![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Arne Östman,1 and Carina Hellberg1*
Ludwig Institute for Cancer Research, Uppsala Branch, Biomedical Center, S-751 24 Uppsala, Sweden,1 McGill Cancer Center, Montreal, Quebec H3G 1Y6, Canada,2 Institute of Molecular Cell Biology, Medical Faculty, Friedrich Schiller University, D-07747 Jena, Germany,3 Cancer Biology Program, Division of Hematology-Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215,4 Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel5
Received 25 August 2003/ Returned for modification 8 October 2003/ Accepted 8 December 2003
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 binding site Y1021, previously implicated in chemotaxis, displayed the largest increase in phosphorylation. The increase in Y1021 phosphorylation was accompanied by increased phospholipase C1 activity and migratory hyperresponsiveness to PDGF. PDGF ß receptor tyrosine phosphorylation in PTP-1B ko MEFs but not in PTP
ko MEFs was also higher than that in control cells. This increase occurred with a site distribution different from that seen after TC-PTP depletion. PDGF-induced migration was not increased in PTP-1B ko cells. In summary, our findings identify TC-PTP as a previously unrecognized negative regulator of PDGF ß receptor signaling and support the general notion that PTPs display site selectivity in their action on tyrosine kinase receptors. |
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Platelet-derived growth factors (PDGFs) are a family of growth factors that stimulate cell growth, survival, and motility. PDGF isoforms act by binding to two structurally related protein tyrosine kinases, the PDGF 
and ß receptors (16). The binding of PDGF to its receptors results in receptor dimerization, promoting phosphorylation in trans between the two receptors in the complex. PDGF-AA forms receptor dimers, PDGF-AB forms and ß receptor dimers, and PDGF-BB forms all combinations of receptor dimers. Two more PDGF dimers, PDGF-CC and PDGF-DD, recently were identified (2, 24, 25) and shown to preferentially signal through receptor and ßß receptor dimers, respectively, but also may activate both receptor types in cells coexpressing and ß receptors (12, 24).
Phosphorylation of tyrosine 857 (Y857) in the catalytic loop of the PDGF ß receptor kinase increases kinase activity (10). In addition, a number of tyrosine residues outside of the catalytic domain are phosphorylated, leading to site-specific recruitment of signal transduction molecules containing SH2 domains to the activated receptor (16); these molecules include adaptor proteins such as Shc and Grb2 and enzymes such as the Src family tyrosine kinases, phosphatidylinositol 3'-kinase (PI 3-kinase), phospholipase C1 (PLC1), and tyrosine phosphatase SHP-2. The interactions occur in a specific manner determined by three to six amino acid residues downstream of the phosphorylated tyrosines.
T-cell PTP (TC-PTP) is a ubiquitously expressed phosphatase (8). The TC-PTP transcript is modified by alternative splicing, giving rise to 45- and 48-kDa spliced forms of TC-PTP (27). The 45-kDa spliced form has been reported to be the major gene product in most human and rodent tissues and cell lines (19). TC-PTP has been implicated in the regulation of growth factor receptor signaling, both at the level of receptor tyrosine phosphorylation and in the regulation of downstream signaling events. The overexpression of a truncated, active form of TC-PTP has been shown to reduce the tyrosine phosphorylation of several proteins in PDGF-stimulated cells (7). Both the epidermal growth factor (EGF) receptor and the adaptor protein p52Shc have been identified as substrates for TC-PTP (38). The association between the EGF receptor and the 45-kDa TC-PTP takes place at the plasma membrane (38), whereas the 48-kDa TC-PTP colocalizes with the EGF receptor in the endoplasmic reticulum (ER) (39). In addition, TC-PTP has been linked to the dephosphorylation of the insulin receptor (11) and acts as a negative regulator of cytokine signaling through dephosphorylation of the Jak family of tyrosine kinases (36).
Regulation of the PDGF ß receptor by tyrosine phosphatases is poorly understood. In addition to SHP-2, several phosphatases, including a low-molecular-weight PTP (PTP-1B) and a receptor-like tyrosine phosphatase (DEP-1), interact with and dephosphorylate the PDGF ß receptor (4, 13, 18, 22). More recently, in-gel PTP assays were used to identify PDGF receptor-associating PTPs and revealed that PTP-PEST and TC-PTP also could be recovered in PDGF receptor immunoprecipitates (26).
Site-selective dephosphorylation of the PDGF ß receptor by SHP-2 and PTP-1B has been demonstrated (5, 21). Analyses of DEP-1 dephosphorylation of PDGF receptors showed less efficient dephosphorylation of the autoregulatory site Y857 than of some SH2 binding sites (22, 32). These findings suggest that phosphatases can modulate specific signaling pathways by selectively dephosphorylating specific tyrosine residues on the PDGF ß receptor and other receptor tyrosine kinases.
It was recently demonstrated that murine embryonic fibroblasts (MEFs) lacking the TC-PTP displayed a defective PDGF-induced IKK/NF-
B activation pathway, whereas PDGF-induced activation of the PI 3-kinase and Erk signaling pathways was unaffected (17). In this study, we further characterized the effect of TC-PTP depletion on PDGF ß receptor signaling. We demonstrate that TC-PTP directly controls PDGF ß receptor phosphorylation in a site-selective manner and also negatively regulates the migratory response to PDGF.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Isolation of PDGF ß receptors from mouse embryos. TC-PTP wild-type (wt) and knockout (ko) embryonic day 14.5 (E14.5) mouse embryos were homogenized in lysis buffer (50 mM Tris, 50 mM NaCl, 0.25% Na deoxycholate, 1% NP-40, 1 mM EGTA, 10 mM NaF, 50 mM disodium ß-glycerophosphate, 1 mM Na3VO4, 0.1% sodium dodecyl sulfate [SDS]). One milligram of total cell extract from each embryo was precleared with protein A-agarose for 1 h at 4°C. PDGF ß receptors then were immunoprecipitated with 2 µg of anti-PDGF ß receptor antibodies at 4°C for 2 h. Protein complexes were washed three times in lysis buffer, resolved by SDS-7% polyacrylamide gel electrophoresis (PAGE), and transferred to polyvinylidene difluoride membranes (Immobilon; Millipore). PDGF ß receptor phosphorylation was detected with monoclonal phosphotyrosine antibody PY99 (1 µg/ml), followed by stripping in 0.4 M NaOH and reprobing with 2 µg of PDGF ß receptor antiserum/ml. Bound antibodies were visualized by enhanced chemiluminescence after incubation with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences).
Cell cultures.
MEF cell lines derived from TC-PTP ko and littermate wt mice were described previously (40). Reexpressing cells were created by transfecting TC-PTP ko MEFs with pcDNA4 vectors containing constructs of either the 45-kDa isoform of mouse wt TC-PTP (17) or a catalytically inactive mutant form of TC-PTP in which the catalytic cysteine is mutated to serine (CS). Two mass cultures stably expressing wt TC-PTP (ko/wt1 and ko/wt2) or the CS mutant (ko/CS1 and ko/CS2) were generated. Additionally, wt TC-PTP and the CS mutant were transiently expressed in TC-PTP ko MEFs by using Lipofectamine/Plus reagent according to the manufacturer's protocol (Invitrogen Life Technologies, Carlsbad, Calif.). PTP ko and wt MEFs (31), PTP-1B ko MEFs (13), and MEFs reconstituted with human wt PTP-1B (PTP-1B wt MEFs) (13) were previously described. All MEFs were grown in Dulbecco minimal essential medium (DMEM) supplemented with 10% fetal calf serum and 1:5,000 plasmocin. Porcine aorta endothelial (PAE) cells stably expressing the PDGF ß receptor (6) and PDGF ß receptors with tyrosine-to-phenylalanine point mutations (34) were previously described. PAE cells were grown in Ham's F-12 medium supplemented with 10% fetal calf serum, 100 U of penicillin/ml, and 100 µg of streptomycin/ml.
Peptide synthesis and generation of site-selective phosphotyrosine antibodies. Peptides corresponding to tyrosine phosphorylation sites of the human PDGF ß receptor (Table 1) were synthesized by 9-fluorenylmethoxy carbonyl chemistry with an Applied Biosystems 433A peptide synthesizer and purified by reverse-phase chromatography followed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to confirm the expected molecular weights. The peptides were conjugated to keyhole limpet hemocyanin by using m-maleimido benzoyl-N-hydroxysuccinimide ester (Pierce), and the conjugates were used to immunize rabbits. The antibodies were affinity purified by passing the antiserum over three consecutive columns of immobilized nonphosphorylated peptide, phosphotyrosine-agarose, and phosphorylated peptide as previously described (9). The peptides were coupled to Sulfolink (Pierce) and phosphotyrosine was coupled to Affigel 10 (Bio-Rad) according to the manufacturers' instructions. The antibodies were eluted from the phosphopeptide column with 4.6 M MgCl2 and immediately diluted in an equal volume of double-distilled H2O. After dialysis against 20 mM HEPES (pH 7.4)- 50 mM NaCl, the antibodies were subjected to ammonium sulfate precipitation. The precipitates were dissolved in phosphate-buffered saline (PBS), and the ammonium sulfate was removed by dialysis against PBS. The antibodies were finally diluted in 50% glycerol and stored at -70°C.
|
Phosphatase assay. Cells were lysed in the buffer described above but supplemented with 10 mM 1,4-dithiothreitol and lacking Na3VO4. Following immunoprecipitation of TC-PTP, the beads were washed three times in lysis buffer and once in phosphatase assay buffer (25 mM imidazole [pH 7.4], 10 mM dithiothreitol, 0.1 mg of BSA/ml). The precipitates were resuspended in assay buffer, and phosphatase activity was determined by using a 32P-labeled peptide (amino acid sequence AEEEIYGEFEAKKKK) as a substrate as previously described (37). Assays were performed in duplicate, and phosphatase activity was expressed as the relative amount of 32P-labeled radioactivity released from the peptide after 7 min of incubation at 30°C.
In vitro PDGF ß receptor dephosphorylation. PAE cells stably expressing the PDGF ß receptor were stimulated with 100 ng of PDGF-BB/ml for 1 h on ice. The cells were lysed in the buffer described above but with the addition of 15 mM iodoacetic acid and 1 mM benzamidine. The phosphorylated receptors were precipitated with WGA-Sepharose, followed by four washes in phosphatase assay buffer. The precipitates were resuspended in a total volume of 100 µl of assay buffer or assay buffer containing various amounts of recombinant TC-PTP (New England Biolabs). The samples were incubated at 30°C for 10 min, receptor dephosphorylation was terminated by the addition of 1 ml of ice-cold lysis buffer including phosphatase inhibitors, and the samples were immediately washed. After the completion of washing, phosphorylation of the different tyrosine residues was detected by immunoblotting as described above.
Inositol phosphate formation. Cells were plated at 105 cells per well in 12-well plates and incubated with serum-free M199 medium supplemented with 1 mg of BSA/ml overnight. After being labeled with 4 µCi of myo-[3H]inositol (Amersham) per well for 24 h, the cells were stimulated with various amounts of PDGF-BB in the presence of LiCl at 37°C for 30 min. Isolation and detection of the released inositol phosphate fraction were performed as described previously (1).
PDGF ß receptor-associated PI 3-kinase activity.
Cells were starved overnight in medium supplemented with 1 mg of BSA/ml and then were stimulated with 50 ng of PDGF-BB/ml at room temperature for 10 min. PDGF ß receptors were immunoprecipitated with 2 µg of anti-PDGF ß receptor antibody at 4°C for 3 h. Aliquots of the corresponding immunoprecipitates were subjected to analysis of PDGF ß receptor levels by immunoblotting. The collected beads were subjected to a kinase reaction in the presence of 20 µCi of [-32P]ATP and with 0.2 µg of presonicated phosphatidylinositol/µl as a substrate as previously described (18). The kinase reaction was performed at 37°C for 30 min and was stopped with 100 µl of chloroform-methanol-HCl (50:100:1 [vol/vol/vol]). Following extraction, the phospholipids were concentrated and applied to a thin-layer chromatography plate (Silica Gel 60; Whatman LK6DF). The plate was developed with 2 M acetic acid-propanol (35:65 [vol/vol]) and exposed to a PhosphorImager.
Cell migration assay. Cell migration was determined by using a 96-well ChemoTX (Neuroprobe) cell migration microplate with a pore size of 3.2 µm. The filters were coated with 50 µg of fibronectin (BD Biochemicals)/ml in PBS for 1 h at room temperature, rinsed twice in distilled water, and air dried. Serum-starved cells were trypsinized to yield single cells, and trypsinization was terminated by the addition of 150 µl of Trasylol/ml of cell suspension. The cells were pelleted and diluted to a final concentration of 2.5 x 106 cells/ml in DMEM supplemented with 1 mg of BSA/ml (DMEM/BSA). The wells of the ChemoTX microplate were filled with DMEM/BSA or with DMEM/BSA supplemented with either 10% fetal bovine serum or various concentrations of PDGF-BB. The filters were placed on the wells to allow contact with the medium, and 50,000 cells were added on top of each filter. The chamber was incubated for 4 h at 37°C in 5% CO2. Cells adhering to the top of the filter were removed, and cells adhering to the bottom of the filter were fixed by 3 min of incubation in 96% ethanol. The filters were washed three times in distilled water, and adherent cells were stained with 0.04% (wt/vol) crystal violet in 4% (vol/vol) ethanol and detected spectrophotometrically by using a Biomek1000 automated laboratory workstation (Beckman) with a 600-nm filter. All experiments were performed in quadruplicate.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
In agreement with a direct effect of TC-PTP on PDGF ß receptor phosphorylation, reconstitution of TC-PTP ko MEFs with wt TC-PTP led to a decrease in PDGF ß receptor phosphorylation (Fig. 2A, left panel). In contrast, receptor phosphorylation in TC-PTP ko cells transfected with the CS mutant was similar to that in parental ko cells (Fig. 2A, left panel). To verify the effect of TC-PTP reconstitution, we transiently expressed wt TC-PTP and the CS mutant in TC-PTP ko MEFs. Analysis of wt TC-PTP expression indicated that the expression of TC-PTP activity corresponding to 27% that seen in wt MEFs (Fig. 2B, right panel) was sufficient to restore PDGF ß receptor phosphorylation to the levels detected in wt MEFs (Fig. 2A, right panel). Expression of the CS mutant led to a slight increase in PDGF ß receptor phosphorylation, presumably due to its ability to act as a substrate trap (Fig. 2A, right panel). Together, the results in Fig. 2 indicate that the PDGF ß receptor is directly dephosphorylated by TC-PTP.
Generation of a panel of site-specific PDGF ß receptor antibodies.
To study the phosphorylation of the individual tyrosine residues of the PDGF ß receptor, we generated antibodies against phosphopeptides derived from the human PDGF ß receptor, corresponding to four phosphorylation sites (Table 1). The designations of the antibodies are based on the sequence of the human receptor. To test the specificity of affinity-purified antibodies, PAE cells stably expressing the PDGF ß receptor were used. As a control for site selectivity, we used a series of PAE cell lines each expressing PDGF ß receptor mutants containing tyrosine-to-phenylalanine point mutations of one or two of the tyrosine residues corresponding to the receptor phosphorylation sites. To obtain maximal receptor tyrosine phosphorylation, the cells were stimulated with 100 ng of PDGF-BB/ml for 1 h on ice. None of the antibodies bound to the unphosphorylated PDGF ß receptor in an immunoblotting assay, whereas all of the antibodies recognized the phosphorylated wt PDGF ß receptor in an immunoblotting assay (Fig. 3). The antibodies also recognized the phosphorylated PDGF receptor, a result which was expected because several phosphorylated tyrosines are conserved between the two PDGF receptors (data not shown). Low-affinity recognition of the PDGF receptor also was detected with the pY771 antibody, despite the fact that this site is not conserved (data not shown). Mutations of individual phosphorylation sites of the PDGF ß receptor to which the antibodies were raised specifically abolished binding, demonstrating that the antibodies are site selective (Fig. 3).
|
|
|
is associated with site-selective effects on PDGF ß receptor phosphorylation.
To investigate the effects of losses of other PTPs on the phosphorylation of selected sites in the PDGF ß receptor, we used MEFs from PTP-1B and PTP ko mice. MEFs from PTP-1B ko mice displayed greater phosphorylation of the PDGF ß receptor following stimulation with PDGF-DD than did MEFs reconstituted with human PTP-1B (Fig. 6A), in agreement with published results (13). In contrast, PTP ko MEFs did not differ in receptor phosphorylation from the corresponding MEFs from wt mice (Fig. 6A). The phosphorylation of the four different sites of the PDGF ß receptor was examined by using these cell lines. The increased phosphorylation induced by a loss of PTP-1B was not evenly distributed over the sites investigated. In contrast to the results for TC-PTP ko MEFs (data from Fig. 4C inserted into Fig. 6B), a loss of PTP-1B led to a marked increase in the phosphorylation of Y579, whereas Y771 was found to be less affected than the other sites (Fig. 6B). As expected from the findings shown in Fig. 6A, a loss of PTP did not affect the phosphorylation of any of the sites investigated (Fig. 6B).
|
PLC1 is activated following binding to Y1021 of the PDGF ß receptor. Stimulation of wt cells with PDGF-BB dose dependently activated PLC1, measured as the production of inositol phosphate (Fig. 7A). Consistent with the observed hyperphosphorylation of Y1021 in TC-PTP ko cells, ligand-induced PLC1 activation was increased in these cells (Fig. 7A). PLC1 expression levels were not affected by deletion of TC-PTP (Fig. 7A, inset).
|
Thus, the large increase in the phosphorylation of Y1021 observed in TC-PTP ko MEFs correlated with an increase in PLC1 activation, whereas the more modest increase in the phosphorylation of Y751 had no observable effect on downstream signaling.
Loss of TC-PTP but not of PTP-1B increases PDGF-induced cell migration.
Overactivation of PLC1 has been linked to an increased chemotactic response following PDGF stimulation (35). Therefore, we investigated the effect of a loss of TC-PTP on cell migration in response to PDGF-BB. TC-PTP ko MEFs displayed a 1.5-fold-higher level of random migration than did wt MEFs. Both cell types displayed an increased migration rate in response to PDGF-BB (Fig. 8A), but the increase was larger for TC-PTP ko MEFs than for wt MEFs (nine- and sixfold increases, respectively, in response to 10 ng of PDGF-BB/ml). This effect was partially reversed in TC-PTP ko cells stably transfected with wt TC-PTP (data not shown). When serum was used as a stimulus, the TC-PTP wt MEFs responded with a greater increase in migration than did the TC-PTP ko MEFs (15- and 4-fold increases, respectively), indicating that the increase seen following PDGF stimulation was not due to increased general cell motility following the loss of TC-PTP (Fig. 8A).
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 and increased cell migration in response to PDGF (Fig. 7 and 8). PTPs that were previously implicated in the control of PDGF receptor phosphorylation include the classical PTPs SHP-1, SHP-2, PTP-1B, PTP-PEST, DEP-1, and LMW-PTP (4, 14, 20, 22, 26, 41). Most of these studies have associated individual PTPs with PDGF receptor signaling after analyses of cells with heterologous PTP expression or by characterization of PTPs coprecipitating with PDGF receptors. In the present study, we identified TC-PTP as a negative regulator of PDGF receptor phosphorylation by analyses of the consequences of PTP depletion.
Both TC-PTP and PTP-1B were found to display site selectivity in dephosphorylating the PDGF ß receptor and, importantly, each phosphatase regulated the phosphorylation of a distinct set of tyrosine residues (Fig. 3 and 6). The notion that different phosphatases regulate the phosphorylation status of different tyrosine residues could account for cell type-specific effects of growth factor stimulation. By regulating the expression and/or activation of tyrosine phosphatases, the cell consequently might be able to modulate growth factor-induced signals and fine-tune its response to the surrounding environment. In addition, regulation of the expression and activity of site-selective PDGF ß receptor-directed phosphatases by other receptors would provide a way for cross talk between different classes of receptors.
In general, the issue of site selectivity in the dephosphorylation of tyrosine kinase receptors by PTPs remains poorly explored. However, studies that have been done so far on PDGF receptor dephosphorylation support the notion that selectivity in dephosphorylation is a common feature. Deletion of the binding site for SHP-2 results in a specific increase in the phosphorylation of Y771 in PDGF and ß receptor heterodimeric complexes (9). Also, DEP-1 dephosphorylation of the PDGF ß receptor displays site selectivity, with Y1021 and Y857 occurring as preferred and nonpreferred sites, respectively (22, 32). Finally, the phosphorylation of regulatory Y857 was dramatically increased after the overexpression of the catalytically inactive form LMW-PTP, suggesting that this site is a preferred site for dephosphorylation by LMW-PTP (5). Support for the general notion of the site-selective action of PTPs was also recently provided by the demonstration that DEP-1 preferentially dephosphorylates Y1349 and Y1365 of the hepatocyte growth factor receptor/c-Met (30).
A loss of TC-PTP resulted in a larger increase in overall PDGF ß receptor phosphorylation than did a loss of PTP-1B. It should be noted, however, that the experimental systems are not identical. TC-PTP ko MEFs were compared to MEFs from littermate wt mice, whereas PTP-1B ko MEFs were compared to reconstituted cells. Furthermore, the immortalization of the MEFs used in this study is likely to affect signaling pathways, thereby affecting the intracellular response to growth factors. However, the fact that the distributions of the increases in tyrosine phosphorylation between the sites differ between TC-PTP and PTP-1B ko MEFs implies that these PTPs regulate different responses to PDGF. Also, the absence of effects of PTP-1B depletion on PDGF-induced migration is in agreement with the results of Haj et al. (13), who detected a selective increase in Erk activation following PDGF ß receptor activation in PTP-1B ko cells. In the context of discussing PTP specificity, it is also noteworthy that depletion of PTP has no effects on PDGF ß receptor phosphorylation (Fig. 6). This observation presents clear evidence that not all PTPs are involved in the control of PDGF receptor signaling.
Phosphorylation of Y1021 and subsequent activation of PLC1 have been linked to PDGF-induced chemotaxis (15, 23, 35). It is therefore noteworthy that TC-PTP ko cells display hyperphosphorylation of Y1021, enhanced PLC1 activation, and an increased migratory response whereas, in contrast, PTP-1B ko cells are characterized by less than a twofold increase in the phosphorylation of Y1021 and no increase in the migratory response to PDGF. These findings support the possibility that site-selective dephosphorylation by PTPs translates into alterations in specific cellular responses. The physiological relevance of these findings should be further explored, e.g., by comparing the patterns of PDGF receptor phosphorylation in tissues where PDGF mediates predominantly proliferative or migratory responses.
TC-PTP also has been linked to the dephosphorylation of other tyrosine kinase receptors, including the EGF and insulin receptors (11, 38, 39). In addition, TC-PTP acts as a negative regulator of cytokine signaling through dephosphorylation of the Jak family of tyrosine kinases (36). The target specificity of TC-PTP is presently unclear, and more studies are required for identification of the functional role(s) of this enzyme.
Recent observations indicate spatially restricted tyrosine kinase dephosphorylation by PTPs. Dephosphorylation of the EGF receptor by the 48-kDa isoform of TC-PTP occurs in the ER, whereas nuclear 45-kDa TC-PTP translocates to the cell periphery, where it dephosphorylates the EGF receptor in response to EGF (38). PTP-1B is also located in the ER, where it dephosphorylates the EGF receptor and the PDGF receptor (14). From this perspective, it will be interesting to elucidate whether the effects on PDGF receptor phosphorylation demonstrated in this study occur on the entire receptor population or on spatially restricted receptor subsets.
PDGF receptor signaling has well-documented important functions in developmental as well as pathological processes (3, 33). The identification of TC-PTP as a negative regulator of PDGF receptor signaling will be important in the further elucidation of the pathophysiological role of the receptor. One obvious topic for future studies is to investigate whether the bone marrow stroma-dependent defect in hematopoiesis seen in TC-PTP ko mice involves perturbed PDGF receptor signaling (40). This possibility is supported by previous studies which implicated PDGF receptor signaling in diseases of the bone marrow stroma.
In conclusion, we have obtained evidence that both TC-PTP and PTP-1B act as distinct site-selective modulators of PDGF ß receptor signaling. Further studies should be aimed at elucidating the physiological relevance of this regulatory mechanism.
|
ACKNOWLEDGMENTS |
|---|
C.H. and A.Ö. received support from the Swedish Cancer Foundation. F.D.B. was supported by Deutsche Forschungsgemeinschaft (grant Bo 1042/4-2).
|
FOOTNOTES |
|---|
Present address: Division of Experimental Clinical Chemistry, Lund University, Malmö University Hospital, SE-205 02 Malmö, Sweden.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Bergsten, E., M. Uutela, X. Li, K. Pietras, A. Ostman, C. H. Heldin, K. Alitalo, and U. Eriksson. 2001. PDGF-D is a specific, protease-activated ligand for the PDGF beta-receptor. Nat. Cell Biol. 3:512-516.[CrossRef][Medline]
3. Betsholtz, C., L. Karlsson, and P. Lindahl. 2001. Developmental roles of platelet-derived growth factors. Bioessays 23:494-507.[CrossRef][Medline]
4. Chiarugi, P., P. Cirri, G. Raugei, G. Camici, F. Dolfi, A. Berti, and G. Ramponi. 1995. PDGF receptor as a specific in vivo target for low M(r) phosphotyrosine protein phosphatase. FEBS Lett. 372:49-53.[CrossRef][Medline]
5. Chiarugi, P., P. Cirri, M. L. Taddei, D. Talini, L. Doria, T. Fiaschi, F. Buricchi, E. Giannoni, G. Camici, G. Raugei, and G. Ramponi. 2002. New perspectives in PDGF receptor downregulation: the main role of phosphotyrosine phosphatases. J. Cell Sci. 115:2219-2232.
6. Claesson-Welsh, L., A. Eriksson, A. Moren, L. Severinsson, B. Ek, A. Östman, C. Betsholtz, and C. H. Heldin. 1988. cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules. Mol. Cell. Biol. 8:3476-3486.
7. Cool, D. E., N. K. Tonks, H. Charbonneau, E. H. Fischer, and E. G. Krebs. 1990. Expression of a human T-cell protein-tyrosine-phosphatase in baby hamster kidney cells. Proc. Natl. Acad. Sci. USA 87:7280-7284.
8. Cool, D. E., N. K. Tonks, H. Charbonneau, K. A. Walsh, E. H. Fischer, and E. G. Krebs. 1989. cDNA isolated from a human T-cell library encodes a member of the protein-tyrosine-phosphatase family. Proc. Natl. Acad. Sci. USA 86:5257-5261.
9. Ekman, S., A. Kallin, U. Engstrom, C. H. Heldin, and L. Rönnstrand. 2002. SHP-2 is involved in heterodimer specific loss of phosphorylation of Tyr771 in the PDGF beta-receptor. Oncogene 21:1870-1875.[CrossRef][Medline]
10. Fantl, W. J., J. A. Escobedo, and L. T. Williams. 1989. Mutations of the platelet-derived growth factor receptor that cause a loss of ligand-induced conformational change, subtle changes in kinase activity, and impaired ability to stimulate DNA synthesis. Mol. Cell. Biol. 9:4473-4478.
11. Galic, S., M. Klingler-Hoffmann, M. T. Fodero-Tavoletti, M. A. Puryer, T. C. Meng, N. K. Tonks, and T. Tiganis. 2003. Regulation of insulin receptor signaling by the protein tyrosine phosphatase TCPTP. Mol. Cell. Biol. 23:2096-2108.
12. Gilbertson, D. G., M. E. Duff, J. W. West, J. D. Kelly, P. O. Sheppard, P. D. Hofstrand, Z. Gao, K. Shoemaker, T. R. Bukowski, M. Moore, A. L. Feldhaus, J. M. Humes, T. E. Palmer, and C. E. Hart. 2001. Platelet-derived growth factor C (PDGF-C), a novel growth factor that binds to PDGF alpha and beta receptor. J. Biol. Chem. 276:27406-27414.
13. Haj, F. G., B. Markova, L. D. Klaman, F. D. Bohmer, and B. G. Neel. 2003. Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatase-1B. J. Biol. Chem. 278:739-744.
14. Haj, F. G., P. J. Verveer, A. Squire, B. G. Neel, and P. I. Bastiaens. 2002. Imaging sites of receptor dephosphorylation by PTP1B on the surface of the endoplasmic reticulum. Science 295:1708-1711.
15. Hansen, K., M. Johnell, A. Siegbahn, C. Rorsman, U. Engstrom, C. Wernstedt, C. H. Heldin, and L. Rönnstrand. 1996. Mutation of a Src phosphorylation site in the PDGF beta-receptor leads to increased PDGF-stimulated chemotaxis but decreased mitogenesis. EMBO J. 15:5299-5313.[Medline]
16. Heldin, C. H., A. Östman, and L. Rönnstrand. 1998. Signal transduction via platelet-derived growth factor receptors. Biochim. Biophys. Acta 1378:F79-F113.[Medline]
17. Ibarra-Sanchez, M. J., J. Wagner, M. T. Ong, C. Lampron, and M. L. Tremblay. 2001. Murine embryonic fibroblasts lacking TC-PTP display delayed G1 phase through defective NF-kappaB activation. Oncogene 20:4728-4739.[CrossRef][Medline]
18. Jandt, E., K. Denner, M. Kovalenko, A. Östman, and F.-D. Böhmer. 2003. The protein-tyrosine phosphatase DEP-1 modulates growth factor-stimulated cell migration and cell-matrix adhesion. Oncogene 22:4175-4185.[CrossRef][Medline]
19. Kamatkar, S., V. Radha, S. Nambirajan, R. S. Reddy, and G. Swarup. 1996. Two splice variants of a tyrosine phosphatase differ in substrate specificity, DNA binding, and subcellular location. J. Biol. Chem. 271:26755-26761.
20. Kazlauskas, A., G. S. Feng, T. Pawson, and M. Valius. 1993. The 64-kDa protein that associates with the platelet-derived growth factor receptor beta subunit via Tyr-1009 is the SH2-containing phosphotyrosine phosphatase Syp. Proc. Natl. Acad. Sci. USA 90:6939-6943.
21. Klinghoffer, R. A., and A. Kazlauskas. 1995. Identification of a putative Syp substrate, the PDGF beta receptor. J. Biol. Chem. 270:22208-22217.
22. Kovalenko, M., K. Denner, J. Sandström, C. Persson, S. Gross, E. Jandt, R. Vilella, F. Böhmer, and A. Östman. 2000. Site-selective dephosphorylation of the platelet-derived growth factor beta-receptor by the receptor-like protein-tyrosine phosphatase DEP-1. J. Biol. Chem. 275:16219-16226.
23. Kundra, V., J. A. Escobedo, A. Kazlauskas, H. K. Kim, S. G. Rhee, L. T. Williams, and B. R. Zetter. 1994. Regulation of chemotaxis by the platelet-derived growth factor receptor-beta. Nature 367:474-476.[CrossRef][Medline]
24. LaRochelle, W. J., M. Jeffers, W. F. McDonald, R. A. Chillakuru, N. A. Giese, N. A. Lokker, C. Sullivan, F. L. Boldog, M. Yang, C. Vernet, C. E. Burgess, E. Fernandes, L. L. Deegler, B. Rittman, J. Shimkets, R. A. Shimkets, J. M. Rothberg, and H. S. Lichenstein. 2001. PDGF-D, a new protease-activated growth factor. Nat. Cell Biol. 3:517-521.[CrossRef][Medline]
25. Li, X., A. Ponten, K. Aase, L. Karlsson, A. Abramsson, M. Uutela, G. Bäckström, M. Hellström, H. Boström, H. Li, P. Soriano, C. Betsholtz, C. H. Heldin, K. Alitalo, A. Östman, and U. Eriksson. 2000. PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor. Nat. Cell Biol. 2:302-309.[CrossRef][Medline]
26. Markova, B., P. Herrlich, L. Ronnstrand, and F. D. Bohmer. 2003. Identification of protein tyrosine phosphatases associating with the PDGF receptor. Biochemistry 42:2691-2699.[CrossRef][Medline]
27. Mosinger, B., Jr., U. Tillmann, H. Westphal, and M. L. Tremblay. 1992. Cloning and characterization of a mouse cDNA encoding a cytoplasmic protein-tyrosine-phosphatase. Proc. Natl. Acad. Sci. USA 89:499-503.
28. Neel, B. G., and N. K. Tonks. 1997. Protein tyrosine phosphatases in signal transduction. Curr. Opin. Cell Biol. 9:193-204.[CrossRef][Medline]
29. Östman, A., and F. D. Böhmer. 2001. Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases. Trends Cell Biol. 11:258-266.[CrossRef][Medline]
30. Palka, H. L., M. Park, and N. K. Tonks. 2003. Hepatocyte growth factor receptor tyrosine kinase met is a substrate of the receptor protein-tyrosine phosphatase DEP-1. J. Biol. Chem. 278:5728-5735.
31. Peretz, A., H. Gil-Henn, A. Sobko, V. Shinder, B. Attali, and A. Elson. 2000. Hypomyelination and increased activity of voltage-gated K(+) channels in mice lacking protein tyrosine phosphatase epsilon. EMBO J. 19:4036-4045.[CrossRef][Medline]
32. Persson, C., U. Engström, S. L. Mowbray, and A. Östman. 2002. Primary sequence determinants responsible for site-selective dephosphorylation of the PDGF beta-receptor by the receptor-like protein tyrosine phosphatase DEP-1. FEBS Lett. 517:27-31.[CrossRef][Medline]
33. Pietras, K., T. Sjoblom, K. Rubin, C. H. Heldin, and A. Östman. 2003. PDGF receptors as drug targets. Cancer Cells 3:439-443.
34. Rönnstrand, L., A. K. Arvidsson, A. Kallin, C. Rorsman, U. Hellman, U. Engstrom, C. Wernstedt, and C. H. Heldin. 1999. SHP-2 binds to Tyr763 and Tyr1009 in the PDGF beta-receptor and mediates PDGF-induced activation of the Ras/MAP kinase pathway and chemotaxis. Oncogene 18:3696-3702.[CrossRef][Medline]
35. Rönnstrand, L., A. Siegbahn, C. Rorsman, M. Johnell, K. Hansen, and C. H. Heldin. 1999. Overactivation of phospholipase C-gamma1 renders platelet-derived growth factor beta-receptor-expressing cells independent of the phosphatidylinositol 3-kinase pathway for chemotaxis. J. Biol. Chem. 274:22089-22094.
36. Simoncic, P. D., A. Lee-Loy, D. L. Barber, M. L. Tremblay, and C. J. McGlade. 2002. The T cell protein tyrosine phosphatase is a negative regulator of janus family kinases 1 and 3. Curr. Biol. 12:446-453.[CrossRef][Medline]
37. Sörby, M., J. Sandström, and A. Östman. 2001. An extracellular ligand increases the specific activity of the receptor-like protein tyrosine phosphatase DEP-1. Oncogene 20:5219-5224.[CrossRef][Medline]
38. Tiganis, T., A. M. Bennett, K. S. Ravichandran, and N. K. Tonks. 1998. Epidermal growth factor receptor and the adaptor protein p52Shc are specific substrates of T-cell protein tyrosine phosphatase. Mol. Cell. Biol. 18:1622-1634.
39. Tiganis, T., B. E. Kemp, and N. K. Tonks. 1999. The protein-tyrosine phosphatase TCPTP regulates epidermal growth factor receptor-mediated and phosphatidylinositol 3-kinase-dependent signaling. J. Biol. Chem. 274:27768-27775.
40. You-Ten, K. E., E. S. Muise, A. Itie, E. Michaliszyn, J. Wagner, S. Jothy, W. S. Lapp, and M. L. Tremblay. 1997. Impaired bone marrow microenvironment and immune function in T cell protein tyrosine phosphatase-deficient mice. J. Exp. Med. 186:683-693.
41. Yu, Z., L. Su, O. Hoglinger, M. L. Jaramillo, D. Banville, and S. H. Shen. 1998. SHP-1 associates with both platelet-derived growth factor receptor and the p85 subunit of phosphatidylinositol 3-kinase. J. Biol. Chem. 273:3687-3694.
This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
