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Previous Article | Next Article 
Molecular and Cellular Biology, May 2005, p. 3690-3703, Vol. 25, No. 9
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.9.3690-3703.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Institute of Molecular Cell Biology, Jena University Hospital, Jena,1 Department of Internal Medicine, Hematology, and Oncology, University of Münster, Münster, Germany2
Received 12 August 2004/ Returned for modification 14 September 2004/ Accepted 16 January 2005
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ABSTRACT |
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promotes complex glycosylation and surface localization. However, PTP coexpression has no effect on the maturation of a surface glycoprotein of vesicular stomatitis virus. The maturation of wild-type FLT-3 is impaired by general PTP inhibition or by suppression of endogenous PTP1B. Enhanced complex formation of FLT-3 ITD with the ER-resident chaperone calnexin indicates that its retention in the ER is related to inefficient folding. The regulation of RTK maturation by tyrosine phosphorylation was observed with other RTKs as well, defines a possible role for ER-resident PTPs, and may be related to the altered signaling quality of constitutively active, transforming RTK mutants. |
INTRODUCTION |
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The receptor tyrosine kinase (RTK) Fms-like tyrosine kinase 3 (FLT-3) is expressed in multiple hematopoietic lineages (21, 22). Constitutively active FLT-3 mutants, notably, versions harboring internal tandem duplications in the juxtamembrane domain (FLT-3 ITD) and versions with point mutations in the kinase activation loop, have been found in approximately 30% of acute myeloid leukemia cases (28, 38). Activated versions of FLT-3 are characterized not only by constitutive signaling but also by a different signaling quality, which is connected to their transforming capacity. Hallmarks of altered signaling are a strong activation of STAT5a and of STAT response genes, pronounced antiapoptotic effects, and the suppression of myeloid cell differentiation (25, 26, 34). The occurrence of active FLT-3 mutants is associated with a poor prognosis in patients with acute myeloid leukemia, and FLT-3 is considered a promising target for therapy (for reviews, see references 33 and 35). Tyrosine kinase inhibitors from different structural families, including AG1296 (39), SU11248 (29), PKC412 (42), and CEP-701 (17), have been shown to inhibit the signaling of activated FLT-3. Some of these compounds are presently in clinical trials.
In our analysis of FLT-3 signaling, we observed inefficient maturation of FLT-3 ITD and its reduced expression at the cell surface. The systematic investigation of these phenomena revealed that the maturation of FLT-3 ITD is impaired by its constitutive kinase activity. Entrapment by the chaperone calnexin, and therefore ER retention, indicates decreased efficiency of folding of FLT-3 ITD. This previously unrecognized mechanism appears to be generally relevant for RTKs and has several testable implications for the mechanism of transformation of constitutively active RTKs and for the cellular roles of protein-tyrosine phosphatases.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Stratagene QuikChange method was used to introduce KA and YF mutations into FLT-3 cDNAs. Note that the numbering of amino acids for wild-type FLT-3 is maintained for FLT-3 ITD, despite the ITD insertion, to allow better comparison.
Primer sequences are available on request.
For the creation of a pSuper PTP1B small interfering (siRNA) expression construct, the sequence 5'-TGG AAG AAG CCC AAA GGA G-3', corresponding to positions 221 to 239 of the human PTP1B cDNA, was used to design oligonucleotides (2) which were cloned into vector pSuper.retro.puro (Oligoengine, Seattle, Wash.). An SHP-1 siRNA expression construct in the same vector was generated in an analogous manner with the sequence 5'-GAG GTG TCC ACG GTA GCT TCC-3', corresponding to positions 145 to 165 of the human SHP-1 cDNA (sequence kindly provided by H. Keilhack, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass.). All constructs were verified by DNA sequence analysis.
A eukaryotic expression construct for vesicular stomatitis virus glycoprotein (VSV-G) was kindly provided by S. Gutkind. An expression construct containing enhanced cyan fluorescent protein (ECFP) fused to an ER-targeting sequence derived from calreticulin, including a KDEL retrieval sequence, was used as an ER localization marker (pECFP-ER). ECFP fused to a Golgi compartment-targeting sequence derived from human ß-1,4-galactosyltransferase was used as a Golgi compartment localization marker (pECFP-Golgi). Both constructs were purchased from BD Biosciences (Heidelberg, Germany). Expression constructs for the protein-tyrosine phosphatases (PTPs) PTP1B and PTP-PEST were kindly provided by A. Ullrich and M. Tremblay, respectively. An EGFP-SHP-1 expression construct was described earlier (37).
Cells and reagents. HEK293 cells were grown in Dulbecco modified Eagle medium (DMEM)-F-12 medium (Gibco) containing 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany). COS-7 cells were grown in DMEM (Gibco) containing 10% FCS. MV4-11 and THP-1 cells, kindly provided by S. Scholl (Department of Internal Medicine, Jena University Hospital), were grown in RPMI 1640 (Biochrom) containing 10% heat-inactivated FCS. Agarose beads linked to lectin from Galanthus nivalis, wortmannin, bis-indolylmaleimide I (BisI), neuraminidase (type V; N2876) from Clostridium perfringens, and brefeldin A were obtained from Sigma (Taufkirchen, Germany). PP1 and geldanamycin were obtained from Alexis (Grünberg, Germany). Biotinylated lectins from Dolichos biflorus (DBA), G. nivalis (GNA), Ricinus communis (RCA120), Glycine max (soybean; SBA), Sambucus nigra (SNA), and Ulex europaeus (UEA) were purchased from Vector Laboratories (Peterborough, United Kingdom). Agarose-linked streptavidin, streptavidin coupled to horseradish peroxidase (HRP), and HRP-coupled RCA120 lectin were obtained from Sigma.
AG1296 was previously described (39). SU11248 was kindly provided by Marie O'Farrell (Sugen, Inc.). MG132 and bp(V)phen were obtained from Calbiochem (Schwalbach, Germany). Redivue PRO-MIX 35S in vitro cell labeling mixture was obtained from Amersham Biosciences (Freiburg, Germany). Peptide N-glycosidase F (PNGase F) was obtained from Roche (Mannheim, Germany). Endo-ß-N-acetylglucosaminidase H (endo-H) and the corresponding reaction buffers were obtained from New England Biolabs (Frankfurt, Germany).
Antibodies to the following were used: FLT-3* (clone 4G8; recognizes the extracellular domain of FLT-3), pan-Erk, and PTP1B (monoclonal) (BD Biosciences); pY591 (3461S; recognizes phosphorylated Y591 in activated FLT-3) (Cell Signaling, Frankfurt, Germany); HA and vinculin (V248) (Upstate, Biomol, Hamburg, Germany); FLT-3 (sc-480), c-Src (sc-18), SHP-1 (sc-287), calnexin (sc-6465), c-Kit (sc-168), and GFP (sc-9996) (Santa Cruz, Santa Cruz, Calif.); and ß-actin, mouse immunoglobulin G2a (IgG2a), mouse IgG1(
), and VSV-G (ascitic fluid) (Sigma). Dichlorotriazenyl amino fluorescein (DTAF)-coupled anti-mouse IgG, Cy3-coupled anti-mouse IgG, and anti-rabbit IgG were obtained from Molecular Probes (Karlsruhe, Germany).
FACS analysis.
MV4-11 and THP-1 cells were indirectly labeled with FLT-3* antibody or mouse IgG1() as an isotype control and with DTAF-coupled goat anti-mouse IgG according to standard protocols. Fluorescence-activated cell sorting (FACS) analysis was performed with a FACSCalibur (Becton Dickinson) instrument. Subsequent data analysis was performed with WinMDI software.
Immunoprecipitation and Western blotting. Vesicle immunoprecipitation was performed as described by Waugh et al. (41). MV4-11 and THP-1 cells were suspended in 4.3 M glycerol in 10 mM Tris-HCl (pH 7.4) for 10 min. Cells then were resuspended in homogenization buffer (0.25 M sucrose, 10 mM Tris-HCl [pH 7.4], 1 mM EGTA, 0.5 mM EDTA, protease inhibitors) and homogenized. ER vesicles were isolated by adding 0.5 µg of anticalnexin antibody and 60 µl of protein G-Sepharose and incubating the mixture at 4°C overnight. Beads were washed three times with washing buffer (0.25 M sucrose, 10 mM Tris-HCl [pH 7.4], 150 mM NaCl) and boiled in 30 µl of 2x sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer. Samples were subjected to SDS-PAGE and immunoblotting for FLT-3 and calnexin.
HEK293 cells were transfected by the calcium phosphate method, and COS-7 cells were transfected with Lipofectamine 2000 (Invitrogen). Cell lysis with lysis buffer containing 0.5% Nonidet P-40 and immunoblotting were performed as previously described (15). For FLT-3-calnexin coimmunoprecipitation, cell lysates were incubated overnight with the corresponding antibody and 1 h with protein A/G-Sepharose beads, and the beads were sedimented and washed three times with 20 mM HEPES (pH 7.5)-150 mM NaCl-10% glycerol-0.1% Triton X-100 (HNGT buffer). Isolation of high-mannose FLT-3 was performed by incubation of cell lysates with 15 µl of immobilized GNA lectin for 2 h and three washes with HNGT buffer. Treatment with PNGase F was performed as previously described (14). For digestion with endo-H, immunoprecipitates from 5 x 106 cells with endogenous FLT-3 expression or 2.4 x 105 transfected cells were subjected to denaturation with 25 µl of denaturation buffer (supplied with the enzyme) at 95°C for 10 min. Then, 25 µl of 2x reaction buffer (supplied with the enzyme) and 0.5 µl of endo-H were added, and the mixture was incubated at 37°C overnight. The reaction was terminated with SDS-PAGE sample buffer, and the reaction products were analyzed by immunoblotting.
Neuraminidase treatment of immunoprecipitates was done after denaturation with SDS-PAGE sample buffer. SDS was diluted to 0.5% with sodium phosphate buffer (pH 7.4; final concentration, 50 mM), and 0.5 µl of neuraminidase was added to a 60-µl sample. The mixture was incubated at 37°C for 2 h.
For lectin pull-down assays, THP-1 cells or FLT-3 KA-overexpressing COS-7 cells were lysed with lysis buffer containing 1% Nonidet P-40. The lysate of 2 x 107 to 2.5 x 107 THP-1 cells or 1.2 x 105 transfected COS-7 cells was mixed with 40 µg of biotinylated lectin and incubated with end-over-end rotation at 4°C overnight. Then, 35 µl of a 1:1 suspension of agarose-coupled streptavidin was added, and incubation was continued for 1 h. The beads were washed three times with HNGT buffer and subsequently extracted with SDS-PAGE sample buffer at 37°C for 30 min. The samples were separated by SDS-7.5% PAGE and subjected to immunoblotting with anti-FLT-3 antibodies. For lectin blotting, FLT-3 was immunoprecipitated from lysates of THP-1 cells or transfected COS-7 cells. Immunoprecipitates corresponding to 1 x 107 to 2 x 107 THP-1 cells or 2.5 x 105 transfected COS-7 cells per lane were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked by incubation with 2% Tween 20 in Tris-buffered saline (TBS) at room temperature for 20 min, washed three times with TBS, and incubated with biotinylated lectin (5 to 20 µg/ml) or HRP-coupled lectin (1 to 5 µg/ml) in TBS containing 1 mM MnCl2, 1 mM MgCl2, and 1 mM CaCl2 at room temperature overnight. The blots were either developed directly (HRP-coupled lectin) or incubated with HRP-conjugated streptavidin (0.2 µg/ml) at room temperature for 1 h and then developed with enhanced chemiluminescence detection.
PTP suppression by siRNA. HEK293 cells were transiently transfected by the calcium phosphate method with 0.4 µg of an expression construct for HA-tagged wild-type FLT-3 plus 4 µg of the corresponding PTP1B siRNA construct per well of a six-well plate. Cells were allowed to express PTP1B siRNA for 4 days and then were lysed. For suppression of SHP-1 expression, THP-1 cells were infected with retroviruses containing the SHP-1 siRNA expression contruct pSuper.retro.puro, produced in the Phoenix Amphopack packaging cell line (kindly provided by G. Nolan, Stanford University). Cells were selected for 10 days with 2 µg of puromycin/ml and expanded. For suppression of PTP1B, THP-1 cells were transfected with siGENOME SMARTpool siRNAs (NM_002827; Dharmacon) by using Nucleofector kit V and optimized protocol V-01 according to the instructions of the supplier (Amaxa GmbH, Cologne, Germany). Three days after transfection, cells were lysed and FLT-3 was immunoprecipitated. PTP1B expression, FLT-3 maturation, and loading (ß-actin or vinculin) were analyzed by immunoblotting.
Inhibitor treatments. Cells were starved overnight in medium containing 0.5% FCS and then treated with kinase or signal transduction inhibitors at the following concentrations: 10 µM AG1296, 1 µM SU11248, 5 µM PP1, 100 nM wortmannin, and 500 nM BisI. For treatment with geldanamycin and brefeldin A, transfected COS-7 cells were allowed to express FLT-3 constructs for 2 days. Cells then were treated for 6 h with either 3 µM geldanamycin or 5 µg of brefeldin A/ml before lysis. For the inhibition of total PTP activity, transfected COS-7 cells were left untreated or were treated with 1 mM bp(V)phen for up to 3 h. In some experiments, cells were preincubated with 25 µM MG132 for 4 h before PTP inhibition.
Pulse-chase labeling. Pulse-chase analysis was carried out as previously described (21). Briefly, MV4-11 cells or transfected COS-7 cells were labeled for 20 min with 200 µCi of Redivue/ml in cysteine- and methionine-free DMEM, with subsequent chasing into DMEM containing 0.1 mM methionine and 0.2 mM cysteine. Immunoprecipitated FLT-3 was analyzed by SDS-PAGE and fluorography.
Immunohistochemistry studies. COS-7 cells were grown on collagen-coated coverslips and transiently transfected with Lipofectamine 2000 (Invitrogen). The cells were allowed to express HA-tagged FLT-3 constructs for 1 day before fixation with 4% paraformaldehyde. After blocking was done with 10% normal goat serum in phosphate-buffered saline (PBS)-1.5% bovine serum albumin (BSA), cells were incubated with FLT-3* antibody (1:100) in PBS-1% BSA overnight, washed, and stained with Cy3-coupled anti-mouse IgG. Coexpression of EGFP-tagged FLT-3 or HA-tagged FLT-3 in COS-7 cells was performed with a number of other proteins. Cells were fixed with methanol at –20°C, quenched with 0.1% NaBH4 at room temperature, blocked as described above, and incubated with pY591 antibody (1:100) in TBS-1% BSA overnight and then with Cy3-coupled anti-rabbit IgG (1:600). For SHP-1 immunostaining, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS, blocked as described above, and incubated with SHP-1 antibody at a dilution of 1:100 and then with Cy3-coupled anti-rabbit IgG as described above. Microscopy was done with a Zeiss LSM 510 confocal laser scanning microscope.
Quantitative analysis. Densitometric analysis of immunoblots was performed with NIH Image 1.61 software. The ratio of the amount of the complex glycosylated (150-kDa) form to the amount of the high-mannose (130-kDa) form was taken as a measure of maturation and is given in arbitrary units. Results are expressed as the mean ± standard error of the mean (SEM). Data were analyzed by using paired two-tailed Student's t tests. A P value of <0.05 was considered significant.
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RESULTS |
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The glycosylation state of the 150-kDa form was analyzed by testing its reactivity with a set of lectins in both pull-down (Fig. 1E) and blotting (Fig. 2) assays. The 150-kDa form selectively interacted with SNA, RCA120, and SBA lectins, indicating the presence of terminal residues of sialic acid, galactose, and N-acetylgalactosamine, respectively, which are absent in the 130-kDa form. Selective reactivity of the 150-kDa form with lectins specific for these sugars was also derived from blotting experiments (Fig. 2). SNA lectin recognizes sialic acid preferentially in a form which is -2,6-linked to subsequent galactose. Thus, SNA lectin reactivity indicates the presence of this terminal disaccharide. As shown before, GNA lectin in both pull-down and blotting assays reacted preferentially with the 130-kDa form. Interaction assays with UEA lectin revealed little reactivity, suggesting that neither of the FLT-3 variants contains significant amounts of fucose. DBA lectin is specific for N-acetylgalactosamine in an -glycosidic linkage. The absence of reactivity with DBA lectin but the reactivity with SBA lectin therefore indicates that the terminal N-acetylgalactosamine residues are engaged in a ß-glycosidic linkage. Treatment of FLT-3 immunoprecipitates from THP-1 cells with neuraminidase resulted in a clear size reduction of the 150-kDa form, further supporting the presence of significant amounts of sialic acid (Fig. 1F). Thus, the carbohydrate part of the 150-kDa form has features which are typical of complex glycosylated glycoproteins (9) and similar to those of oligosaccharide chains in the cell surface epidermal growth factor receptor (45). A tentative structure is indicated in Fig. 2. Taken together, these results indicate that FLT-3 ITD exists in a much higher proportion than wild-type FLT-3 as an ER-bound, immature form containing mannose-rich carbohydrates. Conversely, wild-type FLT-3 exists predominantly as a 150-kDa form containing a larger proportion of carbohydrates with features typical of complex glycosylation.
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PTPs promote and enable FLT-3 maturation. To further establish a relationship of FLT-3 maturation with tyrosine phosphorylation, we explored the possible effects of PTPs. The overexpression of SHP-1, a PTP that is expressed in all hematopoietic lineages (44), resulted in the dephosphorylation of FLT-3 ITD (Fig. 4A and B) and in elevated levels of mature, 150-kDa FLT-3 ITD (Fig. 4A). Both effects were dependent on PTP activity and were not observed with catalytically inactive SHP-1 C455S (Fig. 4A). The 150-kDa form was less efficiently dephosphorylated in these experiments, probably indicating that this form has a localization different from that of SHP-1. The promoting effect of SHP-1 on FLT-3 ITD maturation could also be observed in pulse-chase experiments (Fig. 4C). To explore whether PTP expression has a general effect on cellular glycoprotein maturation, we analyzed the effect of SHP-1 expression on the maturation of VSV-G. VSV-G was previously used to study glycoprotein maturation (23). Since the size difference between mature VSV-G and immature VSV-G is very small, the presence of the immature form can be better assessed by analyzing endo-H sensitivity. In this type of analysis, neither the coexpression of SHP-1 nor that of FLT-3 ITD affected the maturation of VSV-G (Fig. 4D), indicating that both have no general influence on the maturation machinery.
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neither dephosphorylated the receptor nor had an effect on its maturation (Fig. 4F). Thus, not all PTPs can promote maturation, and the effect correlates with the capacity of a PTP to dephosphorylate FLT-3. Conversely, the general inhibition of cellular PTPs by the cell-permeating PTP inhibitor bp(V)phen strongly compromised the maturation of wild-type FLT-3 (Fig. 4G and H). At longer treatment times, FLT-3 protein was lost. In the presence of the proteasome inhibitor MG132, the bp(V)phen-induced loss of FLT-3 was attenuated to some extent (Fig. 4G, lower panel), suggesting that the massive block of FLT-3 maturation upon general PTP inhibition eventually triggered proteasome-mediated degradation. Thus, general PTP inhibition had an effect on FLT-3 maturation similar to the general blockade of transport from the ER to the Golgi compartment by brefeldin A (Fig. 4I). To address more specifically the role of PTPs in maturation, we chose to perform siRNA-mediated suppression of two PTPs. PTP1B is anchored to ER membranes via a C-terminal targeting sequence and may control the phosphorylation status of newly synthesized RTKs (5). Transfection of HEK293 cells with an expression vector directing the synthesis of an siRNA which specifically targets human PTP1B mRNA reduced the expression of endogenous PTP1B by at least 80% (Fig. 4J, left panel). Transiently expressed wild-type FLT-3 clearly matured less efficiently in these cells than in the corresponding control cells. Thus, endogenous PTP1B was limiting for efficient FLT-3 maturation in this setting. Interestingly, the suppression of PTP1B was also associated with hyperphosphorylation of the immature, 130-kDa form of FLT-3 (Fig. 4J, left panel). In an attempt to suppress PTP1B expression in THP-1 cells, we succeeded only in a mild reduction in protein levels (by 35%) compared to the results in mock-treated cells. Still, less efficient maturation of endogenous wild-type FLT-3 could clearly be detected (Fig. 4J, right panel). On the other hand, efficient reduction of endogenous SHP-1 expression had only a rather weak effect on maturation (Fig. 4J, right panel); however, this finding was consistently seen in many experiments. This analysis, which needs to be extended to additional PTPs, indicates the differential importance of endogenous PTPs for FLT-3 maturation.
Reduced folding efficiency causes ER retention of FLT-3 ITD. To explore the mechanism leading to the retarded maturation of FLT-3 ITD, we first considered that its constitutive signaling activity could inhibit components that are important for ER-Golgi compartment translocation and/or complex glycosylation. If this were the case, then it should be possible to assign the maturation block to a selective downstream signaling event. We first tested several inhibitors of signaling pathways which are known to be activated by FLT-3 ITD (for a review, see reference 35). Among them, the protein kinase C (PKC) family inhibitor BisI and the Src family kinase inhibitor PP1 reproducibly but only weakly promoted maturation, whereas the phosphatidylinositol 3-kinase inhibitor wortmannin had no effect (Fig. 5A and B).
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As an obvious possible reason for the inefficient maturation of FLT-3 ITD independent from signaling processes, we considered the inefficient folding of the autophosphorylated kinase. A reduced folding capacity should lead to an enhanced interaction with ER-resident chaperones in the process of ER quality control. The observed ER retention of FLT-3 ITD would be consistent with such a model. An enhanced chaperone dependence of FLT-3 ITD was previously suggested. FLT-3 ITD but not wild-type FLT-3 was shown to associate with heat shock protein 90 (Hsp90), and its activity and expression were dependent on this association (24). In support of this observation, we found that geldanamycin, an inhibitor of Hsp90 function, potently reduced the level of expression of FLT-3 ITD, whereas wild-type FLT-3 was affected to a much lesser extent (Fig. 6A). While this article was in preparation, similar findings were reported by others (7). We therefore tested whether FLT-3 ITD would exhibit an enhanced interaction with the ER chaperone calnexin, a key component of the ER quality control mechanism for glycoproteins (4). FLT-3 ITD indeed associated with calnexin, whereas wild-type FLT-3 and the kinase-inactive FLT-3 ITD K644A mutant interacted with calnexin much less efficiently (Fig. 6B and C). Thus, constitutive phosphorylation of FLT-3 ITD appears to result in inefficient folding, indicated by chaperone dependence and enhanced recognition by the ER quality control system.
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GNNK (30), it was evident that wild-type c-Kit matured properly, while c-KitGNNK existed to a considerable extent in an immature, underglycosylated form (Fig. 6D). Finally, the structurally less related RTK c-Ros also yielded mature and underglycosylated forms upon overexpression. Coexpression with the potent PTP SHP-1 (15) strongly promoted maturation (Fig. 6D). Thus, the regulation of RTK maturation by tyrosine phosphorylation appears to be a more general phenomenon and is not restricted to FLT-3. |
DISCUSSION |
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1 and the ligand GDNF (16). Similar observations have been reported for the insulin receptor in certain rare cases of insulin-resistant diabetes (31). As a common principle, these disease-causing mutations impair the folding efficiency of the kinases, leading to entrapment by the chaperones that are components of the ER quality control. Another class of mutations which lead to proteins with pronounced chaperone interactions are gain-of-function mutations in oncogenic kinases. Such proteins, for example, BCR-Abl, v-Src, and mutated Kit, are dependent on continuous interactions with the chaperone Hsp90, as indicated by their rapid degradation in the presence of the Hsp90-depleting agent geldanamycin or its analogs (12). As we describe here, the transforming RTK FLT-3 ITD interacts with both classes of chaperones, indicating inefficient folding and stability. An enhanced interaction with calnexin in the ER is associated with partial ER retention and reduced surface expression. As a previously unrecognized phenomenon, ER retention of FTL-3 ITD is linked to its constitutive kinase activity and can be overcome by kinase-inactivating mutations, treatment with kinase inhibitors, or coexpression of PTPs. A negative regulatory function of tyrosine phosphorylation for RTK maturation is most likely a more general mechanism, since we observed it also for three other tested members of the RTK family—c-Kit, the PDGFß receptor, and Ros. While this article was in preparation, Lievens et al. (18) reported that highly activated mutant versions of fibroblast growth factor receptor 3 (FGFR-3), which cause a severe form of dwarfism in men, mature very inefficiently. Kinase inactivation, however, restores maturation and surface localization. These findings are consistent with the observations that we report here and support the generality of an inhibitory role of tyrosine phosphorylation in the maturation of RTKs.
For the mechanism of maturation arrest, we considered the possibility that signaling downstream of constitutively active FLT-3 ITD negatively regulates components that participate in the maturation process, for example, molecules of the quality control mechanism or molecules with importance for ER-Golgi vesicle transport. We observed, however, no clear effects of FLT-3 ITD expression on the maturation of VSV-G. Thus, signaling of FLT-3 ITD was obviously without prominent effects on the general glycoprotein maturation machinery. Attempts to link the constitutive signaling activity of FLT-3 ITD with ER retention by testing a panel of signal transduction inhibitors and a panel of YF mutants likewise revealed no clue for a critical signaling event. We observed some weak maturation-promoting effects of the PKC inhibitor BisI, the Src family inhibitor PP1, and a Y589/591F double mutation, which may also be related to impaired interactions with Src family kinases. These potential pathways for the regulation of ER quality control or further steps in FLT-3 maturation deserve further investigation. However, complete inactivity or global dephosphorylation of FLT-3 ITD showed the best correlation with maturation, suggesting that it may be phosphorylation of the RTK itself which reduces folding efficiency and triggers the calnexin interaction. Again, this activity could not be linked to phosphorylation of single sites in our mutational analysis. Instead, the physicochemical effect of phosphorylation on multiple sites may impair folding and potentially also protein stability. Answering these questions will require folding experiments at the level of recombinant FLT-3 protein.
Our findings suggest a novel function for ER-associated PTPs. General PTP inhibition drastically impaired the maturation of wild-type FLT-3 to the point that degradation was initiated. Based on our results, we propose that suppression of the basal activities of newly synthesized RTKs is essential for their release from ER quality control and further processing. Analysis of individual PTPs for their roles in this process is highly warranted. It should be noted that several PTPs have ER localization domains (PTP1B and T-cell PTP) (5, 20) or are enriched in a perinuclear compartment (SHP-1) (37). A role for PTP1B in suppressing the activities of ER-bound RTKs was proposed earlier but has not been linked to RTK maturation (6). A function of PTP1B in this process is supported by our finding of impaired FLT-3 maturation upon siRNA-mediated suppression of endogenous PTP1B expression in HEK293 cells transiently expressing wild-type FLT-3. Also, siRNA-mediated partial reduction of endogenous PTP1B levels in THP-1 cells led to less efficient maturation of endogenous wild-type FLT-3. However, the suppression of SHP-1 had only a weak effect on endogenous FLT-3 maturation, suggesting differential importance of the two PTPs in this process. Further experiments are required to firmly establish the role of PTP1B and to evaluate the role of other PTP family members.
Another interesting implication from our study is the possibility that ER retention of constitutively active RTKs changes the quality of signal transduction by providing access to substrates which are not accessible to surface-bound RTKs. This may be an important aspect of the transforming activities of constitutively active RTKs. Indeed, there are strong qualitative differences in the signaling of FLT-3 ITD compared with ligand-stimulated wild-type FLT-3 which presently cannot be explained (26). ER-retained, constitutively active versions of FGFR-3 are capable of activating the Janus kinase (Jak)/STAT pathway by directly recruiting Jak1, whereas wild-type FGFR-3 cannot activate STAT1 (18). For the RTK Ros, it has been shown that aberrant Golgi compartment localization in glioblastoma cells, conferred by fusion with the Golgi compartment-targeted partner FIG, is transforming (3). Intracellular activation of PDGF receptors by the v-Sis protein has been related to sis-mediated transformation (1, 13). Thus, ER or Golgi compartment retention may emerge as a more general pathway for RTK-mediated pathological signaling and transformation.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB604, A1, and Bo 1043/4-3 to F.-D.B. and Se600/2-3 to H.S.), from the Deutsche Krebshilfe (10-2100-Do2 to F.-D.B.), and from the IZKF Münster (to H.S.).
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FOOTNOTES |
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