBP-3 (Fig. 4DGo).



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  		FIG. 4. Phosphorylation of IGFBP-3 by intact cells and cell membranes. A, Biotinylated IGFBP-3 immobilized on polystyrene tubes was incubated with T-47D membranes and 32P-{gamma}-ATP and subsequently analyzed by SDS-PAGE. The presence of IGFBP-3 was demonstrated with ECL, whereas phosphorylation was demonstrated with autoradiography. Similar experiments are shown in panels B and D where whole cells in addition to membranes are used. PAO indicates the presence (+) or absences (-) of phenylarsine oxide. C, Effect of the addition of IGF-I (1 µg/ml) to the incubation mix of 32P-{gamma}-ATP, membranes and immobilized IGFBP-3 was examined. All incubations were carried out at 30 C for 30 min.

 
Enhanced binding of IGF-I to phosphorylated IGFBP-3
Immobilized biotinylated IGFBP-3 was phosphorylated with T-47D membranes washed and subsequently incubated with 125I-IGF-I. Compared with nonphosphorylated biotinylated IGFBP-3, IGFBP-3 phosphorylated in this manner consistently bound significantly more 125I-IGF-I (Fig. 5AGo). However, the presence of solubilized membrane protein inhibited binding of 125I-IGF-I to both immobilized IGFBP-3 (Fig. 5BGo). This inhibition was apparent when the immobilized IGFBP-3 had been phosphorylated before the addition of the membranes and when nonphosphorylated IGFBP-3 was used. Although part of this inhibition could be due to sequestration of 125I-IGF-I by the type 1 receptor in the soluble membrane preparation, this does not appear to be the whole explanation. A reduction in the availability of 125I-IGF-I would have simply reduced binding to both phosphorylated and nonphosphorylated IGFBP-3 in a similar manner proportionate to the amount of added membrane. However, the pattern of inhibition was different when nonphosphorylated IGFBP-3 was used compared with phosphorylated IGFBP-3. In the case of nonphosphorylated IGFBP-3, some enhancement of binding was apparent at the lower membrane protein concentrations possibly as a result of IGFBP-3 phosphorylation occurring during the incubation.



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  		FIG. 5. Phosphorylation of IGFBP-3 enhances IGF-I binding. A, Biotinylated IGFBP-3 was immobilized on polystyrene tubes and was preincubated with solubilized T-47D membranes in the presence of ATP. After washing, the tubes were then incubated with 125I-IGF-I as described in Materials and Methods. As controls, 125I-IGF-I was incubated with uncoated tubes and tubes coated with biotinylated IGFBP-3 but which were not preincubated with solubilized T-47D membranes. The data represent the mean ± SEM for four separate experiments. The statistical difference was determined using Student’s t test. B, IGFBP-3 was immobilized on polystyrene tubes and preincubated with solubilized T-47D membranes in the presence of ATP (closed histograms). After washing, the tubes were then incubated with 125I-IGF-I. As controls, 125I-IGF-I was incubated with immobilized IGFBP-3 but which were not preincubated with solubilized T-47D membranes (open histograms). Solubilized T-47D membranes were added in various amounts to the reaction mix containing 125I-IGF-I. Statistical differences between the control (no added membrane) and samples with solubilized membrane were determined using Dunnett’s t test. *, P < 0.05.

 
Binding and phosphorylation of glycosylated IGFBP-3
In the initial studies we used E. coli-derived, nonglycosylated IGFBP-3 to determine the molecular mass of the cross-linked IGFBP-3/membrane protein complexes without the added complexity of the carbohydrate moiety. Previous reports have indicated that glycosylation is not critical for membrane binding (14). To confirm this, we investigated whether glycosylated IGFBP-3 was able to compete with nonglycosylated IGFBP-3 for binding to T-47D membranes. When nonbiotinylated glycosylated IGFBP-3 was included in reaction where nonglycosylated, biotinylated IGFBP-3 was incubated with solubilized T-47D membranes, an attenuation of the biotin labeling of the 70- and 30-kDa bands was apparent (Fig. 6AGo). When nonbiotinylated, glycosylated IGFBP-3 was immobilized on polystyrene tubes and incubated with 32P-{gamma}-ATP and solubilized T-47D membranes and subsequently analyzed by SDS-PAGE, phosphorylation of the IGFBP-3 was apparent (data not shown).



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  		FIG. 6. Glycosylated IGFBP-3 competes with nonglycosylated IGFBP-3 for binding and phosphorylation by T-47D cells. A, Nonglycosylated biotinylated IGFBP-3 was incubated with T-47D cell monolayers at 37 C for 1 h in the presence of varying amounts of nonbiotinylated glycosylated IGFBP-3. After cross-linking and SDS-PAGE analysis the biotinylated IGFBP-3 was detected by ECL. B, Glycosylated IGFBP-3, 200 ng, was immobilized on polystyrene tubes and preincubated with solubilized T-47D membranes in the presence of ATP (closed histograms). After washing, the tubes were then incubated with 125I-IGF-I. As controls, 125I-IGF-I was incubated with tubes coated with glycosylated IGFBP-3 but which were not preincubated with solubilized T-47D membranes (open histograms). The data represent the mean ± SEM for four separate experiments. Solubilized T-47D membranes were added in various amounts to the reaction mix containing 125I-IGF-I. Statistical differences between the control (no added membrane) and samples with solubilized membrane were determined using Dunnett’s t test. **, P < 0.01.

 
As was the case with nonglycosylated IGFBP-3, immobilized glycosylated IGFBP-3 which had been preincubated with T-47D cell membranes, bound significantly more 125I-IGF-I than nonphosphorylated IGFBP-3 (Fig. 6BGo). Solubilized membranes also inhibited binding of 125I-IGF-I to glycosylated IGFBP-3 that had been immobilized on polystyrene tubes (Fig. 6BGo).

Effects of dephosphorylation with alkaline phosphatase
To determine whether phosphorylation itself, rather than some other subtle biochemical modification of IGFBP-3, was important in the enhanced IGF-I binding, we incubated immobilized IGFBP-3 with T-47D membranes and subsequently treated the phosphorylated IGFBP-3 with alkaline phosphatase. Treatment of immobilized phosphorylated IGFBP-3 with alkaline phosphatase reduced 125I-IGF-I binding to levels comparable to that seen with nonphsophorylated IGFBP-3 (Fig. 7AGo). Alkaline phosphatase treatment of nonphosphorylated IGFBP-3 immobilized on polystyrene tubes had no affect on the ability of IGFBP-3 to bind 125I-IGF-I (12742.4 ± 384.2 vs. 12101.9 ± 559.7 cpm, n = 9). A similar experiment was undertaken with T-47D cell monolayers. Preincubation with IGFBP-3 enhanced 125I-IGF-I binding to the monolayer approximately 3.5-fold. Treatment with alkaline phosphatase significantly reduced this enhanced binding (Fig. 7BGo). However, even after treatment with alkaline phosphatase, 125I-IGF-I binding to T-47D cell monolayers that had been pretreated with IGFBP-3 remained approximately 3-fold higher than binding to monolayers not exposed to IGFBP-3. The effect of alkaline phosphatase on 125I-IGF-I binding to T-47D cell monolayers preincubated with IGFBP-3 was considerably less marked than that observed on immobilized phosphorylated IGFBP-3 possibly because of binding to nonphosphorylated IGFBP-3 that remained on the cell membrane or the inability of alkaline phosphatase to completely dephosphorylate all the IGFBP-3 in this situation. As a control for these experiments, we used biotinylated IGFBP-3 to determine whether treatment of the cell monolayer with alkaline phosphatase reduced the ability of the monolayer to bind IGFBP-3. There was no significant difference between alkaline phosphatase-treated cell monolayers and buffer-treated cell monolayers to bind biotinylated IGFBP-3 (30.9 ± 2.5 vs. 35.3 ± 1.2 OD units; n = 4; P = not significant).



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  		FIG. 7. The effect of alkaline phosphatase (ALP) treatment on binding of 125I-IGF-I to phosphorylated IGFBP-3. A, IGFBP-3 immobilized on polystyrene tubes was phosphorylated with solubilized T-47D membranes and subsequently treated with ALP as stated in Materials and Methods. Binding of 125I-IGF to the phosphorylated IGFBP-3 and the dephosphorylated IGFBP-3 was compared with binding to nonphosphorylated IGFBP-3. The data represent the mean ± SEM for three separate sets of experiments performed in duplicate. B, Binding of 125I-IGF to T-47D cell monolayers were compared with monolayers that had been incubated with IGFBP-3 and with monolayers that had been incubated with IGFBP-3 and subsequently treated with ALP. The data represent the mean ± SEM for three separate sets of experiments performed in duplicate. The significant differences are shown.

 
Effects of protein kinase inhibitors
A variety of protein kinase inhibitors at various concentrations were tested to provide some insight into the nature of the ectoprotein kinase involved in the phosphorylation of IGFBP-3. Phosphorylation of IGFBP-3 by T-47D membranes was reduced by 47.3 ± 3.5% by H-89, a nonspecific inhibitor of protein kinase A, as well as other kinases. Phosphorylation of IGFBP-3 was also partially inhibited by H-7, a broad range serine-threonine kinase inhibitor that inhibits both protein kinase A and C (Fig. 8Go). No significant inhibition was seen with genistein, a tyrosine kinase inhibitor or PD 98059, a MAPK inhibitor.



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  		FIG. 8. The effects of protein kinase inhibitors on phosphorylation of IGFBP-3 by T-47D cells. The upper panel shows a representative autoradiogram demonstrating the effects of protein kinase inhibitors on phosphorylation of immobilized IGFBP-3. In the lower panel, the data for three separate experiments have been detected as the mean ± SEM expressed as a percent of control where no inhibitor was added. The concentrations of the inhibitors used were 100 nM, 1 µM, 10 µM, and 10 µM for H-89, PD 98059, genistein and H-7, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we have demonstrated using cross-linking that IGFBP-3 bound to multiple proteins on the T-47D cell membrane. This is consistent with other reports using different cell lines where several IGFBP-3 binding sites were also identified (14, 16). The most abundant biotinylated species we observed was the 32-kDa band, which we identified as phosphorylated IGFBP-3. Because this band was enhanced by cross-linking, we speculate that it represents IGFBP-3 that had been associated with a membrane protein, possibly the kinase itself, and subsequently dissociated either by proteolytic activity or by the process of boiling under the reducing conditions before SDS-PAGE. Although protease inhibitors were included in all experiments, it is possible that the inhibition of all proteolytic activity was not complete. Alternatively, the enhancement of the 32-kDa band by DSS treatment itself may have resulted from inactivation of membrane-associated IGFBP-3 protease activity.

In addition to binding to the cell membranes, IGFBP-3 is phosphorylated at the membrane by a process that is inhibited by IGF-I and does not appear to require internalization. Although our solubilized membrane preparation is likely to be contaminated with intracellular membrane components, we also demonstrated that intact cells also have the ability to phosphorylate IGFBP-3. We used biotinylated IGFBP-3 that had been immobilized on streptavidin-coated tubes and intact washed cells that had been treated with an inhibitor of internalization, thus excluding the possibility that the biotinylated IGFBP-3 was internalized and phosphorylated by an intracellular kinase. Although the experiments with intact cells were performed both with cell monolayers and trypsinized cells, we cannot exclude the remote possibility that the phosphorylation was the result of damaged cells exposing internal cell contents. This seems unlikely because the magnitude of the response with intact cells is similar to that seen with cell membranes (see Fig. 4DGo). Furthermore, it is unlikely that the phosphorylation resulted from a secreted protein kinase because no activity was detected in conditioned medium, nor did washing the monolayer affect its ability to phosphorylate IGFBP-3. Although breast cancer cells, including the T-47D cells used in these experiments, secreted IGFBP-3 and a proportion of the IGFBP-3 in breast cancer cell-conditioned medium may be phosphorylated, our assay conditions would not detect this secreted phospho-IGFBP-3 because we analyzed exogenous biotinylated IGFBP-3 retained on streptavidin-coated tubes. Endogenous, secreted IGFBP-3 would lack a biotin label and therefore would not be retained on the streptavidin-coated tubes.

It is not clear whether IGF-I inhibits binding of IGFBP-3 to the membranes or whether IGF-I inhibits the phosphorylation directly. However, it is apparent that IGF-I can bind to the phosphorylated IGFBP-3. Furthermore, phosphorylation occurs without the need for added ATP because it can be detected as an alkaline phosphatase-sensitive change in the pI of IGFBP-3 in the absence of any added ATP (Fig. 3Go). These data indicate that, under normal circumstances, there are sufficient phosphate donors present at the cell surface to allow for phosphorylation of IGFBP-3.

Solubilized membrane inhibited binding of IGF-I to both nonphosphorylated and phosphorylated, immobilized IGFBP-3 (Figs. 5BGo and 6BGo). It is possible that, in this setting with IGFBP-3 immobilized, the IGFBP-3 membrane binding sites are able to bind to IGFBP-3 and prevent the interaction of IGF-I with IGFBP-3. It is also possible that some of this inhibition may have been due to IGF-I binding to the type 1 IGF-I receptor present in the membrane preparation, thereby reducing the availability of IGF-I for binding to IGFBP-3. However, this cannot be the whole explanation because we would expect to get a similar pattern of reduced binding for both phosphorylated and nonphosphorylated IGFBP-3. Rather, enhancement of IGF-I binding is seen with low amounts of membrane with nonphosphorylated IGFBP-3, whereas with phosphorylated IGFBP-3 even these low amounts of membrane inhibit IGF-I binding.

Phosphorylated isoforms of IGFBP-3 have been reported in culture medium of Chinese hamster ovary cells transfected with an IGFBP-3 expression vector and in human skin fibroblasts (18, 19). In the latter cell type, a 4-h incubation with both IGF-I and longR3IGF-I enhanced secretion of phospho-IGFBP-3. Because longR3IGF-I, which does not bind IGFBP-3, was as potent as IGF-I, this enhancement likely involves interaction of IGF-I with the type 1 receptor rather than with IGFBP-3 itself (19). In contrast, we found that IGF-I acutely inhibits phosphorylation of IGFBP-3 by both solubilized membranes (Fig. 4CGo) and intact cells (data not shown).

In IGFBP-3 transfected Chines hamster ovary cells, phosphorylation occurs exclusively on serine residues with Ser111 and Ser113 being the predominating sites (18). These serine residues are located in a consensus phosphorylation site for casein kinase 2 that can also phosphorylate IGFBP-3 in vitro (20). Although we cannot exclude the possibility that IGFBP-3 is phosphorylated by an ectoprotein form of casein kinase 2, it should be noted that consensus phosphorylation sites for other serine-threonine kinases are also present in IGFBP-3 (20). Furthermore, IGFBP-3 phosphorylated by T-47D membranes increased binding of 125I-IGF-I, whereas phosphorylation of IGFBP-3 by casein kinase 2 in vitro had no effect on IGF-I binding (20).

While other investigators have focused on secretion of phosphorylated IGFBP-3 isoforms (18, 19, 20), our novel observations suggest that this phosphorylation occurs at the cell surface. Plasma-derived IGFBP-3 is virtually devoid of phospho-isoforms with less than 1 mol of phosphoserine and no detectable phosphothreonine per mol of IGFBP-3 (20). This would suggest that, under normal circumstances, very little secreted IGFBP-3 is phosphorylated or that secreted phosphorylated IGFBP-3 is rapidly dephosphorylated in the circulation.

Ectoprotein kinases are membrane-associated kinases that are thought to be important in the phosphorylation of extracellular proteins (21), including matrix proteins such as fibronectin and cell adhesion molecules (22, 23). They have been identified on a wide variety of cell types (21, 22, 23, 24). Whereas some ectoprotein kinases are detectable in conditioned medium (24), we were unable to detect any kinase activity directed against IGFBP-3 in T-47D cell-conditioned medium. Although protein kinase inhibitors lack absolute specificity, the results observed with the inhibitors used suggest that this ectoprotein kinase may be a membrane-bound isoform of protein kinase A. However, because these kinase inhibitors are relatively nonspecific, identification of the responsible kinase will require further experimentation. We have also shown that the IGFBP-3 phosphorylation was not restricted to mammary cancer cells but was also apparent with kidney epithelial cells, suggesting that this is also likely to be a generalized phenomenon.

Despite the fact that IGF-I inhibited IGFBP-3 binding and phosphorylation, preincubation of IGFBP-3 with T-47D cells or membranes enhanced 125I-IGF-I binding to immobilized IGFBP-3. This enhancement was reversed by treatment with alkaline phosphatase. Furthermore preincubation of T-47D cells with IGFBP-3 also enhanced 125I-IGF-I binding to the monolayer. This effects was only partially reversed by alkaline phosphastase treatment, suggesting that dephosphorylation of the IGFBP-3 on the membrane did not remove the significant amounts of IGFBP-3 from the membrane. Although we cannot exclude other subtle biochemical alterations of the IGFBP-3 by the T-47D cells, there are several lines of evidence that suggest that phosphorylation is the modification that enhances the affinity of IGFBP-3 for IGF-I. Firstly, after incubation of immobilized IGFBP-3 with T-47D we observed a change in pI that was sensitive to alkaline phosphatase. Secondly, incubation of phosphorylated IGFBP-3 with alkaline phosphatase reduced IGF-I binding to IGFBP-3. Finally, incubation of T-47D cell monolayers with IGFBP-3 enhanced 125I-IGF-I binding to the monolayer, and this effect was partially reduced by treatment of the monolayer with alkaline phosphatase.

The net effect of IGFBP-3 on cellular proliferation in vitro is the sum of the IGF-dependent and IGF-independent effects. These include IGFBP-3 facilitation of IGF-I delivery to the cell membrane receptor (a positive effect), inhibition of IGF binding to the IGF-I receptor (an IGF-I-dependent, negative effect) and the IGF-independent proapoptotic effect. It is unclear from our studies whether IGFBP-3 phosphorylation is important in modulating all, or any, of these effects. Although further studies are required to characterize the nature of the ectoprotein kinase involved in IGFBP-3 phosphorylation, this report should serve to focus attention on role of membrane-associated kinase in the phosphorylation of IGFBP-3.


   Footnotes
 
This work was supported by a grant from the Canadian Institutes for Health Research (CIHR). L.J.M. is a recipient of a CIHR Senior Scientist award and an endowed Research Professorship in Metabolic Diseases.

Abbreviations: DSS, Disucinimidyl suberate; HRP, horseradish peroxidase; IGFBP, IGF binding protein; PAO, phenylarsine oxide; pI, isoelectric point; PMSF, phenylmethylsulfonyl fluoride.

Received January 17, 2003.

Accepted for publication May 7, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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