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Tumor Biology |
Department of Experimental Oncology, Istituto Nazionale Tumori, 20133 Milan [C. C., P. M., M. A. P., I. B.]; Consiglio Nazionale Ricerche Cellular and Molecular Pharmacology Center Department of Pharmacology, University of Milan, 32 Milan [C. P.]; and Istituto FIRC Oncologia Molecolare, Federazione Italiana Ricerca Cancro Institute of Molecular Oncology, 20139 Milan [M. A. P.], Italy
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The knowledge of the availability of inhibitors specific for RET oncoproteins could provide new tools to highlight the physiologically and pathologically activated pathways involved and help in developing new therapeutic strategies for RET-associated diseases. The inhibition of tyrosine kinase activity by small cell-permeable molecules is a promising approach to target oncoproteins and has already reached clinical application for ErbB/HER subgroup of receptors (21 , 22) . Carlomagno et al. (23) have very recently demonstrated that the pyrazolo-pyrimidine PP1 blocks tumorigenesis induced by RET/PTC cytoplasmic oncoproteins because it can inhibit RET enzymatic activity and its transforming effects. We here additionally explored the mechanism of action of the inhibitor specifically showing that PP1 abrogates the cellular morphological modifications induced by the expression of different cytoplasmic or membrane-bound RET oncoproteins in NIH3T3 cells, restoring the characteristic fibroblast actin organization and focal adhesion formation. We also present evidence that along with the inhibition of tyrosine phosphorylation, PP1 induces proteosomal destruction of the activated receptors. In fact, in the presence of the inhibitor, the receptors are rapidly targeted to the degradative pathway, and the intracellular destruction of ubiquitinated RET oncoproteins results accelerated. Taken together, our results suggest that PP1 treatment would represent a promising new strategy to selectively target RET oncogenic products for destruction and could be used to develop new therapeutic techniques for medullary thyroid cancer therapy whose tumors respond very poorly to chemotherapeutic agents.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Arg (TGCCGC) and Met918Thr (ATGACG) was performed. The wild-type (WT) as well as the other RET constructs were cloned in the pRc/CMV expression vector (Invitrogen, Groningen, The Netherlands). The point mutation of the desired codon was confirmed by DNA sequencing. Anti-RET polyclonal antibody (C-19) raised against a peptide corresponding to an amino acid sequence mapping at the COOH terminus of RET was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The following antibodies were also used: mouse antiphosphotyrosine (4G10; Upstate Biotechnology, Inc., Lake Placid, NY); Vinculin (V-4505; Sigma Chemical Co., St. Louis, MO) monoclonal antibody; Tubulin polyclonal antibody (T-3526; Sigma Chemical Co.); TRK polyclonal antibody (Santa Cruz Biotechnology); EGFR polyclonal antibody (sc-03; Santa Cruz Biotechnology, CA); ubiquitin (MMS-258; Babco) monoclonal antibody for Western blots; and ubiquitin monoclonal antibody (FK1, which recognizes only polyubiquitin-protein conjugates; Affiniti BioReagents, Inc.), Cbl (C-15 Santa Cruz Biotechnology) polyclonal antibody; and clathrin (MA1–065, Affiniti BioReagents, Inc.) monoclonal antibody. FITC-labeled phalloidin (Sigma Chemical Co.) was used to detect filamentous actin (final concentration 125 ng/ml). Secondary antibodies were from Santa Cruz Biotechnology.
PP1 inhibitor [4-amino-5-(4-methylphenyl)-7-(-t-butyl)pyrazolo-D-3,4-pyrimidine] was obtained from BioMol Research Laboratories, dissolved in DMSO, and diluted into culture medium at the time of experimentation to a final concentration of 1 µM. PP2, PP3, Genistein, and Herbimycin from Calbiochem (Novacalbiochem Corporation, La Jolla, CA) were dissolved in DMSO and, respectively, diluted into the culture medium to final concentrations of PP2 (1 µM), PP3 (1 µM), Genistein (100 µM), or Herbamycin (0.1 µM).
Lactacystin was purchased from Sigma Chemical Co., dissolved in DMSO, and diluted in culture medium to a final concentration of 2 µM. MG132 was purchased from Calbiochem (Novacalbiochem Corporation), dissolved in DMSO, and then diluted in culture medium to a final concentration of 10 µM. Chloroquine, from Sigma Chemical Co., was dissolved in water and diluted in culture medium to a final concentration of 100 µM. The human recombinant epidermal growth factor was from Life Technologies, Inc. (13247-051) and was diluted into the culture medium to a final concentration of 20 ng/ml.
Cell Culture.
Mouse fibroblast NIH3T3-untransfected cells were grown in DMEM containing 10% calf serum. Cells were transfected as previously described (13)
with pcDNA3 plasmids carrying RETC634R, RETM918T, and RET/PTC3 inserts. Transformation foci were selected in DMEM with 5% calf serum. E25-427 cells, NIH3T3 cells overexpressing the human TRK proto-oncogene, were kindly provided by Dr. Mariano Barbacid and grown in DMEM supplemented with 10% calf serum. HEK 293T cells were grown in DMEM supplemented with 10% FCS and transiently transfected by calcium phosphate precipitation. The human TT cell line was cultured in Ham’s F-12 (Life Technologies, Inc.) supplemented with 15% FCS. The MDA-MB-231 cell line was cultured in RPMI supplemented with 10% FCS.
Immunofluorescence and Confocal Laser Microscopy.
Cells were seeded at low confluence on glass coverslips and cultured for 24 h. Cells were fixed in 4% paraformaldehide and 2% sucrose and then permeabilized with 0.5% Triton X-100 in PBS. Immunostaining with primary antibodies was followed by incubation with rhodamine-conjugated antirabbit (Dako), FITC-conjugated anti-IgG from Jackson Immunoresearch (West Grove, PA). The confocal images were obtained using a Bio-Rad MRC-1024 confocal microscope.
Cell Motility Assay.
Migration of RET-expressing cells was determined using an in vitro model of wound repair as described previously (24)
. The semiconfluent monolayers were scraped with a 10-µm sterile plastic micropipette tip, and cellular debris was removed by washing with serum-free DMEM. The monolayers were incubated in DMEM with or without 1 µM PP1 for 12 h. Cell migration was observed using a phase contrast microscope under x200 magnification. Each condition was examined in duplicate, and the experiments repeated at least three times.
Immunoprecipitation and Western Blot Analysis.
Protein samples were prepared as previously reported (25)
and immunoprecipitated with the specific antisera. Protein concentration was estimated by a modified Bradford assay (Bio-Rad, Munich, Germany). Immune complexes were separated by electrophoresis on 8% SDS-PAGEs, transferred onto nitrocellulose membranes (Millipore Co., Bedford, MA), and analyzed by immunoblotting with the appropriate antibodies. For WCLs, cells were lysed in SDS lyses buffer [62.5 mM Tris-HCl (pH 6.8) and 2% SDS]. Total extracts were separated by SDS-PAGE.
Immunoreactive bands were visualized using horseradish peroxidase-conjugated antirabbit or antimouse antisera (Santa Cruz Biotechnology) and detected with the enhanced chemiluminescence kit (enhanced chemiluminescence detection reagents; Amersham Pharmacia Biotech, Little Chalfort, United Kingdom).
RET Kinase Activity.
To determine the effect of PP1 on the receptor kinase activity, 293T cells were transiently transfected to express RETC634R, RETM918T, PTC3, TRK, and wild-type RET proteins with the calcium-phosphate method. Two days after transfection, cells were lysed in NP40 buffer. Their kinase activity was assessed in vitro by their ability to autophosphorylate and phosphorylate myelin basic protein (MBP; Sigma). Protein extracts were immunoprecipitated with anti-RET or anti-TRK antibodies washed twice with lysis buffer and once with incubation buffer [50 mM HEPES (pH 7.2), 20 mM MnCl2, and 5 mM phenylmethylsulfonyl fluoride] and subjected to kinase assay with MBP as an exogenous substrate [
-32P]ATP and PP1 (1 µM) or PP2 (1 µM), where indicated. After a 15-min incubation at +4°C in 20 µl of the same buffer containing 4 µCi [-32P]ATP diluted with unlabeled ATP to a final concentration of 26 pmol ATP/sample and 50 µM MBP, the reactions were stopped by the addition of reduced Laemmli buffer, and the products were heated at 100°C for 5 min. The radiolabeled RET and MBP proteins were resolved by SDS-PAGE and visualized by autoradiography. The intensity of the bands corresponding to autophosphorylated RET and phosphorylated MBP was quantified by PhosphorImager (Molecular Dynamics) analysis and expressed as the fold increase relative to unstimulated wild-type RET. Control reactions in which RET immunoprecipitates were omitted showed no MBP phosphorylation (data not shown). Immunoprecipitation of equal amounts of RET was verified by anti-RET Western blotting and the quantity measured using 125I-labeled protein A. The intensity of the bands corresponding to RET was quantified by PhosphorImager (Molecular Dynamics) analysis.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, these cells exhibited a bright, rounded morphology with elongated processes very different from the typical phenotype of parental NIH3T3 cells. However, in the presence of PP1 at 1 µM for 12 h, their morphology reverted, becoming similar to the one of untransformed NIH3T3 fibroblasts. As expected, PP1 exerted clear effects on RET/PTC3 cell morphology as well. As a control, parental NIH3T3 cells and NIH3T3-overexpressing TRKA were also examined by phase-contrast microscopy and did not show any significant change upon PP1 treatment (Fig. 1A)
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). When compared with untreated cells, those expressing RETC634R, RETM918T, and RET/PTC3 and treated with 1 µM PP1, exhibited a markedly greater number of actin stress fibers that were well organized with a pattern similar to that seen for RET-negative NIH3T3 cells (Fig. 1B)
. In contrast, parental NIH3T3 cells and NIH3T3-overexpressing TRKA did not show any significant morphological change upon PP1 treatment (Fig. 1B)
. Moreover, PP1 addition also drastically changed subcellular localization of vinculin in RETC634R-, RETM918T-, and RET/PTC3-transformed fibroblasts (Fig. 1C)
. In fact, although in untreated cells we found a consistent cytoplasmic pool of vinculin primarily in the perinuclear region, in PP1-treated cells a significant fraction of vinculin was seen as punctate structures at the cell periphery, reminiscent of focal adhesion plaques (Fig. 1C)
. Therefore, PP1 addition induces actin stabilization and formation of enlarged vinculin-positive focal adhesions similar to the ones observed in parental NIH3T3 cells (Fig. 1C)
, thus reverting the transformed phenotype of RETC634R, RETM918T, and RET/PTC3 fibroblasts. PP1 has no effect on vinculin distribution in TRKA-expressing cells.
An in vitro wound repair model (24)
was used to assess whether the architectural changes induced by PP1 treatment of RET transformed cells could also affect cell motility. Cells were seeded and cultured to 50% confluence, wounded, and the cell culture medium removed. After 12 h from wounding, RETC634R- and RETM918T-expressing cells demonstrated extensive migration into the denuded area (Fig. 2)
. In contrast, NIH3T3-untransfected cells showed no significant migratory potential (Fig. 2)
. When the same experiments were repeated with cells cultured with PP1, we observed that PP1 treatment affected the ability of the RETC634R- and RETM918T-expressing cells to migrate, whereas the motility of parental NIH3T3 cells was not affected (Fig. 2)
. This result provided new information on the effects of PP1 treatment on RET-transformed cells.
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, both RET autophosphorylation activity and transphosphorylation of MBP was impaired. As a control, 293T cells were also transiently transfected with a construct coding for TRKA wild-type protein and the enzymatic activity evaluated. In this case, no significant differences were found when the assay was performed in the presence or absence of PP1 inhibitor (Fig. 3)
. These results clearly demonstrated that PP1 was able to strongly reduce the ability of RET to both auto- and trans-phosphorylate substrates probably by specifically competing for the ATP binding site as already reported (23)
. To further verify that PP1 could fit in the RET ATP binding domain, another inhibitor PP2, which has a chemical structure very similar to that of PP1, was used. PP2 could mimic the results obtained with PP1 reducing both RET autophosphorylation and the phosphorylation of MBP (data not shown). In contrast, other tyrosine kinase inhibitors such as Genistein or Herbamycin, only partially affected RET enzymatic activity (data not shown and Ref. 23
). Taken together, our results additionally suggest that PP1 and PP2 have structures that specifically fit in the ATP binding domain of RET, thus preventing ATP binding.
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. Surprisingly, when we checked for equivalent loading of proteins by reprobing the same blots with anti-RET antibodies, we discovered that even the entire RET protein content of the cell decreased along the time course for both RETC634R and RETM918T (Fig. 4A)
, whereas tubulin levels remained unchanged. Therefore, PP1 was not only able to reduce RET phosphorylation but, unexpectedly, it was also able to cause a progressive loss of RET expression. The effect is specific for RET because PP1 treatment was ineffective on TRKA-overexpressing fibroblasts (Fig. 4B)
. A progressive loss of RET expression was also observed for RET/PTC3 as shown in Fig. 4B
, although the protein level appears clearly reduced only after 18 h. RET protein disappearance was also observed in Western blotting experiments when RETC634R and RETM918T cells were treated with PP2 but not when the same cells were cultured in the presence of PP3 (a negative control of PP2 that is able to inhibit the activity of EGFR kinase) or in the presence of other tyrosine kinase inhibitors such as Genistein or Herbamycin (Fig. 4C)
. Interestingly, a significant decrease in the amount of RET protein along with a decrease in its phosphorylation was detected even when proteins extracted from TT cells (a human MTC cell line) were immunoprecipitated with anti-RET antibodies, and Western blot analysis was performed with anti-RET and anti-pTyr antibodies (Fig. 4D)
. As expected, in this case, the rate of RET protein degradation was slower than in NIH3T3 cells overexpressing the oncoproteins.
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. RET relocalization was observed even when immunofluorescence analysis for RET was performed on RETM918T-transfected cells treated with PP1 (data not shown). To highlight any significant defect in RET oncoprotein biosynthesis in the presence of PP1, we followed the fate of metabolically labeled mutant RET receptors expressed in NIH3T3 cells. Subconfluent cultures were labeled with [35S]methionine-cysteine for 20 min and subsequently chased for varying times. Cells were previously grown for 12 h in the presence or absence of PP1 and then metabolic pulse-chased experiments performed as for untreated cells. As already demonstrated by Bongarzone et al. (26) , the amount of RETC634R and RETM918T increased significantly within the first hour of the chase. In PP1-treated cells, the fate of both RETC634R and RETM918T receptors was comparable with that found in untreated cells during the period of observation, indicating that PP1 did not affect protein biosynthesis (data not shown).
In addition, we examined the cellular proliferation patterns of RETC634R-, RETM918T-, and RET/PTC3-expressing cells in the presence or absence of the inhibitor. One µM PP1 exerted a remarkable inhibitory effect on the growth of RETC634R-, RETM918T-, and RET/PTC3-expressing cells, as already demonstrated by Carlomagno et al. (23)
. Notably, we observed that the cytostatic effect only started after 48 h. In fact, RET-expressing cells resembled the parental cells for the first 2 days, but at later times, no increase in the number of living cells in the presence of PP1 could be detected (Fig. 5)
. This is consistent with the fact that after 48 h after PP1 addition, cells had almost completely lost the expression of the RET oncoproteins, suggesting that RET depletion induces growth arrest. No apoptotic effect was observed as demonstrated by terminal deoxynucleotidyl transferase-mediated nick end labeling analysis when performed on cells treated with PP1 or PP2 (data not shown). This result was confirmed by Western blot analysis using anti-poly(ADP-ribose) polymerase antibodies and performed on whole cell extracts from cells treated with PP1 or PP2 for various times. In fact, no cleavage of poly(ADP-ribose) polymerase, which facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis, was detected (data not shown). The proliferation rate of parental- and TRKA-overexpressing NIH3T3 cells was only partially slowed down in the presence of PP1 (Fig. 5)
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. Lactacystin alone did not significantly modify the amount of RET proteins. RET degradation was also substantially reduced upon incubating cells with another proteosome inhibitor, MG132 (data not shown). Moreover, RET tyrosine-phosphorylated proteins were detected by Western blotting with anti-pTyr antibodies (Fig. 6A)
. The bands corresponding to the RET mutants were not phosphorylated upon treatment with PP1 and lactacystin together, again confirming that PP1 exerts its normal effect by reducing RET phosphorylation but that the proteosomal inhibition was preventing the destruction of the unphosphorylated protein. Taken together, these results strongly indicate that PP1-induced RET degradation is because of proteosomal targeting as the inhibition of the proteosome substantially attenuates the decrease in RET protein levels. Consistent with these observations, chloroquine, a weak base that alkalinizes the lysosome, was ineffective in inhibiting RET degradation (Fig. 6A)
, thus confirming that PP1 treatment most probably targets RET oncoproteins to proteosomal, rather than lysosomal, degradation. The treatment of the cells with chloroquine alone had no effect on RET expression (Fig. 6A)
. Moreover, neither lactacystin nor chloroquine display any detectable effect on TRK expression as detected by Western blotting. On the contrary, consistent with previous data (27)
, chloroquine and, to a less extent, lactacystin, inhibited EGF-mediated degradation of the EGFR in MDA-MB-231 (human breast carcinoma cell line) cells as shown by Western blotting experiments (Fig. 6A
, bottom panel).
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. Moreover, c-Cbl binds to the RET oncoproteins irrespective of PP1 treatment and of RET phosphorylation status. Western blot analysis of both anti-Cbl immunoprecipitates with anti-RET antibodies and anti-RET immunoprecipitates with anti-Cbl (Fig. 6B)
in treated or untreated cells demonstrated that RET and c-Cbl can associate in vivo, suggesting that c-Cbl interaction with activated RET is an early event not affected by PP1. C-Cbl phosphorylation was also studied in untreated cells and cells treated with PP1. Western blot analysis of c-Cbl immunoprecipitates showed that PP1 does not affect c-Cbl phosphorylation status (Fig. 6B
, top panel) but as already demonstrated, dramatically decreased RET phosphorylation and RET protein levels in RETC634R- and RETM918T-transfected cells. In NIH3T3-untransfected cells, c-Cbl phosphorylation was nearly undetectable and did not change upon PP1 treatment (Fig. 6B
, bottom panel).
Ubiquitination of the RET mutants was then studied. Western blot analysis of anti-RET immunoprecipitated receptors, either treated or untreated with PP1 and lactacystin, with a monoclonal antibody against polyubiquitin, indicated that the RETC634R and RETM918T oncoproteins were ubiquitinated. As proteosome inhibition leads to a depletion of free ubiquitin (28, 29, 30)
, the signal corresponding to ubiquitinated RETC634R oncoproteins was more intense when cells were cultured in the presence of lactacystin, although ubiquitinated RET could also be seen for RETC634R cells treated with PP1 alone (Fig. 7A)
. The same results were obtained when analyzing the ubiquitination pattern of RETM918T oncoproteins (data not shown). Additional immunoflorescence studies were performed to confirm that the RETC634R oncoproteins can be ubiquitinated. RETC634R-expressing cells were double stained with anti-RET polyclonal antibodies and monoclonal antipolyubiquitin antibodies (FK1) to detect ubiquitinated RET mutants. Even in these studies, ubiquitinated RETC634R oncoproteins could be observed in untreated cells (Fig. 7B
, top panel, and details in Fig. 7B
), as well as in PP1-treated cells (Fig. 7B
, bottom panel), thus suggesting that PP1 does not promote ubiquitination. Thus, RET oncoproteins are constitutively ubiquitinated, and PP1 treatment does not induce nor affect protein ubiquitination. Taken together, these findings indicate that RET oncoproteins can bind to c-Cbl, can be ubiquitinated, and therefore targeted to proteasomal degradation irrespective of PP1 treatment, but proteosomal degradation is strongly accelerated when cells are treated with PP1.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It would be of great interest to identify inhibitors for RET tyrosine kinase and to develop new therapeutic strategies for RET-associated diseases and, in particular, for MTC, which is the common clinical manifestation of MEN2 syndromes. MTC responds very poorly to chemotherapy, and at present, the only cure involves a total thyroidectomy. Recently, Carlomagno et al. (23) have suggested that PP1 could be used to inhibit RET/PTC-induced tumorigenesis.
A key finding made in the course of the present study, which was aimed to detail the effects of PP1 on RETC634R- and RETM918T-expressing cells, was the unexpected ability of both the two related tyrosine kinase inhibitors, PP1 and PP2, to induce proteosomal destruction of the RET oncoproteins. In fact, although PP1 was shown to inhibit RET oncoprotein kinase activity (our data and Ref. 23
) and to affect RET/PTC3-dependent mitogen-activated protein kinase phosphorylation as early as 2 h after exposure (23)
, the analysis of the long-term effects of PP1 treatment showed that there was a remarkable decrease in the levels of RET oncoproteins after 12 h (Fig. 4A)
, and a clear reversion of the transformed phenotype was observed (Fig. 1)
. Moreover, cells expressing RET oncoproteins treated with PP1 could proliferate as the untreated ones for the first 2 days after PP1 addition, but at later times, a clear cytostatic effect was detectable (Fig. 5)
, and no RET oncoprotein expression was found (Fig. 4A)
. Therefore, our observations extend previous reports on a linkage between the inhibition of RET phosphorylation and cell growth (23)
and suggest that drug-induced receptor destruction clearly contributes to the cytostatic effect of tyrosine kinase inhibitors. Moreover, it is tempting to speculate that a stable phenotypical reversion of RET-transformed cells and growth arrest might be because of the initial dephosphorylation of RET and its consequent depletion.
No studies have been performed to elucidate the way RET receptor becomes physiologically degraded in cells, and no data are available on constitutively activated receptor down-regulation. For ErbB proteins, two major inducible pathways that control their degradation have been well highlighted: ligand-induced rapid endocytosis of ErbB-1 (33) ; and antibody- and oncogenic mutation-induced destruction of ErbB-2 (34) , pathways that are both mediated, at least in part, by the c-Cbl ubiquitin ligase. Cbl proteins have a key role in sorting active RTKs into invaginating pits and function as ubiquitin protein ligases that direct multiubiquitination and down-regulation of RTKs. Cbl-mediated ubiquitination accelerates EGFR endocytosis and its delivery to lysosomes for degradation (33) . Thus, the sorting of RTKs to be internalized is controlled by two types of reversible modification, phosphorylation, and ubiquitination.
The RET mutants associated with MEN2A and MEN2B used in this study are constitutively activated and therefore constitutively phosphorylated. Unexpectedly, consistent with this constitutive phosphorylation status of the RET oncoproteins, we reported that a high proportion of these oncogenic proteins are ubiquitinated and associate with c-Cbl and ubiquitin. Moreover, the treatment of the cells with PP1 does not promote nor modify these processes. In particular, the binding of c-Cbl and RET, which had yet to be demonstrated but has now been shown, is an early event for these oncogenic products that is not affected by PP1-mediated RET dephosphorylation. This is in line with our data showing that none of the RET tyrosine residues phosphorylated and/or relevant for RET-mediated mitogenesis seem to be directly involved in binding c-Cbl (data not shown). Therefore, the ability of RET to bind c-Cbl can be separated from its ability to drive mitogenic signaling and probably none of the RET tyrosine residues whose phosphorylation is impaired by the inhibitor are involved in c-Cbl binding. PP1 might then have a role in inhibiting the RET-mitogenic signaling cascade but not the pathways that control its degradation. Taken together, our results also give rise to intriguing questions on how the fate of RET oncoproteins is determined because their phosphorylation is constitutive as it is their binding to c-Cbl and their ubiquitination.
Interestingly, Citri et al. (35) indicated that the tyrosine kinase inhibitor CI-1033 directed ErbB-2 to a degradative fate mediated by the chaperone destructive system, which is functionally and structurally distinct from the c-Cbl-mediated pathway. The Cbl-mediated pathway requires kinase activity to induce degradation, whereas the chaperone-mediated route is recruited to the kinase domain mainly upon structural perturbation of the ATP-binding pocket of the oncoprotein. PP1 could act on RET oncoproteins through the activation of similar pathways and the hypothesis that PP1 could target the receptor to destruction by inducing such structural perturbation and thus recruiting the stress-inducible machinery is under current study.
In conclusion, we have shown here that the previously reported results on PP1 activity on cytoplasmic RET fusion proteins can be convincingly extended to membrane-bound RET mutants associated with MEN2A and MEN2B syndromes and to sporadic MTCs, emphasizing the potential therapeutic role of this compound. In fact, after PP1-mediated dephosphorylation, RET oncoproteins are rapidly targeted to proteosomal destruction. The interaction with the inhibitor can therefore either block most of the downstream mitogenic signaling, apart from the one driven by c-Cbl so that recycling is prevented and invagination and degradation accelerated, or it can misfold the protein structure, which is then targeted to stress-induced rapid destruction.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Telethon Foundation Grant E0993, the Italian Association for Cancer Research, and the National Counseling of Research Consiglio Nazionale Ricerche Grant 99.00522.PF49, Ministry of Health (RF2001). 
3 To whom requests for reprints should be addressed, at Department of Experimental Oncology–Istituto Nazionale Tumori, Via G. Venezian, 1 20133 Milan, Italy. Phone: 00-39-02-23-90-22-19; Fax: 00-39-02-23-90-27-64; E-mail: italia.bongarzone{at}istitutotumori.mi.it
4 The abbreviations used are: RTK, receptor tyrosine kinase; GDNF, glial cell line-derived neurotrophic factor; PTC, papillary thyroid carcinoma; MTC, medullary thyroid carcinoma; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; WCL, whole cell lysate.
Received 8/26/02. Accepted 3/ 3/03.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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