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Oncogene (2003) 22, 2942–2949 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Protein kinase Ca activation by RET: evidence for a negative feedback mechanism controlling RET tyrosine kinase Francesco Andreozzi1,2,3, Rosa Marina Melillo1,2, Francesca Carlomagno1,2, Francesco Oriente1,2, Claudia Miele1,2, Francesca Fiory1,2, Stefania Santopietro1,2, Maria Domenica Castellone1,2, Francesco Beguinot1,2, Massimo Santoro1,2 and Pietro Formisano*,1,2 1 Dipartimento di Biologia e Patologia Cellulare e Molecolare ‘L. Califano’, Università degli Studi di Napoli ‘Federico II’, via S. Pansini 5, 80131 Napoli, Italy; 2Istituto di Endocrinologia ed Oncologia Sperimentale del C.N.R. Università degli Studi di Napoli ‘Federico II’, via S. Pansini 5, 80131 Napoli, Italy We have studied the role of protein kinase C (PKC) in signaling of the RET tyrosine kinase receptor. By using a chimeric receptor (E/R) in which RET kinase can be tightly controlled by the addition of epidermal growth factor (EGF), we have found that RET triggering induces a strong increase of PKCa, PKCd and PKCf activity and that PKCa, not PKCd and PKCf, forms a liganddependent protein complex with E/R. We have identified tyrosine 1062 in the RET carboxyl-terminal tail as the docking site for PKCa. Block of PKC activity by bisindolylmaleimide or chronic phorbol esters treatment decreased EGF-induced serine/threonine phosphorylation of E/R, while it caused a similarly sized increase of EGFinduced E/R tyrosine kinase activity and mitogenic signaling. Conversely, acute phorbol esters treatment, which promotes PKC activity, increased the levels of E/R serine/threonine phosphorylation and significantly decreased its phosphotyrosine content. A threefold reduction of tyrosine phosphorylation levels of the constitutively active RET/MEN2A oncoprotein was observed upon coexpression with PKCa. We conclude that RET binds to and activates PKCa. PKCa, in turn, causes RET phosphorylation and downregulates RET tyrosine kinase and downstream signaling, thus functioning as a negative feedback loop to modulate RET activity. Oncogene (2003) 22, 2942–2949. doi:10.1038/sj.onc.1206475 Keywords: protein kinase C; tyrosine; phorbol; oncogene; thyroid; familial; papillary; signaling; carcinoma Introduction The GDNF family of neurotrophins comprises four members, collectively designated GFLs (GDNF family ligands): GDNF, Neurturin, Persephin and Artemin (Manie et al., 2001). GFLs function as ligands of a multi-component receptor complex consisting of the *Correspondence: P Formisano; E-mail: [email protected] 3 Current address: Dipartimento di Medicina Sperimentale e Clinica ‘‘G. Salvatore’’, Università degli Studi di Catanzaro ‘‘Magna Graecia’’ Received 8 August 2002; revised 11 February 2003; accepted 12 February 2003 proto-oncogenic receptor tyrosine kinase, RET and a GPI-anchored protein named GFRa. The existence of four different GFRa proteins (GFRa1, GFRa2, GFRa3 and GFRa4) gives different ligands the specificity of binding to RET-GFRa complex. The GFRa receptors are linked to the cell membrane via glycosyl-phosphatidylinositol (GPI)-anchors. This signaling complex is essential for the development of the kidney and some structures of central and peripheral nervous systems (Manie et al., 2001). The RET gene has been associated with several human diseases and genetic syndromes (Jhiang, 2000). Loss-of-function mutations in RET, which result either in the lack of expression or in the expression of a nonfunctional protein, cause defective intestinal innervation, impaired peristalsis and congenital megacolon (Hirschsprung’s disease) (Manie et al., 2001). In papillary carcinomas of the thyroid, chromosomal inversions or translocations cause the fusion of the kinase domain of RET with heterologous proteins containing dimerization motifs. This results in constitutively activated and tyrosine autophosphorylated chimeric RET oncoproteins called RET/PTC (Fagin, 2002). Finally, germline point mutations in RET result in inherited multiple endocrine neoplasia type 2A and 2B (MEN2A, MEN2B) and familial medullary thyroid carcinoma (FMTC). These mutations convert RET into a dominantly transforming oncogene. While cysteine mutations in MEN2A and FMTC activate the RET kinase by inducing disulfide-linked homodimerization, the MEN2B-associated Met918Thr substitution directly targets the RET kinase domain (Ponder, 1999). Signal transduction properties of wild-type and oncogenic RET variants are being elucidated. The cytoplasmic domain of RET contains 14 tyrosines and a longer form, that is generated by alternative splicing, contains two additional tyrosines. Phosphorylated Y1015 and Y1062 are docking sites for several signaling molecules. Phospholipase Cg(PLCg) interacts with activated RET via pY1015. RETpY1062 functions as a multiple docking site being involved in RET binding to the phosphotyrosine-binding (PTB) domains of Shc, IRS1 (insulin receptor substrate 1), FRS2 (FGF receptor substrate 2), RAI and Dok1. The RET inhibition by PKCa F Andreozzi et al 2943 Y1062-associated adaptor proteins strongly contribute to activation of several downstream signaling pathways such as the ras/MAPK or PI3K/AKT. Accordingly, Y1062 is essential for mitogenic, transforming and survival RET signaling (Manie et al., 2001). Eventually, most recently, serine phosphorylation of RET kinase induced by cAMP has been shown to be important for lamellipodia formation, resulting from Rac1 activation (Fukuda et al., 2002). Protein kinase C (PKC) enzymes have emerged as key regulators of receptor tyrosine kinases. PKCs are subdivided into three subfamilies, based on structural properties and requirement of specific cofactors (Jaken, 1996; Newton, 1997; Mellor and Parker, 1998). Conventional PKCs (cPKCs, e.g. a, b, and g) are activated by diacylglycerol (DAG) and calcium; novel PKCs (nPKCs e.g. d, e, Z and y) do not respond to calcium but require DAG for activation; and atypical PKCs (aPKCs e.g. l, z and i) are neither activated by DAG nor by calcium. PKCs might also be regulated by the stimulation of cell surface receptors, such as the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors (Nishizuka, 1988; Seedorf et al., 1995a, b). Several PKC isoforms physically interact with tyrosine kinase receptors, including epidermal growth factor (EGF) (Chen et al., 1996), insulin-like growth factor-I (IGF-I) (Li et al., 1998) and insulin (Formisano et al., 1998; Caruso et al., 2001; Oriente et al., 2001) receptors. In turn, PKC-mediated serine/threonine phosphorylation has been shown to inhibit EGFR (Decker, 1984; Downward et al., 1984; Chen et al., 1996), insulin receptor (Takayama et al., 1984; Caruso et al., 2001), and hepatocyte growth factor receptor (Met) (Gandino et al., 1994) by attenuating their intrinsic tyrosine-kinase enzymatic activity and their autophosphorylation on tyrosine. In this work we show that RET stimulation causes activation of PKC enzymes representative of the different PKC families (a, d and z) PKCa physically associates with RET, this association requiring Y1062; in turn, PKCa activation negatively modulates RET kinase and mitogenic signaling. Materials and methods Materials Media, sera and antibiotics for cell culture were from Life Technologies, Inc. (Grand Island, NY, USA). The lipofectamine reagent, rabbit polyclonal antibodies toward specific PKC isoforms and the PKC assay system (cat. #13161-013) were also purchased from Life Technologies. PKC peptide substrates were from Calbiochem-Novabiochem (La Jolla, CA, USA). Bisindolylmaleimide was from Alexis, Inc. (San Diego, CA, USA). Protein electrophoresis reagents were from Bio-Rad (Richmond, VA, USA). Western blotting and ECL reagents were from Amersham (Arlinghton Heights, IL, USA). All other chemicals were from Sigma (St Louis, MO, USA). Plasmids containing RET/MEN2A (e.g. the Cys634Arg RET mutant), RET/MEN2A-Y1062F (Melillo et al., 2001b), PKCa, PKCd and PKCz have been previously described (Formisano et al., 2000). All RET plasmids encode for RET9 isoform. Cell culture and transfection NIH3T3 and 293 cells were grown in DMEM supplemented with 10% fetal calf serum, 10 000 U/ml penicillin, 10 000 mg/ml streptomycin and 2% l-glutamine, in a humidified CO2 incubator. NIH3T3 cell lines expressing EGFR-RET (E/R) or E/R-Y1062F- and RET/GFRa1 have been previously characterized (Santoro et al., 1994; Carlomagno et al., 1998). Transient transfection of 293 cells was performed by the lipofectamine method according to the manufacturer’s instruction. By using pCAGGS-b-gal as a reporter, transfection efficiency was consistently between 45 and 60%. The expression of the plasmid constructs was routinely controlled by Western blot with specific antibodies. PKC activity assay PKC activity was measured as previously described (Formisano et al., 1998). Briefly, cells were solubilized in 20 mm Tris, pH 7.5, 0.5 mm EDTA, 0.5 mm EGTA, 0.5% Triton X-100, 25 mg/ml aprotinin, 25 mg/ml leupeptin (extraction buffer), and then clarified by centrifugation at 5000 g for 20 min. Lysates were supplemented with the lipid activators (10 mm phorbol 12-myristate 13-acetate, 0.28 mg/ml phosphatidyl serine and 4 mg/ml diolein) and phosphorylation reactions initiated by addition of the substrate solution: 50 mm Ac-MBP(4-14), 20 mm ATP, 1 mm CaCl2, 20 mm MgCl2, 4 mm Tris, pH 7.5 and 10 mCi/ml (3000 Ci/mmol) [g-32P]ATP. The reaction mixtures were further incubated for 10 min at room temperature, rapidly cooled on ice and spotted on phosphocellulose discs. Bound radioactivity was quantitated by liquid scintillation. Activity of the specific PKC isoforms was assayed by using precipitates with specific antibodies. Determination of diacylglycerol intracellular content Diacylglycerol (DAG) content was determined as described by Farese et al. (1988). Briefly, cellular lipids were extracted by chloroform/methanol/water (2 : 1 : 1, by volume) and centrifuged (2000 g for 10 min). DAG mass in lipid extracts was determined by using DAG kinase and [g-32P]ATP to convert DAG into [32P]phosphatidic acid. The latter was purified by thin layer chromatography and DAG mass was estimated by comparing the samples with diolein standards (Preiss et al., 1986). Western blot analysis and immunoprecipitation procedure Cells were solubilized in lysis buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 10 mm EDTA, 10 mm Na4P2O7, 2 mm Na4VO4, 100 mm NaF, 10% glycerol, 1% Triton X-100, 1 mm PMSF, 10 mg/ml aprotinin) for 2 h at 41C. Lysates were clarified at 5000 g for 20 min and protein concentration was determined by a modified Bradford assay (BioRad). Solubilized proteins were separated by SDS–PAGE and transferred on 0.45 mm Immobilon-P membranes (Millipore, Bedford, MA, USA). Upon incubation with the primary and secondary antibodies, immunoreactive bands were detected by enhanced chemiluminescence (ECL) (Amersham) according to the manufacturer’s instructions. Immunoprecipitation of specific PKC isoforms (Formisano et al., 1998) and RET (Santoro et al., 1994) were accomplished as previously described. Phosphorylation in intact cells NIH3T3-E/R cells were incubated in phosphate-free medium containing 100 mCi/ml [32P]orthophosphate for 8 h. EGF (10 nm) and/or TPA (100 ng/ml) were subsequently added for Oncogene RET inhibition by PKCa F Andreozzi et al 2944 15 and 30 min, respectively. Phosphorylation reactions were rapidly quenched on ice and the cells solubilized with 1 ml/dish of lysis buffer. Comparable amounts of proteins were immunoprecipitated with RET antibodies, resuspended in Laemmli buffer, separated by SDS–PAGE and analysed by autoradiography. In order to discriminate between phosphotyrosine and phospho-threonine/serine residues, the gels were incubated with 1 m KOH for 2 h at 551C. The bands corresponding to the chimeric E/R protein were excised from the gels and the radioactivity was quantitated at the b-counter. The amount of phospho-serine/threonine was estimated based on the difference of counts before and after alkali treatment. A densitometric comparison of the bands was also utilized to estimate E/R phosphorylation. Thymidine incorporation The thymidine incorporation assay was accomplished as previously reported (Santoro et al., 1994). Briefly, NIH3T3E/R cells were seeded in six-well plates and, after 18 h, fed with DMEM supplemented with 0.25% BSA. The cells were further incubated for 16 h in the absence or presence of 10 nm EGF, followed by addition of 500 nCi/ml of [3H]thymidine. After 4 h the cells were washed with ice-cold 0.9% NaCl and then with ice-cold 20% TCA followed by solubilization with 1 n NaOH. Radioactivity was quantitated by liquid scintillation counting. Results PKC stimulation by RET NIH3T3 cells expressing a chimeric construct formed by the extracellular ligand-binding domain of the EGFR and the cytosolic portion of RET (E/R) (Santoro et al., 1994) were treated with 10 nm EGF for 15 min and assayed for PKC activity. EGF exposure induced a fivefold increase of PKC activity (Figure 1a). In comparison, untransfected NIH3T3 cells, which express negligible levels of EGFR, displayed no significant increase in PKC activity upon EGF treatment. Acute (60 min) exposure to 100 nm TPA induced a 10–11-fold increase of PKC activity in both cell types. We generated E/R-Y1062F mutant by replacing RET tyrosine 1062 with phenylalanine in the E/R background and stably expressed it in NIH3T3 cells. E/R, E/R-Y1062F displayed comparable expression levels in the selected mass populations (see Figure 3a). EGF-stimulated (but not TPA-stimulated) PKC activity was reduced by >80% in E/R-Y1062F cells compared to E/R (Figure 1a). Pre-exposure to 100 nm bisindolylmaleimide (a concentration able to block PKCa, while having slight, if any, effect on PKCd and PKCz) blunted both EGF- and TPA-induced PKC activity in all cell types (Figure 1a). PKC activity was assayed in isozyme-specific (PKCa, PKCd and PKCz) immunoprecipitates. No PKCbI and bII were detectable in this cell type (data not shown). In ligand-stimulated E/R cells, PKCa, PKCd and PKCz activities were increased by 5-, 4- and 3.5-fold, respectively. The mutation of Y1062 strikingly obstructed the activation of the various PKCs tested (Figure 1b). Oncogene Figure 1 PKC activity in NIH3T3 cells expressing E/R. (a) PKC activity was measured in crude membrane preparations (50 mg/ sample) of parental, and E/R and E/R-Y1062F expressing NIH3T3 cells. Cells were analysed in basal conditions and upon stimulation with 10 nm EGF for 15 min, 100 ng/ml TPA for 30 min, either in the presence or in the absence of pretreatment with 100 nm bisindolylmaleimide for 30 min. Bars represent means7s.d. of four triplicate experiments. (b) PKC activity was assayed in isoformspecific precipitates (200 mg protein/sample) derived from E/R and E/R-Y1062F cells in basal conditions and upon stimulation with EGF, in the absence or in the presence of 15 min pretreatment with 25 mm U73122. Bars represent means7s.d. of four triplicate experiments. (c) NIH3T3 cells expressing GFRa were stimulated with GDNF (10 nm) for 15 min. PKC activity was assayed in isoform-specific precipitates of the cell lysates as described above. Bars represent means7s.d. of four triplicate experiments RET inhibition by PKCa F Andreozzi et al 2945 Similar to EGF in cells expressing the chimeric RET, GDNF (10 nm) induced activation of PKCa, d and z, respectively, by 3-, 2.5- and 2.2-fold in NIH3T3 cells transfected with GDNF receptor a (GFRa) (Figure 1c). A phospholipase C inhibitor, U73122, reduced PKCa activation by >80% in E/R cells and had no effect on PKCd and PKCz activities (Figure 1b). To further study the molecular mechanisms of PKCa activation, we have evaluated the ability of E/R and E/R-Y1062F to interact with PLCg. Both chimeric proteins coprecipitated with PLCg upon EGF stimulation (Figure 2a). In addition, EGF treatment of the cells increased intracellular diacylglycerol (DAG) content by about threefold in E/R and E/R-Y1062F cells and not in mock-transfected NIH3T3 cells (Figure 2b). RET binding to PKC isoforms Figure 2 Activation of PLCg in cells expressing E/R and E/RY1062F. (a) Lysates of parental, E/R and E/R-Y1062F cells (400 mg protein/sample), unstimulated or stimulated with EGF for 15 min, as indicated, were precipitated with anti-RET and blotted with PLCg antibody. The autoradiograph is representative of three independent experiments. (b) Parental, E/R and E/R-Y1062F cells were stimulated with 10 nm EGF for 15 min. Cellular lipids were extracted by chloroform/methanol/water, and DAG mass was measured as described in Materials and methods. Bars represent means7s.d. of four triplicate experiments NIH3T3-E/R cells were stimulated with EGF or left untreated. Cells were harvested and protein lysates were precipitated with anti-RET and immunoblotted with isoform-specific PKC antibodies. PKCa coprecipitated with E/R in EGF-stimulated cells (Figure 3a); conversely, neither PKCd nor PKCz was detectable in E/R precipitates (data not shown). Similarly, E/R was detected in PKCa immunoprecipitates, while absent in PKCd and PKCz precipitates (Figure 3b). Interestingly, EGF treatment failed to induce coprecipitation of E/R-Y1062F with PKCa, demonstrating that Y1062 is essential for RET-PKCa association (Figure 3a). Comparable amounts of chimeric proteins were precipitated by RET antibodies in E/R and E/R-Y1062F cells (Figure 3a) and no difference in PKCa, PKCd and PKCz expression levels was observed in the different cell lines (Figure 3c). PKCa coprecipitation with E/R was evaluated in cells which have been either chronically (24 h) treated with TPA, in order to provoke cPKCs downregulation, or treated with 100 nm bisindolylmaleimide for 30 min. In both cases, EGF exposure of the cells did not induce any significant increase of Figure 3 Co-precipitation of E/R and PKCa. (a) Lysates of parental, E/R and E/R-Y1062F cells (400 mg protein/sample) were precipitated with anti-RET and blotted with either PKCa (top) or RET antibody (bottom). (b) The same lysates were precipitated with isoform-specific PKC antibodies and blotted with RET antibody. (c) Control lysates (50 mg) were blotted with PKC antibodies. (d) E/R cells were treated with either 100 nm bisindolylmaleimide for 30 min or with 100 ng/ml TPA for 24 h; lysates were precipitated with RET Ab and blotted with PKCa Ab. Aliquots of the lysates were directly blotted with PKCa Ab, without prior RET precipitation, to ensure TPA-induced PKC downregulation. These pictures are representative of at least four independent experiments Oncogene RET inhibition by PKCa F Andreozzi et al 2946 PKCa coprecipitation with E/R (Figure 3d), thus indicating that PKCa activation is required for its docking to RET. To confirm the physical association between RET and PKCa we used RET/MEN2A, a naturally occurring oncogenic RET mutant responsible for the familial transmission of the MEN2A cancer syndrome. In the MEN2A mutant, the substitution of an extracellular cysteine (C634) with an arginine causes a ligandindependent dimerization mediated by disulfide bonds and constitutive activation (Santoro et al., 1995). 293 cells were transiently transfected with RET/MEN2A or its Y1062F and Y1015F mutants. Cells expressing comparable protein levels (Figure 4a) were harvested and RET/MEN2A-PKCa association was evaluated. As shown in Figure 4a, PKCa coprecipitated with RET/ MEN2A and only very faintly with Y1062F and Y1015 mutants. PKCd and PKCz, although detectable in total cell lysates, were not revealed in RET/MEN2A precipitates (data not shown), thus confirming that RET selectively binds to PKCa. Interestingly, expression of RET/MEN2A, but not of its mutated counterparts, caused a 5.6-fold increase of PKC activity (Figure 4b). Figure 4 RET/MEN2A binding to and activation of PKC. (a) Lysates (400 mg protein/sample) of 293 cells, mock-transfected or transiently transfected with either RET/MEN2A, RET/MEN2AY1062F or -Y1015F, as indicated, were precipitated with RET Ab and blotted with either PKCa (top) or RET Ab (bottom). Also, blotting with antibodies to P-Y1062 confirmed lack of RET/ MEN2A-Y1062F phosphorylation (not shown). (b) PKC activity was measured in crude membrane preparations (50 mg/sample) of mock-, RET/MEN2A-, RET/MEN2A-Y1062F- or RET/MEN2AY1015F-transfected 293 cells. Bars represent means7s.d. of four triplicate experiments Negative control exerted by PKC on RET kinase Tyrosine autophosphorylation of E/R, a measure of its activity, was assessed in NIH3T3-E/R cells by phosphotyrosine blotting. As expected, E/R tyrosine phosphorylation increased upon EGF treatment in a dose-dependent fashion (Figure 5a). Pretreatment of the cells with bisindolylmaleimide to inhibit PKCa caused a threefold increase of E/R tyrosine phosphorylation in the absence of EGF stimulation and potentiated E/R response to EGF. For example, in the Figure 5 PKC effects on RET tyrosine phosphorylation. (a) E/R cell lysates were immunoprecipitated with RET and blotted with either anti-RET or antiphosphotyrosine. Where indicated, bisindolylmaleimide was added for 30 min and EGF for additional 15 min. Densitometric analysis of five experiments7s.d. is shown. (b) E/R cells were treated with 100 ng/ml TPA prior to 15 min stimulation with 10 nm EGF. Cell lysates were immunoblotted as indicated. Densitometric analysis of five experiments7s.d. is shown Oncogene RET inhibition by PKCa F Andreozzi et al 2947 presence of bisindolylmaleimide, 1 nm EGF induced a E/ R phosphotyrosine content comparable to that obtained with 10 nm EGF in the absence of bisindolylmaleimide. Conversely, acute exposure to TPA, to stimulate PKC, decreased by threefold both basal and EGF-induced E/R tyrosine phosphorylation (Figure 5b). NIH3T3-E/R cells were metabolically labeled with 32 P-orthophosphate. E/R precipitates were separated by Figure 6 PKC effects on RET serine/threonine phosphorylation. (a) 32P-labeled E/R cells were treated with 10 nm EGF and 100 ng/ml TPA as indicated. Cell lysates were immunoprecipitated with RET Ab, separated by SDS–PAGE and autoradiographed. The autoradiograph is representative of four independent experiments. The bar graph represents a densitometric quantitation of 32P content of the bands. (b) E/R cells were exposed to bisindolylmaleimide, EGF or TPA, as indicated. Phospho-serine/threonine content was quantitated as described in Materials and methods, upon alkali treatment of the gels. The bands have been excised from the gel and the amount of phospho-serine/threonine has been estimated based on the difference of counts before and after alkali treatment. Bars represent means7s.d. of four independent experiments SDS–PAGE and analysed by autoradiography to detect overall (both tyrosine and serine/threonine) phosphorylation. EGF and 100 ng/ml TPA increased E/R phosphorylation (Figure 6a). Bands were excised from SDS–PAGE gels before and after treatment with alkali to hydrolyse phosphoserine and phosphothreonine and counted. The amount of phospho-serine/threonine was estimated based on the difference of counts before and after alkali treatment. As shown in Figure 6b, EGF exposure increased serine/threonine phosphorylation of the chimeric protein by about fourfold. Acute treatment with 100 ng/ml TPA (30 min) also induced a sixfold increase of E/R phosphorylation. In the absence of other stimuli, bisindolylmaleimide treatment decreased E/R serine/threonine phosphorylation by twofold, and it returned EGF- and TPA-effects to levels comparable to the basal ones (Figure 6b). EGF stimulation induces DNA synthesis of NIH3T3E/R cells (Santoro et al., 1994). E/R cells were incubated with either 1 or 10 nm EGF in the presence or the absence of 100 nm bisindolylmaleimide for 15 h prior to measuring 3H-thymidine incorporation. As shown in Figure 6, EGF stimulation led to a dose-dependent increase of thymidine incorporation in E/R cells. PKC inhibition by bisindolylmaleimide significantly potentiated the mitogenic response to EGF. Depletion of endogenous cPKCs by 24 h exposure to TPA was similarly accompanied by a 1.6-fold increase of mitogenic response to EGF (Figure 7). Finally, to directly prove RET kinase inhibition by PKCa, 293 cells were transiently cotransfected with RET/MEN2A and either PKCa, PKCd or PKCz expression vectors. Each PKC isoform was overexpressed by 8–10-fold over the basal endogenous levels (Figure 8a). PKC activity assayed in PKCa, PKCd and PKCz transfectants was increased by 5–8-fold with respect to untransfected cells (data not shown). Transfectants expressing similar RET/MEN2A protein levels Figure 7 Effects of PKC inhibition on thymidine incorporation in E/R cells. Thymidine incorporation was assayed in E/R cells treated with either 1 or 10 nm EGF for 16 h as indicated, in the absence or in the presence of preincubation with bisindolylmaleimide (100 nm) or TPA (100 ng/ml) for 24 h. Bars represent means7s.d. of four triplicate experiments Oncogene RET inhibition by PKCa F Andreozzi et al 2948 Figure 8 PKCa effects on RET/MEN2A phosphorylation. Panels a and b: Lysates were harvested from 293 cells, either mocktransfected (lane 1), transfected with RET/MEN2A alone (lane 2) or transiently cotransfected with RET/MEN2A and PKCa (lane 3), PKCd (lane 4) or PKCz (lane 5) and immunoblotted as indicated. Panel a shows a control of the expression of individual PKC isoforms with specific antibodies. In panel b, cell lysates have been immunoprecipitated with RET antibodies. The upper autoradiograph shows the control of RET/MEN2A expression with specific antibodies and the lower autoradiograph has been obtained by blotting RET precipitates with antiphosphotyrosine antibodies. The autoradiographs shown are representative of three independent experiments. Panel c shows the densitometric analysis of phosphoRET/RET ratio. Bars represent means7s.d. of data obtained from three independent experiments were selected (Figure 8b). Antiphosphotyrosine blotting revealed that overexpression of PKCa reduced RET/ MEN2A phosphotyrosine content by at least threefold, as compared to the cells expressing RET/MEN2A alone (Figure 8c). PKCz had only modest effects; PKCd had virtually no effect (Figure 8c). Discussion Growth factor binding triggers RET dimerization, kinase activation and tyrosine autophosphorylation. Oncogene Phosphorylation of Y905 in the catalytic core stabilizes the active conformation of the kinase and facilitates the autophosphorylation of tyrosine residues mainly located in the C-terminal tail, including Y1062 (Iwashita et al., 1996). Once phosphorylated, tyrosine 1062 acts as a multiple docking site recruiting several PTB-domain containing docking proteins (Shc, FRS2, IRS1 and Dok) which, in turn, bind to adapters, like Grb2 and Gab1, or enzymes like phosphatidylinositol 3 kinase (PI3K) and the SHP2 phosphatase, thus activating intracellular signaling events (Manie et al., 2001). Here, by using a RET kinase regulatable by the addition of EGF (E/R) and a constitutively active RET kinase (RET/MEN2A), we show that protein–protein interactions at the level of tyrosine 1062 are likely implicated also in RET signaling termination. Upon activation, RET tyrosine 1062 binds to PKCa and induces PKCa activation. This binding can be either direct or, more likely, indirect and mediated by the multiple protein scaffolds that associate to this residue. The lack of PKCa stimulation by the Y1062F RET mutant can be explained by multiple mechanisms. First of all, Y1062 is required for PKCa binding. In addition, Y1062 may also be necesary to trigger signals activating PKCa. Indeed, by recruiting IRS1, which directly binds to the p85 regulatory subunit of PI3K (Melillo et al., 2001a), and Shc and FRS2, which recruit Grb2–Gab1 complexes, thereby associating with PI3K (Besset et al., 2000; Melillo et al., 2001b), RET Y1062 is crucial for PI3K activation by RET (De Vita et al., 2000; SegouffinCariou and Billaud, 2000). PI3K-generated phosphoinositides can act as endogenous activators of PKC isoforms (Nakanishi et al., 1993; Le Good et al., 1998). It is therefore possible that the impairment of PI3K recruitment determines the lack of PKCs’ activating ability of Y1062F RET mutants. This hypothesis is consistent with our recent finding that IRS1 is involved in mediating PKCa and PKCb activation by insulin (Formisano et al., 2000). Phosphorylated RET tyrosine 1015 directly interacts with phospholipase Cg (PLCg) to enhance DAG production (Borrello et al., 1996). We have observed that block of PLCg activity by U73122 inhibits RET-induced activation of PKCa, and not of PKCd and z. However, RET Y1062F mutants retain the ability to interact with PLCg and nonetheless they fail to activate PKCs, suggesting that PLCg recruitment, though necessary, is not sufficient to trigger PKC activation. By modulating endogenous PKCa with TPA or bisindolylmaleimide or by plasmid transfections, we have found that PKCa promotes RET phosphorylation on threonine/serine residues, phosphoserine content being more abundant than phosphothreonine (not shown). It is likely that PKCa phosphorylates RET directly. Indeed, inspection (at http://scansite.mit.edu/ (Yaffe et al., 2001)) of the intracellular RET sequence revealed three sites (threonine 675 in the juxtamembrane domain, and serines 811 and 819 in the C-terminal lobe of the kinase) confirming the PKCa phosphorylation consensus sequence. Inhibition of RET serine/threonine RET inhibition by PKCa F Andreozzi et al 2949 phosphorylation by bisindolylmaleimide was accompanied by increased RET autophosphorylation on tyrosine. Conversely, significantly decreased RET tyrosine phosphorylation was obtained when PKCa was overexpressed or enhanced by acute phorbol esters treatment, thereby indicating that PKCa activity negatively modulates RET tyrosine kinase activity. In addition, RET mitogenic signaling was enhanced by PKC inhibition and/or downregulation. In conclusion, our findings unveil a negative feedback circuit controlling RET signaling. According to this model, RET activation causes phosphorylation of Y1062 which binds to and leads to the activation of PKCa. PKCa, in turn, promotes RET phosphorylation on serine/threonine and inhibits RET kinase activity thus leading to signaling termination. Acknowledgements This study was supported in part by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), by the EC grants FIGH-CT1999-CHIPS and QLRT-1999-00674, by the Programma Biotecnologie legge 95/95 (MURST 5%), by BioGeM s.c.ar.l. (Biotecnologia e Genetica Molecolare nel Mezzogiorno d’Italia), by the Ministero dell’ Università e della Ricerca Scientifica. The financial support of Telethon — Italy (Grant no. 0896 to FB) is gratefully acknowledged. F Fiory is the recipient of a fellowship of the Federazione Italiana per la Ricerca sul Cancro (FIRC). References Besset V, Scott RP and Ibanez CF. (2000). J. Biol. Chem., 275, 39159–39166. Borrello MG, Alberti L, Arighi E, Bongarzone I, Battistini C, Bardelli A, Pasini B, Piutti C, Rizzetti MG, Mondellini P, Radice MT and Pierotti MA. (1996) Mol. Cell. Biol., 16, 2151–2163. Carlomagno F, Melillo RM, Visconti R, Salvatore G, De Vita G, Lupoli G, Yu Y, Jing S, Vecchio G, Fusco A and Santoro M. 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