Protein-tyrosine Phosphatase-1B (PTP1B) Mediates the Anti-migratory Actions of Sprouty*

Yinges YigzawDagger , Helen M. PoppletonDagger , Nair Sreejayan§, Aviv Hassid§, and Tarun B. PatelDagger

From the Departments of Dagger  Pharmacology and § Physiology, University of Tennessee, The Health Science Center, Memphis, Tennessee 38163

Received for publication, October 9, 2002, and in revised form, October 28, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mammalian Sprouty proteins have been shown to inhibit the proliferation and migration of cells in response to growth factors and serum. In this communication, using HeLa cells, we have examined the possibility that human Sprouty 2 (hSPRY2) mediates its anti-migratory actions by modulating the activity or intracellular localization of protein-tyrosine phosphatases. In HeLa cells, overexpression of hSPRY2 resulted in an increase in protein-tyrosine phosphatase (PTP1B) amount and activity in the soluble (100,000 × g) fraction of cells without an increase in total amount of cellular PTP1B. This increase in the soluble form of PTP1B was accompanied by a decrease in the amount of the enzyme in the particulate fraction. The amounts of PTP-PEST or PTP1D in the soluble fractions were not altered. Consistent with an increase in soluble PTP1B amount and activity, the tyrosine phosphorylation of cellular proteins and p130Cas was decreased in hSPRY2-expressing cells. In control cells, overexpression of wild-type (WT) PTP1B, but not its C215S catalytically inactive mutant mimicked the actions of hSPRY2 on tyrosine phosphorylation of cellular proteins and migration. On the other hand, in hSPRY2-expressing cells, the C215S mutant, but not WT PTP1B, increased tyrosine phosphorylation of cellular proteins and attenuated the anti-migratory actions of hSPRY2. Interestingly, neither WT nor C215S mutant forms of PTP1B modulated the anti-mitogenic actions of hSPRY2. Therefore, we conclude that an increase in soluble PTP1B activity contributes to the anti-migratory, but not anti-mitogenic, actions of hSPRY2.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sprouty (SPRY)1 was originally described as a protein that regulated fibroblast growth factor signaling in Drosophila and inhibited tracheal branching (1). Subsequently, SPRY was reported as an inhibitor of signaling events initiated by the epidermal growth factor (EGF) and fibroblast growth factor receptor tyrosine kinases (2) in Drosophila and shown to modulate wing vein formation. To date, four isoforms of mammalian SPRY proteins have been cloned (1, 3-5). These proteins have a highly conserved C terminus but their N terminus is variable. Like the Drosophila SPRY proteins, a decrease in mouse SPRY2 expression resulted in increased lung branching morphogenesis (3). These findings demonstrated that SPRY proteins have conserved function to modulate respiratory morphogenesis. The ability of mouse SPRY4 to inhibit angiogenesis (6) also demonstrates that the SPRY proteins plays a profound role in regulating tubular morphogenesis.

At the cellular level, we and others have shown that SPRY1 (7), SPRY2 (8), and SPRY4 (6) inhibit migration and proliferation of cells in response to serum and growth factors. However, the mechanisms involved in SPRY-elicited inhibition of cellular proliferation and migration remain to be completely elucidated. SPRY1 and SPRY2 have been shown to decrease Erk activation in response to fibroblast growth factor and vascular endothelium-derived growth factor in vascular endothelial cells (6, 7). However, even though EGF-stimulated cellular proliferation was inhibited by SPRY proteins, the ability of EGF to activate Erk activation was not affected (7). We have also observed that although the activation of Erk by EGF is not altered by overexpression of human SPRY2 (hSPRY2) in HeLa cells,2 the proliferation and migration of these cells in response to EGF is markedly attenuated by hSPRY2 (8). Another level of regulation by SPRY proteins may involve direct interactions with certain signaling molecules. For instance, hSPRY2 has been shown to interact directly with c-Cbl, and this interaction decreases internalization and down-regulation of the EGF receptor (9).

Because the functions of receptor tyrosine kinases can be attenuated by increase in protein-tyrosine phosphatase activities, we investigated the possibility that hSPRY2 may alter the cellular activity of some protein-tyrosine phosphatase(s) and thereby modulate the biological actions of serum or growth factors. In this communication we demonstrate that the amount of soluble protein-tyrosine phosphatase-1B (PTP1B) activity is increased in hSPRY2-expressing cells. This is accompanied by a decrease in protein tyrosine phosphorylation. Expression of PTP1B mimicked the actions of hSPRY2 and inhibited serum-induced cell migration. More importantly, the inhibition of PTP1B activity in hSPRY2-expressing cells by a dominant negative mutant of PTP1B rescued cells from the anti-migratory actions of hSPRY2. These data demonstrate that PTP1B plays an important role in mediating the actions of hSPRY2 on cell migration.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HA-hSPRY2-expressing Cell Lines-- Full-length HA-tagged or red fluorescent protein-tagged hSpry2 were cloned from two expressed sequence tag clones and stable clones of HeLa that express HA-hSpry2 or hSpry2-red fluorescent protein were generated as reported previously (8).

Construction of Recombinant Adenovirus Expressing Wild-type or Mutant PTP1B-- Replication-deficient (E-1 deleted) recombinant type 5 adenovirus-expressing, HA-tagged wild-type, or C215S mutant PTP1B were prepared using the commercial kit, Adeno-X (Clontech, Palo Alto, CA). Plasmids containing wild-type human PTP1B (pJ3H-PTP435) and catalytically inactive C215S mutant PTP1B (pJ3H-PTP435-C215S) were kindly donated by Dr. Jonathan Chernoff (Fox Chase Cancer Institute, Philadelphia, PA). These PTP1B constructs containing an N-terminal hemagglutinin (HA) epitope were used as templates to amplify PCR product expressing the corresponding full-length HA-tagged PTP1B cDNAs (WT and mutant), flanked by NotI and KpnI restriction sites. PCR products were subcloned into the NotI/KpnI site of the pShuttle vector provided in the Adeno-X kit (Clontech). All sequences of PTP1B were verified by DNA sequencing. An I-CeuI/PI-SceI restriction fragment from pShuttle containing the cytomegalovirus-IE promoter/enhancer 5' to the PTP1B cDNA insert and the polyadenylation signal was ligated into adenoviral DNA backbone that was also restricted with I-CeuI and PI-SceI. Following amplification and purification of recombinant viral DNA from bacteria, adenovirus was generated by transfecting PacI-linearized recombinant viral DNA into HEK 293 cells via the use of the lipid transfection agent FuGENE 6 (Roche Diagnostics).

Cell Fractionation-- HeLa cells were lysed by incubation for 15 min on ice with a buffer containing the following: 20 mM Tris-HCl, pH 7.5, 50 mM NaF, 2 mM sodium orthovanadate, 20 mM p-nitrophenyl phosphate, 2 mM EDTA, 2 mM EGTA, 5 mM benzamidine. Lysates were centrifuged for 60 min at 100,000 × g. The supernatants (soluble fraction) and the pellet (particulate fraction) were then separated from each other.

SDS-PAGE and Western Blotting-- Cell lysates were mixed with Laemmli sample buffer and proteins separated on polyacrylamide gels as described by Laemmli (10). Proteins were transferred onto nitrocellulose and incubated in 5% milk in PBS. The membranes were incubated in primary antibody anti-HA (HA.11 from Covance Research Products, Richmond, CA) or anti-PTP1B (BD Biosciences, San Diego, CA) at 1:1000 dilution for 1 h, followed by secondary antibody (goat anti-rabbit immunoglobulin G-horseradish peroxidase, 1:3000 dilution) for 1 h at room temperature. Proteins were detected using an enhanced chemiluminescence kit from Pierce.

Cell Migration Assays-- The modified Boyden chambers (Costar, Corning, NY) were used for monitoring cell migration. Essentially, overnight serum-starved cells were trypsinized and washed twice with serum-free medium containing 0.5% BSA. Cells were plated on the upper side of transwells (35,000 cells/well) in 100 µl of serum-free medium, and 500 µl of the same medium was added to the lower chamber. The cells were allowed to adhere for 1 h. Cells were treated with virus in serum-free medium for 2 h. After 2 h of virus treatment, serum was added (10%) to the lower chamber, and cells were allowed to migrate overnight at 37 °C. At the end of the experiment the transwell inserts were washed with PBS, fixed with methanol/acetone, and stained with hematoxylin. Cells on the upper chamber side of the membrane were removed with cotton swabs, and cells that migrated through the membrane were counted.

Thymidine Incorporation-- Cells were plated in Dulbecco's minimum essential medium containing 10% serum for 6 h at a density of 7 × 104 cells/well in 24-well plates. Then serum was withdrawn for an overnight period, and cells were treated with adenoviral vectors for 2 h to express either EGFP, WT PTP1B, or dominant negative (DN) PTP1B. The medium was exchanged with Dulbecco's minimum essential medium containing 10% serum and cells incubated for an overnight period. [3H]Thymidine, 1.5 µCi (PerkinElmer Life Sciences) was added to each well and cells incubated for 4 h at 37 °C. At the end of this period, plates were placed on ice, and cells were washed three times each with ice-cold PBS, then with ice-cold 10% trichloroacetic acid, and finally with ethanol/ether (2:1). The cells were dissolved with 0.1% SDS in 0.1 N NaOH by incubating overnight at room temperature. Aliquots were counted for 3H and protein concentration determined using the bicinchoninic acid method (11) (Micro BCA protein assay kit from Pierce).

PTP1B Activity Assays-- To monitor PTP1B activity, soluble fractions (100 µg of protein) from control and hSPRY2-expressing cells were used. The PTP1B was immunoprecipitated with anti-PTP1B antibody, and the immunoprecipitates were washed three times with ice-cold PBS and resuspended in 100 µl of buffer containing 20 mM HEPES, pH 7.5, 2.0 mM EDTA, 2.0 mM EGTA, 5 mM benzamidine, 20 µg/ml each of soybean trypsin inhibitor, leupeptin, and aprotinin (12). Equal aliquots of the immunoprecipitates were incubated in a reaction buffer containing 40 mM MES, pH 6.5, and 20 mM dithiothreitol in the presence and absence of 10 mM vanadate. The reactions were initiated by the addition of 32P-labeled RaytideTM. After incubation at room temperature for the indicated times, the reactions were stopped by addition of 1 ml of activated charcoal, and 500-µl aliquots of the supernatant were counted for 32Pi released. The vanadate-sensitive activity is presented as PTP1B activity.

RaytideTM Labeling-- RaytideTM (Oncogene, San Diego, CA) was labeled according to the manufacturer's instructions. Briefly, 0.3 µg/µl Raytide was phosphorylated with 5 units of c-Src (Upstate Biotechnology, Lake Placid, NY) in the presence of 0.3 mM [gamma -32P]ATP (1.1 µCi/nmol), 10 mM MgCl2, in 50 mM HEPES, pH 7.5, 0.1 mM dithiothreitol, 0.015% Brij, and 0.1 mg/ml BSA buffer. The reactions were incubated for 5 h at room temperature. Thereafter, acetylated BSA was added to a final concentration of 7 mg/ml, and the reaction was stopped with ice-cold 20% trichloroacetic acid (final concentration). The BSA/RaytideTM co-precipitate was centrifuged (20,000 × g for 10 min) and washed five times with 500 µl of 40% trichloroacetic acid until the background in the blank reaction (reaction without RaytideTM) was diminished significantly (<= 10% of that in reaction containing RaytideTM). The final RaytideTM/BSA pellet was washed once with acetone (500 µl) and dissolved in 200 mM Tris-HCl, pH 8.0. Aliquots of the 32P-labeled RaytideTM were stored at -80 °C.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Human SPRY2 inhibits the biological actions of a number of growth factors that exert their effects via activation of receptor tyrosine kinases. Because the action of these kinases can be modulated by protein-tyrosine phosphatases (PTPases), we hypothesized that hSPRY2 may alter the activity of PTPases. To address this hypothesis, we utilized the previously described (8) HeLa cell lines that are transfected to express hSPRY2. The Geneticin (G418)-resistant HeLa cells that do not overexpress hSPRY2 protein were used as controls (8). Initially, we monitored the amount of various protein-tyrosine phosphatases in total cell lysates and in the soluble fractions of hSPRY2-expressing cells. As shown in Fig. 1A, the soluble fraction of hSPRY2-expressing cells contained more PTP1B as compared with controls. However, the expression of PTP-PEST and PTP1D were not altered (Fig. 1A); the equal amounts of Erk in the same blots demonstrates that the loading of proteins was the same. Interestingly, the amount of PTP1B in the soluble fraction remained elevated irrespective of whether the cells were grown in serum or serum-free medium for 1 day. Moreover, the total amount of PTP1B in HeLa cells expressing hSPRY2 was not altered (Fig. 1B). Consistent with this latter observation, the increase in soluble PTP1B amount in hSPRY2-expressing cells was accompanied by a decrease in the amount of PTP1B in the particulate fraction (Fig. 1C). The increase in PTP1B in the soluble fraction of cells was also confirmed with other clonal HeLa cell lines expressing hSPRY2-red fluorescent protein (data not shown).


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Fig. 1.   hSPRY2 increases the amount of PTP1B in soluble fractions of Cells. Control and hSPRY2-expressing HeLa cells were grown in 10% fetal bovine serum or serum-starved for 24 h. The cells were then harvested and soluble and particulate fractions derived as described under "Materials and Methods." A, soluble fractions (30 µg of protein each) of the cell lysates were immunoblotted for PTP-PEST, PTP1B, and PTP1D using the commercially available antibodies (BD Biosciences). The same blot was also reprobed with anti-Erk1/2 antibody to verify efficiency of transfer of protein to the nitrocellulose membrane. B, cells were grown as described for A, and Western analysis was performed in total cell lysate for PTP1B content. C, the particulate and soluble fractions were isolated from control and hSPRY2-expressing cells and analyzed for PTP1B content. The same blot was reprobed with anti-Erk1/2 antibody to verify efficiency of transfer of protein to the nitrocellulose membrane. For each panel, a representative of at least three similar experiments is shown.

To determine whether the soluble PTP1B was active, we immunoprecipitated PTP1B from the soluble fractions of control and hSPRY2-expressing cells and monitored its ability to dephosphorylate 32P-labeled RaytideTM. As shown in Fig. 2, PTP1B activity in immunoprecipitates from the soluble fractions of hSPRY2-expressing cells was greater than that in controls. These data (Fig. 2) demonstrate that the soluble PTP1B in the hSPRY2-expressing cells is active. The increase in PTP1B activity in the soluble fraction (Fig. 2) is consistent with the observation that the amount of PTP1B in the soluble fraction of hSPRY2-expressing cells is higher than that in controls (Fig. 1, A and C). Although hSPRY2 expression increased PTP1B protein in the soluble fraction by >= 5-fold (Fig. 1, A and C), the PTP1B activity in immunoprecipitates was increased by ~2-fold. This may be related to the fact that during the immunoprecipitation procedure, despite our efforts to minimize inactivation of PTP1B, oxidation of the critical cysteine (Cys-215) in the catalytic site of PTP1B occurred and decreased activity. It has been demonstrated that the oxidation of this cysteine residue (Cys-215) to cysteine sulfenic acid decreases PTP1B activity (13). Despite this discrepancy, the data in Fig. 2 clearly show that the activity of PTP1B in immunoprecipitates of soluble fractions from hSPRY2-expressing cells is greater than that in controls.


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Fig. 2.   PTP1B activity in the soluble fractions of hSPRY2-expressing cells is elevated. Soluble fractions from control and hSPRY2-expressing cells were derived as described under "Materials and Methods." Aliquots (100 µg of protein each) of the fractions were then subjected to immunoprecipitation using anti-PTP1B antibody. The PTP1B activity in these immunoprecipitates was monitored by measuring the dephosphorylation of 32P-labeled RaytideTM (12,000 dpm) in the presence and absence of 10 mM vanadate as described under "Materials and Methods." The vanadate-sensitive activity is shown. Data are representative of three similar experiments.

An increase in PTP1B activity in the soluble fraction would suggest that tyrosine phosphorylation of cellular proteins may also be altered. Therefore, we compared lysates of hSPRY2-overexpressing cells with control cells to monitor the tyrosine phosphorylation of proteins. As shown in Fig. 3A, tyrosine phosphorylation of several, but not all, proteins in cells expressing hSPRY2 was decreased as compared with control cells. The same observation was made in another clone of hSPRY2-expressing cells. The most significant decrease in tyrosine phosphorylation was observed on proteins of molecular mass of 130 and 66 kDa. Since one of the substrates of PTP1B is known to be p130Cas (14), we examined whether tyrosine phosphorylation of this protein was decreased in cells overexpressing hSPRY2. As shown in Fig. 3B, compared with control, tyrosine phosphorylation of the immunoprecipitated p130Cas was decreased in hSPRY2-overexpressing cells. Note that the amount of p130Cas in the immunoprecipitates was the same (Fig. 3B). Taken together with the data in Fig. 2, the findings in Fig. 3 are consistent with the notion that increase in PTPase activity in hSPRY2-expressing cells would decrease tyrosine phosphorylation of proteins. Moreover, since p130Cas is a substrate for PTP1B (14), the decrease in p130Cas phosphorylation corroborates the findings that soluble PTP1B is increased in SPRY2-expressing cells.


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Fig. 3.   Tyrosine phosphorylation of cellular proteins and p130Cas are decreased in hSPRY2-expressing cells. A, controls and hSPRY2-expressing HeLa cells were maintained in 10% serum, and proteins (20 µg) in total cell lysates were separated by SDS-PAGE and immunoblotted with anti-phosphotyrosine (alpha -Tyr(P) (alpha -pY)) antibody (alpha -Tyr-99(P) from Santa Cruz Biotechnology Inc., Santa Cruz, CA). Arrows depict the bands whose phosphorylation was most notably altered. B, p130Cas from 350 µg of cell lysate protein was immunoprecipitated using anti-p130Cas antibody (BD Biosciences). Proteins in the immunoprecipitates were separated by SDS-PAGE and blotted with anti-phosphotyrosine (alpha -Tyr(P) (alpha -pY)) antibody (alpha -Tyr-99(P)) and anti-p130Cas antibody to ensure equal loading. A representative of three similar blots is shown.

Next, we reasoned that if hSPRY2 was attenuating the migratory and/or proliferative actions of serum or growth factors by increasing PTP1B activity, then antagonizing the activity of PTP1B in hSPRY2-expressing cells should (i) reverse the decrease in tyrosine phosphorylation of proteins and (ii) rescue the cells from the anti-migratory and/or anti-proliferative actions of hSPRY2. To address this possibility, using adenoviral vectors, we expressed either the WT or catalytically inactive mutant (C215S) of PTP1B in control and hSPRY2-overexpressing cells. Adenovirus that would express EGFP was used as a control. The C215S mutant of PTP1B is catalytically inactive but can bind the substrates and thereby protects substrate dephosphorylation by endogenous (wild-type) PTP1B (14-16). Thus, the C215S mutant functions as a DN PTP1B and is referred to as such from here on. Initially, we investigated the effects of expressing either the WT or DN PTP1B on tyrosine phosphorylation of cellular proteins in control and hSPRY2- overexpressing cells. As shown in Fig. 4A, the expression of WT PTP1B decreased tyrosine phosphorylation of cellular proteins in control cells and cells expressing hSPRY2. Note that as shown before (Fig. 3), in cells infected with virus encoding EGFP, the tyrosine phosphorylation of proteins in the hSPRY2-expressing cells was lower than that in control. In control cells, DN PTP1B did not significantly affect tyrosine phosphorylation of cellular proteins, but the tyrosine phosphorylation of proteins in hSPRY2-overexpressing cells was increased by DN PTP1B. It should be noted that the adenovirus-mediated expression of WT and DN PTP1B in control and hSPRY2 containing cells was the same and ~10-fold higher than the levels of endogenous PTP1B (Fig. 4B). Interestingly, analysis of the soluble fraction of cell lysates for the presence of adenoviral-induced PTP1B showed that in hSPRY2-expressing cells, the amounts of both WT and DN PTP1B were greater than that observed in control cells (Fig. 4C). These latter data demonstrate that while overexpression of PTP1B permits the presence of soluble enzyme to be detected in control cells, the overexpressed PTP1B is also subject to changes in cellular localization (i.e. increase in soluble fraction) by hSPRY2.


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Fig. 4.   Expression of WT and DN forms of PTP1B modulates tyrosine phosphorylation of cellular proteins. A, control and hSPRY2-expressing HeLa cells were treated with adenovirus vectors to express EGFP (Control), WT, or DN PTP1B as described under "Materials and Methods." Forty-eight hours later cells were lysed with Laemmli sample buffer, and 30 µg of protein were separated on SDS-PAGE and blotted with anti-phosphotyrosine antibody. The arrows depict the protein bands whose phosphorylation was most markedly altered. The same blot was reprobed for total Erk to verify efficiency of transfer of protein to the nitrocellulose membrane. B, aliquots of the samples from those shown in A (30 µg of protein) were separated by SDS-PAGE, and Western analysis was performed with anti-PTP1B antibody to determine the expression of endogenous and ectopically expressed WT and DN PTP1B. C, control and hSPRY2-expressing HeLa cells were infected with adenovirus to express EGFP or WT PTP1B or DN PTP1B as described in the legend to A and under "Materials and Methods." The soluble fractions from these cell lysates were analyzed for PTP1B content by immunoblotting. For each panel, a representative of at least three similar experiments is shown.

To determine whether the DN PTP1B would rescue the cells from the anti-migratory actions of hSPRY2, the experiment depicted in Fig. 5A was performed. Essentially, cells were infected with adenovirus for 2 h to express EGFP or WT PTP1B or DN PTP1B. The migration of these cells in response to serum was then monitored as described under "Materials and Methods." That the expression of WT and DN PTP1B was similar in these cells was confirmed at the end of the migration protocol as shown in Fig. 4B. In control cells, the expression of WT PTP1B decreased migration of both control and hSPRY2-expressing cells (Fig. 5A). DN PTP1B did not appreciably alter the migration of control cells, indicating that in these cells the endogenous PTP1B does not significantly contribute to cell migration. On the other hand, in hSPRY2-expressing cells, DN PTP1B markedly attenuated the ability of hSPRY2 to inhibit cell migration. The ability of WT PTP1B to inhibit migration and the ability of DN PTP1B to rescue cells from the anti-migratory activity of hSPRY2 clearly show a role for PTP1B in mediating the biological actions of hSPRY2. Since we and others have shown that hSPRY2 also inhibits proliferation of cells (6-8), we investigated whether the expression of PTP1B would protect cells against the anti-mitogenic actions of hSPRY2. As shown in Fig. 5B, neither the WT nor the DN PTP1B altered thymidine incorporation in control and hSPRY2-overexpressing cells.


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Fig. 5.   WT PTP1B mimics the actions of SPRY2, whereas DN PTP1B rescues cells from anti-migratory actions of SPRY2 without altering the actions of hSPRY2 on mitogenesis. A, control and hSPRY2-expressing cells (35,000/well) were placed on the upper side of the transwell in the modified Boyden chambers. The cells were infected for 2 h with adenovirus to express EGFP, WT PTP1B (WT), or DN PTP1B (DN). The lower chambers were then filled with medium containing 10% serum. Cells on the serum side of the membrane were stained with hematoxylin and counted. The mean ± S.E. of three experiments are shown. Significance of differences was assessed by Student's unpaired t test. B, control and hSPRY2-expressing HeLa cells (70,000 cells/well) were infected with adenovirus to express EGFP (Control), WT PTP1B (WT), or DN PTP1B (DN) as described in the legend to A. Cells were grown in serum for 20 h prior to the addition of [3H]thymidine for a period of 4 h. The incorporation of serum-induced [3H]thymidine was monitored as described previously (27). Protein in the trichloroacetic acid extracts was monitored by the bicinchoninic acid method and data corrected for protein amount as described under "Materials and Methods." The means ± S.D. of three experiments are shown. *, p < 0.001 compared with corresponding control, Student's unpaired t test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PTP1B has been reported to be located in the endoplasmic reticulum by a hydrophobic C-terminal 35-amino acid anchoring region (17, 18). For activation of platelets by a variety of agonists that activate calcium-dependent neutral protease, calpain has been shown to cleave the protein above this anchoring region and render it soluble (19). The cleavage of PTP1B results in a 2-fold greater activity of the enzyme and is accompanied by changes in tyrosine phosphorylation of several proteins (19). On SDS-PAGE, the protease-cleaved PTP1B migrates as a 42-kDa species as compared with the full-length enzyme that migrates as a 50-kDa protein. In our experiments, the soluble form of PTP1B was not smaller in size as compared with the particulate enzyme, suggesting that the increase in soluble PTP1B in hSPRY2-expressing cells is not the result of cleavage of the C-terminal anchoring region. Moreover, although the presence of soluble PTP1B was difficult to detect in control cells, the overexpression of both WT and DN PTP1B was accompanied by the presence of PTP1B in the soluble fractions (Fig. 4B). As determined by their migration on SDS-PAGE, these ectopically expressed, soluble, PTP1B species were also not cleaved (Fig. 4B). Interestingly, the presence of full-length (50 kDa) PTP1B in the cytosolic fraction has also been observed in COS7 cells (20). Hence, it would appear that mechanisms other than cleavage of the full-length PTP1B are responsible for changes in its distribution between the endoplasmic reticulum and cytosol. In this context, the ability of hSPRY2 to redistribute PTP1B in cells may provide yet another means of regulating the biological actions of PTP1B. The precise mechanisms by which PTP1B is rendered soluble by hSPRY2 remain to be elucidated and form the subject of future studies.

Consistent with the observation that the amount of PTP1B and activity in the soluble fraction are elevated, we observed that the phosphorylation of several cellular proteins, including p130Cas was also decreased in hSPRY2-overexpressing cells (Fig. 3). Since p130Cas is a critical component of the focal adhesion complex (21), and because its phosphorylation status modulates the migratory response of cells (22-24), the decrease in phosphorylation of p130Cas would contribute to the anti-migratory actions of hSPRY2. We are currently in the process of determining the identity of the other proteins (~66 kDa) whose phosphorylation is decreased.

Among the mechanisms that mediate the actions of SPRY proteins, studies have suggested that SPRY proteins alter the activity of the Ras-Erk pathway by decreasing the activation of Ras (6, 25) or Raf (26) in response to growth factors. However, to date no attention has been given to the role of protein-tyrosine phosphatases in modulating the actions of SPRY proteins. In this respect, this is the first report to show that hSPRY2 modulates the cellular localization and activity of PTP1B in the soluble (cytosolic) fraction of cells. Indeed, our findings that overexpression of WT PTP1B, but not the DN PTP1B, in control cells decreases migration demonstrates that PTP1B plays a profound role in regulating this process. The fact that the DN PTP1B attenuates the anti-migratory actions of hSPRY2 (Fig. 5A) further reinforces the role of PTP1B in the migration of cells. More importantly, the experiments with the DN PTP1B demonstrate that the anti-migratory actions of hSPRY2 are, in part, mediated by an increase in PTP1B activity. Since DN PTP1B only partially rescues cells from the inhibition of migration in hSPRY2-expressing cells, the data in Fig. 5A also suggest that in addition to PTP1B, other mechanisms may also contribute to the anti-migratory actions of hSPRY2 in response to serum. These additional mechanisms remain to be identified. Interestingly, although hSPRY2 inhibits cellular proliferation (6-8), the expression of DN PTP1B did not rescue cells from the anti-mitogenic actions of hSPRY2 (Fig. 5B). These results demonstrate the specificity of the role of PTP1B in the anti-migratory actions of hSPRY2 and also imply that hSPRY2 inhibits cellular proliferation by modulating other mechanisms.

In summary, we have demonstrated that hSPRY2 expression is associated with an increase in PTP1B in the soluble fraction of cells. The experiments with exogenously expressed PTP1B demonstrate that this enzyme plays a profound role in regulating cell migration, but not proliferation, and that the increase in PTP1B activity in hSPRY2-expressing cells contributes to the anti-migratory actions of hSPRY2.

    ACKNOWLEDGEMENT

We thank Dr. Jonathan Chernoff, Fox Chase Cancer Institute, for providing the PTP1B cDNAs.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL48308 (to T. B. P.) and HL063886 (to A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, University of Tennessee, The Health Science Center, 874 Union Ave., Memphis, TN 38163. Tel.: 901-448-6006; Fax: 901-448-4828; E-mail: tpatel@physio1.utmem.edu.

Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M210359200

2 Y. Yigzaw and T. B. Patel, unpublished results.

    ABBREVIATIONS

The abbreviations used are: SPRY, Sprouty; hSPRY2 (arabic numeral following SPRY designates isoform type), human Sprouty 2; EGF, epidermal growth factor; MES, 2-(N-morpholino)ethanesulfonic acid; BSA, bovine serum albumin; PBS, phosphate-buffered saline; HA, hemagglutinin; PTP1B, protein-tyrosine phosphatase-1B; EGFP, enhanced green fluorescent protein; WT, wild-type; DN, dominant negative; Erk, extracellular signal-regulated kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Hacohen, N., Kramer, S., Sutherland, D., Hiromi, Y., and Krasnow, M. A. (1998) Cell 92, 253-263[Medline] [Order article via Infotrieve]
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