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• W2012507368 abstract "Transregulation of the epidermal growth factor receptor (EGFR) by protein kinase C (PKC) serves as a model for heterologous desensitization of receptor tyrosine kinases, but the underlying mechanism remained unknown. By using c-Cbl-induced ubiquitination of EGFR as a marker for transfer from early to late endosomes, we provide evidence that PKC can inhibit this process. In parallel, receptor down-regulation and degradation are significantly reduced. The inhibitory effects of PKC are mediated by a single threonine residue (threonine 654) of EGFR, which serves as a major PKC phosphorylation site. Biochemical and morphological analyses indicate that threonine-phosphorylated EGFR molecules undergo normal internalization, but instead of sorting to lysosomal degradation, they recycle back to the cell surface. In conclusion, by sorting EGFR to the recycling endosome, heterologous desensitization restrains ligand-induced down-regulation of EGFR. Transregulation of the epidermal growth factor receptor (EGFR) by protein kinase C (PKC) serves as a model for heterologous desensitization of receptor tyrosine kinases, but the underlying mechanism remained unknown. By using c-Cbl-induced ubiquitination of EGFR as a marker for transfer from early to late endosomes, we provide evidence that PKC can inhibit this process. In parallel, receptor down-regulation and degradation are significantly reduced. The inhibitory effects of PKC are mediated by a single threonine residue (threonine 654) of EGFR, which serves as a major PKC phosphorylation site. Biochemical and morphological analyses indicate that threonine-phosphorylated EGFR molecules undergo normal internalization, but instead of sorting to lysosomal degradation, they recycle back to the cell surface. In conclusion, by sorting EGFR to the recycling endosome, heterologous desensitization restrains ligand-induced down-regulation of EGFR. epidermal growth factor receptor 4β-phorbol 12-myristate 13-acetate protein kinase C epidermal growth factor hemagglutinin phosphate-buffered saline ubiquitin Chinese hamster ovary wild type transferrin receptor transferrin ubiquitin-protein isopeptide ligase Activation of growth factor receptors by their ligands is followed by the desensitization processes, which can be grouped into homologous and heterologous types (reviewed in Ref. 1Yarden Y. Ullrich A. Annu. Rev. Biochem. 1988; 57: 443-478Crossref PubMed Scopus (1380) Google Scholar). Homologous desensitization is initiated by ligand binding, and it entails endocytic removal of the activated receptors from the cell surface (“down-regulation”). Ligand·receptor complexes are rapidly recruited into clathrin-coated regions of the plasma membrane, which rapidly invaginate to form coated vesicles. Within minutes or less the coated vesicle delivers its content to the sorting early endosome, a peripheral vesicular compartment, whose internal pH is moderately acidic (2Gruenberg J. Maxfield F.R. Curr. Opin. Cell Biol. 1995; 7: 552-563Crossref PubMed Scopus (546) Google Scholar). Sorting of incoming epidermal growth factor receptors (EGFRs)1 to the endosomal carrier vesicle (also called the multivesicular body) depends on the intrinsic tyrosine kinase activity of EGFR, and its default pathway appears to be delivery to the recycling endosome (reviewed in Ref. 3Di Fiore P.P. Gill G.N. Curr. Opin. Cell Biol. 1999; 11: 483-488Crossref PubMed Scopus (117) Google Scholar). The mechanism underlying endosomal sorting has been recently attributed to trans-phosphorylation of the c-Cbl adaptor protein by EGFRs (4Levkowitz G. Waterman H. Zamir L. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (713) Google Scholar). Upon its phosphorylation, the c-Cbl ubiquitin ligase (5Joazeiro C.A.P. Wing S.S. Huang H.-K. Leverson J.D. Hunter T. Liu Y.-C. Science. 1999; 286: 309-312Crossref PubMed Scopus (907) Google Scholar, 6Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar) elevates receptor ubiquitination and thereby targets EGFR to proteasomal/lysosomal degradation in late endocytic compartments (4Levkowitz G. Waterman H. Zamir L. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (713) Google Scholar, 7Lill N.L. Douillard P. Awward R.A. Ota S. Lupher Jr., M.I. Miyake S. Meissner-Lula N. Hsu V.H. Band H. J. Biol. Chem. 2000; 275: 367-377Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Whereas homologous desensitization is driven by the intrinsic kinase activity of EGFR, other protein kinases play a role in heterologous desensitization by a wide variety of stimuli. These include heterologous growth factors such as the platelet-derived growth factor (8Davis J.D. Czech M.P. J. Biol. Chem. 1987; 262: 6832-6841Abstract Full Text PDF PubMed Google Scholar), calcium (9Fearn J.C. King A.C. Cell. 1985; 40: 991-1000Abstract Full Text PDF PubMed Scopus (70) Google Scholar), and 4β-phorbol 12-myristate 13-acetate (PMA; Ref. 10Lund K.A. Lazar C.S. Chen W.S. Walsh B.J. Herbst J.B. Walton G.M. Rosenfeld M.G. Gill G.N. Wiley H.S. J. Biol. Chem. 1990; 265: 20517-20523Abstract Full Text PDF PubMed Google Scholar and references therein). Both calcium and PMA stimulate protein kinase C (PKC), whose major phosphorylation site on the EGFR is threonine 654 (11Hunter T. Ling N. Cooper J.A. Nature. 1984; 311: 480-483Crossref PubMed Scopus (418) Google Scholar). Of the multiple effects of PKC on EGFR, both inhibition of tyrosine kinase activity and deceleration of receptor down-regulation have been attributed to threonine 654, but the disappearance of high affinity EGF binding sites appears independent of this residue (10Lund K.A. Lazar C.S. Chen W.S. Walsh B.J. Herbst J.B. Walton G.M. Rosenfeld M.G. Gill G.N. Wiley H.S. J. Biol. Chem. 1990; 265: 20517-20523Abstract Full Text PDF PubMed Google Scholar). Although trans-regulation by PKC significantly alters signaling downstream to EGFR, the exact mechanism remains unknown. For example, PKC activation causes translocation and stimulation of certain tyrosine phosphatases (12Zhao Z. Shen S.-H. Fisher E.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5007-5011Crossref PubMed Scopus (53) Google Scholar), which may explain why the surface EGFR is desensitized, but upon endocytosis its phosphorylation is significantly enhanced (13Vieira A.V. Lamaze C. Schmid S.L. Science. 1996; 274: 2086-2088Crossref PubMed Scopus (818) Google Scholar). According to an alternative model, PKC affects internalization of EGFR through a mechanism distinct from the one stimulated by EGF binding (14Lin C.R. Chen W.S. Lazar C.S. Carpenter C.D. Gill G.N. Evans R.M. Rosenfeld M.G. Cell. 1986; 44: 839-848Abstract Full Text PDF PubMed Scopus (189) Google Scholar). Further, on the basis of the observation that PMA inhibits internalization and significantly reduces tyrosine kinase activity, it has been concluded that the juxtamembrane domain is involved in the transmission of conformational information from the extracellular ligand binding site to the cytoplasmic kinase domain (10Lund K.A. Lazar C.S. Chen W.S. Walsh B.J. Herbst J.B. Walton G.M. Rosenfeld M.G. Gill G.N. Wiley H.S. J. Biol. Chem. 1990; 265: 20517-20523Abstract Full Text PDF PubMed Google Scholar). One such mechanism may involve inhibition of receptor dimerization (15Kuppuswamy D. Pike J.L. Cell. Signalling. 1991; 3: 107-117Crossref PubMed Scopus (11) Google Scholar), but a recent study proposed that phosphorylation at the juxtamembrane domain stabilizes ligand-induced receptor dimers (16Gulliford T. Ouyang X. Epstein R.J. Cell. Signalling. 1999; 11: 245-252Crossref PubMed Scopus (13) Google Scholar). Thus, despite a consensus that PKC affects several receptor functions, a unifying model is still unavailable. Because earlier works showed that activation of PKC leads to a disappearance of the unoccupied EGFR from the cell surface, but this is not accompanied by receptor degradation (14Lin C.R. Chen W.S. Lazar C.S. Carpenter C.D. Gill G.N. Evans R.M. Rosenfeld M.G. Cell. 1986; 44: 839-848Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 17Beguinot L. Hanover J.A. Ito S. Richert N.D. Willingham M.C. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2774-2778Crossref PubMed Scopus (127) Google Scholar), we suspected that an endocytic mechanism may provide an explanation. To test this model we utilized receptor ubiquitination as a biochemical indication for EGFR sorting to the late endosome (4Levkowitz G. Waterman H. Zamir L. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (713) Google Scholar). The results we present indicate that phosphorylation at threonine 654 diverts internalized EGFR molecules from a degradative fate to a recycling pathway. The emerging regulatory role of PKC in vesicular trafficking of a growth factor receptor may be relevant to other surface molecules whose endocytosis is ligand-mediated. Iodogen, EGF, monensin, and PMA were purchased from Sigma. GF109203X was from Biomol (Plymouth Meeting, PA). Radioactive materials were from Amersham Pharmacia Biotech. The SG565 monoclonal antibody to EGFR was generated in our laboratory. The anti-hemagglutinin (HA) monoclonal antibody was purchased from Roche Molecular Biochemicals. Antibodies to PKC isoforms, phosphotyrosine, and c-Cbl were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The active, doubly phosphorylated form of extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2 (mitogen-activated protein kinase) was detected by using a previously described antibody (18Yung Y. Dolginov Y. Yao Z. Rubinfeld H. Michael D. Hanoch T. Roubini E. Lando Z. Zharhari D. Seger R. FEBS J. 1997; 408: 292-296Crossref PubMed Scopus (113) Google Scholar). Binding buffer contained RPMI 1640 medium supplemented with 0.5% bovine serum albumin and 20 mm HEPES. Solubilization buffer contained 50 mm Tris, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Nonidet P-40, 1 mm EGTA, 1 mmphenylmethylsulfonyl fluoride, 1 mmNa3VO4, 10 μg/ml pepstatin A, 10 μg/ml aprotinin, and 10 μg/ml leupeptin. HNTG buffer contained 20 mm HEPES, pH 7.5, 150 mm NaCl, 0.1% Triton X-100, and 10% glycerol. EGF was labeled with 125I by using the Iodogen reagent. Cells were exposed to the indicated treatments in serum-free Dulbecco's modified Eagle's medium. After treatment, cells were extracted in solubilization buffer and mixed harshly, and lysates were cleared by centrifugation. The EGFR in the lysate supernatants was immunoprecipitated for 2 h at 4 °C. Immunoprecipitates were washed three times with HNTG, resolved by gel electrophoresis, and transferred to a nitrocellulose membrane. Membranes were blocked for 1 h in PBS containing 0.5% Tween-20 and 1% milk and blotted for 2 h with a primary antibody (1 μg/ml) followed by a secondary antibody (0.5 μg/ml) linked to horseradish peroxidase. Immunoreactive protein bands were detected with an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech). We have previously described the construction of c-Cbl in the pCDNA3 expression vector (Invitrogen) containing the HA sequence tag (4Levkowitz G. Waterman H. Zamir L. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (713) Google Scholar). To generate the T654A and K721A mutants of EGFR (threonine 654 mutated to an alanine or lysine 721 mutated to an alanine), we used the Quick-change mutagenesis kit (Stratagene). The ubiquitin-hemagglutinin A (HA-Ub) expression vector was a gift from Dirk Bohmann (EMBL, Heidelberg, Germany). The protocols for transfection of Chinese hamster ovary (CHO) cells were exactly as described (4Levkowitz G. Waterman H. Zamir L. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (713) Google Scholar). The total amount of DNA in each transfection was normalized with an empty pcDNA3 plasmid. Cells pretreated with solvent (ethanol) or with PMA (100 nm) at 37 °C were washed with binding buffer and then incubated for up to 8 min in the presence of a radiolabeled EGF (5 ng/ml). The cells were then put on ice and washed twice with binding buffer, and cellular distribution of the radiolabeled ligand was determined by using a 7-min-long incubation in 0.5 ml of solution of 0.2 m sodium acetate (pH 4.5) containing 0.5 m NaCl. The released radioactivity was considered as cell surface-associated ligand. The remaining radioactivity was solubilized in 100 mm NaOH solution containing 0.1% SDS and considered as internalized ligand. CHO cells were transiently transfected with plasmids encoding a wild type or a mutant EGFR. Forty-eight hours post-transfection cells were treated without or with PMA for 20 min at 37 °C. EGF was then added, and incubation was continued for various time intervals. The medium was then removed, and the cells were washed once with binding buffer and twice with an acidic buffer (2.5 mm KCl, 135 mm NaCl, and 50 mm acetic acid) at room temperature to remove surface-bound ligand (19Lund K.A. Opresko L.K. Starbuck C. Walsh B.J. Wiley H.S. J. Biol. Chem. 1990; 265: 15713-15723Abstract Full Text PDF PubMed Google Scholar). The cells were then washed twice in cold binding buffer and incubated with 0.5 nm125I-EGF for 4 h at 4 °C. The cells were then washed twice and solubilized in 100 mm NaOH solution containing 0.1% SDS, and radioactivity was determined in a γ-counter. CHO cells grown in 48-well plates were transfected with expression vectors encoding a WT or T654A mutant receptor. Forty-eight hours later cells were pretreated at 37 °C with PMA, and 15 min later they were incubated at 4 °C with increasing concentrations of a radiolabeled EGF. Specific binding of the ligand was determined in duplicates after 2 h and analyzed by using the Scatchard method (20Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-672Crossref Scopus (17746) Google Scholar). To follow receptor recycling we used transfected CHO cells and a previously described protocol (21Kornilova E. Sorkina T. Beguinot L. Sorkin A. J. Biol. Chem. 1996; 271: 30340-30346Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Cells were rinsed with ice-cold binding buffer and incubated with 1 ng/ml 125I-EGF at 4 °C for 1 h. Cells were then rinsed twice with cold Dulbecco's modified Eagle's medium and allowed to internalize the ligand for 10 min at 37 °C. Next, cells were rinsed with cold Dulbecco's modified Eagle's medium, and125I-EGF remaining on the cell surface was removed by using a 2.5-min-long wash with a mildly acidic solution (0.2 msodium acetate, 0.5 m NaCl, pH 4.5).125I-EGF-loaded cells were incubated for 1 h at 4 °C with a nonradioactive EGF (100 ng/ml) to saturate surface receptors and then switched to 37 °C for different time intervals to allow for receptor trafficking. At the end of each incubation period, cells were placed on ice, and media were collected to determine the amount of degraded and intact 125I-EGF. This was followed by a 2.5-min-long acid wash (pH 2.8) to determine the amount of surface-bound 125I-EGF. Cells were then solubilized with 1 N NaOH to determine the amount of intracellular 125I-EGF. To separate intact from degraded 125I-EGF products, trichloroacetic acid and phosphotungstic acid were added to the collected medium to final concentrations of 3 and 0.3%, respectively. The mixture was incubated at 4 °C for 30 min and centrifuged to collect precipitates. These were solubilized with 1 N NaOH before counting in a γ-counter, and the supernatants were used to calculate the amount of degraded 125I-EGF. For the effect of PMA treatment, the cells were similarly treated except that preincubation for 20 min was performed with PMA. The amount of recycled125I-EGF was calculated as a fraction of the total cell-associated radioactivity (22Kil S.J. Hobert M. Carlin C. J. Biol. Chem. 1999; 274: 3141-3150Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Twenty-four hours post-transfection cells were plated on coverslips in 6-well plates and assayed 24 h later. Cells were rinsed with Dulbecco's modified Eagle's medium and then pretreated for 20 min at 37 °C without or with PMA before exposure to EGF for 15 min at 37 °C. Cells were fixed for 30 min with 3% paraformaldehyde in PBS. For immunofluorescent labeling, cells were permeabilized for 10 min at 22 °C with PBS containing 1% albumin and 0.2% Triton X-100. Coverslips were then incubated for 1 h at room temperature with a monoclonal antibody to EGFR (10 μg/ml). After washing with PBS, the coverslips were incubated with Cy3-conjugated goat-anti-rabbit F(ab′)2 (Jackson ImmunoResearch Laboratories) for an additional hour. Finally, the coverslips were mounted in Elvanol (Hoechst, Frankfurt) and viewed with a fluorescence microscope (Nikon). For co-localization experiments addressing the late endosomal compartment, cells were pretreated with PMA and then incubated for 20 min with EGF. Following fixation, cells were stained with a rabbit antibody to EGFR or with a murine antibody to lysobisphosphatidic acid (antibody 6C4) (23Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (644) Google Scholar). Antibody detection was performed with a fluoresceine-conjugated goat-anti-rabbit F(ab′)2 or Cy3-conjugated goat-anti-mouse F(ab′)2 antibodies (from Jackson ImmunoResearch Laboratories), respectively. To follow the recycling pathway we used a rhodamine-labeled transferrin (Molecular Probes Inc., Eugene, OR). Lastly, to directly follow EGF, cells were prepared on coverslips as for immunofluorescence analysis. After preincubation with PMA, cells were incubated on ice for 30 min with Texas-red EGF (0.5 μg/ml; from Molecular Probes) and then transferred to 37 °C for 15 min. Fixation was performed with 3% paraformaldehyde in PBS. Microscopic images were obtained using a Bio-Rad MRC-1024 confocal system, using an argon/krypton mixed gas laser, and mounted on a Zeiss Axiovert microscope. CHO cells were selected as a cellular system because these cells express no endogenous EGFR. The receptor was transiently expressed in living cells by using transfection, and cells were treated with EGF subsequent to their exposure to either PMA or to the solvent. Approximately a 50% reduction in the level of the cell surface localized receptor was induced after a 60-min-long exposure to EGF, but pretreatment with PMA decelerated the rate of ligand-induced down-regulation of EGFR (Fig.1 A). PMA alone partially down-regulated EGFR, in agreement with previous reports (17Beguinot L. Hanover J.A. Ito S. Richert N.D. Willingham M.C. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2774-2778Crossref PubMed Scopus (127) Google Scholar, 14Lin C.R. Chen W.S. Lazar C.S. Carpenter C.D. Gill G.N. Evans R.M. Rosenfeld M.G. Cell. 1986; 44: 839-848Abstract Full Text PDF PubMed Scopus (189) Google Scholar). Because down-regulation is the net result of receptor degradation and recycling back to the cell surface, we tested the ability of PMA to affect the ubiquitin-mediated tagging of EGFR to lysosomal degradation (4Levkowitz G. Waterman H. Zamir L. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (713) Google Scholar). Co-expression of a peptide-tagged ubiquitin together with EGFR allowed sensitive detection of ubiquitin adducts, and further enhancement of this process was achieved by simultaneous overexpression of c-Cbl. As expected, EGF-induced ubiquitination of EGFR was significantly enhanced by an overexpressed c-Cbl. However, pretreatment of living cells with PMA remarkably reduced the level of receptor ubiquitination, even in the presence of an overexpressed c-Cbl (Fig.1 B). Ubiquitination levels correlated with the degradation of EGFR; maximal degradation was observed upon EGF treatment of c-Cbl-overexpressing cells. These characteristics were better reflected in an experiment that tested increasing concentrations of PMA, up to 1 μm; at high concentrations PMA alone exerted only a small inhibitory effect on EGFR degradation, but when tested together with EGF we observed remarkable inhibition of both receptor degradation and ubiquitination (Fig. 2 A). Concomitant with these extensive inhibitory effects, PMA also moderately reduced receptor phosphorylation on tyrosine residues (Fig.2 B). Control experiments that tested mitogen-activated protein kinase activation indicated that both EGF and PMA stimulated this kinase cascade (Fig. 2 B, lower panel). Taken together, the results presented in Figs. 1 and 2 indicate that the inhibitory effects of PMA on ubiquitination and degradation of EGFR are functionally linked, suggesting a common mechanism that limits homologous desensitization of EGFR.Figure 2PMA inhibits degradation of EGFR.A, CHO cells were transfected with plasmids encoding EGFR, c-Cbl, and HA-Ub. Forty-eight hours later, cells were left untreated (−) or treated with increasing concentrations of PMA (0.1, 1.0, 10, 100, 500, and 1000 nm), as indicated. Thereafter, cells were untreated or treated for 10 min at 37 °C with EGF (100 ng/ml). Subsequently, cell lysates were prepared, and EGFR was immunoprecipitated and analyzed by immunoblotting with anti-HA or anti-EGFR antibodies. B, whole cell lysates were directly analyzed by immunoblotting with antiphosphotyrosine antibodies (P-Tyr) or with a monoclonal antibody specific to the active, doubly phosphorylated form of mitogen-activated protein kinase.IP, immunoprecipitate; IB, immunoblot.View Large Image Figure ViewerDownload (PPT) Although tumor-promoting phorbol esters act as specific binders and activators of PKC, they may also modulate other signaling pathways. Two lines of evidence implicate PKC in the reduction of receptor ubiquitination and degradation following treatment with PMA. First, inhibition of PKC by using the highly specific antagonist GF109203X abolished the inhibitory effect of PMA on receptor ubiquitination (Fig.3 A). The antagonistic effect of GF109203X on receptor degradation was apparent especially when ubiquitination and degradation were enhanced by an overexpressed c-Cbl (Fig. 3 B), suggesting that PKC action is located upstream to c-Cbl. Because EGF by itself can stimulate PKC in intact cells (24Whitley B. Glaser L. J. Cell Biol. 1986; 103: 1355-1362Crossref PubMed Scopus (46) Google Scholar), we asked whether PKC can limit receptor down-regulation following ligand binding to EGFR. Indeed, blocking PKC with the specific antagonist moderately enhanced ligand-induced ubiquitination and degradation of EGFR, especially in the presence of an overexpressed c-Cbl (Fig.3 C). This observation implies that physiological activation of PKC by EGF, probably through the activation of phospholipase Cγ, can reduce the extent of homologous desensitization. The second line of evidence implicating PKC is presented in Fig.4; chronic down-regulation of the kinase by using prolonged exposure of cells to PMA enhanced EGF-induced ubiquitination of EGFR, in line with a restrictive role of PKC in EGFR ubiquitination. In addition, the effect of a short treatment with PMA was partially impaired in PKC-depleted cells. Control Western blotting experiments confirmed enhanced degradation of both the α and ε isoforms of PKC upon long exposure to PMA (Fig. 4, lower panels). Conceivably, activation of PKC, either directly (by PMA) or indirectly (by EGF), can reduce the extent of receptor ubiquitination and subsequent degradation. The results shown in Figs. Figure 1, Figure 2, Figure 3, Figure 4 implicate PKC in an escape route from receptor degradation, but they leave open the underlying mechanism and the role played by threonine 654. To address the role of this major PKC phosphorylation site, we replaced the threonine with an alanine (mutant denoted T654A). Initial analyses confirmed that the mutant behaved as predicted by previous studies, namely its ligand-induced phosphorylation on tyrosine residues was not affected by PMA, unlike the wild type receptor, whose phosphorylation was reduced upon treatment with PMA (Figs.2 B and 5 B). Likewise, high affinity ligand binding to the wild type receptor was reduced when cells were pretreated with PMA, but this agonist of PKC only minimally affected EGF binding to the T654A mutant (Fig.6 A). Confirmation of the functional characteristics of T654A allowed us to address its ubiquitination and degradation. Evidently, replacement of threonine 654 by an alanine completely abolished the inhibitory effects of PMA on both receptor ubiquitination and receptor degradation (Fig.5 A). First, receptor ubiquitination and degradation were no longer inhibited when cells expressing T654A were exposed to a combination of EGF and PMA, and second, PMA could not abolish degradation of the T654A mutant, as it did in the case of the wild type receptor. Thus, the juxtamembrane domain of EGFR appears to play a major role in the PKC-mediated escape of EGFR from ubiquitination and degradation.Figure 6Threonine 654 of EGFR mediates the effect of PKC on receptor down-regulation and recycling of the EGF· EGFR complex. A, CHO cells were transiently transfected with a WT EGFR expression vector or with a plasmid encoding the T654A mutant (right panel). Forty-eight hours post-transfection duplicate cultures were treated for 20 min at 37 °C without (open symbols) or with PMA (100 nm, closed symbols) prior to performing a binding assay with increasing concentrations of a radiolabeled EGF. The results were analyzed by using the Scatchard method. B, cells were treated as inA, except that after exposure to PMA, or solvent alone, EGF (100 ng/ml) was added, and incubation was continued for the indicated time intervals at 37 °C. Bound EGF was removed, and the level of surface receptors was determined by incubating the cells for 1 h at 4 °C with a radiolabeled EGF. For control we tested a kinase-defective mutant of EGFR (K721A, triangles).C, CHO cells expressing WT EGFR (left panel) or the T654A mutant (right panel) were pretreated for 20 min at 37 °C without (open symbols) or with PMA (100 nm, closed symbols). Thereafter, cells were incubated for 1 h at 4 °C with a radiolabeled EGF (5 ng/ml) followed by incubation at 37 °C for various time intervals. At the end of incubation, cells were rinsed twice with binding buffer and then treated with a low pH buffer that removes surface-bound ligand. The acid-inaccessible internalized ligand is presented as a fraction of total cell-associated radioactivity prior to cell transfer to 37 °C. For control we tested the behavior of a kinase-defective mutant of EGFR (K721A, triangles). D, CHO cells were preincubated at 4 °C for 1 h with a radiolabeled EGF (5 ng/ml) and then switched to 37 °C for 10 min to allow ligand internalization. Sister cultures were pretreated with PMA prior to exposure to EGF (closed symbols). After removing surface-bound ligand, 125I-EGF-loaded cells were incubated for 1 h at 4 °C with a 100-fold excess of an unlabeled EGF. Thereafter, cells were incubated at 37 °C for the indicated periods of time. At the end of incubation, media were collected, and the fraction of degraded 125I-EGF was determined as described under “Experimental Procedures.” Surface-bound and internalized ligand fractions were then assayed. The sum of intact radiolabeled EGF (medium and surface-bound) was expressed as the percentage of total radioactivity at each time point. The average and range (bars) of duplicate determinations is shown. For control we tested a kinase-defective mutant of EGFR (K721A,triangles). Each of the experiments shown was repeated three times.View Large Image Figure ViewerDownload (PPT) Because tyrosine phosphorylation of c-Cbl is essential for ligand-induced ubiquitination and degradation of EGFR (6Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar), our next series of experiments tested the ability of the T654A mutant to engage c-Cbl. As expected, ligand binding stimulated tyrosine phosphorylation of both c-Cbl and EGFR. However, PMA treatment of cells expressing the wild type receptor, but not the T654A mutant, led to a significant reduction in ligand-induced phosphorylation of both the receptor and the substrate (Fig. 5 B). Interestingly, tyrosine phosphorylation of c-Cbl was coupled to its enhanced degradation. Both events were induced by EGF and inhibited by PMA (Fig. 5 B). It is noteworthy that according to a recent report, the macrophage growth factor can elevate ubiquitination of c-Cbl, which is followed by de-ubiquitination and no degradation (25Lee P.S. Wang Y. Dominguez M.G. Yeung Y.G. Murphy M.A. Bowtell D.D. Stanley E.R. EMBO J. 1999; 18: 3616-3628Crossref PubMed Scopus (250) Google Scholar), but EGF-induced proteolysis of c-Cbl has not been reported before. Nevertheless, mutagenesis of threonine 654 of EGFR did not protect c-Cbl from EGF-induced degradation. Taken together, the results presented in Fig. 5 imply that PKC-mediated modification of EGFR at threonine 654 impairs the ability of the modified receptor to engage c-Cbl, and therefore subsequent receptor ubiquitination and degradation are reduced. Two lines of reasoning led us to suspect that modification at threonine 654 alters intracellular routin" @default.
• W2012507368 created "2016-06-24" @default.
• W2012507368 creator A5001348816 @default.
• W2012507368 creator A5021778634 @default.
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• W2012507368 date "2000-08-01" @default.
• W2012507368 modified "2023-10-16" @default.
• W2012507368 title "Threonine Phosphorylation Diverts Internalized Epidermal Growth Factor Receptors from a Degradative Pathway to the Recycling Endosome" @default.
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